Nanomaterials for Cancer Diagnosis (Nanotechnologies for the Life Sciences, Volume 7)

VII

Contents Preface

XVII

List of Authors 1

1.1 1.2 1.3 1.3.1 1.3.1.1 1.3.1.2 1.3.1.3 1.3.2 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.2 1.4.2.1 1.4.2.2 1.4.3 1.4.3.1 1.4.3.2 1.5 1.5.1

XXI

Dendrimers in Cancer Treatment and Diagnosis 1 Srinivasa-Gopalan Sampathkumar, and Kevin J. Yarema Overview 1 Introduction 1

Basic Properties and Applications of Dendrimers 3 Structural Features and Chemical and Biological Properties 3 Basic Features of Dendritic Macromolecules are Inspired by Nature 3 Comparison of the Properties of Dendrimers and Conventional Synthetic Polymers 5 Comparison of the Properties of Dendrimers and Proteins (a Biological Polymer) 6 Dendritic Macromolecules Possess a Wealth of Possible Applications 8 Methods for Dendrimer Synthesis 10 History and Basic Strategies 10 Cascade Reactions are the Foundation of Dendrimer Synthesis 10 Dendrimer Synthesis has Expanded Dramatically in the Past Two Decades 12 Strategies, Cores, and Building Blocks for Dendritic Macromolecules 12 Dendrimers are Constructed from Simple ‘‘Building Blocks’’ 12 The Synthesis of Dendrimers Follows Either a Divergent or Convergent Approach 13 Heterogeneously-functionalized Dendrimers 13 Basic Description and Synthetic Considerations 13 Glycosylation is an Example of Surface Modification with Multiple Bioactivities 14 Dendrimers in Drug Delivery 15 Dendrimers are Versatile Nano-devices for the Delivery of Diverse Classes of Drugs 15

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

VIII

Contents

1.5.2 1.5.2.1 1.5.2.2 1.5.2.3 1.5.3 1.5.3.1 1.5.3.2 1.5.3.3 1.5.4 1.5.4.1 1.5.4.2 1.5.5 1.5.5.1 1.5.5.2 1.5.5.3 1.6 1.6.1 1.6.2 1.6.3 1.6.3.1 1.6.3.2 1.6.3.3 1.6.4 1.6.4.1 1.6.4.2 1.6.5 1.6.5.1 1.6.5.2 1.6.6 1.6.6.1 1.6.6.2 1.6.6.3 1.7

Dendritic Drug Delivery: Encapsulation of Guest Molecules 16 Dendrimers have Internal Cavities that can Host Encapsulated Guest Molecules 16 Using Dendrimers for Gene Delivery 16 Release of Encapsulated ‘‘Pro-drugs’’ 17 Covalent Conjugation Strategies 17 Dendrimers Overcome many Limitations Inherent in Polymeric Conjugation Strategies 17 Dendrimer Conjugates can be Used as Vaccines 19 Release of Covalently-delivered ‘‘Pro-drugs’’ 19 Fine-tuning Dendrimer Properties to Facilitate Delivery and Ensure Bioactivity 20 Delivery Requires Avoiding Non-specific Uptake 20 ‘‘Local’’ Considerations: Contact with, and Uptake by, the Target Cell 21 Drug Delivery: Ensuring the Biocompatibility of Dendritic Delivery Vehicles 22 Biocompatibility Entails Avoiding ‘‘Side Effects’’ such as Toxicity and Immunogenicity 22 Water Solubility and Immunogenicity 22 Inherent and Induced Toxicity 23 Dendrimers in Cancer Diagnosis and Treatment 24 Dendrimers have Attractive Properties for Cancer Treatment 24 Dendrimer-sized Particles Passively Accumulate at the Sites of Tumors 24 Multifunctional Dendrimers can Selectively Target Biomarkers found on Cancer Cells 25 Methods for Targeting Specific Biomarkers of Cancer 25 Targeting by Folate, a Small Molecule Ligand 26 Targeting by Monoclonal Antibodies 26 Dendrimers in Cancer Diagnosis and Imaging 28 Labeled Dendrimers are Important Research Tools for Biodistribution Studies 28 Towards Clinical Use: MRI Imaging Agents 28 Steps Towards the Clinical Realization of Dendrimer-based Cancer Therapies 29 The Stage is now set for Dendrimer-based Cancer Therapy 29 Boron Neutron Capture Therapy 29 Innovations Promise to Speed Progress 30 ‘‘Mix-and-Match’’ Strategy of Bifunctional Dendritic Clusters 30 Towards Therapeutic Exploitation of Glycosylation Abnormalities found in Cancer 31 Towards Targeting Metabolically-engineered Carbohydrate Epitopes 31 Concluding Remarks 33

Contents

Acknowledgments References 33 2

2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.3 2.3.1 2.3.2 2.3.3 2.4 2.4.1 2.4.2 2.4.2.1 2.4.2.2 2.4.2.3 2.5 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.6 2.7

3

3.1 3.2 3.3 3.3.1 3.3.1.1 3.3.1.2 3.3.1.3 3.3.1.4

33

Nanoparticles for Optical Imaging of Cancer Swadeshmukul Santra and Debamitra Dutta Introduction 44 Cancer Imaging Techniques 46

44

Computed Tomography (CT) Scanning 47 Magnetic Resonance (MR) 47 Positron Emission Tomography (PET) 47 Single-photon Emission CT (SPECT) 48 Ultrasonography (US) 48 Optical Imaging 48 Basics of Optical Imaging 48 Optical Imaging Techniques 49 Optical Contrast Agents 50 Nanoparticles for Optical Imaging 51 Why Nanoparticles for Optical Imaging? 51 Development of Nanoparticle-based Contrast Agents 53 Quantum Dots 53 Gold Nanoparticles 57 Dye-doped Silica Nanoparticles 61 Optical Imaging of Cancer with Nanoparticles 65 Active Targeting 65 Passive Targeting 66 Cancer Imaging with Quantum Dots 66 Cancer Imaging with Gold Nanoparticles 68 Cancer Imaging with Dye-doped Silica Nanoparticles 69 Other Nanoparticle-based Optical Contrast Agents 70 Conclusions and Perspectives 70 Acknowledgments 72 References 72 Nanogold in Cancer Therapy and Diagnosis 86 Priyabrata Mukherjee, Resham Bhattacharya, Chitta Ranjan Patra, and Debabrata Mukhopadhyay Introduction 86 Medicinal use of Gold: A Historical Perspective 87 Application of Gold Nanoparticles in Cancer 88 Angiogenesis and Cancer 88 Agents that Inhibit Endothelial Proliferation or Response 90 Agents that Block Activation of Angiogenesis 91 Agents that Block Extracellular Matrix Breakdown 91 Unique Anti-angiogenic Properties of Gold Nanoparticles 91

IX

X

Contents

3.3.1.5 3.3.1.6 3.3.1.7

Gold Nanoparticles Inactivate VEGF165 92 What is the Mechanism of Action? 93 Effect of Gold Nanoparticles on the Activity of VEGF165, VEGF121, bFGF and EGF 94 3.3.1.8 Effect of Gold Nanoparticles on Signaling Events of VEGF165 94 3.3.1.9 Effect of Nanogold on Downstream Signaling events of VEGF165 95 3.3.1.10 Effect of Gold Nanoparticles on Migration of HUVEC Cells 95 3.3.1.11 Effect of Gold Nanoparticles on Angiogenesis in vivo 95 3.3.1.12 Gold Radioisotopes in Cancer Treatment 96 3.3.2 Application of Gold Conjugates in the Treatment of Cancer 96 3.3.2.1 Gold–TNF Conjugate in Cancer Therapeutics 96 3.3.2.2 ‘‘2 in 1’’ System in Cancer Therapeutics 97 3.4 Biocompatibility of Gold Nanoparticles 99 3.4.1 Cellular Adhesion Effects 99 3.4.2 Local Biological Effects 99 3.4.3 Systemic and Remote Effects 99 3.4.4 Effects of the Host on the Implant 100 3.4.5 Addressing the Biocompatibility of Gold Nanoparticles using DNA Microarray Analysis 100 3.4.6 Internalization of Gold Nanoparticles by HUVECs 101 3.4.7 Nanogold Particles do not Alter Global Pattern of Transcription by HUVEC Cells under Serum-free Conditions 101 3.4.8 Nanogold Particles do not Alter the Global Pattern of Transcription by HUVECs in Near-normal Culture Conditions 103 3.5 Synthetic Approaches to Gold Nanoparticles 105 3.5.1 Chemical Methods 105 3.5.2 Physical Methods 105 3.5.3 Biological Methods 106 3.6 Nanotechnology in Detection and Diagnosis with Gold Nanoparticles 106 3.6.1 Cancer Detection 106 3.6.2 Detection in DNA 107 3.6.2.1 Single-mismatch Detection in DNA 107 3.7 Future Direction 109 Acknowledgments 110 References 110 4

4.1 4.2 4.3 4.3.1 4.4 4.4.1

Nanoparticles for Magnetic Resonance Imaging of Tumors 121 Tillmann Cyrus, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza Introduction 121 Magnetic Resonance Imaging (MRI) 121 Targeting Mechanisms 124 Passive versus Active Targeting 124 Superparamagnetic Nanoparticles 126 Ligand-directed Targeting of Iron Oxides 127

Contents

4.4.2 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.6 4.7 4.8

Cell Tracking of Iron Oxides 128 Paramagnetic Nanoparticles 128 Perfluorocarbon Nanoparticles 129 Liposomes 131 Fullerenes 132 Nanotubes 133 Dendrimers 133 Quantum Dots 133 Polymer Nanoparticles 134 Conclusion 135 References 138

5

Magnetic Resonance Nanoparticle Probes for Cancer Imaging 147 Young-wook Jun, Jung-tak Jang, and Jinwoo Cheon Introduction 147 Magnetic Nanoparticle Contrast Agents 150 Silica- or Dextran-coated Iron Oxide Contrast Agents 150 Magnetoferritin 152 Magnetodendrimers and Magnetoliposomes 152

5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.4

6

6.1 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.3 6.3.1 6.3.1.1 6.3.1.2 6.3.2

New Type of Contrast Agent: Non-hydrolytically Synthesized High Quality Iron Oxide Nanoparticles 154 Iron Oxide Nanoparticles in Molecular MR Imaging 157 Infarct and Inflammation 158 Angiogenesis 159 Apoptosis 160 Gene Expression 161 Cancer Imaging 163 Summary and Outlook 166 References 169 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 174 Carola Leuschner Introduction 174 Cancer 175 Conventional Approaches to Cancer/Metastases Detection 177

Current Chemotherapeutic Approaches and their Disadvantages in Cancer Treatments 179 Multidrug Resistance 179 Drug Delivery to Tumors 180 Nanoparticles as Vehicles for Drug Delivery and Diagnosis 181 Targeting Tumor Cells 183 Passive Targeting 183 Active Targeting 185 Detection of Tumors and Metastases using Nanoparticles 186

XI

XII

Contents

6.3.2.1 6.3.2.2 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.5 6.5.1 6.5.2 6.5.3 6.5.3.1 6.5.4 6.5.4.1 6.5.4.2 6.5.4.3 6.6

7

7.1 7.2 7.2.1 7.2.1.1 7.2.1.2 7.2.2 7.2.2.1 7.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.4.1 7.4.4.2 7.4.4.3 7.5 7.6

Nanoparticles for Magnetic Resonance Imaging 186 Targeted Delivery of Nanoparticles to Increase Cellular Uptake for Higher MRI Resolution 188 LHRH and its Receptors 189 The Ligand Luteinizing Hormone Releasing Hormone – LHRH 189 Analogs of LHRH 192 Receptors for LHRH 193 Function–Signal Transduction Pathways 194 LHRH Receptor-mediated Uptake 197 LHRH Receptor Type II 198 LHRH-bound Magnetic Nanoparticles 201 Synthesis and Characterization 201 Treatment using Hyperthermia 202 Treatment using Lytic Peptides 203 Destruction of Metastases through LHRH-SPION-Hecate 203 Detection of Tumors and Metastases 204 Targeted Delivery of SPION Contrast Agents for MRI 204 In Vitro Studies on Receptor-targeted LHRH-SPION Uptake 205 In Vivo Studies on Receptor-targeted LHRH-SPION Uptake 206 Future Outlook 210 Acknowledgments 212 Abbreviations 212 References 213 Carbon Nanotubes in Cancer Therapy and Diagnosis Pu Chun Ke and Lyndon L. Larcom Overview 232

232

SWNT Modification for Solubility and Biocompatibility 234 Chemical Modifications of SWNTs for Solubility 234 Functionalization of SWNTs through Oxidation 235 Functionalization of SWNTs through Covalent Modifications 236 Noncovalent Modifications of SWNTs for Solubility 237 Solubilization of SWNTs Using Lysophospholipids Enables Cellular Studies 239 Diffusion of SWNT–Biomolecular Complexes 247 Gene and Drug Delivery with SWNT Transporters 252 RNA Translocation with SWNT Transporters 253 Gene Transfection with SWNT Transporters 258 Gene Transfection with SWNT Transporters for RNA Interference 261 Drug Delivery with SWNT Transporters 263 Vaccine Delivery by SWNTs 263 Protein Delivery by SWNTs 266 Biosensing by SWNTs 269 Sensing and Treating Cancer Cells Utilizing SWNTs 269 Cytotoxicity of SWNTs 272

Contents

7.7 7.8

Cancers and SWNTs 275 Summary 277 Acknowledgments 278 References 278

8

Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis 285 Kiyotaka Shiba Introduction 285 Nanotubes, Nanowires and Nanorods 285 Carbon Nanotubes 286 Noncarbon Nanotubes 287 Single-wall Carbon Nanohorns 287 Nanorods and Nanowires 288 Self-assembled Nanotubes 289 Cancer Diagnosis 290 Carbon Nanotube-based Detection System 290 Non-carbon Nanotube-based Detection Systems 291 Microcantilevers 292 Nano-tag made of Nanorods 293 Cancer Treatment 293 Carriers for Drug Delivery Systems 294 Imaging Agents 294 Conclusions 295 References 296

8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4 8.4.1 8.4.2 8.5

9

9.1 9.2 9.2.1 9.2.2 9.3 9.3.1 9.3.2 9.3.3 9.4 9.4.1 9.4.2 9.5 9.5.1 9.5.2 9.5.3

Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy 304 Sang Bok Lee and Sang Jun Son Introduction 304

Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications 305 MRI and Therapeutic Application of Magnetic Nanoparticles 305 Biomedical Diagnostic Application of Magnetic Nanoparticles 307 Carbon Nanotubes 310 Carbon Nanotubes for Targeted Cancer Cell Death 310 Carbon Nanotubes for Detection of Cancer Cell 312 Carbon Nanotubes for Targeted Delivery 313 Nanotubes and Nanowires Composed of Artificial Peptides 315 Peptide Nanotubes 315 Peptide-Amphiphile Nanofibers 316 Template-synthesized Nanotubes and Nanorods 318 Differential Functionalization of Nanotube 319 Selective Extraction of Drug Molecules 320 Silica Nanotube Carrier for DNA Transfection 322

XIII

XIV

Contents

9.5.4 9.5.5 9.5.6 9.5.6.1 9.5.6.2 9.5.6.3 9.6

DNA Nanotubes 323 Nanobarcodes for Multiplexing Diagnosis 325 Magnetic Nanotubes 328 Synthesis and Characterization of Magnetic Nanotubes 328 Magnetic Field-assisted Chemical Separation and Biointeraction Drug Uptake and Controlled Release 330 Conclusion 332 References 333

10

Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling 338 Li-Shing Huang, Yuh-Yih Chien, Shu-Hua Chen, Po-Chiao Lin, Kai-Yi Wang, Po-Hung Chou, Chun-Cheng Lin, and Yu-Ju Chen Introduction 338 Fabrication and Biomedical Applications of Nanoparticles 339 Fabrications and Properties of Nanoparticles 339 Metal Nanoparticles in Cancer Diagnosis 343 Principles of Mass Spectrometry 348

10.1 10.2 10.2.1 10.2.2 10.3 10.3.1 10.3.2 10.4 10.4.1 10.4.2 10.4.3 10.4.4 10.5 10.5.1 10.5.2 10.5.3 10.5.4 10.5.5 10.6 10.6.1 10.6.2 10.7

329

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry 348 Affinity Mass Spectrometry 349 Nanoprobe-based Affinity Mass Spectrometry (NBAMS) 350 Preparation of Nanoprobes and Workflow 351 Proof-of-principle Experiment 353 Kinetic Study of the Nanoscale Immunoreaction 356 Detection Limit and Concentration Effect of Nanoprobe-based Immunoassay 356 Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry 359 Selected Protein Profiling from Human Plasma 359 Comparison of Nanoscale and Microscale Immunoassay 359 Suppression of Nonspecific Binding on Magnetic Nanoparticles 361 Enrichment of Target Antigen in Human Plasma 362 Plasma Protein Profiling in Normal Individuals and in Patients 364 Multiplex Assay 364 Workflow of Multiplexed Assay 366 Screening for Patient and Healthy Individuals 367 Future Outlook 369 Acknowledgments 369 References 369

Contents

11

11.1 11.2 11.2.1 11.2.1.1 11.2.1.2 11.2.1.3 11.2.1.4 11.2.1.5 11.2.1.6 11.2.1.7 11.2.2 11.2.2.1 11.2.2.2 11.2.2.3 11.2.2.4 11.2.2.5 11.2.3 11.2.3.1 11.2.3.2 11.2.3.3 11.3

Nanotechnological Approaches to Cancer Diagnosis: Imaging and Quantification of Pericellular Proteolytic Activity 377 Thomas Ludwig Introduction 377 Quantification of Local Proteolytic Activity – an Objective 380 Regulation of Protease Activity 382 Secretion 382 Activation 382 Inactivation 383 Endogenous Protease Inhibitors 383 Glycosylation 384 Oligomerization 384 Protein Trafficking 385 Mechanisms of Confining Proteolytic Activity 385 Membrane-type Matrix Metalloproteinases 386 Cell Surface Receptors for Protease Binding 387 ECM Binding of Proteases 387 Cellular Microdomains 388 The Tumor–Host Conspiracy 388

Local Proteolytic Activity Regulates Complex Cellular Functions 389 Local Proteolytic Activity in Cancer Cell Migration 389 Local Proteolytic Activity and Cell Signaling 389 Functional Insights from Matrix-metalloprotease Deficient Mice 390 Evaluation of Classical Methods for Quantification of Net Proteolytic Activity 391 11.3.1 Functional Detection of Local Proteolytic Activity by In Situ Zymography 392 11.3.2 Tumor Cell Invasion Assays 393 11.3.2.1 Electrical Resistance Breakdown Assay 394 11.3.3 In vivo Detection of Proteolytic Activity 394 11.3.4 Multiphoton Microscopy and Second-harmonic Generation 395 11.3.5 In vitro Detection of Local Proteolytic Activity by Labeled Substrates 396 11.4 Novel Approaches to Local Proteolytic Activity 396 11.5 Conclusions and Perspectives 400 Acknowledgments 401 Abbreviations 401 References 401 Index

413

XV

XVII

Preface Two volumes (6 & 7) in the ten-volume series Nanotechnologies for the Life Sciences have been dedicated to the application of nanotechnology in cancer. The first of these two has captured nanotechnological approaches for the treatment of cancer and is already published. I do hope you had a chance to read it. This is the second of the pair, bringing out the utility of nanotechnology in developing tools and materials for sensitive and early diagnosis of cancer. These two volumes are timely as it is projected that cancer will be the leading cause of death, overtaking heart diseases, in the near future. One of the major goals of the American Cancer Society is early and sensitive detection of cancer. It is astonishing to note that the five year survival rate for breast cancer patients, if the cancer is diagnosed at localized stage, is 97.5%. Currently only 63.5% of breast cancers are diagnosed at the localized stage and there is no sensitive diagnostic tool for detecting micro-metastases, where the survival rate is less than 30%. Having made only limited progress in early and sensitive diagnosis of cancer using traditional methods, a paradigm shift in our approach is required in order to develop imaging agents and diagnostics for detecting cancer in its earliest and pre-symptomatic stage when it can be treated most easily. Needless to say, nanotechnology is offering this new approach and investigations reported so far demonstrate its immense potential. Since these investigations are being published in a very broad range of journals, this book provides a unique collection of consolidated and up-to-date information on nanotechnological diagnostic tools for the detection of cancer. It is my pleasure to present, on behalf of the eminent contributors, the 7th volume in the series, entitled Nanomaterials for Cancer Diagnosis. The uniqueness of nanotechnological approaches in the battle against cancer is a distinct possibility to create technologies for simultaneous diagnosis and treatment of cancer. Therefore, you will find some of the chapters covering both these aspects while focusing primarily on diagnosis. I am grateful to all the contributors who made this book into a comprehensive source of information on the impact of a variety of nanomaterials on a number of diagnostic tools. A book of this magnitude is not possible but for their scholarly contributions. At this point, I also would like to gratefully acknowledge the support of a number of others, especially my employer, family, friends and Wiley VCH publishers for this timely publication.

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

XVIII

Preface

The book is divided into eleven chapters, encompassing a number of diagnostic approaches for cancer through use of a variety of nanomaterials such as dendrimers, quantum dots, gold nanoparticles, dye-doped silica nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs), liposomes, fullerenes, carbon nanotubes, nanowires, nanorods, and so on. The book begins with a chapter reviewing the progress that has been made to date on dendrimers – nano-sized, radially symmetric molecules with well-defined, homogeneous and monodisperse structure consisting of tree-like arms or branches – and dendrimer-based nanomaterials in the diagnosis and treatment of cancer. The authors, Srinivasa-Gopalan Sampathkumar and Kevin J. Yarema from the Johns Hopkins University in Baltimore, USA, have done an excellent job in collating pertinent information on how several different attributes of dendrimers are being utilized to fine-tune their biological activity in order to obtain effective solutions to long standing problems in diagnosis and treatment of cancer. The chapter, Dendrimers in Cancer Treatment and Diagnosis, is valuable for all those interested in acquiring knowledge on this exciting and most promising class of nanomaterials in cancer nanotechnology. The second chapter, Nanoparticles for Optical Imaging of Cancer, has been contributed from the laboratories of University of Central Florida in Orlando, USA. Authors Swadeshmukul Santra and Debamitra Dutta provide a broad overview on various existing cancer-imaging techniques followed by a detailed description on optical imaging in general and nanoparticle-based optical imaging in particular. This chapter will provide readers with all the necessary information that is required for them to obtain a grasp of this exciting and continuously evolving field of nanoparticle-based optical contrast agents. More specifically, readers will have an opportunity to obtain a broader perspective on applications of quantum dots, dye-doped nanoparticles, gold nanoparticles, phosphors & fluorescent polymer particles as potential contrast agents in optical imaging of cancer. Of all the nanomaterials with optical properties, gold nanoparticles are the most widely investigated materials and therefore, a complete chapter is dedicated to the utility of gold nanoparticles in cancer diagnosis. The third chapter, Nanogold in Cancer Therapy and Diagnosis, a contribution from the laboratories of Debabrata Mukhopadhyay from Mayo Clinic Cancer Center in Rochester, USA, brings out the importance of gold nanoparticles in cancer diagnosis and therapy. The authors also have reviewed relevant information on synthetic methods, biocompatibility and the mechanism of action of gold nanoparticles. Moving from nanomaterials-based optical imaging techniques for diagnosis of cancer, the next three chapters describe investigations into utilization of magnetic nanoparticles for enhancing the sensitivity of detection of tumors using magnetic resonance imaging (MRI). Of the existing non-invasive diagnostic tools such as Computed Tomography (CT), Positron Emission Tomography (PET), Single Photon Emission CT (SPECT), Ultrasound (US) and optical imaging, MRI appears to be the most promising and sensitive technique. It is gaining more importance with the discovery of nanomaterials-based contrast agents. Researchers from the Washington University Medical School in St. Louis, USA, contributed the fourth chapter wherein an overview of the MRI technology and principle targeting mechanisms

Preface

are described, followed by a detailed discussion on the application of superparamagnetic and paramagnetic nanoparticles. The chapter, Nanoparticles for Magnetic Resonance Imaging of Tumors, brings out clearly the differences between the superparamagnetic nanoparticles, which are mainly SPIONs and paramagnetic nanoparticles belonging to groups such as liposomes, perfluorocarbon nanoparticles, fullerenes, and others. The chapter contributed by Tillmann Cyrus, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza also provides up-to-date information on rapidly evolving hybrid technologies using quantum dots for non-invasive imaging with MRI and intraoperative direct visualization. While the fourth chapter provides an overview of nanomaterials being developed for MRI, the fifth chapter primarily focuses on superparamagnetic iron oxide nanoparticles. This chapter, Magnetic Resonance Nanoparticle Probes for Cancer Imaging, contributed by Young-Wook Jun, Jung-tak Jang, and Jinwoo Cheon from Yonsei University in Seoul, Korea, reviews recently developed biocompatible magnetic nanoparticles and their utilization in molecular MRI. The final chapter on the application of SPIONs in MRI, the sixth chapter in the book, is from the laboratories of Carola Leuschner, Pennington Biomedical Research Center at Baton Rouge, USA. Being one of my close collaborators and having closely associated with the development of Leutenizing Hormone and Releasing Hormone (LHRH)-conjugated SPIONs, I can confidently say that this chapter is very unique and provides readers with all the information that is available on LHRH-conjugated magnetic nanoparticles. The chapter, LHRH-conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers, provides an elaborate account of the role of LHRH and LHRH receptors in malignant tissue. The central theme of the chapter is a description of comparative advantages of LHRH-SPIONs with other targeting agents in targeting specifically primary tumors and metastases, thereby demonstrating the potential for LHRH-SPIONs in dramatically improving the sensitivity of MRI. Switching gears from nanoparticles, chapter seven written by Pu Chun Ke and Lyndon L. Larcom from Clemson University in Clemson, USA, is a testimony to the ever-increasing number of applications of carbon nanotubes. Not surprisingly, single-walled carbon nanotubes (SWNTs) have already shown promise in cancer diagnosis and therapy and have distinct advantages over multi-walled carbon nanotubes (MWNTs). The chapter, Carbon Nanotubes in Cancer Therapy and Diagnosis, captures up-to-date information available in the literature on functionalization for solubility and biocompatibility, cytotoxicity, gene and drug delivery, and sensing of cancer cells utilizing SWNTs. Following this chapter, Kiyotaka Shiba from the Cancer Institute of the Japanese Foundation for Cancer Research in Tokyo, Japan, has brought out a thorough review on the application of ‘‘non-spherical’’ nanomaterials in cancer treatment and diagnosis. The theme of this 8th chapter, truly reflected in the title Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis, is the current status of carbonaceous as well as non-carbonaceous nanotubes, nanowires, and nanorods with respect to their potential applications in cancer diagnosis and treatment. In the ninth chapter, Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy, authors Sang Bok Lee and Sang Jun Son from the University of Maryland at College Park, USA, describe in detail

XIX

XX

Preface

the importance of carbon nanotubes, nanotubes and nanowires composed of artificial peptides, and template-synthesized silica and magnetic nanotubes in general for various biomedical applications and in particular related to cancer. The authors demonstrate that tubular nanomaterials have several advantages over spherical nanoparticles, especially when multifunctionality is needed as nanotubes have distinctive inner and outer surfaces that can be modified and utilized differently, enabling them to be equipped with the right function in the right position. The last two chapters of this volume are testimony to the breadth of nanotechnological approaches for cancer diagnosis. The penultimate chapter, Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling, provides an overview on a recently developed technique – nanoprobe-based affinity mass spectrometry (NBAMS) – for the screening of normal individual and cancer patients. The chapter is a contribution from the Institute of Chemistry and Genomic Research Center of the Academia Sinica in Taipei, Taiwan, authored by Yu-Ju Chen and co-workers. This chapter provides fundamental principles in mass spectrometry and the detection method in NBAMS, in addition to details of the design, workflow and performance of NBAMS. The final chapter is presented by Thomas Ludwig from Yale University at New Haven, USA. The author points out some of the challenges in cancer diagnosis by focusing on local, nanoscale processes for in vitro and in vivo diagnostics. The chapter, Nanotechnological Approaches to Cancer Diagnosis: Imaging and Quantification of Pericellular Proteolytic Activity, is an interesting chapter on how development of improved nanotechnology enabled methods that are likely to provide real-time high-resolution imaging and quantification of local enzymatic activities will play a major role in the design of new drugs and the understanding of basic principles in tumor cell invasion. This book concludes the two volumes dedicated to cancer nanotechnology. I do realize that there are some topics that have not been covered and also some topics that have been covered are not exhaustive enough. I am hoping that the next edition of this book series will fill these gaps and also will take into consideration suggestions from the readers. I am looking forward to hearing from you. As I end this preface, I am pleased to note that the first six volumes of the ten volume series have already been published and the next four volumes are in print. I am also pleased to know that the Nanotechnologies for the Life Sciences in general is being well received. September 2006 Baton Rouge, USA

Challa S. S. R. Kumar

1

1

Dendrimers in Cancer Treatment and Diagnosis Srinivasa-Gopalan Sampathkumar, and Kevin J. Yarema 1.1

Overview

Dendrimers are nano-sized, radially symmetric molecules with well-defined, homogeneous and monodisperse structure consisting of tree-like arms or branches. Over the past two decades since the term ‘‘dendrimer’’ was formally defined, research interest in these molecules has gradually evolved from a primary focus on overcoming purely synthetic challenges to include aesthetic and theoretical perspectives, and, more recently, with the ongoing flurry of ‘‘nanobiotechnology’’ advances, to develop practical and commercial applications for these elegant nanodevices. Today, a critical mass of knowledge exists to synthetically control the physicochemical properties of dendrimers and thereby govern their ensuing biological behaviors. These fundamental scientific advances, coupled with practical methods to covalently conjugate a wide range of bioactive molecules to the surface of a dendrimer or encapsulate them as guest molecules within void spaces, provide a highly versatile and potentially extremely powerful technological platform for drug delivery. This chapter recaps synthetic advances in dendrimer construction and summarizes the many features of these fascinating macromolecules that endow them with favorable properties for drug delivery applications. Finally, with this enticing technology having matured to the point where it is ready to confront ‘‘real-world’’ challenges, a synopsis is outlined of the prospects for exploiting dendrimer-based nanodevices for one of the most intractable medical challenges, the diagnosis and treatment of cancer.

1.2

Introduction

The discovery, design, and development of anticancer therapeutic agents have proven to be remarkably intractable despite intense efforts at the research and clinical levels over many decades. A brief consideration of the challenges facing an Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

2

1 Dendrimers in Cancer Treatment and Diagnosis

anticancer drug illustrates some of the reasons for frustratingly-slow progress: first the drug must be able to seek out subtle changes that distinguish a transformed cell from the other 200 or so types of healthy cells found in the body and then provide a sufficiently high dose of a toxic agent to kill the cell. The difficulty of this task is amplified by the potential metastasis of cancer cells to widely-spread niches throughout the body, each with unique properties. Furthermore, to successfully cure a patient, each and every cancer cell must be eradicated because even one in a thousand – often harboring latent resistance – can re-grow into a second tumor refractory to therapeutic intervention. Readers of this chapter, contained within a volume devoted to the development of novel cancer therapeutics, do not require convincing of the difficulty of combating cancer and this issue will not be labored here. Instead, this chapter provides a broad overview of dendrimer-based nanotechnologies for the treatment of cancer with a consideration of their synthesis, the encapsulation and covalent attachment of drugs, and various strategies used for tumor specific targeting, imaging, and therapy. The discussion of specific topics begins with a description of the basic properties of dendrimers in Section 1.3 to highlight how these molecules lie at the interface between conventional synthetic polymers and the archetypical nanosized biological polymers, proteins. Section 1.4 briefly outlines the synthesis of dendrimers; exhaustive review articles (referenced therein) provide a wealth of synthetic detail beyond the scope of this discussion. This chapter aims to provide the reader with the knowledge that, by control of design parameters, the attributes of dendrimers can be tuned to incorporate the most desired features of synthetic polymers and proteins and, thereby, gain exquisite control of biological activity. Upon having established that dendrimers are synthetically-tractable, biologicallycompatible nano-devices, their unique suitability for drug delivery will be delineated in some detail in Section 1.5. Specific topics covered include the alternative drug-carrying strategies of encapsulation (Section 1.5.2) and covalent conjugation (Section 1.5.3), as well as design features needed to ensure bioactivity of the drug (Section 1.5.4) and the biocompatibility of the dendrimer (Section 1.5.5). Finally, with the multi-disciplinary set of tools required for dendrimer-based drug delivery now reaching maturity, this area of investigation is undergoing transformation from the developmental stage to ‘‘real-world’’ applications. Accordingly, Section 1.6 discusses the prospects for using dendrimer-based nanotechnologies to overcome arguably the most difficult biomedical problem now faced, the diagnosis and treatment of cancer. In particular, the general properties of dendrimers that make them attractive for cancer treatment will be outlined in Section 1.6.1, with a specific benefit – exploitation of the enhanced permeability and retention effect that allows passive accumulation at the sites of tumors – discussed in Section 1.6.2. The ability of dendrimers to serve as a technological platform for multifunctional nano-devices that include targeting, imaging, and/or cytotoxic modalities is discussed in Section 1.6.3 and their prospects for diagnosis and therapeutic applications are given in Sections 1.6.4 and 1.6.5, respectively. Finally, Section 1.6.6 gives a brief synopsis of innovations that promise to speed progress in the near future.

1.3 Basic Properties and Applications of Dendrimers

Together – while broader in scope than the typical discussion of dendrimers, drug delivery, or cancer therapy – this chapter provides an integrated look at the many considerations required for successful application of dendrimers for cancer therapy. For a more-in-depth consideration of any particular sub-topic, the interested reader is urged to consult the many original research reports and review articles cited throughout.

1.3

Basic Properties and Applications of Dendrimers 1.3.1

Structural Features and Chemical and Biological Properties Basic Features of Dendritic Macromolecules are Inspired by Nature Dendritic structures, characterized by hyperbranched subunits, are widely found in nature. Indeed, the word dendrimer is based on the Greek words ‘‘dendron’’ meaning ‘‘tree’’ or ‘‘branch’’ and ‘‘meros’’ meaning ‘‘part’’ [1, 2]. Taken literally, similarities with dendrimer macromolecules are illustrated by a tree, where the leaves of a tree are maximally displayed on a highly-branched scaffold to maximize their accessibility to the outside world to optimize functions such as light harvesting. The branches of a tree can modify the environment within them, similarly the core/ interior encapsulated within a dendrimer can provide a sheltered microenvironment with tailored chemical properties and reactivities [2]. In addition to actual trees, Nature has scaled dendrimeric structures down to the multi-centimeter level (the intricate neural pathways found in the brain), the millimeter level (ice crystals and snowflakes), and yet further to the micron level (the dendritic outgrowths of neurons). At a molecular, ‘‘nano-size’’ level, dendrimer-like molecules, such as branching polysaccharides, provide an elegant solution to a cell’s need to stably store high energy molecules like monosaccharides; the presence of many chain ends allows the rapid release of large numbers of glucose monomers when needed [3]. Unlike Nature, which provides dendritic structures in a range of sizes from real trees to the namesake molecular nano-sized structures, this chapter focuses exclusively on dendritic macromolecules that are of a synthetically tractable scale and appropriate for cancer therapy. Starburst1 clusters [4], made of poly(amidoamine) (PAMAM) units, are arguably the most-thoroughly characterized and extensivelyutilized dendrimers [5]. A basic characteristic of these molecules is their layered composition – known as ‘‘cascades’’ or ‘‘generations’’ [1] (Fig. 1.1). The overall shapes of dendrimers range from spheres to flattened spheroids (disks) to amoeba-like structures, especially in cases where surface charges exist and give the macromolecule a ‘‘starfish’’-like shape [6]. The exact morphology of a dendrimer depends both on its chemical composition (the chemical composition of PAMAM dendrimers is shown in Fig. 1.1) as well as on the generation number, as exemplified by PAMAM where the lowest generation 1.3.1.1

3

4

1 Dendrimers in Cancer Treatment and Diagnosis

Schematic representation of a generation G4 dendrimer with 64 amino groups at the periphery. This dendrimer starts from an ethylene diamine core; the branches or arms were attached by exhaustive Michael addition to methyl acrylate followed by exhaustive aminolysis of the resulting methyl ester using ethylene diamine [36]. This sequence of reactions was applied in an iterative fashion to increase the level of

Fig. 1.1.

generations. The periphery of successive generations is marked by grey circles, starting from G0, G1, G2, G3 and G4. Of note, distinctive features of dendrimers, including the densely-packed membrane-like arrangement of surface functional groups, the formation of internal cavities, and the condensation into globular structures (not shown), are typically manifest at the G4 stage (and amplified in successive generations, Table 1.1).

1.3 Basic Properties and Applications of Dendrimers

structure (e.g., G0 and G1) have highly asymmetric shapes and posses open structures compared with higher-generation structures that first appear to be disk-like and then progress to increasingly spherical geometries [5] as they assume globular structures with a significant reduction in hydrodynamic volume [7]. In addition to sphere-like dendrimers – based on a ‘‘dot-like’’ core (Fig. 1.1) – increasing interest is developing in cylindrical dendrimers that are based on ‘‘rod-like’’ cores. These interesting spin-off macromolecules have been compared with spaghetti because they can be rigid like the uncooked form of this pasta or highly flexible like the cooked form; these properties can be tuned based on the chemical composition and density of packing of the dendritic branches [6]. Additional features of dendrimers are discussed below, by comparison with the two classes of molecules they are most often likened to, i.e., ‘‘conventional’’ synthetic polymers and, the most extensively studied biological polymer, proteins. 1.3.1.2 Comparison of the Properties of Dendrimers and Conventional Synthetic Polymers Dendrimers have both similarities and differences when compared with traditional polymers. One similarity is the vast diversity in the basic monomeric building blocks used to create both classes of molecules and to provide the final macromolecular products with a wide range of chemical, mechanical, and biological properties. Until recently, polymer chemistry has been focused on the production of linear polymers that often have a degree of branching or crosslinking; this property, however, is dramatically limited by comparison to dendrimers whose entire identity is wrapped up in their hyperbranched character. Interestingly, highly-branched polymers of the same material can be vastly different from conventional polymers of a similar molecular weight and composition; in particular, as dendritic macromolecules progress in size, usually when becoming larger than the third generation (G3), they assume globular structures and occupy considerably smaller hydrodynamic volumes than linear polymers [1]. When dendrimers condense into globular structures, a feat rarely achieved with linear synthetic polymers, their many termini become fixed into an outwards orientation and also form a densely packed, membrane-like surface (Fig. 1.1). This structural arrangement provides numerous attachment points for covalent conjugation of bioactive molecules on the surface as well as enclosed cavities for occlusion of guest molecules within the dendrimer. This tight packing ultimately results in the reaching of a critical branched state – known as the ‘‘starburst effect’’ [4] – where growth of the dendritic macromolecule is dramatically hindered by steric constraints [8] (this state is reached at G10 or G11 for PAMAM, Table 1.1). Dendrimers also have dramatically different rheological properties than conventional polymers; viscosity tends to increase continuously with molecular mass for linear macromolecules whereas the intrinsic viscosity of dendrimers goes through a maximum at approximately the fourth generation and then declines [8, 9]. Finally, dendrimers have a negligible degree of polydispersity because, unlike classical polymerization that is random in nature and produces molecules of various sizes, the size of dendrimers can be carefully controlled during synthesis. Under ideal condi-

5

6

1 Dendrimers in Cancer Treatment and Diagnosis Tab. 1.1. Generation by generation specifications for PAMAM Starburst4 dendrimers. (Adapted from Ref. [5].)

Generation

G0 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

Physical or structural parameter Molecular weight (Daltons)

Diameter (A˚)

Surface groups (xNH2 )

Radius of gyration (A˚)

517 1430 3256 6909 14 215 28 826 58 048 116 493 233 383 467 162 934 720 1 869 780

15 22 29 36 45 54 67 81 97 114 135 167

4 8 16 32 64 128 256 512 1024 2048 4096 8192

4.93 7.46 9.17 11.2 14.5 18.3 22.4 29.1 36.4 46.0 55.2 68.3

tions, preparations of dendrimers are monodisperse, which is to say they have one molecular weight instead of the range, over tens or even hundreds of kDa, often seen for traditional synthetic polymers. Indeed, the homogeneity and uniformity of dendrimers of successive generations becomes strikingly obvious as shown by the tunneling electron microscopy (TEM) images for G5 to G10 PAMAM (Fig. 1.2) [10]. 1.3.1.3 Comparison of the Properties of Dendrimers and Proteins (a Biological Polymer) As discussed above, dendrimers have unusual, often dramatically different, characteristics compared with conventional synthetic polymers. In fact dendritic molecules have often been compared with proteins, which are the workhorse biological polymers. Both classes of macromolecules are globular, are composed of precisely controlled monomeric units, have defined architectures, are of comparable size (Table 1.1), and have surfaces with chemically-reactive sites that can be endowed with biologically-compatible ligands found on proteins (such as glycosylation, Section 1.4.3.2). Moreover, the interior of a dendritic molecule, reminiscent of a protein, can harbor unique microenvironments, providing behaviors like redox chemistry, molecular recognition, ligand and substrate binding, and catalysis [11, 12]. The ability to create and exploit isolated nanoenvironments within a dendritic shell is derived from two main properties of a dendrimer. First, dendritic macromolecules adopt a semi-globular or fully globular character containing internal void

1.3 Basic Properties and Applications of Dendrimers

Transmission electron microscopy (TEM) of PAMAM dendrimers. Dendrimers were positively stained with aqueous sodium phosphotungstate and imaged by conventional TEM: (a) G10, (b) G9, (c) G8, (d) G7, (e) G6, (f ) G5. The scale bars indicate 50 nm, and a

Fig. 1.2.

small amount of G10 has been added as a focusing aid for G6 and G5. (Reprinted with permission from Jackson and coauthors [10]. Copyright 1998 by the American Chemical Society.)

spaces once they reach the fourth generation in size (Fig. 1.1) [8], enabling the encapsulation of protein-like functions, including catalysis [13, 14]. Second, these molecules have molecular flexibility and can undergo deformations, leading to rudimentary ‘‘lock and key’’ molecular recognition of the type vitally important to protein functions [15, 16]. Molecular recognition between molecules is a fundamental process in biology and chemistry without which life could not exist. The concept of molecular recognition, based on complementarity between the receptors and substrates, is very similar to the ‘‘lock and key’’ function first described by Emil Fischer over 100 years ago. In biology, the ‘‘lock’’ is the molecular receptor such as a protein or enzyme and the ‘‘key’’ can be regarded as a substrate such as a drug or ligand that is recognized to give a defined receptor–substrate complex [15]. In proteins, molecular recognition is largely driven by non-covalent forces such as hydrogen bonding, electrostatics, van der Waals forces, p–p interactions, solvent-dependent interactions including hydration forces, and conformational energy [17]; notably, all of these parameters can be controlled in dendrimers through synthetic design. The inherent ability of dendrimers to achieve molecular recognition of biological features, if it can be successfully developed to a level of sophistication where it can be exploited for the recognition of the surface biomarkers that distinguish cancer

7

8

1 Dendrimers in Cancer Treatment and Diagnosis

cells from healthy cells (Section 1.6.3), has important – and extremely beneficial – implications for drug delivery (Section 1.5). Although sharing many superficial features, a close inspection reveals important differences between dendrimers and proteins. For example, remaining on the topic of deformability and flexibility, the linear, folded chain of a protein is more tightly packed but also has a greater potential for flexibility (when a comparison is made between the fully folded and unfolded states of a peptide chain) than is possible for the branches of a dendrimer. Only a small proportion of the potential flexibility of a protein, however, is usually available for ‘‘induced fit’’ interactions because the extensive unfolding of a protein is highly thermodynamically unfavorable. By comparison, although the extensive covalent bond networks within a dendrimer prevents complete unfolding under any condition, this arrangement does provide sufficient flexibility to allow dramatic – albeit somewhat thermodynamically unfavorable – deformations fairly readily [18]. Next, to consider dendrimer surfaces in comparison with proteins, synthetic dendritic macromolecules can be given a significant repertoire of tunable characteristics not found on natural proteins; this feature has greatly propelled the development of practical applications for these molecules. In particular, the surface of a protein contains a relative sparse complement of chemically reactive and accessible functional groups because most amino acid side chains are buried with the globular structure of the protein. By contrast, virtually all of the termini of dendritic branches, which can be customized with a wide range of chemical functionalities (Fig. 1.3), are oriented outward and are highly accessible on the surface of the dendrimer (Fig. 1.1). The consequent ability of a dendrimer to be functionalized with far more surface groups than a protein of comparable size [1, 19] has provided impetus to their widespread use as drug delivery vehicles. 1.3.2

Dendritic Macromolecules Possess a Wealth of Possible Applications

Within the past decade, the success of chemists in synthesizing mimics of natural dendrimers with a plethora of interesting physicochemical properties at the nanoscale has spurred efforts to find practical uses for these versatile nanodevices. Now that efforts to synthesize these molecules have reached fruition, there is a pleasing circularity that certain applications mirror natural processes considering that dendritic molecules were initially inspired by nature. In a dramatic example, a primary function of the leaves of real trees is for light harvesting; now, synthetic dendrimers have been created with highly-efficient light-harvesting antennae as well [8, 20]. Similarly, the dendritic network of hairs found on the Gecko foot that allows amazingly strong attachment to many types of surfaces through van der Walls forces [21] has led to efforts to create new forms of adhesives that are unaffected by the roughness, smoothness, wetness, or other macroscopic properties of the surface while providing strong but reversible adhesion. In addition to these two examples, many novel applications such as the exploitation of organometallic dendrimers as quantum dots for imaging, the solubilization of hydrophilic dyes in

1.3 Basic Properties and Applications of Dendrimers

Structural options for dendrimerbased drug delivery. Dendrimers can be synthesized with neutral surfaces (1) and positive (2) or negative (3) charges at the periphery; moreover, dendritic macromolecules, generally when larger than G3, can harbor non-covalently encapsulated guest/drug molecules [4 and discussion in Section 1.5.2]. An alternative strategy for drug delivery is through covalent conjugation of ligands (‘‘A’’ in 5) to the surface of the dendrimer (Section 1.5.3). The versatility of dendrimers for drug delivery is illustrated by considering that ‘‘A’’ could be a targeting

Fig. 1.3.

ligand (Section 1.6.6.3) and the active drug could be encapsulated within the same macromolecule (6). Synthetic strategies are now available for providing dendritic clusters with extremely high densities of surface ligands (7) and for providing more than one type of surface ligand, either in a random orientation (8), or in blocks (9). The latter dendrimers are now being exploited in sophisticated cancer cell targeting (Fig. 1.4) and drug release (Section 1.5.3.3) strategies where A, B, and C can be any combination of targeting agents, drugs, contrast agents, or functional groups that improve pharmacological properties.

9

10

1 Dendrimers in Cancer Treatment and Diagnosis

apolar dendritic ‘‘solvents’’ [22], use as chemical catalysts, and in electronics as insulated molecular wires, light-emitting diodes, or fiber optics [12, 23, 24] have been reported. Besides their use for drug delivery and cancer therapy, the many emerging chemical, synthetic, research, and industrial uses for dendrimers are outside the scope of this article and will not be discussed further; the interested reader can consult chapter articles [1, 2, 11].

1.4

Methods for Dendrimer Synthesis 1.4.1

History and Basic Strategies

The ability to create homogeneous molecules with defined dendritic architecture and novel physicochemical properties at the sub-nano to nano-size scales occurred in chronological synchrony with the wide-spread application of nanobiotechnology to biology and medicine. Consequently, the parallel development of synthetic chemical methodology and the ever-increasing application of nano-tools in biomedicine triggered an explosive growth in the new field of dendrimer synthesis. This growth is evidenced by a cursory search for ‘‘dendrimers and synthesis’’ in the Web of Science database, which reveals that@2000 articles have been published on this topic since 1986. Clearly, a full discussion is beyond the scope of this report; excellent accounts and review articles on the synthesis of dendrimers by pioneers of the field have appeared at regular intervals [25–32] and are cited throughout this chapter. Nonetheless, a working knowledge of the chemical properties of dendrimers is critical to successfully devise efficacious therapeutic strategies with these versatile, but temperamental, macromolecules (as described in detail in Sections 1.5 and 1.6). Accordingly, we next provide an outline of the basic strategies and building blocks employed in dendrimer synthesis, with an emphasis on families of dendritic molecules that possess special properties – such as possessing cavities in their interiors suitable for host–guest complexation similar to enzyme– substrate complexes or displaying several functional groups on their surface appropriate for sophisticated drug delivery strategies – relevant to the field of biology and medicine. Cascade Reactions are the Foundation of Dendrimer Synthesis Although the term ‘‘dendrimer’’ was coined by Tomalia and coworkers less than two decades ago, the basic cascade or iterative methods that are currently employed for synthesis were known to chemists much earlier. For example, similar schemes form the basis of solid phase peptide synthesis. In turn, biology has long exploited similar iterative strategies in biochemical synthetic pathways; one example is provided by fatty acid biosynthesis [33]. Focusing on dendrimers, these macromolecules are constructed by performing simple chemical reactions in a repetitive or iterative manner by using small building blocks. In 1978 Vo¨gtle and coworkers re1.4.1.1

1.4 Methods for Dendrimer Synthesis

Synthetic approaches to dendrimers. (A) Cascade reaction sequences developed for the synthesis of ‘‘non-skid-chain like’’ polyaza macrocyclic compounds [34]. (B) Divergent approach – synthesis of radially symmetric PAMAM dendrimers using ammonia as the trivalent core; the generations are added at each synthetic cycle (two steps), leading

Fig. 1.4.

to an exponential increase in the number of surface functional groups [36]. (C) Convergent approach – synthesis of dendrons or wedges or branches that will become the periphery of the dendrimer when coupled to a multivalent core in the last step of the synthesis [40].

ported a similar approach, termed as cascade reactions, for the construction of nonskid-chain-like poly-aza macrocyclic molecules with well-defined architectures. Cascade synthesis is defined as ‘‘reactions where a functional group (e.g., amine) is made to react in such a way as to appear twice in the subsequent molecule or product’’ [34] (Fig. 1.4A). In the first step of the synthesis a primary or secondary amine was reacted with excess acrylonitrile in a Michael reaction to obtain a product with two arms [bis(2-cyanoethyl)amines]. In the second step the nitrile groups were reduced using cobalt(ii)/sodium borohydride to generate a new set of primary amine groups on both arms. The newly generated amino groups were then subject to identical reaction sequences iteratively to obtain the desired oligo-amine compounds.

11

12

1 Dendrimers in Cancer Treatment and Diagnosis

In 1985 Newkome and coworkers reported the synthesis of cascade molecules consisting of hydrocarbon core and shell with alcohol groups on the surface. These synthetic efforts were inspired by the Leuwenberg model of arboreal design; hence they named their synthetic macromolecular tree-like molecules ‘‘arborols’’ (Latin: arbor ¼ tree). Interestingly, characterization of these molecules showed they could be considered to be unimolecular micelles possessing cavities for encapsulation [35], a property that foreshadowed today’s efforts to use dendrimers for the delivery of encapsulated small molecule drug candidates (Section 1.5.2). Dendrimer Synthesis has Expanded Dramatically in the Past Two Decades In 1986, Tomalia and coworkers coined the now popular name ‘‘dendrimers’’ (Greek: dendron ¼ branch or tree-like) for radially symmetric branched molecules and reported the application of cascade synthesis for the synthesis of starburst dendrimers [36]. These researchers obtained homogeneous dendrimers by using a synthetic sequence of two simple reactions: (a) exhaustive Michael addition of ammonia to methyl acrylate and (b) exhaustive aminolysis of the resulting tri-ester derivative by ethylene diamine. The acrylate addition and aminolysis were repeated in an iterative manner, with excellent yields in each step, to prepare various molecules with increasing molecular weight and generations (Fig. 1.4B). The products with ester groups at the exterior were defined as G(m þ 0.5) generations and those with amine groups at the exterior were defined as G(m) (Fig. 1.1). This simple methodology is both powerful and versatile as it provided the ability to synthesize dendrimers with various surface properties. For instance, the ester groups could be hydrolyzed to present negatively charged carboxylate functional groups at the periphery or the amine groups could be protonated to present positive charges at the periphery. Electron micrographic studies showed the dendrimer with carboxylate groups of generation, G ¼ 4.5, to be highly monodispersed with a diameter of 88 G 10 A˚, compared with the theoretical value of @78 A˚. These dendrimers, when covalently attached to a polymeric backbone, were called ‘‘starburst polymers’’ or, less commonly, ‘‘cauliflower polymers’’ [7, 28]. 1.4.1.2

1.4.2

Strategies, Cores, and Building Blocks for Dendritic Macromolecules Dendrimers are Constructed from Simple ‘‘Building Blocks’’ In terms of synthesis, dendrimers can be constructed by using simple chemical reactions and building blocks reminiscent to the modular assembly of ‘‘LEGO’’ toys. Due to the ease, simplicity and repetitive nature of the synthetic methods, dendrimers based on organic, inorganic and organometallic molecular building blocks with greater than hundred different compositions are currently known, and new designs continue to be reported at a fast pace. In general, dendrimers consists of three major regions – (a) an initiator core, (b) a shell with extending arms or branches made of building blocks and (c) the exterior or outer-most surface groups on the termini of the branches. 1.4.2.1

1.4 Methods for Dendrimer Synthesis

There are innumerable ways of designing dendrimers [37–39]. For instance, the symmetry of the initiator core (Fig. 1.1) can be varied by using a wide range of molecules, which have included ammonia, a,o-diaminoalkanes, tri-substituted benzene, oligo- or polyalcohols, nucleic acids, amino acids, lipids, carbohydrates, or heteroatoms; many additional permutations are possible, e.g., the number of branching units in the initiator can be increased (tri- or tetra- or higher valency cores have been reported). Once the core moiety has been selected, options for the synthesis of the dendritic branches are equally numerous as various types of building blocks can be used, either singly or in combination with each other in the same dendritic macromolecule. The lengths of the dendritic arms, the nature of the surface, and the display of terminal functional groups can all be customized. 1.4.2.2 The Synthesis of Dendrimers Follows Either a Divergent or Convergent Approach Dendrimers can be synthesized by two major approaches. In the divergent approach, used in early periods, the synthesis starts from the core of the dendrimer to which the arms are attached by adding building blocks in an exhaustive and step-wise manner. This process provides dendrimers with incrementally increasing generation numbers. However, only one type of reaction can be performed at each step, giving a uniform display of only one functional group on the exterior surface; moreover, defects in successive generations can arise due to incomplete reactions or steric hindrance (Fig. 1.4B). In the convergent approach, pioneered by Fre´chet and coworkers [40], synthesis starts from the exterior, beginning with the molecular structure that ultimately becomes the outer-most arm of the final dendrimer (Fig. 1.4C). In this strategy, the final generation number is pre-determined, necessitating the synthesis of branches of the various requisite sizes beforehand for each generation [31]. Small branches or dendrons are synthesized starting from the building blocks containing surface groups; these assemblies are then condensed with a multivalent core. This approach is versatile in the sense that branches of different molecular composition can be linked to a single core molecule, introducing regional variations on the final dendrimer (Fig. 1.3); this strategy also minimizes the introduction of defects at various stages of synthesis. 1.4.3

Heterogeneously-functionalized Dendrimers Basic Description and Synthetic Considerations By simultaneously conjugating appropriate targeting moieties, drugs, and imaging agents to dendritic polymers, ‘‘smart’’ drug-delivery nanodevices can be developed to target, deliver, and monitor the progression of therapy. For example, as will described in greater detail below, a dendrimer intended for cancer therapy needs to be functionalized with the drug itself, display a moiety for targeting to the tumor cells, as well as include surface groups designed to improve the pharmacological 1.4.3.1

13

14

1 Dendrimers in Cancer Treatment and Diagnosis

properties (e.g., to ensure water solubility, avoid non-specific uptake or immunogenicity). Several synthetic strategies – primarily the convergent method discussed above (Section 1.4.2.2) – have been developed that enable multiple species to be added to a dendritic surface in an ordered manner [41] and thereby achieve multiple functionalities within the same dendritic nanodevice (Fig. 1.3). The ability to create multi-functional nanodevices based on dendritic scaffolds, however, remains a challenging endeavor because conjugating several types of different molecules to a dendrimer is likely to change its physicochemical properties and resulting biological activity. Practically, additional synthetic steps required to fine-tune bioactivity and remedy bioincompatibility if it arises may render the whole process costinefficient at best and, more troublesomely, lead to loss of product uniformity, thereby negating a key benefit of dendrimers, i.e., their monodisperse, fully defined nature [42]. 1.4.3.2 Glycosylation is an Example of Surface Modification with Multiple Bioactivities An outstanding demonstration of the synthetic power of decorating the surface of dendrimers with ‘‘interesting’’ molecules comes, once again, by way of comparison of these nanodevices with proteins. Proteins, which have had the opportunity to evolve biocompatibility and systemic functions in multicellular organisms over hundreds of millions of years, have found it advantageous to decorate their surfaces with complex carbohydrates when they are displayed on the cell surface or secreted into the extracellular milieu. In the past few years, it has become clear that these sugars play many key roles in molecular recognition over short distances, such as interactions with the extracellular matrix and with neighboring cells, as well providing system-wide communication (e.g., almost all protein hormones are glycosylated). When developing dendritic nanotools requiring bioactivity similar to that found in proteins, including the ability needed by a drug candidate to seek out and evoke responses at a specific but far-removed cell type in the body, it is wise to learn from nature and consider the inclusion of sugars to be an important design parameter. The ability to provide dendrimers with oligosaccharide coatings has been facilitated by the many functional groups that can be displayed on the surface and function as chemical handles for covalent attachment of a second group. A pioneering example of sugar display on a dendritic scaffold was provided by the unusual ninecarbon sugar sialic acid [43, 44]. This sugar, when displayed on human cells, serves as a critical binding epitope for the influenza virus. The virus, however, does not bind to soluble sialic acid, or sialic acid appended to a conventional polymer. Because these forms of sialic acid do not serve as effective binding elements, they are unable to act as a molecular decoys [45] and prevent the virus from binding to its real target, sialic acid on the human cell. By contrast, when sialic acid was conjugated to the surface of a dendritic polymer, it functioned as an effective and efficient binding decoy [46, 47], opening the door to the development of new diagnostic devices and novel anti-viral therapies [48, 49]. The molecular basis of the preferential recognition of sialic acid by the influenza virus when this sugar was

1.5 Dendrimers in Drug Delivery

displayed on a highly structured dendritic scaffold was traced to the ‘‘cluster glycoside effect’’ [50]. Over the past decade it has become firmly established that carbohydrate-based recognition depends on multiple simultaneous interactions to increase specificity and affinity [45, 51]. The demonstration that dendrimers provide an ideal synthetic platform for the appropriate display of carbohydrates to achieve the cluster glycoside response [52–54], along with improved methodology to synthesize glycoconjugated-dendrimers [43, 55], has driven the expansion of this approach from a single monosaccharide to a sugar-amino acid couple (the Tn antigen, which is N-acetylgalactosamine linked to serine [56]) to disaccharides (lactose [57] and the T-antigen [58]), and, finally, to tetrasaccharides (the sialyl Lewis X epitope [59]).

1.5

Dendrimers in Drug Delivery 1.5.1

Dendrimers are Versatile Nano-devices for the Delivery of Diverse Classes of Drugs

A successful drug must perform the demanding tasks of selectively recognizing and binding to a molecular target, then triggering an appropriate biological response, all the while possessing pharmacological properties that render it ‘‘drug-like’’. In some cases, nature has supplied compounds – such as aspirin or penicillin – that can be used directly as drugs but the more common situation is that many otherwise promising therapeutic agents are not successful in the clinic because of their poor pharmacological properties. The properties of dendrimers, in particular the synthetic ability to provide them with many different biological properties, along with their capacity to carry conjugated surface molecules or encapsulated guest molecules, make them immediately attractive as potential vehicles for drug delivery. Drug delivery efforts are complicated by the diversity of molecules that hold potential therapeutic or diagnostic value; briefly reviewing three classes of drug candidates based on size demonstrates the wide applicability of dendrimers to drug delivery. First, regarding ‘‘small molecules’’, many low molecular weight drug candidates are limited by poor solubility in aqueous environments or, if they are soluble, face rapid elimination from the bloodstream through filtration in the kidney. In the past, efforts have been made to modify the molecule itself, often following the ‘‘rule-of-five’’ guidelines developed by Lipinski to raise awareness of the properties and structural features that render molecules more or less ‘‘drug-like’’ [60]. Dendrimers present an attractive alternative strategy to the redesign of the drug because they allow unfavorable properties of a small molecule, such as insolubility, to be overcome by the larger characteristics of the macromolecule. An approach for improving the pharmacological properties of higher molecular weight drug candidates, analogous to Lipinski’s guidelines for the modification of small molecule drugs, has been applied for protein therapeutics such as recombinant antibodies

15

16

1 Dendrimers in Cancer Treatment and Diagnosis

and protein toxins used in cancer treatment. In these cases, the amino acid sequences of recombinant proteins have been ‘‘humanized’’ by genetic engineering to avoid immunogenicity [61, 62] and their glycosylation patterns have been modified to increase serum half-life [63, 64]. These efforts, undertaken with actual proteins, illuminate design features that can benefit the development of protein mimics, dendrimers. In particular, the ‘‘humanizing’’ experiments show that small changes, such as the substitution of a single amino acid for another, can avoid significant problems like undesired systemic immune responses. In the same manner, small changes in the surface properties of dendrimers, such as the addition of poly(ethylene glycol) (PEG), can avoid unwanted immunogenicity. Finally, even extremely large therapeutic candidates, notably plasmids or naked viral DNAs used for non-viral gene delivery that are well beyond the size of traditional drugs, are also benefiting from dendrimer-assisted delivery. The next section outlines specific approaches for the delivery of both small and large drug candidates by dendrimers. 1.5.2

Dendritic Drug Delivery: Encapsulation of Guest Molecules 1.5.2.1 Dendrimers have Internal Cavities that can Host Encapsulated Guest Molecules The flexible branches of a dendrimer, when constructed appropriately, can provide a tailored sanctuary containing voids that provide a refuge from the outside environment [2] wherein drug molecules can be physically trapped [65] (Figs. 1.1 and 1.3). Encapsulation of hydrophilic, hydrophobic, or even amphiphilic compounds as guest molecules within a dendrimer [66] can be enhanced by providing various degrees of multiple hydrogen bonding sites or ionic interactions [65, 67] or highly hydrophobic interior void spaces [68, 69]. A wide variety of molecules have been successfully encapsulated inside dendrimers. In early experiments, compounds used to demonstrate the ‘‘guest molecule’’ concept included easy-to-visualize dye molecules such as rose bengal [66] and Reichardt’s dye [69] as well as pyridine [65] and peptides [67]. More recently, actual drugs, including 5-fluorouracil [70], 5-amino salicylic acid, pyridine, mefanminic acid and diclofenac [65], paclitaxel [71, 72], docetaxel [73], as well as the anticancer agent 10-hydroxycamptothecin [69], have been successfully encapsulated. Together, these results demonstrate that encapsulation is a general strategy for the delivery of low molecular weight compounds by dendrimers. This method is anticipated to be of particular value when display of the bioactive molecule on the surface of the dendrimer induces unwanted immunogenicity or reduces biocompatibility (Section 1.5.5).

Using Dendrimers for Gene Delivery The delivery of small molecules complexed as guest molecules in internal void spaces of dendrimers is, at least in retrospect, intuitively obvious. By contrast, the delivery of extremely large macromolecules, such as MDa-sized plasmid DNA for non-viral gene therapy, is counter-intuitive because the encapsulation of a ‘‘guest’’ molecule many times the molecular weight of the dendrimer itself appears impos1.5.2.2

1.5 Dendrimers in Drug Delivery

sible. Nonetheless, experimental evidence had demonstrated that gene delivery strategies also benefit from the participation of dendrimers [74]. For example, from its original discovery of efficacy for gene delivery [75], the fractured form of PAMAM, known as Superfect TM , is now a commercially-available transfection agent for in vitro applications [76]. Typical approaches to optimize dendritic gene delivery for in vivo use have involved the surface modification of a PAMAM backbone, either with arginine [77] or hydroxyl groups [78]. Alternatively, the results reported by Kim and coworkers, who demonstrated improved gene delivery with a novel PAMAM-PEG-PAMAM triblock copolymer, show that construction of dendrimers composed of new building blocks is warranted [76]. Although still in their infancy, there are efforts afoot to exploit dendrimers for the delivery of smaller nucleic acids such as antisense oligonucleotides and short interfering RNAs (siRNA); the success of these applications is likely to depend on the continuing development of novel materials for dendrimer synthesis [79]. Release of Encapsulated ‘‘Pro-drugs’’ Once a dendrimer carrying an encapsulated drug reaches the intended site of action, the guest molecule generally must be released to gain bioactivity. Indeed, a concern is that the active drug would ‘‘leak’’ out prematurely, thereby reducing the amount available for the intended therapeutic intervention, or more ominously, result in systemic toxicity. Reassuringly, early experiments showed that the close packing of dendritic branches on the surface of the macromolecule (Fig. 1.1) effectively formed a ‘‘membrane’’ that reduced diffusion to immeasurably slow rates [66]. In other cases, the release of encapsulated guest molecules was relatively faster, occurring over a few hours, apparently through hydrolytic degradation of the dendrimer in aqueous conditions [65]. The observation that guest molecules could be liberated at different rates demonstrated that viable opportunities exist to tailor the release for either slow or rapid delivery (Fig. 1.5). At present, additional control of delivery rates is being sought; for instance, the ability of a dendrimer to instantaneously release its entire drug payload upon reaching its cellular target would be valuable. Promising steps in this direction are being taken by the development of pH-sensitive materials [65], the fine tuning of hydrolytic release conditions, and the selective liberation of guest molecules on the basis of their size or shape [80]. 1.5.2.3

1.5.3

Covalent Conjugation Strategies 1.5.3.1 Dendrimers Overcome many Limitations Inherent in Polymeric Conjugation Strategies The strategy of coupling small molecules to polymeric scaffolds by covalent linkages to improve their pharmacological properties has been under experimental test for over three decades [81–84]. Unfortunately, conventional linear polymers typically used in these efforts are plagued by inherent properties that render them distinctly ‘‘un-drug-like’’, including high polydispersity and size distributions, a

17

18

1 Dendrimers in Cancer Treatment and Diagnosis

Requirements for dendrimer-based, cancer-targeted drug delivery. (a) Dendrimers with multiple surface functional groups (Section 1.4.3) can be directed to cancer cells by tumor-targeting entities that include folate or antibodies specific for tumor-associated antigens (TAAs). (b) The next step is intake into the cell, which in the case of folate

Fig. 1.5.

targeting occurs by membrane receptormediated endocytosis (Section 1.6.3.2). (c) Once inside the cell, the drug generally must be released from the dendrimer, which, for the self-immolative method (Section 1.5.3.3), results in the simultaneous disintegration of the dendritic scaffold (d).

lack of defined structure, and a low density of drug payload per unit volume or mass. Properties of dendrimers that overcome these problems include monodispersity that results in the ability to select the precise sizes of nanoparticle required to a specific application (Table 1.1), a fully defined structure that allows the presentation of attached conjugates in a defined architecture, a high ratio of drug payload to volume, and enhanced control over drug release rates. Unsurprisingly, based on these many beneficial features, a wide range of biologically active molecules have

1.5 Dendrimers in Drug Delivery

already been covalently attached to dendrimers. These conjugates range from small molecule drugs, such as ibuprofen [85], fluorescent and radioactive imaging agents (Section 1.6.4), oligonucleotides, oligosaccharides and peptides, as well as much larger molecules such as monoclonal antibodies (Section 1.6.3). Biologically active molecules attached to dendrimers can have two fundamentally different relationships to the host molecule. In some cases, exemplified by vaccine applications, there is no need to liberate active drug from the dendrimer (indeed, the success of antibody production usually depends on the unique display characteristics achieved by conjugation to the dendrimer). In most cases, however, the conjugated dendritic assembly functions as ‘‘pro-drug’’ where, upon internalization into the target cell, the conjugate must be liberated to activate the drug. Dendrimer Conjugates can be Used as Vaccines Most low molecular weight substances are not immunogenic; consequently, when it is desired to raise antibodies against small molecules, they must be conjugated to a macromolecule. In the past, natural proteins have commonly been used as carriers to generate antibodies to small molecules; now an alternative strategy using dendrimers has been demonstrated. In particular, unmodified PAMAM dendrimers that fail to elicit an antibody response on their own become haptenized upon protein conjugation and generate a dendrimer-dependent antigenic response [86, 87]. A specific example of this technique is provided by the dendrimeric presentation of antigenic HIV peptides, which proved superior to other multimeric presentation strategies, such as conjugation to dextran [88]. Notably, the immunogenicity of dendrimer conjugates is not limited to peptides antigens; in one study antibodies were produced against densely penicilloylated dendrimers that were subsequently used for the diagnostic testing of patients with potential allergy to b-lactam antibiotics [89]. Finally, although carbohydrate-conjugated dendrimers (Section 1.4.3.2) are typically non-immunogenic [1], antibodies can be successfully elicited against cancer-specific oligosaccharides displayed on a dendritic scaffold, offering a method for generation of a new class of cancer vaccines (Section 1.6.6.2). 1.5.3.2

Release of Covalently-delivered ‘‘Pro-drugs’’ Similar to encapsulated guest molecules that generally require release from the void spaces of a dendrimer to gain bioactivity (Section 1.5.2.3), a covalently delivered dendritic conjugate must also be cleaved within the target cell to regenerate the active cytotoxic agent (Fig. 1.5). At the same time, to ensure systemic nontoxicity, the covalent linker must be stable in circulation [90]. Several strategies are being pursued to ensure the successful cleavage and activation of the pro-drug in the target cell or tissue while avoiding systemic release. These include activation by low pH found in endosomal vesicles, installation of enzyme-cleavable ester linkages into the linkers that attach the pro-drug to the dendritic macromolecule, or disulfide bonds that are liberated in the reducing environment of the endoplasmic reticulum, photoactivation, or sensitivity to ultrasound [1]. Briefly returning to the benefits of dendritic clusters over conventional polymers for drug delivery, problems with the delivery of covalent conjugates when conven1.5.3.3

19

20

1 Dendrimers in Cancer Treatment and Diagnosis

tional polymers, such as poly(lactic acid) (PLA) or its copolymer with glycolide (PLGA), are used include a lack of sustained drug release [91]. Generally, these and other linear, randomly oriented polymers have an initial burst where as much as 50% of drug is released followed by a dramatic drop-off. An advantage of dendrimers is that their release rates are more consistent, which has been demonstrated by polylactide-PAMAM dendrimers [91] and dendrimer-platinate [92]. Consistent release from dendrimers is likely an inherent feature of their defined three-dimensional structure as their sites of drug attachment are continuously exposed to solvent, compared with random polymers where conjugated pro-drug moieties can be internalized randomly. The unique architectural features of dendrimers offer additional elegant strategies to gain exquisite control over release of active drug. In particular, the production of dendrimers functionalized with catalytic antibodies [68] has spurred the development of dendrimers capable of ‘‘selfimmolation’’ [93–95]. Self-immolative dendrimers provide an attractive potential platform for multidrug delivery. To briefly explain, these unique assemblies have the ability to release all of their tail units (i.e., the active drug) through a self-immolative chain fragmentation, which is initiated by a single cleavage at the dendrimer’s core [96]. The first generation of dendritic prodrugs was demonstrated by Shamis and coworkers who synthesized doxorubicin and camptothecin as tail units and designed a retro-aldol retro-Michael focal trigger provided by action of the catalytic antibody 38C2 [94]. This method showed a dramatic increase in toxicity to tumor cells upon bioactivation of the pro-drug compared with tests done in the absence of the activating antibody. This technology, when fully developed into a complete chemical adaptor system that combines a tumor-targeting device (Section 1.6.3), a pro-drug, and pro-drug activation trigger, provides a sophisticated platform for future research efforts and the development of drugs for in vivo use [93]. 1.5.4

Fine-tuning Dendrimer Properties to Facilitate Delivery and Ensure Bioactivity Delivery Requires Avoiding Non-specific Uptake From the initial entry into the body, a drug candidate confronts many barriers and diversions on its route to the site of intended bioactivity. Uptake by oral ingestion is ideal for patient comfort and, while still largely speculative for dendrimers [97], there is now evidence that uptake occurs in the rat gut [98]; this route is enticing based on an increasing recognition that nanoparticle uptake across the gut is largely governed by the physicochemical properties and surface chemistries of oral drug delivery vehicles [99]. Typically, to get to the target site in the body, the drug candidate must avoid becoming trapped with the extracellular matrix, which has been shown to hinder cellular uptake and reduce the efficiency of other nanosized delivery vehicles [100]; instead entry into the bloodstream is generally required for transit to the intended site of action. Once in the bloodstream, either by successful navigation of an oral route or through direct injection, dendrimers below a certain size are at risk of filtration 1.5.4.1

1.5 Dendrimers in Drug Delivery

and removal by the kidney. This pitfall, however, can be avoided by ensuring that sufficiently large dendrimers are used. Indeed an important design feature and overriding impetus to use dendrimer delivery vehicles is to prevent the filtration of these drug candidates by the kidney. A second, off-target ‘‘trap’’ for dendrimers has been identified in a study that showed sequestration of dendrimers in the liver and spleen, in part due to their surface properties and in part due to their size [101]. As discussed elsewhere, both of these parameters can be controlled with exquisite sensitivity for dendritic macromolecules, allowing longer residence times in the blood (the longer the serum half-life, the greater the opportunity to reach the intended site of action). ‘‘Local’’ Considerations: Contact with, and Uptake by, the Target Cell Once a dendrimer has successfully entered the bloodstream and has been designed to minimize undue accumulation in non-target organs or tissues, it still faces the challenge of seeking out and interacting with its targeted site of action. The diversity of cell surface targets available for a nanodevice to bind to is vast; here we limit ourselves to specific examples related to cancer (Section 1.6.3). We will jump ahead to the point when a dendrimer has made ‘‘first contact’’ with a cell and reflect on how it interacts with the membrane. In this regard, there are provocative studies with PAMAM polymers that suggest that binding to the cell surface is facilitated by the deformable properties of dendrimers [15, 16, 18] (Section 1.3.1.3). Cellbinding induced deformations, if they prove real, have important implications for drug delivery. For example, the flattened forms of dendrimers lose their internal voids where guest molecules – such as drug payloads – are sequestered [6]. If this step occurs too soon, i.e., outside of the target cell, the drug might be ineffective, whereas if it occurs at the right moment, i.e., in the cytosol for cytosolic-acting drugs, it would provide an additional design parameter to exploit in the drug release process (Section 1.5.2.3). Notably, the deformations proposed to occur upon the interaction of a dendrimer with a cell, where the dendrimer shifts from a canonical ‘‘spherical’’ shape to a flattened disk with a significant loss in volume, have been most-extensively investigated at the dendrimer–mica interface. Clearly, the plasma membrane of a cell shares few biophysical characteristics with an extremely flat and rigid surface of mica, therefore, combined with the thermodynamically unfavorable aspects of the putative shape change, the extrapolation to drug delivery in biological systems should not be overstated. Encouragingly, shape changes also have been observed – but not thoroughly characterized – for dendrimers encountering the air–water interface, which is a better model for biological systems. Regardless of the current lack of concrete information, the intriguing nature of this potential mechanism for cell targeting and drug release merits its discussion here and also warrants further experimental investigation. Once a dendrimer is in contact with a cell, there is strong experimental evidence that the exact surface properties of the dendrimer influence cellular uptake [102]. Therefore, the ability to modulate the chemical properties of a dendrimer provides additional options for controlling the uptake of a dendritic drug delivery device into a cell and even partitioning pro-drug release into specific organelles. To elaborate 1.5.4.2

21

22

1 Dendrimers in Cancer Treatment and Diagnosis

by briefly recapitulating a series of elegant experiment from the Banaszak Holl group, these researchers used a battery of assays, ranging from dye leakage to atomic force microscopy, to demonstrate that G5–G7 PAMAM dendrimers disrupt lipid bilayers and form holes large enough (5–40 nm) to account for dendrimer internalization. Moreover, the hole formation could be tuned by the exact size of the dendrimer, as well as surface chemical properties. To be specific, G7 amineterminated PAMAM initiated hole formation while its G5 counterpart did not. The smaller G5 dendrimer, however, did expand holes at existing defects; by contrast, acetamide terminated G5 PAMAM neither initiated hole formation nor expanded existing defects [102, 103]. The mechanism of hole formation in membranes by PAMAM was proposed to involve the removal of lipid molecules from the membrane to form aggregates consisting of a dendrimer surrounded by lipid molecules [103]. Once inside a cell, there are early indications that the precise properties of a dendrimer can influence subcellular trafficking. Eventually, if these processes can be better understood and controlled, their exploitation for drug delivery will be very attractive considering that some entities, such as dendrimerdelivered ibuprofen, need to only gain access to the cytosol [85], whereas other class of drugs, such as dendrimer-delivered plasmid DNAs, have the moredemanding task of reaching the nucleus [104]. 1.5.5

Drug Delivery: Ensuring the Biocompatibility of Dendritic Delivery Vehicles 1.5.5.1 Biocompatibility Entails Avoiding ‘‘Side Effects’’ such as Toxicity and Immunogenicity To briefly reiterate, properties of dendritic polymers important for drug delivery include negligible polydispersity, a high-density payload of pro-drug, and the ability to selectively release the active form of drug precisely at its intended site of action. Although dendrimers are capable of each of these tasks, their advantages are for naught if the final dendritic complex is not ‘‘biocompatible.’’ Biocompatibility, a broad term with numerous meanings, will be considered here from three perspectives, water solubility, lack of immunogenicity, and toxicity.

Water Solubility and Immunogenicity The first two biocompatibility issues mentioned above, namely water solubility and immunogenicity, are closely related insofar as highly-hydrated macromolecules tend to be less immunogenic. With dendrimers, there are many options available to overcome difficulties that arise in these areas. For example, solubility can be readily adjusted by surface modifications to surface chemistry or by the addition of conjugated ligands (Section 1.5.3, Fig. 1.3). Moreover, dendrimers such as the commonly used G3, G5, and G7 PAMAM clusters are not inherently immunogenic [105]. Derivatized PAMAM such as the G4D-(1B4M-Gd)62 magnetic resonance imaging (MRI) contrast dendrimer, however, can become immunogenic (which is not surprising considering the deliberate efforts to render small molecules immunogenic through presentation on a dendritic scaffold). This problem – 1.5.5.2

1.5 Dendrimers in Drug Delivery

once again tying together the concepts of solubility and immunity – was overcome in one study by conjugation of poly(ethylene glycol) (PEG) to the surface of the dendrimer. Notably, PEG also had the positive effect of decreasing non-specific clearance from the blood, likely due to the increased hydration and resulting solubility of the particle [106]. Inherent and Induced Toxicity A basic issue in drug delivery is the avoidance of non-specific, systemic, or offtarget toxicity. At its simplest this issue, when applied to dendrimers, involves the biological effects of the material used to construct the polymer. Ideally, the building blocks themselves, as well as their degradation products upon delivery and release of the drug payload, are non-toxic. One strategy is to directly use natural biological molecules, such as carbohydrates [59, 107], amino acids and peptides [108], nucleic acids [109–113], or lipids [114, 115] as the building blocks. To provide additional synthetic flexibility, while maintaining biocompatibility, an increasing number of biologically compatible and generally-regarded as safe (GRAS) materials are being used in dendrimer construction. Examples include dendritic polyglycerol [116], melamine [117]; phosphate [118], polyglycerols [39], a polyester dendrimer based on poly(ethylene oxide) that has tunable molecular weights and architectures [84], and dendrimers composed of citric acid and poly(ethylene glycol) [65]. The pioneering PAMAM-based dendrimers illustrate a second issue beyond inherent toxicity of the material or breakdown products, namely ‘‘induced’’ toxicity. The PAMAM family (Table 1.1), although not explicitly designed for biocompatibility, was found to be non-toxic when generations 1 through 5 were tested [105]. Evaluation of G7 dendrimers, however, showed potential biological complications, including dose-dependent toxicity [105], thereby illustrating that, while the basic material of PAMAM is inherently non-toxic, deleterious outcomes could be ‘‘induced’’ by factors such as the size or structure of the nanodevice. Smaller generation, non-toxic, dendrimers are sufficient for some applications but larger clusters are needed to fully exploit the enhanced permeation and retention (EPR) effect important in the treatment of cancer with macromolecular therapeutics (Section 1.6.2); consequently, toxicity cannot simply be avoided by restricting use to small, safe-sized particles. Instead, one strategy devised to avoid toxicity was the re-design of the building blocks of PAMAM-based material [76, 119] while another strategy involved the development of completely new polymeric backbones [120]. The selection of ‘‘safe’’ building blocks to avoid deleterious effects in dendrimer construction is unlikely to prevent all problems. To illustrate, even very safe building blocks, such as amino acids, can be highly toxic or immunogenic when assembled into large macromolecules – in this case proteins – in the ‘‘wrong’’ way. Indeed, the toxicity of dendrimers could be the result of several factors beyond the simple properties of the unloaded scaffold. For instance, with cancer drugs intended to kill cells, systemic toxicity could result if the drug is taken up by the wrong cellular target (i.e., a healthy cell or tissue, rather than a cancer cell or tumor) or if the nanodevice was ‘‘leaky’’ (i.e., if the pro-drug was released systemically before reaching the target cell). Fortunately, many strategies exist for prevent1.5.5.3

23

24

1 Dendrimers in Cancer Treatment and Diagnosis

ing toxicity, including directing a drug to its intended site of action by targeting moieties (Section 1.6.3) and developing sophisticated release strategies (Section 1.5.3.3). Problems that arise from the surface properties of the conjugated dendrimer can be ameliorated by masking the surface with something as simple as PEG or, in more advanced schemes, by coating with sugars or peptides to make glycodendrimers or peptide dendrimers, respectively (discussed in Ref. [121]) to mimic proteins naturally found in circulation (Section 1.4.3.2).

1.6

Dendrimers in Cancer Diagnosis and Treatment 1.6.1

Dendrimers have Attractive Properties for Cancer Treatment

Cancer epitomizes the challenges faced during drug delivery: an anticancer drug must be able to seek out subtle changes that distinguish a transformed cell from the other 200 or so types of healthy cells found in the body and then provide a sufficiently high dose of a toxic agent to selectively kill the cell while not harming its healthy neighbors. Therefore, even though dendrimers can be endowed with many favorable properties for drug delivery (Section 1.5), an ultimate challenge – ergo, a ‘‘real-world’’ test – of these versatile nano-devices will be whether they can successfully meet the formidable tasks of diagnosing and treating of malignant disease. As described in Section 1.7, although significant work remains in several areas, prospects now appear bright for dendrimer-based approaches to cancer treatment. 1.6.2

Dendrimer-sized Particles Passively Accumulate at the Sites of Tumors

To begin the discussion of properties that make dendrimers attractive vehicles for cancer treatment, we revisit the concept that encapsulation (Section 1.5.2) or covalent linkage (Section 1.5.3) of small molecule drug candidates to a dendrimer enhances the pharmacological properties of the drug. In cancer chemotherapy, these desirable size-based features are reinforced by the enhanced permeability and retention (EPR) effect that improves the delivery of macromolecules to tumors. The EPR effect is based on unique pathophysiological features of a solid tumor, such as extensive angiogenesis resulting in hyper-vascularization, limited lymphatic drainage, and increased permeability to lipids and macromolecules. These features, which help ensure adequate nutrient supply to meet the metabolic requirements of rapidly growing tumors [122, 123], can be turned to the tumor’s disadvantage by the use of nano-sized therapeutic agents. The EPR effect was discovered when selective accumulation of the SMANCS conjugate (styrene-maleic anhydride-neocarzinostatin) was observed at the site of tumors while similar accumulation was not seen with neocarzinostatin alone

1.6 Dendrimers in Cancer Diagnosis and Treatment

[124, 125]. The EPR response was subsequently demonstrated for similarly-sized liposomes, thereby establishing that this effect was largely a function of particle size and did not solely depend on the chemical or biophysical properties of the macromolecule. Specifically, in one study optimal tumor delivery occurred for liposomes having a size distribution between 70 and 200 nm in diameter [126]. An independent study showed efficacy for liposomes loaded with daunorubicin in the same size range; specifically, those @142 nm in diameter exhibited an inhibitory effect against Yoshida sarcoma whereas smaller (@57–58 nm) and larger (@272 nm) liposomes had weaker or no effect [127]. Over time, cautionary notes were raised that tempered initial enthusiasm for exploiting the EPR effect for cancer treatment. For example, the porosity of the vasculature in tumors can be highly variable even with a single vessel that can be leaky to one size of particle in one region but not in another [128]. Experimentally addressing this issue was complicated by the size polydispersity of traditional nanoparticles used to exploit the EPR effect, which were typically either lipids or conventional polymers that rendered a significant proportion of intended drug inactive. Fortunately this issue – the ability to match exact and uniform sizes needed to target an individual tumor – is highly tractable with dendrimers because selection of an exactly-sized entity is possible (Table 1.1) compared with the large size distributions that plague liposome and most polymeric materials [42]. The ability to construct monodisperse populations of dendrimers in the size range needed to exploit the EPR effect is an encouraging step towards the passive exploitation of tumor properties. Once the basic issue of size was resolved, however, secondary challenges (and opportunities) arose from observations that the chemical properties of the nano-sized particle can play significant roles in modulating the EPR effect. By way of a specific example, ‘‘conventional’’ polymeric materials showed efficacy at a smaller size range, occurring at ~60 nm for both watersoluble and hydrogel forms of poly(vinyl alcohol) (PVA) [129], whereas almost identically-sized 57 nm egg phosphatidylcholine (EPC)-liposomes were ineffective [127]. As reported above, liposomes about twice this size showed maximal efficacy, so it was not unexpected that the EPC-liposomes were ineffective. Interestingly, however, hydrogenated egg phosphatidylcholine (HEPC)-liposomes in this size range (specifically, 58 nm) were active [127], illustrating that the exact chemical properties of the material is a critical design parameter. In this respect, the many options for dendrimer ‘‘building blocks’’, as well as the ability to further tune surface properties provide many opportunities to endow dendrimers with favorable ‘‘passive’’ properties for tumor targeting. 1.6.3

Multifunctional Dendrimers can Selectively Target Biomarkers found on Cancer Cells Methods for Targeting Specific Biomarkers of Cancer As discussed above, dendrimers can achieve passive EPR-mediated targeting to a tumor simply by control of their size and physicochemical properties. Passive tar1.6.3.1

25

26

1 Dendrimers in Cancer Treatment and Diagnosis

geting, which localizes the nano-particle in the close vicinity of a cancer cell, can be immediately useful for diagnostic purposes (Section 1.6.4) or for the delivery of radioisotopes capable of killing any cell within a defined radius. In general, however, most delivery strategies require that the anticancer agent directly attached to, or be taken up by, the target cell. The ability to append more than one type of functionality to a dendrimer (Fig. 1.3) allows the inclusion of ligands intended to bind specifically to cancer cells in the design of a multi-functional drug-delivery nanodevice (Fig. 1.5). Although a wide range of targeting ligands have been considered, including natural biopolymers such as oligopeptides, oligosaccharides, and polysaccharides such as hyaluronic acid, or polyunsaturated fatty acids [90, 130], discussion here is limited to folate, which is an exemplary small molecule tumor-targeting agent [42], as well as monoclonal antibodies directed against tumor associated antigens (TAAs). Targeting by Folate, a Small Molecule Ligand Folate is an attractive small molecule for use as a tumor targeting ligand because the membrane-bound folate receptor (FR) is overexpressed on a wide range of human cancers, including those originating in ovary, lung, breast, endometrium, kidney and brain [131]. As a small molecule, it is presumed to be non-immunogenic, it has good solubility, binds to its receptor with high affinity when conjugated to a wide array of conjugates, including protein toxins, radioactive imaging agents, MRI contrast agents, liposomes, gene transfer vectors, antisense oligonucleotides, ribozymes, antibodies [131, 132] and even activated T-cells [133]. Upon binding to the folate receptor, folate-conjugated drug conjugates are shuttled into the cell via an endocytic mechanism, resulting in major enhancements in cancer cell specificity and selectivity over their non-targeted formulation counterparts [131, 132]. Recently, folate has been enlisted in an innovative dendrimer-based targeting schemes ([42, 134], Section 1.6.6.1). 1.6.3.2

Targeting by Monoclonal Antibodies Of the many strategies devised to selectively direct drugs to cancer cells, perhaps the most elegant (and demanding!) is the use of monoclonal antibodies that recognize and selectively bind to tumor associated antigens (TAAs) [135–138]. TAA-targeting monoclonal antibodies have been exploited as delivery agents for conjugated ‘‘payloads’’ such as small molecule drugs and prodrugs, radioisotopes, and cytokines [139, 140]. The field of ‘‘immunotherapy’’ envisioned almost a hundred years ago, and given renewed impetus a quarter century ago by the development of monoclonal antibody technologies, has nonetheless progressed erratically over the past two decades as many pitfalls have been encountered [139]. Current prospects remain mixed but hopeful; optimistically, progress marked by commercial interest with companies providing their immunotherapeutic drug candidates with flashy trademarked names, such as ‘‘Armed Antibodies TM’’ [141]. Similarly, the rosy opinion that this field is ‘‘on the verge of clinical fruition’’ has been published recently [142]. Perhaps, more realistically, one recent synopsis 1.6.3.3

1.6 Dendrimers in Cancer Diagnosis and Treatment

holds out ‘‘hope’’ for a major clinical impact for this strategy within the next 10 years [136]. Although a detailed discussion of the many pitfalls encountered in immunotherapy efforts is beyond the scope of this chapter, one key issue – readily addressed by dendrimers – is the requirement that an extremely potent cytotoxic drug be used in targeted antibody therapy. This point is illustrated by the fact that the greatest progress in this field has occurred for immunotoxins, which are antibody–toxin chimeric molecules that kill cancer cells via binding to a surface antigen, internalization and delivery of the toxin moiety to the cell cytosol. In the cytosol, protein toxins, such as those from diphtheria or pseudomonas, catalytically inhibit a critical cell function and cause cell death [143]. The high potency of immunotoxins for killing cancer cells is dramatically illustrated by ricin, where the catalytic activity of this ribosome-inactivating enzyme allows a single immunotoxin conjugate to kill a cell upon successful uptake and trafficking to the site of action [144, 145]. A drawback of immunotoxins is their significant immunogenicity, which limits repeated use [136]; from a broader perspective, their repeated use is made necessary by difficulties in providing a sufficiently high drug load to eradicate all cancer cells despite the high potency of conjugated toxin. An alternative approach of radioimmunotherapy, where high energy radionuclides are conjugated to TAA-targeting antibodies, also shows promise [146] but suffers from indiscriminate toxicity (the surrounding healthy tissues, as well as off-target tissues, become irradiated in addition to the target cancer cells). A third possible approach for immunotherapy, the conjugation of commonly-used small molecule drugs to TAAs, is hindered by the relatively low potency of most low molecular weight therapeutics. To illustrate this point, @10 000 TAAs occur on a typical cancer cell [101], making this number the upper limit for the number of targeting antibodies that can bind to the cell. The widely used anticancer drug cisplatin, to give one example, requires internalization of at least 50
this level of drug molecules for therapeutic efficacy. A numerical analysis of the cisplatin example presented above indicates that each tumor-targeting antibody would have to be modified with a large number of small molecules to be effective as an anticancer drug (in this case, roughly 50 cisplatin molecules upon superficial analysis). Modification of an antibody with multiple radioisotopes, toxins, or even small molecules to increase the efficacy of cell killing, however, diminishes or eliminates the inherent specific antigen-binding affinity of an antibody. Therefore, to maximize drug loading while minimizing the deleterious effects on the biological integrity of the host antibody, an attractive approach is to use a linker molecule, such as a dendrimer, that can be highly conjugated (or internally loaded) with drug while modifying only a single site on the surface of the antibody [147]. Methodology to covalently attach antibodies to dendrimers that preserve the activity of the antigen–antibody binding site [148, 149], e.g., by chemical modification of their carbohydrates and subsequent linkage to PAMAM [150], has opened the door for the inclusion of dendrimers in immunotherapy [151, 152], thereby enhancing the future prospects of this chronically ‘‘almost-there’’ strategy.

27

28

1 Dendrimers in Cancer Treatment and Diagnosis

1.6.4

Dendrimers in Cancer Diagnosis and Imaging Labeled Dendrimers are Important Research Tools for Biodistribution Studies The synthetic ability to attach both a tumor-targeting antibody and a potent payload of anticancer drugs to the same dendritic molecule provides a platform for multifunctional nano-scale drug delivery devices (Fig. 1.5). Before this technology can be applied in the clinic, however, its safety and efficacy must be demonstrated; towards this end, fluorescently-modified dendritic conjugates have been used extensively to characterize cell targeting, surface binding, uptake and internalization, and even sub-cellular localization [85, 151, 152]. The radiolabeled counterparts appropriate for animal studies have allowed detailed examination of the biodistribution of dendrimers. Several radio-isotopes have been conjugated to dendrimers, including 3 H [153], 14 C [105], 88 Y [154], 111 In [154, 155], and 125 I [98, 149, 156–158]. These studies have established that the chemical and physical properties of dendrimers can be tuned to favor distribution to or away from specific organs and, ultimately, to achieve favorable biodistribution to tumors. The methods used in these experiments, however, typically requiring post-administration dissection of the host animal to allow the analysis of organ sequestration and tissue distribution of the radioisotope, are clearly not applicable to clinical practice. Instead, they have served as an important stepping stone along the path towards non- or minimally-invasive diagnostic procedures, which are proceeding mainly by the development of MRI contrast agents. 1.6.4.1

Towards Clinical Use: MRI Imaging Agents Upon successful demonstration of the selective accumulation of dendrimers at the sites of tumors in animal models, a natural extension of this approach was to substitute gadolinium for the previously-tested isotopes or fluorophores. Gadolinium ( 153 Gd) is the best known and most extensively utilized magnetic resonance (MR) contrast agent [159, 160] and has previously been shown to be valuable for the improved diagnosis of cancer [161, 162]. Importantly, the in vivo efficacy of gadolinium is greatly enhanced when used as part of a macromolecular system [159]; in the past, attempts to create macromolecular gadolinium platforms have included the conjugation of chelators for this metal to both proteins [163] and conventional polymers [164]. These efforts have met with mixed (but generally limited) success. By contrast, Kobayashi and Brechbiel report that, by conjugating gadolinium to dendrimers, the unique properties of these polymers, such as exquisite size control, allowed selective targeting and imaging of the kidney, vascular, liver, or tumors [159]. Of note, tumor specific targeting and accumulation of gadolinium contrast agents is possible by use of either the folate receptor [165] or TAAs [159]. A drawback of the initial PAMAM-based MR contrast agents was their long residence time in the body; this problem, however, can be met by modifying both the surface properties [106] and basic chemical composition of the dendrimer. Specifically, diaminobutane (DAB) dendrimer-based chelators were more rapidly excreted from the 1.6.4.2

1.6 Dendrimers in Cancer Diagnosis and Treatment

body, illustrating that the development of clinically-acceptable dendrimer MR platforms is realistic [166]. 1.6.5

Steps Towards the Clinical Realization of Dendrimer-based Cancer Therapies The Stage is now set for Dendrimer-based Cancer Therapy The use of dendrimers for cancer treatment is still in its infancy with few, if any, applications successfully translated to the clinic. Consequently, their use as diagnostic agents constitutes both an important goal in and of itself, and also a valuable ‘‘baby step’’ towards the ultimate goal of curing cancer. As discussed, the process of actual killing cancer cells entails the complicated process of drug uptake followed by release of the drug into the cytoplasm or nucleus and is clearly a more demanding process than cell surface labeling, or even localization to the vicinity of the tumor, sufficient for diagnostic purposes. Nonetheless, in some cases, the transition from imaging to therapy will be closely linked, as evidenced by efforts now underway to combine antibody-targeted MR imaging nanoparticles with the delivery of antiangiogenic genes intended to inhibit the vascularization to the V2 carcinoma model in rabbits [167]. Another promising strategy – boron neutron capture therapy – has undergone impressive development over the past decade and is presented next as a successful demonstration of the promise of dendrimer-based cancer therapies. 1.6.5.1

Boron Neutron Capture Therapy Cisplatin-based therapies illustrate the need for multiple conjugations of small molecules – estimated at 50 for this platinum drug – to a targeting antibody (Section 1.6.3.3). While some efforts are underway to use dendrimeric strategies for platinum drug delivery [168], an even more demanding situation, where thousands of ligands are required per targeting antibody, is provided by boron neutron capture therapy (BNCT). Accordingly, BNCT will be discussed here as an illustrative example of how dendrimers can help overcome high hurdles in the development of innovative cancer therapies. As a brief background, BNCT is based on the nuclear reaction that occurs when boron-10, a stable isotope, is irradiated with low energy (a 0.025 eV) or thermal neutrons to yield alpha particles and recoiling lithium-7 nuclei. A major requirement for the success of BNCT is the selective delivery of a sufficient number of boron atoms (@10 9 ) to individual cancer cells to sustain a lethal 10 B(n, alpha) ! 7 Li capture reaction [169, 170]. Considering that the maximal number of antigenic sites per tumor cell is in the range of 100 000, and more commonly only 1/10 th that level, an a priori calculation suggests that each targeting antibody must be linked to at least 2000, but preferably closer to 5000, boron atoms [101]. Clearly, a single TAA-targeting antibody cannot be directly conjugated at this level and conventional polymers – e.g., polylysine conjugated with @1700 boron derivatives and linked to a targeting antibody – caused the antibody to lose in vivo tumor localizing properties [171]. By contrast, when a 1.6.5.2

29

30

1 Dendrimers in Cancer Treatment and Diagnosis

PAMAM dendrimer was used for polyvalent boron conjugation, the linked antibody maintained immuno-recognition (although in vivo tumor targeting remained problematic because the conjugated dendrimer had a strong propensity to mislocalize in the spleen and liver) [101]. Over the decade since these pioneering efforts were first reported, continued progress has been made to solve problems such as off-target tissue localization, which was traced to the size of the dendrimer and presence of a large number of amine groups on the surface of PAMAM, by exploiting the versatility of dendrimer chemistry. In short, the re-design of boronated, anti-body-targeted dendrimers has culminated in the successful treatment of gliomas in the rat [158, 169, 172] and laid the foundation for translation of this technology into clinical tests in the foreseeable future. 1.6.6

Innovations Promise to Speed Progress ‘‘Mix-and-Match’’ Strategy of Bifunctional Dendritic Clusters Two lessons are immediately apparent from the dedicated efforts to bring dendrimer-based BNCT to fruition. One is that dendritic technologies, while still at an early developmental stage, hold tremendous promise and merit continued investigation. The second is that the coupling of one treatment modality (BNCT) with one targeting strategy (antibodies to a specific type of glioma) required a staggering amount of effort. The growing realization that cancer is hundreds, if not thousands, of unique diseases at the cellular and molecular level, suggests that a commensurate number of therapeutic strategies are needed. The diversity of targeting strategies (which are not limited to folate and TAAs discussed here), coupled with the many ‘‘payload’’ possibilities (beside radioisotopes, boron, and cisplatin discussed here) used to diagnose and kill cancer cells, means that there are literally tens of thousands of individually customized therapies required to fully confront the myriad clinical manifestations of cancer. The sobering reality is that, if each of these customized treatments will require a decade long effort by a large team of researchers and clinicians, the large problem of cancer treatment will not be solved for a long time. Choi and coworkers [134] have come up with an innovative mix-and-match scheme that promises to offset this gloomy prediction. These researchers have recently reported a cancer-targeting strategy that is reminiscent of the antibody– toxin/immunoconjugate strategy where distinct, but linked, entities are used to first recognize and bind and then subsequently modify a cancer cell. Their strategy, however, has great potential to improve on both the ‘‘targeting’’ and ‘‘payload’’ aspects of cancer therapy by, at first seemingly paradoxically, completely dividing these functions into separate dendritic clusters (Fig. 1.6). The key to this approach was to include a DNA ‘‘zipper’’ on each dendrimer that allows the targeting cluster, composed of folate-derivatized PAMAM in proof-of-concept experiments [173], to be readily combined with the imaging or drug-carrying dendrimer by way of the complementary DNA strand [134]. It can be envisioned that the production of libraries of dendrimers targeted to different cancer-specific biomarkers can be pro1.6.6.1

1.6 Dendrimers in Cancer Diagnosis and Treatment

DNA–dendrimer conjugates as potential cancer targeting imaging agents or therapeutics. (Adapted from Ref. [189].) Differentially functionalized dendrimers covalently conjugated to complementary deoxyoligonucleotides can readily form duplex combinatorial nanoclusters that possess

Fig. 1.6.

cancer cell-specific ligands hybridized to an imaging agent or drug. Cell-specific targeting ligands (e.g., folic acid in one study) are appended to Dendrimer A, and Dendrimer B is conjugated with an imaging agent or drug [134].

duced by a ‘‘mix-and-matched’’ strategy by combining ‘‘off-the-shelf ’’ targeting and drug clusters as needed [42]. Development of easily-customizable nanomedicine platforms that exploit the facile duplex DNA formation for the generation of hybrid nano-clusters, thus circumventing the tedious synthesis of multiply-functionalized dendrimers, offers hope that the next ten years will witness rapid expansion of dendrimer technologies that build on the painstaking advances of the past decade. 1.6.6.2 Towards Therapeutic Exploitation of Glycosylation Abnormalities found in Cancer Aberrant glycosylation, where the patterns of complex carbohydrate glycoforms found on the surfaces of cancer cells are dramatically different from those on healthy cells, is a hallmark of cancer [174–178]. Efforts to exploit these changes therapeutically, however, have long been stymied by the difficulty of controlling these complex and diverse molecules in an artificial synthetic setting. Today, with new technologies such as dendrimers that provide a platform for physiologicallyrelevant display of carbohydrates, new vistas are opening up for exploiting these molecules to intervene in malignant disease. Promising – but still early-stage – efforts in this direction include the presentation of oligosaccharides found only in cancer cells [53, 56, 58, 179–181] on a dendritic scaffold (Section 1.4.3.2) for vaccine development (Section 1.5.3.2).

Towards Targeting Metabolically-engineered Carbohydrate Epitopes As discussed above, one area of rapidly-expanding investigation is the abnormal glycosylation associated with the cancer cells; in particular dendrimeric scaffolds provide a unique platform to control the multimeric carbohydrate presentation needed to enact the ‘‘cluster glycoside effect’’ [45, 50, 51], which is crucial for tar1.6.6.3

31

32

1 Dendrimers in Cancer Treatment and Diagnosis

geting diseased tissues found in malignant diseases [1, 24]. Another approach to exploiting glycosylation for the treatment of cancer is through ‘‘chemical biology’’ strategies, such as the ability to express non-natural sialic acids on the cell surface through the use of N-acetylmannosamine (ManNAc) analogs [49, 182, 183] (Fig. 1.7). By appropriate design of the ManNAc analog, sialic acids, which are interesting nine-carbon sugars often overexpressed on cancer cells [175], can be provided with a ‘‘chemical handle’’ – such as a ketone, azide, or thiol [184–186] – for tar-

Chemoselective targeting of drugloaded dendrimers to the cell surface. (A) Overview of sialic acid engineering. (a) A dendrimer can encapsulate and assist the delivery of N-acetylmannosamine (ManNAc) analogs, such as the thiol-containing sugar ‘‘ManNTGc’’ (shown as ‘‘*’’) into a cell (Section 1.5.2). (b) Once inside the cell, ManNTGc can be metabolically converted into CMP-Neu5TGc, a compound that serves as a sugar-nucleotide needed for the glycosylation process (c) where ‘‘Neu5TGc’’ a non-natural form of sialic acid, is installed into cell surface glycoconjugates. Overall, this process replaces

Fig. 1.7.

natural sialic acids, such as ‘‘Neu5Ac’’, with their thiol-containing counterparts (d), which can then be targeted by dendritic assemblies such as the bifunctional ‘‘targeting’’ and ‘‘payload’’ clusters shown in Fig. 1.6. (B) Details of the ‘‘chemoselective ligation reaction’’ required for targeting the appropriately derivatized dendrimeric assembly to the cell. In this case, a maleimideconjugated targeting capsule will selectively interact with the sialic acid-display thiols to covalently bind the dendrimer to the cell surface via thio-ether bond formation.

References

geted delivery of a second agent such as the ricin A-chain used in immunotoxins [187] or small molecule anticancer drugs [188]. Dendrimers offer assistance at several steps in this process of translating early-stage anticancer strategies like ‘‘sialic acid engineering’’ from the laboratory to clinical relevance. An enticing proposition is that the starting material – ManNAc, which like all sugars has notoriously poor pharmacological properties – can be made ‘‘drug-like’’ by encapsulation (or covalent ligation). Subsequently, after display of the target epitope on the cell surface, which is a modified thiol-bearing sialic acid in the case shown in Fig. 1.7, this can benefit from the high local density of dendritic display of maleimide to increase the rate of drug binding to the cell surface, which occurs over an unacceptably long period of several hours for current covalent coupling schemes [188]. This strategy, under evaluation in our laboratory, coupled with a high drug payload on the DNA-hybridized cluster (Fig. 1.6), provides renewed impetus for the already promising application of sugar-based therapeutic approaches to cancer. A particularly attractive aspect of this approach is that @10 8 sialic acids exist on cancer cells, greatly improving prospects to deliver adequate levels of drug to achieve therapeutic efficacy compared with TAA-targeting schemes (Section 1.6.3).

1.7

Concluding Remarks

Dendrimers, chemically-defined entities with tunable biological properties, have advanced over the past two decades to the point where they stand on the cusp of major contributions to the treatment of cancer in a meaningful way. Although, as has been apparent by the many instances cited throughout this chapter where gaps in knowledge still remain and that must be plugged before dendrimers are ready for wide clinical use, their extreme versatility combined with the extensive research efforts now underway are sure to add sophistication to drugs already in use as well as spur the development of entirely new classes of anticancer therapy.

Acknowledgments

Funding was provided by the Whitaker Biomedical Engineering Institute and Department of Biomedical Engineering at The Johns Hopkins University, the Arnold and Mabel Beckman Foundation, and The National Institutes of Health.

References 1 U. Boas, P. M. H. Heegaard,

Dendrimers in drug research. Chem. Soc. Rev. 2004. 33, 43–63. 2 G. M. Dykes, Dendrimers: A review of their appeal and applications. J.

Chem. Technol. Biotechnol. 2001. 76, 903–918. 3 A. Sunder, J. Heinemann, H. Frey, Controlling the growth of polymer trees: Concepts and perspectives for

33

34

1 Dendrimers in Cancer Treatment and Diagnosis

4

5

6

7

8

9

10

11

12

13

14

hyperbranched polymers. Chem. Eur. J. 2000. 6, 2499–2506. ‘‘Starburst2’’ is an official trademark owned by Dow Chemical that covers PAMAM based dendrimers but the term ‘‘starburst’’ is often also used generically to describe the properties of certain dendritic macromolecules. P. K. Maiti, T. C ¸ agin, G. Wang, W. A. Goddard III, Structure of PAMAM dendrimers: Generations 1 through 11. Macromolecules 2004. 37, 6236–6254. ¨ ter, J. P. Rabe, A. D. Schlu Dendronized polymers: Synthesis, characterization, assembly at interfaces, and manipulation. Angew. Chem. Int. Ed. 2000. 39, 864–883. P. G. de Gennes, H. Hervet, Statistics of ‘‘starburst’’ polymers. J. Phys. Lett. 1983. 44, L351–L360. B. Klajnert, M. Bryszewska, Dendrimers: Properties and applications. Acta Biochim. Pol. 2001. 48, 199–208. J. M. J. Fre´chet, Functional polymers and dendrimers: Reactivity, molecular architecture, and interfacial energy. Science 1994. 263, 1710–1715. C. L. Jackson, H. D. Chanzy, F. P. Booy, B. J. Drake, D. A. Tomalia, B. J. Bauer, E. J. Amis, Visualization of dendrimer molecules by transmission electron microscopy (TEM): Staining methods and Cryo-TEM of vitrified solutions. Macromolecules 1998. 31, 6259–6265. D. K. Smith, F. Diederich, Functional dendrimers: Unique biological mimics. Chem. Eur. J. 1998. 4, 1353–1361. S. Hecht, J. M. J. Fre´chet, Dendritic encapsulation of function: Applying Nature’s site isolation principle from biomimetics to materials science. Angew. Chem. Int. Ed. 2001. 40, 74–91. A. Esposito, E. Delort, D. Lagnoux, F. Djojo, J.-L. Reymond, Catalytic peptide dendrimers. Angew. Chem. Int. Ed. 2003. 42, 1381–1383. A. Clouet, T. Darbre, J.-L. Reymond, A combinatorial approach to catalytic peptide dendrimers. Angew. Chem. Int. Ed. 2004. 43, 4612–4615.

15 B. Chen, S. Piletsky, A. P. Turner,

16

17

18

19

20

21

22

23

24

25

High molecular recognition: Design of ‘‘Keys’’. Comb. Chem. High Throughput Screen. 2002. 5, 409–427. C. S. Goh, D. Milburn, M. Gerstein, Conformational changes associated with protein-protein interactions. Curr. Opin. Struct. Biol. 2004. 14, 104–109. P. O. Glantz, T. Arnebrant, T. Nylander, R. E. Baier, Bioadhesion – A phenomenon with multiple dimensions. Acta Odontol. Scand. 1999. 57(5), 238–241. A. Mecke, I. Lee, J. R. Baker Jr, M. M. Holl, B. G. Orr, Deformability of poly(amidoamine) dendrimers. Eur. Phy. J. E Soft Matter 2004. 14, 7–16. D. Farin, D. Avnir, Surface fractality of dendrimers. Angew. Chem. Int. Ed. Engl. 1991. 30, 1377–1379. S. L. Gilat, A. Adronov, J. M. J. Fre´chet, Light harvesting and energy transfer in novel convergently constructed dendrimers. Angew. Chem. Int. Ed. 1999. 38, 1422–1427. K. Autumn, Y. A. Liang, S. T. Hsieh, W. Zesch, W. P. Chan, T. W. Kenny, R. Fearing, R. J. Full, Adhesive force of a single gecko foot-hair. Nature 2000. 405, 681–185. G. M. Dykes, L. J. Brierley, D. K. Smith, T. McGrail, G. J. Seeley, Supramolecular solubilisation of hydrophilic dyes by using individual dendritic branches. Chem. Eur. J. 2001. 7, 4730–4739. R. Esfand, D. A. Tomalia, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today 2001. 6, 427– 436. M. Ozkan, Quantum dots and other nanoparticles: What can they offer to drug discovery? Drug Discovery Today 2004. 9, 1065–1071. D. A. Tomalia, A. M. Naylor, W. A. Goddard III, Starburst dendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem. Int. Ed. Engl. 1990. 29, 138–175.

References 26 H.-B. Mekelburger, W. Jawarek, F.

27

28

29

30

31

32

33

34

35

36

37

Vo¨gtle, Dendrimers, arborols, and cascade molecules: Breakthrough into generations of new materials. Angew. Chem. Int. Ed. Engl. 1992. 31, 1571–1576. J. Issberner, R. Moors, F. Vo¨gtle, Dendrimers: From generations and functional groups to functions. Angew. Chem. Int. Ed. Engl. 1994. 33, 2413–2420. D. Gudat, Inorganic cauliflower: Functional main group element dendrimers constructed from phosphorus- and silicon-based building blocks. Angew. Chem. Int. Ed. Engl. 1997. 36, 1951–1955. M. Fischer, F. Vo¨gtle, Dendrimers: From design to application – A progress report. Angew. Chem. Int. Ed. 1999. 38, 884–905. G. R. Newkome, C. N. Moorefield, F. Vo¨gtle, Dendrimers and Dendrons: Concepts, Syntheses, Applications. 2001, Weinheim, Wiley-VCH. S. M. Grayson, J. M. J. Fre´chet, Convergent dendrons and dendrimers: From synthesis to applications. Chem. Rev. 2001. 101, 3819–3868. I. Gitsov, C. Lin, Dendrimers – Nanoparticles with precisely engineered surfaces. Curr. Org. Chem. 2005. 9, 1025–1051. N. Feuerbacher, F. Vo¨gtle, Iterative synthesis in organic chemistry. Top. Curr. Chem. 1998. 197, 1–18. E. Buhleier, W. Wehner, F. Vo¨gtle, ‘‘Cascade’’- and ‘‘nonskid-chain-like’’ synthesis of molecular cavity topologies. Synthesis 1978. 155–158. G. R. Newkome, Z. Yao, G. R. Baker, V. K. Gupta, Micelles. Part 1. Cascade molecules: A new approach to micelles. A [27]-arborol. J. Org. Chem. 1985. 50, 2003–2004. D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P. Smith, Dendritic macromolecules: Synthesis of starburst dendrimers. Macromolecules 1986. 19, 2466–2468. D. A. Tomalia, H. D. Durst, Genealogically directed synthesis – Starburst cascade dendrimers and

38

39

40

41

42

43

44

45

46

47

hyperbranched structures. Top. Curr. Chem. 1993. 165, 193–313. T. M. Miller, T. X. Neenan, Convergent synthesis of monodisperse dendrimers based upon 1,3,5trisubstituted benzenes. Chem. Mater. 1990. 2, 346–349. R. Haag, J.-F. Stumbe´, A. Sunder, H. Frey, A. Hebel, An approach to core-shell-type architectures in hyperbranched polyglycerols by selective chemical differentiation. Macromolecules 2000. 33, 8158–8166. C. J. Hawker, J. M. J. Fre´chet, Preparation of polymers with controlled molecular architecture. A new convergent approach to dendritic macromolecules. J. Am. Chem. Soc. 1990. 112, 7638–7647. J. R. Morgan, M. J. Cloninger, Heterogeneously functionalized dendrimers. Curr. Opin. Drug Discov. Devel. 2002. 5, 966–973. Y. Choi, J. R. Baker Jr, Targeting cancer cells with DNA-assembled dendrimers: A mix-and-match strategy for cancer. Cell Cycle 2005. 4, 669–671. M. Meldal, P. M. St Hilaire, Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products. Curr. Opin. Chem. Biol. 1997. 1, 552–563. D. Zanini, R. Roy, Synthesis of new a-thiosialodendrimers and their binding properties to the sialic acid specific lectin from Limax flavus. J. Am. Chem. Soc. 1997. 119, 2088–2095. K. J. Yarema, C. R. Bertozzi, Chemical approaches to glycobiology and emerging carbohydrate-based therapeutic agents. Curr. Opin. Chem. Biol. 1998. 2, 49–61. J. J. Landers, Z. Cao, I. Lee, L. T. Piehler, P. P. Myc, A. Myc, T. Hamouda, A. T. Galecki, J. R. J. Baker Jr, Prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers. J. Infect. Dis. 2002. 186, 1222–1230. J. D. Reuter, A. Myc, M. M. Hayes, Z. Gan, R. Roy, D. Qin, R. Yin, L. T. Piehler, R. Esfand, D. A. Tomalia, J. R. Baker Jr, Inhibition of viral

35

36

1 Dendrimers in Cancer Treatment and Diagnosis

48

49

50

51

52

53

54

55

56

57

adhesion and infection by sialic-acidconjugated dendritic polymers. Bioconjugate Chem. 1999. 10, 271–278. D. H. Charych, J. O. Nagy, W. Spevak, M. D. Bednarski, Direct colorimetric detection of a receptorligand interaction by a polymerized bilayer assembly. Science 1993. 261, 585–588. O. T. Keppler, R. Horstkorte, M. Pawlita, C. Schmidt, W. Reutter, Biochemical engineering of the N-acyl side chain of sialic acid: Biological implications. Glycobiology 2001. 11, 11R–18R. J. J. Lundquist, E. J. Toone, The cluster glycoside effect. Chem. Rev. 2002. 102, 555–578. L. L. Kiessling, S. Pohl, Strength in numbers: Non-natural polyvalent carbohydrate derivatives. Chem. Biol. 1996. 3, 71–77. W. B. Turnbull, J. F. Stoddart, Design and synthesis of glycodendrimers. J. Biotechnol. 2002. 90, 231– 255. R. Roy, M. G. Baek, Glycodendrimers: Novel glycotope isosteres unmasking sugar coding. Case study with Tantigen markers from breast cancer MUC1 glycoprotein. J. Biotechnol. 2002. 90, 291–309. R. Roy, Synthesis and some applications of chemically defined multivalent glycoconjugates. Curr. Opin. Struct. Biol. 1996. 6, 692–702. P. R. Ashton, S. E. Boyd, C. L. Brown, N. Jayaraman, J. F. Stoddart, A convergent synthesis of a carbohydrate-containing dendrimer. Angew. Chem. Int. Ed. Engl. 1997. 36, 732–735. T. Toyokuni, S.-i. Hakomori, A. K. Singhal, Synthetic carbohydrate vaccines: Synthesis and immunogenicity of Tn antigen conjugates. Bioorg. Med. Chem. 1994. 2, 1119– 1132. S. Andre´, P. J. Ortega, M. A. Perez, R. Roy, H.-J. Gabius, Lactosecontaining starburst dendrimers: Influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and

58

59

60

61

62

63

64

65

66

67

immunoglobulins) on binding properties. Glycobiology 1999. 9, 1253–1261. R. Roy, M.-G. Baek, K. RittenhouseOlson, Synthesis of N,N 0 bis(acrylamido)acetic acid-based Tantigen glycodendrimers and their mouse monoclonal IgG antibody binding properties. J. Am. Chem. Soc. 2001. 123, 1809–1816. M. M. Palcic, H. Li, D. Zanini, R. S. Bhella, R. Roy, Chemoenzymatic synthesis of dendritic sialyl Lewis(x). Carbohydr. Res. 1997. 305, 433–442. M. S. Lajiness, M. Vieth, J. Erickson, Molecular properties that influence oral drug-like behavior. Curr. Opin. Drug Discov. Devel. 2004. 7, 470–474. N. R. Gonzales, R. De Pascalis, J. Schlom, S. V. S. Kashmiri, Minimizing the immunogenicity of antibodies for clinical application. Tumour Biol. 2005. 26, 31–43. B. Matthey, A. Engert, S. Barth, Recombinant immunotoxins for the treatment of Hodgkin’s disease (Review). Int. J. Mol. Med. 2000. 6, 509–514. K. Viswanathan, S. Lawrence, S. Hinderlich, K. J. Yarema, Y. C. Lee, M. Betenbaugh, Engineering sialic acid synthetic ability into insect cells: Identifying metabolic bottlenecks and devising strategies to overcome them. Biochemistry 2003. 42, 15 215–15 225. M. J. Betenbaugh, N. Tomiya, S. Narang, J. T. A. Hsu, Y. C. Lee, Biosynthesis of human-type N-glycans in heterologous systems. Curr. Opin. Struct. Biol. 2004. 14, 601–606. H. Namazi, M. Adeli, Dendrimers of citric acid and poly (ethylene glycol) as the new drug-delivery agents. Biomaterials 2005. 26, 1175–1183. J. F. G. A. Jansen, E. M. M. de Brabander-van den Berg, E. W. Meijer, Encapsulation of guest molecules into a dendritic box. Science 1994. 266, 1226–1229. U. Boas, S. H. M. So¨ntjens, K. J. Jensen, J. B. Christensen, E. W. Meijer, New dendrimer-peptide hostguest complexes: Towards dendrimers

References

68

69

70

71

72

73

74

75

76

as peptide carriers. ChemBioChem 2002. 3, 433–439. A. Co´rdova, K. D. Janda, Synthesis and catalytic antibody functionalization of dendrimers. J. Am. Chem. Soc. 2001. 123, 8248–8259. M. T. Morgan, M. A. Carnahan, C. E. Immoos, A. A. Ribeiro, S. Finkelstein, S. J. Lee, M. W. Grinstaff, Dendritic molecular capsules for hydrophobic compounds. J. Am. Chem. Soc. 2003. 125, 15 485– 15 489. P. K. Tripathi, A. J. Khopade, S. Nagaich, S. Shrivastava, S. Jain, N. K. Jain, Dendrimer grafts for delivery of 5-fluorouracil. Pharmazie 2002. 57, 261–264. T. Ooya, J. Lee, K. Park, Effects of ethylene glycol-based graft, starshaped, and dendritic polymers on solubilization and controlled release of paclitaxel. J. Controlled Release 2003. 93, 121–127. T. Ooya, J. Lee, K. Park, Hydrotropic dendrimers of generations 4 and 5: Synthesis, characterization, and hydrotropic solubilization of paclitaxel. Bioconjugate Chem. 2004. 15, 1221–1229. J. M. Benito, M. Gomez-Garcia, C. Ortiz Mellet, I. Baussanne, J. Defaye, J. M. Garcia Fernandez, Optimizing saccharide-directed molecular delivery to biological receptors: Design, synthesis, and biological evaluation of glycodendrimer-cyclodextrin conjugates. J. Am. Chem. Soc. 2004. 126, 10 355–10 363. T. Hudde, S. A. Rayner, R. M. Comer, M. Weber, J. D. Isaacs, H. Waldmann, D. F. Larkin, A. J. George, Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium. Gene Ther. 1999. 6, 939–943. J. Haensler, F. C. Szoka Jr, Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 1993. 4, 372–379. T.-i. Kim, H. J. Seo, J. S. Choi, H.-S. Jang, J.-u. Baek, K. Kim, J.-S. Park,

77

78

79

80

81

82

83 84

85

PAMAM-PEG-PAMAM: Novel triblock copolymer as a biocompatible and efficient gene delivery carrier. Biomacromolecules 2004. 5, 2487–2492. J. S. Choi, K. Nam, J.-y. Park, J.-B. Kim, J.-K. Lee, J.-s. Park, Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J. Controlled Release 2004. 99, 445–456. J. H. Lee, Y.-b. Lim, J. S. Choi, Y. Lee, T.-i. Kim, H. J. Kim, J. K. Yoon, K. Kim, J.-s. Park, Polyplexes assembled with internally quaternized PAMAMOH dendrimer and plasmid DNA have a neutral surface and gene delivery potency. Bioconjugate Chem. 2003. 14, 1214–1221. M. Hussain, M. Shchepinov, M. Sohail, I. F. Benter, A. J. Hollins, E. M. Southern, S. Akhtar, A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides. J. Controlled Release 2004. 99, 139–155. J. F. G. A. Jansen, E. W. Meijer, E. M. M. de Brabander-van den Berg, The dendritic box: Shapeselective liberation of encapsulated guests. J. Am. Chem. Soc. 1995. 117, 4417–4418. H. Ringsdorf, Structure and properties of pharmacologically active polymers. J. Polym. Sci. Polym. Symp. 1975. 51, 135–153. H. Bader, H. Ringsdorf, B. Schmidt, Water-soluble polymers in medicine. Angew. Makromol. Chem. 1984. 123/124, 457–485. J. Kopacek, Soluble biomedical polymers. Polym. Med. 1977. 7, 191–221. E. R. Gillies, E. Dy, J. M. J. Fre´chet, F. C. Szoka, Biological evaluation of polyester dendrimer: Poly(ethylene oxide) ‘‘bow-tie’’ hybrids with tunable molecular weight and architecture. Mol. Pharm. 2005. 2, 129–138. P. Kolhe, J. Khandare, O. Pillai, S. Kannan, M. Lieh-Lai, R. M. Kannan, Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload. Biomaterials 2006. 27, 660–669.

37

38

1 Dendrimers in Cancer Treatment and Diagnosis 86 S. C. Lee, R. Parthasarathy, K.

87

88

89

90

91

92

93

94

Botwin, D. Kunneman, E. Rowold, G. Lange, J. Klover, A. Abegg, J. Zobel, T. Beck, T. Miller, W. Hood, J. Monahan, J. P. McKearn, R. Jansson, C. F. Voliva, Biochemical and immunological properties of cytokines conjugated to dendritic polymers. Biomed. Microdevices 2004. 6, 191–202. F. Chaves, J. C. Calvo, C. Carvajal, Z. Rivera, L. Ramirez, M. Pinto, M. Trujillo, F. Guzman, M. E. Patarroyo, Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages. J. Pept. Res. 2001. 58, 307–316. L. J. Cruz, E. Iglesias, J. C. Aguilar, L. J. Gonza´lez, O. Reyes, F. Albericio, D. Andreu, A comparative study of different presentation strategies for an HIV peptide immunogen. Bioconjugate Chem. 2004. 15, 112–120. F. Sa´nchez-Sancho, E. Pe´rezInestrosa, R. Suau, C. Mayorga, M. J. Torres, M. Blanca, Dendrimers as carrier protein mimetics for IgE antibody recognition. Synthesis and characterization of densely penicilloylated dendrimers. Bioconjugate Chem. 2002. 13, 647–653. S. Jaracz, J. Chen, L. V. Kuznetsova, I. Ojima, Recent advances in tumortargeting anticancer drug conjugates. Bioorg. Med. Chem. 2005. 13, 5043–5054. Q. Cai, Y. Zhao, J. Bei, F. Xi, S. Wang, Synthesis and properties of star-shaped polylactide attached to poly(amidoamine) dendrimer. Biomacromolecules 2003. 4, 828–834. N. Malik, E. G. Evagorou, R. Duncan, Dendrimer-platinate: A novel approach to cancer chemotherapy. Anticancer Drugs 1999. 10, 767–776. D. Shabat, R. J. Amir, A. Gopin, N. Pessah, M. Shamis, Chemical adaptor systems. Chem. Eur. J. 2004. 10, 2626–2634. M. Shamis, H. N. Lode, D. Shabat, Bioactivation of self-immolative

95

96

97

98

99

100

101

102

103

dendritic prodrugs by catalytic antibody 38C2. J. Am. Chem. Soc. 2004. 126, 1726–1731. R. J. Amir, N. Pessah, M. Shamis, D. Shabat, Self-immolative dendrimers. Angew. Chem. Int. Ed. 2003. 42, 4494–4499. F. M. H. de Groot, C. Albrecht, R. Koekkoek, P. H. Beusker, H. W. Scheeren, ‘‘Cascade-release dendrimers’’ liberate all end groups upon a single triggering event in the dendritic core. Angew. Chem. Int. Ed. 2003. 42, 4940–4494. A. T. Florence, T. Sakthivel, I. Toth, Oral uptake and translocation of a polylysine dendrimer with a lipid surface. J. Controlled Release 2000. 65, 253–259. ˜ oR. Wiwattanapatapee, B. Carren Go´mez, N. Malik, R. Duncan, Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: A potential oral delivery system? Pharm. Res. 2000. 17, 991–998. A. T. Florence, N. Hussain, Transcytosis of nanoparticle and dendrimer delivery systems: Evolving vistas. Adv. Drug Delivery Rev. 2001. 50, S69–S89. M. Ruponen, P. Honkakoski, S. Ro¨nkko¨, J. Pelkonen, M. Tammi, A. Urtti, Extracellular and intracellular barriers in non-viral gene delivery. J. Controlled Release 2003. 93, 213–217. R. F. Barth, D. M. Adams, A. H. Soloway, F. Alam, M. V. Darby, Boronated starburst dendrimermonoclonal antibody immunoconjugates: Evaluation as a potential delivery system for neutron capture therapy. Bioconjugate Chem. 1994. 5, 58–66. S. Hong, A. U. Bielinska, A. Mecke, B. Keszler, J. L. Beals, X. Shi, L. Balogh, B. Orr, J. R. Baker Jr, M. M. Banaszak Holl, Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: Hole formation and the relation to transport. Bioconjugate Chem. 2004. 15, 774–782. A. Mecke, S. Uppuluri, T. M. Sassanella, D.-K. Lee, A.

References

104

105

106

107

108

109

110

111

112

113

Ramamoorthy, J. R. Baker Jr, B. G. Orr, M. M. Banaszak Holl, Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers. Chem. Phys. Lipids 2004. 132, 3–14. G.-D. Zhang, A. Harada, N. Nishiyama, D.-L. Jiang, H. Koyam, T. Aida, K. Kataok, Polyion complex micelles entrapping cationic dendrimer porphyrin: Effective photosensitizer for photodynamic therapy of cancer. J. Controlled Release 2003. 93, 141–150. J. C. Roberts, M. K. Bhalgat, R. T. Zera, Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst TM dendrimers. J. Biomed. Mater. Res. 1996. 30, 53–65. H. Kobayashi, S. Kawamoto, T. Saga, N. Sato, A. Hiraga, T. Ishimori, J. Konishi, K. Togashi, M. W. Brechbiel, Positive effects of polyethylene glycol conjugation to generation-4 polyamidoamine dendrimers as macromolecular MR contrast agents. Magn. Reson. Med. 2001. 46, 781–788. N. Ro¨ckendorf, T. K. Lindhorst, Glycodendrimers. Top. Curr. Chem. 2001. 217, 201–238. L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Roho, F. Albericio, Peptide and amide bond-containing dendrimers. Chem. Rev. 2005. 105, 1663–1681. M. S. Shchepinov, I. A. Udalova, A. J. Brigman, E. M. Southern, Oligonucleotide dendrimers: Synthesis and use as polylabelled DNA probes. Nucleic Acids Res. 1997. 25, 4447–4454. T. W. Nilsen, J. Grayzel, W. Prensky, Dendritic nucleic acid structures. J. Theoret. Biol. 1997. 187, 273–284. R. H. E. Hudson, M. J. Damha, Nucleic acid dendrimers: Novel biopolymer structures. J. Am. Chem. Soc. 1998. 115, 2119–2124. Y. Suzuki, T. Otomo, H. Okazi, H. Sawai, Synthesis and properties of a new type DNA dendrimer. Nucleic Acids Symp. Ser. 2000. 44, 125–126. Y. Li, Y. D. Tseng, S. Y. Kwon, L. D’Espaux, J. S. Bunch, P. L.

114

115

116

117

118

119

120

121

122

123

McEuen, D. Luo, Controlled assembly of dendrimer-like DNA. Nat. Mater. 2004. 3, 38–42. K. T. Al-Jamal, T. Sakthivel, A. T. Florence, Dendrisomes: Vesicular structures derived from a cationic lipidic dendron. J. Pharm. Sci. 2005. 94, 102–113. S. Takeoka, K. Mori, H. Ohkawa, K. Sou, E. Tsuchida, Synthesis and assembly of poly(ethylene glycol)lipids with mono-, di-, and tetraacyl chains and a poly(ethylene glycol) chain of various molecular weights. J. Am. Chem. Soc. 2000. 122, 7927–7935. H. Frey, R. Haag, Dendritic polyglycerol: A new versatile biocompatible-material. J. Biotechnol. 2002. 90, 257–267. M. F. Neerman, W. Zhang, A. R. Parrish, E. E. Simanek, In vitro and in vivo evaluation of a melamine dendrimer as a vehicle for drug delivery. Int. J. Pharm. 2004. 281, 129–132. A.-M. Caminade, J.-P. Majoral, Nanomaterials based on phosphorus dendrimers. Acc. Chem. Res. 2004. 37, 341–348. X.-Y. Wu, S.-W. Huang, J.-T. Zhang, R.-X. Zhuo, Preparation and characterization of novel physically cross-linked hydrogels composed of poly(vinyl alcohol) and amineterminated polyamidoamine dendrimer. Macromol. Biosci. 2004. 4, 71–75. H. R. Ihre, O. L. Padilla De Jesu´s, F. C. Szoka Jr, J. M. J. Fre´chet, Polyester dendritic systems for drug delivery applications: Design, synthesis, and characterization. Bioconjugate Chem. 2002. 13, 443–452. M. J. Cloninger, Biological applications of dendrimers. Curr. Opin. Chem. Biol. 2002. 6, 742–748. L. S. Heuser, F. N. Miller, Differential macromolecular leakage from the vasculature of tumors. Cancer 1986. 57, 461–464. H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in

39

40

1 Dendrimers in Cancer Treatment and Diagnosis

124

125

126

127

128

129

130

131

132

133

macromolecular therapeutics: A review. J. Controlled Release 2000. 65, 271–284. Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986. 46, 6387–6392. L. W. Seymour, Passive tumor targeting of soluble macromolecules and drug conjugates. Crit. Rev. Ther. Drug Carrier Syst. 1992. 9, 135–187. D. Liu, A. Mori, L. Huang, Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes. Biochim. Biophys. Acta 1992. 1104, 95–101. A. Nagayasu, T. Shimooka, Y. Kinouchi, K. Uchiyama, Y. Takeichi, H. Kiwada, Effects of fluidity and vesicle size on antitumor activity and myelosuppressive activity of liposomes loaded with daunorubicin. Biol. Pharm. Bull. 1994. 17, 935–939. R. K. Jain, Barriers to drug-delivery in solid tumors. Sci. Am. 1994. 271, 58–65. Y. Tabata, Y. Murakami, Y. Ikada, Tumor accumulation of poly(vinyl alcohol) of different sizes after intravenous injection. J. Controlled Release 1998. 50, 123–133. S. Zalipsky, N. Mullah, J. A. Harding, J. Gittelman, L. Guo, S. A. DeFrees, Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 1997. 8, 111–118. J. A. Reddy, V. M. Allagadda, C. P. Leamon, Targeting therapeutic and imaging agents to folate receptor positive tumors. Curr. Pharm. Biotechnol. 2005. 6, 131–150. C. P. Leamon, J. A. Reddy, Folatetargeted chemotherapy. Adv. Drug Delivery Rev. 2004. 56, 1127–1141. E. J. Roy, U. Gawlick, B. A. Orr, D. M. Kranz, Folate-mediated targeting of T cells to tumors.

134

135

136

137

138

139

140

141

142

143

144

Adv. Drug Delivery Rev. 2004. 56, 1219–1231. Y. Choi, T. Thomas, A. Kotlyar, M. T. Islam, J. R. Baker, Synthesis and functional evaluation of DNAassembled polyamidoamine (PAMAM) dendrimer clusters with cancer cellspecific targeting. Chem. Biol. 2005. 12, 35–43. D. Laheru, E. M. Jaffee, Immunotherapy for pancreatic cancer – science driving clinical progress. Nat. Rev. Cancer 2005. 5, 549–467. M. Harris, Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol. 2004. 5, 292–302. M. Z. Lin, M. A. Teitell, G. J. Schiller, The evolution of antibodies into versatile tumor-targeting agents. Clin. Cancer Res. 2005. 11, 129–138. J.-Y. Zhang, Tumor-associated antigen arrays to enhance antibody detection for cancer diagnosis. Cancer Detect. Prev. 2004. 28, 114–118. R. E. Kontermann, Recombinant bispecific antibodies for cancer therapy. Acta Pharmacol. Sin. 2005. 26, 1–9. P. A. Trail, D. H. King, G. M. Dubowchik, Monoclonal antibody drug immunoconjugates for targeted treatment of cancer. Cancer Immunol. Immunother. 2003. 52, 328–337. G. C. McDonald, N. Glover, Effective tumor targeting: Strategies for the delivery of armed antibodies. Curr. Opin. Drug Discov. Devel. 2005. 8, 177–183. S. V. Govindan, G. L. Griffiths, H. J. Hansen, I. D. Horak, D. M. Goldenberg, Cancer therapy with radiolabeled and drug/toxinconjugated antibodies. Technol. Cancer Res. Treat. 2005. 4, 375–392. D. J. FitzGerald, R. Kreitman, W. Wilson, D. Squires, I. Pastan, Recombinant immunotoxins for treating cancer. Int. J. Med. Microbiol. 2004. 293, 577–582. K. Sandvig, S. Grimmer, T. G. Iversen, K. Rodal, M. L. Torgersen, P. Nicoziani, B. van Deurs, Ricin transport into cells: Studies of endocytosis and intracellular

References

145

146

147

148

149

150

151

152

153

transport. Int. J. Med. Microbiol. 2000. 290, 415–420. S. Olsnes, The history of ricin, abrin and related toxins. Toxicon 2004. 44, 361–370. A. Gruaz-Guyon, O. Raguin, J. Barbet, Recent advances in pretargeted radioimmunotherapy. Curr. Med. Chem. 2005. 12, 319–338. J. C. Roberts, Y. E. Adams, D. A. Tomalia, J. A. Mercer-Smith, D. K. Lavallee, Using starburst dendrimers as linker molecules to radiolabel antibodies. Bioconjugate Chem. 1990. 1, 305–308. P. Singh, Terminal groups in Starburst dendrimers: Activation and reactions with proteins. Bioconjugate Chem. 1998. 9, 54–63. H. Kobayashi, N. Sato, T. Saga, Y. Nakamoto, T. Ishimori, S. Toyama, K. Togashi, J. Konishi, M. W. Brechbiel, Monoclonal antibodydendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity. Eur. J. Nucl. Med. Mol. Imaging 2000. 27, 1334–1339. N. Fischer-Durand, M. Salmain, B. Rudolf, A. Vessie`res, J. Zakrzewski, G. Jaouen, Synthesis of metalcarbonyl-dendrimer-antibody immunoconjugates: Towards a new format for carbonyl metallo immunoassay. ChemBioChem 2004. 5, 519– 525. T. P. Thomas, A. K. Patri, A. Myc, M. T. Myaing, J. Y. Ye, T. B. Norris, J. R. Baker Jr, In vitro targeting of synthesized antibody-conjugated dendrimer nanoparticles. Biomacromolecules 2004. 5, 2269–2274. A. K. Patri, A. Myc, J. Beals, T. P. Thomas, N. H. Bander, J. R. Baker Jr, Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem. 2004. 15, 1174–1181. S. S. Nigavekar, L. Y. Sung, M. Llanes, A. El-Jawahri, T. S. Lawrence, C. W. Becker, L. Balogh, M. K. Khan, 3 H Dendrimer nano-

154

155

156

157

158

159

160

particle organ/tumor distribution. Pharm. Res. 2004. 21, 476–483. H. Kobayashi, C. Wu, M.-K. Kim, C. H. Paik, J. A. Carrasquillo, M. W. Brechbiel, Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1B4MDTPA and its conjugation with antiTac monoclonal antibody. Bioconjugate Chem. 1999. 10, 103–111. M. Mamede, T. Saga, H. Kobayashi, T. Ishimori, T. Higashi, N. Sato, M. W. Brechbiel, J. Konishi, Radiolabeling of avidin with very high specific activity for internal radiation therapy of intraperitoneally disseminated tumors. Clin. Cancer Res. 2003. 9, 3756–3762. D. S. Wilbur, P. M. Pathare, D. K. Hamlin, K. R. Buhler, R. L. Vessella, Biotin reagents for antibody pretargeting. 3. Synthesis, radioiodination, and evaluation of biotinylated starburst dendrimers. Bioconjugate Chem. 1998. 9, 813–825. N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J. W. Weener, E. W. Meijer, W. Paulus, R. Duncan, Dendrimers: Relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125 I-labelled polyamidoamine dendrimers in vivo. J. Controlled Release 2000. 65, 133–148. W. Yang, R. F. Barth, G. Wu, A. K. Bandyopadhyaya, B. T. S. Thirumamagal, W. Tjarks, P. J. Binns, K. J. Riley, H. Patel, J. A. Coderre, M. J. Ciesielski, R. A. Fenstermaker, Boronated epidermal growth factor as a delivery agent for neutron capture therapy of EGF receptor positive gliomas. Appl. Radiat. Isot. 2004. 61, 981–985. H. Kobayashi, M. W. Brechbiel, Dendrimer-based macromolecular MRI contrast agents: Characteristics and application. Mol. Imaging 2003. 2, 1–10. S.-E. Stiriba, H. Frey, R. Haag, Dendritic polymers in biomedical applications: From potential to clinical use in diagnostics and therapy. Angew. Chem. Int. Ed. 2002. 41, 1329–1334.

41

42

1 Dendrimers in Cancer Treatment and Diagnosis 161 T. Kaminaga, T. Takeshita, I.

162

163

164

165

166

167

168

Kimura, Role of magnetic resonance imaging for evaluation of tumors in the cardiac region. Eur. Radiol. 2003. 13(Suppl 4), L1–L10. P. de Graaf, F. Barkhof, A. C. Moll, S. M. Imhof, D. L. Knol, P. van der Valk, J. A. Castelijns, Retinoblastoma: MR imaging parameters in detection of tumor extent. Radiology 2005. 235, 197–207. M. D. Ogan, U. Schmiedl, M. E. Mosely, W. Grodd, H. Paajanen, R. C. Brasch, Albumin labeled with Gd-DTPA. An intravascular contrastenhancing agent for magnetic resonance blood pool imaging: Preparation and characterization. Invest. Radiol. 1987. 22, 665–671. L. D. Margerum, B. K. Campion, M. Koo, N. Shargill, J. Lai, A. Marumoto, P. C. Sontum, Gadolinium(III) DO31 macrocycles and polyethylene glycol coupled to dendrimers. Effect of molecular wieght on physical and biological properties of macromolecular magnetic resonance imaging agents. J. Alloys Compd. 1997. 249, 185–190. S. D. Konda, S. Wang, M. Brechbiel, E. C. Wiener, Biodistribution of a 153 Gd-folate dendrimer, generation ¼ 4, in mice with folate-receptor positive and negative ovarian tumor xenografts. Invest. Radiol. 2002. 37, 199–204. H. Kobayashi, S. Kawamoto, S.-K. Jo, H. L. Bryant Jr, M. W. Brechbiel, R. A. Star, Macromolecular MRI contrast agents with small dendrimers: Pharmacokinetic differences between sizes and cores. Bioconjugate Chem. 2003. 14, 388–394. S. Guccione, K. C. Li, M. D. Bednarski, Molecular imaging and therapy directed at the neovasculature in pathologies. How imaging can be incorporated into vascular-targeted delivery systems to generate active therapeutic agents. IEEE Eng. Med. Biol. Mag. 2004. 23, 50–60. K. Onitsuka, M. Fujimoto, H. Kitajima, N. Ohshiro, F. Takei, S. Takahashi, Convergent synthesis of

169

170

171

172

173

174

175

176

platinum-acetylide dendrimers. Chem. Eur. J. 2004. 10, 6433–6446. R. F. Barth, A. H. Soloway, Boron neutron capture therapy of primary and metastatic brain tumors. Mol. Chem. Neuropathol. 1994. 21, 139–154. L. Liu, R. F. Barth, D. M. Adams, A. H. Soloway, R. A. Reisfeld, Bispecific antibodies as targeting agents for boron neutron capture therapy of brain tumors. J. Hematother. 1995. 4, 477–483. F. Alam, A. H. Soloway, R. F. Barth, N. Mafune, D. M. Adams, W. H. Knoth, Boron neutron capture therapy: Linkage of a boronated macromolecule to monoclonal antibodies directed against tumorassociated antigens. J. Med. Chem. 1989. 32, 2326–2330. R. F. Barth, G. Wu, W. Yang, P. J. Binns, K. J. Riley, H. Patel, J. A. Coderre, W. Tjarks, A. K. Bandyopadhyaya, B. T. S. Thirumamagal, M. J. Ciesielski, R. A. Fenstermaker, Neutron capture therapy of epidermal growth factor (þ) gliomas using boronated cetuximab (IMC-C225) as a delivery agent. Appl. Radiat. Isot. 2004. 61, 899–903. A. Quintana, E. Raczka, L. Piehler, I. Lee, A. Myc, I. Majoros, A. K. Patri, T. Thomas, J. Mule´, J. R. Baker Jr, Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharm. Res. 2002. 19, 1306–1310. J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman, R. S. Kerbel, ß1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 1987. 236, 236–239. S. Sell, Cancer-associated carbohydrates identified by monoclonal antibodies. Hum. Pathol. 1990. 21, 1003–1019. A. Kobata, J. Amano, Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours. Immunol. Cell Biol. 2005. 83, 429–439.

References 177 J. W. Dennis, M. Granovsky, C. E.

178

179

180

181

182

Warren, Protein glycosylation in development and disease. BioEssays 1999. 21, 412–421. L. Tong, G. Baskaran, M. B. Jones, J. K. Rhee, K. J. Yarema, Glycosylation changes as markers for the diagnosis and treatment of human disease, in Biochemical and Genetic Engineering Reviews, S. Harding (Ed.), 2003, Intercept Limited, Andover, Hampshire, UK. pp. 199–244. J. R. Allen, C. R. Harris, S. J. Danishefsky, Pursuit of optimal carbohydrate-based anticancer vaccines: Preparation of a multiantigenic unimolecular glycopeptide containing the Tn, MBr1, and LewisY antigens. J. Am. Chem. Soc. 2001. 123, 1890–1897. J. R. Allen, J. G. Allen, X. F. Zhang, L. J. Williams, A. Zatorski, G. Ragupathi, P. O. Livingston, S. J. Danishefsky, A second generation synthesis of the MBr1 (Globo-H) breast tumor antigen: New application of the N-pentenyl glycoside method for achieving complex carbohydrate protein linkages. Chemistry 2000. 6, 1366–1375. V. Kudryashov, P. W. Glunz, L. J. Williams, S. Hintermann, S. J. Danishefsky, K. O. Lloyd, Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to LewisY conjugates in mice. Proc. Natl. Acad. Sci. U.S.A. 2001. 98, 3264–3269. H. Kayser, R. Zeitler, C. Kannicht, D. Grunow, R. Nuck, W. Reutter, Biosynthesis of a nonphysiological

183

184

185

186

187

188

189

sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J. Biol. Chem. 1992. 267, 16 934–16 938. K. J. Yarema, L. K. Mahal, R. E. Bruehl, E. C. Rodriguez, C. R. Bertozzi, Metabolic delivery of ketone groups to sialic acid residues. Application to cell surface glycoform engineering. J. Biol. Chem. 1998. 273, 31 168–31 179. L. K. Mahal, K. J. Yarema, C. R. Bertozzi, Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997. 276, 1125–1128. E. Saxon, C. R. Bertozzi, Cell surface engineering by a modified Staudinger reaction. Science 2000. 287, 2007–2010. S.-G. Sampathkumar, A. V. Li, M. B. Jones, Z. Sun, K. J. Yarema, Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2006. 2, 149–152. L. K. Mahal, K. J. Yarema, G. A. Lemieux, C. R. Bertozzi, Chemical approaches to glycobiology: Engineering cell surface sialic acids for tumor targeting, in Sialobiology and Other Novel Forms of Glycosylation, Y. Inoue, Y. C. Lee, F. A. Troy II (Eds.), 1999, Gakushin Publishing Company, Osaka, Japan, pp. 273–280. D. A. Nauman, C. R. Bertozzi, Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting. Biochim. Biophys. Acta 2001. 1568, 147–154. S.-G. Sampathkumar, K. J. Yarema, Targeting cancer cells with dendrimers. Chem. Biol. 2005. 12, 5–6.

43

44

2

Nanoparticles for Optical Imaging of Cancer Swadeshmukul Santra and Debamitra Dutta 2.1

Introduction

The word ‘‘cancer’’ comes from the Latin word for crab. Historically, an ancient physician from Greece noticed the resemblance of the swollen mass of blood vessels around a malignant tumor to the shape of a crab and so named the disease. The malignant tumor is also seen to adhere to surrounding tissues that it can seize upon in a stubborn manner, similar to a crab. Human cancer consists of more than 200 different diseases [1] in which cells multiply at an exponential growth rate in an uncontrolled fashion. This abnormal growth rate leads to the formation of a lump, called malignant tumor. Gradually the tumor tissue grows and invades the adjacent tissues and organs, obstructing normal physiological functions. In some cases, the cancerous cells can detach from its origin and migrate through circulation to different parts of the body, forming a new tumor site. This is termed as cancer metastasis. Over a period of time, malignant tumors cause malfunctioning of various organs, which turns fatal. According to the American Cancer Society’s annual report [2], about 570 280 people are expected to die of various cancers in the year 2005 in the United States of America (USA). During the last century, cancer has slowly advanced to become the leading cause of death for patients below the age of 85 in USA, despite rapid advances in global cancer research towards the understanding of cancer biology in the past several decades. The formation of malignant tumors is associated with six different cellular characteristics [3, 4], each of which is unique for cancer development. These characteristics are self-sufficiency in growth signals, evading apoptosis (a process by which a cell is ‘‘commanded’’ by the environment to die), insensitivity to anti-growth signals (an inherent mechanism for preventing undesirable cell growth), sustained angiogenesis (a process of growth of new blood vessels), tissue invasion and metastasis and limitless replicative potential. Cancer can develop in any living organ or tissue in the body. The part of the body in which the cancer first develops is referred to as the primary site. The most common cancer developing sites include the skin, lungs, female breasts, prostate, coNanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

2.1 Introduction

lon and rectum, and corpus uteri. The secondary site refers to the body part where metastasized cancer cells grow and form secondary tumors. Even if a cancer has spread to another part of the body, it is always described with respect to the primary site. For example, advanced breast cancer that has spread to the lymph nodes under the arm and to the lungs is always considered breast cancer. The diagnosis of cancer means an attempt to accurately identify the anatomical site of origin of the malignancy and the type of cells involved. The presence of cancer may be preliminarily suspected by some other disease-like symptoms. For example, weight loss and abdominal pain can be caused by stomach cancer or an ulcer. However, to confirm the diagnosis of cancer, a biopsy (removal of tissue for microscopic evaluation) is usually done. Biopsies can provide information about histological type, classification, grade, potential aggressiveness and other information that may help determine the best treatment. A biopsy together with advanced imaging technologies, can not only confirm the presence of cancer, but also can pinpoint the primary and secondary cancer sites. Early cancer diagnosis, in combination with the precise cancer therapies, could eventually save millions of lives. The diagnosis of cancer at the early stage is extremely challenging and has been an active research area of great interest in current times. If the tumor is located near the body’s surface, a tissue sample can be easily retrieved for a biopsy (removal of tissue for microscopic evaluation) and the tissue abnormality can be confirmed at the cellular level. However, if the tumor mass is inaccessible for a biopsy, one has to then rely upon the existing imaging techniques for the detection of the tumor location. Existing diagnostic non-invasive imaging techniques such as Computed Tomography (CT), Magnetic Resonance (MR), Positron Emission Tomography (PET), Single Photon emission CT (SPECT), Ultrasound (US) and optical imaging are effective for macroscopic visualization of tumors. However, none of these techniques are sensitive enough for the diagnosis of abnormalities in the microscopic level. Substantial research efforts are being made for the development of better cancer imaging techniques. Of them, optical imaging has shown a great promise with respect to the image resolution [5]. The feasibility of developing optical imaging technique for the sensitive detection of cancer has been recently demonstrated [6, 7] using nanoparticle-based highly sensitive optical contrast agents. This chapter provides a knowledge base platform to readers interested in learning about nanoparticle technology and its implications in diagnostic cancer imaging. An overview of existing cancer imaging techniques with special emphasis on optical-based imaging techniques has been incorporated. Optical imaging has strong potential in becoming an attractive alternative to existing cancer imaging techniques. Optical imaging is a highly sensitive, non-invasive, non-ionizing, relatively inexpensive and simple technique. With the aid of better contrast agents, this imaging technology could be transferred to a clinical setup for human applications for early cancer diagnosis in the near future. Recent developments which are directly associated with the improvement of optical image contrast, such as the use of sophisticated laser technology, highly sensitive charged-coupled device (CCD) technology and powerful mathematical modeling of light propagation through the

45

46

2 Nanoparticles for Optical Imaging of Cancer

biological systems, have been integrated into the imaging components. However, presently, significantly limited numbers of appropriate in vivo optical contrast agents are available. Therefore, there is a great demand of developing highly sensitive, stable and clinically safe in vivo optical contrast agents. Nanoscience and nanotechnology is an interdisciplinary research area that brings various traditional disciplines such as chemistry, physics, materials science, biomedical, molecular biology, and many others together under one umbrella. Several edited books [8–12] on nanoscience and nanotechnology include the development of various nanoparticles such as metals, semiconductors, etc. for biological applications. Several review articles [5, 7, 9, 13–22] have recently reported advance research highlights on optical-based contrast agents [15–17, 21], some of which emphasize nanoparticle-based optical contrast agents [7, 18–20, 22] for bioimaging. However, none of these articles captured thoroughly recent developments on nanoparticle-based optical contrast agents suitable for cancer imaging. In this chapter, we made every effort to provide extensive details of nanoparticle-based contrast agents developments, including separate sections on nanoparticle design, synthesis strategies, nanoparticle dispersion, surface modification, bioconjugations and cancer imaging applications. We hope that this chapter will be useful for students, teachers, research scientists, general audiences who are interested in learning more about early cancer diagnosis and others. We have attempted to explain each section and sub-section of this chapter in a simple manner so that readers could easily grasp the general strategy of various nanoparticle-based optical contrast agent development. Since the field of cancer imaging is expanding rapidly, we start with an introduction to highlight the contents of this chapter (Section 2.1). Section 2.2 briefly overviews various existing cancer-imaging techniques, allowing readers to understand merits and demerits of these techniques. Section 2.3 describes the basics of optical imaging, optical imaging techniques and optical contrast agents. This chapter provides a clear understanding of the merits and challenges of developing opticalbased imaging techniques and contrast agents. Section 2.4 details recent advances in nanoparticle-based optical contrast agents. While Section 2.4.1 provides the reasons for developing such contrast agents, Section 2.4.2 provides some literature review on the development of nanoparticle-based various contrast agents such as quantum dots, dye-doped nanoparticles and gold nanoparticles. Various cancer imaging applications using nanoparticles are reviewed in Section 2.5. Section 2.6 covers some miscellaneous nanoparticles such as up-converting phosphors, fluorescent polymer particles, etc. that are potential contrast agents. Section 2.7 provides concluding remarks and the perspectives of nanoparticle-based optical imaging of cancers.

2.2

Cancer Imaging Techniques

Some of the major imaging techniques routinely used in hospital setup for cancer imaging in humans are briefly described below.

2.2 Cancer Imaging Techniques

2.2.1

Computed Tomography (CT) Scanning

The detection components of a typical CT scanner include the X-ray tube, detectors, image reconstruction computer and visual display monitor. The X-ray tube generates a beam of X-rays that are made to pass through the body of the patient. The detectors are positioned to absorb the X-rays coming out through the body. While processing the information, the reconstruction computer takes into consideration that X-rays passing through denser tissues like bones are attenuated to a higher extent than those passing through softer tissue such as lungs. Thus the X-ray beams of varying strengths that come out from the body create a differential profile. This profile is measured by the detectors and finally imaged by the display monitor. The complete setup rotates around the patient and acquires about 1000 snapshots for every 360
rotation, when a slice is completed. After each rotation, the information from the detectors is collected together and processed by the computer to construct a two-dimensional image (slice) on the display monitor. Image resolution is on the order of 50–100 mm with data acquisition time varying from 5 to 30 min. Recently, there have been continuing efforts to merge the CT modality with other modules like nuclear imaging modalities, which have limited spatial resolution, to generate better images. 2.2.2

Magnetic Resonance (MR)

In this technique, patients are placed in a strong electromagnetic field that causes the hydrogen atoms of water molecules present in the body fluid to align with the field. A short, powerful radio signal is then sent through the body at a desired level (slice) perpendicular to the original field. Hydrogen atoms with similar frequencies resonate with the radio signal and get excited. When the radio signal is switched off, the excited atoms will release their excitation energy in the form of radio waves and return to their normal state. The time taken for the hydrogen atoms to release their energy is characteristic of the physical properties of the tissue. These radio waves are detected and the time taken is measured and analyzed by a computer to construct an image of the tissues. Usually, it is difficult to distinguish tumors from normal tissues in the body using an MR image. Therefore, patients are injected with contrast agents that selectively highlight the tumors. Standard MR images (1.5 Tesla, the magnetic field strength) provide a spatial resolution of 1 mm, which could be increased to about 10 mm with certain modifications. 2.2.3

Positron Emission Tomography (PET)

In this technique, a positron-emitting isotope ( 11 C or 18 F) is attached to a biological molecule that has an affinity to tumor cells and introduced into the patients. The decaying isotopes emit positrons, which collide with a nearby electron and annihilate to release g-rays. These rays are detected and analyzed by a computer. The po-

47

48

2 Nanoparticles for Optical Imaging of Cancer

sition of the tumor in the body can be located by tracking the density of the positrons in a particular region. 2.2.4

Single-photon Emission CT (SPECT)

This imaging technique is similar to PET, except that the SPECT isotope itself emits a single g-ray instead of a positron. This technique is very inexpensive when compared with PET. However the spatial resolution is not as good as PET. 2.2.5

Ultrasonography (US)

In this form of imaging, ultrasonic sound waves are sent through the body. The waves are partially reflected from the interfaces of different tissues. The intensity of reflected waves depends on the density of the tissues. The time taken for the reflected wave to reach the detector gives a measure of the depth of the tissue location. The advantage of ultrasonography is that the images are generated in real time with very high temporal resolution. However, ultrasonic waves cannot travel through bone and therefore cannot detect tissues behind bony structures such as the brain behind the skull.

2.3

Optical Imaging 2.3.1

Basics of Optical Imaging

Optical imaging is a sensitive, non-invasive, non-ionizing (clinically safe) and relatively inexpensive technique that has strong potential for diagnostic cancer imaging. Two major components are associated with an optical imaging system: an imaging component and an optical contrast-enhancing component (i.e., contrast agent). Recent advances in optical imaging have utilized sophisticated laser technology, highly sensitive charged-coupled device (CCD) technology and powerful mathematical modeling of light propagation through the biological systems; all these developments have formed a solid basis for the imaging component. Molecular fluorescent probes have been successfully used as optical contrast agents for imaging various cancer tissues in the past [5, 15–17, 23–27]. However, the sensitivity of the contrast agent has become the major obstacle in obtaining a highresolution image. Again, in vivo deep tissue optical imaging has been limited because of the low penetration depth of the light in the ultraviolet (UV) and visible spectral range (the approximate tissue penetration depth is about 1–2 mm). Nearinfrared (NIR) light in the spectral range 650–900 nm can, however, penetrate much deeper (up to several centimeters) into the tissue and skull [5, 15]. This is due to the relatively low absorption of tissue components (water and hemoglobin)

2.3 Optical Imaging

in the NIR spectral range. Therefore, the development of an NIR-based optical imaging system has attracted tremendous attention in the cancer imaging community in recent years. For developing optical-based imaging system, it is important to understand how light interacts with biological tissues. Simply, a tissue can interact with light photons by absorption, scattering and reflection. Since biological tissue represents a complex system in terms of light propagation, it is expected that the optical image would be somewhat distorted. A robust mathematical modeling is thus necessary to improve image quality. Again, all biological tissues somewhat autofluoresce upon interaction with the light in the UV and visible spectrum. The tissue autofluorescence originates from the natural tissue fluorescent molecules such as nicotinamide, flavins, collagen, and elastin [25]. To develop a robust optical imaging system it is thus important to address all sorts of light interaction with tissue as well as tissue autofluorescence. 2.3.2

Optical Imaging Techniques

Optical-based imaging methods such as confocal imaging, multiphoton imaging, microscopic imaging by intravital microscopy or total internal reflection fluorescence microscopy have been used traditionally to image fluorescence events that originates in vivo from surface and subsurface region. Recently, advanced imaging technologies that use photographic systems with continuous or intensitymodulated light and tomographic systems have shown great potential for deep tissue imaging. With the aid of highly sensitive contrast agents such as nanoparticles, it may be possible to transfer optical imaging technology to human application. There are several potential optical imaging techniques, such as reflectance fluorescence imaging and fluorescence-mediated molecular tomography (FMT), that use the diffuse component of light for probing molecular events deep in tissue samples. These techniques are briefly described below. In a typical reflectance imaging technique, a simple ‘‘photographic method’’ is used where the light source and the detector reside on the same side of the imaging object (e.g., an animal). This technique is currently used for in vivo assessment of fluorescent dyes [such as green fluorescent proteins (GFP), bioluminescent molecules, etc.]. In a reflectance imaging system, the light source can be either an appropriate laser for the target fluorescent molecules or a white light with the appropriate low-pass filter. The laser excitation source is preferable because it provides a narrow and well-defined spectral window (G3 nm) when compared with white light (G10 nm). A high-sensitivity CCD camera is usually used as detector. Reflectance imaging has been successfully used to image cathepsin B [28], cathepsin D [29], matrix metalloproteinase 2 (MMP-2) [30], using activatable probes that are dark in the native (quenched) state and fluoresce upon interaction with a specific enzyme. This technique has been used for the elucidation of MMP-2 (a biomarker) expression levels in two different breast cancers, MMP-2 positive human HT1080 fibrosarcoma and MMP-2 negative BT20 mammary adenocarcinoma [31]. A NIR probe was activated upon interaction with both the tumors. The level of MMP-2

49

50

2 Nanoparticles for Optical Imaging of Cancer

expression was directly related to the number of probe molecule activation. As expected, great probe activation was found in human HT1080 fibrosarcoma. Reflectance imaging technique has also been used for targeting cell-surface receptors in vivo using peptide-NIR dye conjugate [26, 32] and for studying gene expression [33]. Reflectance imaging is a simple, fast and highly sensitive imaging and screening technique for capturing surface fluorescent events in vivo or in excised tissues. This imaging system can be, inexpensively, made portable for the laboratory bench. However, this technique has several limitations. Firstly, the technique will allow imaging of a few millimeters thick tissue. As a result, the appearance of deeper lesions is significantly blurred. Secondly, quantitative information cannot be extracted from the reflectance imaging technique. For example, the surface appearance of a small structure with high dye loading that is located in a deeper tissue could be similar with a larger structure of low dye loading that is closer to the surface. Fluorescence-mediated molecular tomography is a powerful technique to resolve and quantify deep tissue fluorescence signal. This technique, usually termed as diffuse optical tomography (DOT) utilizes advanced photon sources, a detection system and rigorous mathematical modeling of light propagation in tissue. The DOT technique uses multiple projections and measures light around the boundary of the illuminated body followed by a complex mathematical modeling to construct the three-dimensional tomographic image. This technique has recently been applied clinically for imaging tissue oxy- and deoxy-hemoglobin concentration and blood saturation [34–37]. Based on the same principle, fluorescence molecular tomography (FMT) has been developed where measurements of fluorescent molecular probes at both the emission and excitation wavelengths were considered. The FMT technique has been recently used for imaging cathepsin B activity in deep tissue structure of 9L gliosarcomas [38–40]. An optical imaging system that can image both reflected light and fluorescence light to generate multi-spectral digital imaging of tissue morphology from a large field of view with mm resolution has also been developed [21]. Readers are encouraged to read a few recent review articles [5, 21, 41] that describe the topic on optical imaging techniques. 2.3.3

Optical Contrast Agents

Optical tissue contrast agents are used in biological systems (e.g., cells, tissues, etc.) to enhance the optical contrast by virtue of their contrast enhancing properties (e.g., fluorescence, scattering, etc.). Tissue contrast agents, for example, are capable of reducing the background signal and improving the image resolution. Fluorescent molecular contrast agents, mostly organic fluorescent compounds, possess high extinction coefficient and quantum yield and have the potential to drastically suppress tissue autofluorescence and hence background signal. Effective delivery (loading) of these contrast agents to the target tissue has also been realized to be one of the most important factors for achieving better image contrast, other than its intrinsic fluorescent characteristics (extinction coefficients, quantum yield, etc.). The concentration of contrast agent per unit volume of target tissue would

2.4 Nanoparticles for Optical Imaging

determine the signal strength. Therefore, a higher loading of contrast agent is always desirable for better image resolution and hence in obtaining a sharp marginal contrast between the normal and the pathological (e.g., a tumor) tissues. A few important features of contrast agents have to be kept in mind prior to using or developing new contrast agents for diagnostic cancer imaging. Firstly, contrast agents with the excitation and emission band maxima in the NIR range (650 to 900 nm) are highly preferable for deep tissue imaging. Secondly, contrast agents should have a high extinction coefficient for effective absorption and a high quantum yield for obtaining strong fluorescence signal. Thirdly, they should be photostable and should not have any photo-sensitizing effects (i.e., photodynamic effect causing the damage of cellular DNA and hence cell death; also termed as photosensitized cell death). Fourthly, contrast agents should be hydrophilic so that an aqueous-based formulation can be easily made. Fifthly, contrast agents should have low toxicity so that they can be administered safely. Lastly, for cancer imaging, contrast agents should be attached to appropriate cancer specific delivery systems (e.g., antibodies, peptides, folates, etc.) for targeting. Organic fluorescent contrast agents, although studied extensively for various bioimaging applications [42], starting from cellular to tissues to whole animal fluorescence imaging have, however, several limitations for them to be considered as robust contrast agents. Firstly, organic fluorescent contrast agents (dyes) rapidly undergo photobleaching. As a result, the fluorescence signal fades away when exposed to the excitation light source (particularly when a laser is used for the excitation), limiting sensitive detection of the target. Secondly, fluorescent dyes are usually hydrophobic. To make aqueous-based formulation, chemical modifications (e.g., sodium salt) are often required that sometimes compromises their spectral characteristics. Thirdly, a handful of fluorescent compounds have been shown to possess low toxicity. Lastly, a few dyes have excitation and emission bands in the NIR spectral range (e.g., cyanine dyes). Another class of fluorescent contrast agents is fluorescent proteins. Fluorescent proteins such as green fluorescent protein (GFP) represent one class of imaging marker genes (IMG, artificial genes) with an optical signature where GFP is the transcriptional product of IMG. The GFPbased optical imaging has been successfully used to study various gene expressions in vivo. For example, human and rodent tumor cell lines, transected with GFP, could be visualized in vivo for monitoring tumor growth and metastasis [43–45]. The major drawback of GFP is limited penetration depth since the tissue can highly absorb the green emission of GFP.

2.4

Nanoparticles for Optical Imaging 2.4.1

Why Nanoparticles for Optical Imaging?

Nanoparticle (NP)-based contrast agents present a whole new class of robust nanometer size (between 1 and 100 nm) particulate materials that has strong potential

51

52

2 Nanoparticles for Optical Imaging of Cancer

for optical imaging of cancer. The use of NPs for bioimaging applications has several advantages. Firstly, the sensitivity of the optical imaging could be greatly improved using nanoparticle-based contrast agents. A classic example is the fluorescent quantum dots (Qdots) and their applications in cancer imaging [6, 13, 21, 46–65]. Qdots are usually crystalline cadmium sulfide (CdS) and cadmium selenide (CdSe)-based semiconductor particulate materials. They are small (<5 nm) and bright, having a broad excitation band but a narrow emission band. Dye-doped nanoparticles (NPs) such as dye-doped silica [66–69], dye-doped polymer particles [70–72], present another class of materials for sensitive cancer detection. In dye-doped NPs, each particle carries thousands of dye molecules, thus greatly enhancing the fluorescence signal. Dye-doped NPs are usually smaller (about two to three orders of magnitude) than cells, which make them suitable for cellular application. Gold nanoparticles have been well studied and have also been used for sensitive cancer cell imaging [21, 47, 73–75]. They possess a strong surface plasmon band that originates from efficient light scattering by the nanosize particles. Secondly, nanoparticle-based contrast agents have better photostability than traditional organic dye-based contrast agents. This has tremendous potential for sensitive and real-time monitoring of cancer progression (e.g., monitoring cancer growth and metastasis). Photostable nanoparticles will allow non-invasive imaging of cancer tissue multiple times for monitoring tumor growth and also the effect of cancer drugs during cancer therapy. For example, Qdots are extremely photostable. The effective surface passivation of Qdots with a wide bandgap material such as zinc sulfide (ZnS) or zinc selenide (ZnSe) makes them photostable. In dye-doped NPs, dye molecules remain encapsulated by the particle-matrix that protects them from photobleaching. This is because the particle-matrix can, somewhat, prevent the penetration of oxygen molecules that cause dye degradation. Usually, Qdot materials are more photostable than dye doped NPs. Gold nanoparticles efficiently scatter light and, therefore, do not fade away via photobleaching process [76–78]. Lastly, multiple imaging modalities can be integrated into nanoparticle-based contrast agents (also called as multifunctional nanoparticles [79–88]), making them suitable for imaging using multiple modalities (such as fluorescence, X-ray, MRI, etc. [86, 89, 90]). This would have great importance for in vivo cancer imaging applications. Once labeled with the multimodal contrast agents, tumors could be imaged non-invasively using a CT scan or MRI for the pre-surgical assessment. During the surgical procedure, tumor tissue could be directly visualized in realtime by the optical property (e.g., fluorescence) of the contrast agent. This mode of tumor visualization would provide direct guidance to surgeons for the effective tumor resection, enabling them to demarcate the boundary between the tumor and normal tissues. Nanoparticle surface is usually modified to obtain multiple functional groups/ligands to improve aqueous dispersibility, specific targeting (e.g., cancer targeting), biocompatibility, etc. [6, 20, 66, 67, 84, 86, 89–95]. Nanoparticles-based contrast agents have strong potential for early cancer diagnosis since they are bright and photostable. For in vivo cancer diagnosis, NIR-based nanoparticle contrast agents will be required. The following sub-sections describe

2.4 Nanoparticles for Optical Imaging

in detail the design, synthesis, surface modification, and cancer targeting of various nanoparticle-based contrast agents (e.g., Qdots, dye-doped NPs, gold, etc.). 2.4.2

Development of Nanoparticle-based Contrast Agents

A robust nanoparticle design is the key step for the synthesis of highly sensitive optical contrast agents. In a typical nanoparticle-based optical contrast agent design, the optical core is encapsulated by an intermediate coating followed by an outermost layer containing appropriate functional groups for bioconjugation. Figure 2.1 shows a general schematic representation of nanoparticle design. For cancer labeling, these particles are then attached to cancer cell targeting agents (e.g., folates, antibodies, etc.). The resulting nanoparticles can be used as cancer imaging probes (Fig. 2.2). Quantum Dots Fluorescent Qdots are ultra-small (2–8 nm diameter) semiconductor nanocrystals, having broad absorption band with narrow and symmetric emission band (fullwidth at half-maximum @25–40 nm), that typically emit in the visible to NIR spectral range [7, 18, 96]. Qdots absorption is associated with the promotion of electrons from the conduction band to the valence band when the excitation energy exceeds that band gap energy between two electronic bands (semiconductor band gap), resulting in the formation of an electron–hole pair, called an exciton. In con2.4.2.1

Schematic representation of core– shell nanoparticle design. The nanoparticle core (e.g., quantum dot, gold nanoparticle, dye-doped silica nanoparticle, etc.) is the primary source of an optical signal. The intermediate shell (e.g., ZnS surface passivation followed by polymer/silica coating,

Fig. 2.1.

pure/hybrid silica coating, etc.) is designed to protect the nanoparticle core. The outermost shell contains appropriate surface functional groups (e.g., carboxyls, amines, thiols, etc.) for biomolecule/ligand (e.g., antibodies, peptides, proteins, sugars, folates, etc.) attachment.

53

54

2 Nanoparticles for Optical Imaging of Cancer

Schematic representation showing a typical nanoparticle-based cell-labeling strategy. In this strategy cancertargeting agents (e.g., folates, antibodies, etc.) are coated onto nanoparticle surface. The resulting coated particles are capable of recognizing cancer cells.

Fig. 2.2.

trast to organic fluorescent molecules, the absorption probability of Qdot nanocrystals increases at shorter wavelengths (higher energy), resulting in a broadband absorption spectrum. When the Qdot is smaller than the Bohr exciton radius (which is typically a few nanometers), the quantum confinement effect is observed in such nanocrystals [97]. In this situation, Qdot energy levels are quantized, with values directly related to the Qdot size. Qdot emission is due to a radiative recombination of an exciton, which is characterized by a long lifetime [98] (>10 ns), leading to the emission of a photon in a narrow and symmetric energy band. These spectral characteristics of Qdot materials are different from a typical organic fluorescent molecule with red-tailed broad emission band and short fluorescence lifetimes. In comparison to traditional fluorescent molecules (fluorophores) or fluorescent proteins (e.g., GFP), Qdots have several attractive optical features that are desirable for long-term, multi-target and highly sensitive bioimaging applications. Some of the major optical features of Qdots are described below. (a) Large molar extinction coefficient: Qdots are highly sensitive fluorescent agents (or fluorescent tags) for labeling cells and tissues. Unlike organic fluorescent compounds, Qdots have very large molar extinction coefficients [99], typically of the order of 0.5–5  10 6 m1 cm1 which means that Qdots are capable of absorbing excitation photons very efficiently (the absorption rate is approximately 10– 50 faster than organic dyes). The higher rate of absorption is directly correlated to the Qdot brightness and it has been found that Qdots are approximately 10–20 brighter than organic dyes [100–102], allowing highly sensitive fluorescence imaging.

2.4 Nanoparticles for Optical Imaging

(b) Excellent photostability: Qdots are several thousand times more photostable than organic dyes. This feature allows real-time monitoring of biological processes over a long period. (c) Much longer lifetime: Qdots are highly suitable for time-correlated lifetime imaging spectroscopy. This is possible due to the longer excited state lifetime of Qdots (about one order of magnitude longer than that of organic dyes), allowing effective separation of Qdot fluorescence from the background fluorescence. This will improve the image contrast by reducing the signal-to-noise ratio dramatically [103, 104] in the time-delayed data acquisition mode. (d) Large Stokes shift: Unlike in organic dyes, the excitation and emission spectra of Qdots are well separated (i.e., large Stokes shift value; up to 400 nm, depending on the wavelength of the excitation light). This allows further improvement of sensitivity of the detection by reducing the high autofluorescence background often seen in biological specimens [6]. (e) Multiple targeting capability. The wavelength of Qdot emission is size dependent. This is a unique feature of Qdot materials in comparison to organic fluorescent dyes. The size dependent emission of Qdots allows imaging and tracking of multiple targets simultaneously using a single excitation source. This feature is particularly important in tracking a panel of disease-specific molecular biomarkers simultaneously, allowing classification and differentiation of various complex human diseases [105]. The development of Qdot-based fluorescent probes involves a multi-step process: synthesis, surface capping and bioconjugation. Each step is described below in detail. Qdot Synthesis Qdot nanocrystals are made out of hundreds to thousands of atoms that typically belong to group II and VI elements or group III and V elements in the periodic table. For example, CdSe, CdTe, and ZnSe are group II–VI semiconductor Qdots, whereas InP and InAs Qdots are group III–V semiconductors. The Qdot emission can be continuously tuned from 400 to 2000 nm by changing both the particle size and chemical composition. Herein, we briefly describe two robust synthesis techniques that produce high quality Qdots. Hot Solution-phase Mediated Qdot Synthesis This is most popular technique of synthesizing high quality Qdots. Typically, Qdots are synthesized at elevated temperature in high boiling point non-polar organic solvents. Bawendi’s group have reported [106] the synthesis of highly crystalline and monodisperse (size distribution 8–11%) CdSe Qdots using high-temperature growth solvents/ligands (mixture of trioctylphosphine/trioctylphosphine oxide, TOP/TOPO). A combination of TOPO and hexadecylamine can also be used [96]. The purpose of using hydrophobic organic molecules as mixed solvents or as a solvent/ligand mixture is two-fold. The mixture serves as a robust reaction medium and also coordinates with unsaturated metal atoms on the Qdot surface to prevent the formation of bulk semiconductors. Following a similar synthesis strategy, Qu et al. have reported the formation high

55

56

2 Nanoparticles for Optical Imaging of Cancer

quality CdSe nanocrystals having fluorescence quantum yields as high as 85% at room temperature [107]. Reverse-micelle Mediated Qdot Synthesis The reverse micelle synthesis of high quality CdS:Mn/ZnS core–shell Qdots has been reported [86, 89, 90, 108–110]. Reverse micelles [also called water-in-oil (W/O) microemulsion system] are an isotopic, thermodynamically stable homogeneous mixture of oil, water and surfactant molecules where the surfactant capped (stabilized) water droplets remain uniformly dispersed in the bulk oil phase. The water droplets serve as a tiny reactor (nano-reactor) for the synthesis of Qdots. This is a simple procedure that does not require extreme reaction conditions such as high temperature or high pressure. This is a robust method that allows room temperature synthesis of monodisperse Qdots at normal atmospheric pressure. Yang et al. have reported the synthesis of manganese-doped cadmium sulfide core and zinc sulfide shell (CdS:Mn/ZnS) Qdots using AOT (dioctylsulfosuccinate sodium salt, a surfactant)/heptane (an oil)/water reverse micelle system [90, 108, 109]. The bright yellow emitting CdS:Mn/ZnS Qdots are small (average Qdot size was 3.2 nm) and highly photostable. Qdot Surface Passivation and Aqueous Stabilization Effective surface passivation of the Qdot nanocrystal core with wide bandgap semiconductor materials (shell) is extremely important [100, 111] for the following reasons. For example, with cadmium selenide/zinc sulfide (CdSe/ZnS) core–shell Qdots, the epitaxially matched ZnS layer effectively passivates the surface defects of the CdSe core [100, 108, 112], protects the core from oxidation, prevents leaching of highly toxic Cd 2þ ions, and also drastically improves the quantum yield by reducing surface defects (that act as exciton traps, leading to non-radiative recombination processes). Surface passivation with silica is effective for the CdSe core [90, 113, 114] nanocrystals. TOP/TOPO-capped Qdots prepared using hot solution phase mediated synthesis route are hydrophobic. For biological applications, however, it is necessary to obtain aqueous dispersible Qdots. Therefore, phase transfer from the organic (e.g., toluene, hexanes, chloroform) to aqueous solution is usually performed by surface functionalization with hydrophilic ligands. There are three major routes to surface functionalization. Firstly, the ‘‘cap exchange’’ route that involves replacement of TOP/TOPO capping with bifunctional ligands. The bifunctional ligands [102, 115–118] have two functional moieties, Qdot surface anchoring (e.g., thiol) and hydrophilic moieties (e.g., hydroxyl, carboxyl). Secondly, the formation of a hydrophilic silica shell [86, 89, 90, 101, 108, 119] that encapsulates the Qdot. Lastly, the over-coating of TOP/TOPO-capped Qdots with amphiphilic ‘‘diblock’’ and ‘‘triblock’’ copolymers and phospholipids [6, 64, 120–124]. Notably, Qdots capped with mono-mercapto ligands have short shelf-lives, about a week, due the weak (dynamic) thiol–ZnS interaction [125], although polydentate thiolated ligands (containing more than one thiol groups) afford better stability (from a week to a couple of years) [116, 118, 125]. Applying a silica shell over the Qdots has several advantages with respect to long-term stability (shelf-life) and bio-

2.4 Nanoparticles for Optical Imaging

compatibility. Also, a silica coating remains stable upon pH fluctuation (below pH 8) and a further coating with a multifunctional hybrid silica is possible using appropriate silane reagents [86, 89]. The polymer/phospholipid encapsulation is also robust in terms of long-term stability. Both the silica and the polymer/ phospholipid coating increase the particle size (@20–30 nm). However, Yang et al. reported the W/O microemulsion-mediated synthesis of silica-overcoated CdS:Mn/ ZnS Qdots [90] where the silica shell thickness was approximately 2–3 nm. Qdot Bioconjugation Qdot bioconjugation represents the attachment of biomolecules (e.g., proteins, antibodies, peptides, DNA, etc.) to the Qdot surface, forming a hybrid structure, interfacing both the inorganic and the biological materials, for targeting to biological systems such as cells, tissues, etc. either specifically or nonspecifically. Qdots are comparable or slightly larger than many proteins. For example, a 510 nm green-emitting Qdot size is comparable to GFP and a 650 nm redemitting Qdot size is comparable to DyRed (a red-emitting fluorescent protein) [126]. Medintz et al. have shown that about 15–20 maltose binding proteins (Mr @ 44 kDa) can be conjugated to a single 6-nm Qdot [127]. There are three major ways to attach proteins to Qdot surface. Firstly, using carbodiimide [e.g., EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] coupling chemistry, carboxylated Qdots are covalently conjugated to the protein molecules through the formation of stable amide bond. Secondly, disulfide bonds can be formed between Qdot surface sulfur atoms (from the ZnS surface) and peptides containing cysteine residues [128, 129]. Histidine-expressing proteins [130] or peptides containing polyhistidine residues [131–133] can also be directly attached to the Zn atom on the Qdot surface. Lastly, engineered proteins containing positively charged domains can be non-covalently adsorbed onto the negatively charged Qdot surface via electrostatic interaction [116, 134, 135]. Although various bioconjugation strategies have been tested, none of them can control the ratios of proteins per Qdots. There is certainly a lack of experimental tools with which to discern the orientation of a protein immobilized on a Qdot surface. For specific targeting, it is highly desirable that the delivery proteins (e.g., antibody) are properly oriented and fully functional. The Qdot bioconjugation step is, therefore, extremely important in obtaining success in bioimaging. Gold Nanoparticles For over 30 years, nanometer-sized gold particles have been used to stain cells and tissue samples for electron microscopy. The basic principle of interactions between gold particles and biomolecules, like proteins, has been well studied for immunocytochemical staining applications. Although nanosize metals like gold and silver do not fluoresce they can effectively scatter light due to the collective oscillation of the conduction electrons induced by the incident electric field (light). This is known as ‘‘surface plasmon resonance’’ [20]. Thus, colloidal gold particles exhibit a range of intense colors in the visible and NIR spectral regions. Gold nanoparticles, because of their strong SPR properties, have attracted considerable attention in bioimaging in recent years. The SPR signal originates from 2.4.2.2

57

58

2 Nanoparticles for Optical Imaging of Cancer

the collective oscillation of conduction electrons upon interaction with absorption photons [136]. The SPR frequency depends on various factors, e.g., particle size [137] shape [138–140], dielectric properties [141, 142], aggregate morphology [143–146], surface functionalization [147, 148] and the refractive index of the surrounding medium [149–152]. Gold nanoparticles have high absorption [146, 151, 153] and scattering cross section [154, 155]. For example, the absorption cross section of a 5 nm diameter gold particle is about 3 nm [156] at a wavelength of 514 nm [136], which is about two orders of magnitude higher than that of organic fluorescent molecules at room temperature. The scattering cross section of gold nanoparticles is much larger than polymeric spherical particles of similar size, especially in the red region of the spectrum, i.e., red to NIR range, having potential in deep tissue imaging. For example, using composite core–shell gold (dielectric silica core and gold shell) particles, it is possible to tune the scattering from 600 to 1200 nm [141]. Due to excellent biocompatibility [157–159], gold nanoparticles have been widely used in immunohistochemistry (gold-based staining) and in ultra-sensitive DNA detection assays [146, 160, 161]. However, a few literature reports are available on gold nanoparticle-based cancers imaging. Gold Nanoparticle Synthesis Various methods have been reported for the synthesis of gold nanoparticles. There are two general approaches (so-called ‘‘top-down’’ and ‘‘bottom-up’’) that primarily categorize most reported synthesis strategies. Synthesis of gold nanoparticles by employing laser ablation technique is an example of a ‘‘top-down’’ approach, where the embryonic (nascent) particles are formed from the ionized gold atoms via nucleation and growth processes. The challenge still remains how to stabilize particles in the solution phase. Using a surfactant-based capping agent (sodium dodecylsulfate, an ionic surfactant), Kondow et al. have successfully stabilized ultrafine (@5 nm) particles [162]. The capping agents, in general, control the particle size and size distribution, prevents particle aggregation, and stabilize particle solution (such as in aqueous-based medium). In the ‘‘bottom up’’ approach, gold nanoparticles and gold nanocomposites (e.g., composite of gold and silica) have been chemically synthesized by reducing gold precursors. Various reduction methods have been reported. The major synthesis routes are as follows. (a) Reduction of gold precursors (e.g., hydrochloroauric acid, HAuCl4 ) using appropriate reducing agents, such as citrate [163–166], sodium borohydride [167], ascorbic acid [168], etc. The citrate reduction of the gold(iii) ions has been widely used. While sodium citrate reduces [AuCl 4 ] ions in hot aqueous solution, it forms a colloid. The reported average particle size is about 20 nm. Both the citrate ions and the oxidation products (e.g., acetone dicarboxylate) act as capping agents [163–165]. In conjunction with citrate ions, amphiphile surfactants have also been used that allowed particle size tuning upon varying the gold/stabilizer ratio [166]. A two-phase synthesis of gold nanoparticles (the Brust–Schiffrin method) has been reported in which a phase-transfer agent (tetraoctylammonium bromide) is used to transfer [AuCl 4 ] ions from an aqueous phase to an organic phase (tol-

2.4 Nanoparticles for Optical Imaging

uene) containing alkanethiol stabilizer. The Au(iii) in organic phase is reduced by the addition of aqueous sodium borohydride. The resulting Au clusters are then capped immediately by alkanethiols. The Brust–Schiffrin method produces monodisperse particles (approx 1.4 G 0.4 nm) in the diameter range 1.5–5.2 nm [167, 169]. (b) Microemulsions [170–172], copolymer micelles [173], reversed micelles [172], surfactant, membranes, and other amphiphiles have been widely used for the synthesis of stabilized gold nanoparticles. Wilcoxon and coworkers have studied the synthesis of gold nanoparticles formed in aqueous media and in reverse micelles, using chemical and photolytic reduction [174]. The chemical reduction method was achieved using reduction agents such as hydrazine, sodium borohydride, and metallic sodium. The advantages of using reverse micelles for the synthesis of gold nanoparticles are (i) it produces monodisperse particles, (ii) particles remain coated by surfactant molecules that prevent particle aggregation surrounding the molecule and (iii) the size of the nanoparticles can be easily varied by changing reaction parameters such as concentrations of the reagents, water-tosurfactant molar ratio, temperature, time allowed for ripening of particles, etc. (c) Another popular, long standing, method is the seed mediated route [175]. Indeed, the use of preformed metallic seeds as nucleation centers in nanoparticle synthesis has a long history [175–182]. Various references are available on the seed mediated growth of gold nanoparticles and also of gold nanorods [183–189]. Further nucleation during the ‘‘growth’’ part of the reaction often leads to non-uniform size distribution [190, 191]. The presence of seeds appears to cause additional nucleation (sometimes referred to as secondary nucleation) [184]. The step-bystep particle enlargement is considered more effective than the one-step seeding method to avoid additional nucleation [192]. More recently a modification of seed mediated method was used by Loo et al. [193] to achieve gold coating on the silica nanoparticles. Gold shells were grown using the method of Duff et al. [194]. Briefly, small gold colloid (1–3 nm) was adsorbed onto the aminated (amine functionalized) silica nanoparticle surface. More gold was then reduced onto these colloid nucleation sites using potassium carbonate and HAuCl4 in the presence of formaldehyde. Gold shell particles have been used for whole blood immunoassay [195], photothermal tumor ablation [196] molecular imaging in live cells [197] and for cancer imaging and therapy [198]. (d) Reduction of gold precursors using a combination of appropriate reducing agents and radiation such as UV [188, 199–201], ultrasound [202–207], heat [208–210]. The UV irradiation method has been used to prepare gold nanoparticles [188, 199, 200, 211, 212], including when in synergy with micelles [211] or seeds [188]. The ‘‘gold seed particles’’ are prepared photochemically by UV irradiation, preferably in the presence of a neutral micelle of a non-ionic surfactant, Triton X-100 [poly(oxyethylene) iso-octyl phenyl ether] [201]. The seed particles subsequently grow by successive addition of metal ions and, again, exposure to UV irradiation under the same experimental conditions. Several reports are available on the sonochemical synthesis (chemical synthesis using ultrasound radiation) of gold nanoparticles [202–207]. The general mecha-

59

60

2 Nanoparticles for Optical Imaging of Cancer

nism of reduction is briefly described here. When a solution is exposed to ultrasound radiation of sufficient intensity it produces a cavitation field made up of a large distribution of vapor and gas-filled bubbles, which pulsate continuously. When the pressure inside the bubble falls below the vapor pressure of the liquid the bubble fills with vapor and grows. At a certain point when the pressure turns positive the bubble collapses, resulting in extreme temperatures and pressures in the interior. The localized hot point causes ionization of molecular species present within the interior of the collapsing bubble. For water vapor, this dissociation results in the production of H and OH radicals [213]. The radicals produced in the interior of the bubble can then diffuse into the bulk solution and reduce metal ions, yielding nanosized metallic particles such as gold nanoparticles. Gold has also been fabricated by employing thermolysis [208–210] of organic derivatives of gold. This novel strategy involves the reductive elimination of thiolate ligands with simultaneous attachment of an organic moiety on the growing nuclei [210]. Surface Functionalization and Bioconjugation As mentioned in the previous section, gold nanoparticles have been widely used as immunostaining agents for labeling cell, tissue section, blots, etc. In general, protein conjugated gold nanoparticles are mostly used as labeling probes. Although the actual mechanism of macromolecule (e.g., proteins) binding to gold particles is poorly understood, some of the accepted mechanisms are [214]:

(1) Protein binding via electrostatic (ionic) interaction. Negatively charged gold nanoparticles can bind to positively charged protein domains via electrostatic interactions. (2) Protein binding via hydrophobic interaction. Hydrophobic domains present in the protein structure can interact with the metal surface of the particle. (3) Protein binding via chemical interaction. Protein molecules containing sulfohydryl (aSaH) groups can chemically interact with the gold atoms. This is also called dative binding. Other biomolecules, such as protein A, antibodies, lectins, avidins (or streptavidins), etc., have also been conjugated to gold nanoparticles to be used as sensitive probes. Some of the binding strategies are described below. Protein A–Gold Conjugate Protein A–gold conjugates are generally prepared by adsorbing protein A onto the gold surface. Following a similar method, many other immunoglobulin binding proteins can also be attached to gold nanoparticles. These probes have been used as ‘‘universal’’ probes for labeling cells, tissue sections and various blots. In a typical tissue labeling experiment, primary antibodies are specifically targeted to the tissue antigens. In the following step, protein A– gold conjugates bind to the antibodies. The advantage of this labeling technique is that the same protein A–gold conjugate can be used for various immunochemical procedures.

2.4 Nanoparticles for Optical Imaging

Antibody–Gold Conjugate Antibody–gold conjugated probes are prepared by coating antibodies directly on to the gold nanoparticle surface. These probes have been successfully used for the detection, localization and quantification of antigens on the target specimens. This is a powerful technique for detection of pathogens, intracellular foreign substances, monitoring cellular metabolic processes, etc. Lectin–Gold Conjugate Lectin-coated gold nanoparticle probes have been used for the detection of sugar-binding receptors that are expressed on the cell membranes. Lectin molecules have specific carbohydrate binding sites. In this assay, a specific carbohydrate molecule is sandwiched between the lectin molecule and the cellular receptor. The objective of this assay is to localize glycoproteins, glycolipids, etc. on cell surfaces. Avidin (or Streptavidin)–Gold Conjugate Avidin–gold conjugated probes have been used to localize, detect and quantify biotin molecules. This assay is similar to protein A–gold complex-based assays except that the primary antibodies are attached to biotin molecules. Dye-doped Silica Nanoparticles Amorphous silica (silicon dioxide) nanoparticles that are produced via Stober’s sol–gel [67, 215, 216] or microemulsion technique [66, 68, 91, 93, 217–225] have recently found applications in the area of bioimaging. Unlike Qdots or gold nanoparticles, silica does not have inherent strong fluorescence that can be exploited for sensitive imaging applications. However, silica nanoparticles can be made fluorescent by incorporating fluorescent dye molecules inside the silica matrix (dyedoping). Another approach could be attaching fluorescent dye molecules (via covalent binding) on the silica surface. For bioimaging applications, it is preferable that dye molecules remain encapsulated by the silica matrix for the following reasons. Silica-based nanoparticles exhibit several attractive features, e.g., silica is water dispersible and is resistant to microbial attack. The size of silica particles remains unchanged by changing solvent polarity (i.e., resistant to swelling) and, therefore, silica porosity remains unaltered in a wide selection of solvents, including aqueous-based neutral and acidic solutions. A silica matrix is optically transparent, allowing excitation and emission light to pass through efficiently. Moreover, fluorescent dyes can be effectively entrapped inside the silica particles. The spectral characteristics of the dye molecules remain almost intact. Silica encapsulation provides a protective layer around dye molecules, reducing oxygen molecule penetration (which causes photodegradation of dye molecules) both in air and in aqueous medium (in the latter case dissolved oxygen). As a result, photostability of dye molecules increases substantially compared with bare dyes in solution. Amorphous silica appears to be a biocompatible [22] and non-toxic [23] material, and has potential biological applications. The surface of a silica particle can be easily modified to attach biomolecules such as proteins, peptides, antibodies, oligonucleotides, etc., using conventional silanebased chemistry. For example, carboxylated silica nanoparticles can be covalently 2.4.2.3

61

62

2 Nanoparticles for Optical Imaging of Cancer

attached to the amine groups of proteins, antibodies etc. through the formation of stable amide bond [216]. Peptides containing a cysteine residue (through a aSaH group) can be attached to the aminated silica nanoparticles [89] through (SPDP) coupling chemistry. A general synthesis strategy of fluorescent silica nanoparticles is the incorporation of organic or metalloorganic dye molecules inside the silica matrix [66, 93, 226–231]. For example, a metalloorganic dye, tris(2,2 0 -bipyridyl)dichlororuthenium(ii) (Rubpy), has been entrapped inside silica nanoparticles using a reverse microemulsion-based synthesis approach [66] where the positively charged Rubpy molecules were electrostatically bound to the negatively charged silica matrix. Dyedoped silica-based imaging probes are non-isotopic, sensitive and relatively photostable in the physiological environment. Additionally, the interaction potential of the silica surface can be easily manipulated to facilitate the interaction with cells [232–234]. Due to these novel features, functionalized silica nanoparticles (FSNPs) have found widespread applications in bioanalysis and bioimaging applications. Synthesis There are two reported synthesis routes to dye-doped silica nanoparticles: Stober’s sol–gel method and the reverse microemulsion method. Stober’s Method In a typical Stober’s method, alkoxysilane compounds [e.g., tetraethylorthosilicate (TEOS), tetramethylorthosilicate (TMOS), various TEOS or TMOS derivatives, etc.] undergo base-catalyzed hydrolysis and condensation in an ammonia–ethanol–water mixture, forming a stable alcohol. This method has been widely used for synthesizing both pure and hybrid (when more than one silane compound are used, such as dye-doped silica particles) silica nanoparticles with particle diameters ranging from a few tens to several hundreds of nanometers (sub-micron size). Following Stober’s protocol with a slight modification, fairly monodisperse organic dye doped fluorescent silica nanoparticles have been synthesized. Since organic dyes are normally hydrophobic, doping them inside the hydrophilic silica matrix is not straightforward. Typically, a reactive derivative of organic dye (e.g., amine-reactive fluorescein isothiocyanate, FITC) is first reacted with an amine-containing silane compound (e.g., APTS), forming a stable thiourea linkage. Then FITC conjugated APTS and TEOS are allowed to hydrolyze and condense to form FITC conjugated silica particles. Note that particles so formed will have some amount of bare dye molecules on the particle surface that is covalently attached. These bare dyes, due to their hydrophobic nature, will somewhat compromise the overall particle aqueous dispersibility and, also, they will be prone to photobleaching. Therefore, an additional coating with pure silica is usually applied around the dye-conjugated silica nanoparticles. Using Stober’s method, bulk amounts (kilograms) of silica particles can be easily produced in a typical laboratory setup. Reverse Microemulsion (W/O) Method This method is used for the synthesis of pure silica, as well as inorganic and organic dye-doped silica nanoparticles. Figure 2.3 shows a schematic representation of dye-doped silica nanoparticle synthesis steps. The W/O microemulsion is a robust technique for producing monodisperse

2.4 Nanoparticles for Optical Imaging

Scheme of a water-in-oil (W/O) microemulsion mediated synthesis of dyedoped silica nanoparticles. (A) An immiscible mixture of water and oil (bulk phase). Upon addition of an appropriate surfactant, a W/O microemulsion is formed (B), where each tiny water droplet (nanosize water pool) is stabilized in the bulk oil phase with a surfactant coating (C). In reality each nanosize

Fig. 2.3.

water droplet serves as a nanoreactor for the synthesis of nanoparticles (D). The nanoparticle core (shown as filled circles), intermediate shell (inner ring) and outermost shell (outer ring) of the dye-doped silica nanoparticles are constructed in modular fashion by adding appropriate silane-based reagents at various stages of the synthesis process.

particles in the nanometer size range (tens to a few hundred nanometers). Figure 2.4 shows a typical transmission electron microscopic image of dye-doped silica nanoparticles synthesized using the W/O microemulsion technique. The W/O microemulsion is an isotropic, single-phase system that consists of surfactant, oil (as the bulk phase) and water (as nanosize droplets). Each surfactant-coated water droplet that is stabilized in the oil phase serves as an individual nanoreactor for the synthesis of silica nanoparticles. The water droplets undergo rapid and spontaneous collision and coalescence (fusion followed by separation) processes. As a result droplet contents (e.g., water-soluble reagents) are mixed together and chemical reactions (e.g., precipitation, hydrolysis and condensation reactions, etc.) take place. The surfactant present at the interface of oil and water nanodroplet is responsible for the thermodynamic stability of the W/O microemulsion system. Nucleation and growth processes are carried out inside the confined spherical volume of the nanoreactor. Varying the water-to-surfactant molar ratio and the dynamic properties of the microemulsion system helps to control the size of the nanoparticles.

63

64

2 Nanoparticles for Optical Imaging of Cancer

Typical transmission electron microscopic image of dye-doped silica nanoparticles about 100 nm in size. Particles were synthesized using the water-in-oil (W/O) microemulsion technique. As expected, nanoparticles were highly monodisperse,

Fig. 2.4.

confirming that the W/O microemulsion is a robust technique for the synthesis of dyedoped silica nanoparticles. This technique can be easily adopted to produce other types of nanoparticles such as quantum dots and multimodal nanoparticles.

The fluorescence brightness of dye-doped silica nanoparticles can be improved by incorporating high-quantum-yield organic dyes having large absorption coefficients. In other words, brighter probes will improve the image resolution if encapsulated fluorescent dyes do not experience substantial photobleaching during imaging. Surface Functionalization and Bioconjugation For bioimaging (e.g., cancer imaging), it is highly desirable that dye-doped silica nanoparticles are appropriately surface modified with cancer targeting molecules such as cancer specific antibodies, folates. This surface modification involves a few steps. Firstly, the particle surface should be modified to obtain appropriate functional groups such as, amines, carboxyls, thiols, etc. Secondly, using suitable coupling reagents, nanoparticles are attached to the bio-recognition molecules (such as antibodies, folates, etc.). Lastly, bioconjugated particles are targeted to cancers. Note that all these steps are usually carried out in aqueous-based solutions. A few bioconjugation methods are briefly mentioned below.

(1) Bioconjugation with carboxylated particles. This is one of the most common bioconjugation techniques to immobilize protein molecules on silica nanoparticles. The surface of the nanoparticle is modified to obtain carboxyl groups (aCOOH) by using a carboxylated silane reagent. Biomolecules such as proteins, antibodies, etc. containing free amine functional groups are then covalently attached to the carboxyl functionalized nanoparticle, using carbodiimide-coupling chemistry [235]. (2) Bioconjugation with aminated particles: Many cancer cells overexpress folate receptors. Cancer targeting with folate-conjugated nanoparticles has been

2.5 Optical Imaging of Cancer with Nanoparticles

recently reported [67]. Folates are chemically attached to aminated silica nanoparticles using carbodiimide chemistry. (3) Bioconjugation with avidin–biotin binding: Avidin is a protein molecule that contains four specific binding pockets for biotin molecules. A strong binding affinity exists between avidin and biotin molecules, which is comparable to covalent binding. Avidin-coated nanoparticles are typically attached to biotinylated molecules such as antibodies, proteins, etc. [236]. (4) Bioconjugation through disulfide bonding: Sulfohydryl-modified nanoparticles are conjugated to disulfide-linked oligonucleotides (e.g., DNAs). In this method, oligonucleotides are attached to nanoparticles through di-sulfide bond formation [237]. (5) Bioconjugation using cyanogen bromide chemistry: Nanoparticles with hydroxyl groups (such as silica) can be activated with cyanogen bromide to form a reactive aOCN derivative of the nanoparticles. The OCN derivative then readily reacts with proteins (via amine groups), forming a ‘‘zero-length’’ bioconjugate as there is no spacer between the particle surface and the protein molecule [66].

2.5

Optical Imaging of Cancer with Nanoparticles

Here we discuss the use of nanoparticle-based optical contrast agents in in vitro and in vivo experiments to image cancerous tissues. These nanoparticle-based contrast agents should provide a new gateway to characterize cancer at the molecular level. As we have already realized, these ultra-sensitive and specific probes provide a viable alternative to rapidly and non-invasively image the uptake, distribution and binding of nanoparticles to tumors. To establish the widespread use, it is important to understand the delivery, interaction and recognition mechanism of these contrast agents with cancer cells. Various delivery vehicles with varying specificity have been used to target cancer tissues, mainly for drug delivery applications, some of which are folates, antibodies, lectins, growth factors, cytokines, hormones and low-density lipoproteins. Obviously, most of these carriers can be similarly used for molecular imaging applications. These can be broadly classified [6, 238] as active and passive targeting. 2.5.1

Active Targeting

This refers to the conjugation of targeting ligands to nanoparticles to provide preferential accumulation into the tumor antigens and blood vessels with high affinity and specificity. This relies on specific interactive forces between lectins– carbohydrate, ligand–receptors and antibody–antigens [214]. Lectins can recognize and bind to glycoproteins that occur on the surface of cells. These proteins can bind to certain carbohydrates in a specific manner. Direct

65

66

2 Nanoparticles for Optical Imaging of Cancer

and reverse lectin targeting have made used of this specific interactions to receptors or antigens expressed by the plasma membrane. Folate receptor-based interactions are an excellent example of ligand–receptor based active targeting. Folate receptors are overexpressed on the surface of various cancers like those of the brain, ovary, kidney, breast and lungs. Confocal microscopic studies have demonstrated the selective intake and receptor-mediated endocytosis of folate-conjugated nanoparticles by tumor cells. Antibody-mediated tumor targeting has been performed for detecting the presence of antigenic moieties on the surface of cancer cells. Tumor targeting ligands like monoclonal antibodies are attached to nanoparticles to target the specific receptors. These moieties are minimally present on the surface of normal tissues. Only certain antigens are actually tumor-specific and are referred to as tumorspecific antigens. 2.5.2

Passive Targeting

This mode of targeting particles to tumors includes strategies like using the enhanced permeation and retention (EPR) effect, use of a unique tumor environment and a direct local delivery of imaging agents to tumors. In the EPR strategy, nanoparticles with a hydrophilic surface and diameter < 100 nm are made to accumulate at the tumors. The nanoparticles are engineered to prevent their uptake by the reticuloendothelial system, resulting in faster circulation and enhanced targeting ability in the physiological environment. Various researchers have demonstrated the strategy of exploiting the unique tumor environment to trigger the release of therapeutic drugs. The drug is conjugated to tumor specific ligands and remains inactive till it reaches its target site. On reaching the tumor, the linkages are hydrolyzed either by the enzymes present, or by a change in pH and the drug is released by the nanoparticles. Sometimes the imaging agents can be locally delivered to avoid its systemic circulation. But this is a challenging procedure as it involves the precise delivery using injections or surgical procedures that can be frequently cumbersome. 2.5.3

Cancer Imaging with Quantum Dots

Surface-functionalized quantum dots have been used to image various tumor cells and tissues in in vitro and in vivo experiments. Some of the different cell lines that have been used are human mammary epithelial tumor (MDA-MB-231) [239], human breast cancer (MDA-MB-435S [240], MDA-MD-435 [129], MCF 7 [240], and SK-BR-3 [64]), human prostate cancer [6], squamous carcinoma [19, 241] B16 melanoma (skin cancer) [56], human neuroblastoma (SK-N-SH) [242], colon tumor (SW480) [240], lung tumor (NCI H1299) [240], and bone tumor (Saos-2) [240] cells.

2.5 Optical Imaging of Cancer with Nanoparticles

In 2002, Parak and coworkers used water-soluble [119] siloxane-coated quantum dots of two different sizes (2.8 and 4.1 nm cores), functionalized with thiol and/or amine groups, to label human mammary epithelial tumor cells (MDA-MB-231) [239]. These Qdots emitted at 554 and 626 nm, respectively, and were more photo-stable than ordinary organic dyes. Confocal microscopic images verified the presence of nanocrystals ingested rapidly inside the cells, and not on the surface. The quantum dot crystals were found in the perinuclear region of the cells even after a week. Almost at the same time, Ackerman and coworkers [129] incubated human breast carcinoma MDA-MD-435 cells with peptide coated quantum dots. These cells were then injected into mice to create tumor grafts. The mice were imaged 8–12 weeks after tumor inoculation. Although quantum dot probes were not detected inside the mice, in vitro tests showed that peptide-coated Qdots could specifically target tumor cells. Wu et al. have used streptavidin-conjugated commercial Qdots, QD 560 (emission maximum 560 nm) and QD 608 (emission max. 608 nm), to detect Her2 cancer markers on the surface of human breast cancer cells (SK-BR3) [64]. The nanocrystals effectively labeled the cancer cells with negligible affinity to normal cells. Recently, Nie and coworkers [6] have developed multifunctional nanoparticle probes (2.5 nm radius core protected by a 1-nm TOPO cap with 2-nm polymer coating and 5 nm PEG/affinity ligand shell) for imaging. The quantum dots were used for in vivo imaging to target to tumor sites either through a slow passive targeting process or a more efficient active targeting process. Squamous carcinoma cells have been labeled with quantum dots conjugated to epidermal growth factors (EGF) [19, 241]. These probes with a broad fluorescence peak in the NIR at 770 nm can specifically bind and activate the EGF receptors [241] of cancer cells in C3H mice [19]. Similarly dihydroxylipoic acid (DHLA)-capped quantum dots can be efficiently delivered into B16 melanoma (skin cancer) cells [56]. The melanoma cells were labeled with the Qdots and injected into live mice to track tumor cell extravasation. The Qdots did not pose a detectable threat [56] to the labeled cells or the host animal and behaved as ‘‘inert fluorescent tags.’’ Quantum dots have also been used to detect integrin av subunits in human neuroblastoma cells (SK-NSH) [242] and label mouse lymphocytes (EL-4 cells derived from murine T-cell lymphoma) [243]. In vivo imaging to map sentinel lymph nodes (SLN) in rats and pigs has also been achieved using Qdots [238, 244, 245]. The presence of lymph node metastases is an early warning signal for breast and lung cancer. NIR nanocrystals with oligomeric phosphine coating (for solubility in aqueous buffers) was used to guide a surgeon during cancer surgery. A new quantum dot based tool, called ‘‘Quantum Dot Phagokinetic Track assay’’ has been developed [240] to quantify the invasive potential of different tumor cells. When cancer cells move through a bed of Qdots, they engulf the nanocrystals and leave behind a phagokinetic trail depleted in Qdots. Cells with higher metastatic potential engulf more Qdots and leave a clearer trail than those with weak metastatic potential. Seven different cancerous and non-cancerous cell lines were used

67

68

2 Nanoparticles for Optical Imaging of Cancer

to compare the different invasive potentials, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2. This assay rapidly discriminates between invasive and non-invasive cancer cell lines with greater sensitivity than the conventional Boyden chamber invasion assay. 2.5.4

Cancer Imaging with Gold Nanoparticles

Gold bioconjugates have been used for vital imaging of precancerous and cancerous cells by researchers for in vitro and in vivo experiments. The unique optical property of the metal in the nanosized range has been used for detecting breast carcinoma cells (SK-BR-3) [246] breast cancer markers like HER2, oral epithelial live cancer cells (HOC 313 clone 8 and HSC 3) [73] and neoplastic cervical biopsies [75]. Gold nanocages <40 nm can be used for specific targeting of breast cancer cells [246]. Gold-bioconjugates with surface plasmon resonance peaks at 800 nm were synthesized and used to label breast cancer cells (SK-BR-3) that overexpresses epidermal growth factor receptor 2 (EGFR2 or HER2). Primary antibodies (monoclonal anti-HER2 antibody from mouse) were immobilized on the SK-BR-3 cells. Secondary antibody (e.g., anti-mouse immunoglobulin G or IgG) conjugated gold nanocages were added to the cancer cells bound with anti-HER2 antibodies and imaged. A gold nanoshell based contrast agent [198] has also been used to image breast cancer cells. The nanoshell consists of a dielectric core surrounded by a thin gold shell. The dimensions were maintained to obtain an optical scattering peak in the NIR region at around 800 nm. In this case, anti-HER2 was attached to a PEG linker, and then attached to the nanoshell surface using a sulfur-containing group. The nanoshells successfully detected the HER2-positive SKBr3 breast adenocarcinoma cells. SPR scattering imaging or SPR absorption spectroscopy can be used with antibody-conjugated gold nanoparticles for imaging oral epithelial living cancer cells in vivo and in vitro [73]. Two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3) and a non-malignant cell line (HaCaT) were incubated with the gold nanoparticles. The cell cytoplasm contained dispersed and aggregated forms of the colloidal gold nanoparticles, with non-specific uptake for malignant cells. The gold nanoparticles conjugated to monoclonal anti-EGFR antibodies specifically and homogeneously bound to the cancer cell with enormously greater affinity than to non-cancerous cells. A relatively sharper SPR absorption band with a redshifted maximum was obtained for specific binding compared with that obtained for noncancerous cells. Sokolov and coworkers [75] have reported the use of gold bioconjugates with monoclonal antibodies against EGFR for the real-time vital optical imaging of precancerous cells and tissues. Cervical epithelial cancer cells (SiHa cells) and 3D tissue constructs and normal and cancerous cervical biopsies were used for in vivo

2.5 Optical Imaging of Cancer with Nanoparticles

imaging. They have shown the topical delivery of gold conjugates for imaging the whole epithelium. 2.5.5

Cancer Imaging with Dye-doped Silica Nanoparticles

Dye-doped silica nanoparticles have been used for in vitro imaging of cancer cells. Researchers have demonstrated their use in labeling human leukemia [66, 92, 93], HepG liver cancer [68], human oral carcinoma [67] and lung carcinoma cells [91, 217]. Figure 2.5 shows a confocal image of human lung cancer cells labeled with folate-conjugated FITC-doped 100-nm silica nanoparticles. Tan and coworkers have used 60-nm dye-doped silica nanoparticles, doped with Rubpy dye to label human leukemia cells [66, 92, 93]. Similarly, He and coworkers [68] have reported a method to recognize HepG liver cancer cells using FITC-APTS-doped silica nanoparticles. More recently, FITC-APTS-doped folate-conjugated silica nanoparticles were used to target overexpressed folate receptors in human oral carcinoma [67] and lung carcinoma cells [91, 217]. The dye-doped silica nanoparticles were covalently attached to folic acid molecules by a carbodiimide coupling reaction. The affinity of folate immobilized conjugates for folate receptors on the cancer cell surface were utilized for imaging [67]. The nanoparticles were detected using fluorescent techniques and imaged using a confocal microscope.

Folate conjugated FITC-doped 100nm silica nanoparticles were targeted to human lung cancer cells. Confocal microscopic images (left: fluorescence, right: transmission) showed that folate-conjugated nanoparticles successfully labeled cancer cells. This nanoparticle-based cancer labeling strategy

Fig. 2.5.

could be extended further to image other type of cancer cells that overexpress folate receptors. Note that nanoparticles were taken up by the cancer cells via receptor-mediated endocytosis. The extracellular nanoparticle concentration was 50 mg mL1 and the incubation time was 2 h.

69

70

2 Nanoparticles for Optical Imaging of Cancer

2.6

Other Nanoparticle-based Optical Contrast Agents

Up-converting phosphors, dye-doped polymer particles are also considered as optical nanoparticle probes. However, application of these particles for cancer imaging is yet to be explored. In up-converting phosphor nanoparticles both the absorber and the emitter ions are present in the crystal lattice. The absorber is excited by the energy of the infrared (IR) light source. The absorber transfers this energy to the emitter ion that emits a detectable photon. The up-conversion of light is caused by multiphoton process. The unique feature of converting IR light into visible radiation of the phosphors has shown great promise as a new type of fluorescence reporter for bioanalysis [247, 248]. Their emission bandwidths are generally very narrow (25–50 nm). There are several advantages of using up-converting phosphor technology (UPT) for bioanalysis. First, emission occurs at discrete wavelengths with a large anti-Stokes shift. Second, the emission is strong and does not fade away. Third, excitation wavelength is in the IR range, which reduces the background signal drastically by completely eliminating autofluorescence from the biological samples. Fourth, it has simultaneous detection capability for multiple target analytes. Fifth, the instrumentation requires low cost microscope modifications for IR excitation and visible emission. Zijlmans et al. have reported application of the phosphor probe for the sensitive detection of antigens in tissue sections or on cell membranes [248]. They conjugated phosphor [green-emitting ytterbium/erbium (Y.Yb.Er)O2 S and a blue-emitting ytterbium/thulium (Y.Yb.Tm)2 O2 S] particles to NeutrAvidin (Pierce, Rockford, IL) first and then successfully targeted in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. Various fluorescent polymers particles have been reported for bioimaging applications, such as long-lifetime europium chelate complex-doped 107-nm polystyrene particles [249–251], fluorescein isothiocyanate labeled poly(butyl cyanoacrylate) nanoparticles [252], and fluorescent gelatin NPs [253]. Multimodal (fluorescent and paramagnetic) nanoparticles have been prepared where a NIR dye (Cy5.5) and iron oxide nanoparticles were co-doped in dextran particles. These particles were used in constructing quantitative 3D tomographic NIR fluorescence imaging combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms [254].

2.7

Conclusions and Perspectives

Early diagnosis can prevent cancer and possibly cure cancer patients. However, currently available techniques such as CT scan, MRI scan, PET scan, etc. are not sensitive enough to diagnose cancers at the early stage. The challenge is to develop sophisticated in vivo imaging techniques that could precisely detect the presence of a

2.7 Conclusions and Perspectives

few abnormal cells in the body. Optical-based imaging systems have potential for early cancer diagnosis. To achieve better resolution, a sensitive optical imaging system must be coupled with highly sensitive image-enhancing optical contrast agents. Unlike organic-based dyes, nanoparticle-based contrast agents such as quantum dots, gold nanoparticles, and dye-doped silica nanoparticles have shown great promise for highly sensitive optical imaging of cancers. As described in this chapter, most successful experiments were conducted in vitro using various cancer cell lines. So far, few animal experiments have been performed that demonstrate that quantum dots have strong potential for in vivo cancer imaging. The future of nanoparticle-based in vivo optical contrast agents and cancer imaging will rely upon many factors. Firstly, the toxicity of nanoparticles needs to be thoroughly investigated. There are no straightforward answers to the toxicity of quantum dots, gold- and silica-based nanomaterials. Cadmium ions from cadmium sulfide and cadmium selenidebased quantum dots are highly toxic to cells. Although effective surface passivation with zinc sulfide and further coating with polymer, silica, etc. can reduce the release of cadmium ions, the long-term fate of these materials is highly uncertain, especially under in vivo conditions. Gold- and silica-based materials are biocompatible substances. However, their long-term in vivo behavior in the nanoscale size regime is also uncertain. Again, these particles are not digestible, meaning that if they are not naturally cleared by the body, they will be accumulated somewhere. This might cause additional threats in terms of secondary toxic effect. Secondly, the size of the particle would play a critical role. It is probably better to work with smaller particles, as they can be easily loaded intra-cellularly. Small particles (<10 nm) may be excreted naturally through the body (via kidney and spleen). Despite these advantages, smaller particles would have some limitations with respect to image resolution. As cancer cells divide rapidly, the concentration of particles in daughter cells will be diluted. As a result, image resolution will be compromised with time. Thirdly, development of nanoparticles with NIR excitation and emission would be extremely important for deep tissue imaging. Tumors that are present in a deeper tissue area can be imaged using NIR nanoparticles. A few NIR quantum dots have been tested in animal models recently. However, little work has been performed on the development of NIR silica or gold nanoparticles. Fourthly, nanoparticles with high extinction coefficients, quantum yields and photostability would be highly desirable for sensitive cancer imaging. Quantum dots are likely to be better candidates than gold- and silica-based nanoparticles. Further improvement towards effective surface passivation would be important. The spectral characteristics of gold nanoparticles are highly sensitive to the surfacecapping agents and therefore, robust surface capping would be necessary for stable spectral output. As silica is porous, dye-doped silica nanoparticles would experience photobleaching as well as fluorescence quenching to some extent. Lastly, effective surface modification and bioconjugation for nanoparticle delivery are highly desirable. No matter how robust the nanoparticles are, the ultimate image contrast would also depend on the nanoparticle loading efficiency. The selec-

71

72

2 Nanoparticles for Optical Imaging of Cancer

tion of specific cancer targeting strategies would thus play a crucial role. We envision that this part of the research and development would attract substantial attention in the near future. Nanoparticle-based optical contrast agents have a bright future in cancer imaging. However, the key is how to make them smart in all the aspects mentioned above for human applications. Although there is no straightforward route to such smart particles, research progress in this area over the last few years has been overwhelming.

Acknowledgments

We acknowledge the Particle Engineering Research Center (PERC) at the University of Florida for the entire support. Both SS and DD express sincere thanks to Dr. Parvesh Sharma, PERC, for his generous help during the preparation of the manuscript.

References 1 Tannock, I. F., Hill, R. P. (Eds.),

2 3

4

5

6

7

8

The Basic Science of Oncology, 3rd edn., McGraw-Hill, Health Professions Division, New York, 1998. Cancer Facts & Figures 2005. American Cancer Society, Atlanta, 2005, p. 1. Hanahan, D., Weinberg, R. A., The hallmarks of cancer. Cell 2000, 100, 57–70. Schulz, W. A., Molecular Biology of Human Cancers: An Advanced Student’s Textbook. Springer, New York, NY, 2005, pp. 11–17. Bremer, C., Ntziachristos, V., Weissleder, R., Optical-based molecular imaging: Contrast agents and potential medical applications. Eur. Radiol. 2003, 13, 231–243. Gao, X. H., Cui, Y. Y., Levenson, R. M., Chung, L. W. K., Nie, S. M., In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 2004, 22, 969–976. Medintz, I. L., Uyeda, H. T., Goldman, E. R., Mattoussi, H., Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 2005, 4, 435–446. Nalwa, H. R. (Ed.), Encyclopedia of Nanoscience and Nanotechnology,

9

10

11

12

13

14

15

American Scientific Publishers, Stevenson Ranch, CA, 2004. Feldhein, D. L., Foss, C. A. (Eds.), Metal Nanoparticles: Synthesis, Characterization & Applications, 1st edn., Marcel Dekker, New York, NY, 2001. Schmid, G. (Ed.), Nanoparticles: From Theory to Application, John Wiley & Sons, Hoboken, NJ, 2004. Rotello, V., Nanoparticles: Building Blocks for Nanotechnology, in Nanostructure Science and Technology, Lockwood, D. J. (Ed.), 1st edn., Springer, New York, NY, 2003, 1–300. Niemeyer, C. M., Mirkin, C. A. (Eds.), Nanobiotechnology: Concepts, Applications and Perspectives, John Wiley & Sons, Hoboken, NJ, 2004. Ben-Ari, E. T., Nanoscale quantum dots hold promise for cancer applications. J. Natl. Cancer Institute 2003, 95, 502–504. Brigger, I., Dubernet, C., Couvreur, P., Nanoparticles in cancer therapy and diagnosis. Adv. Drug Delivery Rev. 2002, 54, 631–651. Licha, K., Contrast Agents for Optical Imaging, in Contrast Agents II, Springer, Berlin, Heidelberg, 2002, 1–29.

References 16 Licha, K., Olbrich, C., Optical

17

18

19

20

21

22

23

24

25

26

imaging in drug discovery and diagnostic applications. Adv. Drug Delivery Rev. 2005, 57, 1087–1108. Licha, K., Riefke, B., Ebert, B., Grotzinger, C., Cyanine dyes as contrast agents in biomedical optical imaging. Acad. Radiol. 2002, 9, S320– S322. Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., Weiss, S., Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005, 307, 538–544. Morgan, N. Y., English, S., Chen, W., Chernomordik, V., Russo, A., Smith, P. D., Gandjbakhche, A., Real time in vivo non-invasive optical imaging using near-infrared fluorescent quantum dots. Acad. Radiol. 2005, 12, 313–323. Santra, S., Xu, J. S., Wang, K. M., Tan, W. H., Luminescent nanoparticle probes for bioimaging. J. Nanosci. Nanotechnol. 2004, 4, 590–599. Sokolov, K., Aaron, J., Hsu, B., Nida, D., Gillenwater, A., Follen, M., MacAulay, C., Adler-Storthz, K., Korgel, B., Descour, M., Pasqualini, R., Arap, W., Lam, W., Richards-Kortum, R., Optical systems for in vivo molecular imaging of cancer. Technol. Cancer Res. Treatment 2003, 2, 491–504. Rosi, N. L., Mirkin, C. A., Nanostructures in biodiagnostics. Chem. Rev. 2005, 105, 1547–1562. Smith, P. W., Fluorescence emissionbased detection and diagnosis of malignancy. J. Cell. Biochem. 2002, 54–59. Weissleder, R., Scaling down imaging: Molecular mapping of cancer in mice. Nat. Rev. Cancer 2002, 2, 11–18. Bornhop, D. J., Contag, C. H., Licha, K., Murphy, C. J., Advances in contrast agents, reporters, and detection. J. Biomed. Opt. 2001, 6, 106–110. Becker, A., Hessenius, C., Licha, K., Ebert, B., Sukowski, U., Semmler, W., Wiedenmann, B., Grotzinger,

27

28

29

30

31

32

33

34

35

C., Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat. Biotechnol. 2001, 19, 327–331. Becker, A., Riefke, B., Ebert, B., Sukowski, U., Rinneberg, H., Semmler, W., Licha, K., Macromolecular contrast agents for optical imaging of tumors: Comparison of indotricarbocyanine-labeled human serum albumin and transferrin. Photochem. Photobiol. 2000, 72, 234–241. Weissleder, R., Tung, C. H., Mahmood, U., Bogdanov, A., In vivo imaging of tumors with proteaseactivated near-infrared fluorescent probes. Nat. Biotechnol. 1999, 17, 375–378. Tung, C. H., Mahmood, U., Bredow, S., Weissleder, R., In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000, 60, 4953–4958. Bremer, C., Tung, C. H., Weissleder, R., In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 2001, 7, 743–748. Bremer, C., Bredow, S., Mahmood, U., Weissleder, R., Tung, C. H., Optical imaging of matrix metalloproteinase-2 activity in tumors: Feasibility study in a mouse model. Radiology 2001, 221, 523–529. Achilefu, S., Dorshow, R. B., Bugaj, J. E., Rajagopalan, R., Novel receptortargeted fluorescent contrast agents for in vivo tumor imaging. Investigative Radiol. 2000, 35, 479–485. Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W. S., Cao, Y. A., Negrin, R. S., Contag, C. H., Visualizing the kinetics of tumor-cell clearance in living animals. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12 044–12 049. Hillman, E. M. C., Hebden, J. C., Schweiger, M., Dehghani, H., Schmidt, F. E. W., Delpy, D. T., Arridge, S. R., Time resolved optical tomography of the human forearm. Phys. Med. Biol. 2001, 46, 1117–1130. Pogue, B. W., Poplack, S. P., McBride, T. O., Wells, W. A.,

73

74

2 Nanoparticles for Optical Imaging of Cancer

36

37

38

39

40

41

42

43

Osterman, K. S., Osterberg, U. L., Paulsen, K. D., Quantitative hemoglobin tomography with diffuse near-infrared spectroscopy: Pilot results in the breast. Radiology 2001, 218, 261–266. Benaron, D. A., Hintz, S. R., Villringer, A., Boas, D., Kleinschmidt, A., Frahm, J., Hirth, C., Obrig, H., van Houten, J. C., Kermit, E. L., Cheong, W. F., Stevenson, D. K., Noninvasive functional imaging of human brain using light. J. Cerebral Blood Flow Metab. 2000, 20, 469–477. Pogue, B. W., Poplack, S. P., McBride, T. O., Wells, W. A., Osterman, K. S., Osterberg, U. L., Hemoglobin imaging of breast tumors with near-infrared tomography. Radiology 2000, 214, 609–609. Demchik, L. L., Sameni, M., Nelson, K., Mikkelsen, T., Sloane, B. F., Cathepsin B and glioma invasion. Int. J. Developmental Neurosci. 1999, 17, 483–494. Yan, S. Q., Sameni, M., Sloane, B. F., Cathepsin B and human tumor progression. Biol. Chem. 1998, 379, 113–123. Rempel, S. A., Rosenblum, M. L., Mikkelsen, T., Yan, P. S., Ellis, K. D., Golembieski, W. A., Sameni, M., Rozhin, J., Ziegler, G., Sloane, B. F., Cathepsin-B expression and localization in glioma progression and invasion. Cancer Res. 1994, 54, 6027–6031. Ntziachristos, V., Bremer, C., Weissleder, R., Fluorescence imaging with near-infrared light: New technological advances that enable in vivo molecular imaging. Eur. Radiol. 2003, 13, 195–208. Haugland, R. P. Handbook of Fluorescent Probes and Research Products, Molecular Probes, Eugene, OR, 2003. Yang, M., Baranov, E., Jiang, P., Sun, F. X., Li, X. M., Li, L. N., Hasegawa, S., Bouvet, M., Al-Tuwaijri, M., Chishima, T., Shimada, H., Moossa, A. R., Penman, S., Hoffman, R. M., Whole-body optical imaging of green

44

45

46

47

48

49

50

51

fluorescent protein-expressing tumors and metastases. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1206–1211. Moore, A., Sergeyev, N., Bredow, S., Weissleder, R., A model system to quantitate tumor burden in locoregional lymph nodes during cancer spread. Invasion & Metastasis 1999, 18, 192–197. Moore, A., Marecos, E., Simonova, M., Weissleder, R., Bogdanov, A., Novel gliosarcoma cell line expressing green fluorescent protein: A model for quantitative assessment of angiogenesis. Microvascular Res. 1998, 56, 145–153. Stroh, M., Zimmer, J. P., Duda, D. G., Levchenko, T. S., Cohen, K. S., Brown, E. B., Scadden, D. T., Torchilin, V. P., Bawendi, M. G., Fukumura, D., Jain, R. K., Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nat. Med. 2005, 11, 678–682. Sonvico, F., Dubernet, C., Colombo, P., Couvreur, P., Metallic colloid nanotechnology, applications in diagnosis and therapeutics. Curr. Pharmaceut. Design 2005, 11, 2091–2105. Dressler, C., Minet, O., Novkov, V., Muller, G., Beuthan, J., Combination of bioactive quantum dots with an organic fluorescent dye for two-color labeling of cancer cells – Comparison between laser-induced and lamp-induced fluorescence. Laser Phys. 2005, 15, 536–544. Parungo, C. P., Ohnishi, S., Kim, S. W., Kim, S., Laurence, R. G., Soltesz, E. G., Chen, F. Y., Colson, Y. L., Cohn, L. H., Bawendi, M. G., Frangioni, J. V., Intraoperative identification of esophageal sentinel lymph nodes with near-infrared fluorescence imaging. J. Thoracic Cardiovascular Surgery 2005, 129, 844–850. Ballou, B., Ernst, L. A., Waggoner, A. S., Fluorescence imaging of tumors in vivo. Curr. Med. Chem. 2005, 12, 795–805. Ferrari, M., Cancer nanotechnology: Opportunities and challenges. Nat. Rev. Cancer 2005, 5, 161–171.

References 52 Lyons, S. K., Advances in imaging

53

54

55

56

57

58 59

60

61 62

63

mouse tumour models in vivo. J. Pathol. 2005, 205, 194–205. Jiang, W., Papa, E., Fischer, H., Mardyani, S., Chan, W. C. W., Semiconductor quantum dots as contrast agents for whole animal imaging. Trends Biotechnol. 2004, 22, 607–609. Sage, L., Finding cancer cells with quantum dots. Anal. Chem. 2004, 76, 453A. Chen, G. Y. J., Yao, S. Q., Lighting up cancer cells with ‘‘dots’’. Lancet 2004, 364, 2001–2003. Voura, E. B., Jaiswal, J. K., Mattoussi, H., Simon, S. M., Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat. Med. 2004, 10, 993–998. Wu, X. Y., Harper, T. F., Ng, C. H., Simultaneous detection of HER2 gene amplification and HER2 protein expression in breast cancer cells with semiconductor nanocrystal quantum dots. FASEB J. 2004, 18, C103–C103. Quantum dots target, image cancer cells. Chem. Eng. News 2004, 82, 40–40. Minet, O., Dressler, C., Beuthan, J., Heat stress induced redistribution of fluorescent quantum dots in breast tumor cells. J. Fluorescence 2004, 14, 241–247. Beuthan, J., Dressler, C., Minet, O., Laser-induced fluorescence detection of quantum dots redistributed in thermally stressed tumor cells. Laser Phys. 2004, 14, 213–219. Uren, R. F., Cancer surgery joins the dots. Nat. Biotechnol. 2004, 22, 38–39. Kim, S., Lim, Y. T., Soltesz, E. G., De Grand, A. M., Lee, J., Nakayama, A., Parker, J. A., Mihaljevic, T., Laurence, R. G., Dor, D. M., Cohn, L. H., Bawendi, M. G., Frangioni, J. V., Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 2004, 22, 93–97. Wu, X. Y., Liu, H. J., Liu, J. Q., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N. F., Peale, F.,

64

65

66

67

68

69

70

71

Bruchez, M. P., Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 2003, 21, 452–452. Wu, X. Y., Liu, H. J., Liu, J. Q., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N. F., Peale, F., Bruchez, M. P., Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 2003, 21, 41–46. LaVan, D. A., Lynn, D. M., Langer, R., Moving smaller in drug discovery and delivery. Nat. Rev. Drug Discov. 2002, 1, 77–84. Santra, S., Zhang, P., Wang, K. M., Tapec, R., Tan, W. H., Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal. Chem. 2001, 73, 4988–4993. Santra, S., Liesenfeld, B., Dutta, D., Chatel, D., Batich, C. D., Tan, W. H., Moudgil, B. M., Mericle, R. A., Folate conjugated fluorescent silica nanoparticles for labeling neoplastic cells. J. Nanosci. Nanotechnol. 2005, 5, 899–904. He, X. X., Duan, J. H., Wang, K. M., Tan, W. H., Lin, X., He, C. M., A novel fluorescent label based on organic dye-doped silica nanoparticles for HepG liver cancer cell recognition. J. Nanosci. Nanotechnol. 2004, 4, 585–589. He, X. X., Wang, K. M., Tan, W. H., Li, J., Yang, X. H., Huang, S. S., Li, D., Xiao, D., Photostable luminescent nanoparticles as biological label for cell recognition of system lupus erythematosus patients. J. Nanosci. Nanotechnol. 2002, 2, 317–320. Stsiapura, V., Sukhanova, A., Artemyev, M., Pluot, M., Cohen, J. H. M., Baranov, A. V., Oleinikov, V., Nabiev, I., Functionalized nanocrystaltagged fluorescent polymer beads: Synthesis, physicochemical characterization, and immunolabeling application. Anal. Biochem. 2004, 334, 257–265. Pan, X. Q., Lee, R. J., Ratnam, M., Penetration into solid tumor tissue

75

76

2 Nanoparticles for Optical Imaging of Cancer

72

73

74

75

76

77

78

79

of fluorescent latex microspheres: A mimic of liposome particles. Anticancer Res. 2004, 24, 3005–3008. Ueno, H., Hihara, J., Shimizu, K., Osaki, A., Yamashita, Y., Yoshida, K., Toge, T., Experimental study on fluorescent microspheres as a tracer for sentinel node detection. Anticancer Res. 2005, 25, 821–825. El-Sayed, I. H., Huang, X. H., El-Sayed, M. A., Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer. Nano Lett. 2005, 5, 829–834. Copland, J. A., Eghtedari, M., Popov, V. L., Kotov, N., Mamedova, N., Motamedi, M., Oraevsky, A. A., Bioconjugated gold nanoparticles as a molecular based contrast agent: Implications for imaging of deep tumors using optoacoustic tomography. Mol. Imaging Biol. 2004, 6, 341–349. Sokolov, K., Follen, M., Aaron, J., Pavlova, I., Malpica, A., Lotan, R., Richards-Kortum, R., Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Res. 2003, 63, 1999–2004. Schultz, S., Smith, D. R., Mock, J. J., Schultz, D. A., Single-target molecule detection with nonbleaching multicolor optical immunolabels. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996–1001. Schultz, S., Mock, J., Smith, D. R., Schultz, D. A., Nanoparticle based biological assays. J. Clinical Ligand Assay 1999, 22, 214–216. Yguerabide, J., Yguerabide, E. E., Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications. J. Cell. Biochem. 2001(Suppl.), 71–81. Guo, J., Yang, W. L., Deng, Y. H., Wang, C. C., Fu, S. K., Organic-dyecoupled magnetic nanoparticles encaged inside thermoresponsive PNIPAM microcapsules. Small 2005, 1, 737–743.

80 Veiseh, O., Sun, C., Gunn, J.,

81

82

83

84

85

86

Kohler, N., Gabikian, P., Lee, D., Bhattarai, N., Ellenbogen, R., Sze, R., Hallahan, A., Olson, J., Zhang, M. Q., Optical and MRI multifunctional nanoprobe for targeting gliomas. Nano Lett. 2005, 5, 1003–1008. del Campo, A., Sen, T., Lellouche, J. P., Bruce, I. J., Multifunctional magnetite and silica-magnetite nanoparticles: Synthesis, surface activation and applications in life sciences. J. Magn. Magn. Mater. 2005, 293, 33–40. Kopelman, R., Koo, Y. E. L., Philbert, M., Moffat, B. A., Reddy, G. R., McConville, P., Hall, D. E., Chenevert, T. L., Bhojani, M. S., Buck, S. M., Rehemtulla, A., Ross, B. D., Multifunctional nanoparticle platforms for in vivo MRI enhancement and photodynamic therapy of a rat brain cancer. J. Magn. Magn. Mater. 2005, 293, 404–410. Spasova, M., Salgueirino-Maceira, V., Schlachter, A., Hilgendorff, M., Giersig, M., Liz-Marzan, L. M., Farle, M., Magnetic and optical tunable microspheres with a magnetite/gold nanoparticle shell. J. Mater. Chem. 2005, 15, 2095– 2098. Pellegrino, T., Kudera, S., Liedl, T., Javier, A. M., Manna, L., Parak, W. J., On the development of colloidal nanoparticles towards multifunctional structures and their possible use for biological applications. Small 2005, 1, 48–63. Xie, H. Y., Zuo, C., Liu, Y., Zhang, Z. L., Pang, D. W., Li, X. L., Gong, J. P., Dickinson, C., Zhou, W. Z., Cell-targeting multifunctional nanospheres with both fluorescence and magnetism. Small 2005, 1, 506–509. Santra, S., Yang, H. S., Holloway, P. H., Stanley, J. T., Mericle, R. A., Synthesis of water-dispersible fluorescent, radio-opaque, and paramagnetic CdS:Mn/ZnS quantum dots: A multifunctional probe for bioimaging. J. Am. Chem. Soc. 2005, 127, 1656–1657.

References 87 Kircher, M. F., Mahmood, U., King,

88

89

90

91

92

93

94

95

96

R. S., Weissleder, R., Josephson, L., A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res. 2003, 63, 8122–8125. Huber, M. M., Staubli, A. B., Kustedjo, K., Gray, M. H. B., Shih, J., Fraser, S. E., Jacobs, R. E., Meade, T. J., Fluorescently detectable magnetic resonance imaging agents. Bioconjugate Chem. 1998, 9, 242–249. Santra, S., Yang, H., Stanley, J. T., Holloway, P. H., Moudgil, B. M., Walter, G., Mericle, R. A., Rapid and effective labeling of brain tissue using TAT-conjugated CdS: Mn/ZnS quantum dots. Chem. Commun. 2005, 3144–3146. Yang, H. S., Holloway, P. H., Santra, S., Water-soluble silicaovercoated CdS:Mn/ZnS semiconductor quantum dots. J. Chem. Phys. 2004, 121, 7421–7426. Santra, S., Yang, H., Dutta, D., Stanley, J. T., Holloway, P. H., Tan, W. H., Moudgil, B. M., Mericle, R. A., TAT conjugated, FITC doped silica nanoparticles for bioimaging applications. Chem. Commun. 2004, 2810–2811. Qhobosheane, M., Santra, S., Zhang, P., Tan, W. H., Biochemically functionalized silica nanoparticles. Analyst 2001, 126, 1274–1278. Santra, S., Wang, K. M., Tapec, R., Tan, W. H., Development of novel dye-doped silica nanoparticles for biomarker application. J. Biomed. Opt. 2001, 6, 160–166. Levy, L., Sahoo, Y., Kim, K. S., Bergey, E. J., Prasad, P. N., Nanochemistry: Synthesis and characterization of multifunctional nanoclinics for biological applications. Chem. Mater. 2002, 14, 3715–3721. Wang, Z., Levy, R., Fernig, D. G., Brust, M., The peptide route to multifunctional gold nanoparticles. Bioconjugate Chem. 2005, 16, 497– 500. Gao, X. H., Yang, L. L., Petros, J. A., Marshal, F. F., Simons, J. W., Nie,

97

98

99

100

101

102

103

104

105

106

S. M., In vivo molecular and cellular imaging with quantum dots. Curr. Opin. Biotechnol. 2005, 16, 63–72. Alivisatos, A. P., Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, 933–937. Efros, A. L., Rosen, M., The electronic structure of semiconductor nanocrystals. Annu. Rev. Mater. Sci. 2000, 30, 475–521. Leatherdale, C. A., Woo, W. K., Mikulec, F. V., Bawendi, M. G., On the absorption cross section of CdSe nanocrystal quantum dots. J. Phys. Chem. B 2002, 106, 7619–7622. Dabbousi, B. O., RodriguezViejo, J., Mikulec, F. V., Heine, J. R., Mattoussi, H., Ober, R., Jensen, K. F., Bawendi, M. G., (CdSe)ZnS core-shell quantum dots: Synthesis and characterization of a size series of highly luminescent nanocrystallites. J. Phys. Chem. B 1997, 101, 9463–9475. Bruchez, M., Moronne, M., Gin, P., Weiss, S., Alivisatos, A. P., Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281, 2013–2016. Chan, W. C. W., Nie, S. M., Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281, 2016–2018. Jakobs, S., Subramaniam, V., Schonle, A., Jovin, T. M., Hell, S. W., EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett. 2000, 479, 131–135. Pepperkok, R., Squire, A., Geley, S., Bastiaens, P. I., Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr. Biol. 1999, 9, 269–272. Gao, X. H., Nie, S. M., Molecular profiling of single cells and tissue specimens with quantum dots. Trends Biotechnol. 2003, 21, 371–373. Murray, C. B., Norris, D. J., Bawendi, M. G., Synthesis and characterization of nearly monodisperse CdE (E ¼ S; Se; Te) semi-

77

78

2 Nanoparticles for Optical Imaging of Cancer

107

108

109

110

111

112

113

114

115

116

conductor nanocrystallites. J. Am. Chem. Soc. 1993, 115, 8706–8715. Qu, L., Peng, X., Control of photoluminescence properties of CdSe nanocrystals in growth. J. Am. Chem. Soc. 2002, 124, 2049–2055. Yang, H., Santra, S., Holloway, P. H., Synthesis and application of Mn-doped II–VI semiconductor nanocrystals. J. Nanosci. Nanotechnol. 2005, 5, 1364–1375. Yang, H. S., Holloway, P. H., Cunningham, G., Schanze, K. S., CdS:Mn nanocrystals passivated by ZnS: Synthesis and luminescent properties. J. Chem. Phys. 2004, 121, 10 233–10 240. Yang, H., Holloway, P. H., Efficient and photostable ZnS-Passivated CdS:Mn luminescent nanocrystals. Adv. Functional Mater. 2004, 14, 152–156. Hines, M. A., Guyot-Sionnest, P., Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals. J. Phys. Chem. 1996, 100, 468–471. Chen, F. Q., Gerion, D., Fluorescent CdSe/ZnS nanocrystal-peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Lett. 2004, 4, 1827–1832. Selvan, S. T., Li, C. L., Ando, M., Murase, N., Formation of luminescent CdTe-silica nanoparticles through an inverse microemulsion technique. Chem. Lett. 2004, 33, 434–435. Selvan, S. T., Tan, T. T., Ying, J. Y., Robust, non-cytotoxic, silica-coated CdSe quantum dots with efficient photoluminescence. Adv. Mater. 2005, 17, 1620–1625. Goldman, E. R., Balighian, E. D., Mattoussi, H., Kuno, M. K., Mauro, J. M., Tran, P. T., Anderson, G. P., Avidin: A natural bridge for quantum dot-antibody conjugates. J. Am. Chem. Soc. 2002, 124, 6378–6382. Mattoussi, H., Mauro, J. M., Goldman, E. R., Anderson, G. P., Sundar, V. C., Mikulec, F. V., Bawendi, M. G., Self-assembly of

117

118

119

120

121

122

123

124

CdSe-ZnS quantum dot bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 2000, 122, 12 142–12 150. Mitchell, G. P., Mirkin, C. A., Letsinger, R. L., Programmed assembly of DNA functionalized quantum dots. J. Am. Chem. Soc. 1999, 121, 8122–8123. Uyeda, H. T., Medintz, I. L., Jaiswal, J. K., Simon, S. M., Mattoussi, H., Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores. J. Am. Chem. Soc. 2005, 127, 3870–3878. Gerion, D., Pinaud, F., Williams, S. C., Parak, W. J., Zanchet, D., Weiss, S., Alivisatos, A. P., Synthesis and properties of biocompatible watersoluble silica-coated CdSe/ZnS semiconductor quantum dots. J. Phys. Chem. B 2001, 105, 8861–8871. Osaki, F., Kanamori, T., Sando, S., Sera, T., Aoyama, Y., A quantum dot conjugated sugar ball and its cellular uptake on the size effects of endocytosis in the subviral region. J. Am. Chem. Soc. 2004, 126, 6520–6521. Pellegrino, T., Manna, L., Kudera, S., Liedl, T., Koktysh, D., Rogach, A. L., Keller, S., Radler, J., Natile, G., Parak, W. J., Hydrophobic nanocrystals coated with an amphiphilic polymer shell: A general route to water soluble nanocrystals. Nano Lett. 2004, 4, 703–707. Mattheakis, L. C., Dias, J. M., Choi, Y. J., Gong, J., Bruchez, M. P., Liu, J. Q., Wang, E., Optical coding of mammalian cells using semiconductor quantum dots. Anal. Biochem. 2004, 327, 200–208. Ballou, B., Lagerholm, B. C., Ernst, L. A., Bruchez, M. P., Waggoner, A. S., Noninvasive imaging of quantum dots in mice. Bioconjugate Chem. 2004, 15, 79–86. Dubertret, B., Skourides, P., Norris, D. J., Noireaux, V., Brivanlou, A. H., Libchaber, A., In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 2002, 298, 1759–1762.

References 125 Parak, W. J., Gerion, D., Pellegrino,

126

127

128

129

130

131

132

133

T., Zanchet, D., Micheel, C., Williams, S. C., Boudreau, R., Le Gros, M. A., Larabell, C. A., Alivisatos, A. P., Biological applications of colloidal nanocrystals. Nanotechnology 2003, 14, R15–R27. Jaiswal, J. K., Simon, S. M., Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 2004, 14, 497–504. Medintz, I. L., Clapp, A. R., Mattoussi, H., Goldman, E. R., Fisher, B., Mauro, J. M., Selfassembled nanoscale biosensors based on quantum dot FRET donors. Nat. Mater. 2003, 2, 630–638. Pinaud, F., King, D., Moore, H. P., Weiss, S., Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatinrelated peptides. J. Am. Chem. Soc. 2004, 126, 6115–6123. Ackerman, M. E., Chan, W. C. W., Laakkonen, P., Bhatia, S. N., Ruoslahti, E., Nanocrystal targeting in vivo. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12 617–12 621. Hainfeld, J. F., Liu, W. Q., Halsey, C. M. R., Freimuth, P., Powell, R. D., Ni-NTA-gold clusters target Histagged proteins. J. Struct. Biol. 1999, 127, 185–198. Slocik, J. M., Moore, J. T., Wright, D. W., Monoclonal antibody recognition of histidine-rich peptide encapsulated nanoclusters. Nano Lett. 2002, 2, 169–173. Medintz, I. L., Konnert, J. H., Clapp, A. R., Stanish, I., Twigg, M. E., Mattoussi, H., Mauro, J. M., Deschamps, J. R., A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9612–9617. Medintz, I. L., Trammell, S. A., Mattoussi, H., Mauro, J. M., Reversible modulation of quantum dot photoluminescence using a proteinbound photochromic fluorescence resonance energy transfer acceptor. J. Am. Chem. Soc. 2004, 126, 30–31.

134 Hanaki, K., Momo, A., Oku, T.,

135

136

137

138

139

140

141

142

143

144

Komoto, A., Maenosono, S., Yamaguchi, Y., Yamamoto, K., Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker. Biochem. Biophys. Res. Commun. 2003, 302, 496–501. Goldman, E. R., Anderson, G. P., Tran, P. T., Mattoussi, H., Charles, P. T., Mauro, J. M., Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays. Anal. Chem. 2002, 74, 841–847. Bohren, C. F., Huffman, D. R., Absorption and scattering of light by small particles. Wiley-VCH, Berlin 1983, 3–476. Kreibig, U., Genzel, L., Opticalabsorption of small metallic particles. Surf. Sci. 1985, 156, 678–700. Sarkar, D., Halas, N. J., General vector basis function solution of Maxwell’s equations. Phys. Rev. E 1997, 56, 1102–1112. Murphy, C. J., Jana, N. R., Controlling the aspect ratio of inorganic nanorods and nanowires. Adv. Mater. 2002, 14, 80–82. Jin, R. C., Cao, Y. W., Mirkin, C. A., Kelly, K. L., Schatz, G. C., Zheng, J. G., Photoinduced conversion of silver nanospheres to nanoprisms. Science 2001, 294, 1901–1903. Oldenburg, S. J., Averitt, R. D., Westcott, S. L., Halas, N. J., Nanoengineering of optical resonances. Chem. Phys. Lett. 1998, 288, 243–247. Haynes, C. L., Van Duyne, R. P., Nanosphere lithography: A versatile nanofabrication tool for studies of size-dependent nanoparticle optics. J. Phys. Chem. B 2001, 105, 5599–5611. Novak, J. P., Feldheim, D. L., Assembly of phenylacetylene-bridged silver and gold nanoparticle arrays. J. Am. Chem. Soc. 2000, 122, 3979–3980. Novak, J. P., Nickerson, C., Franzen, S., Feldheim, D. L., Purification of molecularly bridged metal nanopar-

79

80

2 Nanoparticles for Optical Imaging of Cancer

145

146

147

148

149

150

151

152

153

ticle arrays by centrifugation and size exclusion chromatography. Anal. Chem. 2001, 73, 5758–5761. Brousseau, L. C., Novak, J. P., Marinakos, S. M., Feldheim, D. L., Assembly of phenylacetylene-bridged gold nanocluster dimers and trimers. Adv. Mater. 1999, 11, 447–449. Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L., Mirkin, C. A., Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 1997, 277, 1078–1081. Caruso, R. A., Antonietti, M., Solgel nanocoating: An approach to the preparation of structured materials. Chem. Mater. 2001, 13, 3272–3282. Marinakos, S. M., Novak, J. P., Brousseau, L. C., House, A. B., Edeki, E. M., Feldhaus, J. C., Feldheim, D. L., Gold particles as templates for the synthesis of hollow polymer capsules. Control of capsule dimensions and guest encapsulation. J. Am. Chem. Soc. 1999, 121, 8518–8522. Malinsky, M. D., Kelly, K. L., Schatz, G. C., Van Duyne, R. P., Chain length dependence and sensing capabilities of the localized surface plasmon resonance of silver nanoparticles chemically modified with alkanethiol self-assembled monolayers. J. Am. Chem. Soc. 2001, 123, 1471–1482. Cocchini, F., Bassani, F., Bourg, M., Model calculation of the opticalproperties of metallic particles in a dielectric medium. Surf. Sci. 1985, 156, 851–858. Mulvaney, P., Liz-Marzan, L. M., Giersig, M., Ung, T., Silica encapsulation of quantum dots and metal clusters. J. Mater. Chem. 2000, 10, 1259–1270. Underwood, S., Mulvaney, P., Effect of the solution refractive-index on the color of gold colloids. Langmuir 1994, 10, 3427–3430. Hayat, M. A., Colloidal Gold: Principles, Methods and Applications. Elsevier, Burlington, MA, 1989.

154 Sauthier, M. L., Carroll, R. L.,

155

156

157

158

159

160

161

162

163

164

Gorman, C. B., Franzen, S., Nanoparticle layers assembled through DNA hybridization: Characterization and optimization. Langmuir 2002, 18, 1825–1830. El-Sayed, M. A., Some interesting properties of metals confined in time and nanometer space of different shapes. Acc. Chem. Res. 2001, 34, 257–264. Taylor, J. R., Fang, M. M., Nie, S. M., Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles. Anal. Chem. 2000, 72, 1979–1986. Mann, S., Shenton, W., Li, M., Connolly, S., Fitzmaurice, D., Biologically programmed nanoparticle assembly. Adv. Mater. 2000, 12, 147–150. Mrksich, M., A surface chemistry approach to studying cell adhesion. Chem. Soc. Rev. 2000, 29, 267–273. Bright, R. M., Walter, D. G., Musick, M. D., Jackson, M. A., Allison, K. J., Natan, M. J., Chemical and electrochemical Ag deposition onto preformed Au colloid monolayers: Approaches to uniformlysized surface features with Ag-like optical properties. Langmuir 1996, 12, 810–817. Taton, T. A., Lu, G., Mirkin, C. A., Two-color labeling of oligonucleotide arrays via size-selective scattering of nanoparticle probes. J. Am. Chem. Soc. 2001, 123, 5164–5165. Taton, T. A., Mirkin, C. A., Letsinger, R. L., Scanometric DNA array detection with nanoparticle probes. Science 2000, 289, 1757–1760. Mafune, F., Kohno, J. Y., Takeda, Y., Kondow, T., Full physical preparation of size-selected gold nanoparticles in solution: Laser ablation and laserinduced size control. J. Phys. Chem. B 2002, 106, 7575–7577. Turkevitch, J., Stevenson, P. C., Hillier, J., Faraday Soc. 1951, 11, 55. Henglein, A., Giersig, M., Formation of colloidal silver nanoparticles: Capping action of

References

165

166

167

168

169

170

171

172

173

citrate. J. Phys. Chem. B 1999, 103, 9533–9539. Link, S., Wang, Z. L., El-Sayed, M. A., Alloy formation of gold-silver nanoparticles and the dependence of the plasmon absorption on their composition. J. Phys. Chem. B 1999, 103, 3529–3533. Yonezawa, T., Kunitake, T., Practical preparation of anionic mercapto ligand-stabilized gold nanoparticles and their immobilization. Colloids Surf. A-Physicochem. Eng. Aspects 1999, 149, 193–199. Brust, M., Fink, J., Bethell, D., Schiffrin, D. J., Kiely, C., Synthesis and reactions of functionalized gold nanoparticles. J. Chem. Soc., Chem. Commun. 1995, 1655–1656. Kohler, J. M., Wagner, J., Albert, J., Formation of isolated and clustered Au nanoparticles in the presence of polyelectrolyte molecules using a flowthrough Si chip reactor. J. Mater. Chem. 2005, 15, 1924–1930. Brust, M., Walker, M., Bethell, D., Schiffrin, D. J., Whyman, R., Synthesis of thiol-derivatized gold nanoparticles in a 2-phase liquidliquid system. J. Chem. Soc., Chem. Commun. 1994, 801–802. Lin, J., Zhou, W. L., O’Connor, C. J., Formation of ordered arrays of gold nanoparticles from CTAB reverse micelles. Mater. Lett. 2001, 49, 282–286. Chiang, C. L., Controlled growth of gold nanoparticles in AOT/C12E4/ isooctane mixed reverse micelles. J. Colloid Interface Sci. 2001, 239, 334–341. Chen, F. X., Xu, G. Q., Hor, T. S. A., Preparation and assembly of colloidal gold nanoparticles in CTAB-stabilized reverse microemulsion. Mater. Lett. 2003, 57, 3282–3286. Sohn, B. H., Choi, J. M., Yoo, S. I., Yun, S. H., Zin, W. C., Jung, J. C., Kanehara, M., Hirata, T., Teranishi, T., Directed self-assembly of two kinds of nanoparticles utilizing monolayer films of diblock copolymer micelles. J. Am. Chem. Soc. 2003, 125, 6368–6369.

174 Wilcoxon, J. P., Williamson, R. L.,

175 176

177

178

179

180

181

182

183

184

Baughman, R., Optical-properties of gold colloids formed in inverse micelles. J. Chem. Phys. 1993, 98, 9933–9950. Zsigmondy, R., Thiessen, P., Das Kolloide Gold. Verlagsges. Leipzig, 1925. Henglein, A., Formation and absorption spectrum of copper nanoparticles from the radiolytic reduction of Cu(CN)(2)(-). J. Phys. Chem. B 2000, 104, 1206–1211. Teranishi, T., Hosoe, M., Tanaka, T., Miyake, M., Size control of monodispersed Pt nanoparticles and their 2D organization by electrophoretic deposition. J. Phys. Chem. B 1999, 103, 3818–3827. Teranishi, T., Miyake, M., Size control of palladium nanoparticles and their crystal structures. Chem. Mater. 1998, 10, 594–600. Brown, K. R., Natan, M. J., Hydroxylamine seeding of colloidal Au nanoparticles in solution and on surfaces. Langmuir 1998, 14, 726–728. Shirtcliffe, N., Nickel, U., Schneider, S., Reproducible preparation of silver sols with small particle size using borohydride reduction: For use as nuclei for preparation of larger particles. J. Colloid Interface Sci. 1999, 211, 122–129. Schneider, S., Halbig, P., Grau, H., Nickel, U., Reproducible preparation of silver sols with uniform particlesize for application in surfaceenhanced Raman-spectroscopy. Photochem. Photobiol. 1994, 60, 605–610. Brown, K. R., Walter, D. G., Natan, M. J., Seeding of colloidal Au nanoparticle solutions. 2. Improved control of particle size and shape. Chem. Mater. 2000, 12, 306–313. Lu, L. H., Wang, H. S., Zhou, Y. H., Xi, S. Q., Zhang, H. J., Jiawen, H. B. W., Zhao, B., Seed-mediated growth of large, monodisperse core-shell goldsilver nanoparticles with Ag-like optical properties. Chem. Commun. 2002, 144–145. Jana, N. R., Gearheart, L., Murphy, C. J., Evidence for seed-mediated

81

82

2 Nanoparticles for Optical Imaging of Cancer

185

186

187

188

189

190

191

192

193

nucleation in the chemical reduction of gold salts to gold nanoparticles. Chem. Mater. 2001, 13, 2313–2322. Jana, N. R., Gearheart, L., Murphy, C. J., Seed-mediated growth approach for shape-controlled synthesis of spheroidal and rod-like gold nanoparticles using a surfactant template. Adv. Mater. 2001, 13, 1389–1393. Mieszawska, A. J., Zamborini, F. P., Gold nanorods grown directly on surfaces from microscale patterns of gold seeds. Chem. Mater. 2005, 17, 3415–3420. Wei, Z. Q., Zamborini, F. P., Directly monitoring the growth of gold nanoparticle seeds into gold nanorods. Langmuir 2004, 20, 11 301–11 304. Sau, T. K., Pal, A., Jana, N. R., Wang, Z. L., Pal, T., Size controlled synthesis of gold nanoparticles using photochemically prepared seed particles. J. Nanoparticle Res. 2001, 3, 257–261. Pradhan, N., Jana, N. R., Kaushik, M., Tarashankar, P., Seed mediated growth: A convenient way for size control in nanoparticle synthesis. J. Surf. Sci. Technol. 2000, 16, 188– 199. Wiesner, J., Wokaun, A., Anisometric gold colloids – Preparation, characterization, and opticalproperties. Chem. Phys. Lett. 1989, 157, 569–575. Watzky, M. A., Finke, R. G., Nanocluster size-control and ‘‘magic number’’ investigations, experimental tests of the ‘‘living-metal polymer’’ concept and of mechanism-based sizecontrol predictions leading to the syntheses of iridium(0) nanoclusters centering about four sequential magic numbers. Chem. Mater. 1997, 9, 3083–3095. Carrot, G., Valmalette, J. C., Plummer, C. J. G., Scholz, S. M., Dutta, J., Hofmann, H., Hilborn, J. G., Gold nanoparticle synthesis in graft copolymer micelles. Colloid Polym. Sci. 1998, 276, 853–859. Loo, C., Lin, A., Hirsch, L., Lee, M. H., Barton, J., Halas, N., West,

194

195

196

197

198

199

200

201

202

J., Drezek, R., Nanoshell-enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treatment 2004, 3, 33–40. Duff, D. G., Baiker, A., Edwards, P. P., A new hydrosol of gold clusters. 1. Formation and particle-size variation. Langmuir 1993, 9, 2301–2309. Hirsch, L. R., Halas, N. J., West, J. L., Whole-blood immunoassay facilitated by gold-nanoshell-conjugate antibodies. Nanobiotechnol. Protocols 2005, 303, 101–111. O’Neal, D. P., Hirsch, L. R., Halas, N. J., Payne, J. D., West, J. L., Photothermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Lett. 2004, 209, 171–176. Loo, C., Hirsch, L., Lee, M. H., Chang, E., West, J., Halas, N., Drezek, R., Gold nanoshell bioconjugates for molecular imaging in living cells. Opt. Lett. 2005, 30, 1012–1014. Loo, C., Lowery, A., Halas, N., West, J., Drezek, R., Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Lett. 2005, 5, 709–711. Mallick, K., Wang, Z. L., Pal, T., Seed-mediated successive growth of gold particles accomplished by UV irradiation: a photochemical approach for size-controlled synthesis. J. Photochem. Photobiol. A Chem. 2001, 140, 75–80. Zhou, Y., Wang, C. Y., Zhu, Y. R., Chen, Z. Y., A novel ultraviolet irradiation technique for shapecontrolled synthesis of gold nanoparticles at room temperature. Chem. Mater. 1999, 11, 2310–2312. Pal, A., Photoinitiated gold sol generation in aqueous Triton X-100 and its analytical application for spectrophotometric determination of gold. Talanta 1998, 46, 583–587. Reed, J. A., Cook, A., Halaas, D. J., Parazzoli, P., Robinson, A., Matula, T. J., Grieser, F., The effects of microgravity on nanoparticle size distributions generated by the ultrasonic reduction of an aqueous

References

203

204

205

206

207

208

209

210

211

gold-chloride solution. Ultrasonics Sonochem. 2003, 10, 285–289. Pol, V. G., Gedanken, A., CalderonMoreno, J., Deposition of gold nanoparticles on silica spheres: A sonochemical approach. Chem. Mater. 2003, 15, 1111–1118. Okitsu, K., Yue, A., Tanabe, S., Matsumoto, H., Yobiko, Y., Yoo, Y., Sonolytic control of rate of gold(III) reduction and size of formed gold nanoparticles: Relation between reduction rates and sizes of formed nanoparticles. Bull. Chem. Soc. Jpn. 2002, 75, 2289–2296. Okitsu, K., Yue, A., Tanabe, S., Matsumoto, H., Yobiko, Y., Formation of colloidal gold nanoparticles in an ultrasonic field: Control of rate of gold(III) reduction and size of formed gold particles. Langmuir 2001, 17, 7717–7720. Chen, W., Cai, W. P., Zhang, L., Wang, G. Z., Zhang, L. D., Sonochemical processes and formation of gold nanoparticles within pores of mesoporous silica. J. Colloid Interface Sci. 2001, 238, 291–295. Chen, W., Cai, W. P., Liang, C. H., Zhang, L. D., Synthesis of gold nanoparticles dispersed within pores of mesoporous silica induced by ultrasonic irradiation and its characterization. Mater. Res. Bull. 2001, 36, 335–342. Shimizu, T., Teranishi, T., Hasegawa, S., Miyake, M., Size evolution of alkanethiol-protected gold nanoparticles by heat treatment in the solid state. J. Phys. Chem. B 2003, 107, 2719–2724. Teranishi, T., Hasegawa, S., Shimizu, T., Miyake, M., Heatinduced size evolution of gold nanoparticles in the solid state. Adv. Mater. 2001, 13, 1699–1701. Nakamoto, M., Yamamoto, M., Fukusumi, M., Thermolysis of gold(I) thiolate complexes producing novel gold nanoparticles passivated by alkyl groups. Chem. Commun. 2002, 1622–1623. Mossmer, S., Spatz, J. P., Moller, M., Aberle, T., Schmidt, J.,

212

213

214 215

216

217

218

219

220

221

Burchard, W., Solution behavior of poly(styrene)-block-poly(2vinylpyridine) micelles containing gold nanoparticles. Macromolecules 2000, 33, 4791–4798. Meltzer, S., Resch, R., Koel, B. E., Thompson, M. E., Madhukar, A., Requicha, A. A. G., Will, P., Fabrication of nanostructures by hydroxylamine seeding of gold nanoparticle templates. Langmuir 2001, 17, 1713–1718. Grieser, F., in Semiconductor Nanoclusters – Physical, Chemical, and Catalytic Aspects, Kamat, P. V., Meisel, D. (Ed.), 1997, Vol. 103, pp. 57–77. Hermanson, G. T., Bioconjugation Techniques. 1996, 593–604. Stober, W., Fink, A., Bohn, E., Controlled growth of monodisperse silica spheres in micron size range. J. Colloid Interface Sci. 1968, 26, 62– 67. Qhobosheane, M., Zhang, P., Tan, W. H., Assembly of silica nanoparticles for two-dimensional nanomaterials. J. Nanosci. Nanotechnol. 2004, 4, 635–640. Santra, S., Dutta, D., Moudgil, B. M., Functional dye-doped silica nanoparticles for bioimaging, diagnostics and therapeutics. Food Bioproducts Process. 2005, 83, 136–140. Wang, L., Yang, C. Y., Tan, W. H., Dual-luminophore-doped silica nanoparticles for multiplexed signaling. Nano Lett. 2005, 5, 37–43. Lian, W., Litherland, S. A., Badrane, H., Tan, W. H., Wu, D. H., Baker, H. V., Gulig, P. A., Lim, D. V., Jin, S. G., Ultrasensitive detection of biomolecules with fluorescent dyedoped nanoparticles. Anal. Biochem. 2004, 334, 135–144. Tan, M. Q., Wang, G. L., Hai, X. D., Ye, Z. Q., Yuan, J. L., Development of functionalized fluorescent europium nanoparticles for biolabeling and timeresolved fluorometric applications. J. Mater. Chem. 2004, 14, 2896–2901. Bagwe, R. P., Yang, C. Y., Hilliard, L. R., Tan, W. H., Optimization of dye-doped silica nanoparticles prepared using a reverse micro-

83

84

2 Nanoparticles for Optical Imaging of Cancer

222

223

224

225

226

227

228

229

230

emulsion method. Langmuir 2004, 20, 8336–8342. Tan, M. Q., Ye, Z. Q., Wang, G. L., Yuan, J. L., Preparation and timeresolved fluorometric application of luminescent europium nanoparticles. Chem. Mater. 2004, 16, 2494–2498. Ye, Z. Q., Tan, M. Q., Wang, G. L., Yuan, J. L., Novel fluorescent europium chelate-doped silica nanoparticles: Preparation, characterization and time-resolved fluorometric application. J. Mater. Chem. 2004, 14, 851–856. Zhao, X. J., Bagwe, R. P., Tan, W. H., Development of organic-dye-doped silica nanoparticles in a reverse microemulsion. Adv. Mater. 2004, 16, 173–176. Santra, S., Tapec, R., Theodoropoulou, N., Dobson, J., Hebard, A., Tan, W. H., Synthesis and characterization of silica-coated iron oxide nanoparticles in microemulsion: The effect of nonionic surfactants. Langmuir 2001, 17, 2900–2906. Harma, H., Soukka, T., Lovgren, T., Europium nanoparticles and timeresolved fluorescence for ultrasensitive detection of prostate-specific antigen. Clin. Chem. 2001, 47, 561–568. Gao, H. F., Zhao, Y. Q., Fu, S. K., Li, B., Li, M. Q., Preparation of a novel polymeric fluorescent nanoparticle. Colloid Polym. Sci. 2002, 280, 653–660. Makarova, O. V., Ostafin, A. E., Miyoshi, H., Norris, J. R., Meisel, D., Adsorption and encapsulation of fluorescent probes in nanoparticles. J. Phys. Chem. B 1999, 103, 9080–9084. Schlupen, J., Haegel, F. H., Kuhlmann, J., Geisler, H., Schwuger, M. J., Sorption hysteresis of pyrene on zeolite. Colloids Surf. APhysicochem. Eng. Aspects 1999, 156, 335–347. Charreyre, M. T., Yekta, A., Winnik, M. A., Delair, T., Pichot, C., Fluorescence energy-transfer from fluorescein to tetramethylrhodamine covalently bound to the surface of polystyrene latex-particles. Langmuir 1995, 11, 2423–2428.

231 Charreyre, M. T., Zhang, P.,

232

233

234

235

236

237

238

239

240

Winnik, M. A., Pichot, C., Graillat, C., Adsorption of Rhodamine-60 onto polystyrene latex-particles with sulfate groups at the surface. J. Colloid Interface Sci. 1995, 170, 374–382. Liu, X. J., Tan, W. H., A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons. Anal. Chem. 1999, 71, 5054–5059. Cordek, J., Wang, X. W., Tan, W. H., Direct immobilization of glutamate dehydrogenase on optical fiber probes for ultrasensitive glutamate detection. Anal. Chem. 1999, 71, 1529–1533. Fang, X. H., Liu, X. J., Schuster, S., Tan, W. H., Designing a novel molecular beacon for surface-immobilized DNA hybridization studies. J. Am. Chem. Soc. 1999, 121, 2921–2922. Wang, S., Low, P. S., Folate-mediated targeting of antineoplastic drags, imaging agents, and nucleic acids to cancer cells. J. Controlled Release 1998, 53, 39–48. Zhao, X. J., Hilliard, L. R., Mechery, S. J., Wang, Y. P., Bagwe, R. P., Jin, S. G., Tan, W. H., A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15 027–15 032. Tapec, R., Zhao, X. J. J., Tan, W. H., Development of organic dye-doped silica nanoparticles for bioanalysis and biosensors. J. Nanosci. Nanotechnol. 2002, 2, 405–409. Kim, G. J., Nie, S., Targeted cancer nanotherapy. Nanotoday 2005, 8, 28–33. Parak, W. J., Boudreau, R., Le Gros, M., Gerion, D., Zanchet, D., Micheel, C. M., Williams, S. C., Alivisatos, A. P., Larabell, C., Cell motility and metastatic potential studies based on quantum dot imaging of phagokinetic tracks. Adv. Mater. 2002, 14, 882–885. Pellegrino, T., Parak, W. J., Boudreau, R., Le Gros, M. A., Gerion, D., Alivisatos, A. P., Larabell, C. A., Quantum dot-based cell motility assay. Differentiation 2003, 71, 542–548.

References 241 Lidke, D. S., Nagy, P., Heintzmann,

242

243

244

245

246

247

R., Arndt-Jovin, D. J., Post, J. N., Grecco, H. E., Jares-Erijman, E. A., Jovin, T. M., Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 2004, 22, 198– 203. Winter, J. O., Liu, T. Y., Korgel, B. A., Schmidt, C. E., Recognition molecule directed interfacing between semiconductor quantum dots and nerve cells. Adv. Mater. 2001, 13, 1673–1677. Hoshino, A., Hanaki, K., Suzuki, K., Yamamoto, K., Applications of Tlymphoma labeled with fluorescent quantum dots to cell tracing markers in mouse body. Biochem. Biophys. Res. Commun. 2004, 314, 46–53. Soltesz, E., Kim, S., Laurence, R., DeGrand, A., Parungo, C., Dor, D., Cohn, L., Bawendi, M., Frangioni, J., Mihaljevic, T., Intraoperative sentinel lymph node mapping of the lung using near-infrared fluorescent quantum dots. Ann. Thoracic Surgery 2005, 79, 269–277. Parungo, C. P., Colson, Y. L., Kim, S. W., Kim, S., Cohn, L. H., Bawendi, M. G., Frangioni, J. V., Sentinel lymph node mapping of the pleural space. Chest 2005, 127, 1799–1804. Chen, J., Saeki, F., Wiley, B. J., Cang, H., Cobb, M. J., Li, Z. Y., Au, L., Zhang, H., Kimmey, M. B., Li, X. D., Xia, Y., Gold nanocages: Bioconjugation and their potential use as optical imaging contrast agents. Nano Lett. 2005, 5, 473–477. van de Rijke, F., Zijlmans, H., Li, S., Vail, T., Raap, A. K., Niedbala, R. S., Tanke, H. J., Up-converting phosphor reporters for nucleic acid microarrays. Nat. Biotechnol. 2001, 19, 273–276.

248 Zijlmans, H. J. M. A. A., Bonnet, J.,

249

250

251

252

253

254

Burton, J., Kardos, K., Vail, T., Niedbala, R. S., Tanke, H. J., Detection of cell and tissue surface antigens using up-converting phosphors: A new reporter technology. Anal. Biochem. 1999, 267, 30–36. Soukka, T., Harma, H., Paukkunen, J., Lovgren, T., Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticleantibody bioconjugates. Anal. Chem. 2001, 73, 2254–2260. Soukka, T., Antonen, K., Harma, H., Pelkkikangas, A. M., Huhtinen, P., Lovgren, T., Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology. Clin. Chim. Acta 2003, 328, 45–58. Soukka, T., Paukkunen, J., Harma, H., Lonnberg, S., Lindroos, H., Lovgren, T., Supersensitive timeresolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology. Clin. Chem. 2001, 47, 1269–1278. Alyaudtin, R. N., Reichel, A., Lobenberg, R., Ramge, P., Kreuter, J., Begley, D. J., Interaction of poly(butylcyanoacrylate) nanoparticles with the blood-brain barrier in vivo and in vitro. J. Drug Targeting 2001, 9, 209–221. Coester, C. J., Langer, K., Von Briesen, H., Kreuter, J., Gelatin nanoparticles by two step desolvation – A new preparation method, surface modifications and cell uptake. J. Microencapsulation 2000, 17, 187–193. Josephson, L., Kircher, M. F., Mahmood, U., Tang, Y., Weissleder, R., Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes. Bioconjugate Chem. 2002, 13, 554–560.

85

86

3

Nanogold in Cancer Therapy and Diagnosis Priyabrata Mukherjee, Resham Bhattacharya, Chitta Ranjan Patra, and Debabrata Mukhopadhyay 3.1

Introduction

Cancer is the second leading cause of death in the United States. Nearly half of all men and a little over one-third of all women in the United States will develop cancer during their lifetimes. Today, millions of people live with cancer or have had cancer. The uncontrolled division of cells is termed as cancer. Cancer cells are different from normal cells in that they continue to grow, divide and form new abnormal cells. Cancer cells usually accumulate in an organ to form solid tumors; however, non-tumor forming cancer of the fluid connective tissue, blood, also occurs such as leukemia. Cancer cells can travel to other parts of the body from their primary site of growth and this is called metastasis. Cancer can grow in various sites in the human body such as bladder, breast, colon, kidney (renal cell), lung, skin (melanoma), pancreas, prostate and thyroid [1]. Therapies differ considerably, depending upon the site and stage of tumor formation. Besides conventional treatments such as surgery, chemotherapy and radiation several other alternative approaches have been examined, such as laser therapy, photodynamic therapy, gene therapy, stem cell transplantation and anti-angiogenic therapy [2–5]. Due to the highly heterogeneous nature of the disease, the main challenge to cancer therapists today is to deliver drugs that can be specifically targeted to the different ‘‘hallmarks’’ of cancer growth. Unfortunately, common anticancer as well as new generation of anti-angiogenic or anti-stromal agents have several limitations: they are usually associated with high toxicity; several of them are nonspecific and target both normal as well as malignant cells; have poor bioavailability; and last but not least, they have poor half-lives and fast clearance from the body. An ideal therapeutic approach in cancer would be to deliver multi-drugs specifically to the primary tumor, as well as to the site of metastasis and its microenvironment while simultaneously monitoring the prognosis through noninvasive approaches.

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

3.2 Medicinal use of Gold: A Historical Perspective

Recently, several groups, including ours, have shown that nanoparticles possess enormous potential to improve the efficacy of cancer treatment [6]. Hence, our long-term goal is to develop nanotherapeutics with multifunctional capabilities. This method of drug administration might significantly reduce the dosage of the drugs and this in turn may lead to better specificity, low toxicities, better bioavailability, in a targeted manner with real-time detection by non-invasive imaging. Our recent published and preliminary data support at least part of this hypothesis, as we have shown that when conventional anticancer drugs are loaded with goldnanoparticles alone or in combination with other drugs such as an anti-angiogenic agent, the efficacy of each drug was intact or even better when tested in respective assays [7–9]. Our goal is to put together all the components to develop ‘‘smart’’ drug(s) that will target all the ‘‘hallmarks’’ of cancer growth and thereby inhibit tumor progression and metastasis. In this chapter, we emphasize potential targets of the angiogenesis component of the tumor and discuss the current standing with respect to nanotechnology.

3.2

Medicinal use of Gold: A Historical Perspective

Gold has a long history of use [10, 11]. The therapeutic use of gold can be traced back to the Chinese in 2500 bc. They were the first to prepare and use red colloidal gold as the ‘‘drug of longevity.’’ Red colloidal gold is still in use today in India in the form of Ayurvedic medicine for rejuvenation and revitalization during old age under the name of Swarna Bhasma (‘‘Swarna’’ meaning gold, ‘‘Bhasma’’ meaning ash) [12]. Mahdihassan has explored the historical use of gold in eastern traditions, especially in India and China [13]. In India cinnabar-gold is known as ‘‘Makaradhwaja’’. Makaradhwaja means emblem of god of fertility, a drug for vigor of youth [14]. Gold also has a long history of use in the western world as nervine, a substance that could revitalize people suffering from nervous conditions [12]. In the 16 th century gold was recommended for the treatment of epilepsy [15]. At the beginning of the 19 th century gold was the drug of choice for the treatment of syphilis [16]. Several books describing the medicinal use of gold came out at the beginning of 19 th century [17–21]. The discovery by Robert Koch of the bacteriostatic effect of gold cyanide towards the tubercle bacillus marked the beginning of the modern day medicinal use of gold. Following Koch’s discovery, gold therapy for tuberculosis was introduced in the 1920s [22]. The major clinical uses of gold compounds are in the treatment of rheumatic diseases, including psoriasis, juvenile arthritis, palindromic rheumatism and discoid lupus erythematosus [23]. Gold compounds that were mainly used for the treatment of rheumatoid arthritis were gold thiolates (AuSR), where gold is unipositively charged. Sodium aurothiomalate and aurothioglucose are two prime examples of gold thiolates mainly used for rheumatoid arthritis. These drugs are water soluble and administered as deep intramuscular injection. Follow-

87

88

3 Nanogold in Cancer Therapy and Diagnosis

ing such injection these drugs get rapidly adsorbed and, at the same time, gold is rapidly cleared from the circulation and distributed to various organs such as kidney, liver, spleen and so on [22]. The adsorption of gold in the kidney causes nephrotoxicity, a major side effect. Other toxic reactions include mouth ulcers, skin reactions, blood disorder and liver toxicity [23–25]. To overcome the poor pharmacokinetics and toxicity, a second-generation gold drug, auranofin, was introduced in 1985 for arthritis [26–29]. The presence of phosphine ligands makes auranofin more lyophilic with better retention in the circulation. The adsorption of gold in the kidney was also reduced, with significant reduction in the nephrotoxicity. The antitumor activity of cis-platin was discovered in 1969, prompting the discovery of other metal-containing antitumor drugs. Gold has also been included in the search on the basis of three rationales [30–33]: (a) both Pt(ii) and Au(iii) form analogous square-planar complexes with a d 8 configuration of the central ions, (b) analogy to the immunomodulatory effects of gold(i) antiarthritic agents, and (c) complexation of known antitumor agents with gold(i) and gold(iii) to produce compounds with enhanced activity. Auranofin showed activity against HeLa cells in vitro and P388 cells in vivo. This discovery led to the screening of many phosphinecontaining gold drugs, of particular interest are bis(diphos)gold(i) complexes [34, 35]. These complexes showed promising antitumor properties but exhibited cardiovascular toxicity that precluded their use in clinical trials. The mechanism of action of these gold drugs is poorly understood. However, it is thought that, under biological conditions, gold(i) and gold(iii) species are reduced to gold(0), which may be the active species. Hardly any report describes the use of gold nanoparticles as anticancer agent. However, recently we have shown, for the first time, the anti-angiogenic property of gold nanoparticles.

3.3

Application of Gold Nanoparticles in Cancer

The biological application of gold nanoparticles began almost three decades ago in the form of immunogold staining procedure. Since then, gold nanoconjugates have been extensively used to detect cellular components using electron microscopy as an indirect passive component. However, the use of these gold nanoparticles for in vivo drug delivery has never been described. This section discusses the application of ‘‘naked’’ gold nanoparticles as well as in the form of their nanoconjugates in the treatment of cancer. 3.3.1

Angiogenesis and Cancer

Angiogenesis, the formation of new blood vessels from existing one’s, plays an important role in the growth and spread of cancer. New blood vessels ‘‘feed’’ the cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby

3.3 Application of Gold Nanoparticles in Cancer

tissue, spread to other parts of the body, and form solid tumors [36]. In the early 1970s Dr. J. Folkman first showed that solid tumors are angiogenic and require vascularization for growth [37, 38]. The central concept that tumor growth is ‘‘angiogenesis dependent’’ is well accepted today, with more than 2500 scientific reports showing angiogenesis linked to tumor growth. Angiogenesis is tightly regulated by a balance between endogenous proangiogenic growth factors like Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PLGF), Platelet Derived Growth Factor (PDGF), Tumor Growth Factor-beta (TGF-b) and Angiopoietin-1 (Ang-1) and antiangiogenic factors like thrombospondin-1 (TSP-1), somatostatin, endostatin, angiostatin, interleukins, interferons and tissue inhibitors of matrix metalloproteinases (TIMPs) [39, 40]. In this chapter we briefly discuss angiogenesis in relation to cancer to provide a basis for understanding the fabrication and targets for nanodrug synthesis and use. For a detailed review on angiogenesis and cancer please refer to Refs. [1–44]. The angiogenic response in the microvasculature is associated with changes in cellular adhesive interactions between adjacent endothelial cells (ECs), pericytes, fibroblasts, and immune mediators express many different cytokines and growth factors that react with other cells or extra cellular matrix (ECM) components to affect EC migration, proliferation, tube formation, and vessel stabilization. During tumor growth this balance is disrupted and the scale tips towards the tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on ECs (Fig. 3.1). Folkman has propounded the idea that inhibiting angiogenesis could inhibit the growth of tumors [41]. Since then considerable effort has been invested in the discovery of agents that block the development of tumor vasculature. Yet only recently have the differences between the vasculature of the normal tissue and the tumor been realized. The tumor vasculature is strikingly tortuous, vessels have poor pericyte cover and numerous shunts, making it often difficult to distinguish between arterioles and venules. Blood flow through the tumor capillaries is sluggish and sometimes stationary, leading to the entire microenvironment, including red blood cells and endothelium, being highly hypoxic [42–44]. Although the idea of targeting and disrupting tumor vasculature as an anticancer therapy has been around for sometime, experimental evidence of efficacy was absent until Burrows et al., targeted the toxin ricin to the tumor vasculature in a mouse neuroblastoma model [45]. Since then several different antiangiogenic agents have been developed and used to treat cancer. These include drugs that directly target specific molecules involved in neo-vessel formation, such as antibodies to VEGF, or others that indirectly inhibit endothelial cell function like inhibitors of matrix metalloproteinase (MMP) breakdown [46, 47]. Other strategies include the use of cytotoxic drugs such as thalidomide that appear to have both antiangiogenic as well as tumor cell killing properties; however, the specific mechanism of action of these drugs is not known [48, 49]. Camptothecin analogs, 9-amino-20(S)camptothecin, topotecan, CPT-11, are inhibitors of topoisomerase I and also decrease tumor angiogenesis [50]. Paclitaxel, a microtubule inhibitor, has shown anti-proliferative action in in vivo models [51, 52].

89

90

3 Nanogold in Cancer Therapy and Diagnosis

Schematic diagram showing the role of angiogenesis in tumor growth and blood vessel formation.

Fig. 3.1.

Agents that Inhibit Endothelial Proliferation or Response Such targets include endoglin, integrins, dominant negative receptors and agents that prevent the release or activation of FGF. Direct administration of endogenous angiogenic inhibitors such as angiostatin, endostatin, and gene therapy using DNA that encodes for angiogenesis inhibitors, including angiostatin and platelet factor 4, are being evaluated. Administration of synthetic angiogenic inhibitors that specifically prevent endothelial cell division include derivatives of fumagillin, such as TNP-470 [53, 54]; combrestatin phosphate, which induces apoptosis in proliferat3.3.1.1

3.3 Application of Gold Nanoparticles in Cancer

ing endothelial cells and causes tubulin destabilization [55]; and vitaxin, which is a humanized monoclonal antibody to alpha5-beta3 integrin present on endothelial cell surface, and EMD 121974 a small molecule blocker of alpha5-beta3 integrins [56–58]. Agents that Block Activation of Angiogenesis Interferon alpha has demonstrated a 30% response rate in patients with AIDSrelated Kaposi sarcoma and has been shown to be active in the treatment of hemangiomas, chronic myeloid leukemia, myeloma, melanoma, lymphoma and renal cell carcinoma [59–61]. SU5416, a small molecule blocker of VEGF-receptor2 signaling, is in Phase II clinical trial for metastatic colorectal cancer, non-small cell lung cancer and von Hippel-Lindau disease [62]. SU6668, a small molecule blocker of VEGF, FGF and PDGF receptor signaling, is in Phase I trial for selected advanced tumors [63]. Humanized monoclonal antibody to VEGF is now in clinical trials for several cancers, including metastatic renal cancer, advanced prostate cancer, non-small cell lung cancer, colorectal cancer and other solid cancers [46]. 3.3.1.2

Agents that Block Extracellular Matrix Breakdown Clinical trials have been ongoing for seven MMP inhibitors. Marimastat, a synthetic inhibitor that blocks TNF-a convertase, has shown clinical activity when combined with chemotherapy in colorectal, ovarian, prostate, gastric and pancreatic cancer [64]. Phase I studies with AG3340 in combination with other chemotherapeutic agents have been well tolerated in patients with advanced prostate and other solid tumors [65, 66]. Bay12-9566 and MM1270, synthetic MMP inhibitors, have undergone Phase I trials as single agents in pancreatic, ovarian and colorectal cancers [67, 68]. Although studies with antiangiogenic molecules have been elegant and results encouraging yet these advances should be viewed with cautious optimism as side effects that occur in some patients include hypertension, thrombosis, proteinuria, and even fatal hemorrhage. Therefore, selectivity, delivering drugs to specific targets on the tumor endothelium, has remained an obstacle in the development of better antiangiogenic therapy. Gold nanoparticles provide an avenue for targeted delivery of bioactive molecules to the tumor microenvironment by means of binding molecules such as antibodies that are specific for tumor-associated markers. Recent reports have demonstrated the potential utility of gold nanoparticles that served as a payload for delivery of DNA, proteins and imaging compounds to the tumor. 3.3.1.3

Unique Anti-angiogenic Properties of Gold Nanoparticles Gold nanoparticles have been extensively used in biological applications due to their biocompatibility [69], dimensions (<50 nm), ease of characterization [70, 71] and their rich history of surface chemistry that can be easily exploited to better suit the needs of biomedical applications [72]. VEGFs are mitogenic for vascular endothelial cells [73] and act as potent angiogenic factors and blood vessel permeabilizing agents [73–76] in vivo. Four isoforms of VEGF containing 121, 165, 189 and 3.3.1.4

91

92

3 Nanogold in Cancer Therapy and Diagnosis

206 amino acids are produced from a single gene as a result of alternate splicing [76]. The 121 amino acid form of VEGF induces the proliferation of endothelial cells but, in contrast to VEGF 165, lacks the heparin binding ability [77]. Many anti-angiogenic approaches have been developed to block the interaction of VEGFs with their receptors in order to inhibit angiogenesis and tumor growth that include monoclonal antibodies targeting VEGF [78, 79] and the use of soluble decoy receptors blocking the binding of VEGF to its receptors [80]. We have shown, for the first time, that gold nanoparticles bind and inhibit the activity of Vascular Permeability Factor/Vascular Endothelial Growth Factor 165 (VPF/VEGF-165), an endothelial cell (ECs) mitogen and a prime mediator of angiogenesis that plays a tremendous role in pathological neo-vascularization, including rheumatoid arthritis, chronic inflammation and neoplastic disorders. Gold nanoparticles used in this study were prepared by the reduction of aqueous chloroaurate ions with sodium and characterized using UV/Visible spectroscopy (UV/Vis), because of the presence of characteristic surface plasmon resonance band (Fig. 3.2), and transmission electron microscopy (TEM). The TEM image clearly showed that gold nanoparticles @5 nm in diameter were formed by this method (Fig. 3.3). Gold Nanoparticles Inactivate VEGF165 Whether gold nanoparticles had any effect on VEGF165 was tested on VEGf165induced HUVEC proliferation. VEGF165 was pre-incubated with various concentrations of gold nanoparticles (67, 335, 670 nm) overnight at 4
C and then added them to serum-starved HUVECs followed by [ 3 H]-thymidine incorporation. Gold nanoparticles inhibited VEGF165-induced proliferation ( p < 0:0001). However, 3.3.1.5

UV/Vis spectrum of gold nanoparticles obtained by sodium borohydride reduction of tetrachloroauric acid.

Fig. 3.2.

3.3 Application of Gold Nanoparticles in Cancer

Transmission electron micrograph of gold nanoparticles obtained by borohydride reduction of gold salts.

Fig. 3.3.

gold nanoparticles did not inhibit non-heparin binding VEGF121-induced proliferation of HUVEC cells. Importantly, the gold nanoparticles were not toxic to HUVECs, where no inhibition in proliferation was observed in the only nano-gold treated samples compared to the control. Numerous examples of surface modifications of gold nanoparticles with alkanethiols aromatic thiols, and primary amines have been reported [81–85]. Such an ionic/pseudo-covalent reaction may explain the interaction between the gold nanoparticles and VEGF165 that led to the inhibition of its activity. However, the inability of gold nanoparticles to inhibit the activity of VEGF121 suggested that the presence of a heparin-binding domain was necessary to be inactivated by gold nanoparticles. What is the Mechanism of Action? To prove that gold nanoparticles bind to the heparin-binding domain of VEGF165, gold nanoparticles at different concentrations (335, 670 and 1340 nm) were preincubated with VEGF165 overnight at 4
C. VEGF165 was then precipitated from this complex with a saturating concentration of heparin-sepharose. In the absence of gold, all VEGF165 was bound to heparin-sepharose and was detected only in the precipitated fraction while none was detected in the supernatant fraction. Heparin sepharose binds to the heparin binding domain of a heparin binding protein and precipitates it from the solution. The results of the pre-incubation experiments with heparin sepharose conclusively proved that VEGF165 in the supernatant fraction was in the gold-bound form and thus could not interact with the heparin-sepharose. From these results, it was concluded that gold nanoparticles inhibited VEGF165 from binding to heparin-sepharose because gold itself binds to VEGF165 through the heparin-binding domain. Signaling events of VEGF165 leading to proliferation were initiated by its association with cell surface receptors, mainly KDR, where the heparin-binding domain plays an important role. Pre3.3.1.6

93

94

3 Nanogold in Cancer Therapy and Diagnosis

incubation experiments with heparin sepharose clearly proved that blocking the heparin-binding domain of VEGF165 gold nanoparticles inhibited its association with KDR and, thereby, inhibited its activity. Direct binding of VEGF165 to gold nanoparticles was confirmed by XPS analysis. The presence of a single Au 4f7=2 peak at 83.9 eV clearly demonstrated only one form of Au present in solution and that it is Au(0) [86]. The presence of two sulfur peaks, at 162.7 and 167.1 eV, represented two chemically distinct sulfur species. The peak at 162.7 eV was assigned to gold-bound sulfur and that at higher BE was assigned to oxidized sulfur species such as in sulfones. The nitrogen 1s peak at 399.6 eV was likely due to unionized, non-protonated nitrogen [87]. However, the possibility of gold nanoparticles binding to VEGF165 through nitrogen as well could not be ruled out. Hence it was concluded that the direct binding of VEGF165 to gold nanoparticles occurred through sulfur and/or nitrogen, both present in the heparin-binding domain of VEGF165. We have described earlier that angiogenesis plays a central role in the growth and progression of tumor [69, 70]. This process is also important for the promotion and maintenance of other diseases like neoplasia and rheumatoid arthritis [71]. As there are several reports that indicate that gold salts can retard the progression of rheumatoid arthritis [72], we reasoned that gold nanoparticles might also inhibit angiogenesis. Because VEGF/VPF [73, 74] and bFGF [75] are two critical cytokines for the induction of angiogenesis, we investigated whether nontoxic novel gold nanoparticles [76] being used at present in several biomedical applications can inhibit the functions of these two important proangiogenic growth factors. 3.3.1.7 Effect of Gold Nanoparticles on the Activity of VEGF165, VEGF121, bFGF and EGF The effects of gold nanoparticles on the activity of VEGF165, VEGF121, bFGF and EGF were tested on two different cell lines, namely HUVEC (for VEGF165 and VEGF121) and 3T3 (for EGF and bFGF). The gold nanoparticles inhibited VEGF165-induced proliferation of HUVECs- and bFGF-induced proliferation of 3T3 cells but did not inhibit either VEGF121- or EGF-induced proliferation. Gold nanoparticles are not toxic to either cell line as no inhibition in proliferation was observed in the only nanogold treated samples compared with the control. The inhibition of proliferative activity of two heparin binding growth factors, namely VEGF165 and bFGF, by nanogold and its inability to inactivate non-heparin binding growth factors, namely VEGF121 and EGF, clearly suggested that the heparinbinding domain of VEGF165 and bFGF played the crucial role for their interactions with nanogold and, hence, inactivation.

Effect of Gold Nanoparticles on Signaling Events of VEGF165 With the addition of 335–670 nm nanogold, VEGF165-induced phosphorylation of VEGFR-2 was profoundly inhibited. However, at 67 nm nanogold @40% inhibition of phosphorylation was evident [ from densitometry quantitation using NIH Image software, p < 0:0001]. Almost complete inhibition of VEGFR-2 phosphory3.3.1.8

3.3 Application of Gold Nanoparticles in Cancer

lation was observed at concentrations of 335 and 670 nm nanogold. The results of receptor phosphorylation clearly suggested that nanogold binds directly to VEGF165 and inhibited its interaction with cell surface receptors hence inhibiting phosphorylation. Effect of Nanogold on Downstream Signaling events of VEGF165 To further support the hypothesis that nanogold binds to VEGF165 and inactivates its signaling capability, an intracellular calcium release experiment in HUVECs was performed. A gold concentration of 67 nm gave @34% inhibition (determined by comparing the length of the upstroke of VEGF165 induced sample to the nanogold-treated samples) and complete inhibition was observed at 335–670 nm ( p < 0:05). The VEGF receptors on the HUVECs were still functional as evidenced by an increase in calcium release comparable to the control when VEGF was added after 300 s. These observations also clearly demonstrated that gold nanoparticles bind directly to VEGF165 and inhibited its signaling events but did not perturb the receptor functions. 3.3.1.9

Effect of Gold Nanoparticles on Migration of HUVEC Cells Nanogold inhibited VEGF165-induced migration of HUVECs. Rho A activity was also completely inhibited at 670 nm nanogold, further confirming the hypothesis that nanogold binds directly with the heparin binding growth factor through the heparin binding domain and inhibits its signaling activity. 3.3.1.10

Effect of Gold Nanoparticles on Angiogenesis in vivo The efficacy of nanogold in inhibiting VEGF165-induced permeability and angiogenesis in vivo was tested in a nude mouse ear model. Ad-VEGF injected mice treated with nanogold developed lesser edema than mice treated with Ad-VEGF only. As shown, 30 min after injection of Evan’s blue dye into the tail vein of these treated mice, a decrease in permeability was also observed. In the MOT model, less fluid accumulation in the peritoneal cavity was observed in nanogold treated samples than in controls. Taken together, our results showed that gold nanoparticles selectively inhibited VEGF165- and bFGF-induced proliferation of HUVECs and fibroblasts, respectively. In a similar fashion, nanogold also inhibited the activity of placental growth factor (PlGF) (unpublished results). Gold nanoparticles directly bind heparinbinding growth factors, presumably through cysteine residues of the heparinbinding domain, and inhibit growth factor mediated signaling. Gold binds strongly with thiols and amines [85–87]. Such an ionic/pseudo-covalent reaction may explain the interaction between the gold nanoparticles and VEGF that led to a decrease in its proliferative activity. Gold nanoparticles also inhibit VEGF-induced angiogenesis and permeability in vivo as well as causing lesser ascites fluid accumulation in a MOT model where VEGF/VPF activity is primarily responsible for fluid accumulation in the peritoneal cavity. Here, we mention, briefly, the toxicity of nanogold. Because metal poisoning is associated with renal and hepatic toxicities, we determined the effect of nanogold administration on liver and renal func3.3.1.11

95

96

3 Nanogold in Cancer Therapy and Diagnosis

tion tests. Normal mice were injected with same dose of nanogold as in MOT model intraperitoneally for 7 consecutive days. On day 8, the mice were sacrificed and serum was collected. There were no significant differences between serum levels of creatinine, blood urea nitrogen, bilirubin alkaline phosphatase, ALT (Alanine Aminotransferase) and AST (Aspartate Aminotransferase) between the nanogold treated and untreated control animals. This finding is significant because growth factor-mediated proliferation and angiogenesis have a central role in many pathological conditions, including neoplasia, rheumatoid arthritis and chronic inflammation. The low production cost and relative ease in modifying nanogold make them a feasible choice in future biomedical applications. Gold Radioisotopes in Cancer Treatment Radioisotopes of gold have also long been used for the treatment of different types of cancer. A cumulative survival of 96.3% was achieved in the treatment of stage Ia ovarian cancer with radiogold [88]. In addition, Ehrlich ascites tumor has been prevented by intraperitoneal injection of colloidal 198 Au [89]. In the treatment of limited epithelial carcinoma of the ovary, 104 patients received intraperitoneal radiocolloid [90]. Fifty six of these patients also received external beam radiation therapy to the pelvis. Five year actuarial no-evidence-of-disease survival rates were 95% for stage Iai, 82% for stage Iaii, 73% for stage Ib, 67% for Ic, 67% for Iia, 67% for Iib without gross residual tumor (GRT) and so on. Radioactive gold has also been used to prevent hepatic metastasis by intravenous administration [91]. Recently, gold nanoparticles have been used to increase radiotherapy in mice [92]. Mice bearing subcutaneous EMT-6 mammary carcinomas received a single intravenous injection of 1.9-nm gold particles (up to 2.7 g-Au per kg body weight). The gold content of the tumor elevated by this treatment to 7 mg-Au per g in tumor. The mice were then subjected to 250 kVp X-ray therapy for several minutes. The one-year survival rate was found to be 86% versus 20% with X-rays alone and 0% with gold alone. 3.3.1.12

3.3.2

Application of Gold Conjugates in the Treatment of Cancer

The biological application of gold nanoparticles began in 1971 when Faulk and Taylor invented the immunogold staining procedure [93]. Since then gold nanoconjugates have been extensively used to detect cellular components using electron microscopy. Mostly gold nanoconjugates were seen as passive components, used only to visualize different cellular components. However, the use of these gold nanoparticles for in vivo drug delivery has never been described. Recently, gold nanoconjugates have been used as more active components that can interfere with the biological activities [94–98]. Gold–TNF Conjugate in Cancer Therapeutics Recently, colloidal gold, a sol composed of nanoparticles of Au 0 , has been used as a therapeutic for the treatment of cancer as well as an indicator for immunodiagnos3.3.2.1

3.3 Application of Gold Nanoparticles in Cancer

tics. However, the use of these gold nanoparticles for in vivo drug delivery has not been described. The development of a colloidal gold (cAu) nanoparticle vector that targets the delivery of tumor necrosis factor (TNF) to a solid tumor growing in mice has been reported [99]. The optimal vector, designated PT-cAu-TNF, consists of molecules of thiol-derivatized PEG (PT) and recombinant human TNF that are directly bound onto the surface of the gold nanoparticles. Following intravenous administration, PT-cAu-TNF rapidly accumulates in MC-38 colon carcinoma tumors and shows little to no accumulation in the livers, spleens (i.e., the RES) or other healthy organs of the animals. Tumor accumulation was evidenced by a marked change in color of the tumor as it acquired the bright red/purple of the colloidal gold sol and was coincident with the active and tumor-specific sequestration of TNF. Finally, PT-cAu-TNF was less toxic and more effective in reducing tumor burden than native TNF since maximal antitumor responses were achieved at lower doses of drug. ‘‘2 in 1’’ System in Cancer Therapeutics Recently, our group has reported the fabrication of a ‘‘2 in 1’’ system containing functional anti-angiogenic agent and cytotoxic drug. Angiogenesis, the formation of new blood vessels from a pre-existing vessel, is a necessary step for tumor growth and metastasis [99–102]. This provides the basis for anti-angiogenic therapy – depriving tumor cells the nutrients essential for their growth by blocking their blood supply. Although this treatment may restrict tumor growth, antiangiogenic therapy alone might not be enough to prevent tumor growth and cause increased cell killing. Therefore, to achieve maximal therapeutic benefit it is necessary to use anti-angiogenic agents in combination with other modalities such as anticancer drugs [103]. One of the problems in the use of anticancer drugs is that they have short half-lives, sometimes with extensive systemic toxicity [104]. When delivered in a gold-conjugated form, these molecules might have increased retention in the body with reduced systemic toxicity. VEGF-antibody attached on gold nanoparticles may facilitate delivery of the drugs to the site of tumor, as many tumors are known to produce VEGF [105, 106]. We hypothesize that use of a cytotoxic agent such as gemcitabine along with VEGF antibody will not only allow tumor cell killing but also give rise to more ‘‘normalized’’ blood vessels, resulting in better drug delivery [107]. This study employed a nano-composite system consisting of a nanogold core, bearing a functional anti-angiogenic molecule, VEGF antibody-2C3 (AbVF), and an anticancer drug, gemcitabine. This provides a unique ‘‘2 in 1’’ system where two components with different functions have been attached onto a single gold core, yet the functional activity of the individual components are retained. To attach multiple components onto a single gold core, it is essential to determine the saturation concentration of the individual components [108]. The saturation concentration of AbVF was determined after incubating it with gold nanoparticles with increasing concentrations of AbVF for 30 min followed by treatment with 100 mL of 10% NaCl solution to test for aggregation [109]. Gemcitabine was similarly attached to nanogold. Without any protection, gold nanoparticles aggre3.3.2.2

97

98

3 Nanogold in Cancer Therapy and Diagnosis

gate immediately upon addition of a 10% NaCl solution. The extent of aggregation, however, should decrease with increasing protection of the nanogold surface either by antibody or by gemcitabine. Increased stabilization of nanogold dispersions was observed with increasing concentrations of AbVF. In the absence of antibody but in the presence of NaCl, gold nanoparticles completely aggregated and, as expected, their absorbance dropped to zero [109]. Next, the saturation concentration of gemcitabine on gold nanoparticles was determined, similarly as above. Gold nanoparticles were incubated with different concentrations of gemcitabine for 30 min and their UV/Vis spectra were recorded. Absorbance of nanogold gradually increased with increasing concentrations of gemcitabine until it reached a maximum at 20 mg mL1 . According to the Mie theory, the observed shift in Ymax with an increase in plasmon resonance coincides with a rising dielectric constant of the medium surrounding the gold nanoparticles [110]. At doses higher than 20 mg mL1 , the absorbance of the nanocomposite gradually decreased, alongside a gradual increase in redshift in Ymax . This relationship, along with a broadening of the spectra, may be attributed to the aggregation of gold nanoparticles upon the addition of gemcitabine beyond the 20 mg mL1 limit. To attach both gemcitabine and AbVF on nanogold surface, incubation experiments were carried out below the 50% saturation concentration for AbVF and gemcitabine. Gold nanoparticles were first incubated for 30 min at room temperature with 10 mg mL1 of gemcitabine followed by another 30 min incubation with 2 mg mL1 of VEGF antibody. Aggregation tests were performed to confirm the attachment of both the components. The functional activities of gemcitabine and AbVF were tested by in vitro cell culture assays. The activity of gemcitabine was tested on 786-O cells using a BrdU proliferation assay. The observed activity of gemcitabine combined with AbVF on nanogold surfaces was comparable to gemcitabine alone at the same concentration. In the control experiments with gold or AbVF alone or gold-AbVF, no inhibition 786-O proliferation was observed. Therefore, the inhibition in proliferation was due to the presence of gemcitabine within the nanocomposite and the presence of AbVF on the same nanocomposite did not exert any negative influence on the activity of gemcitabine. The functional activity of AbVF was determined on VEGF165 induced calcium release in HUVEC cells. HUVECs release calcium into the cytoplasm when induced with VEGF165 [6]. AbVF attached to nanogold surfaces with gemcitabine inhibited calcium release more efficiently than the same dose of AbVF alone. At 10 ng mL1 , almost no inhibition of VEGF165-induced calcium release was observed in the samples of AbVF alone but a moderate inhibition and delay was observed with AbVF in the nanocomposite form. At 20 ng mL1 , moderate inhibition was observed in samples with only AbVF but complete inhibition of calcium release was observed for the nanocomposite. This suggested an optimal threshold concentration requirement for VEGF to be able to induce calcium release. To further confirm that the inhibition in calcium release was due to AbVF, the nanogold surface was blocked with gemcitabine and control mouse IgG. VEGF165-induced calcium release was not inhibited by this nanocomposite. From

3.4 Biocompatibility of Gold Nanoparticles

the calcium release assay, it was concluded that AbVF retained its functional activity in the AbVF–gold–gemcitabine nanocomposite form and gemcitabine did not had no negative influence on the activity of VEGF antibody. This study was a unique example of a ‘‘2 in 1’’ system where anti-angiogenic molecule, VEGF antibody and cytotoxic drug (gemcitabine) were attached on the same nanogold surface, with potential implication in cancer therapeutics.

3.4

Biocompatibility of Gold Nanoparticles

The biocompatibility of nanoparticles in general is discussed in detail elsewhere [110–114]. Here, recent efforts addressing the issue related to gold nanoparticles will be discussed briefly. The issue of biocompatibility arises when any foreign body is introduced inside the human body for medical purposes or otherwise [111–114]. In most general terms biocompatibility can be defined as ‘‘the ability of a material to perform with an appropriate host response in a specific application’’ or ‘‘the exploitation by materials of the proteins and cells of the body to meet a specific performance goal’’. Interactions between the foreign body and the host can be classified in four general categories (a) cellular adhesion effects, (b) local biological effects, (c) systemic and remote effects, and (d) effects of the host on the implant. 3.4.1

Cellular Adhesion Effects

The interaction of the foreign body, direct or indirect, weak or strong, specific or non-specific, receptor mediated or non-mediated with the host may influence the biocompatibility of the foreign body. 3.4.2

Local Biological Effects

Local biological effects include cell viability, cell mitotic function such as proliferation, cell cycle phases, etc., plasma membrane integrity, toxicity and modification or normal wound healing. 3.4.3

Systemic and Remote Effects

Systemic and remote effects include systemic toxicity (such as kidney failure, liver failure, etc.), elevation of unusual components in the blood, allergic, pyrogenic, carcinogenic and teratogenic responses.

99

100

3 Nanogold in Cancer Therapy and Diagnosis

3.4.4

Effects of the Host on the Implant

This mainly includes immune responses, such as inflammation, fibrosis, etc., around the implant, physical and mechanical effects, stability and biological degradation processes. 3.4.5

Addressing the Biocompatibility of Gold Nanoparticles using DNA Microarray Analysis

Nanotechnology is a rapidly evolving multidisciplinary branch of science involving systems and/or device manufacturing at the nano-scale level [115, 116]. Nanoparticle-aided strategies have been implemented to address a diverse array of issues, including drug delivery, targeting and monitoring of molecules/cells/ tissues, and development of novel drug regimens [117–119]. A modified variety of nanoparticles, known as quantum dots, which are highly fluorescent semiconductor particles measuring roughly the size of an average protein, have been developed and shown to be valuable for monitoring metastatic tumor cells in an animal model [120]. These fluorescent quantum dots are reported to be active for over a month inside the body, allowing one to monitor migration and localization of the probe over a long period of time [121, 122]. Conceptually, nanoparticles can have effects, both desired and unintended, on an organism through acting directly and/or indirectly on individual cells. Since all investigations employing nanotechnology were performed related to specific questions, efforts to assess potential global effects remain virtually unanswered. However, some recent reports involving nanotechnology have generated widespread concerns regarding the effect of nanoparticles on the ecosystem and ultimately to human health [123–125]. Several recently published papers showed the toxicological effects of carbon nanotubes [126, 127]. It is, however, not known if all nanoparticles have similar biological effects; it is also not known if the same nanoparticle will have the same effect on different organisms. In fact, the overall consensus among scientists working on various aspects of nanoparticles is that very little is known about the potential impacts of nanoparticles on health and environment. Evidently, a thorough evaluation of the underlying issues needs to be systematically performed to distinguish reality from mere extrapolation of inadequate data. Metallic gold is known to be biocompatible; however, facts regarding the biocompatibility of gold nanoparticles are rare. Recently efforts have been made to address the biocompatibility issue of gold nanoparticles. Significant damage to the cell membrane was observed by cationically modified gold nanoparticles and they were shown to be moderately toxic, whereas anionic particles were quite non-toxic. A series of gold nanoparticles with various surface modifications were tested recently for uptake and acute toxicity in human leukemia cell lines. The authors concluded that although some precursor of nanoparticles might cause toxicity, the nanoparticles themselves are not toxic, further strengthening our findings [128]. The

3.4 Biocompatibility of Gold Nanoparticles

effects observed in those cases may be due to the presence of the surface modifier and not because of the gold nanoparticles. To find out the effect of bare gold nanoparticles, our laboratory has focused on the genotropic effects of gold nanoparticles on normal human cell lines. We hypothesize that the overall physiology of a cell is equivalent to its global transcriptional profile. Consequently, we speculate that the expression signature of a cell in the presence or in absence of a given nanoparticle can generate valuable information regarding the potential effect of a nanoparticle on the target cell. However, a given nanoparticle can, possibly, interact with cellular components directly and elicit an effect or interact with non-cellular components first, which, in turn, may produce an effect on the target cell (an indirect effect). These so-called ‘‘direct’’ and ‘‘indirect’’ effects, if any, will need to be distinguished to ascertain the full spectrum of effects of nanoparticles on target cells (or on nontarget cells). We have previously shown that gold nanoparticles effectively blocked the effect of vascular endothelial growth factor (VEGF)-mediated signaling by HUVECs (Human Umbilical Vein Endothelial Cells) and also inhibited angiogenesis in vivo in a mouse ovarian tumor model [10]. We have evaluated the potential direct effect of gold ‘‘naked’’ nanoparticles on HUVECs. 3.4.6

Internalization of Gold Nanoparticles by HUVECs

To discern the effect of any nanoparticles on any cell lines, it is important to know the location of the nanoparticles, whether most of them are outside or inside the cells, and to find out if the nanoparticles change size upon internalization. To address the localization of gold nanoparticles, transmission electron microscopy was performed after incubating HUVECs with gold nanoparticles for 1 and 24 h under serum-free conditions and in the presence of serum. TEM clearly demonstrated the internalization of gold nanoparticles at both time points under such conditions, and no significant difference on the distribution of the gold nanoparticles was observed (Fig. 3.4). The size of gold nanoparticles did not alter significantly upon internalization. The distribution of gold nanoparticles under serum-free conditions and in the presence of serum was similar. No significant difference in the distribution of gold nanoparticles at 1 and 24 h was observed, confirming that 1 h incubation was enough for gold nanoparticles to be taken up HUVECs. 3.4.7

Nanogold Particles do not Alter Global Pattern of Transcription by HUVEC Cells under Serum-free Conditions

Since serum contains a host of heparin-binding domain containing proteins, including growth factors, with which nanogold can potentially interact and block their functions [9], which, in turn, can have an (indirect) effect on cells in culture, it was necessary to exclude serum (FBS) in an attempt to evaluate a direct effect of gold nanoparticles on HUVECs. Therefore, cells were first cultured in serumcontaining media until they were about 80% confluent, after which the cells were

101

102

3 Nanogold in Cancer Therapy and Diagnosis

Internalization of gold nanoparticles by HUVECs, (a) internalization under serumfree conditions after 4 h incubation; (b) and (c) higher magnification images of (a).

Fig. 3.4.

(d) Internalization of gold nanoparticles in the presence of serum after 4 h incubation; (e) and (f ) higher magnification images of (d).

washed repeatedly with PBS to get rid of serum. The cells were then incubated in serum-free media for 24 h, after which gold nanoparticles were added and the culture continued for 1 and 24 h, respectively. Total RNA extracted from such cells was subjected to microarray analysis. The expression level of genes determined through microarray was subjected to Scatter plot analysis. The results indicated a strikingly similar pattern of gene expression at both 1 and 24 h by cells cultured with or without nanogold particles. The correlation coefficients were 0.98 for 1 h and 0.97 for 24 h, respectively. These observations strongly attest to the similarities

3.4 Biocompatibility of Gold Nanoparticles

of gene expression, indicating that 5-nm gold nanoparticles have no effect on gene expression by HUVECs. It is possible that a small number of genes truly undergoing differential expression in the presence of gold nanoparticles were missed by the Scatter plot analysis. To eliminate this possibility, the microarray-based expression results were manually checked and seven genes were selected to show expression difference of at least 1.5 fold and having expression values of >300 either for control or for cells treated with gold. RT-PCR was performed for all these genes to validate their mRNA expression status in cells cultured with and without gold nanoparticles. The results showed no significant difference in the mRNA expression levels of any of the seven genes, supporting the observation made through Scatter plot analysis.

3.4.8

Nanogold Particles do not Alter the Global Pattern of Transcription by HUVECs in Near-normal Culture Conditions

The lack of an effect of gold nanoparticles on HUVECs as observed under serumfree conditions could be due to the possibility that cells exposed to serum-free media preclude any potential effect of the gold nanoparticles on cell transcription. To eliminate this possibility, the first step was omitted completely (culturing cells in serum-free media for 24 h before adding gold particles) and confined incubation of cells with gold for up to 4 h. Briefly, cells were first grown in the presence of serum to near confluency, then repeatedly washed with serum-free media, nanogold particles were then added followed by incubation in serum-free media for 1 and 4 h. RNA isolated from cells at indicated times (1 and 4 h after exposure to gold particles) was subjected to microarray analysis as above. Scatter plot analysis of the data showed no effect of gold particles on the global transcriptional pattern of HUVECs even when the cells were not exposed to serum-free conditions for a long time. Correlation coefficients obtained from these experiments were 0.995 for 1 h and 0.994 for the 4 h, further supporting this observation. The data was also manually scanned to identify cases of differentially expressed genes not evident in the Scatter plot analysis. Nine genes were identified based on the criteria mentioned earlier. RT-PCR was performed for these genes to validate their mRNA expression level in cells cultured with and without nanogold for 1 and 4 h. The RT-PCR results did not suggest any difference in the mRNA expression levels of these genes in HUVECs not exposed to serum-free conditions prior to adding nanogold in the culture, supporting the results from Scatter plot analysis. Thus, all of the experiments suggested a lack of a direct effect of gold nanoparticles on the transcriptional program of HUVECs. Conceptually, any agent, physical, chemical or biological, can have an effect on a cell/organism; the magnitude of the effect, however, will depend on a diverse array of known and unknown parameters. Whether such an effect can be measured by

103

104

3 Nanogold in Cancer Therapy and Diagnosis

standard means depends on the resolution and sensitivity of a given method. We have previously shown changes in the level of gene expression due to the effect of sound waves on cultured cells [128, 129]. Thus, measurement of gene expression can document subtle intracellular changes caused by the extracellular environment of a cell. DNA-microarray has rapidly become a useful tool for measuring gene expression at the global level – this is, probably, because the resolution of the microarray can be very high (the one used in this study contains probes for virtually all known and predicted human genes) and the sensitivity is within an acceptable range. However, with such a tool, we did not observe any effect (positive or negative) of gold nanoparticles on the global transcriptional pattern of HUVEC cells. Some genes selected by manual screening of the microarray data appeared to be differentially expressed between HUVEC cells cultured with and without gold nanoparticles. However, repeated RT-PCR failed to document differential expression for any of these genes. Thus, both RT-PCR and microarray-based results strongly suggest a lack of effect of gold nanoparticles on mRNA expression. Recently, we have demonstrated that nanogold particles can not only bind to VEGF but also can inhibit VEGF-dependent HUVEC proliferation and ascites fluid accumulation in a mouse ovarian tumor model, illustrating a unique mechanism of deactivation of heparin binding growth factors by gold nanoparticles on a complex biological process [7]. Note that VEGF has cysteine residues on its heparinbinding domain, which can easily become conjugated with gold nanoparticles through covalent interaction, causing sequestration and/or inactivation of the growth factor [8]. Since, the experiments reported herein were not VEGFdependent, no such indirect effect of nanogold should be expected (in agreement with our previous reports where we did not see any toxic effects of gold nanoparticles on HUVEC). The results presented in this study enabled some important inferences. First, although the results could not rule out subtle effects of 5-nm gold nanoparticles or effects beyond the time points investigated under conditions tested, 5-nm naked gold nanoparticles with no surface modifications may indeed have no direct effect on human cells. Second, gold nanoparticles of smaller size were expected to be more ‘‘active’’ than particles of larger size; however, it would be interesting to see if nanoparticles of different sizes with no surface modification have any significant direct effect on biological processes. Finally, since 5 nm nanoparticles appeared to have no effect (toxic or non-toxic) on HUVECs, such particles may be ideal to realize at least some of the promises of nanotechnology, assuming a similar effect of such particles on other types of cells. Notably, the potential genotropic effects of gold nanoparticles have been evaluated in this study; however, such particles may also exert non-genotropic effects (such as membrane protein cycling) on target cells. Such a possibility will need to be addressed to evaluate comprehensively the direct effect of nanoparticles on cell metabolism. We believe our approach provides a general experimental strategy for screening a diverse array of nanoparticles for their potential global effects on any type of target cells/tissues under diverse experimental conditions.

3.5 Synthetic Approaches to Gold Nanoparticles

3.5

Synthetic Approaches to Gold Nanoparticles

Even though this chapter focuses on the application of nanogold in cancer therapy and diagnosis, it would be incomplete without a description of the synthesis of gold nanoparticles, since the synthesis procedure may effect the properties of the gold nanoparticles and, hence, their application in the treatment of cancer. Here we describe briefly the general synthesis process for the preparation of gold nanoparticles. There are basically two synthetic methods: chemical and physical. 3.5.1

Chemical Methods

In 1857, Faraday first reported the synthesis of gold nanoparticles by reduction of chloroaurate ions by phosphorous in carbon disulfide [130]. Among all the methods that are applied for the reduction of HAuCl4 to gold nanoparticles, the citrate reduction and borohydride reduction methods are most commonly used. The citrate reduction method, first introduced by Turkevitch in 1951, yields AuNPs approximately 20 nm in diameter [131]. Recently, it was shown that the size of AuNPs formed by the citrate reduction method can be controlled by the use of a stabilizer along with the reducing agent and by manipulating the ratio of gold salts to reducing agent/stabilizer [132]. In 1994, Brust and Schiffrin introduced a novel method for the synthesis of thiol-capped gold nanoparticles using sodium borohydride as a reducing agent [133]. Nanoparticles so-formed are thermally stable, air stable, with reduced dispersity and a size ranging from 1.5 to 5 nm. Spontaneous reduction of gold salts to gold nanoparticles by an inorganic matrix such as fumed silica has also been reported [134, 135]. These matrices not only reduce the gold salts to gold nanoparticles but also provide them with a solid support. Such conjugates exhibit interesting catalytic properties. 3.5.2

Physical Methods

The most common physical methods for the synthesis of gold nanoparticles include UV, irradiation, NIR irradiation, sonochemical method, radiolysis and thermolysis. UV irradiation can improve the quality of the gold nanoparticles formed when it is used in the presence of micelles or seeds [136–139]. Near-IR irradiation produces a large size growth of thiol-stabilized gold nanoparticles [140]. The rate of AuCl4  reduction in aqueous solution as well as sizes of gold nanoparticles formed can be controlled by using an ultrasonic field of 200 kHz [141, 142]. Radiolysis has also been used to control the sizes of gold nanoparticles or when they are synthesized in the presence of radicals [143, 144]. Gold nanoparticles have also been produced by the thermolysis of gold salts [144, 145].

105

106

3 Nanogold in Cancer Therapy and Diagnosis

3.5.3

Biological Methods

Biological methods for the synthesis of nanoparticles have been reported recently. Some living microorganisms, such as fungus, can reduce metal salts to metal nanoparticles. Both extracellular and intracellular synthesis of metal nanoparticles have been reported using fungus [146, 147]. Cadmium sulfide nanoparticles has also been synthesized [148].

3.6

Nanotechnology in Detection and Diagnosis with Gold Nanoparticles

Nanotechnology [149] combined with biology is the most advanced technology both from an academic viewpoint and for commercial applications. The integration of nanotechnology with biology and medicine is expected to produce major advances in molecular diagnostics, therapeutics, molecular biology, and bioengineering [150–158]. Optical [159] and magnetic [160] properties of metals, semiconductors, polymers and magnetic nanoparticles, and quantum dots (small devices that contain free electrons and have typical dimensions between nanometers to micrometers – these tiny crystals glow when stimulated by ultraviolet light, e.g., ZnS, ZnSe, CdSe, CdTe, PbSe, etc.) are being used for DNA detection, cancer diagnostics, drug discovery and clinical therapy in biomedical research [121, 154, 161– 179]. Among inorganic nanoparticles and quantum dots, colloidal gold nanoparticles have attracted significant research and practical attention in biology. Due to fascinating colors, size-dependent properties and dimensional similarities to biomacromolecule, these colloidal gold nanoparticles are widely used in biomedical research such as DNA detection [150, 177(b), 180], highly sensitive diagnostics [177(a), 181], thermal ablation and radiotherapy enhancement [182], as well as drug and gene delivery [183]. For instance, antibody-modified gold nanoparticles, when used for detection of prostate specific antigen, had an almost one million-fold higher sensitivity than a conventional ELISA-based assay [164]. Near-infrared radiation absorbing gold–silica nanoshells have been prepared and evaluated for thermal ablation of tumors after systemic administration [92, 182]. 3.6.1

Cancer Detection

Sayed et al. have reported that binding gold nanoparticles to a specific antibody for cancer cells could make cancer detection much easier using a simple microscope [177(a)]. Their technique for detection of cancer cells is very simple, clean, faster, economically very cheap and eco-friendly/non-toxic. They proposed that the very good scattering and light absorption properties of gold nanoparticles could be used to easily distinguish healthy from cancerous

3.6 Nanotechnology in Detection and Diagnosis with Gold Nanoparticles

cells with the help of a very simple microscope. Many cancer cells have a protein, known as Epidermal Growth Factor Receptor (EFGR), all over their surface, while healthy cells typically do not express the protein as strongly. By conjugating, or binding, the gold nanoparticles to an antibody for EFGR, suitably named antiEFGR, they were able to get the nanoparticles to attach themselves to the cancer cells. When they added the conjugated nanoparticle solution to healthy cells and cancerous cells, they observed with a simple microscope that the whole cancer cell shines. In contrast, healthy cells did not bind to the nanoparticles specifically and, consequently, they did not shine. In this promising technique, they found that the gold nanoparticles have a 600% greater affinity for cancer cells than for noncancerous cells. The gold nanoparticles that worked the best were 35 nm in size. Researchers tested their technique using cell cultures of two different types of oral cancer and one nonmalignant cell line. The shape of the strong absorption spectrum of the gold nanoparticles is also found to distinguish between cancer cells and noncancerous cells. 3.6.2

Detection in DNA

Several studies suggest that nanoparticle-based technology could enable sensitive detection of sequence variation in DNA [180]. Early studies demonstrated arraybased DNA discrimination by allele-specific oligonucleotide hybridization using gold nanoparticle reporters containing bound oligonucleotides for hybridization to complementary targets [180(a)]. Detection was achieved by silver enhancement, resulting in deposition of silver on the surface of the nanoparticles, which enabled scanometric detection to define the location of the gold nanoparticles on an array [180(b)]. Attachment of a dye close to the surface of the nanoparticles via linkage to the oligonucleotide also enabled detection after silver enhancement by surfaceenhanced Raman spectroscopy (SERS) [180(c)]. Single-mismatch Detection in DNA Maxwell et al. have demonstrated the design and feasibility of bio-conjugated gold nanoparticles for recognizing and detecting specific DNA sequences in a single step [150]. According to their method, colloidal gold nanocrystals 2.5 nm in size have been used to develop a new class of nanobiosensors that can recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. The 2.5-nm gold nanoparticles function as both a nano-scaffold and a nano-quencher (efficient energy receptor). They proposed that single-stranded DNA is conformationally flexible. Conversely, two oligonucleotide molecules (oligos) are self-assembled in a constrained conformation (arch-like structure) on each gold particle (2.5 nm in diameter) A T6 spacer (six thymines) is inserted at both the 3 0 and 5 0 ends to reduce steric hindrance. In the assembled (closed) state, the fluorophore is quenched by the nanoparticle because of the stable arch-like structure. Upon target binding, the constrained conformation opens, the fluorophore leaves the surface because of the 3.6.2.1

107

108

3 Nanogold in Cancer Therapy and Diagnosis

structural rigidity of the hybridized DNA (double stranded), and fluorescence is restored. In the open state, the fluorophore is separated from the particle surface by about 10 nm. In this novel method gold nanoparticles represent a new class of universal fluorescence quenchers that are substantially different from DABCYL (4,4 0 -dimethylaminnophenylazobenzoic acid) and should find new applications in molecular engineering and biosensor development and homogeneous bioassays that are not possible by DABCYL. In another method, Dubertret et al. have shown that gold-quenched nucleic acid probes (gold–oligonucleotide–dye) can be more sensitive than other probes currently available for mismatch detection. With their developed probes a point mutation can be selectively detected in the presence of random sequences even if only 1 out of 50 sequences has one mutation. They have developed a hybrid material composed of a 25-nucleotide long single-stranded DNA (ssDNA) molecule, a 1.4 nm diameter gold nanoparticle, and a fluorophore (an organic dye) that is highly quenched by nanoparticle through a distance-dependent process. This hybrid material can form the hairpin structure that brings the fluorophore and the gold particle in close proximity (within a few angstroms). In this conformation, the gold cluster quenches the fluorescence of the dye. Through sequence-specific hybridization to a single-stranded target DNA, the hairpin structure changes to a rod-like structure (on the right), which maintains the fluorophore and the quencher far apart and thus restores the fluorescence. They reported that 1.4-nm diameter gold nanoparticles are better than a conventional fluorescence quencher, such as DABCYL, because gold nanoparticles quench fluorescence as much as 100 better and have higher quenching efficiency than dyes emitting in the near-infrared region. As well the above examples of the use of gold nanoparticles in cancer and DNA detection, numerous recent reports relate the biological applications of gold nanoparticles in imaging and in therapy [8, 184–193]. A few are described here very briefly. Chen et al. have demonstrated a novel nanoshell-based all-optical platform technology for integrating cancer imaging and therapy applications [184]. Immunotargeted nanoshells are engineered to both scatter light in the NIR enabling optical molecular cancer imaging, and to absorb light, allowing selective destruction of targeted carcinoma cells through photothermal therapy. They have shown that dual imaging/therapy immunotargeted nanoshells can be used to detect and destroy breast carcinoma cells that overexpress HER2, a clinically relevant cancer biomarker. Bioconjugated with antibodies, gold nanocages (<40 nm) have also been used for specific targeting of breast cancer cells [185]. These gold nanocages have a moderate scattering cross-section of @8.10 A˚ @ 10–16 m 2 but a very large absorption cross-section of @7.26 A˚ @ 10–15 m 2 , suggesting their potential use as a new class of contrast agents for optical imaging. Surface-enhanced Raman scattering (SERS) studies have been reported on indocyanine green (ICG) on colloidal silver and gold nanoparticles, demonstrating a

3.7 Future Direction

novel optical probe for applications in living cells [186]. The ICG gold nanoprobe delivers spatially localized chemical information from its biological environment by employing SERS in the local optical fields of the gold nanoparticles. Applications of nanostructures including gold nanoparticles in biodiagnostics have been thoroughly reviewed recently [187]. Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. Huang et al. have developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor [188]. They have developed a sandwich assay with gold nanoparticles for a major enhancement in sensitivity of PSA detection. The assay enables the detection of total PSA levels at clinically relevant concentrations. Gold nanoparticles can be used as an amplifying payload strategy for adenoviral cancer gene therapy [189]. Colloidal gold nanoparticles can be used as a versatile platform for developing tumor targeted cancer therapies [8, 190–192]. On the basis of theoretical and experimental results, a new dynamic mode – the bubbles-overlapping mode (BOM) – has been reported for the selective killing of cancer cells to whose cell membrane nanoparticles conjugated with specific antibodies have been attached [192].

3.7

Future Direction

Nanotechnology can be used in cancer detection and diagnosis. To improve cancer treatment, detection of cancer at an early stage is a very critical step. Presently, detection and diagnosis of cancer usually depend on molecular changes in cells and tissues that are detected by a doctor’s physical examination or imaging expertise. Nanotechnology is uniquely promising as an early detection tool for cancer treatment for several reasons: To successfully detect cancer at its earliest stages, scientists must be able to detect molecular changes even when they occur only in a small percentage of cells. This means the necessary tools must be extremely sensitive. The potential for nanostructures to enter and analyze single cells suggests they could meet this need. Many nanotechnology tools will make it possible for clinicians to run tests without physically altering the cells or tissue they take from a patient. This is important because the samples clinicians use to screen for cancer are often in limited supply. Scientists would like to perform tests without altering cells, so they can be used again if further tests are needed. Other technologies will focus on improved methods of reading the genetic code on single strands of DNA to detect errors that may contribute to cancer. Scientists believe nanopores, tiny holes that allow DNA to pass through one strand at a time, will make DNA sequencing more efficient. As DNA passes through a nanopore, scientists can monitor the shape and electrical properties of each base on the strand. Because these properties are unique for each of the four bases that make up the genetic code, scientists can use the passage of DNA through a nanopore to

109

110

3 Nanogold in Cancer Therapy and Diagnosis

decipher the encoded information, including errors in the code known to be associated with cancer. To detect cancer, scientists can design beads containing quantum dots to bind to sequences of DNA that are associated with cancer. When the quantum dots are stimulated with light, they will emit their unique barcodes, or labels, making the critical, cancer-associated DNA sequences visible. The vast number of possible combinations of quantum dots means that scientists can create many unique labels, which can be used at the same time. This will allow scientists to look simultaneously at numerous regions of DNA. This will be important in the detection of cancer, which results from many different changes within a cell. These technologies are in various stages of discovery and development. Experts believe that quantum dots, nanopores and other devices for detection and diagnosis may be available for clinical use in 5–15 years. Therapeutic agents are expected to be available within a similar time frame. Devices that integrate detection and therapy could be used clinically in about 15 or 20 years.

Acknowledgments

This work was partly supported by NIH CA-78383 and HL-70567 and also by a grant from the American Cancer Society to DM. The authors are grateful to D. Lecy for her help in revising the manuscript.

References 1 American Cancer Society: Cancer Facts

2

3

4

5

6

and Figures 2005. Atlanta, GA, American Cancer Society, 2005. D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3(5), 380–387. E. J. Lee, J. L. Jameson. Gene therapy of pituitary diseases. J. Endocrinol. 2005, 185(3), 353–362. A. Perillo, G. Ferrandina, L. Pierelli, G. Bonanno, G. Scambia, S. Mancuso. Stem cell-based treatments for gynecological solid tumors. Eur. Rev. Med. Pharmacol. Sci. 2005, 9(2), 93–102. M. Dhanabal, M. Jeffers, W. J. Larochelle. Anti-angiogenic therapy as a cancer treatment paradigm. Curr Med. Chem. Anti-Canc Agents. 2005, 5(2), 115–130. P. Mukherjee, R. Bhattacharya, D. Mukhopadhyay. Gold nanoparticles

bearing functional anti-cancer drug and anti-angiogenic agent: a ‘‘2 in 1’’ system with potential application in cancer therapeutics. J. Biomed. Nanotechnol. 2005, 1, 1–5. 7 R. Bhattacharya, P. Mukherjee, Z. Xiong, A. Atala, S. Soker, D. Mukhopadhyay. Gold nanoparticles inhibit VEGF165-induced proliferation of HUVEC cells. Nano Lett. 2004, 4(12), 2479–2481. 8 G. F. Paciotti, L. Myer, D. Weinreich, D. Goia, N. Pavel, R. E. McLaughlin, L. Tamarkin. Colloidal gold: A novel nanoparticle vector for tumor directed drug delivery. Drug Deliv. 2004, 11(3), 169–183. 9 P. Mukherjee, R. Bhattacharya, P. Wang, L. Wang, S. Basu, J. A. Nagy, A. Atala, D. Mukhopadhyay, S. Soker. Antiangiogenic properties of gold nanoparticles. Clin. Cancer Res. 2005, 11(9), 3530–3534.

References 10 G. J. Higby. Gold in medicine:

11

12

13

14

15

16

17 18

19

20

21

22 23

A review of its use in the West before 1900. Gold Bull. 1982, 15, 130–140. R. G. Buckley, A. M. Elsome, S. P. Fricker, G. R. Henderson, B. R. Theobald, R. V. Parish, B. P. Howe, L. R. Kelland, Antitumor properties of some 2-[(dimethylamino)methyl]phenylgold(III) complexes, J. Med. Chem. 1996, 39, 5208–5214. D. G. Richards, D. L. McMillin, E. A. Mein, C. D. Nelson. Gold and its relationship to neurological/ glandular conditions. Int. J. Neurosci. 2002, 112, 31–53. S. Mahdihassan. Tan, cinnabar, as drug of longevity prior to alchemy. Am. J. Chin. Med. 1984, 12, 50–54. S. Mahdihassan. Cinnabar-gold as the best alchemical drug of longevity, called Makaradhwaja in India. Am. J. Chin. Med. 1985, 13, 93–108. O. Temkin. The Failing Sickness. A History of Epilepsy from the Greeks to the beginnings of Modern Neurology. Baltimore, Johns Hopkins Press. S. P. Fricker. Medical uses of gold compounds: past, present and future. Gold Bull. 1996, 29, 53–59. J. C. Burnett. Gold as a remedy in disease, Gould, London 1895. H. Eichhorst. Handbook of Practical Medicine. Volume 1, Diseases of the Circulatory and Respiratory Apparatus. William Wood and Company, New York, 1886. S. O. L. Potter. A Compend of Material Medica, Therapeutics, and Prescription Writing, with Especial Reference to the Physiological Actions of Drugs. Sixth Edition 1902. P. Blakiston’s Sons & Co, Philadelphia, 1894. H. A. Hare. A Text Book of Practical Therapeutics. 14 th edition, Lea & Febiger, Philadelphia, 1912. S. T. Gerber. Stedman’s Practical Medical Dictionary. Williams & Wilkins, Baltimore, 1942. Koch, R. Dtsch. Med. Wochenstr, 1890, 16, 756–757. D. A. Gordon, in Textbook of Rheumatology, W. W. Kelly, E. D. Harris, D. Ruddy, C. B. Sledge

24

25

26 27

28

29

30

31

32 33

34

(Eds.), W. B. Saunders, New York, 1989, Ch. 48, p. 804. D. T. Felson, J. J. Anderson, R. F. Meenan. The comparative efficacy and toxicity of second-line drugs in rheumatoid arthritis. Results of two metaanalyses. Arthritis Rheum. 1990, 33, 1449–1461. C. F. Shaw III. Gold-based therapeutic agents. Chem. Rev. 1999, 99, 2589– 2600. D. T. Hill, B. M. Sutton. Cryst. Struct. Commun. 1980, 9, 679. Y. Y. Tong, A. J. L. Pombierro, D. L. Hughes, R. L. Richards, Synthesis and properties of the gold(I) complexes with bulky thiolates [Au(SR)](6) and [Au(SR)(PPH(3))] (RbC(6)H(2)ME(3)-2,4,6 or C6H2PR3I-2,4,6), (R ¼ C6 H2 ME3 -246 or C6H2PR3I-2,4,6), and the molecular-structure of [AU(SC6H2PR3I-2,4,6)(PPH(3)], Transition Met. Chem. 1995, 20, 372–375. M. Nakamoto, W. Hiller, H. Schmidbaur, Preparation and crystal structures of gold(I) complexes of a series of benzothiolates-pair formation by gold-gold interactions of (Benzenethiolate)(triphenylphosphane) gold(I), Chem. Ber. 1993, 126, 605–610. R. Bau, Crystal structure of the antiarthritic drug gold thiomalate (myochrysine): A double-helical geometry in the solid state, J. Am. Chem. Soc. 1998, 120, 9380–9381. C. F. Shaw III, in Metal Compounds in Cancer Therapy, S. P. Fricker (Ed.), Chapman & Hall, London, 1994, p. 46. I. Haiduc, C. Silvestru. Rhodium, iridium, copper and gold antitumor organometallic compounds (review). In Vivo, 1989, 3, 285–293. S. J. Berners-Price, P. J. Sadler, Struct. Bonding 1988, 70, 27. P. J. Sadler, M. Nasr, V. L. Narayanan, in Platinum Coordination Complexes in Cancer Chemotherapy, M. P. Hacker, E. B. Douple, I. H. Krakoff (Eds.), Martinus Nijhoff Publishing, Boston, 1984, p. 209. S. J. Berners-Price, P. S. Jarret, P. J. Sadler, 31P NMR Studies of

111

112

3 Nanogold in Cancer Therapy and Diagnosis

35

36

37 38

39

40

41

42

43

44

45

[Aum(pdppe)]2þ antitumor complexes. Conversion into [A@(dppe)@]þ induced by thiols and blood plasma, Inorg. Chem. 1987, 26, 3074–3077. R. G. Buckley, A. M. Elsome, S. P. Fricker, G. R. Handerson, B. R. C. Theobald, R. V. Parish, B. P. Howe, L. R. J. Kelland. Antitumor properties of some 2-[(dimethylamino)methyl]phenylgold(III) complexes. Med. Chem. 1996, 39, 5208–5214. F. Bussolino, A. Mantovani, G. Persico. Molecular mechanisms of blood vessel formation. Trends Biochem. Sci. 1997, 22(7), 251–256. J. Folkman. Tumor angiogenesis. Adv. Cancer Res. 1974, 19(0), 331–358. J. Folkman. Tumor angiogensis: Role in regulation of tumor growth. Symp. Soc. Dev. Biol. 1974, 30(0), 43–52. M. K. Gupta, R. Y. Qin. Mechanism and its regulation of tumor-induced angiogenesis. World J. Gastroenterol. 2003, 9(6), 1144–1155. P. Nyberg, L. Xie, R. Kalluri. Endogenous inhibitors of angiogenesis. Cancer Res. 2005, 65(10), 3967– 3979. R. Langer, H. Conn, J. Vacanti, C. Haudenschild, J. Folkman. Control of tumor growth in animals by infusion of an angiogenesis inhibitor. Proc. Natl. Acad. Sci. U.S.A. 1980, 77(7), 4331–4335. M. A. Konerding, E. Fait, A. Gaumann. 3D microvascular architecture of pre-cancerous lesions and invasive carcinomas of the colon. Br. J. Cancer. 2001, 84(10), 1354–1362. A. Eberhard, S. Kahlert, V. Goede, B. Hemmerlein, K. H. Plate, H. G. Augustin. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: Implications for antiangiogenic tumor therapies. Cancer Res. 2000, 60(5), 1388–1393. D. M. McDonald, P. L. Choyke. Imaging of angiogenesis: From microscope to clinic. Nat. Med. 2003, 9(6), 713–725. F. J. Burrows, P. E. Thorpe. Eradication of large solid tumors in mice with an immunotoxin directed

46 47

48

49

50

51

52

53

54

against tumor vasculature. Proc. Natl. Acad. Sci. U.S.A. 1993, 90(19), 8996–9000. L. M. Ellis. Bevacizumab. Nat. Rev. Drug Discov. 2005, Suppl, S8–9. F. Mannello, G. Tonti, S. Papa. Matrix metalloproteinase inhibitors as anticancer therapeutics. Curr. Cancer Drug Targets. 2005, 5(4), 285–298. R. Neubert, A. C. Nogueira, D. Neubert. Thalidomide derivatives and the immune system. I. Changes in the pattern of integrin receptors and other surface markers on T lymphocyte subpopulations of marmoset blood. Arch. Toxicol. 1993, 67(1), 1–17. M. F. McCarty. Thalidomide may impede cell migration in primates by down-regulating integrin beta-chains: Potential therapeutic utility in solid malignancies, proliferative retinopathy, inflammatory disorders, neointimal hyperplasia, and osteoporosis. Med. Hypotheses. 1997, 49(2), 123–131. J. J. O’Leary, R. L. Shapiro, C. J. Ren, N. Chuang, H. W. Cohen, M. Potmesil. Antiangiogenic effects of camptothecin analogues 9-amino20(S)-camptothecin, topotecan, and CPT-11 studied in the mouse cornea model. Clin. Cancer Res. 1999, 5(1), 181–187. N. Klauber, S. Parangi, E. Flynn, E. Hamel, R. J. D’Amato. Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol. Cancer Res. 1997, 57(1), 81–86. D. Belotti, V. Vergani, T. Drudis, P. Borsotti, M. R. Pitelli, G. Viale, R. Giavazzi, G. Taraboletti. The microtubule-affecting drug paclitaxel has antiangiogenic activity. Clin. Cancer Res. 1996, 2(11), 1843–1849. B. E. Turk, Z. Su, J. O. Liu. Synthetic analogues of TNP-470 and ovalicin reveal a common molecular basis for inhibition of angiogenesis and immunosuppression. Bioorg. Med. Chem. 1998, 6, 1163–1169. T. Yoshida, Y. Kaneko, A. Tsukamoto, K. Han, M. Ichinose, S. Kimura. Suppression of hepatoma

References

55

56

57

58

59

60

61

growth and angiogenesis by a fumagillin derivative TNP470: Possible involvement of nitric oxide synthase. Cancer Res. 1998, 58, 3751–3756. Y. Zheng, J. Hua, K. G. Pinney, C. M. Garner, R. R. Kane, J. A. Prezioso, D. J. Chaplin, K. Edvardsen. Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis. Int. J. Cancer. 2004, 111(4), 604–610. G. C. Tucker. Alpha v integrin inhibitors and cancer therapy. Curr. Opin. Investig. Drugs. 2003, 4(6), 722–731. J. A. Posey, M. B. Khazaeli, A. DelGrosso, M. N. Saleh, C. Y. Lin, W. Huse, A. F. LoBuglio. A pilot trial of Vitaxin, a humanized antivitronectin receptor (anti alpha v beta 3) antibody in patients with metastatic cancer. Cancer Biother. Radiopharm. 2001, 16(2), 125–132. T. Taga, A. Suzuki, I. GonzalezGomez, F. H. Gilles, M. Stins, H. Shimada, L. Barsky, K. I. Weinberg, W. E. Laug. Alpha v-Integrin antagonist EMD 121974 induces apoptosis in brain tumor cells growing on vitronectin and tenascin. Int. J. Cancer 2002, 98(5), 690–697. L. M. Evans, L. M. Itri, M. Campion, R. Wyler-Plaut, S. E. Krown, J. E. Groopman, H. Goldsweig, P. A. Volberding, S. B. West, R. T. Mitsuyasu, et al. Interferon-alpha 2a in the treatment of acquired immunodeficiency syndrome-related Kaposi’s sarcoma. J. Immunother. 1991, 10, 39–50. M. A. Fischl, D. M. Finkelstein, W. He, W. G. Powderly, P. L. Triozzi, R. T. Steigbigel. A phase II study of recombinant human interferon-alpha 2a and zidovudine in patients with AIDS-related Kaposi’s sarcoma. AIDS Clinical Trials Group. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 1996, 11, 379–384. A. K. Morris, A. W. Valley. Overview of the management of AIDS-related Kaposi’s sarcoma. Ann Pharmacother. 1996, 30, 1150–1163.

62 L. Rosen, M. Mulay, A. Mayers, et al.

63

64

65

66

67

68

69

70

71

Phase I dose-escalating trial of SU5416, a novel angiogenesis inhibitor in patients with advanced malignancies. J. Clin. Oncol. 1999, 18, 161a. K. Yorozuya, T. Kubota, M. Watanabe, H. Hasegawa, S. Ozawa, M. Kitajima, L. M. Chikahisa, Y. Yamada. TSU-68 (SU6668) inhibits local tumor growth and liver metastasis of human colon cancer xenografts via anti-angiogenesis. Oncol. Rep. 2005, 14(3), 677–682. W. P. Steward. Marimastat (BB2516): Current status of development. Cancer Chemother. Pharmacol. 1999, 43(suppl), S56–S60. G. Wilding, E. Small, M. Collier, et al. A phase I pharmacokinetic evaluation of the matrix metalloprotease (MMP) inhibitor AG3340 in combination with mitoxantrone and prednisone in patients with advanced prostate cancer. J. Clin. Oncol. 1999, 18, 323a. J. D’Olimpio, K. Hande, M. Collier, et al. Phase I study of the matrix metalloprotease inhibitor AG3340 in combination with paclitaxel and carboplatin for the treatment of patients with advanced solid tumors. J. Clin. Oncol. 1999, 18, 160a. R. Goel, H. Hirte, P. Major, et al. Clinical pharmacology of the metalloproteinase (MMP) and angiogenesis inhibitor Bayer 12-9566 in cancer patients. J. Clin. Oncol. 1999, 18, 160a. M. Eatock, J. Cassidy, J. Johnson, et al. A phase I study of the matrix metalloproteinase inhibitor MM1270 (previously termed CGS270231) with 5FU and folinic acid. J. Clin. Oncol. 1999, 18, 209a. W. Risau. Angiogenesis and endothelial cell function. Arzneimittelforschung, 1994, 44, 417–417. P. Carmeliet, R. K. Jain. Angiogenesis in cancer and other diseases. Nature 2000, 407, 249–257. Z. Szekanecz, G. Szegedi, A. E. Koch. Angiogenesis in rheumatoid arthritis: Pathogenic and clinical

113

114

3 Nanogold in Cancer Therapy and Diagnosis

72

73

74

75 76

77

78

79

80

significance. J. Invest. Med. 1998, 46, 27–41. T. Pincus, G. Ferraccioli, T. Sokka, A. Larsen, R. Rau, I. Kushner, F. Wolfe. Evidence from clinical trials and long-term observational studies that disease-modifying anti-rheumatic drugs slow radiographic progression in rheumatoid arthritis: Updating a 1983 review. Rheumatology 2002, 41, 1346–1356. D. R. Senger, C. A. Perruzzi, J. Feder, H. F. Dvorak. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 1986, 46, 5629–5632. D. W. Leung, G. Cachianes, W. J. Kuang, D. V. Goeddel, N. Ferrara. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989, 246, 1306–1309. W. Risau. Mechanisms of angiogenesis. Nature 1997, 386, 671–674. L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Sershen, B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas, J. L. West. Nanoshell-mediated nearinfrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13 549–13 554. A. Kumar, S. Mandal, P. R. Selvakannan, R. Pasricha, A. B. Mandale, M. Sastry, Langmuir 2003, 19, 6277. S. Basu, J. A. Nagy, S. Pal, E. Vasile, I. A. Eckelhoefer, V. S. Bliss, E. J. Manseau, P. S. Dasgupta, H. F. Dvorak, D. Mukhopadhyay. The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor. Nat. Med. 2001, 7, 569–574. D. Fambrough, K. McClure, A. Kazlauskas, E. S. Lander. Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 1999, 97, 727–741. H. Zeng, D. Zhao, D. Mukhopadhyay. Flt-1-mediated down-regulation of endothelial cell

81

82

83

84

85

86

87

proliferation through pertussis toxinsensitive G proteins, beta gamma subunits, small GTPase CDC42, and partly by Rac-1. J. Biol. Chem. 2002, 277, 4003–4009. L. Wang, H. Zeng, P. Wang, S. Soker, D. Mukhopadhyay. Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J. Biol. Chem. 2003, 49, 48 848–48 860. A. Pettersson, J. A. Nagy, L. F. Brown, C. Sundberg, E. Morgan, S. Jungles, R. Carter, J. E. Krieger, E. J. Manseau, V. S. Harvey, I. A. Eckelhoefer, D. Feng, A. M. Dvorak, R. C. Mulligan, H. F. Dvorak. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab. Invest. 2000, 80, 99–115. J. A. Nagy, K. T. Herzberg, J. M. Dvorak, H. F. Dvorak. Pathogenesis of malignant ascites formation: Initiating events that lead to fluid accumulation. Cancer Res. 1993, 53, 2631–2643. R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger, C. A. Mirkin. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 1997, 277, 1078–1081. A. Ulman, An Introduction to Ultrathin Organic Films: From Langmuir–Blodget to Self-assembly, Academic Press, New York, 1991. A. Lon, J. R. Porter, J. I. David, S. L. Westcott, M. Graupe, R. S. Czernuszewicz, N. J. Halas, R. T. Lee. Alkanethiol molecules containing an aromatic moiety self-assembled onto gold clusters, Langmuir 1998, 14, 7378. S. R. Johnson, S. D. Evans, S. W. Mahon, A. Ulman. Alkanethiol molecules containing an aromatic moiety self-assembled onto gold clusters, Langmuir 1997, 13, 51– 57.

References 88 H. J. Buchsbaum, W. C. Keettel.

89

90

91

92

93

94

95

96

97

98

Radioisotopes in treatment of stage Ia ovarian cancer. Natl. Cancer Inst. Monogr. 1975, 42, 127–128. E. E. Rogoff, R. Romano, E. W. Hahn. The prevention of Ehrlich ascites tumor using intraperitoneal colloidal 198Au. Dose vs. size of inoculum. Radiology 1975, 114, 225–226. R. D. Pezner, K. R. Stevens Jr, D. Tong, C. V. Allen. Limited epithelial carcinoma of the ovary treated with curative intent by the intraperitoneal installation of radiocolloids. Cancer 1978, 42, 2563–2571. A. E. Alfanso, A. Hassan, B. Gardner, S. Stein, J. Patti, N. A. Solomon, J. McCarthy, J. Steigman, Prevention of hepatic metastases by intravenous radioactive gold, Cancer Res. 1978, 38, 2740–2744. J. F. Hainfeld, D. N. Slatkin, H. M. Smilowitz. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 2004, 49, N309–N315. W. P. Faulk, G. M. Taylor. An immunocolloid method for the electron microscope. Immunochemistry 1971, 8, 1081–1083. A. M. Gobin, D. P. O’Neal, D. M. Watkins, N. J. Halas, R. A. Drezek, J. L. West. Near infrared laser-tissue welding using nanoshells as an exogenous absorber. Lasers Surg. Med. 2005, 37, 123–129. S. J. Oldenberg, R. D. Averitt, S. L. Westcott, N. J. Halas, Nanoengineering of optical resonances, Chem. Phys. Lett. 1998, 228, 243–247. R. D. Averitt, S. L. Westcott, N. J. Halas, Linear optical properties of gold nanoshells. J. Opt. Soc. Am. B 1999, 16, 1824–1832. S. J. Oldenburg, J. B. Jackson, S. L. Westcott, N. J. Halas, Light scattering from dipole and quadrupole nanoshell antennas. Appl. Phys. Lett. 1999, 75, 1063–1065. D. P. O’Neal, L. R. Hirsch, N. J. Halas, J. D. Payne, J. L. West. Photothermal tumor ablation in mice using

99

100

101

102

103

104

105

106

107

108

near infrared-absorbing nanoparticles. Cancer Lett. 2004, 209, 171. D. R. Senger, S. J. Galli, A. M. Dvorak, C. A. Perruzzi, V. S. Harvey, H. F. Dvorak. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983, 219, 983–985. H. C. Lichtenbeld, N. Ferarra, R. K. Jain, L. L. Munn. Effect of local antiVEGF antibody treatment on tumor microvessel permeability. Microvasc. Res. 1999, 57, 358–362. P. J. Keck, D. D. Hauser, G. Krivi, K. Sanzo, T. Warren, J. Feder, D. T. Conolly. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989, 246, 1309–1312. N. Ferrara, K. Houck, L. Jakeman, D. W. Leung, Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr. Rev. 1992, 13, 18–32. R. K. Jain, P. Carmeliet. Vessels of death or life? Sci. Am. 2001, 285, 38–45. M. A. Moses, H. Brem, R. Langer, Advancing the field of drug delivery: Taking aim at cancer. Cancer Cell 2003, 4(5), 337–341. A. S. Zahr, M. de Villiers, M. V. Pishko, Encapsulation of drug nanoparticles in self-assembled macromolecular nanoshells, Langmuir 2005, 21, 403–410. R. A. Brekken, P. J. Overholser, V. A. Stastny, J. Waltenberger, J. D. Minna, P. E. Thorpe, Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice, Cancer Res. 2000, 60, 5117–5124. H. Okino, R. Maeyama, T. Manabe, T. Matsuda, M. Tanaka, Trans-tissue, sustained release of gemcitabine from photocured gelatin gel inhibits the growth of heterotopic human pancreatic tumor in nude mice, Clin. Cancer. Res. 2003, 9, 5786–5793. C. Mangeney, F. Ferrage, I. Aujard, V. Marchi-Artzner, L. Jullien, O. Ouari, E. D. Rekai, A. Laschewsky, I.

115

116

3 Nanogold in Cancer Therapy and Diagnosis

109

110

111

112

113

114

115

116

117

118

119

120

121

Vikholm, J. W. Sadowski, Synthesis and properties of water-soluble gold colloids covalently derivatized with neutral polymer monolayers. J. Am. Chem. Soc. 2002, 124, 5811–5821. S. R. Johnson, S. D. Evans, S. W. Mahon, A. Ulman, Alkanethiol molecules containing an aromatic moiety self-assembled onto gold clusters, Langmuir 1997, 13, 51–57. R. A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Austin, TX, 2003. B. Jonathan, Biological Performance of Materials: Fundamentals of Biocompatibility, 3rd edition, Marcel Dekker, New York, 1999. D. F. Williams (Ed.), Fundamental Aspects of Biocompatibility, Vols I, II, CRC Press, Boca Raton, FL, 1981. J. L. Braybrook (Ed.), Biocompatibility Assessment of Medical Devices and Materials, John Wiley and Sons, New York, 1997. H. S. Frederic, D. L. Christiansen, Biomaterials Science and Biocompatibility, Springer Verlag, New York, 1999. A. P. Alivisatos, Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, 933–937. M. C. Daniel, D. Astruc, Gold nanoparticles: Assembly, supramolecular chemistry, quantum-sizerelated properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 2004, 104, 293–346. D. F. Emerich, C. G. Thanos, Nanotechnology and medicine. Expert Opin. Biol. Ther. 2003, 3, 1–9. P. L. Brannon, J. O. Blanchette, Nanoparticle and targeted systems for cancer therapy. Adv. Drug Delivery Rev. 2004, 56, 1649–1659. M. Ferrari, Cancer nanotechnology: Opportunities and challenges. Nat. Rev. Cancer 2005, 5, 161–171. G. Y. J. Chen, S. Q. Yao, Lighting up cancer cells with ‘‘dots’’. Lancet 2004, 364, 2001–2003. E. B. Voura, J. K. Jaiswal, H. Mattoussi, S. M. Simon, Tracking metastatic tumor cell extravasation

122

123

124

125

126

127

128

129

130

131

with quantum dot nanocrystals and fluroscence emission-scanning microscopy. Nat. Med. 2004, 10, 993–998. X. Gao, Y. Cui, R. M. Levenson, L. W. Chung, S. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 2004, 22, 959–960. R. F. Service, Nanotechnology grows up. Science 2004, 304, 1732– 1734. D. Vergano, Nano proponents square off against specter of ‘gray goo’. USA Today, September 27, 2004. V. L. Colvin, The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 2003, 21, 1166–1170. E. Oberdorster, Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environ. Health Perspect. 2004, 112, 1058–1062. A. D. Maynard, P. A. Baron, M. Foley, A. A. Shvedova, E. R. Kisin, V. Castranova, Exposure to carbon nanotube material: Aerosol release during the handling of unrefined single-walled carbon nanotube material. J. Toxicol. Environ. Health A 2004, 67, 87–107. J. Parvizi, C. C. Wu, D. G. Lewallen, J. F. Greenleaf, M. E. Bolander, Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression. J. Orthop. Res. 1999, 17, 488. H. Hatano, H. J. Siegel, H. Yamagiwa, J. T. Bronk, R. T. Turner, M. E. Bolander, G. Sarkar, Identification of estrogen-regulated genes during fracture healing, using DNA microarray. J. Bone Miner. Metab. 2004, 22, 224. M. Faraday, Experimental relations of gold (and other metals) to light. Philos Trans. 1857, 147, 145–181. J. Turkevitch, P. C. Stevenson, J. Hiller. Nucleation and growth process in the synthesis of colloidal gold, Discuss. Faraday. Soc. 1951, 11, 55.

References 132 T. Yonezawa, T. Kunitake, Practical

133

134

135

136

137

138

139

140

preparation of anionic mercapto ligand stabilized gold nanoparticles and their immobilization. Colloids Surf. A: Physicochem. Eng. Asp. 1999, 149, 193. M. Brust, M. Walker, D. Bethel, D. J. Schiffrin, R. J. Whyman. Synthesis of thiol derivatized gold nanoparticles in a two phase liquidliquid system. J. Chem. Soc., Chem. Commun. 1994, 801. P. Mukherjee, C. R. Patra, A. Ghosh, R. Kumar, M. Sastry, Characterization and catalytic activity of gold nanoparticles synthesized by autoreduction of aqueous chloroaurate ions with fumed silica, Chem. Mater. 2002, 14, 1678. K. Esumi, A. Suzuki, A. Yamahira, K. Torigoe, Role of poly(amidoamine) dendrimers for preparing nanoparticles of gold, platinum and silver, Langmuir 2001, 16, 2604. S. Mossmer, J. P. Spatz, M. Moeller, T. Aberle, J. Schmidt, W. Burchard, Solution behaviour of poly(styrene)-block-poly(2vinylpyridine) micelles containing gold nanoparticles, Macromolecules 2000, 33, 4791. T. K. Sau, A. Pal, N. R. Jana, Z. L. Wang, T. Pal, Size controlled synthesis of gold nanoparticles using photochemically prepared seed particles, J. Nanopart. Res. 2001, 13, 257. S. Meltzer, R. Resch, B. E. Koel, M. E. Thompson, A. Madhukumar, A. A. G. Requicha, P. Will, Fabrication of nanostructures by hydroxylamine seeding of gold nanoparticle templates, Langmuir 2001, 17, 1713. K. Mallick, Z. L. Wang, T. Pal, Seed mediated successive growth of gold nanoparticles accomplished by UVirradiation: A photochemical approach for size controlled synthesis, J. Photochem. Photobiol. 2001, 140, 75. Y. Zhou, C. Y. Wang, Y. R. Zhu, Z. Y. Chen, A. Novel ultraviolet irradiation technique for shape controlled synthesis of gold

141

142

143

144

145

146

147

148

149

150

151

nanoparticles at room temperature, Chem. Mater. 1999, 11, 2310. Y. Niidome, A. Hori, T. Sato, S. Yamada, Enormous size growth of thiol passivated gold nanoparticles induced by near IR laser light. Chem. Lett. 2000, 310. A. Henglein, D. Meisel, Radiolytic control of the size of colloidal gold nanoparticles, Langmuir 1998, 14, 7392. E. Gachard, H. Remita, J. Khatouri, B. Keita, L. Nadjo, J. Belloni, Radiation induced and chemical formation of gold clusters, New J. Chem. 1998, 1257. M. Nakamoto, M. Yamamoto, M. Fukusumi, Thermolysis of gold(I) thiolate complexes producing novel gold nanoparticles passivated by alkyl groups, Chem. Commun. 2002, 1622. T. Shimizu, T. Teranishi, S. Hasegawa, M. Miyake, Size evolution of alkanethiol-protected gold nanoparticles by heat treatment in the solid state, J. Phys. Chem. B 2003, 107, 2719. P. Mukherjee, A. Ahmad, D. Mandal, S. Senapati, S. R. Sainkar, M. I. Khan, R. Ramani, R. Parischa, P. V. Ajayakumar, M. Alam, M. Sastry R. Kumar, Angew. Chem. Int. Ed. 2001, 40, 3585. P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, M. I. Khan, R. Kumar, M. Sastry, Chem. Biochem. 2002, 3, 461. A. Ahmad, P. Mukherjee, D. Mandal, S. Senapati, M. I. Khan, R. Kumar, M. Sastry, J. Am. Chem. Soc. 2002, 124, 12 108. R. Feynman, There’s plenty of room at the bottom, Science 1991, 254, 1300–1301. D. J. Maxwell, J. R. Taylor, S. Nie, Self-assembled nanoparticle probes for recognition and detection of biomolecules, J. Am. Chem. Soc. 2002, 124, 9606–9612. D. A. LaVan, D. M. Lnn, R. Langer, Moving smaller in drug discovery and delivery, Nat. Rev. Drug. Discov. 2002, 1, 77–84.

117

118

3 Nanogold in Cancer Therapy and Diagnosis 152 W. C. W. Chan, D. J. Maxwell, X.

153

154

155

156

157

158

159

160

161

Gao, R. E. Bailey, M. Y. Han, S. Nie, Curr. Opin. Biotechnol. Luminescent quantum dots for multiplexed biological detection and imaging, 2002, 13, 40–46. C. M. Niemeyer, Nanoparticles, proteins, and nucleic acids: Biotechnology meets materials science, Angew. Chem., Int. Ed. 2001, 40, 4128–4158. O. V. Salata, Applications of nanomaterials in biology and medicine. J. Nanobiotechnol. 2004, 2, 3. W. Fu, D. Shenoy, J. Li, C. Crasto, G. Jones, C. Dimarzio, S. Sridhar, M. Amiji, Biomedical applications of gold nanoparticles functionalized using hetero-bifunctional poly(ethylene glycol) spacer, Mater. Res. Soc. Symp. Proc. 2005, 845, AA5.4.1 C. Zandonella, Cell nanotechnology: The tiny toolkit, Nature 2003, 423, 10–12. J. L. West, N. J. Halas, Applications of nanotechnology to biotechnology: Commentary, Curr. Opin. Biotechnol. 2000, 11, 215–217. S. K. Sahoo, V. Labhasetwar. Nanotech approaches to drug delivery and imaging, Drug Disc. Today 2003, 8, 1112–1120. W. J. Parak, D. Gerion, T. Pellegrino, D. Zanchet, C. Micheel, C. S. Williams, R. Boudreau, M. A. Le Gros, C. A. Larabell, A. P. Alivisatos, Biological applications of colloidal nanocrystals, Nanotechnology 2003, 14, R15–R27. Q. A. Pankhurst, J. Connolly, S. K. Jones, J. Dobson, Applications of magnetic nanoparticles in biomedicine, J. Phys. D: Appl. Phys. 2003, 36, R167–R181. (a) M. Bruchez, M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013–2016. (b) W. C. W. Chan, S. M. Nie, Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science 1998, 281, 2016–2018. (c) S. Wang, N.

162

163

164

165

166

167

168

Mamedova, N. A. Kotov, W. Chen, J. Studer, Antigen/antibody immunocomplex from CdTe nanoparticle bioconjugates, Nano Lett. 2002, 2, 817–822. (a) C. Mah, I. Zolotukhin, T. J. Fraite, J. Dobson, C. Batich, B. J. Byrne, Microsphere-mediated delivery of recombinant AAV vectors in vitro and in vivo, Mol. Therapy 2000, 1, S239. (b) D. Panatarotto, C. D. Prtidos, J. Hoebeke, F. Brown, E. Kramer, J. P. Briand, S. Muller, M. Prato, A. Bianco, Immunization with peptide-functionalized carbon nanotubes enhances virus-specific neutralizing antibody responses, Chem. Biol. 2003, 10, 961–966. R. L. Edelstein, C. R. Tamanaha, P. E. Sheehan, M. M. Miller, D. R. Baselt, L. J. Whitman, R. J. Colton, The BARC biosensor applied to the detection of biological warfare agents, Biosensors Bioelectron. 2000, 14, 805–813. J. M. Nam, C. C. Thaxton, C. A. Mirkin, Nanoparticles-based bio-bar codes for the ultrasensitive detection of proteins, Science 2003, 301, 1884–1886. R. Mahtab, J. P. Rogers, C. J. Murphy, Protein-sized quantum dot luminescence can distinguish between ‘‘straight’’, ‘‘bent’’, and ‘‘kinked’’ oligonucleotides, J. Am. Chem. Soc. 1995, 117, 9099–9100. (a) J. Ma, H. Wong, L. B. Kong, K. W. Peng, Biomimetic processing of nanocrystallite bioactive apatite coating on titanium, Nanotechnology 2003, 14, 619–623. (b) A. de la Isla, W. Brostow, B. Bujard, M. Estevez, J. R. Rodriguez, S. Vargas, V. M. Castano, Nanohybrid scratch resistant coating for teeth and bone viscoelasticity manifested in tribology, Mater. Resr Innovat. 2003, 7, 110–114. J. Yoshida, T. Kobayashi, Intracellular hyperthermia for cancer using magnetite cationic liposomes, J. Magn. Magn. Mater. 1999, 194, 176–184. R. S. Molday, D. MacKenzie, Immunospecific ferromagnetic iron

References

169

170

171

172

173

174

175

176

dextran reagents for the labeling and magnetic separation of cells, J. Immunol. Methods 1982, 52, 353–367. R. Weissleder, G. Elizondo, J. Wittenburg, C. A. Rabito, H. H. Bengele, L. Josephson, Ultrasmall superparamagnetic iron oxide: Characterization of a new class of contrast agents for MR imaging, Radiology 1990, 175, 489–493. W. J. Parak, R. Boudreau, M. L. Gros, D. Gerion, D. Zanchet, C. M. Micheel, S. C. Williams, A. P. Alivisatos, C. A. Larabell, Cell motility and metastatic potential studies based on quantum dot imaging of phagokinetic tracks, Adv. Mater. 2002, 14, 882–885. R. M. Penner, Hybrid electrochemical/chemical synthesis of quantum dots, Acc. Chem. Res. 2000, 33, 78–86. X. Zhao, L. R. Hilliard, S. J. Mechery, Y. Wang, R. P. Bagwe, S. Jin, W. Tan, A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15 027–15 032. X. Gao, S. Nie, Molecular profiling of single cells and tissue specimens with quantum dots, Trends Biotechnol. 2003, 21, 371–373. X. Wu, H. Liu, J. Liu, K. N. Haley, J. A. Treadway, J. P. Larson, N. Ge, F. Peale, M. P. Bruchez, Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots, Nat. Biotechnol. 2003, 21, 41–46. B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, A. Libchaber, In vivo imaging of quantum dots encapsulated in phospholipid micelles, Science 2002, 298, 1759– 1762. Y. W. Jun, Y. M. Huh, J. S. Choi, J. H. Lee, H. T. Song, S. Kim, S. Yoon, K. S. Kim, J. S. Shin, J. S. Suh, J. Cheon, Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis

177

178

179

180

181

via magnetic resonance imaging, J. Am. Chem. Soc. 2005, 127, 5732– 5733. (a) I. H. El-Sayed, X. Huang, M. A. El-Sayed, Surface plasmon resonance scattering and absorption of antiegfr antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer, Nano Lett. 2005, 5, 829–834. (b) B. Dubertret, M. Calame, A. J. Libchaber, Singlemismatch detection using goldquenched fluorescent oligonucleotides, Nat. Biotechnol. 2001, 19, 365–370. I. L. Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Quantum dot bioconjugates for imaging, labeling and sensing. Nat. Mater. 2005, 4, 435–446. X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, S. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots, Nat. Biotechnol. 2004, 22, 969–975. (a) C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhofff, A DNA based method for rationally assembling nanoparticles into macroscopic materials, Nature 1996, 382, 607–609. (b) T. A. Taton, C. A. Mirkin, R. L. Letsinger, Scanometric DNA array detection with nanoparticle probes, Science 2000, 289, 1757–1760. (c) Y. C. Cao, R. Jin, C. A. Mirkin, Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection, Science 2002, 297, 1536–1540. (d) J. M. Nam, Savka I. Stoeva, C. A. Mirkin, Bio-bar-codebased DNA detection with PCR-like sensitivity, J. Am. Chem. Soc. 2004, 126, 5932–5933. (e) P. Fortina, L. J. Kricka, S. Surrey, P. Grodzinski, Nanobiotechnology: The promise and reality of new approaches to molecular recognition, Trends Biotechnol. 2005, 168–173. (a) K. K. Jain, Nanotechhnology in clinical laboratory diagnostics, Clin. Chim. Acta 2005, 358, 37–54. (b) C. M. Goodman, C. D. McCusker, T. Yilmaz, V. M. Rotello, Toxicity of gold nanoparticles functionalized with cationic and anionic side chains,

119

120

3 Nanogold in Cancer Therapy and Diagnosis

182

183

184

185

186

187

188

Bioconjugate Chem. 2004, 15, 897– 900. L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Shersen, B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas, J. L. West, Nanoshell-mediated nearinfrared thermal therapy of tumors under magnetic resonance guidance, Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13 549–13 553. M. Thomas, A. Klibanov, Conjugation to gold nanoparticles enhances polyethylenimines transfer of plasmid DNA into mammalian cells, Proc. Natl. Acad. Sci., U.S.A. 2003, 100, 9138–9143. C. Loo, A. Lowery, N. Halas, J. West, R. Drezek, Immunotargeted nanoshells for integrated cancer imaging and therapy, Nano Lett. 2005, 5, 709–711. J. Chen, F. Saeki, B. J. Wiley, H. Cang, M. J. Cobb, Z.-Y. Li, L. Au, H. Zhang, M. B. Kimmey, X. D. Li, Y. Xia, Gold nanocages: Bioconjugation and their potential use as optical imaging contrast agents, Nano Lett. 2005, 5, 473–477. J. Kneipp, H. Kneipp, W. L. Rice, K. Kneipp, Optical probes for biological applications based on surfaceenhanced Raman scattering from indocyanine green on gold nanoparticles, Anal. Chem. 2005, 77, 2381–2385. N. L. Rosi, C. A. Mirkin, Nanostructures in biodiagnostics, Chem. Rev. 2005, 105, 1547–1562. L. Huang, G. Reekmans, D. Saerens, J.-M. Friedt, F. Frederix, L. Francis,

189

190

191

192

193

S. Muyldermans, A. Campitelli, C.-V. Hoof, Prostate-specific antigen immunosensing based on mixed selfassembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays, Biosensors Bioelectron. 2005, 21, 483–490. M. Everts, J. L. Leddon, R. J. Kok, M. A. Preuss, C. L. Millican, D. E. Nikles, D.-T. Johnson and D.-T. Curiel, Gold nanoparticles as an amplifying payload strategy for adenoviral cancer gene therapy, Mol. Therapy 2005, 11, 342. G. F. Paciotti, L. Myer, D. G. I. Kingston, T. Ganesh, L. Tamarkin, Colloidal gold nanoparticles: A versatile platform for developing tumor targeted cancer therapies, Nanotech 2005, NSTI Nanotechnology Conference and Trade Show, NSTI, Anaheim, CA, 2005, 1, 7–10. G.-F. Paciotti, L.-D. Myer, T.-H. Kim, S. Wang, H.-R. Alexander, D. Weinreich, L. Tamarkin, Colloidal gold: A novel colloidal nanoparticle vector for tumor-directed drug delivery, Clin. Cancer Res. 2001, 7, 3673S–3674S. V. P. Zharov, R. R. Letfullin, E. N. Galitovskaya, Microbubblesoverlapping mode for laser killing of cancer cells with absorbing nanoparticle clusters, J. Phys. D: Appl. Phys. 2005, 38, 2571–2581. E. E. Connor, J. Mwamuka, A. Gole, C.-J. Murphy, M. D. Wyatt, Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity, Small 2005, 1, 325–327.

121

4

Nanoparticles for Magnetic Resonance Imaging of Tumors Tillmann Cyrus, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza 4.1

Introduction

Molecular imaging with nanoparticulate agents represents a novel tool that will allow the detection of tumors and metastases in early stages that evade conventional imaging technologies. Several of these nanoparticulate agents are also undergoing development to deliver drugs locally, thus increasing therapeutic effects at the tumor site while minimizing systemic adverse effects that are often limitations of conventional chemotherapy. The combination of molecular imaging and local delivery of therapeutic agents is unique to nanoparticulate agents and regarding tumor detection and treatment will likely change the current medical paradigm of ‘‘see and treat’’ to a ‘‘detect and prevent’’ strategy. An overview of this technology is given here, focusing on particles that are available for non-invasive magnetic resonance imaging (MRI). Principle targeting mechanisms are described, followed by superparamagnetic nanoparticles, which create negative or dark contrast effects on MRI, and paramagnetic nanoparticles, which produce positive or bright contrast effects. Superparamagnetic nanoparticles all contain magnetic elements, mostly iron oxides, and are sub-classified according to molecular modifications and size. Paramagnetic nanoparticles differ substantially and thus are presented according to their principal groups, such as liposomes, perfluorocarbon nanoparticles, fullerenes, and others. Finally, hybrid technologies using quantum dots are presented, which allow non-invasive imaging with MRI and intraoperative direct visualization. As these technologies are rapidly evolving, particular emphasis is given to the most current developments.

4.2

Magnetic Resonance Imaging (MRI)

Originally developed as a noninvasive method using a magnetic field and radio waves to generate detailed images of the inside of the human body, MRI is based Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

122

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

on the principles of nuclear magnetic resonance (NMR). Atoms consist of protons, electrons, and often neutrons. All of these particles possess a spin. Depending on its composition, some nuclei have a net spin that, when placed in an external magnetic field ðB0 Þ, aligns with the external magnetic field, somewhat like a magnet with a north and south pole would. For a hydrogen nucleus, for example, there are two possible energy states: the lower energy configuration is parallel to the external magnetic field (e.g., N-S-N-S), and the higher energy state is anti-parallel (e.g., N-N-S-S). For a given nuclei, the energy of separation between spin states depends on the magnitude of B0 . The parallel state is slightly favored, on the order of a few parts per million, and is described by Boltzmann statistics (as a function of the energy of separation and temperature). Thus, for about every one million nuclei, there is one extra aligned with the B0 , field resulting in a net magnetization pointing in the direction of the main magnetic field; these spinning nuclei do not perfectly align with B0 but rather precess about it at their resonance frequency, also called the Larmor Frequency, which is defined as the product of the gyromagnetic ratio ðgÞ of the nucleus and the external magnetic field. Typically, these individual ‘‘spins’’ are thought of collectively as a net magnetic vector, aligned with B0 . Individual particles can undergo a transition from the lower to the higher energy state by absorbing a photon, and vice versa by emitting one. In MRI, the required frequency for this photon is in the radiofrequency (RF) range at the resonance or Larmor frequency, which is the product of the gyromagnetic ratio and B0 . When exposed to RF energy at this frequency the nuclei change spin states and, when thought of en masse, can be represented by a net magnetic vector, which ‘‘rotates’’ away from the longitudinal axis, but returns to alignment once the external RF transmission is ceased. The rate of this return to equilibrium (known as longitudinal or spin–lattice relaxation) is exponential and can be described by the time constant T1. When the net magnetic vector is aligned with B0 , an MR signal cannot be measured. But with the net magnetic vector ‘‘tipped’’ out of alignment, the component of the magnetization vector in the transverse plane (Mxy) can be measured as an MR signal (using an external RF antenna such as a coil of wire tuned to the correct resonance frequency). Since the individual spins comprising the net vector experience slightly different magnetic milieus, the spins have slightly different precessional frequencies and, thus, the magnitude of Mxy (transverse magnetization) decays over time (independently of longitudinal relaxation). This signal decay is also exponential, with time constant T2. The detected signal, created by exciting the spins out of longitudinal alignment with B0 , is the source for MR image generation is based on the absorption and emission of energy in the radiofrequency range of the electromagnetic spectrum. Hence, the signal in MRI results from the difference between the energy absorbed by the spins that make a transition from the lower energy state to the higher energy state and the energy emitted by the spins that simultaneously make a transition from the higher energy state to the lower one. Although highly sensitive, NMR can only be performed on isotopes whose natural and biological abundance is high enough to be detected. The hydrogen in water

4.2 Magnetic Resonance Imaging (MRI)

is an ideal atom is this respect. Its nucleus contains a single proton and has a large magnetic moment. Of the three hydrogen isotopes, the natural abundance of 1 H is 99.985%. The 1 H nuclei in water have an MR signal, and the human body consists primarily of water. While bones only contain about 22% water, muscle is 75% and blood up to 83% water, explaining the advantage of MR in imaging soft tissues including tumors. To generate an image, the MRI machine applies an RF pulse ðB1 Þ at the specific resonance frequency of hydrogen. This excitation pulse is localized toward the area of the body that is of interest by simultaneously superimposing a secondary magnetic field, which is a function of position (i.e., gradient magnetic field). This gradient magnetic field causes the resonance frequency of hydrogen nuclei to vary as a function of position within the MRI magnet; thus, a finite ‘‘slice’’ of interest can be excited to give MR signal since the nuclei can absorb energy (to change spin state) only at their resonance frequency. As described earlier, once this RF excitation pulse is turned off, the 1 H nuclei begin to return to equilibrium with B0 . But while some transverse magnetization remains, the signal that will ultimately be reconstructed into the MR image can be detected. This signal is already localized upon excitation, via ‘‘slice selection,’’ in one dimension. The remaining two dimensions are encoded by quickly switching on and off other gradient magnetic fields before (phase encoding) and during (frequency encoding) the readout of the generated signal. By repeating this process multiple times, data is collected until the signal is completely spatially encoded and the final image can be reconstructed. The signal intensity for each location in the image (volume element, or voxel) is dependent on multiple things, including the number of 1 H nuclei present (proton density), and the T1 and T2 of the tissue present. T1 relaxation is characterized by the longitudinal return of the net magnetization to its ground state in the direction of the main magnetic field. It also depends on the main magnetic field strength, i.e., higher magnetic fields are generally associated with longer T1. T2 relaxation occurs when spins in the high and low energy state exchange energy but do not lose it to the surrounding area. Thus, transverse magnetization is lost. In pure water, T1 and T2 are approximately the same, but in biological tissues T2 is considerably shorter. These differences are exploited in conjunction with imaging sequence timing parameters to obtain either T1- or T2-weighted images. Similarly to other imaging technologies, MR imaging benefits from the usage of contrast agents. Conventional MR contrast agents work by altering the local magnetic field and relaxation parameters in the tissue being examined. Superparamagnetic iron oxide particles, for instance, generate image contrast by shortening the MR relaxation times with a predominant effect on apparent T2 relaxivity, which leaves dark contrast effects (i.e., signal voids; Fig. 4.1). Paramagnetic particles such as gadolinium chelates, however, produce bright contrast in T1-weighted MR images (Fig. 4.2). The advent of nanoparticulate contrast agents afforded a dramatic improvement in imaging. These agents, which are described in this chapter, can passively or actively target epitopes within the body, enhance imaging, and, in some cases, even deliver drugs locally.

123

124

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

T2-weighted MRI. (A) Conventional MRI and (B) MRI obtained 24 h after the administration of lymphotrophic superparamagnetic nanoparticles. The accumulation of the iron-containing

Fig. 4.1.

superparamagnetic nanoparticles leads to a homogeneous decrease in signal intensity in a lymph node in the left iliac region (arrow). (Reproduced with permission from Ref. [27].)

4.3

Targeting Mechanisms 4.3.1

Passive versus Active Targeting

Nanoparticulate agents concentrate within a site by passive and/or active targeting mechanisms (Fig. 4.3). Passive targeting refers to the accumulation within or the extravasation through a tumor neovasculature distinguished by a dismorphic, fenestrated architecture [1]. Vascular endothelial and basic fibroblast growth factors produced by tumors accelerate angiogenic sprouting with endothelial pores between 200 and 780 nm in diameter that entrap or permit extravasation of macro-

T1-weighted MRI. (A) Conventional MRI and (B) MRI obtained 30 min after the administration of perfluorocarbon nanoparticles. C32 tumor in an athymic nude

Fig. 4.2.

mouse (arrow in A). Signal enhancement of angiogenic vasculature as detected by an b 3 targeted paramagnetic nanoparticles (B).

4.3 Targeting Mechanisms

Schematic illustration of passive and active targeting with nanoparticles. (A) Passive targeting, via the enhanced permeability and retention (EPR) effect due to pathological endothelialization of angiogenic vessels in tumors, allowing the leakage of very small nanoparticles into the subendothelium and

Fig. 4.3.

into the space between tumor cells. (B) Active targeting with very small ligand-targeted nanoparticles. These particles bind respective epitopes on tumor cells. (C) Active targeting with ligand targeted nanoparticles with nominal diameters > 150 nm.

molecules and nanoparticles [2, 3], dependent on size. In many tumors, ineffective lymphatic drainage further contributes to the passive, local retention of nanoparticles [4]. This combination enhances the permeability and retention (EPR) effect [4, 5]. In some situations phagocytic cells, naturally responsible for particle clearance, ingest the agent while migrating into the tumors core. Particles are removed from the circulation in a size-dependent hierarchy by the lung (largest), spleen, liver, and bone marrow (smallest). Opsonization (i.e., biological tagging) with blood proteins such as immunoglobulins, complement proteins, or non-immune serum factors can enhance macrophage recognition, phagocytosis, and further accelerate circulatory clearance. While complement fixation promotes liver sequestration, the presence of antibody Fc receptors favors splenic removal.

125

126

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

Active targeting encompasses ligand-directed, site-specific accumulation of nanoparticulate agents. Antibodies, peptides, polysaccharides, aptamers, and drugs, may be utilized to home nanoparticles specifically to cellular biomarkers. These ligands may be attached covalently (i.e., chemical conjugation) or non-covalently (e.g., avidin–biotin interactions) to the contrast agent. Further surface chemistry modifications, such as incorporation of surface poly(ethylene glycol) or crosslinking of surfactants, are employed to delay rapid systemic destruction or clearance of the agents. Homing-ligands increase contrast signal and therapeutic uptake in target organs beyond the concentrations achieved with passive approaches – benefits usually achieved with active ingredient dosages orders of magnitude less than what would be given in the clinic today.

4.4

Superparamagnetic Nanoparticles

Superparamagnetic iron oxide nanoparticles generate contrast by shortening the MR relaxation times with a predominant effect on T2 relaxivity leaving dark or negative contrast effects (Fig. 4.1). These particles are categorized, based upon nominal diameter, into superparamagnetic iron oxides (SPIO, 50 to 500 nm) and ultrasmall superparamagnetic iron oxides (USPIO, <50 nm). The variation in size results in different physicochemical and pharmacokinetic properties. SPIO nanoparticles with maghemite or magnetite cores have to be coated with molecules such as dextran, phospholipids, or poly(amino acids) to prevent aggregation. These particles passively localize to normal liver and spleen parenchyma, causing a loss of signal on MR images [6–12]. In contrast, pathological changes in the liver, such as benign tumors, primary hepatic carcinoma, or metastases, have decreased reticuloendothelial system (RES) function, thus avoiding absorption of SPIO particles and retaining their intrinsic signal intensity on MR images. The resulting MR images depict decreased MR signal intensity of the normal liver and spleen tissues and enhanced contrast of focal disease areas. Modifying SPIOs to allow targeting of asialoglycoprotein receptors on hepatocytes can further enhance absorption by native liver cells. These nanoparticles are coated with asialofetuin [13] or galactose [14, 15]. A water-dispersible oleic acid (OA)-pluronic-coated iron oxide magnetic nanoparticle formulation, which can be loaded with high doses of hydrophobic anticancer agents like doxorubicin, has recently been developed [16]. The formulation increased the average particle diameter of the iron oxide particles from 9.3 to 193 nm, but had no effect on the magnetic properties of the core. These nanoparticles demonstrated sustained intracellular drug retention and dose-dependent antiproliferative effect over a period of two weeks in vitro. Ultrasmall particles of iron oxide (USPIOs) with a mean diameter of 10 to 50 mm are cleared slower by the RES than SPIOs, thus increasing their intravascular half-life [17]. Moreover, their small size allows them to migrate through interendothelial junctions and capillary pores or fissures [18, 19] to potentially accumu-

4.4 Superparamagnetic Nanoparticles

late in the vascular wall. Such USPIOs are phagocytosed by macrophages in atherosclerotic plaque of WHHL rabbits in quantities sufficient to be detected by MRI [20], and have been shown to detect atherosclerotic plaque in vivo [21]. Extravasation of USPIOs has been imaged by MRI in the setting of capillary damage associated with infections [22] and inflammation [23], human ischemic stroke [23], angiogenesis associated with atherosclerosis [21], and neovascularity induced by solid tumors [24–26]. 4.4.1

Ligand-directed Targeting of Iron Oxides

Monocrystalline iron oxide nanoparticles (MION) have been developed to address the limitations of passive tissue accumulation and phagocytosis by macrophages and the RES system. These particles have an average core diameter of 3 nm and can be directly coupled to homing ligands to specifically target epitopes in the tissue of interest. An early clinical trial was based on MION without such ligands at the time [27]. Starting in 1999, patients with prostate cancer were selected to receive MION, delivering 2.6 mg kg
1 body weight. The investigators were able to demonstrate non-invasive detection by MRI of clinically occult lymph node metastases. Dextran-coated MION coupled to human holo-transferrin (Tf-MION) have been used to visualize transgene expression in a gliosarcoma mouse model in vivo [28]. In these experiments, the cellular uptake of MION was increased approximately 500% relative to control cells following overexpression of engineered transferrin receptor. In other experiments MION have been used to indirectly assess angiogenesis through estimates of blood volume distribution in brain tumors [29]. The targeting efficiency of iron oxide particles was further improved with the development of dextran crosslinked iron oxide (CLIO) particles [30]. These have since been used with various ligands, including e-selectin [31], a peptide sequence from the transactivator protein (Tat) of HIV-1 [32–35], and annexin V [36]. Another example of targeted iron oxide nanoparticles are SPIO coupled to humanized biotinylated monoclonal antibody (Herceptin) that was targeted to the human Her-2/neu (c-erb B-2) tyrosine kinase receptor expressed by human breast cancer cell lines [37]. This formulation yielded contrast proportional to the expression level of Her-2/neu receptors. Other investigators have employed polyacrylamide to encapsulate 10–15 crystals of iron oxide within the particle matrix, for blood pool imaging of gliomas [38]. Future applications of these nanoparticles may include direct targeting of ligands. The combination of superparamagnetic and non-superparamagnetic nanoparticle technologies such as near-infrared fluorescent (NIRF) probes, which can be targeted to different proteases in tissues [39, 40], has also been employed. Upon phagocytosis by macrophages, detection is facilitated by light scattering with charge-coupled device (CCD) cameras [41] or fluorescence-mediated tomography (FMT) [42, 43]. CLIO-NIRF nanoprobes have been used to coincidently localize axillary and brachial lymph nodes by MRI and NIRF imaging following intravenous

127

128

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

injection into C57BL/6 mice [44]. The utilization of CLIO-NIRF for non-invasive preoperative and subsequently intraoperative localization of brain tumors has also been established. In experiments with a Fisher 344 gliosarcoma model with stably green fluorescence protein expressing 9L glioma cells, non-invasive brain tumor localization of the superparamagnetic particles with MRI was followed later by intraoperative direct visualization of the fluorescent activity [45]. In conjunction with T2-weighted imaging, magnetite particles can be used to augment targeting through external field placement and create vibratory release of drug at localized sites [46]. One early agent pursuing this development path targets luteinizing hormone releasing hormone (LHRH) overexpressing breast cancer cells and releases a lytic peptide load when the particles fracture in response to magnetic field changes [47]. 4.4.2

Cell Tracking of Iron Oxides

Magnetic labeling of individual cells prior to reaching their target location allows advantage to be taken of the strong effects of iron oxide on T2 relaxation while increasing spatial resolution due to diminished background disturbance. The concept of cellular tracking became feasible once augmentation of cytoplasmic endocytosis of iron oxide particles was accomplished. An early example of this technology resulted from the exploitation of cell surface proteins, such as the transferrin receptor [48]. In these experiments, dextran-coated MION were incubated with CG-4 rat central nervous system glial precursor cells in vitro and then the cells were grafted into the spinal cord of rats. The migration, implantation, and remyelination process was imaged non-invasively by MR and corroborated by histology 10–14 days after transplantation. Subsequently, MION coated with various transfection agents have been used to nonspecifically label human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor cells, and human cervical carcinoma cells [49]. Non-invasive tracking of human mesenchymal and neuronal stem cells has also been reported for iron oxide nanoparticles encapsulated within cationic dendrimers [50]. CD8þ T-cells have been labeled with CLIO derivatized with monoclonal antibodies in a mouse melanoma model [51], and stem cell homing to bone marrow has been tracked after labeling with CLIO coupled with HIV-Tat peptides. Particle uptake has been increased over 100-fold using such specific targeting conjugates [33, 52].

4.5

Paramagnetic Nanoparticles

Paramagnetic nanoparticles produce ‘‘bright’’ contrast in T1-weighted MR images (Fig. 4.2). This allows subtle anatomical features to be discerned near the contrast agent. Nanoparticles can carry large paramagnetic payloads on the surface to provide adequate signal for noninvasive MR imaging at clinically relevant field

4.5 Paramagnetic Nanoparticles

Schematic illustration of a perfluorocarbon nanoparticle. Imaging agents such as gadolinium, targeting ligands, and drugs are covalently bound into the phospholipid monolayer. Phospholipids and drugs

Fig. 4.4.

within the nanoparticle surfactant exchange with lipids of the target membrane through a convection process after successful targeting. (Nominal particle diameter is 250 nm.)

strengths. Linking these nanoparticles with ligands to specific epitopes facilitates active targeting. Perfluorocarbon emulsions, dendrimers, fullerenes, and liposomes are the most prevalent paramagnetic nanoparticle technologies currently in various stages of development for clinical applications. 4.5.1

Perfluorocarbon Nanoparticles

The large lipid surface area of perfluorocarbon (PFC) nanoparticles can be functionalized with magnetic labels for imaging, with homing ligands for specific tissue targeting, and with hydrophobic drugs for local delivery of treatment (Fig. 4.4). Due to the nominal size of the PFC nanoparticles (250 nm diameter), their utility is geared toward vascular-accessible targets, such as thrombosis, atherosclerosis, restenosis, and other angiogenic-dependent diseases, such as most tumors. Access beyond the circulation is sterically precluded, which prevents the unintended targeting of other cell types expressing the same biomarkers. Each nanoparticle can be coupled to several diverse ligands to afford polyvalent binding, which can amplify the signal from either microscopic pathology, where epitope concentrations are low, or where biologic temporal variation requires multiple biosignature recognition for robustness. Binding to multiple ligands also enhances binding avidity and improves targeting, which also reduces the particle dissociation rate from the biomarker and provides more persistent MR contrast for convenient imaging in a clinical setting. PFC nanoparticle emulsion technology offers the particular advantage of multimodal imaging. The inherent acoustic contrast of PFC nanoparticles allows detection of these emulsions when targeted and bound in vivo or in vitro due to the

129

130

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

markedly decreased speed of sound of the PFC core versus the surrounding waterlike media [53, 54]. The high 19 F concentrations of the perfluorocarbon provide a unique signal, which can be used for both imaging and spectroscopy. In addition, the integration of fluorine imaging with proton imaging provides an independent validation of the targeted contrast, quantitative assessment of biomarker concentration, and dosimetry of local therapeutic delivery. For MRI imaging, each bound paramagnetic nanoparticle delivers 50 000 to 90 000þ gadolinium ions [55, 56]. These can be detected with low-resolution scans in routine clinical practice. The relaxivity of paramagnetic nanoparticles allows the calculation of ‘‘ionic relaxivity’’ with respect to absolute Gd concentration, and the determination of ‘‘molecular relaxivity’’ referring to the collective relaxivity of the entire paramagnetic nanoparticle. For applications with a 1.5 T scanner, perfluorocarbon nanoparticles have an ionic relaxivity of @30 mm
1 s
1 and a molecular relaxivity of greater than 2  10 6 mm
1 s
1 , which is many fold greater than the ionic and molecular relaxivity of Gd-DTPA alone (@5 mm
1 s
1 ) [57]. Due to this extraordinarily high relaxivity a voxel with as few as 100 pm of nanoparticles can be detected conspicuously with a contrast-to-noise ratio (CNR) of 5 [58]. Consequently, even single layers of cells can be imaged [59]. Various molecular epitopes, including high-density epitopes such as fibrin in thrombi and very sparse biomarkers such as integrins in neovascular beds have been targeted with perfluorocarbon nanoparticles. In angiogenic states, a v b 3 integrin is expressed on the luminal surface of activated endothelial cells and smooth muscle cells but not on mature quiescent cells. Nanoparticles targeted to a v b 3 -integrin sensitively detect angiogenic endothelium at 1.5 T in New Zealand White rabbits bearing 12d Vx-2 tumors (<1.0 cm) [60]. In these experiments, the expression of a v b 3 -integrin was obtained with MRI molecular imaging and paralleled by immuno-histochemical staining, which revealed an asymmetric distribution along the border of the tumor capsule where the popliteal fossa mass abutted the muscle border. The MRI signal from tumor vasculature was enhanced by 126% within 2 h of injection of a v b 3 -integrin-targeted nanoparticles. Additional in vivo competition-blockade studies diminished targeted signal enhancement by more than 50%, which supports specificity of the a v b 3 -targeted paramagnetic agent. In different experiments, an b 3 -integrin-targeted nanoparticles were administered systemically into athymic mice implanted with human melanoma xenografts (C-32, ATCC). The MR signal enhancement from the targeted angiogenic vasculature was apparent in 0.5 h and became progressively more prominent through the next 2 h (177%). Here too, in vivo competition studies and immuno-histology corroborated the specific localization of an b 3 -targeted nanoparticles (Fig. 4.5). Recent studies have demonstrated the unique drug delivery capability of these agents to locally deliver antiangiogenic therapy through a process we term ‘‘contact facilitated drug delivery’’. Atherosclerotic rabbits were treated with an b 3 -targeted paramagnetic nanoparticles including 0 or 0.2 mol% fumagillin. MRI signal enhancement averaged over all imaged slices from the renal artery to the diaphragm at 2-h post injection provided quantitative assessments of neovascular proliferation within the aortic wall. MR baseline aortic wall signal enhancement produced

4.5 Paramagnetic Nanoparticles

Fig. 4.5. (A) Full slice baseline T1 -weighted MR image (axial view) of an athymic nude mouse. (B) Full slice T1 -weighted MR image (axial view) of athymic nude mouse 1 h after injection of targeted PFC nanoparticle. (C) Enlarged section of MR image (B). T1 -weighted

signal enhancement of angiogenic vasculature of early tumor at 1 h as detected by an b 3 targeted paramagnetic nanoparticles (arrow). [Reproduced with permission from G. M. Lanza et al. J. Nucl. Cardiol. 11(6) (2004) 733– 743.]

by an b 3 -targeted paramagnetic nanoparticles was heterogeneously distributed throughout the great vessel and averaged 16:7 G 1:1% and 16:7 G 1:6% in animals receiving fumagillin or control nanoparticles, respectively. Reassessment 7 days later of residual aortic angiogenic activity with an b 3 -targeted paramagnetic nanoparticles (no drug) revealed that MR signal enhancement in rabbits given an b 3 targeted fumagillin nanoparticles was markedly reduced (2:9 G 1:6%; p < 0:05), whereas the aortic wall enhancement from the control animals was unchanged (18:1 G 2:1%). The combination of MR molecular imaging with drug delivery permits local therapeutic concentrations to be estimated, i.e., rational drug dosing, by quantitative MR 19 F spectroscopy [58, 59]. Hence, targeted paramagnetic nanoparticles can serve as a platform to diagnose, treat, and monitor therapy. 4.5.2

Liposomes

Liposomes were first described in 1963 following the demonstration that phospholipids in water form closed bilayer vesicles with an aqueous core [61, 62]. The size of these liposomes can reach >700 nm, dependent on the size of the core components and the number of bilayers. Hydrophilic payloads may be stored in the inner aqueous core, hydrophobic ones can be embedded in the phospholipid bilayer and small proteins may be carried on the outer surface. The clinical potential of liposome technology was previously hampered by the short circulation halflives secondary to rapid phagocytosis by macrophages of the reticuloendothelial

131

132

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

systems. However, the adoption of surface PEGylation and phospholipid crosslinking has helped to reduce the rapid in vivo destruction and clearance of liposomes [63–65]. Numerous studies have investigated various liposome formulations in their potential to target and treat cancer in animal models (reviewed in Ref. [66]). Using catheter-based techniques, liposome formulations were shown to deliver genes in vivo [67, 68] and, more recently, taking advantage of developments in molecular biology, liposome-mediated drug and gene transfer has been demonstrated for lipid prodrug liposomes [69], and small interfering RNA (siRNA) cationic liposomes [70, 71]. Several studies in humans have led to the approval of liposomal formulations based on doxorubicin, (DoxilTM , ALZA Corporation, Tibotec Therapeutics, NJ) for treatment ovarian cancer and AIDS-related Kaposi’s sarcoma, and daunorubicin (DaunoXome2, Gilead Sciences Ltd., UK) for the treatment of advanced Kaposi’s sarcoma. Many more applications are being investigated in phase II trials [72, 73]. Generally, these liposomal agents accumulate in tumor tissues via passive targeting using the EPR effect. In the treatment of brain tumor models, direct injection and convection-enhanced delivery (CED) to overcome the blood– brain barrier have been employed [74, 75]. Because many cancer cells overexpress certain receptors compared with healthy tissues, avenues of active targeting with liposomes that have been fused with agonists to these receptors have been explored. For instance, liposomes have been fused with low-density lipoprotein (LDL) particles, thereby enhancing uptake into cancer cells, which frequently overexpress these receptors [76], and haloperidol-associated stealth liposomes have been used successfully to deliver genes to breast cancer cells [77]. MRI facilitated in vivo monitoring of a liposomal doxorubicin formulation utilizing MnSO4 as the paramagnetic metal was recently investigated in a murine flank tumor model. This novel thermal-sensitive liposomal formulation took advantage of the relaxivity of Mn, which is similar to Gd. Visualization was possible within minutes following IV liposomal infusion as the temperature-sensitive particles entered the heated tumor, ruptured, and released MnSO4 locally [78]. Rabbit adenocarcinoma neovasculature has been imaged with a newly developed an b 3 -targeted paramagnetic UV polymerized liposome. In these experiments, adequate tumor contrast was appreciated 24 h after injection [79], reflecting both the prolonged circulatory persistence of the nanovesicles and the time required to extravasate adequately into the tumor. After incorporation of cationic phospholipids into the polymerized bilayer, delivery of a mutant Raf gene to the neovasculature of M21-L melanoma was accomplished in athymic WEHI mice [80]. ATPmu-Raf, the mutant antisense gene, resulted in apoptosis of the tumor-associated endothelium and ultimately induced tumor regression. 4.5.3

Fullerenes

Fullerenes, also referred to as buckyballs due to their characteristic geodesic structure, have unique physical, electrochemical, and photochemical properties [81]. These unique geodesic structures can be formulated to encapsulate gadolinium

4.6 Quantum Dots

atoms for imaging with MRI and have been functionalized for usage as receptor agonists and antagonists, free radical scavengers, and bactericidal agents [82]. Prolonged circulation, up to 48 h, has been demonstrated following intravenous injection for water-soluble gadolinium endohedral metallofullerenes [83, 84]. Recently, fullerene formulations have been developed that can influence proton relaxivities by manipulation of the pH [85]. A remaining challenge is the homing of paramagnetic buckyballs to important biochemical epitopes for molecular imaging at clinically relevant field strengths [86] as well as elucidation of their interaction with blood constituents, biodistribution, metabolism, and safety. 4.5.4

Nanotubes

Ultrashort single-walled carbon nanotubes (20–100 nm), which are linear superparamagnetic ‘‘molecular magnets’’, have been loaded with gadolinium ions for possible usage as MRI contrast agents. The relaxivities (@170 mm
1 s
1 ) of these nanotubes are greater than for other gadolinium-based contrast agents currently in clinical use [87]. Studies are ongoing regarding the ideal length for biomedical applications, biocompatibility, and exploitation of the tube exterior to anchor targeting ligands and or drugs. 4.5.5

Dendrimers

Dendrimers are hyperbranched, structurally well-defined polymers. They are based upon polyamines, polyamides, or polyaryl-ether subunits, and have varying core structures such as carbohydrate or calixarene [88]. Paramagnetic polyamidoamine (PAMAM) and diaminobutane (DAB) dendrimers have been developed for MRI applications. Several spherically branched molecules [89] have been evaluated in several animal models to detect intratumoral vasculature [90] and lymph vessels [91–93] with high-resolution MRI on clinical 1.5 T MRI scanners. Blood pool imaging has been accomplished with conjugated dendrimers [94, 95]. However, conjugation with antibodies for targeting has tended to unfavorably alter their pharmacokinetics [96]. Recently, improved kinetics have been obtained with small molecule, e.g., folate, based targeting applications [97, 98]. Future formulations need to address the question of toxicity, which can be high, especially for dendrimers with cationic surface groups and amino-terminated dendrimers [99, 100]. The most promise for medical applications has been shown for anionic PAMAM dendrimers and hydroxy- and methoxy-terminated dendrimers [101–103].

4.6

Quantum Dots

Semiconductor nanocrystals, also known as quantum dots, absorb light over a very broad spectral range, depending on the particle composition and size (typically 2–8 nm in diameter). This allows for the excitation of a broad spectrum of colors using

133

134

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

a single excitation laser wavelength [104]. When conjugated to targeting proteins such as transferrin or antibodies, these nanocrystals can bind to surface receptors on cells [105], be incorporated by cells [106], and even nuclear localization has been demonstrated [104]. Thus, their use for in vitro diagnostic applications has been extended to include in vivo small animal imaging applications. Recently, in vivo cancer targeting has been demonstrated in a mouse model growing human prostate cancer [107]. In these experiments, antibodies against prostate-specific membrane antigen were conjugated to nanocrystals, allowing for active targeting. Currently, linking quantum dots to therapeutic, e.g., anticancer, agents is being investigated for applicability. Unlike other nanoparticles, which can carry targeting ligands and therapeutic agents, semiconductor nanocrystals, due to their small size, will likely have to be conjugated to other nanoparticles to achieve specific targeting, while allowing for real-time imaging and drug delivery. The first such multimodality probes have recently been developed by integrating quantum dots with superparamagnetic nanoparticles [108, 109]. The main concerns with quantum dot technology, however, are biocompatibility, biodistribution, metabolism and biosafety. Nanocrystals can contain elements such as cadmium and indium and various techniques have been employed to make these particles more water-soluble and safer. One method is to modify the particles by growing a silica layer onto the surface of the quantum dots [110] or coating the dots with amphiphilic polymers [111, 112]. Another avenue of protecting the nonbiocompatible elements of the quantum dots involves conjugation with targeting molecules [111, 112]. While polymer-encapsulated nanocrystals may be biocompatible for immediate imaging, the issue of toxicity during breakdown and elimination of these particles from the body requires further study.

4.7

Polymer Nanoparticles

Nanoparticles formulated from poly(hydroxy acids) such as the copolymer of poly(lactic acid) (PLA) and poly(d,l-lactide-co-glycolide) (PLGA) have been investigated for localized drug and gene delivery [113, 114]. These particles are biodegradable, like absorbable suture materials, and result in a similar inflammatory response during degradation [115]. Drugs can be encapsulated into the nanoparticles during the emulsification process and are later released during the decay of the nanoparticle. Depending on size, the entire nanoparticle can be incorporated into cells through phagocytosis. Degradation kinetics, and thus drug release, can be adjusted by changing copolymer composition and molecular weights [116]. In recent experiments, transferrin targeted nanoparticles containing doxorubicin afforded prolonged survival in a murine prostate cancer model [117]. The particle size averaged 220 nm and the treatment was given as a single direct intratumoral injection into the subcutaneously grown prostate cancer in athymic nude mice. Sustained release of drug was observed, as compared with the paclitaxel Cremophor1 EL formulation, and complete regression of tumor was observed with the higher nanoparticle associated paclitaxel dosage (24 mg kg
1 ) compared

4.8 Conclusion

with (in order of decreasing survival) lower dosage (12 mg kg
1 ), non-transferrin targeted paclitaxel nanoparticles, paclitaxel Cremophor1 EL, or Cremophor1 EL alone and control nanoparticles. These researchers took advantage of the overexpression of transferrin receptors by many tumors, yet the nanoparticles were still injected directly into the tumor in these experiments and systemic application may prove less effective since many normal tissues express transferrin receptors [118]. Recently shell-crosslinked nanoparticles derivatized with gadolinium were studied for their capability for polyvalent targeting [119]. These particles demonstrated large ionic relaxivities (@39 mm
1 s
1 ) and a high molecular relaxivity of 20 000 mm
1 s
1 . Such particles have been conjugated with folate and shown to result in higher tumor uptake in small tumors overexpressing the folate receptor than controls [120]. Furthermore, folate nanoparticle uptake in the smaller tumors was competitively inhibited by excess folate, supporting ligand-based targeting. 4.8

Conclusion

Nanoparticulate agents, such as superparamagnetic agents, perfluorocarbon nanoparticle emulsions, liposomes, fullerenes, dendrimers, are being intensively studied for various clinically relevant applications, particularly for magnetic resonance imaging (Table 4.1). The detection of tumor or metastasis development in the early stages appears feasible with nanoparticles targeted to unique or abundant epitopes on tumor tissues (Table 4.2). Nanoparticle-facilitated signal enhancement Tab. 4.1.

Nanoparticle classification.

Nanoparticles

Ref.

Superparamagnetic SPIO USPIO MION CLIO PAM Magnetite

16, 37 24–26 27, 28, 49 44, 45, 51 38 46, 47

Nanoparticles

Ref.

Paramagnetic PFC

53–60

Liposomes

63, 66, 70–80, 121

Fullerenes

83–86

Nanotubes

87, 122

Magneto-fluorescent NIRF

39–45

Dendrimers

89–95, 97–99, 102, 103

Fluorescent Quantum dots

107, 112

Polymer nanoparticles

114, 117, 119, 120

SPIO – Superparamagnetic iron oxides; USPIO – ultrasmall superparamagnetic iron oxides; MION – monocrystalline iron oxide nanoparticles; CLIO – crosslinked iron oxide nanoparticles; PAM – polyacrylamide magnetic nanoparticles; NIRF – near-infrared fluorescent nanoparticles; PFC – liquid perfluorocarbon nanoparticles.

135

Species/model

In vitro In vitro Mice Rats Mice In vitro Human trial Rats Mice Rats Mice Rabbits Mice In vitro

SPIO

SPIO

USPIO

USPIO

MION

MION

MION

CLIO

CLIO

PAM

Magnetite

PFC

Quantum dots

Quantum dots

Human SK-BR-3 breast cancer cells

Subcutaneously implanted human prostate cancer

Vx-2 tumors

LHRH expressing human breast cancer cells

9L gliomas

Melanoma

9L gliosarcoma

Prostate cancer

Human cervical carcinoma cells

Transferrin receptor expressing 9L gliosarcoma

Breast cancer

VEGF-expressing Mel57 (human melanoma) tumors in mouse brains

Human Her-2/neu receptor expressing breast cancer cells

Breast and prostate cancer

Cancer

MRI applications of nanoparticles for cancer diagnosis and treatment.

Nanoparticle

Tab. 4.2.

A

A

A

A

P

Cellular labeling

A

P

Cellular labeling

A

P

P

A

P

Targeting: active (A); passive (P)

N/A

N/A

N/A

Lytic peptide

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Doxorubicin

Treatment

112

107

60

47

38

51

45

27

49

28

25, 26

24

37

16

Ref.

136

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

Mice In vitro In vitro Rats Rabbits Mice Mice Mice rats In vitro In vitro Mice

Polymer nanoparticles

Liposomes

Liposomes

Liposomes

Liposomes

Liposomes

Liposomes

Dendrimers

Dendrimers

Dendrimers

Melanoma and breast cancer cell lines Healthy CD-1 mice

Folate receptor expressing KB tumor cells

Human skin carcinoma Rat prostate carcinoma

Melanoma B16 tumors

M21-L melanomas

V2 squamous cell carcinoma

Subcutaneously implanted rat fibrosarcoma

MCF-7 human breast cancer cells

CV1-P kidney cell line

Subcutaneously implanted human prostate cancer

P

A

P

A

A

A

P

A

A

A

Doxorubicin

Methotrexate

N/A

Doxorubicin

Systemic injection. Mutant Raf gene

N/A

Doxorubicin delivered upon tumor heating

N/A (reporter gene delivery)

Methotrexate

Intratumoral injection Paclitaxel

102

98

90

121

80

79

78

77

76

117

4.8 Conclusion 137

138

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

will allow the imaging of tumors in size ranges that are undetectable with conventional imaging protocols. Importantly, delivery of drugs by nanocarriers specifically to tumor tissues will allow for localized therapy in dosage ranges that cannot be achieved with conventional systemic chemotherapy due to side effects. Ushering in the era of ‘‘personalized medicine’’ these nanoparticulate technologies will ultimately allow physicians to diagnose disease in asymptomatic individuals and treat pathology early and custom-tailored to the individual patient.

References 1 S. Sharma, M. C. Sharma, C.

2

3

4

5

6

Sarkar, Morphology of angiogenesis in human cancer: A conceptual overview, histoprognostic perspective and significance of neoangiogenesis, Histopathology 46 (2005) 481–489. M. Dellian, B. Witwer, H. Salehi, F. Yuan, R. Jain, Quantitation and physiological characterization of angiogenic vessels in mice: Effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenvironment, Am. J. Pathol. 149 (1996) 59–71. S. K. Hobbs, W. L. Monsky, F. Yuan, W. G. Roberts, L. Griffith, V. P. Torchilin, R. K. Jain, Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 4607–4612. Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. H. Maeda, L. W. Seymour, Y. Miyamoto, Conjugates of anticancer agents and polymers: Advantages of macromolecular therapeutics in vivo, Bioconjugate Chem. 3 (1992) 351–362. R. C. Semelka, J. K. Lee, S. Worawattanakul, T. C. Noone, R. H. Patt, S. M. Ascher, Sequential use of ferumoxide particles and gadolinium chelate for the evaluation of focal liver lesions on MRI, J. Magn. Reson. Imaging 8 (1998) 670–674.

7 J. Scott, J. Ward, J. A. Guthrie, D.

8

9

10

11

12

13

Wilson, P. J. Robinson, MRI of liver: A comparison of CNR enhancement using high dose and low dose ferumoxide infusion in patients with colorectal liver metastases, Magn. Reson. Imaging 18 (2000) 297–303. M. Nakayama, Y. Yamashita, K. Mitsuzaki, T. Yi, A. Arakawa, K. Katahira, Y. Nakayama, M. Takahashi, Improved tissue characterization of focal liver lesions with ferumoxide-enhanced T1 and T2weighted MR imaging, J. Magn. Reson. Imaging 11 (2000) 647–654. K. Imam, D. A. Bluemke, MR imaging in the evaluation of hepatic metastases, Magn. Reson. Imaging Clin. N. Am. 8 (2000) 741–756. H. Kato, M. Kanematsu, H. Kondo, S. Goshima, M. Matsuo, H. Hoshi, N. Moriyama, Ferumoxide-enhanced MR imaging of hepatocellular carcinoma: Correlation with histologic tumor grade and tumor vascularity, J. Magn. Reson. Imaging 19 (2004) 76–81. T. Araki, SPIO-MRI in the detection of hepatocellular carcinoma, J. Gastroenterol. 35 (2000) 874–876. H. Nakamura, N. Ito, F. Kotake, Y. Mizokami, T. Matsuoka, Tumordetecting capacity and clinical usefulness of SPIO-MRI in patients with hepatocellular carcinoma, J. Gastroenterol. 35 (2000) 849–855. M. Stiskal, H. C. Schwickert, F. Demsar, T. P. Roberts, D. Szolar, R. Weissleder, R. C. Brasch, Contrast enhancement in experimental radiation-induced liver injury:

References

14

15

16

17

18

19

20

21

Comparison of hepatocellular and reticuloendothelial particulate contrast agents, J. Magn. Reson. Imaging 6 (1996) 286–290. R. Weissleder, P. Reimer, A. S. Lee, J. Wittenberg, T. J. Brady, MR receptor imaging: Ultrasmall iron oxide particles targeted to asialoglycoprotein receptors, Am. J. Roentgenol. 155 (1990) 1161–1167. P. Reimer, R. Weissleder, T. J. Brady, A. E. Yeager, B. H. Baldwin, B. C. Tennant, J. Wittenberg, Experimental hepatocellular carcinoma: MR receptor imaging, Radiology 180 (1991) 641–645. T. K. Jain, M. A. Morales, S. K. Sahoo, D. L. Leslie-Pelecky, V. Labhasetwar, Iron oxide nanoparticles for sustained delivery of anticancer agents, Mol. Pharmaceutics 2 (2005) 194–205. R. Weissleder, G. Elizondo, J. Wittenberg, C. A. Rabito, H. H. Bengele, L. Josephson, Ultrasmall superparamagnetic iron oxide: Characterization of a new class of contrast agents for MR imaging, Radiology 175 (1990) 489–493. E. M. Renkin, Multiple pathways of capillary permeability, Circulation Res. 41 (1977) 735–743. V. W. M. van Hinsbergh, Endothelial permeability for macromolecules: Mechanistic aspects of pathophysiological modulation, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 1018– 1023. S. G. Ruehm, C. Corot, P. Vogt, S. Kolb, J. F. Debatin, Magnetic resonance imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits, Circulation 103 (2001) 415–422. M. E. Kooi, V. C. Cappendijk, K. B. J. M. Cleutjens, A. G. H. Kessels, P. J. E. H. M. Kitslaar, M. Borgers, P. M. Frederik, M. J. A. P. Daemen, J. M. A. van Engelshoven, Accumulation of ultrasmall superparamagnetic particles of iron oxide in human atherosclerotic plaques can be detected by in vivo

22

23

24

25

26

27

28

magnetic resonance imaging, Circulation 107 (2003) 2453–2458. J. Gellissen, C. Axmann, A. Prescher, K. Bohndorf, K. P. Lodemann, Extra- and intracellular accumulation of ultrasmall superparamagnetic iron oxides (USPIO) in experimentally induced abscesses of the peripheral soft tissues and their effects on magnetic resonance imaging, Magn. Resonan. Imaging 17 (1999) 557–567. A. Saleh, M. Schroeter, C. Jonkmanns, H.-P. Hartung, U. Modder, S. Jander, In vivo MRI of brain inflammation in human ischaemic stroke, Brain 127 (2004) 1670–1677. W. Leenders, B. Kusters, J. Pikkemaat, P. Wesseling, D. Ruiter, A. Heerschap, J. Barentsz, R. M. de Waal, Vascular endothelial growth factor-A determines detectability of experimental melanoma brain metastasis in GD-DTPA-enhanced MRI, Int. J. Cancer 105 (2003) 437–434. K. Turetschek, T. P. Roberts, E. Floyd, A. Preda, V. Novikov, D. M. Shames, W. O. Carter, R. C. Brasch, Tumor microvascular characterization using ultrasmall superparamagnetic iron oxide particles (USPIO) in an experimental breast cancer model, J. Magn. Reson. Imaging 13 (2001) 882–888. K. Turetschek, S. Huber, E. Floyd, T. Helbich, T. P. L. Roberts, D. M. Shames, K. S. Tarlo, M. F. Wendland, R. C. Brasch, MR imaging characterization of microvessels in experimental breast tumors by using a particulate contrast agent with histopathologic correlation, Radiology 218 (2001) 562–569. M. G. Harisinghani, J. Barentsz, P. F. Hahn, W. M. Deserno, S. Tabatabaei, C. H. van de Kaa, J. de la Rosette, R. Weissleder, Noninvasive detection of clinically occult lymph-node metastases in prostate cancer, New Engl. J. Med. 348 (2003) 2491–2499. R. Weissleder, A. Moore, U. Mahmood, R. Bhorade, H.

139

140

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

29

30

31

32

33

34

35

Benveniste, E. A. Chiocca, J. P. Basilion, In vivo magnetic resonance imaging of transgenic expression, Nat. Med. 6 (2000) 351–355. J. F. Dunn, M. A. Roche, R. Springett, M. Abajian, J. Merlis, C. P. Daghlian, S. Y. Lu, M. Makki, Monitoring angiogenesis in brain using steady-state quantification of DeltaR2 with MION infusion, Magn. Resonan. Med. 51 (2004) 55–61. D. Hogemann, L. Josephson, R. Weissleder, J. P. Basilion, Improvement of MRI probes to allow efficient detection of gene expression, Bioconjugate Chem. 11 (2000) 941– 946. H. W. Kang, L. Josephson, A. Petrovsky, R. Weissleder, A. J. Bogdanov, Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture, Bioconjugate Chem. 13 (2002) 122–127. A. M. Koch, F. Reynolds, M. F. Kircher, H. P. Merkle, R. Weissleder, L. Josephson, Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells, Bioconjugate Chem. 14 (2003) 1115– 1121. L. Josephson, C.-H. Tung, A. Moore, R. Weissleder, High-efficiency intracellular magnetic labelling with novel superparamagnetic-Tat peptide conjugates, Bioconjugate Chem. 10 (1999) 186–191. C. H. Dodd, H. C. Hsu, W. J. Chu, P. Yang, H. G. Zhang, J. D. J. Mountz, K. Zinn, J. Forder, L. Josephson, R. Weissleder, J. M. Mountz, J. D. Mountz, Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivatorpeptide-derived superparamagnetic nanoparticles, J. Immunol. Methods 256 (2001) 89–105. M. Lewin, N. Carlesso, C.-H. Tung, X.-W. Tang, D. Cory, D. T. Scadden, R. Weissleder, Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells, Nat. Biotechnol. 18 (2000) 410–414.

36 E. A. Schellenberger, A. J.

37

38

39

40

41

42

43

44

Bogdanov, D. Hogemann, J. Tait, R. Weissleder, L. Josephson, Annexin V-Clio: A nanoparticle for detecting apoptosis by MRI, Mol. Imaging 1 (2002) 102–107. D. Artemov, N. Mori, B. Okollie, Z. M. Bhujwalla, MR molecular imaging of the Her-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles, Magn. Resonan. Med. 49 (2003) 403–408. B. A. Moffat, G. R. Reddy, P. McConville, D. E. Hall, T. L. Chenevert, R. R. Kopelman, M. Philbert, R. Weissleder, A. Rehemtulla, B. D. Ross, A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI, Mol. Imaging 2 (2003) 324–332. C.-H. Tung, U. Mahmood, S. Bredow, R. Weissleder, In vivo imaging of proteolytic enzyme activity using a novel molecular reporter, Cancer Res. 60 (2000) 4953–4958. C. Bremer, C.-H. Tung, R. Weissleder, In vivo molecular target assessment of matrix metalloproteinase inhibition, Nat. Med. 7 (2001) 743–748. U. Mahmood, C.-H. Tung, A. Bogdanov, Jr, R. Weissleder, Nearinfrared optical imaging of protease activity for tumor detection, Radiology 213 (1999) 866–870. V. Ntziachristos, R. Weissleder, Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation, Optics Lett. 26 (2001) 893–895. V. Ntziachristos, A. G. Yodh, M. Schnall, B. Chance, Concurrent MRI and diffuse optical tomography of breast after indocyanine green enhancement, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 2767–2772. L. Josephson, M. F. Kirchner, U. Mahmood, Y. Tang, R. Weissleder, Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes, Bioconjugate Chem. 13 (2002) 554–560.

References 45 M. F. Kircher, U. Mahmood, R. S.

46

47

48

49

50

51

52

King, R. Weissleder, L. Josephson, A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation, Cancer Res. 63 (2003) 8122–8125. C. S. Kumar, C. Leuschner, E. E. Doomes, L. Henry, M. Juban, J. Hormes, Efficacy of lytic peptidebound magnetite nanoparticles in destroying breast cancer cells, J. Nanosci. Nanotechnol. 4 (2005) 245–249. C. Leuschner, C. S. S. R. Kumar, W. Hansel, J. Hormes, Targeting breast cancer cells and their metastases through luteinizing hormone releasing hormone (LHRH) receptors using magnetic nanoparticles, J. Biomed. Nanotechnol. 1 (2005) 229–233. J. W. M. Bulte, S.-C. Zhang, P. van Gelderen, V. Herynek, E. K. Jordan, I. D. Duncan, J. A. Frank, Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 15 256–15 261. J. A. Frank, B. R. Miller, A. S. Arbab, H. A. Zywicke, E. K. Jordan, B. K. Lewis, L. H. Bryant, Jr, J. W. M. Bulte, Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents, Radiology 228 (2003) 480–487. J. W. M. Bulte, T. Douglas, B. Witwer, S.-C. Zhang, B. K. Lewis, P. van Gelderen, H. Zywicke, I. D. Duncan, J. A. Frank, Monitoring stem cell therapy in vivo using magnetodendrimers as a new class of cellular MR contrast agents, Acad. Radiol. 9 (2002) S332–S335. M. F. Kircher, J. R. Allport, E. E. Graves, V. Love, L. Josephson, A. H. Lichtman, R. Weissleder, In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors, Cancer Res. 63 (2003) 6838–6846. P. Wunderbaldinger, L. Josephson, R. Weissleder, Tat peptide directs

53

54

55

56

57

58

enhanced clearance and hepatic permeability of magnetic nanoparticles, Bioconjugate Chem. 13 (2002) 264–268. G. M. Lanza, K. D. Wallace, M. J. Scott, W. P. Cacheris, D. R. Abendschein, D. H. Christy, A. M. Sharkey, J. G. Miller, P. J. Gaffney, A. Wickline, A novel site-targeted ultrasonic contrast agent with broad biomedical application, Circulation 94 (1996) 3334–3340. G. M. Lanza, D. R. Abendschein, C. S. Hall, M. J. Scott, D. E. Scherrer, A. Houseman, J. G. Miller, S. A. Wickline, In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles, J. Am. Soc. Echocardiography 13 (2000) 608–614. S. Flacke, S. Fischer, M. J. Scott, R. J. Fuhrhop, J. S. Allen, M. McLean, P. Winter, G. A. Sicard, P. J. Gaffney, S. A. Wickline, G. M. Lanza, Novel MRI contrast agent for molecular imaging of fibrin: Implications for detecting vulnerable plaques, Circulation 104 (2001) 1280–1285. G. M. Lanza, C. H. Lorenz, S. E. Fischer, M. J. Scott, W. P. Cacheris, R. J. Kaufmann, P. J. Gaffney, S. A. Wickline, Enhanced detection of thrombi with a novel fibrin-targeted magnetic resonance imaging agent, Acad. Radiol. 5(Suppl. 1) (1998) S173–S176. P. M. Winter, S. D. Caruthers, X. Yu, S.-K. Song, J. Chen, B. Miller, J. W. M. Bulte, J. D. Robertson, P. J. Gaffney, S. A. Wickline, G. M. Lanza, Improved molecular imaging contrast agent for detection of human thrombus, Magn. Resonan. Med. 50 (2003) 411–416. A. M. Morawski, P. Winter, K. C. Crowder, S. D. Caruthers, R. J. Fuhrhop, M. J. Scott, J. D. Robertson, D. R. Abendschein, G. M. Lanza, S. A. Wickline, Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI, Magn. Resonan. Med. 51 (2004) 480–486.

141

142

4 Nanoparticles for Magnetic Resonance Imaging of Tumors 59 G. M. Lanza, X. Yu, P. M. Winter,

60

61

62

63

64

65

66

67

68

D. R. Abendschein, K. K. Karukstis, M. J. Scott, L. K. Chinen, R. W. Fuhrhop, D. E. Scherrer, S. A. Wickline, Targeted antiproliferative drug delivery to vascular smooth muscle cells with a magnetic resonance imaging nanoparticle contrast agent: Implications for rational therapy of restenosis, Circulation 106 (2002) 2842–2847. P. M. Winter, S. D. Caruthers, A. Kassner, T. D. Harris, L. K. Chinen, J. S. Allen, E. K. Lacy, H. Zhang, J. D. Robertson, S. A. Wickline, G. M. Lanza, Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel an b3 targeted nanoparticle and 1.5 Tesla magnetic resonance imaging, Cancer Res. 63 (2003) 5838–5843. A. D. Bangham, Membrane models with phospholipids, Progr. Biophys. Mol. Biol. 18 (1968) 29–36. M. J. Hope, M. B. Bally, L. D. Mayer, A. S. Janoff, P. R. Cullis, Generation of multilamellar and unilamellar phospholipid vesicles, Chem. Phys. Lipids 40 (1986) 89–107. A. Gabizon, D. Papahadjopoulos, Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 6949–6953. M. C. Woodle, D. D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta 1113 (1992) 171–199. A. S. Ulrich, Biophysical aspects of using liposomes as delivery vehicles, Biosci. Rep. 22 (2002) 129–150. K. J. Harrington, K. N. Syrigos, R. G. Vile, Liposomally targeted cytotoxic drugs for the treatment of cancer, J. Pharm. Pharmacol. 54 (2002) 1573–1600. E. G. Nabel, G. Plautz, G. J. Nabel, Site-specific gene expression in vivo by direct gene transfer into the arterial wall, Science 249 (1990) 1285– 1288. V. J. Pompili, D. Gordon, H. San, Z. Yang, D. W. M. Muller, G. J. Nabel, E. G. Nabel, Expression and function

69

70

71

72

73

74

of a recombinant PDGF B gene in porcine arteries, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 2254–2264. S. S. Jensen, T. L. Andresen, J. Davidsen, P. Hoyrup, S. D. Shnyder, M. C. Bibby, J. H. Gill, K. Jorgensen, Secretory phospholipase A2 as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids, Mol. Cancer Ther. 3 (2004) 1451–1458. M. Nogawa, T. Yuasa, S. Kimura, M. Tanaka, J. Kuroda, K. Sato, A. Yokota, H. Segawa, Y. Toda, S. Kageyama, T. Yoshiki, Y. Okada, T. Maekawa, Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer, J. Clin. Invest. 115 (2005) 978–985. J. Yano, K. Hirabayashi, S.-i. Nakagawa, T. Yamaguchi, M. Nogawa, I. Kashimori, H. Naito, H. Kitagawa, K. Ishiyama, T. Ohgi, T. Irimura, Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer, Clin. Cancer Res. 10 (2004) 7721–7726. S. U. Gnad-Vogt, R.-D. Hofheinz, S. Saussele, S. Kreil, A. Willer, F. Willeke, L. Pilz, R. Hehlmann, A. Hochhaus, Pegylated liposomal doxorubicin and mitomycin C in combination with infusional 5fluorouracil and sodium folinic acid in the treatment of advanced gastric cancer: Results of phase II trial, AntiCancer Drugs 16 (2005) 435–440. D. Katsaros, M. V. Oletti, I. A. Rigault de la Longrais, A. Ferrero, A. Celano, S. Fracchioli, M. Donadio, R. Passera, L. Cattel, C. Bumma, Clinical and pharmacokinetic phase II study of pegylated liposomal doxorubicin and vinorelbine in heavily pretreated recurrent ovarian carcinoma, Ann. Oncol. 16 (2005) 300–306. C. Mamot, J. B. Nguyen, M. Pourdehnad, P. Hadaczek, R. Saito, J. R. Bringas, D. C. Drummond, K. Hong, D. B. Kirpotin, T. R.

References

75

76

77

78

79

80

81

McKnight, M. S. Berger, J. W. Park, K. S. Bankiewicz, Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery, J. NeuroOncol. 68 (2004) 1–9. R. Saito, J. R. Bringas, T. R. McKnight, M. F. Wendland, C. Mamot, D. C. Drummond, D. B. Kirpotin, J. W. Park, M. S. Berger, K. S. Bankiewicz, Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging, Cancer Res. 64 (2004) 2572–2579. K. Amin, K.-Y. Ng, C. Brown, M. Bruno, T. Heath, LDL induced association of anionic liposomes with cells and delivery of contents as shown by the increase in potency of liposome dependent drugs, Pharmaceutical Res. 18 (2001) 914–921. A. Mukherjee, T. K. Prasad, N. M. Rao, R. Banerjee, Haloperidolassociated stealth liposomes: A potent carrier for delivering genes to human breast cancer cells, J. Biol. Chem. 280 (2005) 15 619–15 627. B. L. Viglianti, S. A. Abraham, C. R. Michelich, P. S. Yarmolenko, J. R. MacFall, M. B. Bally, M. W. Dewhirst, In vivo monitoring of tissue pharmacokinetics of liposome/ drug using MRI: Illustration of targeted delivery, Magn. Resonan. Imaging 51 (2004) 1153–1162. D. A. Sipkins, D. A. Cheresh, M. R. Kazemi, L. M. Nevin, M. D. Bednarski, K. C. Li, Detection of tumor angiogenesis in vivo by an b3 targeted magnetic resonance imaging, Nat. Med. 4 (1998) 623–626. J. D. Hood, M. Bednarski, R. Frausto, S. Guccione, R. A. Reisfeld, R. Xiang, D. A. Cheresh, Tumor regression by targeted gene delivery to the neovasculature, Science 296 (2002) 2404–2407. S. R. Wilson, Biological applications of fullerene derivatives: A brief overview, in The Fullerene Handbook, Kadish, K., Ruoff, R. (Eds.), Wiley, New York, 2000, pp. 437–465.

82 S. Bosi, T. Da Ros, G. Spalluto,

83

84

85

86

87

88

89

90

M. Prato, Fullerene derivatives: An attractive tool for biological applications, Eur. J. Med. Chem. 38 (2003) 913–923. L. Qingnuan, X. Yan, Z. Xiaodong, L. Ruili, D. Qieqie, S. Xiaoguang, C. Shaoliang, L. Wenxin, Preparation of (99m)Tc-C(60)(OH)(x) and its biodistribution studies, Nucl. Med. Biol. 29 (2002) 707–710. M. Mikawa, H. Kato, M. Okamura, M. Narazaki, Y. Kanazawa, N. Miwa, H. Shinohara, Paramagnetic watersoluble metallofullerenes having the highest relaxivity for MRI contrast agents, Bioconjugate Chem. 12 (2001) 510–514. E. Toth, R. D. Bolskar, A. Borel, G. Gonzalez, L. Helm, A. E. Merbach, B. Sitharaman, L. J. Wilson, Watersoluble gadofullerenes: Toward highrelaxivity, pH-responsive MRI contrast agents, J. Am. Chem. Soc. 127 (2005) 799–805. R. D. Bolskar, A. F. Benedetto, L. O. Husebo, R. E. Price, E. F. Jackson, S. Wallace, L. J. Wilson, J. M. Alford, First soluble M@C60 derivatives provide enhanced access to metallofullerenes and permit in vivo evaluation of Gd@C60 [C(COOH)2]10 as a MRI contrast agent, J. Am. Chem. Soc. 125 (2003) 5471–5478. B. Sitharaman, K. R. Kissell, K. B. Hartman, L. A. Tran, A. Baikalov, I. Rusakova, Y. Sun, H. A. Khant, S. J. Ludtke, W. Chiu, S. Laus, E. Toth, L. Helm, A. E. Merbach, L. J. Wilson, Superparamagnetic gadonanotubes are high-performance MRI contrast agents, Chem. Commun. (2005) 3915–3917. U. Boas, P. M. H. Heegaard, Dendrimers in drug research, Chem. Soc. Rev. 33 (2004) 43–63. B. Misselwitz, H. Schmitt-Willich, W. Ebert, T. Frenzel, H. J. Weinmann, Pharmacokinetics of Gadomer-17, a new dendritic magnetic contrast agent, Magma 12 (2001) 128–134. C. Fink, F. Kiessling, M. Bock, M. P. Lichy, B. Misselwitz, P. Peschke,

143

144

4 Nanoparticles for Magnetic Resonance Imaging of Tumors

91

92

93

94

95

96

N. E. Fusenig, R. Grobholz, S. Delorme, High-resolution threedimensional MR angiography of rodent tumors: Morphologic characterization of intratumoral vasculature, J. Magn. Reson. Imaging 18 (2003) 59–65. G. Staats, E. Spuntrup, A. Bucker, J. Vazquez-Jimenez, O. J. Liakopoulos, D. Pfluger, S. Grosskortenhaus, B. Misselwitz, R. W. Gunther, Interstitial T1weighted MR lymph fistulography with Gadomer-17 in an experimental animal model, ROFO-Fortsch. Gebiet Rontgenstrahlen Bildgebenden Verfah. 175 (2003) 275–281. M. G. Torchia, B. Misselwitz, Combined MR lymphangiography and MR imaging-guided needle localization of sentinel lymph nodes using Gadomer-17, Am. J. Roentgenol. 179 (2002) 1561–1565. B. Misselwitz, H. Schmitt-Willich, M. Michaelis, J. J. Oellinger, Interstitial magnetic resonance lymphography using a polymeric T1 contrast agent: Initial experience with Gadomer-17, Invest. Radiol. 37 (2002) 146–151. N. Sato, H. Kobayashi, A. Hiraga, T. Saga, K. Togashi, J. Konishi, M. W. Brechbiel, Pharmacokinetics and enhancement patterns of macromolecular MR contrast agents with various sizes of polyamidoamine dendrimer cores, Magn. Reson. Med. 46 (2001) 1169–1173. H. Kobayashi, N. Sato, A. Hiraga, T. Saga, Y. Nakamoto, H. Ueda, J. Konishi, K. Togashi, M. W. Brechbiel, 3D-micro-MR angiography of mice using macromolecular MR contrast agents with polyamidoamine dendrimer core with reference to their pharmacokinetic properties, Magn. Reson. Med. 45 (2001) 454–460. H. Kobayashi, C. Wu, M.-K. Kim, C. Paik, H., J. A. Carrasquillo, M. W. Brechbiel, Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1B4M-

97

98

99

100

101

102

103

DTPA and its conjugation with AntiTac monoclonal antibody, Bioconjugate Chem. 10 (1999) 103–111. G. A. Wiseman, E. Kornmehl, B. Leigh, W. D. Erwin, D. A. Podoloff, S. Spies, R. B. Sparks, M. G. Stabin, T. Witzig, C. A. White, Radiation dosimetry results and safety correlations from 90 Y-ibritumomab tiuxetan radioimmunotherapy for relapsed or refractory non-Hodgkin’s lymphoma: Combined data from 4 clinical trials, J. Nucl. Med. 44 (2003) 465–474. A. Quintana, E. Raczka, L. Piehler, I. Lee, A. Myc, I. Majoros, A. Patri, T. Thomas, J. Mule, J. J. Baker, Design and function of a dendrimerbased therapeutic nanodevice targeted to tumor cells through the folate receptor, Pharm. Res. 19 (2002) 1310– 1316. N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J. W. Weener, E. W. Meijer, W. Paulus, R. Duncan, Dendrimers: Relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo, J. Controlled Release 65 (2000) 133– 148. N. Bourne, L. R. Stanberry, E. R. Kern, G. Holan, B. Matthews, D. I. Bernstein, Dendrimers, a new class of candidate topical microbicides with activity against herpes simplex virus infection, Antimicrob. Agents Chemother. 44 (2000) 2471–2474. H. R. Ihre, O. L. Padilla De Jesus, F. C. J. Szoka, J. M. J. Frechet, Polyester dendritic systems for drug delivery applications: Design, synthesis, and characterization, Bioconjugate Chem. 13 (2002) 443–452. O. L. Padilla De Jesus, H. R. Ihre, L. Gagne, J. M. J. Frechet, F. C. J. Szoka, Polyester dendritic systems for drug delivery applications: In vitro and in vivo evaluation, Bioconjugate Chem. 13 (2002) 453–461. G.-P. Yan, B. Hu, M.-L. Liu, L.-Y. Li, Synthesis and evaluation of gado-

References

104

105

106

107

108

109

110

111

112

linium complexes based on PAMAM as MRI contrast agents, J. Pharm. Pharmacol. 57 (2005) 351–357. M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Semiconductor nanocrystals as fluorescent biological labels, Science 281 (1998) 2013–2016. M. Dahan, S. Levi, C. Luccardini, P. Rostaing, B. Riveau, A. Triller, Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking, Science 302 (2003) 442–445. D. S. Lidke, P. Nagy, R. Heintzmann, D. J. Arndt-Jovin, J. N. Post, H. E. Grecco, E. A. JaresErijman, T. M. Jovin, Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction, Nat. Biotech. 22 (2004) 198–203. X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, S. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots, Nat. Biotech. 22 (2004) 969–976. D. Wang, J. He, N. Rosenzweig, Z. Rosenzweig, Superparamagnetic Fe2O3 Beads-CdSe/ZnS quantum dots core-shell nanocomposite particles for cell separation, Nano Lett. 4 (2004) 409–413. H. Gu, R. Zheng, X. Zhang, B. Xu, Facile One-pot synthesis of bifunctional heterodimers of nanoparticles: A conjugate of quantum dot and magnetic nanoparticles, J. Am. Chem. Soc. 126 (2004) 5664–5665. M. A. Correa-Duarte, Y. Kobayashi, R. A. Caruso, L. M. Liz-Marzan, Photodegradation of SiO2-coated CdS nanoparticles with silica gels, J. Nanosci. Nanotechnol. 1 (2001) 95–99. W. C. W. Chan, S. Nie, Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science 281 (1998) 2016–2018. X. Wu, H. Liu, J. Liu, K. N. Haley, J. A. Treadway, J. P. Larson, N. Ge, F. Peale, M. P. Bruchez, Immunofluorescent labeling of cancer marker

113

114

115

116

117

118

119

120

Her2 and other cellular targets with semiconductor quantum dots, Nat. Biotechnol. 21 (2003) 41–46. I. Fishbein, M. Chorny, S. Banai, A. Levitzki, H. D. Danenberg, J. Gao, X. Chen, E. Moerman, I. Gati, V. Goldwasser, G. Golomb, Formulation and delivery mode affect disposition and activity of tyrphostinloaded nanoparticles in the rat carotid model, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 1434–1439. H. Cohen, R. J. Levy, J. Gao, I. Fishbein, V. Kousaev, S. Sosnowski, S. Slomkoswski, G. Golomb, Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles, Gene Therapy 7 (2000) 1896–1905. J. M. Anderson, M. S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres, Adv. Drug Delivery Rev. 28 (1997) 5–24. S. Y. Lin, K. S. Chen, H. H. Teng, M. J. Li, In vitro degradation and dissolution behaviours of microspheres prepared by three low molecular weight polyesters, J. Microencapsulation 17 (2000) 577–586. S. K. Sahoo, W. Ma, V. Labhasetwar, Efficacy of transferrin-conjugated paclitaxel-loaded nanoparticles in a murine model of prostate cancer, Int. J. Cancer 112 (2004) 335–340. K. Gatter, G. Brown, I. Trowbridge, R. Woolston, D. Mason, Transferrin receptors in human tissues: Their distribution and possible clinical relevance, J. Clin. Pathol. 36 (1983) 539–545. J. L. Turner, D. Pan, R. Plummer, Z. Y. Chen, A. K. Whittaker, K. L. Wooley, Synthesis of gadoliniumlabeled shell crosslinked nanoparticles for magnetic resonance imaging applications, Adv. Functional Mater. 15 (2005) 1248–1254. R. Rossin, D. Pan, K. Qi, J. L. Turner, X. Sun, K. L. Wooley, M. J. Welch, 64Cu-Labeled folateconjugated shell cross-linked nanoparticles for tumor imaging and

145

146

4 Nanoparticles for Magnetic Resonance Imaging of Tumors radiotherapy: Synthesis, radiolabeling, and biologic evaluation, J. Nucl. Med. 46 (2005) 1210–1218. 121 X.-B. Xiong, Y. Huang, W.-l. Lu, X. Zhang, H. Zhang, T. Nagai, Q. Zhang, Enhanced intracellular delivery and improved antitumor

efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic, J. Controlled Release 107 (2005) 262–275. 122 C. R. Martin, P. Kohli, The emerging field of nanotube biotechnology, Nat. Rev. Drug Discov. 2 (2003) 29–37.

147

5

Magnetic Resonance Nanoparticle Probes for Cancer Imaging Young-wook Jun, Jung-tak Jang, and Jinwoo Cheon 5.1

Introduction

Inorganic nanoparticles exhibit unique optical, magnetic, and electronic properties arising from nanoscale quantum effect and are emerging as key materials for nextgeneration device applications [1]. When these nanoparticles are applied to biological systems, they have the potential to improve modern imaging techniques, such as X-ray imaging, computed tomography (CT), near-IR fluorescence imaging, positron emission spectroscopy (PET), and magnetic resonance imaging (MRI) [2–4]. Although such modern imaging techniques provide excellent anatomical information of living objects, they have difficulties in detecting molecular and cellular events. Considering that most biological processes and disease attacks are related to molecular and cellular events, precise observations of detailed biological processes are important [5, 6]. Inorganic nanoparticles, upon conjugation with biomolecular markers, can report such molecular and sub-cellular events. In addition, their nanoscale materials properties can lead to the significant enhancements in detection sensitivity and resolution. Semiconductor quantum dots are a good example of such nanoparticle probes. Quantum dots exhibit better fluorescence properties, such as high quantum yield, multicolor emissions by single excitation, and high photo-stability, than conventional organic fluorophores [7–9] Such enhanced properties have allowed the imaging of cell signaling, cell evolution, cell trafficking, and in vivo cancer detection [10–13]. In contrast, magnetic nanoparticles with unique superparamagnetism can serve as excellent probes in magnetic resonance imaging (MRI). MRI detects subtle change in the relaxation time of the proton nuclear spins of water molecules, which make up from 70 to 90% of most tissues. Although this is one of the most powerful medical diagnostic tools due to its non-invasive nature, multidimensional tomographic capabilities, and high spatial resolution [14], MRI has a lower sensitivity than other tools [6]. Such weakness can be significantly improved by using magnetic nanoparticle probes [15, 16]. Under an applied magnetic field, magnetized nanoparticles generate an induced magnetic field, which perturbs the magNanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

148

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

MR contrast effects of magnetic nanoparticles. Under an applied magnetic field (M), magnetic nanoparticles are magnetized and generate an induced magnetic field (dM),

Fig. 5.1.

which perturbs the magnetic relaxation processes of the proton in water molecules, which is reflected as dark MR contrast.

netic relaxation processes of the protons in water molecules surrounding the magnetic nanoparticles. This leads to a shortening of the spin–spin relaxation time (T2) of the proton, which results in darkening of the MR images (Fig. 5.1). According to the outer sphere spin–spin relaxation formula of solvent protons by solute magnetic particles, the spin–spin relaxation time of the proton is given by Eq. (1), where gI is the gyromagnetic ratio of protons in water, m is the molarity of magnetic nanoparticles, r is their radius, NA is Avogadro’s number, m is the magnetic moment of the nanoparticle, os and oI are the respective Larmor angular precession frequencies of the solute electronic and water proton magnetic moments, the functions jn ðo; tÞ are spectral density functions, and t ð¼ r 2 =DÞ is the time scale of fluctuations in the particle–water proton magnetic dipolar interaction arising from the relative diffusive motion (D) of a particle and water molecules [17].   1 32pNA ½M
2 2 ¼ g m f6:5j2 ðos ; tÞ þ 1:5j1 ðoI ; tÞ þ j1 ð0; tÞg T2 405000rD 1

ð1Þ

Therefore, T2 is shortened by increasing the magnetic moment of the nanoparticles. Recently, Cheon, Suh, and coworkers experimentally demonstrated such magnetic moment effects on T2 by elucidating the correlated nanoscale effects of iron oxide nanoparticles between size, magnetism, and T2 relaxivity [18]. Figure

5.1 Introduction

Nanoscale size effects of iron oxide nanoparticles on magnetism and induced magnetic resonance (MR) signals. (a) TEM images of Fe3 O4 nanocrystals of 4, 6, 9, and 12 nm. (b) Size-dependent T2-weighted MR images of iron oxide nanoparticles in aqueous solution at 1.5 Tesla. (c) Size-dependent Fig. 5.2.

changes from red to blue in color-coded MR images based on T2 values. (d) Graph of 1/T2 relaxivity versus iron oxide nanoparticle size. (e) Magnetization of iron oxide nanoparticles measured by a SQUID magnetometer. (From Ref. [18], with permission.)

5.2(a) shows transmission electron microscopic images of highly monodispersed iron oxide nanoparticles, 4, 6, 9, and 12 nm. These magnetic nanoparticles exhibit size-dependent magnetic moments and as the nanoparticle size is increased from 4 to 6, 9, and to 12 nm, the mass magnetization at 1.5 T changes from 25 to 43, 80, and 102 emu (g-Fe)1 (Fig. 5.2e). Such a trend is clearly reflected in the T2weigthed MR images. The 1/T2 relaxivity gradually increases from 56 to 106, 130, and to 190 m1 s1 , which is imaged by the gradual change of the MR contrast from white to black through gray (Fig. 5.2b–d). Such effect of magnetic nanoparticles on MR contrast gives them the ability to report various biological events. For example, magnetic nanoparticles >30 nm have been used for phagocytosis imaging [19, 20]. When phagocytes uptake magnetic nanoparticles, they are imaged as dark contrast. But tumor cells without phagocytic ability are imaged as white contrast. By utilizing such an effect, liver metastasis [21, 22], spleen [23], and lymph node detection [24] have been performed. In contrast, when smaller nanoparticles (e.g., 10 nm) can easily escape from phagocytes, magnetic nanoparticles, upon conjugation with a target specific biomolecule, can detect target tissues through molecular interactions between

149

150

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

nanoparticle–biomolecule conjugates and molecular markers expressed from target tissues [24, 25]. Various types of clinically benign iron oxide based magnetic nanoparticles [e.g., superparamagnetic iron oxide (SPIO)] have been explored, and imaging has been reported of infarct [26–28], angiogenesis [29], apoptosis [30], gene expression [31, 32], and cancer [18, 33–36]. However, MR signal sensitivity and specificity of nanoparticle probes to the target tissue are still unsatisfactory for clinical applications and further efforts are needed to make them better. This chapter briefly reviews recently developed biocompatible magnetic nanoparticles and their utilization in molecular MRI.

5.2

Magnetic Nanoparticle Contrast Agents

For their successful utilization as molecular MR contrast agents, magnetic nanoparticles must fulfill the following requirements: (a) uniform and high superparamagnetic moment, (b) high colloidal stability under physiological conditions (e.g., high salt concentration and pH changes), (c) the ability to escape the reticuloendothelial system (RES), (d) low toxicity and biocompatibility, and (e) possession of functionality to be linked to biologically active species (e.g., nucleic acid, proteins). Since these properties are highly related to their size, stoichiometry, and surface structures, various types of iron oxide nanoparticles have been developed. 5.2.1

Silica- or Dextran-coated Iron Oxide Contrast Agents

For conventional MR contrast agents, iron oxide nanoparticles are synthesized through the precipitation of iron oxide in an aqueous solution containing ferrous salt by adding an alkaline solution [37]. Such iron oxide nanoparticles are usually insoluble as-is and a coating material is required for them to be soluble in aqueous media. In early attempts to make them water soluble, silica was used as a coating material [38]. The size of the core magnetic iron oxide can vary between 4 to 10 nm and the total particle size varies from 10 nm to 1 mm, including coating materials. Since these nanoparticles have a broad size distribution, further size sorting procedures, including differential centrifugation and dialysis, are required. A representative silica-coated iron oxide contrast agent is AMI-121 (generic name: Ferumoxsil), which is now commercially available as Lumirem1 (Guerbet) and Gastromark1 (Advanced Magnetics) (Table 5.1). The core is @10 nm-sized polycrystalline iron oxide and the hydrodynamic size is @300 nm. This agent is delivered orally and used for abdomen MRI [39]. Although silica-coated iron oxide nanoparticles are reasonably stable in aqueous media, they tend to aggregate in blood and, therefore, are inadequate for blood injection. To enhance the colloidal stability of iron oxide nanoparticles, another type of coating agent, dextran or carbodextran, has been developed [40] (Table 5.1). Since dextran possesses high colloidal stability against harsh physiological condi-

5.2 Magnetic Nanoparticle Contrast Agents Tab. 5.1.

Various silica- or dextran-coated iron oxide contrast agents. Coating material

Magnetization T2 relaxivity Blood half-life (MC1 sC1 ) at 1.5 T (emu gC1 )

Silica

N/A

72

<5 min

78

98

@6 min

151

3 min

Agent

Iron oxide Total core size size (nm) (nm)

AMI-121[a] [36], Lumiren, Gastromark

10

@300

AMI-25[a] [38], Feridex Endorem

5–6

80–150 Dextran

SHU 555A[a] [39], @4.2 Resovist

@62

Carbodextran N/A

AMI-227[a] [41], Combidex, Sinerem

4–6

20–40

Dextran

69.8

53

>24 h

MION [42–44], CLIO

2.8

10–30

Dextran

60–68

69

@10 h

a Commercialzed.

tions, dextran-coated iron oxide nanoparticles can have good stability. Such nanoparticles are prepared through a co-precipitation method from aqueous solution containing ferrous salt and dextran by adding an alkaline solution [37]. There are three representative dextran-coated iron oxide nanoparticles: AMI-25 [Feridex1 (Berlex Lab.) and Endorem1 (Guerbet)], SHU 555A [Resovist1 (Schering)], and AMI-227 [Combidex1 (Advanced Magnetics) and Sinerem1 (Guerbet)]. AMI-25 is composed of a @5-nm iron oxide core and dextran coating materials. The total size is between 80 and 150 nm with a T2 relaxivity of @98.3 mm1 s1 [41]. SHU 555A has an @4.2-nm iron oxide core coated with carbodextran, with total size of @62 nm; Resovist has a higher T2 relaxivity of 151.0 mm1 s1 [42] and has no known side effects after fast intravenous injection [43]. These magnetic contrast agents are generally trapped and accumulated by the reticuloendothelial cells in the liver with a short blood half-life of less than 10 min and, therefore, are used for liver imaging [20]. Compared with these two iron oxide contrast agents, AMI-227 has similar iron oxide core size (@5 nm) but a smaller overall size of 20–40 nm. Although AMI-227 has lower T2 contrast effects (T2 relaxivity of @53 mm1 s1 ), its smaller size provides a much higher blood half-life of @24 h, which enables MR angiography and lymph node detection [44]. Smaller dextran-coated iron oxide nanoparticles, including monocrystalline iron oxide (MION) and its derivative, crosslinked iron oxide (CLIO) are composed of a @2.8-nm core iron oxide and dextran shell with a total size of 10–30 nm [45–47].

151

152

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

Synthesis scheme of magnetoferritin. Removal of ferrihydrite from native ferritin produces apoferritin and subsequent formation of magnetite nanoparticles inside apoferritin affords magnetoferritin. (From Ref. [49], with permission.)

Fig. 5.3.

Since these nanoparticles are relatively small and have a long blood half-life, and their surface can be readily linked with biologically active molecules, they are useful for in vivo molecular MRI of biological targets [45] (Section 5.3.1). 5.2.2

Magnetoferritin

Ferritin is a well-known iron storage protein used to sequester and store iron, which is composed of a @6 nm hydrated iron oxide, ferrihydrite (5Fe2 O3 9H2 O), core and polypeptide apoferritin shell [48]. Ferritin has been used as an efficient synthesizer for other magnetic materials [49–51]. For example, magnetically less useful ferrihydrite can be replaced by iron sulfide or magnetite nanoparticles (Fig. 5.3) [49]. Researchers have also utilized magnetoferritin as contrast agents for MRI. Magnetoferritin possesses a reasonably high T2 relaxation of 157 mm1 s1 [52]. Although magnetoferritin is expected to have high biocompatibility and colloidal stability in the blood when considering that they mimic naturally-occurring ferritin, actual results are contradictory. These magnetoferritin particles are rapidly cleared from the blood circulation (blood half-life of less than 10 min) by the reticuloendothelial system in the liver, spleen, and lymph nodes [52]. Therefore, magnetoferritins are only suitable for liver, spleen, and lymph-node detection rather than molecular imaging. 5.2.3

Magnetodendrimers and Magnetoliposomes

The unique pore structures and multiple functional end-groups of dendrimers make them useful as host materials in drug and gene delivery. Similarly, dendrimers can efficiently deliver magnetic nanoparticles to cells. Bulte, Frank, and coworkers have demonstrated carboxy-terminated dendrimer (G ¼ 4.5) coated iron oxide contrast agents [53, 54]. Typically, magnetodendrimers are synthesized through the pH-controlled reaction of a ferrous salt and a trimethylamine oxide

5.2 Magnetic Nanoparticle Contrast Agents

Schematics and TEM images of (a, b) magnetodendrimers and (c, d) magnetoliposomes. (From Refs. [54 and 55], with permission.)

Fig. 5.4.

oxidant in a methanol/water mixture containing polyamidoamine dendrimers (Fig. 5.4a). The core size of the magnetodendrimers is 7–8 nm and they tend to aggregate to oligomers 20–30 nm in size (Fig. 5.4b). Magnetodendrimers show enhanced magnetic properties [saturation magnetism: @94 emu (g-Fe)1 ] and a high T2 relaxivity of 200–406 m1 s1 [53], compared with those of dextran-coated MION. Since dendrimers can be efficiently transfected to cells without any transfection agents, these magnetodendrimers can be used as labelers for cellular MR imaging and trafficking [53]. Similarly, liposomes, which are also widely used for drug and gene delivery, can be good coating materials to solubilize iron oxide nanoparticles. Liposomes have a bilayer assembly of surfactant molecules with a hydrophilic head and a hydrophobic tail. As shown in Fig. 5.4(c), the hydrophilic ends of the inner layer surfactants encapsulate the iron oxide nanoparticles and the hydrophilic head of the outer layer surfactant make them soluble in water. Bulte, Frank, and coworkers have reported that such magnetoliposome can be utilized for bone marrow MR contrast agents [55]. The iron oxide core size of the magnetoliposomes is @16 nm, and the entire size is @40 nm (Fig. 5.4d) with a T2 relaxivity of @240 mm1 s1 .

153

154

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

5.2.4

New Type of Contrast Agent: Non-hydrolytically Synthesized High Quality Iron Oxide Nanoparticles

With the exception of MION and CLIO, previously developed iron oxide MR contrast agents undergo rapid uptake by the reticuloendothelial systems (RES) and, therefore, are effectively for liver, spleen, and lymph node detection. Consequently, researchers have encountered difficulties when they are utilized for molecular MRI [19–23]. Successful molecular imaging requires high-performance magnetic nanoparticle systems that exhibit excellent magnetic properties, the ability to escape from the RES, and possess active functionality that can be linked with biologically active molecules [15]. Since magnetic properties of nanoparticles depend highly on the materials properties such as size, shape, stoichiometry, and crystallinity [18, 56, 57], it is critical to be able to control such properties. However, conventional waterphase protocols, which have been widely adopted for superparamagnetic iron oxide (SPIO) contrast agents, generally lack precise size-controllability and monodispersity, and afford poor crystallinity and non-stoichiometric compositions [37]. In contrast, nonhydrolytic high-temperature growth methods allow one to have sizecontrollability, high single crystallinity, and good stoichiometry [18, 57–60]. For example, the nanoparticle size can be easily controlled from 4 to @20 nm with a very narrow size distribution (s < 8%) by controlling the growth conditions [18, 57]. One difficulty that must be overcome before their utilization as MR contrast agents is obtaining water-solubility since nonhydrolytically synthesized iron oxide nanoparticles are soluble only in organic media. Various surface modification methods have been developed, including bifunctional ligand [18, 36, 61], micellular [62, 63], polymer [64–66], and siloxane-linking procedures [67, 68]. For example, nonhydrolytically synthesized nanoparticles can be transferred to aqueous media by overcoating the nanoparticles with poly(ethylene glycol)-(PEG)-ylated phospholipid micelles. Such a micellular coating strategy has been demonstrated with quantum dots [69], and Bao and coworkers have successfully extended this strategy to make water-soluble iron oxide nanoparticles (Fig. 5.5) [63]. The PEGylated nanoparticles can be further linked to cellular transfection Tat peptides and utilized for MR cellular labeling [63]. Bawendi and coworkers have proposed another approach to transfer iron oxide nanoparticles from organic to aqueous media by coating them with polymeric phosphine oxide ligands [70]. These polymeric ligands can tightly bind to the iron oxide nanoparticle surface through multidentate bondings (Fig. 5.6). The siloxane linkage to a metal oxide surface is efficient and strong. Zhang and coworkers have successfully applied this strategy for the synthesis of water-soluble iron oxide nanoparticles [71]. Refluxing toluene solution containing triethoxysilylterminated PEG ligands and nonhydrolytically synthesized iron oxide nanoparticles provides iron oxide nanoparticles with high colloidal stability in aqueous media (Fig. 5.7). The major advantage of these nonhydrolytic synthesized iron oxide nanoparticles, as mentioned above, is the precise size control with high monodispersity.

5.2 Magnetic Nanoparticle Contrast Agents

(a) Synthetic scheme and (b) TEM image of poly(ethylene glycol) (PEG)-ylated iron oxide nanoparticles. (From Ref. [63], with permission.)

Fig. 5.5.

Multidentate phosphine oxide ligand approach for the synthesis iron oxide nanoparticles. (a) The phosphine oxide functional groups bind to the surface of iron

Fig. 5.6.

oxide and exposed PEG groups make them water soluble. (b) Iron oxide nanoparticles dissolved in water. (From Ref. [70], with permission.)

155

156

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

Siloxane–poly(ethylene glycol) (PEG) coated iron oxide nanoparticles. Silanization of terminal ethoxysilane groups of the PEG ligand on top of iron oxide nanoparticles induces the formation of PEG-coated iron oxide nanoparticles. (From Ref. [71], with permission.)

Fig. 5.7.

Cheon, Suh, and coworkers have demonstrated such advantages for the synthesis of iron oxide MR contrast agents [18, 36, 61]. As shown by transmission electron microscopy (Fig. 5.8), nanoparticles obtained are @9 nm, with a narrow size distribution (s < 8%). HR-TEM and X-ray analyses show that the nanoparticles are single-crystalline stoichiometric Fe3 O4 . Water-soluble iron oxide nanoparticles (WSIO) are then obtained by introducing 2,3-dimercaptosuccinic acid (DMSA) ligand onto the nanoparticle surface. This ligand endows the surface with high

Fig. 5.8.

TEM image of @9-nm Fe3 O4 nanoparticles.

5.3 Iron Oxide Nanoparticles in Molecular MR Imaging

(a) Schematic of 2,3dimercaptosuccinic acid (DMSA)-coated iron oxide nanoparticles. The carboxylic ends of DMSA bind to the surface iron oxide nanoparticles and are further stabilized through interligand disulfide crosslinkages. Remaining free thiol can be used for further

Fig. 5.9.

conjugation for biomolecules such as antibody. (b) Solubility test of as-synthesized (as-syn) and DMSA-coated iron oxide nanoparticles (WSIO ¼ water-soluble iron oxide nanoparticles). (c) Stability test of WSIO in various concentrations of NaCl solution. (From Ref. [18], with permission.)

water-phase stability through (a) carboxylate chelate bonding to iron and (b) disulfide crosslinkages between the ligands (Fig. 5.9a) [18]. Furthermore, the remaining free thiol group of the ligand can be used to attach target-specific biomolecules. Soobtained Fe3 O4 nanocrystals with the DMSA ligand are fairly stable in water and phosphate-buffered saline (PBS) up to an NaCl concentration of 250 mm without any aggregation. These nanoparticles have been utilized as MR probes, upon conjugation with cancer targeting antibody, not only for the in vitro detection of cancer cells but also for in vivo imaging of cancer implanted in a mouse [18, 36] (Section 5.3.5).

5.3

Iron Oxide Nanoparticles in Molecular MR Imaging

When iron oxide nanoparticles are conjugated with biologically active materials (e.g., antibody), the resulting iron oxide–biomolecule conjugates possess dual functionalities of both the MR contrast enhancers and molecular recognition capability. These conjugates act as molecular imaging probes that can efficiently report on various molecular/biological events occurring in region-of-interest targets (Fig.

157

158

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

Fig. 5.10.

Nanoparticle-assisted molecular MR imaging of biological systems.

5.10). Molecular MRI studies utilizing such iron oxide–biomolecule conjugates include the imaging of inflammation [72, 73], infarct [26–28], angiogenesis [29], apoptosis [30], gene expression [31, 32], b-amyloid plaques [74], and cancer [18, 33–36]. 5.3.1

Infarct and Inflammation

For imaging of infarcts and inflammations, monocrystalline iron oxide (MION) nanoparticles are conjugated with specific antibodies through electrostatic interactions or covalent linkages by the reaction of potassium periodate-activated surface hydroxyl groups with lysine residues of antibodies. For example, R11 D10 antimyosin Fab was electrostatically conjugated to hydroxyl groups on MION surfaces for cardiac infarct imaging [27]. Since infarcted cardiac cells have increased porosity compared with normal cells, iron oxide–antimyosin Fab conjugates can efficiently be transported into damaged cells and recognize myosin. Figure 5.11 shows T2-weighted MR images of a mouse with cardiac infarct after injection of MION-R11 D10 antimyosin Fab conjugates. The infarcted region is clearly observed as dark MR images, while no contrast effect was seen when unconjugated MIONs were administered. Such a targeting effect of MION-R11 D10 antimyosin Fab conju-

5.3 Iron Oxide Nanoparticles in Molecular MR Imaging

Fig. 5.11. MR detection of (a, b) cardiac infarct and (c, d) inflammation of a mouse. After intravenous injection of iron oxide– antimyosin antibody, dark MR contrast in the infarcted area is observed. Similarly,

inflammation is not imaged at the control mouse but an inflammation site is clearly shown as dark contrast. (From Refs. [27 and 72], with permission.)

gates was evaluated through ex vivo immunohistological analyses through Prussian blue staining. Weissleder and coworkers further extended this strategy for the detection of inflammation by conjugating MIONs with polyclonal human immunoglobulin G. MION-IgG conjugates consistently detected the area of inflammation in T2-weighted spin-echo MR images (Fig. 5.11c,d), which was also further confirmed by a histological Prussian blue staining study [72]. 5.3.2

Angiogenesis

Angiogenesis is a fundamental growth process of new blood vessels for development, reproduction, and wound repair. This process is also related to the progression of tumor growth. Therefore, the development of anti-angiogenic agents can be a potential pathway to efficient cancer treatment. Imaging of angiogenesis is also related to the cancer diagnosis and the evaluation of anti-cancer agents. Several molecular markers are involved in angiogenesis: vascular endothelial growth factor (VEGF), fibroblast growth factors (FGG), platelet-derived endothelial cell growth

159

160

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

Fig. 5.12. In vitro MR detection of E-selectin stimulated by interleukin-1b. (a) Without interleukin 1b, a white MR image is obtained from HUVEC treated only with CLIO– anti-E-selectin antibody. (b) In contrast, after

interleukin 1b and CLIO–anti-E-selectin are sequentially dosed to HUVEC, E-selectin expression is clearly imaged as a dark MR image. (From Ref. [29], with permission.)

factor (PD-EDGF), Tie-2 receptor, integrin, and E-selectin [75]. Among the various angiogenesis markers, VEGF, Tie 2 receptor, and integrin have been extensively studied [76–78]. Bogdanov and coworkers have reported that E-selectin expression in human endothelial cells can be imaged by using crosslinked iron oxide (CLIO)– monoclonal anti-human E-selectin antibody conjugate MR contrast agent [29]. When only CLIO–antibody conjugates are treated to endothelial cells with a low E-selectin expression level, an MR contrast effect is hardly detected (Fig. 5.12a). In contrast, treatment of cells with interleukin-1b, which stimulates the expression of E-selectin, followed by nanoparticle–antibody dosing results in a significant MR contrast effect (Fig. 5.12b). 5.3.3

Apoptosis

Apoptosis is an active process of programmed self-destruction of cells. In the early stages of apoptosis, the redistribution of phosphatidylserine in the cell membrane occurs and the detection of such processes can be an indicators of the programmed

5.3 Iron Oxide Nanoparticles in Molecular MR Imaging

Fig. 5.13. (a) MR imaging of apoptosis using the SPIO–C2 domain of synaptotagmin I. (b) T2-weighted MR images of (b, 1) water, (b, 2) SPIO–C2 conjugate, (b, 3) SPIO–BSA control conjugate, (b, 4) SPIO only treated

apoptotic cells, and (b, 5) SPIO–C2 conjugate treated normal cells. In vivo MR images of tumors implanted in a mouse (c) before and (d) after drug treatment. (From Ref. [30], with permission.)

cell death. Representative binding proteins to phosphatidylserine are annexin V and synaptotagmin I. Although imaging of apoptosis using these antibodies has been already performed through radio-isotope techniques [79], the spatial resolution is only @1–3 mm and needs improvement. Brindle and coworkers have shown that conjugates of the SPIO and C2 domain of synaptotagmin I (C2-SPIO) can detect apoptotic cells through MRI with @0.1 mm resolution [30]. While there are no MR signals for nonspecific SPIO (BSA-SPIO) [Fig. 5.13b(3)], SPIO-only treated apoptotic cells [Fig. 5.13b(4)], and C2-SPIO treated normal cells [Fig. 5.13b(5)], C2SPIO treated apoptotic cells [Fig. 5.13b(2)] clearly show dark MR contrast with a significant change in T2 (DT2 ¼ @90%). Further extension of this strategy to an in vivo animal study was also successful. When C2-SPIO was intravenously injected to drug-treated tumor-bearing mice, the nanoparticle conjugates are able to detect apoptotic regions with a significant MR signal change (Fig. 5.13c,d). 5.3.4

Gene Expression

Gene expression is an emerging field in biomedical sciences, and the imaging of such expression is of importance. Although several approaches to detect in vivo gene expression have been performed through optical [80, 81] and radioisotope imaging techniques [82], there have been limitations such as low-penetration depth of light for optical imaging and poor spatial resolution of radioisotope imaging. Weissleder and coworkers have demonstrated that MR detection of transgene

161

162

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

5.3 Iron Oxide Nanoparticles in Molecular MR Imaging

expression of engineered transferrin receptor (ETR) in tumors is possible by using MION-transferrin (MION-Tf ) contrast agents [32]. When MION-Tf is treated to the cells with various ETR expression levels, a gradual decrease in T2 is observed as the ETR expression levels of cells are increased due to proportional binding of MION-Tf conjugates to the expressed ETR. These workers also determined whether ETR expression can be detected in in vivo live mice with ETR positive tumors and ETR negative tumors. The results show that the MR contrast effect is only observed for ETR positive tumors (Fig. 5.14b–d). Ex vivo MRI of excised tumors shows more dramatic differences between these two tumors (Fig. 5.14e,f ). 5.3.5

Cancer Imaging

Non-invasive detection of cancer in its early stages is of great interest since early detection of cancer can significantly increase the survival rate of patients. With conventional MRI techniques, the present detectable size of cancer is roughly @1 cm 3 . If nanoparticle contrast agents can specifically recognize cancer cells through molecular interaction, selective enhancement of the MR signal of cancer cells can provide one with a way to clearly distinguish cancer from normal tissues. Tiefenauer and coworkers have reported that the detection of cancers is possible through such molecular recognition of superparamagnetic iron oxide (SPIO) nanoparticle– antibody conjugates [35]. Conjugation of poly(glutamic acid-lysine-tyrosine)-coated nanoparticles with anti-carcinoembryonic antigen (CEA) antibody is performed through a conventional sulfo-MBS crosslinking method. In the T2-weighted MR images, dark contrast is imaged at the CEA-expressed tumors, although the contrast difference is not highly pronounced (Fig. 5.15). Artemov and coworkers have utilized another approach to detect cancer cells [34]. Since the avidin–biotin interaction is strongly binding, avidin conjugated SPIO can efficiently detect biotinylated cancer specific antibodies that bind to the cancer cells (Fig. 5.16a). Their in vitro fluorescence-assisted cell sorting analyses and MRI confirm cancer detection (Fig. 5.16b). Au-565 cells with high expression of HER2/neu cancer markers are imaged as dark MR contrast, while no contrast effect is obtained from MDAMB-231 cells with low HER2/neu expression. Recently, Cheon, Suh, and coworkers have achieved highly efficient cancer targeting by using high quality, small-sized water-soluble iron oxide (WSIO) nanoparticle–antibody conjugates [36]. The WSIO nanoparticles have high magnetic momentum [@100 emu (g-Fe)1 ] and small hydrodynamic size (@9 nm), which are advantageous for both in vitro and in vivo cancer imaging. When these _________________________________________________________________________________ H Fig. 5.14. In vivo and ex vivo imaging of engineered transferrin receptor (ETR) expression in tumors. (a) Schematic of MRI, (b, c) in vivo imaging of ETR(þ) and ETR() tumor implanted mouse before (b) and after

(c, d) the injection of MION-transferrin. Ex vivo imaging of excised ETR(þ) and ETR() tumors (e) and their color maps based on T2 (f ). (From Ref. [32], with permission.)

163

164

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

MR detection of carcinoembryonic antigen (CEA) overexpressed tumors by using iron oxide–CEA antibody conjugates. MR images before (a) and after (b) injection of SPIO–CEA antibody. (From Ref. [35], with permission.)

Fig. 5.15.

nanoparticles are conjugated with Herceptin, they successfully detect cancer cells (SK-BR-3) as dark MR image (Fig. 5.17b) through molecular interaction between nanoparticle surface-bound Herceptin and HER2/neu cancer markers, compared with non-treated (Fig. 5.17a) and WSIO-irrelevant conjugate treated cells (Fig. 5.17c). This MRI result is also confirmed by an optical technique where vivid green fluorescence from the fluorescein (FITC) is clearly observed only for FITC-WSIOHerceptin probe conjugate treated cells (Fig. 5.17d,e). Furthermore, WSIO nanoparticles enable the detection of various cell lines with different levels of HER2/

Fig. 5.16. In vitro MR detection of HER2/neu overexpressed cancer cells by using avidin-coated SPIO and biotinylated Herceptin. (a) Schematic and (b) MR images of SPIO–avidin conjugate treated cells (AU-565, MDA-MB-231, MCF-7). (From Ref. [34], with permission.)

5.3 Iron Oxide Nanoparticles in Molecular MR Imaging

Fig. 5.17. In vitro cancer detection using water-soluble iron oxide (WSIO)–Herceptin conjugates. MR images of (a) non-treated, (b) WSIO–Herceptin treated, (c) WSIOirrelevant antibody treated breast cancer cells (SK-BR-3). (d) MR image of WSIO–Herceptin

conjugate treated cell lines with increasing expression levels of HER2/neu receptors: Bx-PC-3, MDA-MB-231, BT-474, and NIH3T6.7 cell lines. Control conjugates are treated to Bx-PC-3 cell lines. (From Ref. [36], with permission.)

165

166

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

neu cancer marker expression: Bx-PC-3, MDA-MB-231, BT-474, and NIH3T6.7 cell lines, which are arranged in the order of increasing HER2/neu expression level. T2-weighted MR signals of the cell lines treated with WSIO–Herceptin probe conjugates become darker as the expression level of the HER2/neu receptors is increased (Fig. 5.17f ). Notably, the MR signal intensities of the cell lines treated with WSIO–Herceptin probe conjugates show a marked difference from that of control conjugates, indicating excellent specific binding efficiency of the probe conjugates. These magnetic probes have been successfully extended to the in vivo detection of cancer cells implanted in mouse [36]. When these WSIO–Herceptin conjugates are intravenously injected into a mouse, they successfully reach and recognize HER2/neu receptors overexpressed from cancer cells which results in a significant MR contrast effect in the tumor sites, with a @20% decrease in T2 compared with control experiments (Fig. 5.18a–c). In high-resolution MR images of WSIO– Herceptin conjugate treated mouse measured at 9.4-T MRI, a dark MR image initially appears near the bottom region of the tumor and then gradually grows and spreads to the central and upper region of the tumor as time elapses (Fig. 5.18d). They found that such a time-dependent MR signal change reveals the heterogeneous pattern of the intratumoral vasculatures, where the bottom side of the tumor has well-developed vascular structures.

5.4

Summary and Outlook

Although there has been much progress in the development of magnetic nanoparticle contrast agents for molecular MRI in the past few years, their successful utilization is limited to in vitro systems, except for a few in vivo cases. The main difficulties lie in their poor MR contrast effects and limited stability under in vivo conditions. The MR signal enhancing effect of conventional iron oxide-based nanoparticles is unsatisfactory, compared with other diagnostic tools such as fluorescence and PET, and needs to be improved. Therefore, it is important to develop new types of magnetic nanoparticle contrast agents that can significantly improve contrast effects. Since nanoparticles with higher magnetization values provide stronger MR contrast effects, the development of novel nanoparticles with superior magnetism is the first prerequisite. Concurrently, since the MR contrast effects of nanoparticles are strongly correlated with a material’s characteristics in terms of their size, shape, composition, single crystallinity, and magnetism, it is important to have a good nanoparticle model system that can clearly describe the relationship between nanoscale material characteristics and MR contrast effects (Fig. 5.19a). The next required step is to impart high colloidal stability and biocompatibility to the magnetic nanoparticles. As described in previous sections, various coating materials have been developed for such applications, but it is still necessary to develop general and more reliable protocols for tailoring nanoparticle surfaces with desired coating materials. Since a smaller overall size is advantageous for escaping the re-

5.4 Summary and Outlook

Fig. 5.18. In vivo MRI of cancer targeting events of WSIO–Herceptin conjugates. Color maps of T2-weighted MR images of cancer cell implanted (NIH3T6.7) mice at different temporal points (pre-injection, immediate post, 4 h) after the intravenous injection of (a) WSIO-irrelevant antibody control conjugates and (b) WSIO–Herceptin probe conjugates. (c) T2 versus time after the injection of WSIOantibody conjugates in (a) and (b) samples. (d) T2*-weighted MR images of cancer cell implanted (NIH3T6.7) mouse at 9.4 Tesla and

their color maps at different temporal points after probe conjugate injection. Tumor area is circled with white dotted lines. A dark MR image gradually grows and spreads from the bottom region of the tumor to the central and upper region of the tumor (red circles). (e) Fluorescence immunohistochemical analyses of an excised tumor slice. Endothelial vessels were stained with Rhodamine-anti-CD31 (red fluorescence) and probe conjugates were stained with FITC-anti-human IgG (green fluorescence). (From Ref. [36], with permission.)

ticuloendothelial system, the coating materials should be as small as possible while possessing high colloidal stability without any aggregation under physiological conditions (Fig. 5.19b). The toxicity of magnetic nanoparticles is also a very important issue that needs to be resolved before clinical utilization. Although iron oxide nanoparticles have been regarded as clinically benign materials, potential cytotoxicity arising from their size, shape, and coating materials should also be examined, along with

167

168

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

Nanoparticle-assisted molecular MRI. (a) Modern molecular chemistry approach enables the tailored synthesis of high quality magnetic nanoparticles with desired sizes and monodispersity. (b) Then, evaluation and optimization of materials properties such as magnetism, hydrodynamic size, and colloidal and biostabilities are important. (c) When these nanoparticles are conjugated

Fig. 5.19.

to biomolecules, the resulting nanoparticle– biomolecule conjugates possess the capabilities of both MR contrast effects and molecular recognition of target biosystems. (d) This enables molecular MRI that can report various biological events, such as pinpointing cancer diagnosis, cell migration, cell signaling, and genetic developments, with high sensitivity and specificity.

systematic guidelines for the nano-toxicity evaluation of newly developed novel nanoparticles. Once novel nanoparticles with highly enhanced magnetism, small size, and high colloidal and biostability are developed, significant improvements in MR detection sensitivity and target specificity are expected (Fig. 5.19c,d). This can bring huge advances in current cancer diagnosis and biomedical imaging fields. For example, highly enhanced MR contrast of a biological target through the molecular recognition of such nanoparticle contrast agents will promise in vivo diagnosis of early-stage cancer with sub-millimeter dimension. In addition, many unrevealed

References

biological processes, such as in vivo pathways of cell evolutions, cell differentiations, cell-to-cell interactions, molecular signaling pathways, could be precisely monitored at the molecular level by using next-generation nanoparticle contrast agents.

References 1 Alivisatos, A. P. Semiconductors

2

3

4 5

6

7

8

9

10

clusters, nanocrystals, and quantum dots, Science 1996, 271, 933–937. LaVan, D. A., Lynn, D. M., Langer, R. Moving smaller in drug discovery and delivery, Nat. Rev. Drug. Discov. 2002, 1, 77–84. LaConte, L., Nitin, N., Bao, G. Magnetic nanoparticle probes, Nanotoday 2005, 8, 32–38. Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47. Weissleder, R., Mahmood, U. Molecular imaging, Radiology 2001, 219, 316–333. Massoud, T. F., Gambhir, S. S. Molecular imaging in living subjects: Seeing fundamental biological processes in a new light, Gene Dev. 2003, 17, 545–580. Wu, X., Liu, H., Liu, J., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N., Peale, F., Bruchez, M. P. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots, Nat. Biotechnol. 2003, 21, 41–46. Bruchez, Jr., M., Moronne, M. M., Gin, P., Weiss, S., Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013. Warren, C. W. C. and Nie, S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science 1998, 281, 2016. Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., Weiss, S. Quantum dots for live cells, in vivo imaging, and diagnostics, Science 2005, 307, 538–544.

11 Bruchez, M., Moronne, M., Gin, P.,

12

13

14

15

16

17

18

19

Weiss, S., Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013–2016. Chan, W. C. W., Nie, S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science 1998, 281, 2016–2018. Han, M., Gao, X., Su, J. Z., Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules, Nat. Biotechnol. 2001, 19, 631–635. Mitchell, D. G. MRI Principles, W.B. Saunders Company, Philadelphia, 1999. Bulte, J. W. M., Kraitchman, D. L. Iron oxide MR contrast agents for molecular and cellular imaging, NMR Biomed. 2004, 17, 484–499. Wang, Y.-X. J., Hussain, S. M., Krestin, G. P. Superparamagnetic iron oxide contrast agents: Physicochemical characteristics and applications in MR imaging, Eur. Radiol. 2001, 11, 2319–2331. Koenig, S. H., Keller, K. E. Theory of 1/T1 and 1/T2 NMRD profiles of solutions of magnetic nanoparticles, Magn. Reson. Med. 1995, 34, 227– 233. Jun, Y.-w., Huh, Y.-M., Choi, J.-s., Lee, J.-H., Song, H.-T., Kim, S., Yoon, S., Kim, K.-S., Shin, J.-S., Suh, J.-S., Cheon, J. Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging, J. Am. Chem. Soc. 2005, 127, 5732–5733. Hamm, B., Staks, T., Taupitz, M., Maibauer, R., Speidel, A., Huppertz, A., Frenzel, T., Lawaczeck, R., Wolf,

169

170

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

20

21

22

23

24

25

26

27

28

K. J., Lange, L. J. Magn. Reson. Imag. 1994, 4, 659–668. Reimer, P., Tombach, B. Hepatic MRI with SPIO: Detection and characterization of focal liver lesions, Eur. Radiol. 1998, 8, 1198–1204. McLachlan, S. J., Morris, M. R., Lucas, M. A., Fisco, R. A., Eakins, M. N., Fowler, D. R., Scheetz, R. B., Olukotun, A. Y. J. Magn. Reson. Imag. 1994, 4, 301–307. Weissleder, R. Liver MR imaging with iron oxides: Toward consensus and clinical practice, Radiology 1994, 193, 593–595. Bengele, H. H., Palmacci, S., Rogers, J., Jung, C. W., Crenshaw, J., Josphson, L. Biodistribution of an ultrasmall superparamagnetic iron oxide colloid, BMS 180549, by different routes of administration, Magn. Reson. Imag. 1994, 12, 433–442. Suwa, T., Ozawa, S., Ueda, M., Ando, N., Kitajima, M. Magnetic resonance imaging of esophageal squamous cell carcinoma using magnetite particles coated with anti-epidermal growth factor receptor antibody, Int. J. Cancer 1998, 75, 626–634. Kresse, M., Wagner, S., Pfefferer, D., Lawaczeck, R., Elste, V., Semmler, W. Targeting of ultrasmall superparamagnetic iron oxide (USPIO) particles to tumor cells in vivo by using transferrin receptor pathways, Magn. Reson. Med. 1998, 40, 236–242. Krieg, F. M., Andres, R. Y., Winterhalter, K. H. Superparamagnetically labelled neutrophils as potential abscess-specific contrast agent for MRI, Magn. Reson. Imag. 1995, 13, 393–400. Weissleder, R., Lee, A. S., Khaw, B. A., Shen, T., Brady, T. J. Antimyosinlabeled monocrystalline iron oxide allows detection of myocardial infarct: MR antibody imaging, Radiology 1992, 182, 381–385. Kraitchman, D. L., Heldman, A. W., Atalar, E., Amado, L. C., Martin, B. J., Pittenger, M. F., Hare, J. M., Bulte, J. W. M. In vivo magnetic resonance imaging of mesenchymal

29

30

31

32

33

34

35

36

stem cells in myocardial infarction, Circulation 2003, 107, 2290–2293. Kang, H. W., Josephson, L., Petrovsky, A., Weissleder, R., Bogdanov, A., Jr. Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture, Bioconjugate Chem. 2002, 13, 122–127. Zhao, M., Beauregard, D. A., Loizou, L., Davletov, B., Brindle, K. M. Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent, Nat. Med. 2001, 7, 1241–1244. Ho¨gemann, D., Josephson, L., Weissleder, R., Basilion, J. P. Improvement of MRI probes to allow efficient detection of gene expression, Bioconjugate Chem. 2000, 11, 941–946. Weissleder, R., Moore, A., Mahmood, U., Bhorade, R., Benveniste, H., Chiocca, E. A., Basilion, J. P. In vivo magnetic resonance imaging of transgene expression, Nat. Med. 2000, 6, 351–355. ¨hne, G., Tiefenauer, L. X., Ku Andres, R. Y. Antibody-magnetite nanoparticles: In vitro characterization of a potential tumor-specific contrast agent for magnetic resonance imaging, Bioconjugate Chem. 1993, 4, 347–352. Artemov, D., Mori, N., Okollie, B., Bhujwalla, Z. M. MR molecular imaging of the Her-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles, Magn. Reson. Med. 2003, 49, 403–408. Tiefenauer, L. X., Tschirky, A., Iwhne, G., Andres, R. Y. In vivo evaluation of magnetite nanoparticles for use as a tumor contrast agent in MRI, Magn. Reson. Imag. 1996, 14, 391–402. Huh, Y.-M., Jun, Y.-w., Song, H.-T., Kim, S., Choi, J.-s., Lee, J.-H., Yoon, S., Kim, K.-S., Shin, J.-S., Suh, J.-S., Cheon, J. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals, J. Am. Chem. Soc. 2005, 127, 12 387– 12 391.

References ¨ gren, C. E., Johansson, C., 37 Sjo

38

39 40

41

42

43

44

45

46

Naevestad, A., Sontum, P. C., Briley-Saebo, K., Fahlvik, A. K. Crystal size and properties of superparamagnetic iron oxide (SPIO) particles, Magn. Reson. Imag. 1997, 15, 55–67. Hahn, P. F., Stark, D. D., Lewis, J. M., Saini, S., Elizondo, G., Weissleder, R., Fretz, C. J., Ferrucci, J. T. First clinical trial of a new superparamagnetic iron oxide for use as an oral gastrointestinal contrast agent in MR imaging, Radiology 1990, 175, 695–700. Bach-Gansmo, T. Acta Radiol. [Suppl] 1993, 387, 1–30. Weissleder, R., Elizondo, G., Josephson, L., Compton, C. C., Fretz, C. J., Stark, D. D., Ferrucci, J. T. Experimental lymph node metastases: Enhanced detection with MR lymphography, Radiology 1989, 171, 835–839. Weissleder, R., Stark, D. D., Engelastad, B. L., Bacon, B. R., Compton, C. C., White, D. L., Jacobs, P., Lewis, J. Superparamagnetic iron oxide: Pharmacokinetics and toxicity, Am. J. Roentgenol. 1989, 152, 167–173. Reimer, P., Rummeny, E. J., Daldrup, H. E., Balzer, T., Tombach, B., Berns, T., Peters, P. E. Clinical results with resovist: A Phase 2 clinical trial, Radiology 1995, 195, 489–496. Grubnic, S., Padhani, A. R., Revell, P. B., Husband, J. E. Comparative efficacy of and sequence choice for two oral contrast agents used during MR imaging, Am. J. Roentgenol. 1999, 173, 173–178. Bartolozzi, C., Lencioni, R., Donati, F., Cioni, D. Abdominal MR: Liver and pancreas, Eur. Radiol. 1999, 9, 1496–1512. Moore, A., Marecos, E., Bogdanov, A., Weissleder, R. Tumoral distribution of long-circulating dextran-coated iron oxide nanoparticles in a rodent model, Radiology 2000, 214, 568–574. Palmacci, S., Josephson, L. U.S. Patent 1993, 5,262,176.

47 Shen, T., Weissleder, R., Papisov,

48

49

50

51

52

53

54

55

56

M., Bogdanov, A., Brady, T. J. Monocrystalline iron oxide nanocompounds (MION): Physicochemical properties, Magn. Reson. Med. 1993, 29, 599–604. Ford, G. C. Ferritin: Design and formation of an iron-storage molecule, Philos. Trans. R. London Ser. B. 1984, 304, 551. Meldrum, F. C., Heywood, B. R., Mann, S. Magnetoferritin: In vitro synthesis of a novel magnetic protein, Science 1992, 257, 522–523. Bulte, J. W. M., Douglas, T., Mann, S., Frankel, R. B., Moskowitz, B. M., Brooks, R. A., Baumgarner, C. D., Vymazal, J., Strub, M.-P., Frank, J. A. J. Magn. Reson. Imag. 1994, 4, 497–505. Meldrum, F. C., Wade, V. J., Nimmo, D. L., Heywood, B. R., Mann, S. Synthesis of inorganic nanophase materials in supramolecular protein cages, Nature 1991, 349, 684–687. Bulte, J. W. M., Douglas, T., Mann, S., Vymazal, J., Laughlin, P. G., Frank, J. A. Initial assessment of magnetoferritin biokinetics and proton relaxation enhancement in rats, Acad. Radiol. 1995, 2, 871–878. Bulte, J. W. M., Douglas, T., Witwer, B., Zhang, S.-C., Strable, E., Lewis, B. K., Zywicke, H., Miller, B., Gelderen, P., Moskowitz, B. M., Duncan, I. D., Frank, J. A. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells, Nat. Biotechnol. 2001, 19, 1141–1147. Strable, E., Bulte, J. W. M., Moskowitz, B., Vivekanandan, K., Allen, M., Douglas, T. Synthesis and characterization of soluble iron oxidedendrimer composites, Chem. Mater. 2001, 13, 2201–2209. Bulte, M., Cuyper, J. W., De., M., Despres, D. and Frank, J. A. Shortvs. long-circulating magnetoliposomes as bone marrow-seeking MR contrast agents, J. Magn. Reson. Imag. 1999, 9, 329–335. Cheon, J., Kang, N.-J., Lee, S.-M., Lee, J.-H., Yoon, J.-H., Oh, S. J.

171

172

5 Magnetic Resonance Nanoparticle Probes for Cancer Imaging

57

58

59

60

61

62 63

64

65

Shape evolution of single-crystalline iron oxide nanocrystals, J. Am. Chem. Soc. 2004, 126, 1950–1951. Sun, S., Zeng, H. Size-controlled synthesis of magnetite nanoparticles, J. Am. Chem. Soc. 2002, 124, 8204–8205. Redl, F. X., Black, C. T., Papaefthymiou, G. C., Sandstrom, R. L., Yin, M., Zeng, H., Murray, C. B., O’Brien, S. P. Magnetic, electronic, and structural characterization of nonstoichiometric iron oxides at the nanoscale, J. Am. Chem. Soc. 2004, 126, 14 583–14 599. Park, J., An, K., Hwang, Y., Park, J.-G., Noh, H.-J., Kim, J.-Y., Park, J.-H., Hwang, N.-M., Hyeon, T. Ultralarge-scale syntheses of monodisperse nanocrystals, Nat. Mater. 2004, 3, 891–895. Jana, N. R., Chen, Y., Peng, X. Sizeand shape-controlled magnetic (Cr, Mn, Fe, Co, Ni) oxide nanocrystals via a simple and general approach, Chem. Mater. 2004, 16, 3931–3935. Song, H.-T., Choi, J.-s., Huh, Y.-M., Kim, S., Jun, Y.-w., Suh, J.-S., Cheon, J. Surface modulation of magnetic nanocrystals in the development of highly efficient magnetic resonance probes for intracellular labeling, J. Am. Chem. Soc. 2005, 127, 9992–9993. Pileni, M.-P. Nat. Mater. 2003, 2, 145–150. Nitin, N., LaConte, L. E. W., Zurkiya, O., Hu, X., Bao, G. Functionalization and peptide-based delivery of magnetic nanoparticles as an intracellular MRI contrast agent, J. Biol. Inorg. Chem. 2004, 9, 706–712. Yee, C., Kataby, G., Ulman, A., Prozorov, T., White, H., King, A., Rafailovich, M., Sokolov, J., Gedanken, A. Self-assembled monolayers of alkanesulfonic and -phosphonic acids on amorphous iron oxide nanoparticles, Langmuir 1999, 15, 7111–7115. Harris, L. A., Goff, J. D., Carmichael, A. Y., Riffle, J. S., Harburn, J. J., Pierre, T. G. St., Saunders, M. Magnetite nanoparticle

66

67

68

69

70

71

72

73

74

dispersions stabilized with triblock copolymers, Chem. Mater. 2003, 15, 1367–1377. Burke, N. A. D., Stover, H. D. H., Dawson, F. P. Magnetic nanocomposites: Preparation and characterization of polymer-coated iron nanoparticles, Chem. Mater. 2002, 14, 4752–4761. Lu, Y., Yin, Y., Mayers, B. T., Xia, Y. Modifying the surface properties of superparamagnetic iron oxide nanoparticles through a sol-gel approach, Nano Lett. 2002, 2, 183–186. Santra, S., Tapec, R., Theodoropoulou, N., Dobson, J., Hebard, A., Tan, W. Synthesis and characterization of silica-coated iron oxide nanoparticles in microemulsion: The effect of nonionic surfactants, Langmuir 2001, 17, 2900–2906. Dubertret, B., Skourides, P., Norris, D. J., Noireaux, V., Brivanlou, A. H., Libchaber, A. In vivo imaging of quantum dots encapsulated in phospholipid micelles, Science 2002, 298, 1759–1762. Kim, S.-W., Kim, S., Tracy, J. B., Jasanoff, A., Bawendi, M. G. Phosphine oxide polymer for watersoluble nanoparticles, J. Am. Chem. Soc. 2005, 127, 4556–4557. Kohler, N., Fryxell, G. E., Zhang, M. A Bifunctional poly(ethylene glycol) silane immobilized on metallic oxide-based nanoparticles for conjugation with cell targeting agents, J. Am. Chem. Soc. 2004, 126, 7206– 7211. Weissleder, R., Lee, A. S., Fischman, A. J., Reimer, P., Shen, T., Wilikinson, R., Callahan, R., Brady, T. J. Polyclonal human immunoglobulin G labeled with polymeric iron oxide: Antibody MR imaging, Radiology 1991, 181, 245–249. Schmitz, S. A., Taupitz, M., Wagner, S., Wolf, K.-J., Beyersdorff, D., Hamm, B. J. Magn. Reson. Imag. 2001, 14, 355–361. Wadghiri, Y. Z., Sigurdsson, E. M., Sadowski, M., Elliott, J. I., Li, Y., Scholtzova, H., Tang, C. Y., Aguinaldo, G., Pappolla, M., Duff, K., Wisniewski, T., Turnbull, D. H.

References

75 76

77

78

Detection of Alzheimer’s amyloid in transgenic mice using magnetic resonance microimaging, Magn. Reson. Med. 2003, 50, 293–302. Risau, W. Mechanisms of angiogenesis, Nature 1997, 386, 671–674. Veikkola, T., Karkkainen, M., Claesson-Welsh, L., Alitalo, K. Regulation of angiogenesis via vascular endothelial growth factor receptors, Cancer Res. 2000, 60, 203–212. Kim, I., Kim, H. G., Si, J. N., Kim, J. H., Kwak, H. J., Koh, G. Y. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3 0 -kinase/Akt signal transduction pathway, Circ. Res. 2000, 86, 24–29. Saeed, M., Wendland, M. F., Engelbrecht, M., Sakuna, H., Higgins, C. B. Value of blood pool contrast agents in magnetic resonance

79

80

81

82

angiography of the pelvis and lower extremities, Eur. Radiol. 1998, 8, 1047–1053. Lahorte, C. M., Vanderheyden, J. L., Steinmetz, N., Van de Wiele, C., Dierckx, R. A., Slegers, G. Apoptosis-detecting radioligands: Current state of the art and future perspectives, Eur. J. Nucl. Med. Mol. Imag. 2004, 31, 887–919. Shah, K., Weissleder, R. Molecular optical imaging: Applications leading to the development of present day therapeutics, NeuroRx. 2005, 2, 215–225. Gross, S., Piwnica-Worms, D. Spying on cancer: Molecular imaging in vivo with genetically encoded reporters, Cancer Cell 2005, 7, 5–15. Haberkorn, U., Altmann, A. Functional genomics and radioisotopebased imaging procedures, Ann. Med. 2003, 35, 370–379.

173

174

6

LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers Carola Leuschner 6.1

Introduction

Despite new discoveries of drugs and treatment combinations for cancer the mortality rate has not improved in recent decades. There is an urgent need to improve treatment and imaging in patients diagnosed with distant metastatic cancers. Cancer as a confined disease is treatable today; in contrast, distant metastatic disease outcome has not improved over the past 30 years and most patient succumb to this severe, devastating form of the disease. Furthermore, new drugs and contrast agents need to be developed to monitor the efficacy of response to treatments. Nanotechnology for treatment and diagnostics has been the focus of developmental research in the past 10 years and continues to grow exponentially. This thriving field of research focuses on materials that are on the nanometer scale, opening up new avenues that can lead to the development of highly specific compounds for treatment and imaging. Several reviews focus on the fabrication of nanoparticles for various applications, including encapsulation of drugs, and mainly summarize the latest developments for untargeted nanoparticles that are distinguishable by different coating materials, nanomaterials and various sizes and shapes. Nanoparticles open new opportunities to treat cancers by hyperthermia, to encapsulate drugs like doxorubicin, camptotecin or radiolabeled compounds that can be functionalized for higher specificity to reduce systemic exposure, and to deliver specifically nanoparticles to tumors, metastases peripheral organs and the brain. The specific targeting of tumor cells and metastases has been more intensely under investigation. Several receptors overexpressed in cancer cells have been targeted. Although with different success with respect to specificity (some of these targets are also expressed in peripheral tissue), targeting of tumor cells can be surface binding only or by receptor-mediated endocytotic uptake of delivered compounds, which was observed to various extents, depending on the type of receptor. Among targeting moieties on cancer cells, tumors and metastases the receptors for luteinizing hormone releasing hormone (LHRH) have been studied in detail since 1980.

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

6.2 Cancer

The importance of LHRH receptors as specific targeting moiety for cancer treatments and diagnosis has become evident as peripheral organs express little or no receptors; however, most human cancer cells that belong to cancers of the highest incidence and death rates have tested positive for LHRH receptors. Most importantly, this chapter overviews specific targeting of nanoparticles using LHRH receptors for specific delivery, higher efficiency and faster accumulation of nanoparticles within tumor cells. Current applications in nanotechnology targeting the LHRH receptor for treatment and imaging will be explained and the latest findings and discoveries will be summarized for this highly important receptor. Several reviews and book chapters have been published over the past decade that summarize LHRH receptors and their function in gonadal and pituitary tissues. This chapter tries to connect the current knowledge of LHRH and LHRH receptors in malignant tissue to specifically target nanoparticles through LHRH receptors to primary tumors and metastases. The current literature regarding LHRH receptors in malignancies will be reviewed and the latest findings in the development of targeted treatment and diagnosis will be discussed, with emphasis on nanoparticle construction and their consequences in vitro and in vivo.

6.2

Cancer

Cancer is the second leading cause of death, with 10.9 million newly diagnosed cases worldwide in 2005, claiming a total of 6.7 million lives [1]. In the USA, one in four deaths are caused by cancer [2]. Despite new drugs and treatment combinations for cancer the mortality rate has not improved in recent decades. Of 1 372 910 new diagnosed cases for all cancers in 2005 in the USA 570 380 Americans died [2]. The highest mortality rate of all occurring deaths in the United States in men and women was lung cancer, claiming the life of 28% of patients [3]. Since 1974 the survival rate has increased by only 13% as more sensitive diagnostic techniques have made very early detection of cancers possible. The yearly cancer death rates in men and women over the past 70 years has not improved despite new treatment regimens [1, 3]. The reason for this poor development becomes apparent in looking at the 5-year survival rates of cancer patients (Table 6.1, [4–29]). Cancer death is dependent on the disease stage, whether the tumor is confined to the organ of origin and can be resected surgically, treated with radiation therapy or with systemic chemotherapeutics. The table clearly depicts the problem occurring in the distant, metastatic disease of cancer, which significantly reduces the 5-year survival rate of the patients to single digits compared with the organconfined disease stage (Table 6.1). This serious situation can be explained for prostate and breast cancers as examples: The second leading cause of cancer death in men is prostatic adenocarcinoma, and in women mammary carcinoma. Both cancers develop mixed popula-

175

176

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers Tab. 6.1. Occurrence of cancer by organ, survival rates in local and distant metastatic disease and their LHRH-receptor expression.

Cancer type

New cases

Deaths

Localized disease

LHRHreceptors expressed (%)

49

N.D.

5-year survival rate (%) Metastatic disease

2.1

Ref.

Lung

186 550

165 130

Prostate

230 110

29 900

34

100 (86% discovered)

86

4–7

Breast

217 440

40 580

23

97

52

8–12

Colon

106 370

56 730

9

90 (only 38% discovered)

Yes

13, 14

Pancreas

31 860

31 270

1.6

16

67

9, 15, 16

Ovary

25 580

16 090

30

94 (only 29% discovered)

80

17–21

Leukemia

33 440

23 300

64

Non-Hodgkin’s lymphoma

54 370

19 410

56

84

Yes

23

Esophagus

14 250

13 300

2.2

29.1

Yes

24

Liver

18 920

14 270

1.9

16.3

Yes

25

Uterine corpus (Endometrium) (Cervix)

40 320 10 370

7 090 3 710

26 17

96 92

80

18, 19, 26

Urinary bladder

60 240

12 710

6

94

Melanoma

55 100

7 910

13.8

96.7

Yes

22, 27

Kidney

35 710

12 480

9.1

89

Yes

28

Brain

18 400

12 690

Yes

29

1 368 030 newly diagnosed cancer cases in the USA in 2004, 563 700 Americans die. The numbers are stated for the most frequent cancer types for men and women combined [3]. Survival rates are given for patients with localized (malignancy entirely confined to the organ of origin) and distant (malignancy has invaded lymph nodes or organs remote from the primary tumor) disease.

6.2 Cancer

tions of hormone (estrogen/androgen) dependent and independent cells, which are poorly to well differentiated, and have variable proliferation rates. The preferred treatment for men with organ-confined disease is radical prostatectomy, while in women it is mastectomy. Initially, patients are treated with radiation and/or chemotherapy (cyclophosphamide, doxorubicin, 5-fluorouracil) [30, 31], in addition to hormone ablation. Although androgen ablation in prostate cancer patients (LHRH agonists, Leuprolide) [32] leads to a reduction of the primary tumor, and to its partial regression, within 2 years the disease can re-emerge in a poorly differentiated, androgen-independent form, after which there is no therapy available to prolong the patient’s life [33]. In breast cancer patients estrogen ablation is achieved by tamoxifen treatment [34] or ovariectomy. Surgical removal of the primary tumor is invasive and has side effects, depending on the area of resection, on the tumor (encapsulated or invasive), and can even lead to increased proliferation of metastases as single metastatic cells and dormant cancer cells start to proliferate [35]. At the time of diagnosis up to 70% of prostate cancer patients [36, 37] and 40% of breast cancer patients have already developed occult metastases [38]. Bone and lymph node metastases occur in 26% of prostate adenocarcinomas [39] and in 23% of breast cancer patients [38]. More than 70% of patients die from skeletal metastases [40]. Often removal of the primary tumor can promote metastatic growth, and dormant cancer cells can become secondary, often more aggressive, tumors [35]. Current treatments are palliative and can only prolong the life of patients, they do not cure patients. Often, high doses of chemotherapeutics administered systemically are necessary to achieve the pharmacological effective concentration. Even to maintain a state of remission in the disease a high dose chemotherapy is required over a long period [41]. Because of the high toxicity and poor specificity of currently used drugs, an increase in chemotherapeutic dosages is not desirable. The administration of high drug concentrations has disadvantages regarding side effects as chemotherapeutics not only destroy malignant tumors but also healthy tissue and organs, which eventually forces an end to such treatment. Often, patients in recurrent disease do not respond to chemotherapy. This is due to an acquired drug-resistance, which decreases the efficacy of chemotherapy. Dormant metastatic disease is not treatable with chemo- or radiation-therapy as these destroy only fast proliferating cells [38, 42, 43]. Currently used chemotherapeutic drugs are systemically active and cannot target single dormant cancer cells, or slow growing tumors [38, 44], because most chemotherapeutic drugs are not selective for cancer cells and destroy only rapidly dividing cells. 6.2.1

Conventional Approaches to Cancer/Metastases Detection

Sensitive non-invasive detection and monitoring tools are needed for early tumor detection and for monitoring disease progression, efficacy of treatment regimens and responses to treatments. Higher resolution and improved imaging techniques are needed to detect single, disseminated cancer cells or even small cancer cell

177

178

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

clusters in peripheral organs, bones and lymph nodes at the earliest stage before development of secondary tumors. Current detection methods are invasive and lack the sensitivity to detect micrometastases. Clinically, metastases have been quantified by counting macroscopic nodules from biopsy specimen or tumor cell colonies in histological sections. The detection rates with these methods is 1–2% [45–47]. These approaches are invasive and lack the sensitivity necessary to detect micrometastases or single disseminated cells and cell clusters in peripheral organs. The use of immunocytochemistry techniques increased the detection rate of bone metastases in bone marrow aspirates up to 30% [48–50]. RT-PCR techniques (reverse transcriptase polymerase chain reaction) have been developed to detect cytokeratin 18 in bone marrow, with a sensitivity of a single cell in 2
10 7 bone marrow cells [51]. Although these techniques are superior to histological examinations they involve invasive procedures and can take up to 7 days for a final diagnosis. Non-invasive detection methods include X-ray, Mammography, Ultrasound, Magnetic Resonance Imaging (MRI), and nuclear imaging techniques such as Positron Emission Tomography (PET), SPECT (single-photon emission tomography) and Computed Tomography (CT). PET, SPECT and CT techniques require radiochemicals, which can have significant systemic toxicity. Unlike mammography and ultrasound, MRI and CT are independent of tissue depth. They exploit pure tissue– energy interactions and do not require isotopes. Mammography has high false positive rates (70%), whereas MRI is costly and ultrasound has a low spatial resolution and is dependent on tissue depth. The accuracy for detection of tumors from breast cancer by mammography was 36%, by ultrasound 33% and by MRI 77% [52, 53]. Contrast agents like ferrofluids enhanced the detection of tumors by 35% compared with unenhanced MRI (6%) or dynamic CT (14%). Intraoperative ultrasound detected liver metastases as small as 5 mm [54] and reduced false-positive diagnosis [55]. MRI can distinguish metastases from hemangiomas and cysts and is potentially valuable for the detection of occult metastatic disease in the subcentimeter size [56, 57]. Micrometastases as small as 2 mm were detected; PET imaging for pelvic lymph nodes detected metastases as small as 6 mm [58]. Clinically used contrast agents for MRI are gadolinium chelates and iron oxide based particles (superparamagnetic iron oxide nanoparticles ¼ SPIONs) [55, 59]. SPIONs have important advantages over gadolinium chelates: they have low toxicity (in some instances toxicities were reported at concentrations more than 100-fold above the clinically effective dosage [59]) and their detection limit in MRI is in the subnanomolar range, exceeding Gd imaging by a factor of 100 [60]. The accumulation and binding of the nanoparticles to the target cells was either absent or nonspecific and, therefore, insufficient and limited the diagnostic applications to liver, spleen, and bone marrow, all of which are dependent on the reticulo-endothelial system (RES). In the past decade, contrast agents that enhance MR imaging have been used for imaging liver [61, 62], spleen [61, 62], gastrointestinal tracts, cardiovascular diseases, cancer [63, 64] and even lymph node metastases [65–67]. The contrast agent accumulated in healthy tissue and enhanced the resolution of MRI between malignant and healthy tissue [65–67].

6.2 Cancer

6.2.2

Current Chemotherapeutic Approaches and their Disadvantages in Cancer Treatments

Currently used chemotherapeutic drugs are systemically active, have severe side effects and do not target specifically tumors or single dormant cancer cells. Most current chemotherapeutics interfere with the proliferation machinery of fast growing cells – they are not effective on slow growing tumors or dormant cancer cells [38, 43, 44]. Most chemotherapeutic drugs are not selective for cancer cells and do not discriminate between healthy and diseased tissue. However, because they interfere with the proliferation machinery of the cancer cells they show a relatively high efficacy on fast proliferating cells: Cyclophosphamide (Cytoxan) destroys genetic material that controls tumor cell growth; methotrexate and 5-fluorouracil (5FU) are antimetabolites that interfere with cancer cell division; antimicrotuble reagents prevent cell division by acting on the microtubules, among them are paclitaxel (Taxol), docetaxel (Taxotere), vincristine (Oncovin), vinblastine (Velban); doxorubicin (Adriamycin) is a tumor antibiotic. To be effective these drugs need to enter, and accumulate in, the tumor cells. High systemic dosages are required to reach therapeutically effective concentrations at the tumor site, which causes severe side effects and peripheral tissue damage. Among the side effects are destruction of bone marrow cells, which impairs the production of erythrocytes, causes cardiotoxicity, nephrotoxicity, hepatotoxicity and hematotoxicity. Because bone marrow cells produce erythrocytes for oxygen transport, patients become anemic as a result of bone marrow destruction. Hematotoxicity includes the destruction of platelets needed for blood clotting and leukocytes to fight infections. Immediate side effects are nausea, alopecia, and fatigue. Longer lasting effects are the increased risk for infections, which persists until the immune system has recovered from the chemotherapy (4–6 weeks). Multidrug Resistance High-dose chemotherapy does not necessarily cure the patient. Many patients relapse. Cancer can recur after high-dose chemotherapy, often with a lack of response to further chemotherapy, which in turn leads to terminal disease even after several years of apparent disease-free state. This phenomenon is defined as multidrug-resistance (MDR) and is attributed to multiple mechanisms [68, 69]. Most cancers, like colon, kidney, breast, ovarian, prostatic, lung cancers, overexpress the p-glycoprotein gene (also known as the multidrug resistance gene). Its gene product is the protein p-glycoprotein (Pgp), which is located in membranes, Golgi apparatus and nucleus [70, 71]. Pgp is a transmembrane efflux pump that actively excretes cytotoxic drugs of different molecular structure through an ATPase mechanism [72], thus decreasing intracellular drug concentrations. Modulators for Pgp pumps have been reported and include Verapamil, a compound that competes with the drug efflux pump. During differentiation and progression, cancer cells can acquire the ability to remove the administered drug through such a pump mechanism. Other mechanisms include alteration of enzymatic activities like topoisomerase or glutathione 6.2.2.1

179

180

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

S-reductase activity, altered apoptosis regulation, altered transport or alteration of intracellular drug distribution due to increased sequestration of cytoplasmatic vesicles [73]. The severity of side effects during administration of chemotherapeutic drugs and the occurrence of MDR emerges as non-responsive or refractory disease to chemotherapeutic drugs. Drug Delivery to Tumors Tumor morphology is differently organized with normal tissue, to meet the physiological requirements for fast growth. Table 6.2 summarizes some of the key characteristics of tumor tissue compared with normal tissue. Consequently, tumor tissue shows certain characteristics that distinguishes it from normal tissue. Malignant tissues have adapted to their increased requirement of nutrients and oxygen by the following characteristics: angiogenesis, tortuous vasculature, leaky basement membranes, lack of lymphatic system (Table 6.2). Fast growing tumors require high amounts of nutrients, which need to be transported to the fast growing tissues and metabolic waste products need to be eliminated at a higher rates than with normal cells. Newly growing tumors can only meet their nutrient requirements until an average tumor volume of 2 mm 3 , beyond that the tumoral diffusion limit restricts further growth [74]. Further tumor growth requires the formation of neovasculature, a process called angiogenesis. The highly increased ‘‘fuel’’ requirement leads to specific morphologic characteristics in tumors vasculature, which is tortuous, chaotic and lacks the hierarchic 6.2.2.2

Tab. 6.2.

Differences between tumors and normal tissues. Normal tissue

Tumor tissue

Cell growth

Low proliferation

High proliferation

Vasculature

No or little angiogenesis Normal proliferation None

High angiogenesis Chaotic High proliferation of endothelial cells Occlusions

Not hypoxic

Hypoxic

Low permeability

High permeability

Lymphatic system

Functional

Lacking

Interstitial pressure

Normal

High

Pore size (nm)

2–60

100–780

6.3 Nanoparticles as Vehicles for Drug Delivery and Diagnosis

branching pattern found in normal vasculature [75–77]. The fast proliferating tumor cells causes occlusion of the capillary vasculature, which leads to hypoxia and eventually necrosis of tumor tissue [77]. The basement membranes of tumor vasculature is often aberrant, leading to leakiness and increased permeability [76, 78] to counterbalance the high oxygen and nutrient requirements for the fast proliferating tumors [79]. In addition the lack of lymphatic drainage system [80, 81] in conjunction with aberrant cell proliferation and occlusion of capillary vessels increases drastically the interstitial pressure in tumor tissue, which is filled with hyaluronate and proteoglycan-containing fluids. Ideally, removal of the drug from the interstitial into the intracellular compartment of the tumor cell would create the necessary concentration gradient to the plasma to allow further extravasation of the drug. Increased interstitial pressure in the tumor tissue interferes with extravasation of the drugs, causing an increase in drug concentration in the tumor interstitium. The dense packing of tumor cells further reduces diffusion of compounds in solid tumors [41]. This phenomenon is explained by the enhanced permeability and retention effect (EPR) and is characterized by the accumulation of a compound in the interstitium. At this stage, further delivery of drug to the tumor cells is stagnant when the accumulation of the drug in the interstitium exceeds the drug concentration in the plasma [82, 83].

6.3

Nanoparticles as Vehicles for Drug Delivery and Diagnosis

The delivery of drugs using nanoparticles can improve current drug administration and treatment. Presently, nanoparticles are under development that can be used for hyperthermic destruction of tumor cells, may incorporate drugs that are slowly released, and at the same time protect the pharmaceutical compound from destruction, alteration in the plasma, and, moreover, opens a possibility to deliver highly lipophilic compounds. Delivery of drugs through nanoparticles needs to take into account the in vivo distribution of systemically injected particles. The biodistribution and circulation time of contrast agents is determined by several factors, including size, charge, and surface chemistry [84, 85] (Fig. 6.1). Charged particles are rapidly coated with plasma proteins, known as opsonization, aggregate and have an increased phagocytotic index [8, 86]. Opsonized particles and nanoparticles exceeding a diameter of 100 nm are easily recognized by macrophages of the reticuloendothelial system (RES) and preferentially delivered into liver, spleen, lymph nodes, microglia and bones, and reside and accumulate within the macrophages in these organs as fast as 5 min [87–93]. During this process nanoparticles are removed from the circulation and the drug is inaccessible to treat tumor tissues. Nanoparticles can be specifically designed for increased uptake by macrophages that deliver them through the RES system of liver (Kupffer cells),

181

182

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

spleen, lymph nodes (perivascular macrophages), nervous system (microglia) and bones (osteoclasts) [87, 88]. This clearance can occur within 0.5–5 min [90], thus removing the active nanoparticles from the circulation and prevent their access to the tumor tissue. Coating of nanoparticles with polymers alters their distribution in vivo. For example, a hydrophilic coating increases the circulation time by preventing interaction with the RES. Nanoparticles with a hydrophilic coating, neutral charge and that are <100 nm show characteristics that render them unrecognizable by the macrophage system [94]. These particles then access the tumor tissue through their hyperpermeable vasculature, accumulate in the interstitial compartment and are retained therein due to the lack of a functional lymphatic drainage (EPR effect) [83, 84, 95, 96]. If nanoparticles are biodegraded in the interstitium the drug can be released and enter the tumor cells through diffusion. Nanoparticles can enter the tumor cells from the interstitial tumoral compartment. Depending on the surface and ligand characteristics the particles can enter by different pathways, among which are pinocytosis or endocytosis [97–99]. Figure 6.1 illustrates the different routes nanoparticles may encounter in vivo, depending on their size. In summary, nanoparticles for the delivery of drugs to tumors offer an attractive possibility for avoiding obstacles that occur during conventional systemic drug ad-

Fig. 6.1.

In vivo distribution of nanoparticles by size.

6.3 Nanoparticles as Vehicles for Drug Delivery and Diagnosis

ministration. However, new obstacles come into play, when nanoparticles are introduced into the system. The route of administration, particle size and composition, degradable coatings, biologically acceptable coatings, endocytosis properties, and stability in physiological salinity all determine the fate of nanoparticles. Nanoparticles injected into biological systems should not agglomerate to avoid not only macrophage uptake but, most importantly, thrombosis. The accumulation of nanoparticles at the target can be enhanced by either attaching ligands onto the surface of the nanoparticles or, in the case of magnetic nanoparticles, by using an external magnetic field. 6.3.1

Targeting Tumor Cells

Cancer therapy, ideally, should only destroy malignant cells, whether they are singly spread in the periphery or confined in an organ as a tumor. The conjugation of drug molecules with tumor specific ligands could facilitate targeting to the tumor cells and avoid uptake from normal cells: the normal cell would be spared and remain undestroyed whereas the tumor cells could accumulate and concentrate drug molecules, reaching the pharmacologically required concentration. This approach could overcome drug resistance, and side effects to vital organs can be eradicated or minimized through reduced systemic exposure. Malignant cells can be destroyed more effectively: even tumor cells inaccessible to surgery can be destroyed. The specific targeting of drug molecules can lower the plasma concentration and, at the same time, increase the efficacy through concentration of the drugs at the tumor cells. The overall goal of active targeting is to increase the specificity of a drug to the tumor cells, to increase the concentration of the compound at the target cells and to incorporate and retain the drug or compound within the tumor cells (e.g., in the case of contrast agents). High efficiency internalizing and recycling receptors could improve cellular uptake through receptor-mediated endocytosis. This process is highly specific and efficient in cells expressing sufficient numbers of receptors and having sufficient receptor capacities. Nanoparticles can be directed to the tumors by passive or active targeting. Passive targeting can be achieved by changing the size/hydrophobicity or other physicochemical characteristics of the newly designed nanoparticles to target the reticulo-endothelial system. Active targeting involves the direction of magnetic particles by using an external magnetic field or by using ligand-conjugated nanoparticles. Passive Targeting Passive targeting can be achieved by using nanoparticles of less than 100 nm size and free of charge, which determines the delivery to the tumors and metastases in peripheral organs through enhanced circulation time and at the site of the tumor cells through increased incorporation and concentration. The circulation time is 6.3.1.1

183

184

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

enhanced by hydrophilic coating of nanoparticles, whereas a hydrophobic coating ensures delivery to the liver and spleen. Examples for the delivery of nanoparticles through the RES include Endorem and AMI25, dextran-coated iron oxide nanoparticles of 62–150 nm diameter, which accumulated in liver and spleen after intravenous injection and have been clinically used for liver diagnostics as they accumulate up to 80% in the liver [100–102, 62]. Bone marrow imaging for tumor detection has been studied with AMI25 iron particles, which were delivered through the RES to the bone marrow [63, 64, 67]. Recently, occult lymph node metastases from prostate, bladder, breast, renal, penile, rectal and testicular cancer patients were diagnosed using dextran-coated nanoparticles (hydrodynamic size 30 nm) [65, 66]. This application exploits the difference between normal and malignant lymph nodes by delivering iron oxide nanoparticles through macrophages. In malignant lymph, nodes macrophages cannot infiltrate the tumor cells. Consequently, malignant lymph nodes do not accumulate macrophage delivered iron oxide nanoparticles. Other examples of passive targeting include polyacrylcyanoacrylate particles loaded with insulin, which have been orally administered into rats and were incorporated via the Peyer’s patches in the intestinal lining into the lymphatic system [103], and the delivery of cucurbitan to cervical lymph nodes [104]. Gadolinium lipid nanoemulsions (70–90 nm) are an example of nanoparticles with increased circulation time. The nanoemulsion was synthesized from hydrogenated phosphatidylcholine, and gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), which had been coated with polyoxyethylene to create a hydrophilic moiety. Bioavailability was determined for different routes of administration. Intravenous injection was advantageous over i.p. injections with respect to bioavailability, tumor retention and increased accumulation. Tumor accumulation was 49.7 mg-Gd per g-tumor at 24 h compared with 21 mg-Gd per g-tumor at 12 h in the i.p. treated groups. However, intravenous administration resulted in higher Gd accumulation in liver and spleen, lung and kidney compared with i.p. injected groups. Repeated injection with a two-fold enriched formulation led to 100 mg-Gd per g-wt.-tissue [105]. The increased efficacy was due to prolonged circulation of the particles, reduced interaction with the RES, and reduced excretion of the compound Gd and increased retention in the tumor tissue. Polymeric nanoparticles (28 nm) prepared through polymer–metal complex formation between cisplatinum (CDDP) and poly(ethylene glycol)-poly(glutamic acid) block copolymers were tested for their efficacy in delivering cisplatinum to tumors. Lewis lung carcinoma bearing mice were injected i.v. with free CDDP (4 mg kg1 ) or CDDP/m. CDDP/m had prolonged blood circulation time, and accumulated in the tumors 20-fold higher compared with free CDDP, whereas the accumulation in normal tissue was reduced [106]. These data clearly show the advantages of drug encapsulation and coating and size selection of nanoparticles with respect of increased bioavailability, increased target accumulation, and reduced accumulation in normal organs. In the above applications the accumulation and binding of nanoparticles to the target cells was either absent or non-specific and, therefore, insufficient, and limited the diagnostic applications to liver and lymph nodes.

6.3 Nanoparticles as Vehicles for Drug Delivery and Diagnosis

Not only drugs could be delivered to the tumors and cancer cells, but nanoparticles like iron oxide nanoparticles can also be used for imaging procedures. Active Targeting In the past 5 years more attention has been paid to the identification and development of specific antigens and receptors that are unique to cancer cells. The ideal targeting moiety should be expressed on all tumor cells, including metastatic cells in the periphery, but not on healthy, normal cells. Targeting of nanoparticles or drugs has to meet the following characteristics: 6.3.1.2

 

   

Specificity – to exclusively target cancer cells. Efficiency – faster uptake through receptor-mediated endocytosis, systemic delivered compounds concentrate on tumor cells, no involvement of peripheral tissues, faster delivery to tumor cells. Stability – the ligand should not be degraded. Functionality – receptor-mediated endocytosis, specific binding to tumor cells through receptors, facilitating increased uptake. Neutral surface coating – nanoparticles should not be recognized by macrophages. Increased retention through compartmentalization.

The decoration of nanoparticles with tumor targeting molecules, such as hormones, growth factors etc., can increase the efficacy of a treatment regimen by increasing the concentration of the drug at the tumor site, reducing the requirement of systemically administered drug for therapeutic efficacy, and can facilitate sustained intracellular retention of the drug. Antibodies for targeting malignancies have been approved and include Anti-CD 20 (Rituxan2, nonHodgkins lymphoma), Anti-Her-2 (Herceptin2, metastatic breast cancer), Anti CD 33 (Mylotarg2 myelogenous leukemia), and Gefitinib (Iressa2, non-small cell lung cancer) [107]. Other targets under intense investigation include antiangiogenesis compounds to specifically destroy tumor vasculature and eventually starve the tumor tissue. Angiogenesis is a complex process and the number of potential targets includes compounds for limiting endothelial proliferation, expression of angiogenesis inhibitors, and a decrease of angiogenesis stimulatory factors often secreted by tumor cells. The most prominent candidate for targeting vasculature is the vascular endothelial growth factor (VEGF) and an integrin for which antibodies have been developed for the use in conjugation in vascular imaging and tumor vasculature specific drug delivery. Several specific receptors are overexpressed on tumor cells and have been identified and targeted with drug–ligand complexes or ligand–nanoparticle constructs. Some examples, which include lactoferrin [108, 109], ceruloplasmin [108, 109] and insulin [110], have been studied as targeting moieties; the ligands had strong surface binding on tumor cells, yet were not phagocytosed. High efficiency internalizing receptors could improve cellular uptake through receptor-mediated endo-

185

186

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

cytosis and increase dramatically the specificity of uptake in cancer cells, avoiding uptake in peripheral and healthy tissue. Ligands that are endocytosed and recycled include transferrin [111–115], folate [116, 117], TGF alpha [118], nerve growth factor [119], Her2/neu [120], somatostatin, bombesin [121], steroid hormones, CD-8 [122], and HIV-1 tat [123, 124]. This chapter reviews in detail the use of Luteinizing Hormone Releasing Hormone (LHRH) as ligand to target the cancer cells. The following outline describes the characteristics and function of LHRH and its receptors regarding tumor treatment and detection. Drugs delivered in conjugation with LHRH, studied over the past 20 years, include doxorubicine conjugates, studied intensely by Schally and co-investigators [22–24, 121, 125–129], lytic peptides conjugated to LHRH [8, 130], CPT-11 conjugated to LHRH and to pokeweed antiviral protein [159, 160]. Conjugates of lytic peptides or doxorubicin/doxorubicin derivatives with LHRH specifically target and destroy prostate, ovarian and breast cancer cells and their metastases, all of which express LHRH receptors in vitro and in vivo [22–24, 121, 125–130]. Metastases and disseminated cells derived from breast cancer xenografts of estrogen-independent MDA-MB-435S.luc cells, in the presence and absence of the primary tumor, have been detected, characterized and successfully targeted by CG/LHRH–lytic peptide conjugates [161–164, 318]. Schally et al. have treated several cancers with LHRH– doxorubicin conjugates and have developed a drug, which is currently in Phase III clinical trials (personal communication). These studies clearly demonstrate that tumors can be specifically targeted in vivo in experimental xenograft-bearing mice and rats. Moreover, in the above experiments none of the reported side effects of the free compounds were observed, suggesting increased specificity, targeting and efficacy with decreased systemic exposure. In studies using LHRH cytotoxic conjugates of doxorubicin the anterior pituitary did not show any permanent damage – complete recovery of pituitary function was observed [128, 129]. 6.3.2

Detection of Tumors and Metastases using Nanoparticles Nanoparticles for Magnetic Resonance Imaging The MRI signal in magnetic particles is created through the interaction of total water signal and the magnetic properties of longitudinal (T1) and transversal (T2) relaxivity. MRI resolution can be increased by: (a) extending the scan time, (b) using high efficiency coils, (c) increasing field strengths, (d) increasing accumulation of contrast agent in cells or tissue, (e) compartmentalization of particles within the target cells, and (f ) retention of particles. To acquire the best image the signal-to-noise ratio must be optimized. An increase in field strength can cause neurological side effects like seizure, and/or cardiovascular effects. Clinically, MRI is conducted at 1.5 T, which is without side effects and well tolerated. MRI requires long imaging times, which can last up to 1 h. To enhance MRI sensitivities, contrast agents must accumulate in the target 6.3.2.1

6.3 Nanoparticles as Vehicles for Drug Delivery and Diagnosis

cells, without leaking into surrounding tissue, at sufficient concentrations to increase relaxation times and must be retained within the tissue during the imaging procedure. In MRI, the signal can be amplified through an increase in intracellular concentration of the contrast agent [131, 132]. Clinically used contrast agents are paramagnetic gadolinium chelates (DTPA ¼ diethyl tetraminepentaacetic acid) or ferrofluids. The use of superparamagnetic iron-based particles for magnetic resonance imaging has several advantages [133]: Image enhancement with regard to relaxation time is greater in superparamagnetic particles – the change in T1 relaxation time is seven-fold, T2 relaxation time 16-fold, compared with gadolinium DTPA [134]. Gd is highly water-soluble and it is readily excreted, which allows only a short time for imaging and reduces cellular uptake [135, 136]. Superparamagnetic particles are less likely to aggregate because they lack remnant magnetization [137] and retain their physical characteristics even when chemically inert materials are attached [138]. Magnetite can adsorb or attach chemically to inert material without changing the characteristics of T2 relaxivity [138]. To significantly reduce relaxation time, gadolinium chelates have to be administered at high concentrations [139]. In vitro, increased Gd payloads resulted in increased T1 relaxivities and increased the resolution of fibrin clots [137]. In addition, superparamagnetic iron oxide particles have a much more favorable ratio of efficacy dose to LD50 : 1/2400 compared with 1/50 for Gd-DTPA contrast agents [140]; in some instances toxicities were reported at concentrations 100-fold above the clinically effective dosage [139]. Monocrystalline Fe requires up to 1 mg of Fe per kg of tissue for MRI [141, 142]. In liver, up to 2.5 mg-Fe per kg of tissue showed a linear relationship with T2 relaxation [143]. Best imaging was obtained at 5–50 mg-Fe per kg of tissue sample [144]. A four-fold increase of the dose of superparamagnetic iron particles resulted in a decrease of signal loss by a factor of 10 [145]. Iron oxide nanoparticles are biologically safe and show no toxicity [139]. They are metabolized into elemental iron and oxygen by hydrolytic enzymes and the iron joins the normal body stores and is subsequently incorporated into hemoglobin. Iron homeostasis is controlled by absorption, excretion and storage. Acute toxicity has not been observed, in rats or human clinical trials. The administered iron was excreted over a period of four weeks [146]. Renal function, hepatic parameters, serum electrolytes, lactate dehydrogenase remained unchanged from baseline parameters after treatment with ferrofluids [146]. The elevation of serum iron levels persisted for a maximum of 48 h and caused no symptoms. In rats, 250 mg per kg of iron particles injected intravenously caused no side effects, in mice 350 mg kg1 were well tolerated [147]. In the past decade, contrast agents that enhance MRI have been tested for various applications, such as imaging of liver, spleen, gastrointestinal tracts, cardiovascular diseases and cancer detection. Administered systemically, the accumulation of unconjugated nanoparticles has been confined to liver, spleen, and bone marrow, all of which are dependent on the reticulo-endothelial system (RES), and did not directly accumulate in the tumor cells.

187

188

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

In these applications, the biodistribution of nanoparticulate contrast agents was exclusively determined by size, charge and surface chemistry (Fig. 6.1) [84, 85]. Systemically administered nanoparticles that are non-specific were incorporated by the MPS system, or they enter cells through endocytotic pathways involving clathrin-coated pit formation, sequestration and accumulation in vacuolar or lysosomal compartments of the cells [98, 99, 148]. Endocytotic cellular up-take of dextran-coated iron-oxide nanoparticles has been shown for tumor cells in vitro to range from 0.01 to 100 ng of iron per 10 6 cells within 1 h. In contrast macrophage uptake was about nine times higher [149, 150]. For example, occult lymph node metastases from prostate, bladder, breast, renal, penile, rectal and testicular cancer patients have been diagnosed using dextran-coated nanoparticles (hydrodynamic size 30 nm) [65, 66]. This application exploited the difference between normal and malignant lymph nodes by delivering iron oxide nanoparticles through macrophages. In malignant lymph nodes, macrophages could not infiltrate the tumor cells. Consequently, malignant lymph nodes did not accumulate macrophagedelivered iron oxide nanoparticles, and so they were not labeled. The accumulation and binding of the nanoparticles to the tumor cells was either absent or non-specific and therefore insufficient and limited the diagnostic applications to liver and lymph nodes with limited sensitivity. 6.3.2.2 Targeted Delivery of Nanoparticles to Increase Cellular Uptake for Higher MRI Resolution Targeted delivery of nanoparticles can enhance the cellular accumulation of the contrast agent within the cancer cells, can increase the retention and thereby improve the resolution and specificity in imaging modalities. The MRI signal can be amplified through an increase of the intracellular concentration of the contrast agent [131, 132]. Relaxation times in the acquisition of magnetic resonance imaging, i.e., the contrast of the image, increase with larger particles [151]. Compartmentalized particles and clusters have larger relaxation rates than free dispersed particles [152]. If the target cells are sufficiently loaded with iron oxide particles the MRI resolution can be as high as 25 mm [153]. This task requires specifically designed particles that concentrate in the target cells. A pre-requisite of the target cells is that they express high efficiency, internalizing, and functional receptors on their surface, to promote accumulation of the particles in the target cells, without leaking into surrounding tissue. Particle accumulation can be increased by targeting specific receptors on the cancer cells to facilitate receptor-mediated endocytosis of the delivered iron particle or to bind and accumulate the particles on the cell surface. Ideally, the injected nanoparticles should not be recognized by macrophages. Examples for targeted uptake include dextran-coated monocrystalline iron oxide nanoparticles (MIONs) linked to transferrin [115] or to folate [116, 117]. Antibody-coupled paramagnetic liposomes targeting integrin aVb3 of endothelial vascular cells greatly increased the MRI of angiogenic vasculature in rabbit carcinoma [154]. The aVb3-conjugated nanoparticles increased the signal enhancement

6.4 LHRH and its Receptors

post-injection much faster than untargeted nanoparticle delivery (2 versus 24 h) [155, 156]. Dextran-coated nanoparticles have been conjugated to target Her2/neu receptors [158]. Folate-linked iron oxide nanoparticles accumulated in folate receptor expressing KB cells [116] and reached a maximal level of 75 pg-Fe per cell in BT20 breast cancer cell incubation and were poorly incorporated by macrophages [117]. CD-8 conjugated crosslinked iron oxide nanoparticles have been incorporated into T cells after 1 h of incubation at concentrations up to 100 pg-Fe per cell [122]. LHRH iron oxide nanoparticles coated with a silica shell specifically targeted and accumulated in MC-F7 human breast cancer cells in vitro [157]. Basically, the main purpose for targeted delivery of drugs or contrast agents is the improved specificity for a particular cell type, reduced requirement of the compound necessary to gain the desired effect, and the possibility to directly incorporate the desired compound into the target cells, and a sustained retention of the particles within the target cells. The design of nanoparticles carrying specific ligands reduces and avoids the uptake in non-target cells and tissue.

6.4

LHRH and its Receptors 6.4.1

The Ligand Luteinizing Hormone Releasing Hormone – LHRH

Luteinizing hormone releasing hormone (LHRH), also referred to as gonadotropin releasing hormone (GnRH), was purified 30 years ago and its isolation was honored with the Nobel Prize of Medicine that the laureates Schally and Guellerin received in 1977 for their discovery of peptide hormones in the brain. LHRH is excreted from the arcuate nucleus of the hypothalamus into the pituitary gland in a pulsatile manner [165]. In gonadotrophic cells of the adenohypophysis, LHRH stimulates the release of luteinizing hormone and follicle stimulating hormone, which are released into the main body circulation and regulate the production of sex hormones (estradiol and testosterone) and, thereby, the function of normal reproduction in mammals, humans and mice. This relationship is called the pituitary/gonadal axis (Fig. 6.2). The pulsatile release of LHRH is crucial for maintaining its function on the pituitary/gonadal axis and ensuring the optimal function of reproductive organs and maintaining fertility [166–168]. Sustained administration of LHRH or its analogs to patients suppressed steroid hormone production and downregulated and desensitized the LHRH receptor in the pituitary [166, 167]. This observation was exploited for treatments of hormone-dependent cancers; steroid hormone suppression using Leuprolide, which binds to the LHRH receptor, stopped the release of testosterone and inhibited the proliferation of the cancers. Over the last 30 years more than a thousand LHRH agonists and

189

190

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

Fig. 6.2.

Gonadal/pituitary axis and interaction of LHRH with gonadal function.

antagonists have been developed, and are used in the clinic to treat hormonedependent cancers [169] (Table 6.3). The common mechanism of action is the ablation of hormones by administration of these pituitary acting antagonists. Suppression of gonadal steroid secretion reduced the growth of these hormonedependent tumors (Fig. 6.2). Hormone ablation was considered to be the only mechanism of action for LHRH agonists, but recent identification of a second LHRH receptor and the expression of LHRH receptors in malignancies explained why LHRH also had a direct effect on the proliferation and metastatic behavior of cancers [12, 170–173]. In 1971, type I LHRH was isolated the pituitary of pigs and its structure was revealed [174]. The gonadotropic hormone LHRH is also expressed in gonadal peripheral tissue like Leydig cells in the testes and ovarian cells as well as in numerous malignancies. The decapeptide LHRH is produced in numerous species with astonishing homology during evolution. To date 23 different forms have been identified with highly conserved sequences [170–181]. All these peptides consist of 10 amino acids and share at least 50% sequence identity, with the main differences occurring in amino acids 5–8 (Table 6.3) [170, 182–184]. The different isoforms of LHRH were named after the species of its isolation. Two different forms of LHRH coexist in vertebrates, LHRH I and LHRH II. LHRH I (mLHRH) has been isolated from the hypothalamus in pigs [174]; and

6.4 LHRH and its Receptors Tab. 6.3.

Amino acid sequences of LHRH forms and synthetic analogs [170].

LHRH forms

Naturally occurring LHRH 1

2

3

4

mLHRH (LHRH-I)

pGlu

His

Trp

cLHRH (LHRH II)

pGlu

His

lLHRH (LHRH III)

pGlu

rLHRH cfLHRH

5

6

7

8

9

10

Ser Tyr

Gly

Leu Arg

Pro Gly

NH2

Trp

Ser His

Gly

Trp

Tyr

Pro Gly

NH2

His

Trp

Ser His

Asp

Trp

Lys

Pro Gly

NH2

pGlu pGlu

His His

Trp Trp

Ser Tyr Ser His

Gly Gly

Leu Trp Leu Pro

Pro Gly Pro Gly

NH2 NH2

LHRH agonists pGlu Lupron[a] (TAP)

His

Trp

Ser Tyr

D Leu

Leu Arg

Pro NEt

Zoladex[a] (Zeneca)

pGlu

His

Trp

Ser Tyr

D Ser (tBu)

Leu Arg

Pro Gly

NH2

Supprelin[a] (Roberts)

pGlu

His

Trp

Ser Tyr

D His Leu Arg (ImBzl)

Pro Gly

NH2

Synarel[a] (Searle)

pGlu

His

Trp

Ser Tyr

D Nal

Leu Arg

Pro Gly

NH2

Triptorelin[a] (Ferring)

pGlu

His

Trp

Ser Tyr

D Trp

Leu Arg

Pro Gly

NH2

Buserelin[a] (Hoechst)

pGlu

His

Trp

Ser Tyr

D Ser (tBu)

Leu Arg

Pro NEt

LHRH antagonists D Nal D Cpa D Pal Ser Tyr Centroelix[a] (Asta Medica)

D Cit

Leu Arg

Pro D Ala NH2

Ganirelix[a] (Organon)

D Nal D Cpa D Pal Ser Tyr

D hArg Leu D hArg Pro D Ala NH2 (Et)2 (Et)2

Abarelix[b] (Praecis)

D Nal D Cpa D Pal Ser N Me D Asn Tyr

Leu Lys (iPr)

Pro D Ala NH2

Antide[c] (Ares Seronon)

D Nal D Cpa D Pal Ser Lys (Nic)

D Cit

Leu Lys (iPr)

Pro D Ala NH2

Teverelix[d] (Ardana)

D Nal D Cpa D Pal Ser Tyr

D hCit

Leu Lys (iPr)

Pro D Ala NH2

Fe 200486[c] (Ferring)

D Nal D Cpa D Pal Ser Aph D Aph (Hor) (Cba)

Leu Lys (iPr)

Pro D Ala NH2

Nal-Glu[c] (NIH)

D Nal D Cpa D Pal Ser Arg

Leu Arg

Pro D Ala NH2

D Glu (AA)

[a] ¼ Market, [b] ¼ Phase I, [c] ¼ Phase II, [d] ¼ Phase III

191

192

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

LHRH II (cLHRH) has been isolated from chicken [182] and from the forebrain of the teleost fish, the lamprey eel. LHRH III (lLHRH) has also been identified and characterized [183–185]. LHRH II has been conserved from fish to humans [170, 182–184]. In humans, LHRH II is synthesized in several extrapituitary tissues like kidney, bone marrow, and prostate at significant higher levels [186] and in the female reproductive tract such as placenta, endometrium, breast, ovary and granulosa cells [187–189]. LHRH hormones have manifold functions besides reproductive maintenance, including neuroendocrine (growth hormone release in fish), paracrine (placenta and gonads), autocrine (LHRH neurons, immune cells, breast and prostate cancer cells), neurotransmitter in central and peripheral nervous system (symphathic ganglion, mid brain), energy and feeding behavior [175–178, 184, 190–194]. Direct effects of LHRH agonists and antagonists have been studied – most importantly, the antiproliferative effects of LHRH I on hormone-dependent and -independent tumor cells, including gonadal cancers like ovarian, endometrial, prostate and breast and non-gonadal cancers like melanoma. In endometrial, ovarian, breast and prostate cancers in vitro proliferation was inhibited by agonists and antagonists of LHRH I in a time and dose dependent manner [195–197]. Interestingly, the effects on LHRH agonists/antagonists seem to be different in tumor tissue of the reproductive tract, where LHRH antagonists have agonistic effects on the LHRH receptor type II [195]. Further functions of LHRH in gonadal malignancies and melanoma were antimitogenic, through effects on IGF-I, EGF and c-fos expression and antimetastatic-affecting matrix metalloproteinases, adhesion molecules, and plasminogen activator in prostate cancer cells [198]. 6.4.2

Analogs of LHRH

The highly conserved sequence of the amino (pGlu His Trp Ser) and carboxy (Pro Gly NH2 ) termini suggests that these amino acids are critical for receptor binding and activation. The amino terminus is involved in receptor binding and activation, whereas the carboxy terminus contains regions for receptor binding only. Structural changes in these particular amino acids created agonists and antagonists that had been clinically exploited for the stimulation of hormone production, for contraception and treatment of hormone-dependent cancers and diseases [170]. Substitution of amino acids at the amino terminal produced compounds with analogous antagonistic properties, including Cetrorelix and Ganirelix, which are currently marketed. Agonists on the market include Lupron, Zoladex, Sepprelin, Triptorelin and Buserelin (for sequences see Millar et al. 2004 [170]). Substitution of amino acid 6 by D-Gly enhances activity in the pituitary receptor [170] (Table 6.3). Table 6.3 lists amino acid sequences for various agonists and antagonists. Analogs of LHRH are applied for hormonal ablation, whereas antagonists (like Cetrorelix) seem to have antiproliferative effects on ovarian and endometrial cancers rather than the agonist Triptorelin.

6.4 LHRH and its Receptors

6.4.3

Receptors for LHRH

LHRH affects the proliferation and metastatic potential of many cancers directly [171–173]. Autocrine/paracrine loops were identified and studied in breast cancer [199, 200], prostate cancer [201], ovarian cancer [202] and melanoma cell lines [172]. LHRH receptors not only are expressed in gonadal malignancies like prostate [4, 5, 203], breast [10], ovarian [204–208], endometrial cancers [26, 209], but have also been detected in several non-gonadal malignancies, such as laryngeal cancers [24] renal [28], pancreatic [15, 16], brain [29], melanoma [22, 27] liver [25], colon [13, 14, 210] and recently in non-Hodgkin’s lymphoma [23] (Table 6.1). These findings suggest that there may be a connection between cancer and LHRH receptor expression. LHRH receptors are very widespread among malignancies rather than healthy tissues. Consequently, LHRH seems to be a very appropriate candidate for specific targeting of malignancies. Most tumors overexpress LHRH receptors in much higher concentrations than normal tissue (where they can be entirely absent) [125, 211]. Non-malignant tissue of lung, liver, skeletal muscle, pancreas, other visceral organs and kidney express little or no LHRH receptors [11, 203, 212, 213]. Although LHRH-receptor transcripts detected in breast and ovarian cancers are identical to those in pituitary [214] the receptors may differ functionally. In binding studies [12], pituitary LHRH-receptors showed high affinity for agonists such as buserelin (nanomolar Kd ), whereas most LHRH-receptors in extrapituitary sites, including malignancies, have low affinity (micromolar Kd ) but show higher receptor capacities (Table 6.4). For example, LHRH receptors were found in 57% of patients with pancreatic cancers and 67% in pancreatic inflammatory disease, compared with normal tissue with only 9% [16]. Pancreatic cancers showed low-affinity binding sites for LHRH in the membrane, and high-affinity binding sites in the nuclei [9]. In melanoma cells the LHRH receptor type I has high affinity/low capacity [172]. LHRH receptors have been cloned from mouse, rat, sheep, cow and human with an amino acid sequence homology of >85% [213–222]. The nucleotide sequence of LHRH receptors from breast, ovarian, endometrial and prostate cancers is identical to that of the pituitary receptor [5, 197, 213–215, 223–228]. The LHRH receptor belongs to the large superfamily of the seventransmembrane domain receptors that bind G-proteins (G-protein binding receptors, GPBR) (Fig. 6.3) [229–232]. Typically, GPBRs contain a C-terminal cytoplasmatic tail, which is lacking in the LHRH receptor type I. This C-terminal tail is phosphorylated by ligand binding and facilitates the binding of b-arrestins, G protein activation, desensitization and internalization of the receptor, and downregulation via endocytosis and processing [233]. The internalization kinetics of LHRH-receptor type I [234] showed a 20-fold increase in uptake rate constants of the bound radioligand within 1 h compared with kinetics in wild-type TSH receptor, another member of the GBP receptor family (with cytoplasmatic tail).

193

194

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers Tab. 6.4.

LHRH-receptor binding affinities and capacities.

Cell type

aT3-1[a] PC-3[a] PC-3[b] LNCaP[b] MCF-7[c] MDA-MB-435S[c] Hec1B NIH-OVCAR3

High affinity LHRH-receptor Bmax (fmol/ 106 cells)

Kd (nM)

231.9 G 53.7 8.6 G 3.3 97.2 G 10 355.3 G 19 307 G 21 64.6 G 10 72 49

0.867 G 0.26 0.185 G 0.08 15.3 G 1.5 12.8 G 2.5 10.8 G 3.1 14.38 G 1.8 1.5 2.3

Low affinity LHRH-receptor Bmax (pmol/ 106 cells)

Kd (mM)

118.2 G 24 203 G 31 138 G 26 298 G 36 42 73

0.625 G 0.022 0.438 G 0.016 0.358 G 0.041 0.936 G 0.054 3.1 2.4

a Yang

[160] passage # not reported. [130] – # 20. c Leuschner unpublished data – # 237. d Gru ¨ ndker [279]. aT3-1 (mouse pituitary, endogenous LHRH receptors, Kaiser [280]); Hec1B [d] endometrial cancer cell line; NIH-OVCAR3 [d] ovarian cancer cell line. b Leuschner

6.4.4

Function–Signal Transduction Pathways

Signal transduction pathways activated by binding of LHRH to a tumor’s receptor are quite different to the classical pituitary binding. The physiological significance of this remains under investigation (Fig. 6.4). In the pituitary, binding of LHRH to its receptor (type I) initiates coupling to Gaq/11 binding followed by activation of phospholipase C (PLC), and hydrolysis

Schematic of the human LHRH-receptor as an example of a seven-transmembrane receptor G protein binding receptor [170].

Fig. 6.3.

6.4 LHRH and its Receptors

Signal transduction pathways activated by LHRH-receptor binding in pituitary, gonadal and cancerous tissue. (1) Unknown signaling cascade leading to reduction of cell proliferation; (2) signal transduction pathway in pituitary for gonadal function; (3) signaling cascades for LHRH receptor I in malignancies: (3a) signaling resulting in decrease of cell proliferation

Fig. 6.4.

through MAP kinase pathway, (3b) signaling interferes with EGF receptor expression, (3c) signaling cascade that result in reduction of cell cycle and reduction of DNA synthesis, (3d) signaling cascade that reduces apoptosis. Abbreviations are defined at the end of this chapter. (Based on Ref. [207] with modification from Refs. [173, 206, 244, 245, 253, 281].)

of phosphoinositide to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). These secondary messengers mobilize calcium from intracellular stores and activate PKC (protein kinase C) [235, 236]. LHRH also activates PLA2 (phospholipase A2), PLD and MAP-kinases (mitogen-activated protein), providing signals for gene expression for gonadotropin synthesis and secretion [235, 236]. The physiological function for this difference is unresolved. Binding of LHRH to gonadal and receptors on cancer cells activates different signal transduction cascades. In malignant tissue, LHRH receptors have been suggested to regulate cell proliferation and the metastatic potential in tumors. LHRH receptor expression in tumors is associated with the phosphorylation of the epidermal growth factor receptor: the activated EGF receptor phosphorylates a protein corresponding to the LHRH receptor [237, 238]. Growth factor receptor expression can be reduced in the presence of LHRH most likely by phosphotyrosine phospha-

195

196

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

tase (PTP), which in turn is connected to the G-protein ai in tumors [239, 240] (Fig. 6.4). In cancer cells, LHRH receptor coupling occurred to multiple G proteins and the activation of multiple effectors has been reported. In prostate cancer cells, LHRH I binding facilitates the coupling of Gai protein [241], and human gynecological cancer coupling of Gaq and Gai was demonstrated in ovarian and uterine cancer cells [20, 240]. Gai coupling is prevalent in cancers, such as in ovarian [239, 240, 242], uterine leiomyosarcomas, uterine endometrial carcinomas and human prostate cancers [241]. Coupling to Gai protein is poorly understood and it is suggested that Gai activation underlies the antiproliferative effects of LHRH in cancers [240, 243]. Gai coupling can be activated by both agonist and antagonists of LHRH, suggesting the involvement of an additional form of LHRH receptor distinct from the pituitary receptor type [239, 241]. Antiproliferative action in choriocarcinoma, benign prostate hyperplasia and human embryonic kidney cells, all of which express LHRH receptor type I, was demonstrated with agonists and some antagonists through activation of Gai coupling. The signal transduction resulted in activation of caspase and trans-membrane transfer of phosphatidylserine to the outer membrane, JNK and p38 [244]. In hormone-dependent tumors, binding of LHRH to its receptor decreased cyclic AMP levels, which is followed by an activation of phosphotyrosine phosphatase (PTP). This event interferes with the mitogenic signal transduction pathways, which normally activate growth factor cascades such as MAP kinase. The transcription is affected downstream, which include genes such as matrix metalloproteinases, urokinase plasminogen activator (genes for metastatic potency), c-fos (a protooncogene), and cell adhesion molecules. The overall result is reduction of proliferation and interference with the metastatic potential of the cancer cells. Antagonistic LHRH analogs inhibited Gaq/11-based signaling, suggesting that their antiproliferative effect on reproductive tumors could be dependent on ligandselective activation on a Gai form of LHRH-receptor I through stress-activated protein kinase pathways (SAP-K) [244]. In hormone-dependent and -independent prostate cancers the antiproliferative effects of LHRH I were attributed through EGF and IGF receptor reduction initiated by different molecular mechanisms, dependent on hormone requirements of these types of cancer. In hormone-dependent prostate cancer cells LHRH I counteracts the stimulatory effects of epidermal growth factor (EGF) by decreasing the EGF receptors and insulin-like growth factors (IGF) [245, 281]. In breast, ovarian and endometrial cancers LHRH can bind to either Gaq or Gai, the latter inhibiting proliferation. Emons et al. have found that endometrial and ovarian cancer cells PTP (phosphotyrosine phosphatase) are activated by LHRH/ LHRH-receptor action, which counteracts the phosphorylation of EGF receptors, affects the growth factor signal transduction cascade (MAP-K, c-fos), and interferes with cell proliferation [239, 246–248] (Fig. 6.4). Positive correlation between EGF receptor activation and LHRH receptors were observed in various LHRH receptor expressing cancers [6, 24]. Consequently, EGF receptors can be downregulated to-

6.4 LHRH and its Receptors

gether with LHRH receptors through LHRH-analogs or LHRH-targeting treatments [6, 249–251]. LHRH can influence apoptosis in cancer cells through activation of nuclear factor kB [252, 253]: LHRH agonists can activate the cJun N-terminal kinase/activator protein 1 (AP-1) pathway, protein kinase C (PKC) or mitogen-activated protein kinase (MAPK/ERK) [20]. In contrast, when the antagonist cetrorelix was tested in ovarian and endometrial cancer cells the antiproliferative effects were not mediated through LHRH receptor type I, suggesting binding to, therefore, LHRH receptor type II. The signal transduction pathway of LHRH II is still unknown [20]. 6.4.5

LHRH Receptor-mediated Uptake

Extensive internalization and downregulation of the receptor via endocytosis and processing has been studied and described in detail for the pituitary receptor [233]. Receptor internalization is highly influenced by the second intracellular loop of the seven-transmembrane receptor [254]. The COOH-terminal tail, which is lacking in the pituitary LHRH-receptor, determines the internalization and the recycling kinetics and causes resistance of the receptor to desensitization. Several examples of this observation have been reported. For example, deletion of the cytoplasmatic tail in TSH receptors decreased the internalization [255]. The chicken LHRH receptor showed rapid internalization that was dependent on the cytoplasmatic tail [256]. As mentioned earlier the pituitary LHRH receptor (type I) lacks the cytoplasmatic tail; however, the receptor still internalized and recycled, and is resistant to desensitization [257, 258]. These turnovers are much slower than with other GPCRs: While angiotensin II internalized 50–60% [259] and GRP receptors up to 80% [260, 261] of their agonists within 1 h, the LHRH receptor type I internalized only 26% [254]. This agrees with results for the LHRH-II receptor in the marmoset and rhesus monkey containing a Cterminal, cytoplasmatic tail that facilitated a more rapid internalization [262, 263]. Receptor-mediated endocytosis can be described with the four-compartment model [264] that includes (1) receptor endocytosis from cell surface to endosome, (2) recycling from endosome to plasma membrane, (3) receptor movement from endosome to lysosome for degradation, and (4) delivery of de novo synthesized receptors to the plasma membrane (Fig. 6.5). The classical pathway involves GPCR kinases, b-arrestin, clathrin-coated pits and GTPase dynamin. As a representative mode for receptor internalization and recycling the process is described here for the type I receptor. Despite the lack of cytoplasmatic tail the LHRH receptor type I displays distinct characteristics described for other GPCRs that internalized via a clathrin-dependent mechanism, appeared to be independent of b-arrestin levels and recycled through an acidified endosomal compartment (Fig. 6.5). LHRH incubation showed internalization of the ligand–receptor complex and redistributed into vesicular compartments [234]. The internalization was tempera-

197

198

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

Fig. 6.5.

Receptor-mediated endocytosis and recycling of the receptor.

ture and time dependent and was suppressed at 4  C; internalization was observed after 20 min and reached its maximum after 1–2 h. The uptake mechanisms through LHRH receptors are cell type dependent and dependent on the type of LHRH receptor. In HeLa cells, internalization of the receptor was independent of dynamin; clathrin mediation was involved in the internalization of the receptor [265]. Receptor recycling to the plasma membrane in HEK 293 cells (human embryonic cells) was observed as early as 15 min and completed after 1 h. The intracellular pool of receptors was decreased by 68% due to reappearance of receptors at the plasma membrane. The reappearance of plasma membrane receptors was clearly due to recycling of the receptor rather than by de novo synthesis. Recycling of the receptors caused a loss of 10% of the initially detected surface receptors. When kinetics of LHRH and TSH (retained cytoplasmatic tail) receptors were compared, significantly lower rates were determined in HEK 293 cells for the endocytotic rate (16-fold), recycling (2.5-fold) and degradation rates (30-fold) for the LHRH receptor [234]. 6.4.6

LHRH Receptor Type II

The existence of two LHRH isoforms in human tissue led to a search for a second LHRH receptor in human tissue. These studies were supported by the following

6.4 LHRH and its Receptors

observations: Effects on LHRH agonists/antagonists were different in tumor tissue of the reproductive tract, where LHRH antagonists have agonistic effects on the LHRH receptor type II. A recent study on human endometrial cancers and ovarian cancer cell lines demonstrated that the proliferation of these human cancer cells was dose and time dependent, but independent of the LHRH receptor I, suggesting the existence of a second LHRH receptor [206]. LHRH type II has been found in ovarian cancer cell lines [266] and had antiproliferative activity. In human malignant tumors in vitro, LHRH I agonists and antagonists inhibited the cell proliferation in a dose and time dependent manner. LHRH type II has antiproliferative effects on gynecological cancers to a much higher efficacy than triptorelin [267]. RTPCR and Southern Blot analysis showed the expression of LHRH receptor II mRNA in ovarian and endometrial cancer cell lines [266]. In addition, these cell lines responded to LHRH II with reduction in proliferation, to a much higher degree than with LHRH I or triptorelin [267]. In breast cancer cells the antiproliferative effects of Triptorelin (a LHRH-antagonist) and a LHRH II analog inhibited epidermal growth factor induced signal transduction and restored sensitivity to tamoxifen [313]. Recently, in a clinical study, primary and metastatic stromal sarcoma tested positive for LHRH receptor I and II expression using immunohistochemistry methods [209]. Interestingly, recurrent tumors had the highest levels in LHRH receptor I and II expression, suggesting a link between malignancy and LHRH receptors types [209]. Studies on human prostate cancer cells in vitro showed the existence of an 80-kDa protein that specifically bound to LHRH II in addition to the conventional LHRH I receptor. In this study, efficacy and function was studied and the authors found that a novel LHRH II antagonist completely inhibited Ca increase and induced, at high concentrations, cell death through apoptotic processes [268]. In ovarian and endometrial cancers, LHRH II is a more potent inhibitor for cell proliferation [252], receptors of type II have a higher sensitivity to LHRH II ligand [269, 270], and LHRH agonists and antagonists inhibited tumor growth [272]. Type II LHRH receptor has been cloned from monkeys and was highly selective to LHRH II [212, 263, 271]. To date, a functional form of the LHRH receptor type II has not been found in humans, although several observations suggest its existence. Therefore, it is referred to as a putative receptor. Millar found that the type II receptor was more widely distributed: mRNA for LHRH receptor-type II was found in numerous pituitary, brain, mammary glands, uterus, testes, prostate, seminal vesicles, thyroid, heart, pancreas and adrenal tissue, including ovarian and endometrial cancers [193, 212, 242, 268, 273]. In prostate cancer, a novel LHRH II binding protein was detected recently [268], suggesting the existence of a type II receptor in prostate cancer cells. In human pituitary and brain, immunoreactivity to the LHRH receptor type II was demonstrated [212]. Humans may be particularly unusual with respect to the LHRH control of their reproductive axis in that they possess two distinct LHRH precursor genes, on chromosomes 8p11-p21 and 20p13, but only one conventional LHRH receptor subtype

199

200

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

(type I LHRH receptor) encoded within the genome, on chromosome 4. A disrupted human type II LHRH receptor gene homologue is present on chromosome 1q12. In humans, on chromosome 1 and 14 in various tissues a gene for the LHRH receptor II was found, which is not expressed because it contained a premature stop codon [275, 276]. Evidence in support of this concept is available following the characterization of the chromosomal loci encoding the human type II LHRH receptor homologue, a rat type II LHRH receptor gene remnant (on rat chromosome 18) and a mouse type II LHRH ligand precursor gene remnant (on mouse chromosome 2) [277]. The lack of the C-terminal tail causes the type I LHRH receptor to be slowly internalized and to be resistant to desensitization [257, 258]. In contrast, type II LHRH receptors are rapidly internalized and desensitized, contain the c-Terminal tail, and activated a different signal transduction cascade [257, 278]. The LHRH type II specifically induces an intracellular Ca 2þ increase that was inhibited by triptorelix in prostate cancers [268]; LHRH receptor type II was found in monkeys, prostate cancer cells show a Gai linked activation signaling cascade [241]. In ovarian cancer the LHRH-receptor II coupled to the Gaq/11 and activated extracellular signal regulated kinase (ERK1/2), but differs in activating p38 and mitogen-activated protein (MAP) kinase [19]. The above-summarized results and the fact that endometrial, ovarian, breast and prostate cancer cells have two types of LHRH binding sites (low- and high-affinity/ low- and high-capacity) indicate the existence of a second LHRH receptor type. Only the high-affinity/low-capacity receptor type resembles the LHRH receptor type I form of the pituitary. There are reasons to believe that the low affinity binding site on these cancer cells is the putative LHRH receptor II. mRNA for the LHRH receptor type II is expressed in human endometrial and ovarian cancer cells. LHRH II was more potent in its antiproliferative effect than LHRH I agonists [19]. Leung et al. found similar results in ovarian cancers [273]. In pancreatic cancers the low-affinity receptor was located in the cell membrane, whereas the highaffinity receptor was in the nuclear membrane [9] (Table 6.4). In summary, LHRH is a most appropriate ligand for targeting cancers, as there are receptors overexpressed in gonadal tissue, multiple cancers and pituitary. In addition, the LHRH receptor signal transduction pathway in tumor cells is different to normal cells in that the classical PLC/PKC pathway from pituitary is not activated by LHRH binding to a LHRH receptor in a tumor cell; multiple G protein subgroups can be activated. The LHRH binding activates signals to mitogenic signal transduction pathways related to growth factor receptors and tyrosine kinase activities as well as the activation of antimetastatic signals [247, 281]. The expression of LHRH II in malignant cells and tumors could be exploited by targeting even more specifically malignancies, increasing uptake through activation of tumor-specific signal transduction cascades and improving the intracellular delivery of nanoparticles conjugated with LHRH to highest efficiency. The next section reviews the use of nanoparticles linked to LHRH for magnetic resonance imaging, treatment and drug delivery, with emphasis on specific properties of the in vivo use of these nanoparticles.

6.5 LHRH-bound Magnetic Nanoparticles

6.5

LHRH-bound Magnetic Nanoparticles 6.5.1

Synthesis and Characterization

The nanoparticles referred to here are superparamagnetic iron oxide nanoparticles (SPIONs) that were fabricated, by wet chemical synthesis, with amine groups on their surface, which facilitated the binding of LHRH through carbodiimide activation. The binding of LHRH or Hecate to SPIONs was confirmed by FTIR (Fouriertransformation infrared) spectroscopy and HPLC analysis. The SPION surface was saturated by the LHRH peptide, as FTIR spectral analysis of peptide bound magnetite nanoparticles revealed the absence of characteristic bands of aNH2 (3200 and 1625 cm1 ) present in unbound nanoparticles [297, 300]. The LHRH-SPIONs were monodisperse under transmission election micrographs and had an average diameter of 10–20 nm. Both free SPIONs and LHRH-SPIONs retained the cubic spinel structure. Moreover, the particles remained superparamagnetic, regardless of binding to the ligand (Fig. 6.6). Unconjugated SPIONs showed a higher saturation magnetization than LHRH–SPIONs (Ms ¼ 72:1 compared with 30.6 emu g1 ). The coercivity for LHRH-SPIONs was greater than for SPIONs alone (Fig. 6.6). Zeta potential measurements revealed that SPIONs were positively charged (z ¼ 28:5 G 1:93 mV), whereas LHRH-SPIONs were almost neutral (z ¼ 2:2 G 0:58 mV). Similar values were reported by Shieh et al. [301] for nanoparticles of similar surface chemistry (z ¼ 23:3 mV). Magnetic data were calculated per g of powder of both SPIONs and LHRH-SPIONs. In another study LHRH conjugated nanoshells were prepared for treatment with hyperthermia. To increase the iron particle concentration within the tumor tissue,

(A) TEM micrograph of LHRH-SPION nanoparticles. Inset: electron diffraction pattern. (B) Hysteresis and saturation magnetization at 300 K for LHRH-SPIONs (dashed line) and SPION nanoparticles (solid line).

Fig. 6.6.

201

202

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

Fig. 6.7.

Nanoclinics for hyperthermia treatment [157].

nanoparticles were decorated with LHRH to target the cancer cells and increase the cellular accumulation. Bergey et al. have developed a specific targeting device [157]. The particle consisted of a silica shell (7 nm thick) that housed inside a magnetic core of Fe2 O3 (18 nm diameter), a two-photon optical probe and, on the outside, carried the ligand LHRH (DLys6) as targeting moiety; the optical probe served as a tracking device. The final particle diameter ranged between 20 and 50 nm – small enough to diffuse into tissue without destroying the cells. The ligands were covalently attached to the silica surface through spacer molecules (Fig. 6.7). In this nanoclinic, the ligands consisted of an analog of LHRH that has higher binding affinity to the LHRH receptor due to exchange of amino acid 6 into D-Lys. 6.5.2

Treatment using Hyperthermia

Tumor cells are more sensitive to temperatures above 42  C than normal cells [282, 283] and can be destroyed by increasing locally the temperature to 41–42  C for 30 min [284–287]. Magnetic particles generate heat when exposed to an alternating magnetic field (AMF) by hysteresis loss [287–289]. These exposures require high power to elicit heat. The increase of temperature occurs during the thermal loss resulting from reorientation of the magnetism of the magnetic material with low electrical conductivity [288]. The size of the magnetic particle, which can be controlled by appropriate synthesis methods, determines the heating potential, as nanoparticulate size compared with micro size and degree of dispersion reduces the required AC power [290, 291]. Specific absorption power rates (SAR) have been determined in suspensions of magnetite nanoparticles of various size and coating. SAR of dextran ferrites were 180–210 W (g-Fe)1 (120 nm), sonicated dextran ferrites ranged from 12 to 240 W (g-Fe)1 , uncoated ferro-suspension 0–45 W (g-Fe)1 (6–10 nm). Ultrasonification caused dispersion of agglomerated particles and can destroy in

6.5 LHRH-bound Magnetic Nanoparticles

part the dextran coating. Carboxymethyldextran magnetite particles (130 nm) had an SAR of 90 W (g-Fe)1 . SAR data allow an estimate of the amount of particles necessary to heat human tissues [292, 293]. Ferrofluids directly injected into tumor tissue destroyed tumor cells after exposure to the magnetic field [289]. This effect was highly specific and cell destruction was only observed in cells containing the magnetic particles [294]. This process is known as magnetocytolysis. Similar to the diagnostic magnetic resonance imaging approach, an increase in intracellular particle number and concentration increases the efficacy of tumor destruction through hyperthermia. In vitro, LHRH expressing human breast cancer cells, MCF-7, and the receptor negative ovarian cancer cell line were incubated with nanoclinics linked to LHRH or nanoclinics alone. The LHRH-nanoclinic uptake was time dependent, and nanoshells accumulated within 30 min. In contrast, a human ovarian cancer cell line that did not express LHRH receptors did not incorporate LHRH-nanoshells. LHRH decorated nanoparticles were eight times more potent in destroying MCF-7 cells. The number of lysed cells was linearly dependent on the magnetic field exposure time and the concentration of nanoparticles. The same nanoshells without LHRH resulted in comparable cell lysis in MCF-7 and UCI 107 cells. Upregulation of LHRH receptors through EGF increased magnetocytolysis two-fold, whereas downregulation of receptors decreased the treatment effect 2.5-fold. Several crucial observations were made that support the high specificity of these ligand-conjugated nanoclinics: (a) the LHRH-nanoclinics specifically entered the receptor-expressing MCF-7 cells; (b) nanoclinics without the ligand only incorporated particles to a significantly lower amount and were independent of the cell type used; and (c) LHRH-receptor positive MCF-7 cells incorporated higher amounts of LHRH-nanoclinics when LHRH receptors were upregulated through EGF, and lower amounts after downregulation of receptors. These data showed the importance of highly selective nanoclinic uptake. These studies demonstrated specific targeting of cancer cells and incorporation of magnetic nanoparticles conjugated to LHRH and showed in vitro accumulation of magnetic nanoparticles through receptor-mediated endocytosis. These results suggest that specific targeting can increase the efficacy of hyperthermic therapy and keep other organs unaffected. 6.5.3

Treatment using Lytic Peptides Destruction of Metastases through LHRH-SPION-Hecate Recent studies on LHRH-SPIONs conjugated with Hecate, a membrane destroying peptide [6, 295, 315–317], resulted in tumor destruction and elimination of lung metastases in nude mice bearing LHRH receptor expressing MDA-MB-435S.luc breast cancer xenografts. Tumor bearing mice were injected once a week for 3 weeks with LHRH-SPION-Hecate conjugates in the presence and absence of LHRH, SPION-Hecate or saline. At necropsy tumor cells were determined in lung homogenates, and iron contents were also determined. In mice treated with 6.5.3.1

203

204

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

LHRH-SPION-Hecate destroys lung metastases in MDA-MB-435S.luc tumor-bearing mice; co-injection with LHRH or Hecate-SPIONs does not destroy lung metastases (A). Iron is detectable in targeted treatment groups after 1 week (B).

Fig. 6.8.

LHRH-SPION-Hecate, tumor cell death occurred in primary tumors and lung metastases. Unconjugated SPION Hecate did not reduce the number metastatic cells in lungs (Fig. 6.8) or destroy the primary tumors when compared with saline controls. Iron oxide particles in the treated organs were still detectable 1 week after injections, suggesting that LHRH-SPION-Hecate may be useful in imaging and treating the cancer, most importantly serving as a monitoring tool for treatment response [318]. 6.5.4

Detection of Tumors and Metastases Targeted Delivery of SPION Contrast Agents for MRI The development of iron oxide nanoparticles as a contrast agent for MR images requires high accumulation of nanoparticles in the cells of the target tissue. Relaxation times in the acquisition of magnetic resonance imaging, i.e., the contrast of the image, is increased with larger particles [151]. Compartmentalized particles and clusters have larger relaxation rates than free dispersed particles [152]. Highly efficient receptors were required on human breast cancer cells to increase the iron accumulation within the cells. Furthermore, the particles were designed to provide 6.5.4.1

6.5 LHRH-bound Magnetic Nanoparticles

target specificity, a long circulation time and low opsonization to escape macrophage recognition. The following outline describes the development of LHRH-targeted nanoparticles to human breast cancer cells. The increase of cellular accumulation of iron oxide nanoparticles in LHRH receptor positive human breast cancer cells and their metastases iron oxide nanoparticles were developed, which carried LHRH as targeting moiety. Human breast cancer cells overexpress LHRH receptors. Recently, the targeted destruction of primary tumors and metastases had been shown in human breast cancer cells, MDA-MB-435S.luc, both in vitro and in vivo [8, 295– 299]. In Vitro Studies on Receptor-targeted LHRH-SPION Uptake The accumulation of ligand conjugated and unconjugated SPIONs was compared in MDA-MB-435S.luc, TM4 mouse Sertoli cells to determine whether the iron accumulation was specific for individual receptors on the target cells (Fig. 6.9A). In 6.5.4.2

(A) Iron uptake from SPIONs, LHRH-SPIONs and LHRH-SPIONs plus LHRH in LHRH receptor expressing breast cancer cells (MDA-MB-435S.luc) and receptor

Fig. 6.9.

negative mouse Sertoli cells (TM4). (B) Saturation kinetics in MDA-MB-435S.luc cells. (C) Incorporation of SPIONs or LHRHSPIONs into THP-1 macrophages [297].

205

206

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

LHRH receptor overexpressing human breast cancer cells LHRH-SPIONs accumulated quickly and reached an intracellular iron concentration of 452 pg cell1 whereas free SPIONs reached 51 pg cell1 as the highest concentration (Fig. 6.9B). Blocking of the LHRH receptor significantly prevented LHRH-SPION uptake, suggesting that specific receptor-mediated uptake was the mechanism of uptake. Maximal concentrations within the breast cancer cells were reached within 1 h, whereas low temperature (4  C) prevented receptor-mediated iron uptake to less than 1.1 pg cell1 . The mouse Sertoli cell line showed, at all concentrations, iron uptake between 29 and 37 pg cell1 irrespective of LHRH conjugation (Fig. 6.9A). Most importantly, macrophages incorporated significantly less LHRH-SPIONs (85 pg cell1 ) than free SPIONs (246 pg cell1 ) (Fig. 6.9C), suggesting that LHRH provided a coating as well a targeting moiety. This was expected as LHRH-SPIONs were neutral, while SPIONs were positively charged. Others have shown that neutral nanoparticles are less easily incorporated by macrophages than are charged nanoparticles, predicting an increased circulation time in vivo [86, 297–299]. TEM sections from human breast cancer cells in vitro showed that LHRHSPION accumulation in the breast cancer cells resulted in cluster formation in the cytosol and the nucleus. In contrast, no iron particles were observed in cells incubated at 4  C [297–299]. Compared with other ligand conjugated nanoparticles LHRH-SPION uptake was highly efficient and resulted in the highest iron contents in LHRH receptor expressing cancer cells. Dextran-coated nanoparticles <10 nm in diameter resulted in 0.9–1.3 pg-Fe per cell [120], whereas Her2/neu targeting of the same cells increased the uptake by a factor of 2.6 pg-Fe per cell. Folatelinked iron oxide nanoparticles reached a maximal level of 75 pg-Fe per cell in BT20 breast cancer cell incubation [117]. CD-8 conjugated crosslinked iron oxide nanoparticles were incorporated into T cells after 1 h of incubation at concentrations up to 100 pg-Fe per cell [122]. The targeted up-take of nanoparticles in cancer cells has been demonstrated for ferrofluids: dextran-coated Monocrystalline Iron Oxide Nanoparticles (MIONs) linked to transferrin resulted in a 10-fold increase in accumulation in rat glioma cells compared with free MIONs [111]. In contrast, human fibroblasts did not incorporate transferrin-linked MIONs; the particles were only bound to the membranes, but not internalized [302]. LHRH iron oxide nanoparticles coated with a silica shell specifically targeted and accumulated in MC-F7 human breast cancer cells in vitro [157]. Anionic maghemite chelate dimercaptosuccinic acid particles were incorporated in macrophages at up to 6 pg-Fe per cell, and in HeLa cancer cells up to 40 pg-Fe per cell, and showed saturation kinetics [152]. HIV-1 tat dextran-coated SPIONs were incorporated into CD34 cells up to 30 pg cell1 [303, 304]. In Vivo Studies on Receptor-targeted LHRH-SPION Uptake When SPIONs and LHRH-SPIONs were injected intravenously into human breast cancer (MDA-MB-435S.luc) xenograft-bearing nude mice, up to 59.1% of iron particles accumulated in tumors of LHRH-SPION injected mice compared with 8% in groups injected with SPIONs (Fig. 6.10A, B). Lung tissue from tumor-bearing 6.5.4.3

6.5 LHRH-bound Magnetic Nanoparticles

Fig. 6.10. Relative iron distribution in human breast cancer xenograft-bearing mice after injection of SPIONs and LHRH-SPIONs. (A) Iron contents in organs and tumors after LHRH-SPION injection, (B) after SPION

injection (100% ¼ 5 mg-Fe per mouse). TEM micrographs (C) from lung section with metastases after LHRH-SPION injection and (D) after SPION injection [297].

mice, which contained significant numbers of metastatic breast cancer cells, accumulated up to 20% LHRH-SPIONs and only 2% SPIONs (Fig. 6.10A, B). Neither lungs nor livers of normal mice accumulated LHRH-SPIONs, suggesting that LHRH-SPIONs were not incorporated into macrophages in vivo as predicted from low in vitro macrophage uptake (Fig. 6.10). LHRH-SPIONs were either incorporated into tumors or metastatic cells or they were excreted as observed in normal mice. The high accumulation of SPIONs in the livers suggested that these particles were incorporated by macrophages and transported therein to the liver, where they accumulated to 55%. Unconjugated SPIONs accumulated to 8% in tumors ( p < 0:00006), 55% in the livers, ( p < 0:00006), 2% in lungs, ( p < 0:031), and 3.3% in kidneys, ( p < 0:33) (Fig. 6.10B). When lung sections from LHRH-SPION- and SPION-injected mice were stained for iron, only lungs from mice injected with LHRH-SPIONs tested positive [298, 299]. The iron contents of lungs correlated directly with the number of metastatic cells (Fig. 6.11) [297]. The average iron content per lung cancer cell was calculated to be 77.8 G 23.5 pg Fe per metastatic cell in lungs. Lung sections from tumorbearing mice showed iron clusters in groups after LHRH-SPION treatment. No iron clusters were observed in SPION-injected groups (Fig. 6.10C, D). The specific targeting of tumors and metastases and the low accumulation in the livers seemed to be a combination of ligand conjugation and physical characteristics of the LHRH-SPIONs. The LHRH-SPIONs had a sufficiently long circulation

207

208

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

Linear relationship between the metastatic cells in lungs and the iron content in tissue [297].

Fig. 6.11.

time, similar to albumin-coated particles with neutral zeta potential [305]. Most important, neutral particles were less prone to accumulate in liver (20%) [86], because they showed the lowest opsonization and were, therefore, not recognized by macrophages of the RES. Coating nanoparticles with short peptides increased the colloidal stage and decreased the non-specific binding to fibronectin, an effect that was similar to PEG coating [274, 306]. It is highly likely that LHRH served as coating itself, considering the low liver accumulation of LHRH-SPIONs in normal (non-tumor) bearing mice. The biodistribution and circulation time of contrast agents is determined by several factors, such as size, charge, and surface chemistry [84, 85]. Nanoparticles of hydrodynamic size greater than 50 nm, and hydrophilic surface and high charges are eliminated from the circulation within minutes by phagocytotic cells of the reticulo-endothelial system (RES). Therefore, the accumulation of such particles was observed in liver, spleen, bone marrow, and were less likely to accumulate in other organs such as breast tissue, lymph nodes, intestinal tissue or gonads [301, 307, 308]. Coating iron oxide nanoparticles of <50 nm diameter with poly(ethylene glycol) (PEG), sialic acid, neuraminic acid, mucin or glycophorin increased the circulation time [309]. Dextran-coated nanoparticles of <50 nm were detected to 82% in the livers of rats after intravenous injection [310]. Ligand decoration with LHRH was highly superior in increasing SPION concentration in target cells lowering liver accumulation. Other in vivo studies delivered PEG-coated particles decorated with carcinoembryonic antigen, which resulted in 1% tumor, 54% liver and 22% spleen accumulation [309]. Transferrin-decorated MION particles accumulated in rat glioma cells, passing the blood–brain barrier, and were detectable in xenografts [311, 312]. Subcellular Distribution of LHRH and SPIONs in Tissues, Metastases and Tumors The subcellular distribution of LHRH-SPIONs in tumors showed clusters of submicrosize, which were absent in tumors of mice injected with SPIONs alone [296].

6.5 LHRH-bound Magnetic Nanoparticles

TEM images of the MDA-MB-435S.luc tumors, lungs, liver and kidneys confirmed previous observations. Sections containing LHRH-SPIONs were compared with those of mice injected with unconjugated SPIONs [296–298]. Iron oxide nanoparticles were detected in the cytoplasm of the tumor cells. The regions containing the nanoparticles were identified as iron in energy dispersive X-ray spectroscopy analysis; large nanoparticle clusters were formed in the breast cancer xenografts of nude mice injected with LHRH-SPIONs, whereas single nanoparticles were observed in the tumors of the mice injected with SPIONs. At higher magnification these clusters appeared to be monodisperse [296–298]. Similar observations were made in lung sections of tumor-bearing mice; lungs from mice injected with LHRH-SPIONs with metastases had iron particle clusters in their metastases (Fig. 6.10C). These clusters were absent in lung metastases of mice with SPION injections (Fig. 6.10D) [296–298]. TEM images from livers showed high iron accumulation in SPION injected groups, and no visible iron accumulation in kidneys of either LHRH-SPION or SPION injected groups [296]. In summary, the LHRH-SPION particles accumulated at high concentrations in cells expressing functional LHRH receptors. Neutral particles are less prone to accumulate in the liver due to escape from macrophages of the RES. LHRH-SPIONs were poorly recognized and incorporated into macrophages, which improves iron accumulation in tumors and metastases. Metastases incorporate specifically LHRH-SPIONs. Iron cluster formation suggests a good possibility to increase the relaxation times during MRI. LHRH has a dual function: coating and targeting. Enhancement of Magnetic Resonance Images in Tumors and Metastases Preliminary magnetic resonance images of tumors and lung sections containing metastases have also been obtained from the 7 T animal MRI and were analyzed using acquisition of multiple orders of intermolecular multiple-quantum coherence (iMQC) in a multi-CRAZED sequence. The multi-CRAZED sequence is an imaging pulse sequence that consists of 90-{delay t}-{gradient pulse, area GT}-Y{gradient pulse, area nGT}-{delay TE}-180-{delay(TE-nt)}acquisitions, that uses the difference in echo timing to acquire multiple echoes and results in up to five echoes that are separated by t, described as DQC (þ/2 quantum coherence), SQC (þ/1 quantum coherence) and ZQC (zero-quantum coherence) (Fig. 6.12 [314]). Tumors and lung sections containing metastatic cells have been analyzed using different acquisition modalities. The images in Fig. 6.13(A, B) were obtained from tumor pieces of 1–2 cm of tumor samples with LHRH-SPION nanoparticles, while the images at the bottom correspond to 1–2 cm of tumor sample from mice, injected with saline. Due to the heterogeneity of tumor tissue, significant contrast differences are needed to reveal the presence of tumors or the attachment of the magnetic nanoparticles. Hence, classical MRI, such as the T2 images shown in Fig. 6.13(A) [314], did not reveal significant contrast differences between the tumors with or without LHRH-SPIONs, i.e., they did not overcome tissue heterogeneity. Increased contrast was observed in the T2* image acquisition. Multi-CRAZED images show the contrast for each

209

210

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

The multi-CRAZED sequence uses its echo timing to acquire multiple echoes that result in up to five echoes that are separated by t, described as DQC (G2 quantum coherence), SQC (G1 quantum coherence) and ZQC (zero-quantum coherence) [314]. The þSQC (þ1 or single-quantum coherence) and MSQC (1 or single-quantum coherence) Fig. 6.12.

images have primarily conventional contrast; the DQC (þ2-quantum), ZQC (0-quantum) and MDQC (2-quantum) coherences have contrast from subvoxel variations in magnetization density or resonance frequency. Additional gradient pulses permit image acquisition in the standard way (single-line acquisition shown here) [314].

of the five images and resulted in enhanced contrast in LHRH-SPION-containing tumors for DQC and ZQC images (Fig. 6.13C). Similar results were obtained with sections from lungs containing metastases from mice injected with LHRH-SPIONs or saline (Fig. 6.14 [319]). This may be due to the increased concentration of the LHRH particles and formation of clusters, as shown in the TEM image in Fig. 6.10(C). Such particle clusters should give rise to increased contrast due to increased magnetic moments, and their effects on the surrounding water molecules [314].

6.6

Future Outlook

In summary, LHRH is a most appropriate ligand for targeting cancers, as there are receptors overexpressed in gonadal tissue, multiple cancers, their metastases and pituitary. In addition, the LHRH receptor signal transduction pathway in tumor cells is different to normal cells in that that the classical PLC/PKC pathway from the pituitary is not activated by LHRH binding to a LHRH receptor in a tumor cell, and multiple G protein subgroups can be activated. The LHRH binding activates signals to mitogenic signal transduction pathways related to growth factor receptors and tyrosine kinase activities as well as the activation of antimetastatic sig-

6.6 Future Outlook

Fig. 6.13. (A) Conventional T2 scans obtained from tumor sites in nude mice inoculated with saline (bottom image) and LHRH-SPIONs (top image). Tumor pieces ca. 1–2 cm. (B) Presence of LHRH-SPIONs revealed by T2* MRI of nude mice xenograft after injection of saline (bottom image) and LHRH-SPIONs

(top image) using multi-CRAZED analysis [314]. (C) Multi-CRAZED images of a mouse breast tumor embedded with nanoparticles (left) and without nanoparticles (right): 0-quantum (ZQC image); 1-quantum (SQC image); 2-quantum (DQC image); 1-quantum (MSQC image); 2-quantum (MDQC image) [314].

nals, which can be exploited in designing the LHRH targeting of nanoparticles for cancer treatment [247, 281]. The expression of LHRH II in malignant cells and tumors could be exploited by targeting even more specifically malignancies, increasing uptake through activation of tumor specific signal transduction cascades, and improving specifically tar-

211

212

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

Multi-CRAZED images from metastases containing lung tissue from tumor-bearing mice. Images (a)–(e) are lung sections from saline controls, and (f )–(j) are lung sections from mice injected with LHRH-SPIONs. (a) and (f ) are 0-quantum

Fig. 6.14.

(ZQC image); (b) and (g) are 1-quantum (SQC image); (c) and (h) are 2-quantum (DQC image); (d) and (i) are 1-quantum (MSQC image); (e) and (j) are 2-quantum (MDQC image) [319].

geted and intracellular delivery of nanoparticles conjugated with LHRH with highest efficiency. These characteristics could lead to a new era of targeted imaging and treatment and increase the potential of nanoparticle applications in the future. Acknowledgments

The author thanks Dr. Challa Kumar and Dr. Franz Josef Hormes, Center for Advanced Microstructures and Devices, for fruitful discussion and enthusiasm in our collaborations. The support of Dr. William Hansel, Pennington Biomedical Research Center is gratefully acknowledged. The expertise in preparing TEM images of Dr. Jikou Zhou, Lawrence Livermore Laboratory, Berkeley, Dr. Wole Soboyejo, Princeton University, and the preparation of MR images by Dr. Warren Warren, Duke University, are gratefully acknowledged. Daniel Lazarro helped in the preparation of some figures. Abbreviations

AP-1 CT EGF EGF-R FSH GnRH

Activator protein 1 Computed tomography Epidermal growth factor Epidermal growth factor receptor Follicle stimulating hormone Gonadotropin-releasing hormone

References

GPCR Jak-Stat JNK LHRH MAPK MDR MRI MMP Nrf2 NF-kB PET PI-3 K PKC PTP RPTK RT-PCR SAP-K SPECT SPIONs TSH TRH uPa

G protein-coupled receptor Janus kinase signal transducers and activators of transcription cJun N-terminal kinase Luteinizing hormone releasing hormone Mitogen-activated protein kinase Multidrug resistance Magnetic resonance imaging Matrix metallo proteinases Nuclear factor erythroid 2-related factor 2 Nuclear factor kB Positron emission tomography Phosphatidylinositol 3-kinase Protein kinase C Phosphotyrosine phosphatase Phosphotyrosine Kinase Receptor Reverse transcriptase polymerase chain reaction Stress activator activated protein kinase Single-photon emission tomography Superparamagnetic iron oxide nanoparticles Thyroid stimulating hormone Thyrotropin releasing hormone Urokinase plasminogen activator

References 1 Global Cancer Statistics, www.cancer 2

3 4

5

6

.org, 2002. A. Jemal, T. Murray, E. Ward, A. Samuels, R.C. Tiwari, A. Ghafoor, E.J. Feuer, M.J. Thun. Cancer statistics, CA Cancer J. Clin. 2005, 54, 10–30. Cancer Facts and Figures 2004, American Cancer Society, www.acs.org. A. Qayum, W. Gullick, R. Clayton, K. Sikora, J. Waxman. The effects of gnrh analogues in prostate cancer are mediated through tumor receptors, Br. J. Cancer. 1990, 62, 96–99. G. Halmos, J. Arencibia, A. Schally, R. Davis, D. Bostwick. High incidence of receptors for luteinizing hormone-releasing hormone (LHRH) and LHRH receptor gene expression in human prostate cancers, J. Urol. 2000, 163(2), 623–629. C. Leuschner, F. Enright, B. Gawronska-Kozak, W. Hansel.

Human prostate cancer cells and xenografts are targeted and destroyed through LHRH receptors, Prostate. 2003, 56, 239–249. 7 B. Straub, M. Muller, H. Krause, M. Schrader, C. Goessl, R. Heicappell, K. Miller. Increased incidence of luteinizing hormonereleasing hormone receptor gene messenger RNA expression in hormone-refractory human prostate cancers. Clin. Cancer Res. 2001, Aug 7(8), 2340–2343. 8 C. Leuschner, C. Kumar, W. Hansel, J. Hormes. Targeting breast cancer cells and their metastses through luteinizing hormone releasing hormone (LHRH) using magnetic nanoparticles, J. Biomed. Nanotechnol. 2005, 1, 1–5. 9 B. Szende, G. Srkalovic, J. Timar, J. Mulchahey, J. Neill, K. Lapis, A. Csikos, K. Szepeshazi, A. Schally.

213

214

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

10

11

12

13

14

15

16

17

Localization of receptors for lHRH in pancreatic and mammary cancer cells, Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4153–4160. K. Baumann, L. Kiesel, M. Kaufmann, G. Bastert, B. Runnebaum. Characterization of binding sites for a GnRH-agonist (buserelin) in human breast cancer biopsies and their distribution in relation to tumor parameters, Breast Cancer Res. Treat. 1993(25), 1, 37–46. M. Fekete, J.L. Williff, A.V. Schally. Characteristics and distribution of receptors for [D-Trp6]luteinizing hormone releasing hormone, somatostatin, epidermal growth factor and sex steroids in 500 biopsy samples of human breast cancer, J. Clin. Lab. Anal. 1989, 3, 137–147. K. Eidne, C. Flanagan, R. Millar. Gonadotropin releasing hormone binding sites in human breast carcinoma, Science. 1985, 229, 989–999. A. Ben-Yehudah, S. Yarkone, A. Nechushtan, R. Belostotsky, H. Lorberboum-Galski. Linker based LhRH-PE chimeric proteins inhibit cancer growth in nude mice, Med. Oncol. 1999, 16, 38–45. A. Ben-Yehudah, D. Prus, H. Lorberboum-Galski. IV administration of L-GNRH-PE66 efficiently inhibits growth of colon adenocarcinoma xenografts in nude mice. Int. J. Cancer 2001, 92, 263–268. M. Fekete, A. Zalatnai, A. Comaru Schally, A. Schally. Membrane receptors for peptides in experimental and human pancreatic cancers, Pancreas. 1989, 4, 521–528. H. Friess, M. Buchler, L. Kiesel, M. Kruger, H. Beger. LHRH receptors in the human pancreas: Basis for antihormonal treatment ductas carcinoma of the pancreas, Int. J. Panreatol. 1991, 10, 151–159. G. Emons, O. Ortmann, M. Becker, G. Irmer, B. Springer, R. Laun, F. Holzel, K. Schulz, A. Schally, High affinity binding and direct antiproliferative effects of LHRH

18

19

20

21

22

23

24

analogs I human ovarian cancer cell lines, Cancer Res. 1993, 53, 5439–5446. P. Volker, C. Grundker, O. Schmidt, K.D. Schulz, G. Emons. Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: Frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues. Am. J. Obstet. Gynecol. 2002, 186, 171–179. C. Grundker, A. Gunthert, R. Millar, G. Emons. Expression of gonadotropin releasing Hormone II receptor in human endometrial and ovarian cancer cells and effects of gnrh II on tumor cell proliferation, J. Clin. Endocrinol. Metab. 2002, 87, 1427–1430. C. Grundker, G. Emons. Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer, Reprod. Biol. Endocrinol. 2003, 65, 1–7. C. Verschraegen, S. Westphalen, W. Hu, E. Loyer, A. Kudelka, P. Volker, J. Kavanagh, M. Steger, K. Schulz, G. Emons. Phase II study of cetrorelix, a luteinizing hormonereleasing hormone antagonist in patients with platinum-resistant ovarian cancer, Gynecol. Oncol. 2003, 90(3), 552–559. G. Keller, A. Schally, T. Gaiser, A. Nagy, B. Baker, G. Westphal, G. Halmos, J. Engel. Human malignant melanomas express receptors for luteinizing hormone releasing hormone allowing targeted therapy with cytotoxic luteinizing hormone releasing hormone analogue, Cancer Res. 2005, 65, 5857–5863. G. Keller, A. Schally, T. Gaiser, A. Nagy, B. Baker, G. Halmos, J.B. Engel. Receptors for LHRH expressed in human non-Hodgkins lymphomas can be targeted for therapy with the cytotoxic LHRH analogue AN 207, Eur. J. Cancer. 2005, 41, 2196–2202. L. Krebs, X. Wang, A. Nagy, A. Schally, P. Prasad, C. Liebow. A conjugate of doxorubicin and and analog of lHRH shows increased

References

25

26

27

28

29

30

31

32

33

efficacy against oral and laryngeal cancers, Oral Oncol. 2002, 38, 657–663. S. Gong, G. Zhao, H. Zhao, W. Lu, G. Liu, P. Zhu. Ability of luteinizing hormone releasing hormonePseudomonas aeruginosa exotoxin 40 binding to LHRH receptor on human liver cancer cells, World J. Gastroenterol. 2004, 10(19), 2870–2873. A. Imai, T. Ohno, K. Lida, T. Fuseya, T. Fueui, T. Tamaya. Presence of gnrh receptor and its messenger ribonucleic acid in endometrial carcinoma and endometrium, Gynecol. Oncol. 1994, 55, 144–148. R. Moretti, M. Montagnani, J. von Groeninghen, P. Limonta. Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells, J. Clin. Endocrinol. Metab. 2002, 87, 3791–3797. N. Sion-Vardi, J. Kaneti, T. SegalAbramson, J. Giat, J. Levy, Y. Sharoni. Gonadotropin-releasing hormone specific binding sites in normal and malignant renal tissue, J. Urol. 1992, 148(5), 1568–1570. J. van Groeninghen, L. Kiesel, D. Winkler, M. Zwirner. Effects of LHRH in nervous system tumours, Lancet. 1998, 352, 372–373. M. De Lena, C. Brambilla, A. Morabito, G. Bonadonna. Adriamycin plus vincristine compared to and combined with cyclophosphamide, methotrexate and 5-fluorouracil for advanced breast cancer, Cancer. 1975, 35, 1108–1115. J. Bull, D. Tormey, S. Li, P. Carbone, G. Falkson, J. Blom, E. Perlin, R. Simon. A randomized comparative trial of adriamycin versus methotrexate in combination drug therapy, Cancer. 1978, 41, 1649–1657. R. Santen. Endocrine treatment of prostate cancer, J. Clin. Endocrinol. Metab. 1992, 75, 685–689. J. Emmet, L. Greene, A. Papantoniou. Endocrine therapy in carcinoma of the prostate gland: 10 year survival studies, J. Urol. 1960, 83, 471–484.

34 R. O’Regan, V. Jordan. Tamoxifen to

35

36

37

38

39

40

41

42

43

44

raloxifene and beyond, Semin. Oncol. 2001, 28, 260–273. M. O’Reilly, L. Holmgren, Y. Shing, C. Chen, R. Rosenthal, M. Moses, W. Lane, Y. Cao, E. Sage, J. Folkman. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma, Cell. 1994, 79, 315–328. S. Eccles, G. Box, W. Court, J. Sandle, C. Dean. Preclinical models for the evaluation of targeted therapies of metastatic disease, Cell Biophys. 1994, 24–25, 279–291. H. Walter. Simultaneous occurrence of malignant tumors and systemic diseases, Radiol. Clin. 1951, 20, 405–512. K. Pantel, M. Otte. Occult micrometastasis: Enrichment, identification and characterization of single disseminated tumour cells, Semin. Cancer Biol. 2001, 11, 327–337. C. Mettlin, G. Murphy, R. Ho, H. Menck. The National Cancer data base report on longitudinal observations on prostate cancer, Cancer. 1996, 77, 2162–2166. C. Galasko. The anatomy and pathways of skeletal metastases, in Bone Metastases, L. Weiss, A.H. Gilbert (Eds.), Boston, GK Hall, 1981, pp. 49–63. S. Jang, M. Wientjes, D. Lu, J. Au. Drug delivery and transport to solid tumors, Pharm. Res. 2003, 20, 1337–1350. S. Honig. Hormonal therapy and chemotherapy, in Diseases of the Breast, J.R. Harris, M.E. Lippman, M. Morrow, S. Hellman (Eds.), Lippincott-Raven Publishers, Philadelphia, 1996, pp. 669–734. K. Pantel, R. Cote, O. Fodstadt. Detection and clinical importance of micrometastatic disease, J. Natl. Cancer Inst. 1999, 91, 1113–1124. S. Braun, K. Pantel. Biological characteristics in micrometastatic cancer cells in bone marrow, Cancer Metastasis Rev. 1999, 18, 75–90.

215

216

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 45 S. Braun, K. Pantel, P. Muller, W.

46

47

48

49

50

51

52

53

Janni, F. Hepp, C. Kentenich, S. Gastroph, A. Wischnik, T. Dimpfl, G. Kindermann, G. Riethmuller, G. Schlimok. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer, N. Engl. J. Med. 2000, 342, 525–533. I. Diel, B. Krempien, M. Kaufmann, S. Costa, R. Goerner, D. von Fournier, G. Bastert. Ergebnisse von Beckenkammbiopsien von 475 Patientinnen mit primaerem und metastasiertem Mammakarzinom, Tumor Diagn. Ther. 1992, 13, 85–90. K. Morikawa, S. Walker, M. Nakajima, S. Pathak, J. Jessup, I. Fidler. Influence of organ environment on the growth selection and metastasis of human colon carcinoma cells in nude mice, Cancer Res. 1988, 48, 6863–6871. K. Pantel, R. Cote, O. Fodstadt. Detection and clinical importance of micrometastatic disease, J. Natl. Cancer Inst. 1999, 91, 1113–1124. K. Pantel, V. Mueller, M. Auer, N. Nusser, N. Harbeck, S. Braun. Detection and clinical implications of early systemic tumor cell dissemination in breast cancer, Clin. Cancer Res. 2003, 9, 6326–6334. D. Dearnaley, J. Sloane, M. Ormerod, K. Steele, R. Coombes. Clink Hmc, Powles TJ, Ford HT, Neville AM: Increased detection of mammary carcinoma cells in marrow smears using antisera to epithelial membrane antigen, Br. J. Cancer. 1981, 44, 85–90. P. Tschentscher, C. Wagener, M. Neumaier. Sensitive and specific cytokeratin 18 reverse transcriptasepolymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA, Clin. Chem. 1997, 43, 2244–2250. M. Robsen ME. K. Offit. Breast MRI for women with hereditary cancer risk. JAMA. 2004, 292, 1368. E. Warner, D. Plewes, K. Hill, P. Causer, J. Zubovits, R. Jong, M.

54

55

56

57

58

59

60

61

Cutrara, G. DeBoer, M. Yaffe, S. Messner, W. Meschino, C. Piron, S. Narod. Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination, JAMA. 2004, 292(11), 1317–1325. K. Hagspiel, K. Neidl, A. Eichenberger, W. Weder, B. Marincek. Detection of liver metastases: Comparison of superparamagnetic iron oxideenhanced and unenhanced MR imaging at 1.5 T with dynamic CT, intraoperative US, and percutaneous, US. Radiol. 1995, 196(2), 471–478. O. Clement, N. Siauwe. Liver imaging with ferrumxodides. Top. Magn. Res. Imaging. 1998, 9, 167– 182. E. Morris, L. Schwartz, D. Dershaw. MR imaging of the breast in patients with occult primary breast carcinoma, Radiology. 1997, 205, 437–440. C. Schorn, U. Fischer. MRI of the breast in patients with metastatic disease of unknown primary, Eur. Radiol. 1999, 9, 470–473. J. De Jong, J. Pruim, P. Elsinga, W. Vaalburg, H. Mensink. Visualization of prostate cancer with 11C choline positron emmision tomography, Eur. Urol. 2002, 442, 18–23. Y. Wang, S. Hussain, G. Kresting. Superparamagnetic iron oxide contrast agents: Physicochemical characteristics and applications in MR imaging, Eur. Radiol. 2001, 11, 2319–2331. R. Weissleder, G. Elizondo, J. Wittenberg, C. Rabito, H. Bengele, L. Josephson. Ultrasmall superparamagnetic iron oxide: Characterization of a new class of contrast agents for MR imaging, Radiology. 1990, 175(2), 489–493. R. Weissleder, D. Stark, B. Engelstad, B. Bacon, C. Compton, D. White, P. Jacobas, J. Lewis. Superparamagnetic iron oxide: Pharmacokinetics and toxicity, Am. J. Roentgenol. 1989, 152, 167–173.

References 62 B. Bonnemain. Superparamagnetic

63

64

65

66

67

68

69

agents in magnetic resonance imaging: Physicochemical characteristics and clinical applications, J. Drug Target. 1998, 6, 167–174. H. Daldrup, T. Link, S. Blasius, A. Strozyk, S. Konemann, H. Jurgens, E. Rummeny. Monitoring radiation induced changes in bone marrow histopathology with ultra small superparamagnetic iron oxide (USPIO) enhanced MRI, J. Magn. Reson. Imag. 1999, 9, 643–652. B. Van de Berg, F. Lecouvert, J. Kanku, J. Jamart, B. Van Beers, B. Maldague, J. Malghem. Ferrumoxides enhanced quantitative magnetic resonance imaging of the normal and abnormal bone marrow. Preliminary assessment, J. Magn. Reson. Imag. 1999, 9, 322–328. M. Harisinghani, J. Barentsz, P. Hahn, W. Deserno, S. Tabatabaei, C. Hulsbergen, J. Rosette, R. Weissleder. Noninvasive detection of clinically occult lymph node metastases in prostate cancer, N. Engl. J. Med. 2003, 348, 2491– 2499. M.G. Harisinghani, M.A. Saksena, P.F. Hahn, B. King, J. Kim, M.T. Torabi, R. Weissleder. Ferumoxtran10-enhanced MR lymphangiography: Does contrast enhanced imaging alone suffice for accurate lymph node characterization? Amer. J. Roentgenol. 2006, 186, 144–148. J. Mintorovich, K. Shansi, Eovist. Injection and Reovist injection, Two liver specific contrast agents for MRI, Oncol. Supp. 2000(3), 14, 37–40. M. Kavellaris, D. Kuo, C. Burkhart, D. Regl, M. Norris, M. Haber, S. Horwitz. Taxol resistant epithelial ovarian tumors are associated with altered expression of specific beta tubulin isotypes, J. Clin. Invest. 1997, 100, 1282–1293. M. Lehnert, S. Emerson, W. Dalton, R. de Guili, S. Salmon. In vitro evaluation of chemosensitizers for clinical reversal of p-glycoprotein associated: Taxol resistance. J. Natl. Cancer Inst. Mongr. 1993, 15, 63–67.

70 N. Baldini, K. Scotlandi, M. Serra,

71

72

73

74

75

76 77

78

T. Shikita, N. Zini, A. Ognibene, S. Santi, R. Ferracini, N.M. Maraldi. Nuclear immunolocalization of P-glycoprotein in multidrugresistant cell lines showing similar mechanisms of doxorubicin distribution. Eur. J. Cell Biol. 1995, 68, 226–239. A. Molinari, A. Calcabrini, S. Meschini, A. Stringaro, D. Del Bufalo, M. Cianfriglia, G. Arancia. Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. Int. J. Cancer. 1998, 75, 885–893. J. Hamada, T. Tsuruo. Characterization of the ATPase activity of the 170 to 180 kilodalton membrane glycoprotein associated with multi-drug resistance: the 170 to 180 kilodalton membrane glycoprotein is an ATPase, J. Biol. Cheml. 1988, 263, 1454–1458. R. Krishna, L. Mayer. Multidrug resistance (MDR) in cancermechanisms reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur. J. Cancer Sci. 2000, 11, 265–283. A. Jones, A. Harris. New developments in angiogenesis: a major mechanism for tumor growth and target for therapy, Cancer J. from Sci. Am. 1998, 4, 209–217. J. Murray, J. Carmichael. Targeting solid tumours: Challenges, disappointments and opportunities. Adv. Drug Delivery Rev. 1995, 17, 117–127. R. Jain. Molecular regulation of vessel maturation. Nat. Med. 2003, 9, 685–93. T.P. Padera, B.R. Stoll, J.B. Tooredman, D. Capen, E. di Tomaso, R.K. Jain. Pathology: Cancer cells compress intratumour vessels. Nature 2004, 427, 695. K. Weindel, J.R. Moringlane, D. Marme, H.A. Weich. Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid: the key to angiogenesis? Neurosurgery 1994, 35, 439–449.

217

218

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 79 S.P. Olesen. Rapid increase in blood

80

81

82

83

84

85

86

87

88

89

brain barrier permeability during svere hypoxia and metabolic inhibition. Brain Res. 1986, 368, 24–29. Y. Boucher, M. Leunig, R.K. Jain. Tumor angiogenesis and interstitial hypertension, Cancer Res. 1996, 56, 4264–4266. G. Helmlinger, P.A. Netti, H.C. Lichtenbeld, R.J. Melder, R.K. Jain. Solid stress inhibits the growth of multicellular tumor spheroids, Nat. Biotechnol. 1997, 15, 778–783. H. Maeda. The enhanced permeability and retention effect in tumor vasculature, the key role of tumor sensitive macromolecular drug targeting, Adv. Enzyme Regul. 2001, 41, 189–207. H. Maeda. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: A review, J. Controlled Release. 2000, 65, 271–284. E. Seneterre, R. Weissleder, D. Jaramillo, P. Reimer, A. Lee, T. Brady, J. Wittenberg. Bone marrow: Ultrasmall superparamagnetic iron oxide for MR imaging, Radiology. 1991, 179(2), 529–533. D. Pouliquen, I. Lucet, C. Chouly, R. Perdrisot, J. Le Jeune, P. Jallet. Liver-directed superparamagnetic iron oxide: Quantitation of T2 relaxation effects. Magn. Reson. Imag. 1993, 11, 219–228. C. Chouly, D. Pouliquen, J. Lucet, J. Jeune, P. Jallet. Development of superparamagnetic nanoparticles for MRI: Effect of particle size, charge and surface nature on biodistribution, J. Microencapsul. 1996, 13, 245–255. J. Kreuter. Drug targeting with nanoparticles. Eur. J. Drug Metab. Pharmacokine. 1994, 19, 253–256. L. Araujo, R. Lobenberg, J. Kreuter. Influence of the surfactant concentration of the body distribution of nanoparticles, J. Drug Target. 1999, 6, 373–385. V. Lenaerts, J. Nagelkerke, T. Van Berkel, P. Couvreur, L. Grislain, M. Roland, P. Speiser. In vivo uptake

90

91

92

93

94

95

96

97

98

99

of polyisobutyl cyanoacrylate nanoparticles by rat liver Kupffer, endothelial, and parenchymal cells. J. Pharm. Sci. 1984, 73, 980–982. P. Couvreur, B. Kante, V. Lenaerts, V. Scailteur, M. Roland, P. Speiser. Tissue distribution of antitumor drugs associated with polyalkylcyanoacrylate nanoparticles, J. Pharm. Sci. 1980, 69, 199–202. J. Panyam, V. Labhasetwar. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Del. Rev. 2003, 55, 329– 347. M. Ferrari. Cancer nanotechnology: opportunities and challenges, Nat. Rev. 2005, 5, 161–171. J. Vasir, V. Labhasetwar. Targeted drug delivery in cancer therapy, Technol. Cancer Res. Treatment. 2005, 4, 363–374. A. Chonn, S. Semple, P. Cullis. Separation of large unilamellar liposomes from blood components by a spin column procedure: Towards identifying plasma proteins which mediate liposome clearance in vivo, Biochim. Biophys. Acta. 1991, 1070, 215–222. F. Yuan. Transvascular drug delivery in solid tumors, Semin. Radiat. Oncol. 1998, 8, 164–175. Y. Noguchi, J. Wu, R. Duncan, J. Strohalm, K. Ulbrich, T. Akaike, H. Maeda. Early phase tumor accumulation of macromolecules: A great difference in clearance rate between tumor and normal tissues, Jpn. J. Cancer Res. 1998, 89, 307–314. R. Weissleder, H. Cheng, A. Bogdanova, A. Bogdanov. Magnetically labeled cells can be detected by MR imaging, J. Magn. Reson. Imag. 1997, 7, 258–263. T. Yeh, W. Zhang, S. Ildstad, C. Ho. Intracellular labeling of T-cells with superparamagnetic contrast agents, Magn. Reson. Med. 1993, 5, 617–625. U. Schoepf, E. Marecos, R. Melder, R. Jain, R. Weissleder. Intracellular magnetic labeling of lymphocytes for in vivo trafficking studies,

References

100

101

102

103

104

105

106

107

108

109

Biotechniques. 1998, 24(4), 642–646, 648–651. R. Weissleder, D. Stark, B. Engelstad, B. Bacon, C. Compton, D. White, P. Jacobs, J. Lewis. Superparamagnetic iron oxide: Pharmacokinetics and toxicity, Am. J. Roentgenol. 1989, 152, 167–173. P. Reimer, E. Rummeny, H. Daldrup, T. Balzer, B. Tomach, T. Berns, P. Peters. Clinical results with Resovist: A phase 2 clinical trial, Radiology. 1995, 195, 489–496. P. Robinson. Imaging liver metastases: Current limitations and future prospects, Br. J. Radiol. 2000, 73, 234–241. Y. Nishioka, H. Yoshino. Lymphatic targeting with nanoparticulate system, Adv. Drug Delivery Rev. 2001, 47, 55–64. K. Yang, Y. Wen, L. Li, C. Wang, S. Hou, C. Li. Preparation of cucurbitacinBE polylactic acid nanoparticles for targeting cervical lymph nodes, Hua Xi Kou Qiang Yi Xue Za Zhi. 2001, 19, 347–350. T. Watanabe, H. Ichikawa, M. Fukumori. Tumor accumulation of gadolinium in lipid nanoparticles intravenously injected for neutron capture therapy in cancer, Eur. J. Pharmaceut. Biopharmaceut. 2002, 54, 119–124. N. Nishiyama, S. Okazaki, H. Cabral, M. Miyamoto, Y. Kato, Y. Sugiyama, K. Nishio, Y. Matsumura, K. Kataoka. Novel cisplatinincorporated polymeric micelles can eradicate solid tumors in mice, Cancer Res. 2003, 63, 8977–8983. R. Abou-Jawde, T. Choueiri, C. Alemany, T. Mekhail, An overview of targeted treatments in cancer, Clin. Therapeut. 2005, 25, 2121– 2137. A. Gupta, A. Curtis. Lactoferrin and ceruloplsmin derivatized superparamagnetic iron oxide nanoparticles for targeting cell surface receptors, Biomaterials. 2004, 25, 3029–3040. A. Gupta, M. Gupta. Synthesis and surface engineering of ironoxide nanoparticles for biomedical

110

111

112

113

114

115

116

117

118

119

applications, Biomaterials. 2005, 26, 3995–4021. A. Gupta, C. Berry, M. Gupta, A. Curtis. Receptor mediated targeting of magnetic nanoparticles using insulin as a surface ligand to prevent endocytosis, IEEE Trans. Nanobiosci. 2003, 2, 256–261. Z. Qian, H. Li, H. Sun, K. Ho. Targeted drug delivery via transferring receptor mediated endocytosis pathway, Pharmacol. Rev. 2002, 54, 561–587. A. Moore, J. Basilion, E. Chiocca, R. Weissleder. Measuring transferring receptor gene expression by NMR imaging, Biochim. Biophys. Acta. 1998, 1402, 239–249. A. Moore, L. Josephson, R. Bhoreade, J. Basilion, R. Weissleder. Human transferring receptor gene as a marker gene for MR imaging, Radiology. 2001, 221, 244–250. C. Berry, S. Charles, S. Wells, M. Da.by, A. Curtis. The influence of transferring stabilized magnetic nanoparticles on human dermal fibroblasts in culture, Int. J. Pharm. 2004, 269, 211. D. Hogemann, L. Josephson, R. Weissleder, J. Basilion. Improvement of MRI probes to allow efficient detection of gene expression, Bioconjugate Chem. 2000, 11(6), 941–946. H. Choi, S. Choi, R. Zhou, H. Kung, I. Chen. Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery, Acad. Radiol. 2004, 11, 996–1004. Y. Zhang, N. Kohler, M. Zhang. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake, Biomaterials. 2002, 23, 1553–1561. N. Tanji, K. Aoki, M. Yokoyama. Growth factors: Roles in andrology, Arch. Androl. 2001, 47, 1–7. M. Boyle, M. Lawman, A. Gee, M. Young. Nerve growth factor: A chemotactic factor for polymorphonuclear leukocytes in vivo, J. Immunol. 1985, 134, 564–568.

219

220

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 120 M. Funovics, B. Kapeller, C.

121

122

123

124

125

126

127

Hoeller, H. Su, R. Kunstfeld, S. Puig, K. Macfelda. MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents, Magn. Reson. Imag. 2004, 22, 843-M. A. Nagy, A. Schally. Targeting cytotoxic conjugates of somatostatin, luteinizing hormone releasing hormone and bombesin to cancers expressing their receptors: A smarter chemotherapy, Curr. Pharmaceut. Design. 2005, 11, 1167–1180. M. Kircher, U. Mahmood, R. King, R. Weissleder, L. Josephson. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation, Cancer Res. 2003, 63, 8122–8125. L. Josephson, C. Tung, A. Moore, R. Weissleder. High efficiency intracellular magnetic labeling with novel superparamagnetic-tat peptide conjugates, Bioconjugate Chem. 1999, 10, 186–191. C. Dodd, H. Hsu, W. Chu, P. Yang, H. Zhang, J. Mountz, K. Zinn, J. Forder, L. Josephson, R. Weissleder, J. Mountz, J. Mountz. Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivatorpeptide-derived superparamagnetic nanoparticles, J. Immunol. Methods. 2001, 256, 89–105. A. Schally, A. Nagy. Cancer chemotherapy based on targeting of cytotoxic peptide conjugates to their receptors on tumors, Eur. J. Endocrinol. 1999, 141(1), 1–14. A. Nagy, A. Schally. Targeting cytotoxic luteinizing hormone releasing hormone analogs to breast, ovarian, endometrial and prostate cancers, Biol. Reprod. 2005, 73, 851–859. L. Chatzistamou, A. Schally, A. Nagy, K. Szepeshazi, G. Halmos. Effective treatment of metastatic MDA-MB 435 human estrogen independent breast carcinomas with a targeted cytotoxic analogue of

128

129

130

131

132

133

134

135

136

luteinizing hormone releasing hormone AN-207, Clin. Cancer Res. 2000, 6, 4158–4168. M. Kovacs, A. Schally, B. Csernus, R. Busto, Z. Rekasi, A. Nagy. Targeted cytotoxic analogue of LHRH only transiently decreases the gene expression of pituitary receptors for LHRH, J. Neuroendocrinol. 2002, 14, 5–13. M. Kovacs, A. Schally, A. Nagy, M. Koppan, K. Groot. Recovery of pituitary function after treatment with a targeted cytotoxic analog of LHRH, Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1420–1425. C. Leuschner, F. Enright, B. Gawronska-Kozak, W. Hansel. Human prostate cancer cells and xenografts are targeted and destroyed through LHRH receptors, Prostate. 2003, 56, 239–249. P. Foster-Gareau, C. Heyn, A. Alejski, B.K. Rutt. Imaging single mammalian cells with a 1.5 T clinical MRI scanner, Magn. Reson. Med., 2003, 49, 968–971. K.A. Hinds, J.M. Hill, E.M. Shapiro, M.O. Laukkanen, A.C. Silva, C.A. Combs, T.R. Varney, R.S. Balaban, A.P. Koretsky, C.E. Dunbar. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells, Blood 2003, 102, 867–872. A. Merbach, E. Toth. The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, 2001, Wiley, Chichester. C. Simonsen, L. Ostergaard, P. Vestergaard-Poulsen, L. Rohl, A. Bjornerud, C. Gyldensted. CBF and CBV measurements by USPIO bolus tracking: Reproducibility and comparison with Gd based values, J. Magn. Reson. Imag. 1999, 9, 342–347. R. Low. MR imaging in liver using gadolinium chelates, Magn. Reson. Imag. Clin. N. Am. 2001, 9, 372–374. C. Jung, P. Jacobs. Physical and chemical properties of superparamagnetic iron oxide MR contrast

References

137

138

139

140

141

142

143

144

agents: ferrumoxides, ferumoxtran, ferumoxsil, Magn. Reson. Imag. 1995, 13, 661–674. S. Flacke, S. Fischer, M. Scott, R. Fuhrhop, J. Allen, M. McLean, P. Winter, G. Sicard, P. Gaffney, S. Wickline, G. Lanza. Novel MRI contrast agent for molecular imaging of fibrin: Implications for detecting vulnerable plaques, Circulation. 2001, 104, 1280–1285. T. Suwa, S. Ozawa, M. Ueda, N. Ando, M. Kitajima. Magnetic resonance imaging of esophageal squamous cell carcinoma using magnetite particles coated with antiepidermal growth factor receptor antibody, Int. J. Cancer. 1998, 75, 626–634. Y. Wang, S. Hussain, G. Kresting. Superparamagnetic iron oxide contrast agents: Physicochemical characteristics and applications in MR imaging, Eur. Radiol. 2001, 11, 2319–2331. D. Pouliquen, R. Perdrisot, A. Ermias, S. Akoka, P. Jallet, J. Le Jeune. Superparamagnetic iron oxide nanoparticles as a liver MRI contrast agent: Contribution of microencapsulation to improved biodistribution, Magn. Reson. Imag. 1989, 7, 619–627. R. Weissleder, A. Lee, B. Khaw, T. Shen, T. Brady. Antimyosin labeled monocrystalline iron oxide allows detection of myocardial infarct: MR antibody imaging, Radiology. 1992, 182, 281–285. R. Weissleder, A. Lee, A. Fischman, P. Reimer, T. Shen, R. Wilkinson, R. Callahan, T. Brady. Polyclonal human immunoglobulin G labeled with polymeric iron oxide: Antibody MR imaging, Radiology. 1991, 181, 245–249. P. Moroz, H. Pardoe, S. Jones, T. St Pierre, S. Song, B. Gray. Arterial embolization hyperthermia: Hepatic iron particle distribution and its potential determination by magnetic resonance imaging, Phys. Med. Biol. 2002, 47, 1591–1602. H. Pardoe, W. Chua-anusorn, T. St Pierre, J. Dobsen. Detection

145

146

147

148

149

150

151

152

limits for ferromagnetic particle concentrations using magnetic resonance imaging based proton transverse relaxation rate measurements, Phys. Med. Biol. 2003, 48, N89–N95. S. Schmitz, S. Coupland, R. Gust. Superparamagnetic iron oxide enhanced MRI of artherosclerotic plaques in Watanabe rabbits, Invest. Radiol. 2000, 35, 460–471. A. Luebbe, C. Bergeman, H. Riess, F. Schriever, P. Reichardt, K. Possinger, M. Matthia, B. Doerken, F. Herrmann, R. Guertler, P. Hohenberger, N. Haas, R. Sohr, B. Sander, A. Lemke, D. Ohlendorf, W. Huhnt, D. Huhn. Clinical experiences with magnetic drug targeting: A Phase I study with 4epidoxorubicin in 14 patients with advanced solid tumors, Cancer Res. 1996, 56, 4686–4693. B. Bacon, D. Stark, C. Park, S. Saini, E. Groman, P. Hahn, C. Compton, J. Ferrucci. Ferrite particles: A new magnetic resonance imaging contrast agent. Lack of acute or chronic hepatotoxicity after intravenous administration, J. Lab. Clin. Med. 1987, 110, 164–171. D. Schafer. Coupling dynamics and membrane dynamics during endocytosis, Curr. Opin. Cell Biol. 2002, 14, 76–81. A. Moore, R. Weissleder, A. Bogdanov. Uptake of dextrancoated monocrystalline iron oxides in tumor cells and macrophages, J. Magn. Reson. Imag. 1997, 7, 1140– 1145. A. Moore, E. Marecos, A. Bogdanov, R. Weissleder. Tumoral distribution of long-circulating dextran coated iron oxide nanoparticles in a rodent model, Radiology. 2000, 214, 568–574. E. Shapiro, S. Skrtic, K. Sharer, J. Hill, C. Dunbar, A. Koretsky. MRI detection of single particles for cellular imaging, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10 901–10 906. C. Billotey, C. Wilhelm, M. Devaud, J. Bacri, J. Bittoun, F. Gazeau. Cell internalization of anionic maghemite nanoparticles: Quantitative effect on

221

222

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

153

154

155

156

157

158

159

160

magnetic resonance imaging, Magn. Reson. Med. 2003, 49, 646–654. J. Bulte, I. Duncan, J. Fank. In vivo tracking of magnetically labeled cells following transplantation, J. Cereb. Blood Flow Metab. 2002, 22, 899–907. D. Sipkins. Detection of tumor angiogenesis in vivo by aVb3-targeted magnetic resonance imaging, Nat. Med. 1998, 4, 623–626. P. Winter. Nanotechnology in cancer drug delivery, in Nanofabrication for Biomedical Applications, J. Hormes, C. Kumar, C. Leuschner (Eds.), WileyVCH, Weinheim, 2004. P. Winter, S. Caruthers, A. Kassner, T. Haris, L. Chinen, J. Allen, E. Lacy, H. Zhang, J. Robertson, S. Wickline, G. Lanza. Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel alpha (nu)beta3-targeted nanoparticle and 1.5 tesla magnetic resonance imaging, Cancer Res. 2003, 263, 5838–5843. E. Bergey, L. Levy, X. Wang, L. Krebs, M. Lal, K. Kim, S. Pakatchi, C. Liebow, P. Prasad. DC Magnetic field induced magnetocytolysis of cancer cells targeted by LH-RH magnetic nanoparticles in vitro, Biomed. Microdevices. 2002, 4, 293–299. M. Funovics, B. Kapeller, C. Hoeller, H. Su, R. Kunstfeld, S. Puig, K. Macfelda. MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents, Magn. Reson. Imaging. 2004, 22, 843-M. S. Dharap, Y. Wang, J. Khandare, B. Qiu, S. Gunaseelan, P. Sinko, S. Stein, A. Farmanfarmaian, T. Minko. Tumor specific targeting of an anticancer drug delivery system by LHRH peptide, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12 962–12 967. W. Yang, M. Wieczorck, M. Allen, T. Nett. Cytotoxic activity of gonadotropin releasing hormone Pokeweed antiviral protein conjugates in cell lines expressing GnRH receptors, Endocrinology. 2003, 144, 1456– 1463.

161 C. Leuschner, F. Enright, B.

162

163

164

165

166

167

168

169

170

Gawronska, J. Keener, W. Hansel. Lytic peptide conjugated to luteinizing hormone kills breast cancer cells in vivo and in vitro. AACR-NCI-EORTC International conference, Molecular Targets and Cancer Therapeutics. 2001, Miami, FL, Abstract 489. C. Leuschner, F. Enright, P. Melrose, W. Hansel. Targeted destruction of androgen-sensitive and insensitive prostate cancer cells and xenografts throughluteinizing hormone receptors, Prostate. 2001, 46, 116–125. B. Gawronska, C. Leuschner, F. Enright, W. Hansel. Effects of a lytic peptide conjugated to beta hCG on ovarian cancer studies in vitro and in vivo, Gynecol. Oncol. 2002, 85, 45–52. C. Leuschner, F. Enright, B. Gawronska, W. Hansel. Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo, Breast Cancer Res. Treatment. 2003, 78, 17–27. G. Fink. Gonadotropin secretion and its control, in The Physiology of Reproduction, E. Knobil, J. Neill (Eds.), Raven Press, New York, 1988, 1349–1377. S. Kalra. Mandatory neuropeptide steroid signaling for the preovulatory luteinizing hormone releasing hormone discharge, Endocrine Rev. 1993, 14, 507–538. A. Schally. Hypothalamic hormones from neuroendocrinology to cancer therapy, Anticancer Drugs. 1994, 5, 115–130. S. Stojilkovis, K. Catt. Expression and signal transduction pathways of gonadotropin releasing hormone receptors, Recent Progr. Hormone Res. 1995, 30, 161–205. A. Schally, A. Comaru Schally, A. Nagy, M. Kovacs, K. Szepashizai, A. Plonowski, J. Varga, G. Halmos. Hypothalamic hormones and cancer, Frontiers Neuroendocrinol. 2001, 22, 248–291. R. Millar, A. Lu, A. Pawson, C. Flanagan, K. Morgan, S. Maudsley. Gonadotropin releasing hormone

References

171

172

173

174

175

176

177

178

179

receptors, Endocrine Rev. 2004, 25, 235–275. P. Limonta, R. Moretti, M. Marelli, M. Motta. The biology of gonadotropin hormone releasing hormone: Role in the control of tumor growth and progression in humans, Fontiers Neuroendocrinol. 2003, 24, 279–295. R. Moretti, M. Montagnani, J. von Groeninghen, P. Limonta. Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells, J. Clin. Endocrinol. Metab. 2002, 87, 3791–3797. R.M. Moretti, M.M. Marelli, J.C. van Groeninghen, M. Motta, P. Limonta. Inhibitory activity of luteinizing hormone releasing hormone on tumor growth and progression. Endocrine Relat. Cancers, 2003, 10, 161–167. H. Matsuo, Y. Baba, R. Nair, A. Arimura, A. Schally. Structure of the porcine LH and FSH releasing hormone. I. The proposed amino acid sequence, Biochem. Biophys. Res. Commun. 1971, 43, 1334–1339. J. King, R. Millar. Structural and functional evolution of gonadotropin releasing hormone, Int. Rev. Cytol. 1987, 106, 149–182. J. King, R. Millar. Evolutionary aspects of gonadotropin releasing hormone and its receptor, Cell Mol. Neurobiol. 1995, 15, 5–23. R. Millar, B. Troskie, Y. Sun, T. Ott, I. Wakefield, D. Myvurth, A. Pawson, J. Davidson, A. Katz, J. Hapgood, N. Illing, W. Heinstein, S. Sealfon, R. Peter, E. Terasawa, J. King. Plasticity in the structural and functional evolution of GnRH: A peptide for all seasons. Advances in comparative endocrinology, Proc. XIII International Congress of Comparative Endocrinology. Yokohama, Japan, 1997, pp. 15–27. N. Sherwood. The GnRH family of peptides, Trends Neurosci. 1987, 10, 129–132. M. Jiminez-Linan, B. Rubin, J. King. Examination of guinea pig luteinizing hormone releasing hormone gene

180

181

182

183

184

185

186

187

reveals a unique decapeptide and existence of two transcripts in the brain, Endocrinology. 1997, 138, 423–430. M. Yoo, H. Kang, H. Choi, J. Kim, B. Troskie, R. Millar, H. Kwon. Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp8]GnRH) in frog brain, Mol. Cell Endocrinol. 2000, 164, 197–204. B. Adams, J. Tello, J. Erchegeyi, C. Warby, D. Hong, K. Akinsanya, G. Mackie, W. Vale, J. Rivier, N. Sherwood. Six novel gonadotropin releasing hormones are encoded as triplets on each of two genes in a protochrodate, Ciona interstinalis. Endocrinology. 2003, 144, 1907–1919. E. Dubois, M. Zandbergen, J. Peute, H. Goos. Evolutionary development of three gonadotropin releasing hormone systems in vertebrates, Brain Res. Bull. 2002, 57, 413–418. W. Yu, S. Karanth, S. Sower, A. Parlow, S. McCann. The similarity of FSH releasing factor to lamprey gonadotropin releasing hormone III (GnRH III), Proc. Soc. Exp. Biol. Med. 2002, 224, 87–92. K. Miyamoto, Y. Hasegawa, M. Nomura, M. Igarashi, K. Kangawa, H. Matsue. Identification of the second gonadotropin releasing hormone in chicken hypothalamus evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin releasing hormones in avian species, Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 3874–3878. S. Sower, Y. Chiang, S. Lovas, J. Conlon. Primary structure and biological activity of a third gonadotropin releasing hormone from the lamprey brain, Endocrinology. 1993, 132, 1125–1131. R. White, J. Eisen, T. Kasten, R. Fernald. Second gene for gonadotropin releasing hormone in humans, Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 305–309. S. Kang, C. Tai, P. Nathwani, P. Leung. Differential regulation of two forms of GnRH messenger RNA in

223

224

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

188

189

190

191

192

193

194

195

196

197

human granulose luteal cells, Endocrinology. 2001, 142, 182–192. K. Choi, N. Auersperg, P. Lleung. Expression and antiproliferative effect of a second form of gonadotropin releasing hormone in normal and neoplastic ovarian surface epithelial cells, J. Clin. Endocrinol. Metab. 2001, 86, 5075–5078. S. Khosravi, P. Leung. Differential regulation of gonadotropin releasing hormone (GnRH I) and GnRH II messenger ribonucleic acid by gonadal steroids in humban granulose luteal cells, J. Clin. Endocrinol. Metab. 2003, 88, 663–372. G. Emons, A. Schally. The use of luteinizing hormone releasing hormone agonists and antagonists in gynaecological cancers, Hum. Reprod. 1994, 9, 1364–1379. S. Sealfon, H. Weinstein, R. Millar. Molecular mechanisms of ligand interaction with the gonadotropin releasing hormone receptor, Endocrinol. Rev. 1997, 18, 180–205. A. Hsueh, J. Schaeffer. Gonadotropin releasing hormone as a paracrine hormone and neutrotransmitter in extra-pituitary sites, J. Steroid Biochem. 1985, 23, 757–764. R. Millar. GnRH II and type II GnRH receptors, Trends Endocrinol. Metab. 2003, 14, 35–43. J. Temple, R. Millar, E. Rissman. An evolutionarily conserved form of gonadotropin-releasing hormone coordinates energy and reproductive behavior, Endocrinology. 2003, 144(1), 13–19. G. Emons, O. Ortmann, K. Schulz, A. Schally. Growth inhibitory actions of lHRH on tumor cells. Trends Endocrinol. Metab. 1997, 8, 355–362. G. Emons, S. Weis, O. Ortmann, C. Grundker, K. Schulz. LHRH might act as a negative autocrine regulator of proliferation of human ovarian cancer, Eur. J. Endocrinol. 2000, 142, 665–670. D. Dondi, P. Limonta, R. Motetti, M. Marelli, E. Garattini, M. Motta. Antiproliferative effects of LHRH agonists on human androgen

198

199

200

201

202

203

204

205

independent prostate cancer cell line DU145: Evidence for an autocrine inhibitory LHRH loop, Cancer Res. 1994, 54, 4091–4095. R.M. Moretti, M. Montagnani Marelli, J.C. van Grohnignhen, M. Motta, P. Limonta. Inhibitory activity of luteinizing hormone releasing hormone on tumor growth and progression, Endocrine Relat. Cancer. 2003, 10, 161–167. W. Miller, W. Scott, R. Morris, H. Fraser, R. Sharp. Growth of human breast cancer cells inhibited by a luteinizing hormone releasing hormone agonist, Nature. 1985, 313, 231–233. K. Eidne, C. Flanagan, R. Millar. Gonadotropin releasing hormone binding sites in human breast carcinoma, Science. 1985, 229, 989–999. D. Dondi, P. Limonta, R. Moretti, M. Montagnani Motta. Antiproliferative effects of lutei rele agonists on human androgen independent prostate cancer cell line DU 145: Evidence for an autocrineinhibitory loop, Cancer Res. 1994, 54, 4091–4095. G. Emons, S. Weiss, O. Ortmann. LHRH might act as a negative autocrine regulator of proliferation of human ovarian cancer, Eur. J. Endocrinol. 2000, 142, 665–670. A. Tieva, P. Stattin, P. Wikstrom, A. Bergh, J. Damber. Gonadotropinreleasing hormone receptor expression in the human prostate, Prostate. 2001, 47(4), 276–284. G. Emons, O. Ortmann, M. Becker, G. Irmer, B. Springer, R. Laun, F. Holzel, K. Schulz, A. Schally, High affinity binding and direct antiproliferative effects of LHRH analogs I human ovarian cancer cell lines, Cancer Res. 1993, 53, 5439–5446. A. Gunthert, C. Grundker, B. Bottcher, G. Emons. Luteinizing hormone-releasing hormone (LHRH) inhibits apoptosis induced by cytotoxic agent and UV-light but not apoptosis mediated through CD95 in human ovarian and endometrial cancer cells,

References

206

207

208

209

210

211

212

213

Anticancer Res. 2004, 24(3a), 1727–1732. C. Grundker, L. Schlotawa, V. Viereck, N. Eicke, A. Horst, B. Kairies, G. Emons. Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor, Eur. J. Endocrinol. 2004, 151, 141–149. C. Grundker, G. Emons. Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer, Reprod. Biol. Endocrinol. 2003, 7, 1, 65. C. Verschraegen, S. Westphalen, W. Hu, E. Loyer, A. Kudelka, P. Volker, J. Kavanagh, M. Steger, K. Schulz, G. Emons. Phase II study of cetrorelix, a luteinizing hormonereleasing hormone antagonist in patients with platinum-resistant ovarian cancer, Gynecol. Oncol. 2003, 90(3), 552–559. O. Reich, F. Nogales, S. Regauer. Gonadotropin-releasing hormone receptor expression in endometrial stromal sarcomas: An immunohistochemical study, Mod. Pathol. 2005, 18(4), 573–576. R. Eaveri, A. Ben-Yehudah, H. Lorberboum-Galski. Surface antigens/ receptors for targeted cancer treatment: The GnRH receptor/binding site for targeted adenocarcinoma therapy, Curr. Cancer Drug Targets. 2004, 4(8), 673–687. J. Reubi. Peptide receptors as molecular targets for cancer diagnosis and therapy, Endocrine Rev. 2003, 24, 389–427. R. Millar, S. Lowe, D. Conklin, A. Pawson, S. Maudsley, B. Troskie, T. Ott, M. Millar, G. Lincoln, R. Sellar, B. Faurholm, G. Scobie, R. Kuestner, E. Terasawa. A novel mammalian receptor for the evolutionarily conserved type II GnRH, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9636–9641. L. Chi, W. Zhou, A. Prikhozan, C. Flanagan, K. Davidson. Cloning and characterization of the human gonadotropin releasing hormone

214

215

216

217

218

219

220

221

receptor, Mol. Cell. Endocrinol. 1993, 91, R1–6. S. Kakar, L. Musgrove, D. Devor, J. Sellers, J. Neill. Cloning sequencing and expressison of human GnRH receptor, Biochem. Biophys. Res. Commun. 1992, 189, 289–295. M. Tsutsumi, W. Zhou, R. Miller, P. Mellon, J. Roberts, C. Flanagan, K. Dong, B. Gillo, S. Sealfon. Cloning and functional expression of a mouse gonadotropin releasing hormone receptor. Mol. Endocrinol. 1992, 6, 1163–1169. J. Reinhart, L. Mertz, K. Catt. Molecular cloning and expression of CDNA encoding the murine gonadotropin-releasing hormone receptor, J. Biol. Chem. 1992, 267, 21 281–21 284. U. Kaiser, D. Zhao, G. Cardona, W. Chin. Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor, Biochem. Biophys. Res. Commun. 1992, 189, 1645–1652. K. Eidne, R. Sellar, G. Couper, L. Anderson, P. Taylor. Molecular cloning and characterization of the rat pituitary gonadotropin releasing hormone (GnRH) receptor, Mol. Cell. Endocrinol. 1992, 90, R5–R9. M. Perrin, L. Bilezekjian, C. Hoeger, C. Donaldson, J. Rivier, Y. Haas, W. Vale. Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary tumor cell line, Biochem. Biophys. Res. Commun. 1993, 191, 1139–1144. J. Brooks, P. Taylor, P. Saunders, K. Eidne, W. Struthers, A. McNeilly. Cloning and sequencing of the sheep pituitary gonadotropin releasing hormone receptor and changes in expression of its mRNA during the estrous cycle, Mol. Cell. Endocrinol. 1993, 94, R23–R27. N. Illing, G. Jacobs, I. Becker, C. Flanagan, J. Davidson, R. Millar. Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin releasing hormne receptor reveal differences

225

226

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

222

223

224

225

226

227

228

229

230

in primary structure and ligand specificity amon mammalian receptors. Biochem. Biophys. Res. Commun. 1993, 196, 745–751. S. Kakar, C. Rahe, J. Neill. Molecular cloning sequencing and characterizing the bovine receptor for gonadotropin releasing hormone (GnRH), Domest. Anim. Endocrinol. 1993, 10, 335–342. S. Kakar, W. Grizzle, J. Neill. The nucleotide sequence of human GnRH receptors in breast and ovarian tumors are identical with that found in the pituitary, Mol. Cell Endocrinol. 1994, 106, 145–149. S. Sealfon, R. Millar. Functional domains of the gondotropin releasing hormone receptor, Cell Mol. Neurobiol. 1995, 15, 25–42. S. Sealfon, R. Millar. The gonadotropin releasing hormone receptor: Structural determinations and regulatory control, Human Reprod Update. 1995, 1, 216–230. G. Halmos, A. Nagy, N. Lamharzi, A. Schally. Cytotoxic analogs of luteinizing hormone releasing hormone bind with high affinity to human breast cancers, Cancer Lett. 1999, 136, 129–136. G. Irmer, C. Burger, R. Muller, O. Ortmann, U. Peter, S. Kakar, J. Neill, K. Schulz, G. Emons. Expression of the messenger RNAs for luteinizing hormone-releasing hormone (LHRH) and its receptor in human ovarian epithelial carcinoma, Cancer Res. 1995, 55(4), 817–822. P. Limonta, D. Dondi, R. Moretti, R. Maggi, M. Motta. Antiproliferative effects of luteinizing hormonereleasing hormone agonists on the human prostatic cancer cell line LNCaP, J. Clin. Endocrinol. Metab. 1992, 75(1), 207–212. S. Stojilkovic, J. Reinhart, K. Catt. GnRH receptors: Structure and signal transduction pathways, Endocrine Rev. 1994, 15, 462–499. E. Neer. Heterotrimeric G proteins: Organizers of transmembrane signals, Cell. 1995, 80, 249–257.

231 C. Heldin. Dimerization of cell

232

233

234

235

236

237

238

239

surface receptors in signal transduction, Cell. 1995, 80, 213–223. K. Hsieh, T. Martin. Thyrotropin releasing hormone and gonadotropin releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate binding proteins Gq and G11, Mol. Endocrinol. 1992, 6, 1673–1681. E. Loumaye, K. Catt. Agonist induced regulation of pituitary receptors for gonadotropin releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs. J. Biol. Chem. 1983, 258, 12 002–12 009. M. Vrecl, L. Anderson, A. Hanyaloglu, A. McGregor, A. Groarke, G. Milligan, P. Taylor, K. Eidne. Agonist induced endocytosis and recycling of the gonadotropin releasing hormone receptor: Effect of b-arrestin on internalization kinetics, Mol. Endocrinol. 1998, 12, 1818–1829. S. Kraus, Z. Naor, R. Seger. Intracellular signaling pathways mediated by the gnrh receptor, Arch. Med. Res. 2001, 32, 499–509. Z. Naor, D. Harris, S. Shacham. Mechanism of gnrh receptor signaling: Combinatorial cross talk of ca and protein kinase C. Frontiers Neuroendocrinol. 1998, 19, 1–19. C. Liebow, M. Lee, A. Kamer, A. Schally. Regulation of luteinizing hormone releasing hormone by heterologous and autologous receptor stimulated tyrosine phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2244–2248. C. Grundker, P. Volker, K. Schulz, G. Emons. Luteinizing hormonereleasing hormone agonist triptorelin and antagonist cetrorelix inhibit EGFinduced c-fos expression in human gynecological cancers, Gynecol. Oncol. 2000, 78(2), 194–202. G. Grundker, P. Volker, G. Emons. Antiproliferative signaling of lHRH inhuman endometrial and ovarian cancer cells through G protein alpha(I) mediated activation of phosphotyrosine phosphatase,

References

240

241

242

243

244

245

246

Endocrinology. 2001, 142, 2369– 2380. A. Imai, H. Tkagi, S. Horibe, T. Fuseya, T. Tamaya. Coupling of gonadotropin releasing hormone receptor to Gi protein in human reproductive tract tumors, J. Clin. Endocrinol. Metab. 1996, 81, 3249–3253. M. Marelli, R.M. Moretti, D. Dondi, M. Motta, P. Limonta. Luteinizing hormone releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgenindependent prostate cancer cells. Endocrinology. 1999, 140, 5250– 5256. C. Grundker, K. Schulz, A.R. Gunthert, G. Emons. Luteinizing hormone-releasing hormone induces nuclear factor kappaB-activation and inhibits apoptosis in ovarian cancer cells. J. Clin. Endocrinol. Metab. 2000, 85, 3815–3820. H. Everest, J. Hislp, T. Harding, J. Uney, A. Flynn, R. Millar, C. McArdle. Signalling and antiproliferative effects mediated by GnRH receptors after expression in breast cancer cells using recombinant adenovirus, Endocrinology. 2001, 142, 4663–4672. S. Maudsley, L. Davidson, A. Pawson, R. Chan, R. Lopez de Maturana, R. Millar. Gonadotropin releasing hormone antagonists promote proapoptotic signaling in peripheral reproductive tumor cells by activating a Gai coupling state of the type I GNRH receptor, Cancer Res. 2004, 64, 7533–7544. P. Limonta, R.M. Moretti, M.M. Marelli, M. Motta. The biology of gonadotropin hormone-releasing hormone: Role in the control of tumor growth and progression in humans. Front Neuroendocrinol. 2003, 24, 279–295. C. Grundker, A.R. Gunthert, S. Westphalen, G. Emons. Biology of the gonadotropin-releasing hormone system in gynecological cancers. Eur. J. Endocrinol. 2002, 146, 1–14.

247 G. Emons, V. Muller, O. Ortmann,

248

249

250

251

252

253

254

G. Grossmann, U. Tautner, B. von Stuckrad, K. Schulz, A. Schally. Luteinizing hormone releasing hormone agonist triptorelin antagonizes signal transduction and mitogenic activity of epidermal growth factor in human ovarian and endometrial cancer cell lines, Int. J. Oncol. 1996, 9, 1129–1137. M. Lee, C. Liebow, A. Kramer, A. Schally. Effects of epidermal growth factor and analogues of luteinizing hormone releasing hormone and somatostatin on phosphorylation of tyrosine residues of specific substrates in various tumors, Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1656–1660. L. Krebs, X. Wang, H. Pudavar, E. Bergey, A. Schally, A. Nagy, P. Prasad, C. Liebow. Regulation of targeted chemotherapy with cytotoxic lutenizing hormone-releasing hormone analogue by epidermal growth factor, Cancer Res. 2000, 60(15), 4194–4199. A. Nagy, A.V. Schally. Cytotoxic analogs of luteinizing hormone releasing hormone (LHRH); a new approach to targeted chemotherapy. Drug Future 2002, 27, 389–370. J.M. Arencibia, A.V. Schally, M. Krupa, A.M. Bajo, A. Nagy, K. Szepeshazi, A. Plonowski. Targeting of doxorubicin to ES-2 human ovarian cancers in nude mice by linking to an analog of luteinizing hormonereleasing hormone improves its effectiveness. Int. J. Oncol. 2001, 19, 571–577. G. Emons, C. Grundker, A.R. Gunthert, S. Westphalen, J. Kavanagh, C. Verschraegen. GnRH antagonists in the treatment of gynecological and breast cancers. Endocrine Relat. Cancer, 2003, 10, 291–299. C. Grundker, A. Huschmand Nia, G. Emons. Gonadotropin-releasing hormone receptor-targeted gene therapy of gynecologic cancers. Mol. Cancer Ther. 2005, 4, 225–231. K. Arora, A. Sakai, K. Catt. Effects of second intracellular loop mutations

227

228

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers

255

256

257

258

259

260

261

262

on signal transduction and internalization of the gonadotropin releasing hormone receptor, J. Biol. Chem. 1995, 270, 22 820–22 826. D. Nussenveig, M. Heinflink, M. Gershengorn. Agonist-stimulated internalization of the thyrotropinreleasing hormone receptor is dependent on two domains in the receptor carboxyl terminus, J. Biol. Chem. 1993, 268, 2389–2392. A. Pawson, A. Katz, Y. Sun, J. Lopes, N. Illing, R. Millar, J. Davidson. Contrasting internalization kinetics of human and chicken gonadotropin releasing hormone receptors mediated by c-terminal tail, J. Endocinol. 1998, 156, R9–R12. C. McArdle, J. Franklin, L. Green, J. Hislop. Signaling cycling and desensitization of gnrh receptors, J. Endocrinol. 2002, 173, 1–11. A. Heding, M. Vreel, J. Bogerd, A. McGregor, R. Sellar, P. Taylor, K. Eidne. GnRH receptors with intracellular carboxyl terminal tails undergo acute desentitization of total inosito phosphate production and exhibit accelerated internalization kinetics, J. Biol. Chem. 1998, 273, 11 472–11 477. L. Hunyady, A. Baukal, T. Balla, K. Catt, Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction, J. Biol. Chem. 1994, 269, 24 798–24 804. R. Benya, Z. Fathi, J. Battey, R. Jensen. Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization, J. Biol. Chem. 1993, 268, 20 285–20 290. R.T. Jensen, Z. Fathi, J. Battey, R. Jensen. Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization. J. Biol. Chem. 1993, 268, 20 285–20 290. R. Millar, S. Lowe, D. Conklin, A. Pawson, S. Maudsley, B. Troskie, T. Ott, M. Millar, G. Lincoln, R. Sellar, B. Faurholm, G. Scobie, R. Kuestner, E. Terasawa, A. Katz. A

263

264

265

266

267

268

269

novel mammalian receptor for the evolutionarily conserved type II GnRH, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9636–9641. J. Neill, L. Duck, J. Sellers, L. Musgrove. A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates, Biochem. Biophys. Res. Commun. 2001, 282, 1012–1018. J. Koenig, J. Edwardson. Endocytosis and recycling of G protein coupled receptors, Trends Pharmacol. Sci. 1997, 18, 276–287. J. Hislop, H. Everest, A. Flynn, T. Harding, J. Uney, B. Troskie, R. Millar, C. McArdle. Differential internalization of mammalian and non-mammalian gonadotropin releasing hormone receptors: Uncoupling of dynamin dependent internalization from mitogen activated protein kinase signaling, J. Biol. Chem. 2001, 276, 39 685–39 694. C. Grundker, A. Gunthert, R. Millar, G. Emons. Expression of GnRH II receptor in human endometrial and ovarian cancer cells and effects of GnRH II on tumor cell proliferation, J. Clin. Endocrinol. Metab. 2002, 87, 1427–1430. C. Grundker, A.R. Gunthert, M. Hellriegel, G. Emons. Gonadotropin-releasing hormone (GnRH) agonist triptorelin inhibits estradiol-induced serum response element (SRE) activation and c-fos expression in human endometrial, ovarian and breast cancer cells. Eur. J. Endocrinol. 2004, 151, 619–628. K. Maiti, Y. Oh, J. Moon. Differential effects of gonadotropin releasing hormone I and II on prostate cancer cell signaling and cell death, J. Clin. Endocrinol. Metab. 2005, 90, 4287– 4298. N. Illing, B. Troskie, C. Nahornia, J. Hapgood, R. Peter, R. Millar. Two gonadotropin releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus), Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 2526–2531.

References 270 L. Wang, J. Bogerd, H. Choi, J.

271

272

273

274

275

276

277

278 279

Seong, J. Soh, S. Chun, M. Blomenrohr, B. Troskie, R. Millar, W. Yu, S. McCann, H. Kwon. Three distinct types of GnRH receptor characterized in the bull frog, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 361–366. A. Wang, J. Li, K. Maiti, W. Kim, H. Kang, J. Seong, H. Kwon. Preferential ligand selectivity of the monkey type II gonadotropin releasing hormone receptor for GnRH 2 and its analogs, Mol. Cell Endocrinol. 2003, 209, 33–42. G. Emons, O. Ortmann, K.D. Schulz, A.V. Schally. Growth inhibitory actions of LHRH on tumor cells, Trends Endocrinol. Metab. 1997, 8, 355–362. P. Leung, C. Cheng, X. Zhu. Multifactorial role of GnRH I and GnRH II in the human ovary, Mol. Cell Endocrinol. 2003, 202, 145– 153. S.-F. Cheng, L.-K. Chau. Colloidal gold-modified optical fiber for chemical and biochemical sensing. Anal. Chem. 2003, 75, 16. B. Faurholm, R. Millar, A. Katz. The genes encoding the type II gonadotropin releasing hormone receptor and the ribonucleoprotein RBM8A in human overlap in two genomic loci, Genomics. 2001, 78, 15–18. J. Neill. GnRH and GnRH receptor genes in the human genome, Endocrinology. 2002, 143, 737–743. A. Pawson, K. Morgan, S. Maudsley, R. Millar. Type II gonadotrophinreleasing hormone (GnRH-II) in reproductive biology, Reproduction. 2003, 126(3), 271–278. S. Acharjee, K. Maiti. Cells. 2002, 14, 101–107. C. Grundker, P. Volker, F. Griesinger, A. Ramaswamy, A. Nagy, A. Schally, G. Emons. Antitumor effects of the cytotoxic luteinizing hormone-releasing hormone analog AN-152 on human endometrial and ovarian cancers xenografted into nude mice, Am. J. Obstet. Gynecol. 2002, 187, 528–537.

280 U. Kaiser, P. Conn, W. Chin.

281

282

283

284

285

286

287

288

Studies of gonadotropon releasing hormone (GnRH) action using GnRH receptor expressing pituitary cell lines, Endocrinol. Rev. 1997, 18, 46–70. R. Moretti, M. Marelli, D. Dondi. LHRH agonists interfere with the stimularory actions of egf in human prostate cancer cell lines LNCaP and DU 145, J. Clin. Endocrinol. Metab. 1996, 81, 3930–3937. M. Hiraoka, S. Jo, K. Akuta, Y. Nishimura, M. Takahashi, M. Abe. Radiofrequency capacitive hyperthermia for deep-seated tumors. II. Effects of thermoradiotherapy. Cancer. 1987, 60, 128–135. R. Cavaliere, E.C. Ciocatto, B.C. Giovanella, C. Heidelberger, R.O. Johnson, M. Margottini, B. Mondovi, G. Moricca, A. RossiFanelli. Selective heat sensitivity of cancer cells. Biochemical and clinical studies. Cancer. 1967, 20, 1351–1381. P. Wust, B. Hildebrandt, G. Sreenivasa, B. Rau, J. Gellermann, H. Riess, R. Felix, P.M. Schlag. Hyperthermia in combined treatment of cancer. Lancet Oncol. 2002, 3, 487–497. A. Jordan, R. Scholz, P. Wust, H. Faehling, R. Felix. Magnetic fluid hyperthermia (MFH): Cancer treatment with AC magnetic field induced excitation of biocompatible superparamagnetic nanoparticles. J. Magn. Magn. Mater. 1999, 201, 413–419. I. Hilger, R. Hergt, W.A. Kaiser. Effects of magnetic thermoablation in muscle tissue using iron oxide particles: An in vitro study. Invest. Radiol. 2000, 35, 170–179. M. Shinkai, M. Matsui, T. Kobayashi. Heat properties of magnetoliposomes for local hyperthermia. Jpn. J. Hyperthermic Oncol. 1994, 10, 168– 177. R. Hiergeist, W. Andrae, N. Buske, R. Hergt, I. Hilger, U. Richter, W. Kaiser. Application of magnetite ferrofluids for hyperthermia. J. Magn. Magn. Mater. 1999, 201, 420–422.

229

230

6 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 289 W. Schutt, C. Gruttner, U. Hafeli,

290

291

292

293

294

295

296

297

M. Zborowski, J. Teller, H. Putzar, C. Schumichen. Applications of magnetic targeting in diagnosis and therapy – possibilities and limitations: A mini-review. Hybridoma. 1997, 16, 109. I. Hilger, K. Fruhauf, W. Andra, R. Hiergeist, R. Hergt, W.A. Kaiser. Heating potential of iron oxides for therapeutic purposes in interventional radiology. Acad. Radiol. 2002, 9, 198–202. R.E. Rosenzweig. Heating magnetic fluid with alternating magnetic field. J. Magn. Magn. Mater. 2002, 252, 370–374. A.V. Brusentsov, V.V. Gogosov, T.N. Buntsova, A.V. Sergeev, N.Y. Jurchenko, A. Kuznetsow, O. Kuznetsow, L.I. Shumakov. Evaluation of ferromagnetic fluids and suspensions for the site-specific radiofrequency-induced hyperthermia of MX11 sarcoma cells in vitro. J. Magn. Magn. Mater. 2001, 225, 113–119. A. Jordan, R. Scholz, P. Wust, H. Fahling, J. Krause, W. Wlodarczyk, B. Sander, T. Vogl, R. Felix. Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. Int. J. Hyperthermia 1997, 13, 587–605. A. Halbreich, J. Rodger, J.N. Pons, D. Geldwerth, M.F. Da Silva, M. Roudier, J.C. Bacri, Biochimie 1998, 80, 379. W. Hansel, C. Leuschner, F. Enright. Targeted destruction of prostate cancers and their metastases by lytic peptide conjugates. Mol. Cell Endocrinol, 2005, in the press. J. Zhou, C. Leuschner, C. Kumar, J. Hormes, W. Soboyejo: Subcellular accumulation of magnetic nanoparticles in breast tumors and metastases. Biomaterials, 2006, 27, 2001–2008. C. Leuschner, C. Kumar, W. Hansel, J. Zhou, W. Soboyejo, J. Hormes. Ligand conjugated superparamagnetic iron oxide nanoparticles for early detection of metastases.

298

299

300

301

302

303

304

305

Breast Cancer Res. Treatment, 2006, 99, 2, 163–176. C. Leuschner, C. Kumar, J. Hormes, W. Hansel. Targeting breast cancer cells and their metastases through luteinizing hormone releasing hormone receptors using magnetic nanoparticles. J. Biomed. Nanotechnol., 2005, 2, 229–233. C. Leuschner, C. Kumar, M. Urbina, J. Zhou, W. Soboyejo, W. Hansel, F. Hormes. The use of ligand conjugated superparamagnetic iron oxide nanoparticles (SPION) for early detection of metastases. NSTI Nanotechnol. Technical Proc., 2005, 1, 5–6. C. Kumar, C. Leuschner, E. Doomes, L. Henry, M. Juban, J. Hormes. Efficacy of lytic peptide bound magnetite nanoparticles in destroying breast cancer cells, J. Nanosci. Nanotechnol. 2004, 4, 245–249. D. Shieh, F. Chen, C. Su, C. Yeh, M. Wu, Y. Wu, C. Tsai, D. Wu, D. Chen, C. Chou. Aqueous dispersions of magnetite nanoparticles with NHþ surfaces for magnetic manipulations of biomolecules and MRI contrast agents, Biomaterials. 2005, 26, 7183–7191. C. Berry, S. Charles, S. Wells, M. Daby, A. Curtis. The influence of transferring stabilized magnetic nanoparticles on human dermal fibroblasts in culture, Int. J. Pharm. 2004, 269, 211. L. Josephson, C. Tung, A. Moore, R. Weissleder. High efficiency intracellular magnetic labeling with novel superparamagnetic-tat peptide conjugates, Bioconjugate Chem. 1999, 10, 186–191. C. Dodd, H. Hsu, W. Chu, P. Yang, H. Zhang, J. Mountz, K. Zinn, J. Forder, L. Josephson, R. Weissleder, J. Mountz, J. Mountz. Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivatorpeptide-derived superparamagnetic nanoparticles, J. Immunol. Methods. 2001, 256, 89–105. M. Roser, D. Fischer, T. Kissel. Surface-modified biodegradable

References

306

307

308

309

310

311

312

313

albumin nano- and microspheres. II: Effect of surface charges on in vitro phagocytosis and biodistribution in rats, Eur. J. Pharm. Biopharm. 1998, 46, 255–263. F. Pineaud, D. King. Bioactivation and cell targeting of semiconductor CdSe, J. Am. Chem Soc. 2004, 126, 6115–6123. D. Pouliquen, J. Le Jeune, R. Perdrisot, A. Ermias, P. Jallet. Iron oxide nanoparticles for use as an MRI contrast agent: Pharmacokinetics and metabolism, Magn. Reson. Imag. 1991, 9, 275–283. S. Saini, D. Stark, P. Hahn, J. Wittenberg, T. Brady, J. Ferrucci. Ferrite particles: A superparamagnetic MR contrast agent for the reticuloendothelial system, Radiology. 1987, 162, 211–216. L. Tiefenauer, A. Tschirky, G. Kuhne, R. Andres. In vivo evaluation of magnetite nanoparticles for use as a tumor contrast agent in MRI, Magn. Reson. Imag. 1996, 14, 391–402. R. Weissleder, D. Stark, B. Engelstad, B. Bacon, C. Compton, D. White, P. Jacobs, J. Lewis. Superparamagnetic iron oxide: Pharmacokinetics and toxicity, Am. J. Roentgenol. 1989, 152, 167–173. A. Moore, L. Josephson, R. Bhorade, J. Basilion, R. Weissleder. Human transferrin receptor gene as a marker gene for MR imaging, Radiology. 2001, 221, 244–250. A. Moore, E. Marecos, A. Bogdanov, R. Weissleder. Tumoral distribution of long-circulating dextran coated iron oxide nanoparticles in a rodent model, Radiology. 2000, 214, 568– 574. A.R. Gunthert, C. Grundker, A. Olota, J. Lasche, N. Eicke, G. Emons. Analogs of GnRH-I and

314

315

316

317

318

319

GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen. Eur. J. Endocrinol. 2005, 153, 613–625. K.L. Shannon, R.T. Branca, G. Galiana, S. Cenzano, L.S. Bouchard, W. Soboyeyo, W. Warren. Simultaneous acquisition of multiple orders of intermolecular multiple-quantum coherence images in vivo. Magn. Reson. Imaging. 2004, 22, 1407–1412. M. Zaleska, G. Bodek, B. Jana, W. Hansel, A.J. Ziecik. Targeted destruction of normal and cancer cells through lutropin/choriogonadotropin receptors using hecate-bCG conjugate. Exp. Clin Endocrinol. Diabetes. 2003, 111, 146–153. G. Bodek, N.A. Rahman, M. Zaleska, R. Soliymani, H. Lankinen, W. Hansel, I. Huhtaniemi, A.J. Ziecik. A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGb conjugate. Breast Cancer Res. Treat. 2003, 79, 1–10. C. Leuschner, W. Hansel. Membrane disrupting lytic peptides for cancer treatments. Curr. Pharm. Des. 2004, 10, 2299–2310. C. Leuschner, C. Kumar, W. Hansel, F.J. Hormes. Targeting breast cancers and metastases with LHRH and a lytic peptide bound to iron oxide nanoparticles. Clin. Cancer Res. 2005, 11, B 262, 9097s. J. Meng, G. Galiana, T. Branca, J. Zhou, C. Leuschner, C. Kumar, J. Hormes, T. Otiti, A. Beye, W. Warren, W.O. Soboyejo, LHRHfunctionalized superparamagnetic ironoxide nanoparticles (SPIONs) for contrast enhancement in MRI, 2006, in preparation.

231

232

7

Carbon Nanotubes in Cancer Therapy and Diagnosis Pu Chun Ke and Lyndon L. Larcom 7.1

Overview

Integrating nanomaterials and biomedicine is a recent endeavor that aims to provide highly localized detection and treatment for cancers and diseases. Understanding the intricate interplay of nanomaterials and biological components and biological systems necessitates combining materials science, nanotechnology, physics, chemistry, biology, and biomedicine. The impact of this grand exploration touches areas of fundamental science and engineering, health care, and environmental control and protection. Recent advances in our understanding of carcinogenesis have led us to consider taking advantage of the special characteristics of both cancer cells and nanomaterials to detect and treat tumors. Integrating cancer therapy and nanomaterials captivates scientific imagination, offers new hope for human well-being, and unavoidably invites controversy and debate as experienced by many scientific and technological developments during their infancy. Carbon nanotubes (CNTs) are mosaics of carbon atoms synthesized into cylinders of single or multiple layers [1]. The astonishingly simple and plain structures of CNTs transcend the most important classes of nanomaterials owing to their unsurpassed stiffness, easy accommodation of chemical and biological versatilities, and rich quantum electronic properties [2–7]. Since their discovery a mere decade ago, CNTs have orchestrated a broad array of applications, including novel composites [8, 9], electrochemical sensors [10, 11], field emission [12, 13], memory, and nanoscale devices [14–16], just to name a few. In the rapidly growing field of biotechnology, CNTs have been utilized as platforms for ultrasensitive recognition of antibodies (Fig. 7.1, left) [17], as nucleic acids sequencers [18], and as bioseperators, biocatalysts [19], and ion channel blockers (Fig. 7.1, right) [20] for facilitating biochemical reactions and biological processes. Within the realm of nanomedicine, an emerging field of utilizing nanomaterials and nanostructures for medicine, CNTs have been transformed into scaffolds for neuronal and ligamentous tissue growth for regenerative interventions of the central nervous system and orthopedic sites [21–23], substrates for detecting antibodies associated with human autoimmune diseases with high specificity [24], and carriers of contrast agent aquated Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

7.1 Overview

(Left) Scheme for specific recognition of 10E3 mAb with an SWNT device coated with a U1A antigen–Tween conjugate [17]. (8 National Academy of Sciences.) (Right)

Fig. 7.1.

Docking simulation shows a capped SWNT of 0.9 nm in diameter fitting into a selective Kchannel [20]. (8 The American Society for Biochemistry and Molecular Biology, Inc.)

Gd 3þ -ion clusters for greatly enhanced magnetic resonance imaging [25]. Once covalently or noncovalently attached by nucleic acids (DNA or RNA including shortstranded RNA for gene silencing) [26, 27], vaccines (e.g., B cell epitopes) [28], and proteins [29, 30], CNTs have shown to be effective gene and drug delivery vectors that may someday co-exist with or challenge conventional viral and particulate systems [31]. Selective destruction of cancer cells using functionalized CNTs as transporters and near-infrared (NIR) heat inducing agents, as reported by Dai’s group [32], highlights research towards nanomedicine for cancer diagnosis and therapy. To propel research in nanomedicine, the new ‘‘Nanotechnology for Cancer Diagnosis and Therapy’’ initiative was announced by the National Cancer Institute (NCI) in September 2005 [33]. Nanoscience with its implications for biomedicine has been highlighted as one of the ‘‘Priority Areas’’ of the National Science Foundation (NSF) [34] and in the ‘‘Roadmap’’ of the National Institutes of Health (NIH) [35]. It is not beyond our wildest dream that within the next decade or so we might be able to determine to what extent the atomic world obeys the laws of quantum mechanics, resolve the passionate debate between Drexler and Smalley on ‘‘the fat fingers’’ of nanobots and molecular assemblies [36], and witness how nature selectively embraces the ‘‘smart’’ creatures such as CNTs, buckyballs, dendrimers, quantum dots, and other nanoparticles offered by mankind. This chapter reviews, for the first time to our knowledge, the state-of-the-art in cancer diagnosis and therapy from the perspective of single-walled carbon nanotubes (SWNTs). Through the introduction of these early developments we wish to present a panorama of how SWNTs can be used for biomedicine. There are many reasons for using SWNTs instead of multiwalled carbon nanotubes (MWNTs) as our model systems. They resemble nucleic acids in physical dimensions and present excellent platforms for biosensing and biocompatibility, as compared with viral

233

234

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

and particulate gene and drug delivery vectors. They possess an enormously high aspect ratio, which allows for efficient permeation of their attached drug loads through tissues and cells. Their large surface areas are able to carry many biomolecules to their respective targets for sensing, sequencing, and therapy. Their stability, flexibility, and non-immunogenicity may prolong the circulation and availability of attached drugs. Their hydrophobicity may facilitate interaction with cell membranes, and the rich electronic properties of SWNTs may open up new routes and methodologies for probing and imaging the states of cancerous tissues and cells. Because nanotechnology is such a rapidly changing field and because nanomedicine is still in its very infancy, it is beyond our ability to grasp and thus to convey all of the exciting frontiers and aspects of this new science. Instead, we will elucidate only a few of these innovations in this chapter, which is organized into the following sections: Section 7.1 gives an overview; Section 7.2 covers SWNT modification for solubility and biocompatibility; Section 7.3 deals with diffusion of SWNT–biomolecular complexes; Section 7.4 covers gene and drug delivery with SWNT transporters; Section 7.5 deals with sensing and treating cancer cells utilizing SWNTs; Section 7.6 covers the cytotoxicity of SWNTs; Section 7.7 deals cancers and SWNTs; and Section 7.8 provides a summary.

7.2

SWNT Modification for Solubility and Biocompatibility

Pristine SWNTs in aqueous solution form bundles due to the hydrophobic interactions, van der Waals attractions, and p-stacking among individual tubes. Bundle formation presents a major hurdle for the applications of SWNTs in biological systems and medicine. Solutions to this problem include two major routes, covalent and noncovalent modifications of SWNTs. Covalent modification of SWNTs involves esterification or amidation of acid-oxidized nanotubes and side-wall covalent attachment of functional groups [37–41]. These covalent schemes are often characterized by uncertainties in determining reaction efficacy and by undesirable modifications in the physical and chemical properties of SWNTs [28, 42, 43]. In comparison, the noncovalent modifications of SWNTs employ nonspecific attachment of proteins [17, 30], linear bio- and synthetic polymers [DNA, RNA, poly(vinyl pyrrolidone), polystyrene sulfonate], and surfactants [sodium dodecyl sulfate (SDS) etc.] [42–49]. Many surfactants, organic solvents, and residues, however, cause cytotoxicity [50] and/or other side effects that limit the biocompatibility of SWNTs. Developing alternative schemes is thus crucial for facilitating the full range of biological and biomedical applications for SWNTs and their derivatives. 7.2.1

Chemical Modifications of SWNTs for Solubility

Chemical modification of SWNTs is an emerging research area in the fields of materials science and nanotechnology, and is pertinent to the biological applications

7.2 SWNT Modification for Solubility and Biocompatibility

of SWNTs when it is deemed necessary to attach fluorescent tags, drugs, and proteins. These modifications include the functionalization of oxidized SWNTs and covalent modification of SWNTs. Organic chemistry plays a major role in these approaches. Functionalization of SWNTs through Oxidation Functionalization of SWNTs utilizing acidic media (e.g., 3:1 mixture of concentrated sulfuric to concentrated nitric acids) may open the ends of the SWNTs and create functionalities suitable for further derivatization at these acid-induced defect sites. Long-chain alkylamines may be coupled with the carboxylic groups present on the surface of oxidized SWNTs. The coupling of organic acids with amines is a well-known reaction, but tends to proceed only at relatively high temperatures. This reaction is often facilitated by converting the acids into a more reactive moiety, usually a mixed anhydride R(CO)O(CO)R 0 or acid chloride RCOCl. The acid chloride is generally highly reactive, hydrolyzes upon exposure to air (or the humidity therein), and degrades back to carboxylic acid. Activation of the carboxyl moieties with thionyl chloride and their subsequent reaction with amines are feasible. Alternatively, oxidized SWNTs can be condensed directly with amines. Typically, the solubility of such modified SWNTs is approximately 0.5 mg mL
1 in organic solvents such as tetrahydrofuran or dichlorobenzene [51]. However, the electronic properties of these functionalized SWNTs are often deviated or even lost. In most cases, the SWNTs are greatly shortened [52]. Functionalization of SWNTs creates charge polarity to the tubes and may lead to their separation from pristine SWNT clusters in polar solvents. This separation is desirable since it breaks the persistent hydrophobic forces and van der Waals interactions among the tubes and renders SWNTs soluble, thus enabling their further incorporation by biological systems. This functionalization process also facilitates the characterization and purification of single SWNTs. The presence of the functional addends at the defect sites of oxidized CNTs can be monitored by either Raman or IR spectroscopy, which easily identify amide- or ester-bond formation. Regarding biosensing, oxidized SWNTs may, for example, react with an alkyl thiol with a subsequent chemical attachment to gold particles [52–54]. These SWNT– gold particle complexes can be used to construct microelectrode arrays. Potential applications of these microelectrode arrays include developing bio-electrochemical sensors and designing novel molecular-recognition electrodes [51]. One scheme that could lead to gene sequencing and recognition is the attachment of peptide nucleic acids (PNA) via N-hydroxysuccinimide (NHS) esters formed on carboxylated SWNTs, as demonstrated by Williams et al. [55] in 2002 (Fig. 7.2). There are several rationales for using PNA to construct such complexes. First, PNA molecules are uncharged DNA analogues that can be hybridized with complementary DNA, and these PNA attached SWNTs may be incorporated into larger electronic devices by recognition-based assembly. PNA treated SWNTs may also serve in biological systems as probes based on sequence-specific attachment. Second, PNA is compatible with the most convenient solvents such as DMF, though less soluble in aqueous solutions. Third, PNA is not susceptible to enzy7.2.1.1

235

236

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Attachment of DNA to carbon nanotubes. (a, b) N-Hydroxysuccinimide (NHS) esters formed on carboxylated SWNTs are displaced by PNA, forming an amide linkage. (c) A DNA fragment with a singlestranded, ‘‘sticky’’ end hybridizes by base-

Fig. 7.2.

pairing to the PNA–SWNT. (d, e) Atomic-force microscope (tapping mode) images of PNA– SWNTs. SWNTs appear as bright lines; the paler strands represent bound DNA. Scale bars: 100 nm; nanotube diameters: (d) 0.9 and (e) 1.6 nm [55]. (8 Nature Publishing Group.)

matic degradation due to its synthetic status. Lastly, PNA–DNA duplexes are thermally more stable than their DNA–DNA counterparts because PNA is neutral and does not induce electrostatic repulsion. The higher thermal stability of PNA–DNA suggests that shorter sticky ends are required for room-temperature hybridization, which reduces nonspecific electrostatic interactions with metallic electrodes, or with silicon oxide, which is desirable for lithography [55]. Functionalization of SWNTs through Covalent Modifications Apart from oxidation, SWNTs also can be treated with dichlorocarbene, which can be induced by chloroform–sodium hydroxide interaction or phenyl(bromodichloromethyl)mercury [56, 57]. SWNTs react with molecular fluorine at 150–600  C, and these derivatized SWNTs can be retrieved with hydrazine [51]. SWNTs functionalized through this procedure dissolve well in polar solvents, although the fluorine atoms may be substituted by alkyl groups for higher solubility [58]. Fluorination 7.2.1.2

7.2 SWNT Modification for Solubility and Biocompatibility

can also shorten the SWNT to less than 50 nm [51, 59], which has broad implications should the permeation of nuclear membrane pores by SWNTs and their derivatives be desirable. SWNTs may be functionalized through aryl diazonium chemistry [60], and be derivatized with CaC bonds by the electrochemical reduction of various diazonium salts. In addition, because the oxidative coupling of amines can result in their attachment to SWNT surfaces [51, 61], these nanotube-bound amine moieties have great versatility for use as gene and drug carriers. Another covalent SWNT functionalization is derived from the chemical processing of fullerenes [62], which is accomplished by the addition of nitrenes, carbenes, or radicals. Functionalized SWNTs may also be constructed via electrophilic addition by adding chloroform to the side walls of SWNTs during a mechanochemical reaction with AlCl3 . Hydroxyl groups may replace the chlorine atoms, which then may be esterified to yield corresponding esters [51]. This method not only produces soluble SWNTs but also adds free functional groups for further linking to biomacromolecules. Based on the methodology of 1,3-dipolar cycloaddition of azomethine ylides, Prato et al. have devised a route for covalently attaching pyrrolidine rings substituted with chemical functions to the side walls of SWNTs [63, 64]. They chose the triethylene glycol group as the N-substituent of the a-amino acid due to its high solubilizing power. These SWNTs were soluble in chlorinated solvents, acetone, and alcohols but insoluble in diethyl ether or hexane. In particular, the solubility of these functionalized SWNTs in dichloromethane or chloroform was as high as 50 mg mL
1 , suggesting an average covalent attachment of one pyrrolidine ring to every one hundred carbon atoms of the SWNTs. Moreover, the covalent attachment of pyrrolidine rings to pristine SWNTs quenches their signature NIR band of van Hove transitions [51, 64]. Water-soluble SWNTs can also be obtained by exposing the tubes to N-substituted a-amino acid with a terminated amino tertbutoxycarbonyl (Boc) protected group and paraformaldehyde. Upon treatment with gaseous hydrochloric acid, the N-Boc groups can be removed by treating the SWNTs with hydrochloric acid, which further releases the corresponding ammonium salts from the water-soluble SWNTs [51, 65]. 7.2.2

Noncovalent Modifications of SWNTs for Solubility

Noncovalent solubilization of SWNTs has recently gained recognition partly due to the growing concern over the covalent schemes, including their induced alteration to and/or the loss of the mechanical and electronic properties of SWNTs. Noncovalent schemes are also attractive because of their relative ease of releasing load from the tubes, though this release may sometimes act against the purpose of application, causing uncertainties in quantifying gene and drug transfection. This route of solubilization schemes is unlimited to the field of organic chemistry and often embraces the contributions from researchers in materials sciences, biochemistry, and biophysics. Smalley’s group first proposed the idea that SWNTs could be solu-

237

238

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Binding model of a (10,0) SWNT wrapped by a poly(T) sequence of ssDNA. (a) The right-handed helical structure shown here is one of several binding structures found, including left-handed helices and linearly adsorbed structures. In all cases, the bases (red) orient to stack with the surface of the

Fig. 7.3.

nanotube, and extend away from the sugar– phosphate backbone (yellow). (b) The DNA wraps to provide a tube within which the SWNT can reside, hence converting it into a water-soluble object [49]. (8 Nature Publishing Group.)

bilized in aqueous media of amphiphilic molecules such as surfactants [45]. The hydrophobic moieties of the surfactants were hypothesized to interact through wrapping with the hydrophobic surfaces of the SWNTs. This interaction was understood to be facilitated by the van der Waals attractions between the surfactants and the SWNTs in close proximity. Zheng et al. extended this simple scheme to include biomacromolecules such as single-stranded DNA (ssDNA) for the solubilization of SWNTs and sorting of metallic nanotubes from semiconducting ones (Fig. 7.3) [49]. The underlining binding mechanism is the hydrophobic interaction between the nitrogenous bases of the ssDNA and the side walls of the SWNTs, attributed by the p-stacking of the nitrogenous bases and the p-electrons of the carbon atoms on the SWNTs. Poly(A) and poly(C) have lower dispersion efficiencies than poly(T), possibly because these nucleotides tend to strongly self-stack in solution and therefore have smaller free energies for binding to SWNTs. In addition to using nucleic acids, a few groups, most notably Dai’s group at Stanford, have dispersed SWNTs in solutions of peptides and proteins such as bovine serum albumin (BSA) and streptavidin [17, 30]. These research efforts have formed the basis for creating biocompatible nanomaterials for biosensing and therapeutics. However, a direct comparison of the solubility from the noncovalent methods with that from the covalent schemes is unavailable, since most noncovalent studies were conducted through visualization rather than quantification.

7.2 SWNT Modification for Solubility and Biocompatibility

Recently, our group reported the solubilization of SWNTs using RNA polymers [66]. The structure of this RNA resembles that of ssDNA except that it possesses a hydroxyl group instead of a hydrogen atom at the 2 0 of their nucleotides. This structural difference, though seemingly minute, opens the door for many exciting applications, including using RNA as catalysts for inorganic particle growth and organic chemical reactions, and as agents for RNA interference (RNAi) for gene silencing. Using single-molecule fluorescence microscopy our group observed that poly(rU) RNA molecules, when exposed to SWNTs immobilized on a quartz substrate, bound to SWNTs instead of randomly distributing on the substrate. The surface property of this substrate changed from hydrophilic to hydrophobic during the chemical deposition of SWNTs. This suggests that p-stacking may dominate the hydrophobic interaction when nucleic acids are noncovalently attached to SWNTs (Figs. 7.4a, b) [46]. The binding induced changes in Raman spectra are shown in Fig. 7.4(c). The samples were prepared using the same protocol as described for fluorescence imaging except that the labeling was omitted to prevent the strong fluorescence signal of labeled poly(rU) from masking the Raman signal of SWNTs. The Raman spectra were excited with a 647.1 nm laser line and the laser power was maintained at approximately 1 mW to avoid heating. A Leica microscope equipped with a 50 dry objective (NA ¼ 0:75) was used to focus the laser beam at various spots on the pristine and poly(rU)-bound isolated SWNTs. The Raman scattered light was collected using an ISA Triax 550 spectrometer equipped with a liquid-nitrogen cooled CCD. The bottom spectrum corresponds to pristine SWNTs, while the top spectrum corresponds to poly(rU) bound isolated SWNTs. The frequency positions of the RBM, D, and G bands were determined from a Lorentzian line shape analysis. The characteristic RBM was observed between 190–290 cm
1 , while the G band frequency was centered at 1593 cm
1 . No significant shift in the peak positions was found in the spectrum of poly(rU) bound SWNT as compared with that of the pristine SWNTs, possibly because the charge transfer from the poly(rU) molecules to the SWNT was too minute to discern. The Raman spectrum of SWNT-poly(rU) hybrids exhibits a noticeable decrease in the intensity of the RBM band and a small enhancement in the D band intensity compared with the corresponding peaks of the spectrum for isolated SWNTs alone. These changes in Raman spectra are indicative of the effective binding of poly(rU) with SWNTs [46]. 7.2.2.1 Solubilization of SWNTs Using Lysophospholipids Enables Cellular Studies [67] This section details a new approach for solubilizing SWNTs using single-chained phospholipids, or lysophospholipids. This approach is promising because it enables cellular studies using SWNTs and other nanomaterials. The potential applications of lysophospholipid solubilized SWNTs and their biofunctionalized derivatives include in vivo imaging, biosensing, and gene and drug delivery for biomedicine. We first obtained pristine SWNTs using the arc-deposition method. The average diameter of the SWNTs was approximately 1.4 nm measured by Raman spectros-

239

240

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

(a, b) Fluorescence images of SWNTpoly(rU) hybrids. Individual poly(rU) molecules are visible as small blobs in (b) due to the relatively low concentration. SWNTs in both panels are over 50 mm long. (c) Raman spectra collected at a laser excitation wavelength of 647.1 nm from pristine (bottom) and poly(rU)

Fig. 7.4.

bound isolated SWNTs (top) on silicon substrates. Plots are normalized with respect to the amplitudes of the G bands. The asterisks at 305 cm
1 correspond to the Raman lines of the silicon substrates [46]. (8 American Institute of Physics.)

copy, with an average molecular weight of 1  10 6 Dalton (Da), estimated from TEM. To measure the solubility provided by pure phospholipids, SWNTs were dispersed in phosphate-buffered saline (PBS, pH 7.4) containing phospholipids of varying amounts. After sonication for 1 h at room temperature, SWNTs were found to be completely solubilized by LPC 18:0 (Fig. 7.5), and by cell growth mediums (Fig. 7.6a). The weight ratio of solubilized SWNTs to LPC was approximately 1:10, corresponding to a molar ratio of 1:20 000 at saturation (Fig. 7.6b), indicating their high binding capacity. Comparable solubility of SWNTs was also obtained with LPA 16:0 (Fig. 7.5), and LPG 18:0 (Fig. 7.5), based on the same treatments. Approximately half of the soluble SWNTs were sedimented at 16 060g RCF at tip, based on measured optical densities (Fig. 7.6c), indicating their heterogeneous size

7.2 SWNT Modification for Solubility and Biocompatibility

Structures of lysophospholipids LPC 18:0, LPA 16:0, and LPG 16:0 and SDS. LPC: lysophosphatidylcholine; LPA: lysophosphatidic acid; LPG: lysophosphatidylglycerol. The numbers 18 and 0 in LPC 18:0 denote the total

Fig. 7.5.

number of carbon atoms and the total number of double bonds, respectively, contained in the sum of the fatty acyl chains [67]. (8 American Chemical Society.)

distribution. From elution volumes measured with chromatography, we estimated that the SWNT-LPC and the SWNT-LPA complexes had an average molecular weight of 14  10 6 Da and 18  10 6 Da, respectively (Fig. 7.6d). Since each SWNT carries approximately 20 000 LPC molecules and the molecular weight of each LPC is 523.7 Da, the ‘‘molecular weight’’ of each SWNT-LPC complex is estimated at 11:5  10 6 Da, which agrees with our measured value of 14  10 6 Da. The discrepancy is mainly caused by the heterogeneous size distribution of the SWNTs and the occurrence of small SWNT bundles. Figure 7.6(e) compares SWNT solubility for LPC, LPG, and the surfactant SDS (Fig. 7.5), a routine solvent for SWNTs [45]. At 6177g RCF at tip, on a per molecule basis, LPC is approximately 2:5 more effective than SDS in dispersing SWNTs in PBS. At 16060g RCF at tip, LPC is approximately 10 more effective than SDS in dispersing SWNTs, possibly because the resulting micelles differ in size. This difference might be because LPC has a bulkier head group for interfacing with water and a longer acyl chain for binding with SWNTs. SWNTs with LPG are slightly more soluble than with SDS. Solubilization of SWNTs with lysophospholipids was also more effective than with nucleic acids [47, 49, 68], and was far more effective than with proteins [29, 39]. The aqueous SWNT-lysophospholipid solutions were exceptionally stable for months at room temperature, a promising feature in regards to possible applications in both biology and medicine.

241

242

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Characteristics of SWNTs solubilized with phospholipids. (a) Comparison of SWNT solubility with different aqueous phospholipid solutions and cell growth mediums, RPMI supplemented with 10% fetal bovine serum (FBS) and nutrient broth (NB). SWNTs (1 mg) were treated with phospholipids (10 mg) in PBS (1 mL) or growth medium (1 mL). After bath sonication at room temperature for 1 h and centrifugation for 3 min at 6177g, the absorbance was observed at 360 nm as a measure of the amount of SWNTs solubilized. The label ‘‘rel O.D.’’ on the y-axis denotes relative optical density. (b) LPC solubilized SWNTs. SWNTs (1 mg) were treated with increasing concentrations of LPC 18:0, sonicated, and centrifuged as above, with an absorbance observed at 360 nm. Filled triangles: SWNTs with LPC; filled squares: LPC

Fig. 7.6.

alone as a control for micelles. The latter showed negligible absorbance at 360 nm. (c) Sedimentation of LPC-solubilized SWNTs, prepared as above with SWNTs (1 mg) and LPC (10 mg). (d) Size-exclusion chromatography measurements of SWNT-LPC 18:0 (centrifuged at 16 060g for 3 min) and SWNT-LPA 16:0 complexes (centrifuged at 6177g for 3 min). A linear relationship between compound log(molecular weight) and elution volume in mL was established using xylene cyanole FF with blue color (538.6 Da), horse heart myoglobin with reddish color (16951.5 Da), and blue dextran with blue color (2  10 6 Da). (e) Comparison of SWNT solubility in LPC, LPG, and SDS solutions, prepared from SWNTs (1 mg) and detergents (10 mg) as above [67]. (8 American Chemical Society.)

To probe the mechanism of SWNT-lysophospholipid binding, zwitterionic LPC and net negatively charged LPG at physiological pH were bound to SWNTs and imaged with TEM (Figs. 7.6a–c). One can see the formation of areas of tightly packed lysophospholipids in the dark/grey areas, our termed ‘‘lipid phase’’, in

7.2 SWNT Modification for Solubility and Biocompatibility

TEM images of SWNT-LPC (a and c) and SWNT-LPG (b) complexes. Recorded with an Hitachi 7600 and stained with uranyl acetate. Numbers 1–4 in (a) and (c) correspond to (1) an isolated SWNT in the vacuum phase, (2) an LPC striation on an SWNT/SWNT bundle, (3) possibly an LPC micelle on the substrate in the lipid phase, and (4) an uncoated SWNT bundle in the vacuum phase. Note the less organized and wider striations of SWNT-LPG complexes in (b), as compared with those in (a) and (c) for SWNT-

Fig. 7.7.

LPC. Scale bar (a–c): 20 nm. (d) Hypothesized microscopic binding modes of LPC and LPG with SWNTs. The lysophospholipids are shown as truncated triangles, their head groups are at the base of the triangles, and the SWNTs are the inner circles and tubes. The left-hand side illustrates the proposed lipid spiral wrapping along the tube axis while the right-hand side shows their possible binding around the circumference of the tubes [67]. (8 American Chemical Society.)

Figs. 7.7(a–c). The light/blank areas in Figs. 7.7(a–c) correspond to lysophospholipid free regions or our termed ‘‘vacuum phase’’. In the lipid phase SWNTs are wrapped by striations of @5 nm for LPC and 5–7 nm for LPG. Such organizations were previously reported for the binding of SDS and synthetic lipids on CNTs [69]. However, in the vacuum phase (Figs. 7.7a, c) SWNTs are practically naked, indicating that the binding of lysophospholipids to SWNTs is controlled by the local lysophospholipid environment rather than by specific interactions between lysophospholipids and SWNTs. Neither LPC nor LPG binds to SWNTs in the vacuum phase, while both coat SWNTs in the lipid phase. LPC on an SWNT or an SWNT bundle displays such a consistent organized pattern along the tube(s) that striations remain approximately the same size (Figs. 7.7a, c). By contrast, the binding of LPG to SWNTs in the lipid phase does not follow the same pattern (Fig. 7.7b). The size and orientation of the

243

244

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Schematic representations of the mechanisms by which surfactants help disperse SWNTs. (a) SWNT encapsulated in a cylindrical surfactant micelle; right-hand side: cross section; left-hand side: side view.

Fig. 7.8.

(b) Half-cylinder adsorption of surfactant molecules on a SWNT. (c) Random adsorption of surfactant molecules on an SWNT [42]. (8 American Chemical Society.)

striations change along the axis of SWNTs. These differences could be related to the different lysophospholipid organizations shown in their respective backgrounds. Figures 7.7(a) and (c) show that the lipid phase of LPC is composed of many large objects of @5 nm, which are probably micelles, while the lipid phase of LPG is homogeneous, most probably composed of individual lysophospholipids. Another major difference in the binding of LPC vs. LPG in the lipid phase is the shape of the striations. The crests of striations are approximately 0.2 nm above the surface of SWNT(s) for SWNTs, while the clefts almost touch the surface of SWNT(s) for LPC. There has been an ongoing debate on the binding mode of amphiphilic surfactants and cylindrical SWNTs [42, 43, 69]. Surfactants are hypothesized in the literature as adopting one of three configurations: (a) micelles; (b) half-cylinders; and (c) random adsorption (Fig. 7.8) [42]. The periodic wrapping in the lipid phase, as observed in our experiment, strongly suggests that the microscopic binding mode is the ‘‘half-cylinders’’ (Fig. 7.7d). Note that the macroscopic arcs formed by lysophospholipids along the SWNT axis are not semi-spherical but are extended arcs, because the ends of lysophospholipid tails within an arc can not occupy the same point and must be offset along the tube axis. Furthermore, LPC on SWNTs (Fig. 7.7a, c) exhibits macroscopic spirals possibly resulting from the wrapping of deformed lipid-composed long half-cylinder(s). In some cases, the step of the spiral is equal to the width of the half-cylinder; thus a single half-cylinder can provide a complete coating of the tube surface. For SWNT bundles, we observed binding with a larger step corresponding to double spiral wrapping. We also noticed that, in some cases, the ring binding mode and the rings were tilted with respect to the tube axis. As long as the tube diameter remains static, the wrapping mode is conserved along the tube. However, the wrapping mode may change when the bundle size varies (Fig. 7.7a). In contrast, the TEM images with LPG (Fig. 7.7b) exhibit alterations of the wrapping mode down the tube axis. In addition, the average width

7.2 SWNT Modification for Solubility and Biocompatibility

of the LPG striations is noticeably larger than that of LPC (Fig. 7.7b vs. Fig. 7.7a) owing to a net negative charge of LPG head groups. A macroscopic configuration, like LPG head groups in consecutive half-cylinders, paired and in contact with weakly polarizable SWNT surfaces is energetically unfavorable. To reduce the repulsion between LPG head groups, the SWNT-LPG system must pay an ‘‘energy penalty’’ by distorting and/or broadening the striations (Fig. 7.7d, lower section). Indeed, our experimental data revealed that LPC bound better to SWNTs than LPG (Fig. 7.6e). To confirm our observation, we tested double-chained phospholipids for their SWNT solubility. The phospholipids used were dimyristoylphosphatidylcholine (PC 24:0), which is zwitterionic at physiological pH; and 1,2-dioleoylphosphatidylglycerol (PG 36:2) and 1,2-dipalmityolphosphatidylethanolamine (PE 32:0), both of which are negatively charged at physiological pH. None of the above phospholipids provided good solubility for SWNTs. From a geometrical perspective the three-dimensional (3D) structures of lysophospholipids and detergent molecules can be approximated as cones (Fig. 7.7d). The packing of conical objects normally results in spherically shaped objects, e.g., micelles, due to their curvophilicity. In contrast, double-chained phospholipids are considered to be curvophobic [70], their 3D structures are approximated as cylinders and their packing assumes bilayers. Obviously, wrapping around a cylindrical object, i.e., an SWNT, is geometrically preferential for curvophilic lysophospholipids but not for curvophobic phospholipids. The geometrical considerations further support the microscopic binding mode shown in Fig. 7.7(d). ‘‘Half-cylinder’’ wrapping is the only microscopic mode that results in semi-spherical curvature along the SWNT axis and SWNT circumference. In addition to the packing consideration, we calculated the average number of LPC necessary for coating an average SWNT, assuming tight packing and the LPC head group size of 0.6 nm. We found that ‘‘half-cylinder’’ binding will result in a lipidsto-tube ratio of 21 000:1 – in excellent agreement with our experimentally estimated ratio of 20 000:1. Free lysophospholipids and micelles in SWNT-lysophospholipid solution were removed by filtration through 100 kDa Microcon (Amicon, Inc) centrifugation tubes and washed four times. The resulting lysophospholipid-free and micellefree SWNT-LPC complexes were tested by in vivo bioassay using colon cancer (CACO-2) and macrophage (THP-1) cell lines. Each cell line was incubated in its own eight-well chamber slide (LabTek) for 48 h at 37  C in a CO2 incubator. Before treatment, cell adhesion was checked by differential interference contrast (DIC) microscopy using a Zeiss 135 Axiovert inverted microscope. Treatments ranging from 5 to 40 ppm of lysophospholipid-free and micelle-free SWNT-LPC complexes were added to each adherent cell line and incubated for 3 h at 37  C in a CO2 incubator. After incubation, control and treated cells were fixed with 4% paraformaldehyde for 30 min, washed in PBS and subjected to an APO-BrdU TUNEL assay (Invitrogen). This assay detects the onset of apoptosis by fluorescent detection of nuclear DNA fragments or DNA breaks. Cells were labeled with deoxythymidine analogue 5-bromo-2 0 -deoxyuridine-5 0 -triphosphate (BrdUTP) followed by the addi-

245

246

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Fixed CACO-2 cells incubated with SWNT-LPC for 3 h and examined for apoptosis by APO-BrdU TUNEL assay. Panels (a, b, e, f ) show control cells and (c, d, g, h) show cells that have been incubated with 20 ppm of SWNT-LPC. The merged areas shows cells with intact plasma and nuclear membranes in control (f ) and in treatments (h). PI staining

Fig. 7.9.

revealed intact cell nuclei in control (a) and treated cells (c). Neither control (b) nor treated cells (d) appear apoptotic since there was an absence of anti-BrdU antibody labeled staining in the FITC channel. The DIC channel shows intact plasma membranes in both control (e) and treated cells (g). Scale bars: 20 mm.

tion of Alexa-Fluor 488 labeled anti-BrdU antibody. Propidium iodide (PI) was used to image the total DNA content of cells. The prepared cells were imaged using a Zeiss 510 LSM confocal fluorescence microscope. Our bioassays showed no loss of cell viability (Figs. 7.9 and 7.10) when both colon cancer cells (CACO-2) and macrophage (THP-1) cell lines were treated with 20–40 ppm of lysophospholipid-free and micelle-free SWNT-LPC. CACO-2 cell nuclei remained unaffected by treatment of 20 ppm SWNT-LPC (Fig. 7.9c), which was also the case for the macrophage THP-1 cell line treated with 40 ppm SWNT-LPC (Fig. 7.10c). Cell plasma membranes remained intact in both CACO-2 cells (Fig. 7.9e) and THP-1 cells (Fig. 7.10e), and the onset of apoptosis by Apo-BrdU TUNEL assay was not detected in either cell line (Fig. 7.9d and Fig. 7.10d). Motile THP-1 cells treated at 40 ppm SWNT-LPC exhibited elongated cell bodies (Fig. 7.10h), which is thought to be due to SWNT-LPC disruption of plasma membranes or cytoskeleton. SWNTs, otherwise a collection of hydrophobic synthetic nanoparticles, have been solubilized in aqueous lysophospholipid solutions with extended stability. The biocompatibility of lysophospholipids is unsurpassed since they occur naturally in the cell membrane. The signaling capacity of lysophospholipids and the electronic property of SWNTs may be combined for disease detection. The strong absorbance of isolated SWNTs in NIR [44] can be utilized for noninvasive imaging and sensing. Furthermore, since the head groups of lysophospholipids can be functional-

7.3 Diffusion of SWNT–Biomolecular Complexes

Fig. 7.10. Confocal images of fixed macrophages (THP-1) incubated with SWNTLPC for 3 h and examined for apoptosis by APO-BrdU TUNEL assay. Panels (a, b, e, f ) are control cells and (c, d, g, h) are treated cells that have been incubated with 40 ppm of lysophospholipid-free and micelle-free SWNTLPC. The merged areas show cells with intact plasma and nuclear membranes in control (f )

and in treated cells (h). The treated cells have become elongated. PI staining revealed intact cell nuclei in control (a) and treated cells (c). Neither control (b) nor treated cells (d) appear apoptotic since there was an absence of antiBrdU antibody labeled staining in the FITC channel. The DIC channel shows intact cell membranes in control (e) and treatments with elongated cell bodies (g). Scale bars: 20 mm.

ized with tags such as quantum dots, antioxidants, and monoclonal antibodies, this solubility method opens the door for utilizing nanomaterials for in vivo imaging, cancer diagnosis and therapy, and novel nanomedicines.

7.3

Diffusion of SWNT–Biomolecular Complexes

The enhanced elucidation of gene function in human health has led to the need for more effective and robust means for delivering therapeutic genes to target cells. While no single gene transfection method developed to date has been found to be optimal for all cells, SWNTs offer a promising alternative since they can be treated or functionalized for biocompatibility [30]. Recent biochemical assays and theoretical studies have indicated that the nonspecific binding of DNA with SWNTs is realized through the p-stacking between the bases of DNA and the p-electrons of carbon atoms as well as the hydrophobic interaction between the bases of DNA and the sidewalls of SWNTs [49, 71]. Helical wrapping of ssDNA on SWNTs is energetically favorable and sequence dependent [49, 71, 72]. While current efforts on interfacing SWNTs with nucleic acids have focused almost exclusively on DNA,

247

248

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

equal attention should be paid to RNA due to its biological significance. For example: (a) messenger RNAs (mRNAs) are the physical gene transcripts that are translated to functional proteins at the ribosomal level; (b) some RNAs act as enzymes to catalyze various cellular biochemical pathways, they serve as mediators for various organic reactions [73–76] and are often utilized as templates for the growth of inorganic-particles [77]; and (c) small interfering RNAs (siRNAs), which disrupt mRNAs prior to translation, will cause the silencing of a specific posttranscriptional gene’s expression. The use of siRNA is a powerful research tool that has a pivotal role in deciphering the functions and interactions of thousands of unknown genes [78]. Albumin, in contrast, is one of the most abundant serum proteins present in the mammalian circulatory system, contributing over 80% of the colloid osmotic pressure [79]. Indeed, the nonspecific binding of SWNTs to BSA has been elucidated as a general phenomenon [17]. In this section, we introduce the first study by our group on the diffusion of SWNT-poly(rU) and SWNTBSA hybrids, two of the most elementary synthetic biomolecular complexes. This study provides a physical guidance to gene delivery using SWNT as transporters. Diffusion refers to the process by which particles intermingle as a result of their kinetic energy of random motion. It plays a central role in the transportation of small particles across cell membranes, in cytoplasm, and in nuclei. For the applications of gene and drug delivery, the innate hydrophobicity of SWNTs causes the tubes to aggregate or clump, presenting a major challenge for cellular studies. The addition of nucleic acids or proteins mediates the separation of pristine SWNTs through p-stacking and/or by decreasing the degree of hydrophobic interactions, thus markedly enhancing the solubility of the nanotubes. Further, nucleic acids and proteins can be labeled with fluorescent dye molecules and, consequently, SWNTs can be visualized when bound with these biomolecules. Singlemolecule fluorescence microscopy (SMFM) has been utilized for the diffusion studies of phospholipids in biomembranes [80, 81]. Superior to confocal microscopy, wide-field SMFM simultaneously collects significant statistics without averaging or losing molecular individuality [80–82]. Unlike scanning electron microscopy (SEM) and crystallography, SMFM is non-invasive and is, therefore, suitable for in vitro and in vivo studies of biological processes. In SMFM, the trajectories of fluorescently labeled biomolecules can be followed at nanometer resolution. Based on the displacement of the centre of mass (COM) of a molecule, the mean-square-displacement (MSD) [83] of the molecule can be obtained with Eq. (1), where x i and yi are the positional coordinates of the COM of the molecule in frame i, and n denotes the frame number with time interval Dt. MSDðDtÞ ¼ hðxiþn
x i Þ 2 þ ð yiþn
yi Þ 2 i

ð1Þ

The diffusion coefficient D of the molecule undergoing a two-dimensional (2D) random walk is given as D ¼ MSD=4Dt. For diffusion studies, pristine SWNTs were first synthesized using the arcdeposition method [84]. The average diameter of the SWNTs was 1.4 nm, as confirmed by Raman spectroscopy and transmission electron microscopy. The sample

7.3 Diffusion of SWNT–Biomolecular Complexes

of SWNT-poly(rU) hybrids was prepared by modifying the protocol used by Zheng et al. [49], employing the following procedure. A mixture of poly(rU) (0.2 mg mL
1 , lyophilized polyuridylic acid, Midland, 500–2600 nucleotides) and SWNTs (0.01 mg mL
1 ) in TE buffer (10 mm Tris, 1 mm EDTA, pH 7.3) was first placed on ice and probe-sonicated at 10 W for 30 min (VC 130 PB, Sonics). The resultant SWNT-poly(rU) suspension was then centrifuged at 5000 rpm for 10 min to pellet the poly(rU)-wrapped and bare SWNTs. The supernatant of unbound poly(rU) was removed and the pellet resuspended in TE buffer. The sample was then labeled with OliGreen (Molecular Probes, absorption 500 nm, emission 520 nm), which fluoresces strongly only when bound to nucleic acids, and which specifically exhibits a large fluorescence enhancement when bound to the U bases in poly(rU). SWNT-BSA hybrids were prepared by the same procedure as that for the SWNTpoly(rU) hybrids, except labeling since BSA was pre-labeled with TRITC (Sigma, absorption 550 nm, emission 570 nm). Binding of SWNTs and poly(rU) was analyzed by SEM (S4700, Hitachi), and a drop of the solution containing poly(rU) (0.2 mg mL
1 ) and SWNTs (0.01 mg mL
1 ) in TE buffer was dispersed on a silicon substrate after probe-sonication. The substrate was left to dry overnight then coated with chromium prior to imaging. An SEM analysis revealed that the average length of the SWNTs was approximately 400 nm with a standard deviation of 50 nm for the 50 hybrids examined. As shown in Fig. 7.11, poly(rU) molecules are seen bound at (a) the end or (b) midpoint of an SWNT(s). In Fig. 7.11(c), possibly two SWNT-poly(rU) hybrids are joined together. Figure 7.11 suggests that poly(rU) molecules remain bound to SWNTs even in dry conditions. However, the poly(rU) appear mostly as globules and no helical wrapping of poly(rU) on SWNTs is evident due to the possible lack of hydrogen bonds with water molecules [66]. Diffusing poly(rU), SWNT-poly(rU), and SWNT-BSA were imaged on an EPI fluorescence microscope. The excitation source of the microscope was a mercury lamp (PTI). Fluorescence was collected with a water immersion objective (Olympus, 60, NA ¼ 1:2) that was then focused onto a CCD camera (Roper, Cascade 512B). The imaging of poly(rU) and SWNT-poly(rU) was excited with blue light while that of SWNT-BSA was excited with green light. Approximately 100 poly(rU), SWNT-poly(rU), and SWNT-BSA hybrids were tracked, respectively, at a rate of six frames per second. The intensity of possible photoluminescence for isolated SWNTs [44, 85, 86] is expected to be much weaker than the fluorescence of the ultrasensitive OliGreen dye (approximately five bases of poly(rU) per dye) with a wavelength shifting to NIR. Our control experiments confirmed this phenomenon in which isolated SWNTs or SWNT hybrids were not visible without fluorescence labeling. Figure 7.12(a) shows a fluorescence image sequence of an SWNT-poly(rU) hybrid diffusing in TE buffer with a time interval of 1.5 s. Figure 7.12(b) displays the corresponding 2D time trajectory of the SWNT-poly(rU) hybrid. The time interval between the consecutive steps is 0.167 s. The diamonds in the trajectory represent the COMs of the hybrid in individual image frames. The histogram of the bootstrap diffusion coefficients [87] as an ensemble was calculated for 1000 sam-

249

250

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

SEM images of a poly(rU) molecule bound at (a) the end and (b) midpoint of an SWNT(s); (c) possibly, two SWNT-poly(rU) hybrids joined together. Scale bar: 100 nm. The SWNTs are seen individually or in very small bundles (a 10 nm in diameter). Tertiary structures of the poly(rU) may prevent their

Fig. 7.11.

nitrogenous bases from fully binding to the SWNT sidewalls. However, limited by the instrument resolution (@2.5 nm), these fine structures were not clearly resolvable. The background structure is due to the chromium coating used to enhance image contrast [66]. (8 American Institute of Physics.)

(a) Fluorescence sequence of an SWNT-poly(rU) hybrid diffusing in TE buffer (1-4). Time interval: 1.5 s; (b) corresponding random walk of the SWNT-RNA hybrid in (a). Time interval: 0.167 s. Scale bars: 1 mm [66]. (8 American Institute of Physics.)

Fig. 7.12.

7.3 Diffusion of SWNT–Biomolecular Complexes

Fig. 7.13. Bootstrap histograms of the diffusion coefficients of SWNT-poly(rU) (solid bars) and SWNT-BSA (hollow bars) in TE buffer. Inset: bootstrap histogram of the diffusion coefficient of poly(rU) in TE buffer.

For each case, approximately 100 hybrids were tracked with 1000 instances of bootstrap sampling. Also illustrated are the Gaussian fitting curves for the three cases [66]. (8 American Institute of Physics.)

pling instances based on a linear fitting of the MSD versus time interval Dt. The stability/resolution of the imaging system was calibrated to be 5  10
5 mm 2 s
1 with fluorescence beads immobilized on glass slides. As shown in Figure 7.13, the diffusion coefficients DSWNT-polyðrUÞ and DSWNT-BSA of the SWNT-poly(rU) and the SWNT-BSA hybrids were measured to be 0:374 G 0:045 and 0:442 G 0:046 mm 2 s
1 , respectively. The numbers after ‘‘G’’ give the standard deviations of the means. By comparison, the mean diffusion coefficient DpolyðrUÞ of poly(rU) alone (inset of Fig. 7.13) was found to be 0:661 G 0:110 mm 2 s
1 , almost twice that for the SWNT-poly(rU) hybrids due to their different conformations [66]. The diffusion coefficient of a small particle in solution, according to Einstein’s relation, can be described by D ¼ kB T=kdrag, where kB is the Boltzmann constant, T is the temperature of the solution, and kdrag is the drag coefficient. For a rodlike particle, kdrag z hd, where h is the viscosity of the solution, and d is the longest dimension of the particle [88], which is approximately 400 nm for the SWNTs used in our diffusion experiment. The SWNT-poly(rU) hybrids, compared with SWNTBSA, exhibit a relatively smaller mean diffusion coefficient that may be induced by the overhangs of the poly(rU) extruding from the ends of the SWNTs (refer to Fig. 7.11a). While the radius of gyration RG of BSA is merely 3 nm [89], as opposed to an order of tens of nanometers for the RG of poly(rU), the diffusion of SWNTBSA is less affected. Here DD is defined as the full-width at half-maximum for the distribution of diffusion coefficient. By Gaussian fitting, DDpolyðrUÞ is found to be 0.235 mm 2 s
1 (inset of Fig. 7.13), a broad distribution attributed to the length variation (500–2600 nucleotides), the instability, and the conformational changes of the poly(rU) molecules (in transient linear stretches, globules, and other tertiary structures). DDSWNT-polyðrUÞ and DDSWNT-BSA for the two hybrids obtained from the Gaussian fittings in Fig. 7.13 are 0.092 and 0.094 mm 2 s
1 , respectively. In compar-

251

252

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

ison with poly(rU), the narrower distributions of the diffusion coefficients for the two hybrids possibly result from the rod-like structure of the SWNTs, which is expected to dominate the diffusion of the hybrids. Following Einstein’s relation and based on the rule of error analysis, it can be derived that Sd ¼ dSD =D, where Sd and SD are the standard deviations of d and D, and d and D are the means of d and D, respectively. Using the measured diffusion coefficients of SWNT-poly(rU), it can be calculated that the standard deviation Sd of the length of SWNT (d) is approximately 48 nm. This deviation agrees with the SEM observation, which found a 12.5% deviation for the length distribution of the SWNT-poly(rU) hybrids. In that regard, a single-molecule diffusion study can be used as a measure for the structural information of SWNTs and their hybrids [66]. The measured mean diffusion coefficients suggest that a SWNT hybrid would take approximately 1–2 min to diffuse across a cell 10 mm in diameter, consistent with the known diffusion coefficients of DNA and proteins within the cell’s cytoplasm [89]. Faster diffusion can be achieved with SWNT hybrids prepared using extended probe-sonication. In future diffusion studies, the persistence length [90], a parameter that describes bending stiffness and flexibility, will be measured for SWNTs. The effect of the binding of nucleic acids and proteins on the persistence length of SWNTs will be also evaluated. These studies will provide knowledge on the flexibility of SWNTs in comparison with biomacromolecules, such as flexible double-stranded DNA, semiflexible actin filaments, or rigid microtubules. These diffusion studies on SWNTs can be extended to cell membranes and nuclei where hindered diffusion [81, 88] and reptation [88, 91, 92] may be significant. These studies facilitate, from a physical viewpoint, the applications of gene transfection using SWNTs as transporters.

7.4

Gene and Drug Delivery with SWNT Transporters

Gene delivery and transfection, or the introduction and expression of foreign DNA, continue to draw strong interest because of their potential for gene therapy and disease prevention potential [93]. One of the greatest challenges for gene delivery is the physicochemical properties of DNA such as its negative charge and hydrodynamic volume. Transporters for DNA must be developed for gene delivery and gene transfection. Solutions to this problem have included viral (retroviral, lentiviral, and adenoviral) vectors and non-viral transfection vectors, including cationic lipids [94], polyethylenimine (PEI), and other cationic polymers [93, 95]. The viral transfection vectors have proven to be the most effective due to their natural ability to introduce foreign genetic information into cells. However, these viral vectors often provoke immune responses from cells, preventing successful gene delivery. Non-viral transfection vectors can avoid immune responses but are often hindered by low efficiency rates for nuclear membrane penetration and gene expression. Even though a few of the cationic polymers such as PEI boost relatively high efficiency rates for non-viral vectors, they exhibit cytotoxicity [96].

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.14. EPI-fluorescence (A) and confocal microscopy (B) images of 3T3 cells incubated at 37  C with 1 and 5 mM concentration of CNT 1, respectively. EPI fluorescence microscopy images of 3T6 cells incubated at 37  C

with 1 (C, D) and 5 mM (E, F) concentration of CNT 2. The nucleus is stained with DAPI (C and E) [97]. (8 The Royal Society of Chemistry.)

Since none of the current transfection vectors are ideal, alternative vectors must be sought through the exploration of new materials and methods, and SWNTs are one possibility. They possess versatile electronic and mechanical properties and have found numerous applications in both nanotechnology and materials science. Because SWNTs have a large surface area, stability, flexibility, and biocompatibility, they have excellent prospects for effective drug and gene delivery, and therapies [28]. Recently, Pantarotto et al. showed that fluorescently labeled SWNTs that covalently bind with bioactive peptides can penetrate cellular and nuclear membranes (Fig. 7.14) [97]. However, the mechanism for the translocation of SWNTs has yet to be determined and, therefore, can only be speculated upon. Shi Kam et al. have reported the appearance of SWNT–streptavidin conjugates within promyelocytic leukemia and T cells via the endocytosis pathway (Fig. 7.15) [98]. 7.4.1

RNA Translocation with SWNT Transporters

To explore the possibilities of SWNTs as transporters for gene delivery and transfection, we conducted a novel examination of the translocation of nucleic acid RNA polymer poly(rU) into breast cancer cells (MCF7). The non-specific binding

253

254

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

(Top) Synthesis and schematic of various SWNT conjugates. (i) EDC, 5-(5-aminopentyl)thioureidyl fluorescein, phosphate buffer; (ii) EDC, biotin-LC-PEOamine, phosphate buffer; (iii) fluoresceinated streptavidin. (Bottom) Confocal images of cells after incubation in solutions of SWNT conjugates: (a) after incubation in 2; (b) after

Fig. 7.15.

incubation in a mixture of 4 (green due to SA) and the red endocytosis marker FM 4-64 at 37  C (image shows fluorescence in the green region only); (c) same as (b) with additional red fluorescence shown due to FM 4-64 stained endosomes; and (d) same as (b) after incubation at 4  C [98]. (8 American Chemical Society.)

mechanism for SWNTs and poly(rU) in our study, as opposed to the covalent binding scheme by Pantarotto et al. and Shi Kam et al. in their respective peptide and protein delivery studies [97, 98], may offer more options and flexibility for the release of the load carried by SWNTs upon delivery. The sectioning property of confocal fluorescence microscopy in our study allows for axial discrimination of fluorescently labeled SWNT-poly(rU) hybrids on cell membranes, within either the cytoplasm, or the nucleus. In addition to confocal fluorescence imaging of SWNT-poly(rU) incubated with MCF7 breast cancer cells, we performed radioisotope labeling, cell enumeration, and an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, which measures

7.4 Gene and Drug Delivery with SWNT Transporters

cellular metabolic activity through absorption. These additional studies quantitatively evaluate the direct cellular uptake and the cytotoxicity of SWNTs. In our studies, SWNT bundles were synthesized using the arc-deposition method, with the dominant SWNT diameter of @1.4 nm [84]. SWNT bundles were probe-sonicated (VC 130 PB, Sonics & Materials) at 8 W for 90 min on ice in 10% FBS/PBS buffer. The SWNTs at a concentration of 0.4 mg mL
1 were filtered through a 0.45 mm filter before incubating with MCF7. Both the control cells and the cells treated with SWNTs were enumerated at 24, 48, and 72 h. The cell growth medium was RPMI supplemented with 10% FBS and 1% penicillin streptomycin. After the designated time of incubation, cells were released by trypsin-EDTA treatment (5 min) and counted with hemocytometry. Precautions were also taken to prevent contamination in preparing the SWNTs, including filtration to remove the bacteria and sterilization of the instruments in contact with the sample. The MTS assay was performed after treating MCF7 cells with various concentrations of SWNTs (0, 0.0125, 0.025, 0.05, 0.1, 0.125, 0.25, 0.5, and 1 mg mL
1 ). After 24 h of incubation, the growth medium was removed from the 96-well-plate and PBS (200 mL) was used to wash the cells twice. RPMI-1640 medium without phenol red (approx. 200 mL) and MTS (25 mL) were added to each cell well, followed by incubation at 37  C for 3 h. The absorbance in each well was measured using a spectrophotometric plate reader (Benchmark Microplate Reader, Bio-Rad) at a wavelength of 490 nm. The translocation of SWNTs into cells was confirmed by radioisotope labeling assay. SWNTs were probe-sonicated in RPMI-1640 growth medium and incubated with radioactive [methyl- 3 H]thymidine overnight. The radioactively labeled SWNTs were collected by centrifugation and re-suspended in PBS before incubating with MCF7 cells. After incubation, the cells were released by trypsin-EDTA and recollected as pellet by centrifugation. The pellet of MCF7 cells was thoroughly washed with PBS buffer twice to remove excess SWNTs and thymidine bound on the cell surface. The scintillation counts (Beckman Coulter, LS6500) read from the MCF7 cells, as opposed to those from the supernatants and the PBS washing buffer, yielded the translocation efficiencies of the radioactively labeled SWNTs at 1.3%, 6.3%, 10.7%, and 15.4% for incubation times of 0.5, 1, 2, and 4 h, respectively [67]. This result confirms that SWNTs alone can penetrate through cellular membranes with an increased efficiency over time, and concurs with the general understanding that cell membranes intake small hydrophobic particles. For the imaging experiment, the SWNT-poly(rU) hybrids were prepared as follows. A mixture of poly(rU) (0.5 mg mL
1 ) and SWNTs (0.125 mg mL
1 ) in TE (10 mm Tris-HCl, 1 mm EDTA) buffer was probe-sonicated. From scanning electron microscopy and single-molecule diffusion studies, the prepared SWNTpoly(rU) hybrids were characterized in small bundles or isolated form with an average length of 400 nm [66]. To visualize the SWNT-poly(rU) hybrids, poly(rU) was fluorescently labeled with PI, which has an excitation peak at 535 nm and an emission peak at 617 nm. PI intercalates between the base pairs of nucleic acids, it is membrane impermeable, and is generally excluded by viable cells. To achieve fluorescent labeling, PI

255

256

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

(0.05 mL mL
1 ) in TE buffer was incubated with SWNT-poly(rU) hybrids at a volume ratio of 1:20 for at least 15 min. Excess PI and unbound poly(rU) were removed by cassette dialysis (Slide-A-Lyzer, 10,000 MWCO, Pierce) for a total of 36 h with three changes of buffer solution. Approximately 10 000 MCF7 cells were deposited in each well of an eightchambered slide to form a sparsely distributed layer of cells to ensure good exposure of cell membranes to SWNT-poly(rU) hybrids. Cells were directly cultured on the chamber glass slide using RPMI-1640 growth medium at 37  C under 5% CO2 . All cells were incubated for 24 h until an approximate 60% confluence was achieved. Twenty mL of PI, PIþpoly(rU), or PI-labeled SWNT-poly(rU) hybrids (0.05 mL mL
1 ) was added to the chamber slide, and the cells were incubated for 2, 3, or 4 h. After incubation, the cells were washed twice with growth medium (200 mL) and kept in PBS (400 mL) before imaging with the confocal fluorescence microscope (LSM510, Zeiss, objective NA ¼ 1:3, oil). Fluorescence from PI was collected in the TRITC channel, and the background was recorded from the bright-field channel of the microscope. The confocal fluorescence images of the two controls, PI and PIþpoly(rU), showed little to no fluorescence after incubation and the MCF7 cells appeared elongated and healthy (Fig. 7.16). In contrast, the PI control (Fig. 7.16a) showed one instance of intense fluorescence from a PI stained circular unhealthy cell. The minimal fluorescence intensity surrounding the cellular membrane for the PIþpoly(rU) (Fig. 7.16b) was probably due to electrostatic interactions between the charged poly(rU) and cellular membrane. The confocal fluorescence images of the MCF7 cells that were incubated with the SWNT-poly(rU) hybrids for 2 h showed translocation into the cytoplasm without accumulation of SWNT-poly(rU) hybrids on the outside of the cellular membrane. Figure 7.17 shows MCF7 cells appearing to retain the SWNT-poly(rU) hybrids after an incubation of 3 h. All the images were taken from a stack of slices scanned through the cells with a full scanning depth of approximately 10 mm and

Control experiments showing little to no uptake of (a) PI, except in the dying circular cell, and (b) PIþpoly(rU). Scale bar ¼ 10 mm [68]. (8 American Chemical Society.) Fig. 7.16.

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.17. Confocal fluorescence images of MCF7 cells incubated with 0.05 mL mL
1 of PI-labeled SWNT-poly(rU) for 3 h. Images (a)–(h) were acquired at different depths (z ¼ 0:5; 1:51; 2:52; 3:53; 4:54; 5:55; 6:56; 7:57

mm), across the z-axis normal to the chamber slide surface. The arrows point to the fluorescent spots where large SWNT-poly(rU) hybrids are localized. Scale bar ¼ 10 mm [68]. (8 American Chemical Society.)

a scanning step of 1.01 mm. Smaller red spots with low intensities as shown in Figs. 7.17(a–e) are thought to be small soluble SWNT-poly(rU) hybrids and/or PIlabeled poly(rU) dissociated from SWNTs after translocation. Their gradual disappearance (Figs. 7.17f–h) implies the uneven distribution of SWNT-poly(rU) hybrids in cytoplasm and/or possibly the defocusing of the hybrids. Figure 7.17(b) shows the uptake of an SWNT-poly(rU) hybrid in the cytoplasm, as indicated by the arrow. Figure 7.17(d) shows an instance of an SWNT-poly(rU) hybrid appearing in the vicinity of cell membrane; however, whether the exact location of the hybrid is within or on the cell membrane is unclear. In Figs. 7.17(f ) and (g), the fluorescent spot was observed to co-localize with the nucleus, which supports the possibility that the SWNT-poly(rU) hybrid could have penetrated through the nuclear membrane. The observed fluorescence in Figs. 7.17(f ) and (g) could also be from PI-labeled poly(rU) released from SWNTs due to dissociation kinetics and the change of pH between cytosol and nucleus [68]. The released nucleic acids may be used as templates for transfection at a later stage. Energetically, the fluorescence in the nucleus is unlikely to be from either free PI or PI dissociated from poly(rU) and then re-intercalated with the host

257

258

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

DNA or RNA. However, the binding dynamics of SWNTs and nucleic acids are beyond the scope of this chapter. The strong fluorescence in Figs. 7.17(f ) and (g) fades gradually [Figs. 7.17(e) and (h)], which further indicates that the SWNTpoly(rU) hybrid was localized within the cell. From the depth information, we estimate that this large SWNT-poly(rU) hybrid is less than 2 mm long. This length is consistent with that of the SWNTs obtained with probe sonication, confirming that the observed fluorescence was from the SWNT-poly(rU) hybrid. After 4 h of incubation the SWNTs were still present within the MCF7 cytoplasm and nucleus [68]. The uptake of the SWNTs-poly(rU) is hypothesized to be a result of amphipathic properties of both the cellular membrane and the SWNT-poly(rU) hybrids. Due to thermal agitation, lateral diffusion of phospholipids within the biomembrane may contribute to the translocation of SWNT-poly(rU) hybrids by allowing hydrophobic interactions between the hydrocarbon chains and SWNTs within the bilayer. The uptake of the SWNTs by the nuclear membrane is attributed to passive ratchet diffusion [99]. Cell mitosis might also play a significant role in the internalization of the SWNT-poly(rU) hybrids. During cell division, the nuclear envelope breaks down into multiple small vesicles early in mitosis, which may allow the translocation of SWNT-poly(rU) hybrids. In the telophase, the last mitotic stage, the nuclear envelop reforms, possibly incorporating the SWNT-poly(rU) hybrids. Once the SWNT-poly(rU) hybrids are within the MCF7 cells, it is possible that endosomes in the cytoplasm store SWNTs after poly(rU) translocation. In summary, we have demonstrated the delivery of RNA polymer using SWNTs as transporters [68]. Translocation of SWNTs into MCF7 cells was confirmed by radioisotope labeling assay. Based on the sectioning property of confocal microscopy, fluorescently labeled SWNT-poly(rU) hybrids were found across the cellular and the nuclear membranes of MCF7 cells while the controls were excluded. Both cell growth and MTS studies have shown no cytotoxicity in either MCF7 breast cancer cells or d2C keratinocytes for concentrations up to 1 mg mL
1 . These studies show the potential of using SWNTs as transporters for gene delivery, but further biophysical and biochemical studies must be conducted to better decipher the mechanisms of SWNT translocation across cellular and nuclear membranes. Assays on gene transfection using SWNT transporters need to be performed to move towards the final goal of gene therapy and disease prevention. 7.4.2

Gene Transfection with SWNT Transporters

Limited literature is available on gene transfection with CNT transporters, with the only proofs of concept being reported by Pantarotto et al. [100] and Liu et al. [101]. The efficiencies of DNA transfection in mammalian cells were not optimal in these preliminary studies, yet they showed at least one order of magnitude enhancement over DNA alone. Pantarotto [100] revealed that both SWNTs and MWNTs were covalently modified by pyrrolidine rings, each bearing a free amino-terminal oligoethylene glycol moiety attached to the nitrogen atom. The concentration of the functional groups

7.4 Gene and Drug Delivery with SWNT Transporters

was 0.55 and 0.90 mmol g
1 for functionalized-SWNTs and MWNTs, respectively. The presence of these functional groups led to a much increased solubility of the CNTs in aqueous solution. The electrostatic interaction of positively charged ammonium functionalized CNTs with the negatively charged phosphate backbone of plasmid DNA was confirmed by TEM. Plasmid DNA molecules, encoded with marker gene (b-galactosidase; b-gal) were seen adopting spherical, toroidal, or supercoiled structures 15–300 nm in diameter when exposed to the positively charged groups [100]. The varying degree of plasmid condensation could be related to the charge density, the hydrophobic character of the interaction, and the number of plasmid DNA molecules in the condensate [102]. Tighter packing of functionalized SWNTs was noticeable in regions of larger CNT bundles. The interaction of functionalized CNTs with HeLa cells was also visualized by TEM. HeLa cells were incubated with ammonium functionalized CNTs at a concentration of 2.5 mg mL
1 for 1 h and the mixture was then embedded in an epoxy resin. Solidified resin was sliced by an ultramicrotome and the slices were examined by TEM. CNTs were spotted inside the cells; higher magnifications provided more convincing evidence of their internalization. Nuclear localization and the translocation of CNTs through plasma cell membranes were also seen. The mechanism hereby was believed to be the binding of the cationic functional groups on the CNTs to the cell membranes, which could be facilitated by spontaneous insertion of the CNTs across the cell membranes. Subsequent translocation and diffusion of the functionalized CNTs within the intracellular space could occur following these nonendocytotic processes [100]. The ability of ammonium-functionalized SWNTs to enter cells and potentially reach their nuclei resulted in the delivery of plasmid DNA to CHO cells. Hypothetically, these functionalized SWNTs entered the cells via a spontaneous mechanism in which the tubes pierced through the cell membranes due to their enormous aspect ratio. This model is consistent with molecular dynamics simulations which predicted that hydrophobic CNTs with hydrophilic functional groups could spontaneously insert into a lipid bilayer. Both the hydrophobicity of SWNTs and the rapid lateral diffusion of lipids in the membrane bilayer could contribute to the uptake of SWNT-plasmid DNA complexes [100]. The rate of gene transfection depended on the charge ratio of the ammonium groups on the SWNT surface to the phosphate groups of the DNA backbone. Gene expression efficiencies 5–10 higher than those without the presence of SWNTs were obtained when the charge ratio was maintained within the range of 2:1 to 6:1. Gene expression also increased with incubation time of up to 3 h and decreased thereafter. Although the measured transfection efficiency was far from optimal, functionalized SWNTs seem to offer ample opportunities for chemical and biological modifications than do cationic macromolecules such as peptides, dendrimers, and liposomes. The other significant advantage that functionalized SWNTs have over conventional gene and drug delivery methods is their significantly reduced cytotoxicity [100]. In the study conducted by Liu et al. [101], dendritic polyethylenimine (PEI), a most efficient and popular polymer for gene delivery, was grafted onto MWNTs to

259

260

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

anchor DNA. Carboxylic acid groups were first created on the side walls of the MWNTs by heating the tubes at reflux in 3 m nitric acid. The carboxylic acid groups were then transformed into acyl chloride groups by consecutive treatments of thionyl chloride and ethylenediamine [26, 57]. PEI was then grafted onto MWNTs based on the activated monomer mechanism or the activated chain mechanism. Protonated aziridine monomers or the terminal iminium ion groups of propagation chains were transferred to amines on the surface of the MWNTs to form PEIg-MWNTs complexes. DNA encoded with pCMV-Luc gene was then immobilized securely onto the complexes through strong electrostatic interactions arising from the amines. The formation of DNA-PEI-g-MWNT complexes was confirmed by the total inhibition of DNA migration in gel electrophoresis [101]. The PEI obtained by cationic polymerization of aziridine had a dendritic structure that contained primary, secondary, and tertiary amines with a molar ratio of about 1:2:1 [103]. The polymer chemistry of the PEI should not have been affected by their grafting onto the surface of MWNTs. Grafted PEI with a high content of primary, secondary, and tertiary amines could function as anchor points for the immobilization of DNA onto the surface of MWNTs. Consequently, the migration of DNA was totally inhibited in gel electrophoresis when the weight ratio of PEI-gMWNTs to DNA was about 4:1. By comparison, the control experiments using MWNTs and NH2 -MWNTs showed little inhibition on the migration of DNA, even at a high weight ratio of 100:1. This is because the nonspecific adsorption of PEI on the surface of MWNTs did not facilitate the secure anchoring of DNA, possibly due to weak interactions between the PEI and MWNTs [101]. The PEI-g-MWNTs gene transporters created in the Liu study [101] yielded more encouraging results than the Pantarotto scheme [100], which used functionalized SWNTs. Figure 7.18 details a head-to-head comparison of transfection efficiency in human embryonic kidney 293 cells for DNA-PEI (25 kDa), DNA-PEI-g-MWNTs, and DNA alone. Under the optimal conditions, the transfection efficiency using PEI-g-MWNTs was three times higher than that using PEI, and, remarkably, four orders of magnitude higher than that using naked DNA. Transfection efficiencies using PEI-g-MWNTs in COS7 and HepG2 cells, under optimal conditions, were consistently 2 12 times greater than efficiencies using PEI, and were much higher than efficiencies using DNA [101]. The mechanisms proposed and speculated upon in the literature for CNT uptake or their derivatives by cells include phagocytosis, endocytosis, insertion, or passive diffusion. Liu et al. [101] attributed DNA uptake to endocytosis because PEI-gMWNTs, once labeled with fluorescein isothiocyanate and left incubating in cells for 1 h, significantly reduced their presence in cells with increasing temperature. The high transfection efficiency obtained using PEI-g-MWNTs was perhaps due to the secure anchoring of DNA onto the surface of MWNTs. The proton-sponge effect of the grafted PEI could allow the DNA-PEI-g-MWNTs complexes to escape easily from highly acidic endosomes or other vesicles in cells. The larger complexes of DNA-PEI-g-MWNTs could have improved the proton-sponge effect of PEI and facilitated a more effective sedimentation onto the cells [101].

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.18. Transfection efficiency of PEI-g-MWNTs for DNA delivery relative to that of PEI and naked DNA in 293 cells. The level of pCMV-Luc gene expression is given in RLU per mg of protein for quadruplicate runs [101]. (8 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

7.4.3

Gene Transfection with SWNT Transporters for RNA Interference

In addition to its fundamental roles in gene transcription and translation, RNA has been used for two decades to reduce (or interfere with) expression of targeted genes in various systems such as plants, fungi and higher organisms. This process, known as RNAi, is a natural defense mechanism that is thought to have evolved to protect organisms from viral diseases. Many viruses have a genetic blueprint made from RNA instead of DNA. The genetic information of many viruses is held in the form of double-stranded RNA and an enzyme known as Dicer first chops the double-stranded RNA of invaded viruses into small segments of genetic code of approximately 22 bases long. These segments, known as siRNAs, are surprisingly stable and, unlike ssDNA or RNA anti-sense oligonucleotides, do not need extensive modification to survive in tissue culture media or even within living cells. These newly formed siRNA then separate into single strands and some bind to intact lengths of single-stranded viral RNA. Finally, proteins target this tagged viral RNA and destroy it. As a result, RNAi shuts off key viral genes, potentially nipping infections in the bud. In theory, RNAi can be used to treat any disease such as cancer, which is an overactive gene or genes, or HIV, a virus with no cure and vaccine. In one of the most exciting developments thus far, Dai’s group reported gene silencing with SWNT delivered siRNA [104]. Phospholipids (PL) were adsorbed

261

262

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

onto SWNTs and the head groups of the PL were covalently linked by singlechained poly(ethylene glycol) (PEG, MW ¼ 2000) with terminal amine or maleimide groups (PL-PEG-NH2 or PL-PEG-maleimide). The PL-PEG bound to SWNTs via van der Waals and hydrophobic interactions between the fatty alkyl chains of the PL and the side walls of the SWNTs. Although double-chained PL molecules do not solubilize SWNTs, the presence of the PEG greatly increased the hydrophilic moiety of the PL-PEG-SWNT complexes, rendering SWNTs soluble in an aqueous solution. The amine or maleimide terminal on the PL-PEG immobilized on SWNTs was used to conjugate biofunctionalities such as DNA and siRNA. A crosslinker, sulfosuccinimidyl 6-(3 0 -[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP), was attached to a thiol-containing biomolecule (X) to form SWNT-PL-PEG-SS-X (1-X). The biomolecule X was either 1-DNA (15-mer DNA with fluorescence label Cy3) or 1-siRNA. No disulfide linkage was employed in the control experiments [104]. In the study by Dai’s group, biomolecules DNA or siRNA were transported by functionalized SWNTs into mammalian cells. SWNTs and their derivatives, after the cellular internalization of biomolecules, were believed to be allocated in endosomes and/or lysosomes. The disulfide bonds linking the biomolecules and the PLPEI complexes were cleaved and the biomolecules were released by enzymes in lysosomal compartments whose pH (@5) is significantly lower than that of the cytosol (pH 7.2). Translocation of SWNT complexes across the cell membranes was believed to result from endocytosis [104]. Active releasing of endocytosed species from endosomes or lysosomes allows molecule cargoes to reach their intended destinations, thus preventing degradation inside the endosomes or lysosomes [105]. No effect on cell viability or proliferation was found by the presence of SWNTs in this study. Both 1-siRNA and 2-siRNA were prepared where the siRNA was capable of silencing the gene encoding lamin A/C protein present inside the nuclear lamina of cells. HeLa cells were again used to incubate with 1-siRNA (50–500 nm) for up to 24 h. The concentration of SWNTs was 10 nm and cells of 40 000 per well in the presence of 5% FBS were fixed 48–72 h later. The cells were then stained with antilamin and fluorescently labeled secondary antibodies. The commercial transfecting agent lipofectamine (1 mg L
1 ) was used for comparison with the SWNT delivery system. Minimal fluorescence was observed in the experiment (Fig. 7.19b) compared with that of the untreated control (Fig. 7.19a), indicating a significant reduction in the expression of lamin A/C proteins by 1-siRNA. The potency of RNAi was characterized using flow cytometry in the order 1-siRNA, 2-siRNA, and lipofectaminesiRNA, for a given concentration of siRNA (Fig. 7.19c). The higher silencing potency with 1-siRNA than 2-siRNA is possibly due to the release of siRNA from SWNTs by breaking their linking disulfide bonds. This released and subsequently more effective siRNA can then readily escape from endosomes or lysosomes. This two-fold increase in the silencing potency using 1-siRNA compared with lipofectamine was attributed to the high surface area of SWNTs for siRNA cargo loading, the high intracellular transporting ability of SWNTs, and the high degree of

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.19. Confocal microscopy RNAi assay for (a) untreated control HeLa cells and (b) cells incubated with 1-siRNA, the latter showing a much weaker fluorescence than (a) due to silencing of the expression of lamin protein by RNAi. (c) Silencing efficiency of lipofectaminsiRNA (left-hand bars), 2-siRNA (middle bars)

and 1-siRNA (right-hand bars) for 50 and 500 nm siRNA concentrations. Cells were fixed and stained with anti-lamin and a fluorescently labeled secondary antibody prior to analysis. The confocal images were captured at similar experimental settings for (a) and (b) [104]. (8 American Chemical Society.)

endosome/lysosome escape owing to the breakage of the disulfide bond. Excellent siRNA delivery and silencing of the luciferase gene were also observed [104]. This study has shown great promise for using SWNT as molecular transporters for gene therapy. 7.4.4

Drug Delivery with SWNT Transporters

This section reviews recent developments in drug delivery with SWNT transporters, and highlights the research by Pantarotto et al. on immunization with peptide-functionalized SWNTs for enhancing virus-specific neutralizing antibody responses [28], and the research by Dai’s group on intracellular protein transport for inducing apoptosis and on the highly sensitive electronic detection of antibodies associated with human autoimmune diseases [106]. Vaccine Delivery by SWNTs Developing new and effective delivery approaches is of paramount importance for administering protective antigens. SWNTs could serve as excellent transporters for 7.4.4.1

263

264

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

the effective utilization of antigens that have previously been unable to induce adequate or appropriate responses, thus providing significant means of enhancing and modulating immune responses. In vaccine delivery, conserving the conformation of antigen is crucial. This conservation, in turn, yields the induction of antibody responses with the right specificity. Therefore, the presence of SWNTs should not change the biofunctionality of the antigen when they are linked together. Pantarotto et al. evaluated the capacity of mono- and bis-derivatized SWNTs to present B cell epitope from the VP1 coat protein of FMDV [107]. The knowledge obtained from this study served to enhance the neutralizing potential of the B cell epitope. The chemical route for obtaining water-soluble SWNTs is based on the methodology described in Section 7.2.1.2. SWNTs were functionalized with a pyrrolidine ring through the 1,3-dipolar cycloaddition of azomethine ylides [108]. The aminoderivatized SWNTs were subsequently used to covalently link a series of peptides by different strategies. In one approach, B cell epitope, a peptide corresponding to the sequence 141-159 derived from VP1 protein of FMDV, was coupled to functionalized SWNTs, employing a selective chemical ligation to obtain the mono-conjugate 2 [28]. In another approach, functionalized SWNTs were covalently attached to two FMDV peptides to yield the bis-conjugate 3. The latter approach involved a derivatization of the free amino groups of SWNT 1 using an excess of Boc-Lys(Boc)-OH activated with DIC (diisopropylcarbodiimide) and HOBt (1-hydroxybenzotriazole) in DMF, and subsequent cleavage of Boc (tertbutyloxycarbonyl) with TFA (trifluoroacetic acid). The free amino groups were neutralized with DIEA (diisopropylethylamine) and coupled with N-succinimidyl 3-maleimidopropionate in DMF [108]. Excess reagent was removed by adding an amino-PEGA resin. The N-terminal acetylated FMDV 141-159 peptide, with an additional cysteine, was dissolved in water and linked to the two maleimido moieties [109]. Unreacted peptides were removed using a scavenger resin, and reverse-phase HPLC confirmed a peptide covalent bond formation to the SWNTs. The solubilities of the mono-conjugate 2 and the bis-conjugate 3, were 18.0 and 12.5 mg mL
1 , respectively [28]. The activity of the FMDV 141-159 peptide was confirmed using a monoclonal assay. Anti-FMDV 141-159 peptide mAb was presented to a covalently linked antimouse Fcg antibody. SWNTs alone did not react with mAb, while free peptides, mono-conjugates 2, and bis-conjugates 3 all interacted with mAb in increasing masses [28]. However, an analysis of the three sensorgrams showed no difference in the association rate constants and only a small decrease of the dissociation rate constant for the bis-conjugate 3, which might be ascribed to the avidity of the bivalent reagent [28]. Thus both mono- and bis-conjugates preserve the conformation of their attached peptides. An immunization protocol was tested on the coupling of nonimmunogenic peptides to SWNTs. Anti-peptide antibody responses were measured by ELISA using BSA-conjugated FMDV141-159 peptide as antigen (Fig. 7.20 left). The sensitivity of the ELISA was greater than that for the free peptides. The anti-FMDV 141159 antibody response slightly increased when OVA was injected with the monoconjugate 2. Using the bis-conjugate 3 as an immunogen (Fig. 7.20 left) also

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.20. (Left-hand side): Anti-peptide antibody responses following immunization with peptides and peptide–SWNTs. Groups of BALB/c mice were co-immunized intraperitoneally with OVA and free FMDV 141-159 peptide, mono-conjugate 2, or bisconjugate 3 in Freund’s adjuvant emulsion. Serum samples collected 2 weeks after the booster immunization (on day 14 post priming) were screened by ELISA for the presence of antibodies using FMDV 141-159 peptide conjugated to BSA (solid bars), control peptide conjugated to BSA (open bars), or

SWNT 1 functionalized with maleimido group without peptide (hashed bars) as solidphase antigens. Data represent mean of log10 (antibody titers) from five mice per group. (Right-hand side): Neutralization indices of serum samples of immunized mice. Serum samples were collected 2 weeks after the boost of mice co-immunized intraperitoneally with OVA and free FMDV 141-159 peptide, monoconjugate 2, or bis-conjugate 3 in Freund’s emulsion. Data represent the mean of antibody titers from five mice per group [28]. (8 Elsevier Science Ltd.)

significantly elevated the anti-peptide antibody titers. Antibody responses were specific to the FMDV 141-159 peptides but not to the functional groups linking peptides to the SWNTs, because serum antibodies showed little reactivity to control peptides conjugated to BSA (Fig. 7.20 left). Both the peptide conjugates and OVA were sampled by antigen-presenting cells. Assistance from the OVA-specific T helper cells enabled the B cells to produce antibodies that recognized the FMDV 141-159 peptides. No anti-SWNT antibodies were detected (Fig. 7.20 left) [28]. The bystander help provided by the carrier (OVA) should be directed to the covalently

265

266

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

attached peptides and not to the SWNTs. This study indicated that SWNTs alone do not possess intrinsic immunogenicity. Mono-conjugate 2 elicited virus-neutralizing antibody responses in mice (Fig. 7.20 right), which were significantly higher than those induced by the bisconjugate 3 or the control free peptide. An increased number of epitopes at the surface of SWNTs enhanced the immunogenicity of the attached peptides while decreasing the neutralizing titers. It was hypothesized that both copies of the FMDV peptides of the bis-conjugate 3 assumed different conformations than that of their native state, thus failing to invoke antibody responses with the correct specificity [28]. The preferential induction of peptide-specific antibodies with enhanced virus neutralizing capacity using mono-conjugate 2, coupled with the nonresponsiveness to the attached SWNTs, demonstrated the presentation of the attached peptides in vivo, suggesting their possible use as antigen delivery systems since no anti-carbon antibodies were elicited that could influence immune responses to their attached peptides. Protein Delivery by SWNTs The Shi Kam and Dai study [106] explored protein delivery with SWNTs. The proteins used, including streptavidin (SA), protein A (SpA), BSA, and cytochrome c (cytc), were fluorescently labeled and bound to the side walls of SWNTs via a noncovalent and nonspecific mechanism (Section 7.2.2). Translocation of these proteins in mammalian cell lines, including HeLa, NIH-3T3 fibroblast, HL60, and Jurkats, cells was observed by confocal fluorescence microscopy. Endocytosis, an energy dependent internalization mechanism based on the engulfing of the foreign particles, was believed to be responsible since the uptake showed a distinct temperature dependence. For example, incubating the cells in protein–SWNTs conjugates at 4  C yielded little uptake [106]. Cellular uptake of large proteins (molecular weight > 80 kDa) was poor, while the binding and intracellular protein transport by SWNTs appeared general for small- and medium-sized proteins [106]. Figure 7.21 compares the fluorescence level detected for cells incubated with proteins alone, and for cells incubated with protein–SWNTs; this suggests that while proteins in solutions were unable to traverse across cell membranes, SWNTs were able to transport protein cargoes inside cells possibly due to their hydrophobicity and high aspect ratio. SWNT-protein conjugates, once internalized within the cells, were found co-localized with red endocytosis endosome marker FM 4-64 [98], suggesting the confinement of the conjugates in endosomal lipid vesicles. Once within cells, the endosomes could fuse with lysosomes, and cause the degradation of the internalized species in the acidic lysosomes. To prevent lysosomal degradation, chloroquine was added to the cell medium during the incubation of cells in protein–SWNT conjugates to trigger an endosomal release of internalized molecules into the cell cytoplasm. Chloroquine is a membrane-permeable base that can localize inside endosomes and cause increases in pH. The resulting osmotic pressure led to swelling and eventual rupture of the endosomal compartments (Fig. 7.22). This phenomenon suggests that it is possible to create biological functionality for these internalized ‘‘cargo’’ molecules. 7.4.4.2

7.4 Gene and Drug Delivery with SWNT Transporters

Fig. 7.21. Cell cytometry data for untreated HL60 cells (labeled as ‘‘control’’), and for HL60 cells incubated in solutions of fluorescently labeled SA and SpA (a), and

treated with the respective fluorescent protein– SWNT conjugates (b). Cells were incubated in protein–SWNTs solutions for 2 h [106]. (8 American Chemical Society.)

HeLa and NIH-3T3 cell lines, known to undergo cyt-c-induced apoptosis [110], were used for intracellular transport of cyt-c with SWNTs and for apoptosis assay. After cell incubation in the cyt-c-SWNT conjugates, apoptosis was analyzed using fluorescently (FITC) labeled Annexin V. Annexin V-FITC is an efficient marker for early stage apoptosis as it binds to the phospholipid flipped from the inner to the outer leaflet of the plasma membrane during apoptosis. For NIH-3T3 cells in the presence of chloroquine and incubated, respectively, with cyt-c-SWNT conjugates and with cyt-c alone, the degree of Annexin V staining was characterized by both confocal microscopy (Fig. 7.23a, b) and cell flow cytometry (Fig. 7.23c). Significantly higher percentages of apoptosed cells were observed when incubated with cyt-c-SWNT conjugates than with cyt-c alone [106]. Figure 7.23(c; inset) shows the

Fig. 7.22. Endosomal rupture. Cells were incubated with (a) cyt-c-SWNT conjugate and (b) cyt-c-SWNT þ 100 mm of chloroquine at 37  C and 5% CO2 . Confocal images were taken immediately after incubation and washing.

These images indicate the release of protein– SWNT conjugates from the endosome: overall green color across the cell in (b) vs. green individualized spots inside the cells in (a) [106]. (8 American Chemical Society.)

267

268

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Apoptosis induction by cytochrome c cargos transported inside cells by SWNTs. (a) Confocal image of NIH-3T3 cells after 3 h incubation in 50 mm cytochrome c alone (no SWNT present) and 20 min staining by Annexin V-FITC (green fluorescent). (b) Cell images after incubation in 50 mm cytochrome c-SWNTs in the presence of 100 mm chloroquine and after Annexin V-FITC staining. (c) Cytometry data of the percentage of cells undergoing early stage apoptosis (as stained by Annexin V-FITC) after exposure to 100 mm chloroquine only (labeled ‘‘untreated’’),

Fig. 7.23.

SWNT þ 100 mm of chloroquine, 10 mm cytc þ 100 mm chloroquine, 10 mm cyt-cSWNT þ 100 mm chloroquine, and cyt-c-SWNT without chloroquine. Inset: representative confocal image of the blebbing of the cellular membrane (stained by Annexin V-FITC) as the cell undergoes apoptosis. Note that PI costaining was used, and all data shown here excluded PI-positive cells and were recorded 4– 5 h after exposure to chloroquine. The level of PI staining for all cells was a normal @4–6% out of @10 000 [106]. (8 American Chemical Society.)

blebbing of the cellular membrane stained by Annexin V-FITC, a phenomenon associated with cells undergoing apoptosis. To investigate the effect of endosomal release on the efficiency of apoptosis induction by cyt-c transported by SWNTs, cells were incubated in cyt-c-SWNT in both the presence and absence of chloroquine. Higher degrees of apoptosis were consistently observed for cells treated with cyt-c-SWNT in the presence of chloroquine (Fig. 7.23c). This higher degree of apoptosis is due to the more efficient endosomal releasing of proteins, suggesting that the cytochrome c bound and transported inside cells by SWNT carriers remained biologically active for apoptosis induction [106]. However, it is unclear if the functionality of cytochrome c was ob-

7.5 Sensing and Treating Cancer Cells Utilizing SWNTs

tained after detaching from the SWNTs, or if it remained effective even when proteins were still bound to the SWNTs. Biosensing by SWNTs In a separate study by Dai’s group [17], SWNTs were employed as biosensors for potential medical diagnostic and biological applications. In this scheme (Fig. 7.1 left), SWNTs were coated with U1A antigen-Tween conjugates and the immobilized antigens were recognized by 10E3 mAbs. The U1A RNA splicing factor is a prominent autoantigen target in systemic lupus erythematosus and mixed connective tissue disease, and detection of its autoantibodies by ELISA forms a clinical fluorescence assay. With SWNT sensors, binding can be monitored in real-time electronically without labeling, with the resulting sensitivity at a remarkable 1 nm or less. Similar results were obtained with two other U1A-specific mAbs. In contrast, two different mAbs (3E6 and 6E3) specifically used for TIAR (a structurally related but different RNA binding protein autoantigen) failed to recognize the U1A splicing factor. This result compares favorably with fluorescence-based detection of immobilized antigens, where the limit of detection was found to be 2.3 nm [17]. In addition to offering a higher sensitivity, Dai’s scheme also permitted sample preparation and detection to be performed in solution without protein denaturation. This study serves as a proof of concept that SWNT devices can detect clinically and biologically important interactions, with a potential for screening assays of mAb panels for use as reagents or therapeutics. Since the human proteome contains more than 300 000 different protein isoforms, methods for high-throughput screening of mAbs are crucial for their identification. Furthermore, multiplex analysis of autoantibodies may be performed with arrays of SWNT devices to diagnose patients with autoimmune diseases such as HIV, complementing or overtaking other recently developed techniques such as planar array-based methods [17]. 7.4.4.3

7.5

Sensing and Treating Cancer Cells Utilizing SWNTs

A milestone in the possible use of SWNTs to diagnose and treat cancer was recently reported by Dai et al. [32]. They demonstrated how several unique characteristics of SWNTs could be combined to serve the purpose of selectively causing cancer cell destruction. SWNTs functioned as multifunctional biological transporters for cargoes such as Cy3-DNA, various phospholipids, and folate moiety. The destruction of the cancer cells was due to the strong absorbance of translocated SWNTs in the NIR range (700–1100 nm) because of distinct first and second van Hove optical transitions (Fig. 7.24) [44]. Ex vitro studies showed an increase to approximately 70  C after heating SWNTs of various concentrations for 2 min with continuous 808 nm laser light [32]. Heating was caused from optical stimulation of electronic excitations of SWNTs that was transferred to molecular vibration energies and heat. The transfer to molecular

269

270

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Semiconducting SWNTs electronic structure and density diagram of electronic states. Optical emission and excitation transitions are represented by solid arrows; dashed arrows show non-radiative relaxation of

Fig. 7.24.

the electron in the conduction band and of the hole in the valence band before emission [86]. (8 American Association for the Advancement of Science.)

vibration energies and heat has another interesting effect if pulsed NIR laser light is discharged. The pulsed optical stimulation of SWNTs seemed to cause the release of their cargoes, as demonstrated with confocal fluorescent images of Cy3DNA-SWNT complexes (Fig. 7.25). Perhaps more important than the demonstration of SWNT complex translocation is the preservation of cell viability. As previously shown, SWNT complexes below certain concentrations have no adverse affect on cell viability, and biological systems like cells experience limited or no damage because they are completely transparent to continuous NIR light. These two noninvasive aspects were combined to induce the destruction of cancer cells. Once cancer cells internalized SWNT complexes and were exposed to continuous NIR laser light, they experienced excessive heating and eventual death [32]. After exposure to this treatment, cells appeared round with aggregated SWNT complexes appearing within the growth medium (Fig. 7.26 left). Another key issue involving cell viability in this experiment is the ability of cancer cells to selectively internalize SWNTs, thus ensuring that they can destroy harmful cells while preserving healthy ones. Specifically, SWNTs were conjugated with various phospholipids (PL), a poly(ethylene glycol) (PEG) moiety, and terminal folic acid (FA) group that provided specificity for this selective internalization (Fig. 7.27). Additionally, the preparation of cancer cells required some ingenuity

7.5 Sensing and Treating Cancer Cells Utilizing SWNTs

Fig. 7.25. NIR laser light excitation of Cy3DNA-SWNT complexes causing DNA release and nuclear translocation. Confocal image of HeLa cells after 12 h incubation with 2.5– 5 mg L
1 Cy3-DNA-SWNT complexes and

radiation with six NIR pulses at 808 nm and intensity of 1.4 W cm
2 for 10 s. Yellow coloring represents colocalization of green Cy3DNA and red DRAQ5 in cell nuclei [32]. (8 National Academy of Sciences.)

Fig. 7.26. Image of dead and aggregated cells after internalization of DNA-SWNT and continuous NIR laser radiation exposure for 2 min at 1.4 W cm
2 . The dead cells show round and aggregated morphology 24 h after laser activated cell death. Black aggregates of

SWNTs are released from dead cells and could visually be seen floating. Raman data and SEM image of black aggregates (36 000) confirm aggregates are SWNTs [32]. (8 National Academy of Sciences.)

271

272

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Chemical structures of the cancer cell targeting scheme. (a) PL-PEG-FA and PLPEG-FITC are conjugated with SWNTs and synthesized by conjugating PL-PEG-NH2 with FA or FITC. (b) Diagram of selective internalization of PL-PEG-FA-SWNTs into Fig. 7.27.

folate-starved cells with increased folate receptors (FR). (c) No internalization of PLPEG-FA-SWNTs into normal cells without folate receptors [32]. (8 National Academy of Sciences.)

that included folate moiety starvation to increase the number of folate receptors on the membranes of these cells. SWNTs conjugated with folate moiety were selectively internalized by cancer cells with folate receptor markers, and then bombarded with continuous NIR laser, which heated these internalized SWNTs, which in turn destroyed the cancer cells. Whereas the folate-starved cancer cells were destroyed (Fig. 7.28), healthy cells without SWNTs complexes were transparent to the laser light and remained viable [32]. Though much more research is necessary, this study clearly demonstrated the amazing transporting potential for functionalized SWNTs to deliver both drugs and cancer therapies due to their unique chemical and optical properties.

7.6

Cytotoxicity of SWNTs

Toxicity has been a controversial issue, shadowing the science of nanotechnology. An improved understanding of the cytotoxicity of SWNTs will undoubtedly benefit research on the biological and biomedical aspects of nanomaterials. An early study by Shvedova et al. determined that transition metal catalysts such as iron and nickel at SWNT concentrations of 0.06 mg mL
1 and higher were toxic to human epidermal keratinocytes [111]. Indeed, inhaling SWNTs, perplexingly, caused the

7.6 Cytotoxicity of SWNTs

Fig. 7.28. Images of SWNT complexes internalized by folate-starved cells. (Top): Confocal image showing the round cell morphology of dead cells. (Bottom): Confocal fluorescence image of SWNTs carrying two cargoes, PL-PEG-FA and PL-PEG-FITC,

internalized within folate-starved cancer cells. The green FITC fluorescence represents the internalization of SWNT with FA and FITC cargoes [32]. (8 National Academy of Sciences.)

growth of granulomas in the lungs of rats, and in the total absence of such pulmonary biomarkers as inflammation, cell proliferation, and cytotoxicity [112, 113]. Another study by Colvin’s group using the buckyball C60 , a cousin of SWNTs, revealed that nano-C60 had a greater toxicity to human skin cells than C60 (OH)24 – the fullerene molecules containing most additional chemical groups on their surfaces. However, fullerene molecules with a higher degree of surface modification were far less toxic due to their inability to generate oxygen radicals [114]. Colvin et al. also found that the toxicity of water-soluble SWNTs to human dermal fibroblasts decreased as the functionalization of the tubes increased [115]. Their specific func-

273

274

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Cell growth curves for MCF7 cells without (control; left-hand bars) and with SWNTs (0.04 mg mL
1 ). Incubation time was 72 h [68]. (8 American Chemical Society.) Fig. 7.29.

tionalizations included SWNT-phenyl-SO3X ratios of 18, 41, and 80 and SWNTphenyl-(COOH)2 with a carbon/-phenyl-(COOH)2 ratio of 23. The cytotoxicity of unfunctionalized SWNTs was 200 parts per billion (ppb), ten times lower than unfunctionalized fullerenes, and modified SWNTs were found to be noncytotoxic. Therefore, while cell death did increase with dose concentration, it did not exceed 50% for concentrations of modified SWNTs up to 2000 ppm [115]. Pantarotto et al. supported this supposition, consistently finding that 90% of fibroblasts remained alive when incubated with SWNTs of 5 mm [97]. Shi Kam et al. have reported that promyelocytic leukemia cells were viable when the SWNT concentrations were below 1.25 mm [98]. Figure 7.29 shows cell growth for MCF7 cells both with and without SWNTs (0.4 mg mL
1 ) [68]. A similar result was observed for d2C keratinocytes cells (data not shown). In both cases, no significant difference in growth was observed for SWNTs treated MCF7 or d2C cells over a 3 day period. The cell count for the MCF7 cells with SWNTs showed only a minimal decrease in the number of cells for day 2 and day 3; however, the overall trend mirrored the cell count for the control MCF7 cells. An MTS assay for cellular metabolism (Fig. 7.30) on the absorbance of MCF7 cells was performed on cultures without SWNTs (control) and with SWNTs of various concentrations. Absorbance was similar for the control cells (0.631) and for those of various concentrations of SWNTs (0.607 to 0.631). The highest concentrations of SWNTs (1 and 0.5 mg mL
1 ) have the largest drop in absorbance (0.024 and 0.021, respectively). However, at concentrations lower than 0.5 mg mL
1 , the absorbances have variances of less than 0.01. Our results suggest that SWNT of a concentration as high as 1 mg mL
1 has no effect on metabolism and cell viability, suggesting some general trends in biological responses to nanoparticles. Nonetheless, notably, a comprehensive risk assessment of SWNTs should go beyond cytotoxicity and account for such crucial issues as exposure rates, uptake mechanisms, and cellular transport of SWNTs and their derivatives. These studies formulate the practice of nanomedicine, guide scientific and technological advancement, and have profound impacts on workplace protection and environment control.

7.7 Cancers and SWNTs

Fig. 7.30. MTS assay of MCF7 cell absorbance vs. SWNT concentration. The control cell absorbance was measured to be 0.630. Cell absorbance ranges from 0.607 to 0.631 for SWNT concentrations of 0.0125–1 mg mL
1 [68]. (8 American Chemical Society.)

7.7

Cancers and SWNTs

An estimated almost 1.4 million new cases of cancer will be diagnosed in 2005 and over 1500 people will die of the disease per day. Despite intensive research, progress in treatment of the disease has been slow and very few significant advances have been made since President Nixon declared his ‘‘War on Cancer’’ and passage of the National Cancer Act in 1971. Traditional chemotherapy and radiotherapy protocols result in actual cures only infrequently and are often accompanied by serious side effects, some of which are permanent. Since therapy for metastatic cancer requires a method that will destroy cancer cells distant from the primary neoplasm, an effective therapy must involve widespread, but specific tumor cell destruction. There are several causes for the failure of current therapies to cure cancer. While cancer cells are more sensitive than their healthy counterparts to radiation and the drugs used for chemotherapy, the differential between the two is not sufficiently large to allow destruction of all of the cancer cells without damaging the healthy ones. Frequently, even in cases where most of the cancer cells are destroyed, some transformed cells escape detection and survive to establish secondary tumors – sometimes years later. In the process of destroying the tumor, cells of the immune system can be damaged, leading to immunosuppression and eventually tolerance of the tumor. Patients can suffer permanent damage, resulting in neuropathies and arthropathies. A dose that will destroy the tumor, yet not severely damage the host is difficult to determine; and in many cases, it may be impossible to obtain. In addition, cells surviving an initial attack will be stimulated to mutate,

275

276

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

resulting in a more aggressive phenotype. Current evidence indicates that metastatic tumor cells have several mutations. Usually, at least one of these involves a gene coding for DNA repair. This facilitates further mutation and allows the cells to evolve mechanisms for evading destruction. The aim of our current research is to overcome these complications by using nanotubes to target tumor cells with greater specificity so that physical, chemical and genetic therapies can selectively destroy tumor cells, allowing more effective killing without damaging sensitive healthy tissue. As previously described, the work of Dai et al. [106] represents the first major step toward the direct application of SWNTs for cancer therapy. Here, cells were killed by laser irradiation of engulfed SWNTs, raising the temperature to 70  C. Internalization of the SWNTs was stimulated by starving the cells for folate and conjugation of the SWNTs with folic acid. While the studies were performed in a somewhat artificial system in vitro, modifications of the technique could make this approach a valuable contribution to the anti-tumor armantarium. The value of hyperthermia as an anti-cancer therapy has long been recognized and current studies indicate that it can lead to significant improvement in response when combined with radiation or chemotherapy. In these studies only temperatures in the range 41–43  C are required. Van der Heijden et al. [116] found that raising the temperature to only 43  C was synergistic with four different drugs for the killing of four different human bladder transitional carcinoma cell lines. Raaphorst and Yang [117] found that a temperature of only 41  C sensitized human cells to killing with cisplatin. The mechanism of enhanced killing involved inhibition of DNA repair, and increased intracellular drug concentrations [118]. In a recent study of sixty eight patients with advanced cervical cancer, Westermann et al. [119] achieved complete remission in 90% of the patients by combining hyperthermia with both radiation and chemotherapy. After 538 days, 74% of the patients remained alive without signs of recurrence. Here the temperature was raised to 41  C. Hence only moderate heating by laser irradiation of intracellular SWNTs could enhance tumor destruction. The synergistic interaction of hyperthermia and chemotherapy could be utilized if SWNT drug delivery vehicles were also to serve as infrared targets for heating. Another use of SWNTs for enhancing radiation therapy has been suggested by Yinghuai et al. [120], who have successfully attached C(2)B(10) carborane cages to SWNTs. These could be used to increase concentrations of boron in tumor cells relative to blood and other organs, resulting in sensitization of the tumors for neutron capture therapy. An obvious method for targeting SWNTs to tumor cells is to attach antibodies specific for the tumor cell surface. Cancer therapy with monoclonal antibodies alone has had minimal success; the best results having been found with trastuzumab (Herceptin) for treatment of her2/nu positive breast cancers [121] and with antibodies against CD20 for non-Hodgkin’s lymphoma (Rituxan, Zevalin, Bexxar [122]). While the coupling of monoclonals with radioisotopes or toxins (e.g., ricin) is also being explored, a problem with these approaches is that the antibodies are usually produced in mice, causing them to be recognized as foreign by the

7.8 Summary

immune system of the patient and destroyed. Conjugating antibodies to SWNTs could extend their lifetimes while targeting the SWNTs to the site of the primary tumor and to metastatic sites. Binding of a toxin to the nanotube or laser heating would then increase the effectiveness of killing. Anti-angiogenesis therapies are also being explored. While these produced promising results during early development, at present they have had limited effectiveness clinically. Recent data indicate the anionic phospholipids, particularly phosphatidylserine, become exposed on the external surface of the vascular endothelial cells in tumors. An antibody produced against these phospholipids localizes in the tumor vasculature and slows the growth of tumors in mice [123]. This effect can be enhanced by pretreatment with docetaxel [124]. Conjugation of this type of antibody to SWNTs would serve to target the SWNTs to the tumor. In this case, blood supply to the tumor would be inhibited by the antibody while additional destruction of the tumor vasculature and the surrounding tissue could be done with laser heating. The possible uses of SWNTs as delivery vehicles for genes or siRNAs are too numerous to be reviewed here. Transfection of the tumor suppressor gene p53 should be attempted, since this is the most commonly mutated tumor suppressor gene in human cancers. Mutated forms of p53 appear in almost 50% of some tumor types. Both transfection of the p53 gene and transfection of siRNA against the apoptosis inhibitor bcl-2 should drive tumor to self-destruction. Transfection of siRNA the pglycoprotein coded for by the multidrug resistance gene MDR1 would restore sensitivity of resistant cancer cells to chemotherapeutic drugs. It is possible to attach multiple moieties to SWNTs because of their structure. This will allow almost limitless possibilities for combinations of targeting ligands and inducers of apoptosis or necrosis.

7.8

Summary

Iijima could not possibly have imagined, in 1991 [125], the subsequent flurry of enchanting scientific endeavors surrounding CNTs whose impact seems to have increasingly touched every corner of our life. Perhaps no other nanostructure or nanomaterial invented so far has attracted this grand level of attention and fervent study. Much remains to be done to offer high sensitivity and localized treatment for cancers and diseases with functionalized and thus intelligent nanostructures and nanomaterials. The realization of this dream demands joint efforts by researchers in the fields of materials science, physical science, and life science, as nature never intends to favor the description of any specific discipline. It is to our best hope that, after reading this chapter, you may start to envision shuttling biomolecules and drug loads through fluidic cell membranes, reporting complex pathogenic pathways, sensing the pH, ionic strength, and temperature of nanoscale bioenvironment, detecting, treating, and preventing cancers and diseases, all with this synthetic wonder material called CNT.

277

278

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

Acknowledgments

The authors thank J. M. Moore for assistance. One of the authors, P. C. Ke, dedicates this chapter to his loved ones.

References 1 Dresselhaus, M. S., Dresselhaus,

2

3

4

5

6

7

8

G., Avouris, P. Eds., Carbon Nanotubes: Synthesis. Structure, Properties, and Applications, 2001, Springer-Verlag, Berlin. Dalton, A. B., Collins, S., Munoz, E., Razal, J. M., Ebron, V. H., Ferraris, J. P., Coleman, J. N., Kim, B. G., Baughman, R. H. Super-tough carbon-nanotube fibres. Nature 2003, 423, 703. Barone, P. W., Baik, S., Heller, D. A., Strano, M. S. Near-infrared optical sensors based on single-walled carbon nanotubes. Nat. Mater. 2005, 4, 86–92. Cherukuri, P., Bachilo, S. M., Litovsky, S. H., Weisman, R. B. Nearinfrared fluorescence microscopy of single-walled carbon nanotubes in phagocytic cells. J. Am. Chem. Soc. 2004, 126, 15 638–15 639. Azamian, B. R., Davis, J. J., Coleman, K. S., Bagshaw, C. B., Green, M. L. Bioelectrochemical single-walled carbon nanotubes. J. Am. Chem. Soc. 2002, 124, 12 664–12 665. Belavoine, F., Schultz, P., Richard, C., Mallouh, V., Ebbesen, T. W., Mioskowski, C. Helical crystallization of proteins on carbon nanotubes: A first step towards the development of new biosensors. Angew. Chem. Int. Ed. 1999, 38, 1912–1915. Baughman, R., Zakhidov, A., de Heer, W. Carbon nanotubes – the route toward applications. Science 2002, 297, 787–792. Biercuk, M. J., Llaguno, M. C., Radosavljevic, M., Hyun, J. K., Johnson, A. T., Fischer, J. E. Carbon nanotube composites for thermal management. Appl. Phys. Lett. 2002, 80, 2767–2769.

9 Kempa, K., Kimball, B., Rybczynski,

10

11

12

13

14

15

16

J., Huang, Z. P., Wu, P. F., Steeves, D., Sennett, M., Giersig, M., Rao, D. V. G. L. N., Carnahan, D. L., Wang, D. Z., Lao, J. Y., Li, W. Z., Ren, Z. F. Photonic crystals based on periodic arrays of aligned carbon nanotubes. Nano Lett. 2003, 3, 13–18. Zhao, Y. D., Zhang, W. D., Chen, H., Luo, Q. M. Direct electron transfer of glucose oxidase molecules adsorbed onto carbon nanotube powder microelectrode. Anal. Sci. 2002, 18, 939–941. Gooding, J. J., Erokhin, P., Hibbert, D. B. Parameters important in tuning the response of monolayer enzyme electrodes fabricated using self-assembled monolayers of alkanethiols. Biosens. Bioelectron. 2000, 15, 229–239. de Heer, W. A., Chatelain, A., Ugarte, D. A carbon nanotube fieldemission electron source. Science 1995, 270, 1179–1180. Rinzler, A. G., Hafner, J. H., Nikolaev, P., Lou, L., Kim, S. G., Tomanek, D., Nordlander, P., Colbert, D. T., Smalley, R. E. Unravelling nanotubes: Field emission from an atomic wire. Science 1995, 269, 1550–1553. Bachtold, A., Hadley, P., Nakanishi, T., Dekker, C. Logic circuits with carbon nanotube transistors. Science 2001, 294, 1317–1320. Yao, Z., Ch. Postma, H. W., Balents, L., Dekker, C. Crossed nanotube intramolecular junctions. Nature 1999, 402, 273–276. Fuhrer, M. S., Nygard, J., Shih, L., Forero, M., Yoon, Y. G., Mazzoni, M. S. C., Choi, H. J., Ihm, J., Louie,

References

17

18

19

20

21

22

23

24

25

S. G., Zettl, A., McEuen, P. L. Crossed nanotube junctions. Science 2000, 288, 494–497. Chen, R. J., Bangsaruntip, S., Drouvalakis, K. A., Shi Kam, N. W., Shim, M., Li, Y., Kim, W., Utz, P. J., Dai, H. J. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4984–4989. Wang, J., Liu, G., Jan, M. R., Zhu, Q. Electrochemical detection of DNA hybridization based on carbonnanotubes loaded with CdS tags. Electrochem. Commun. 2003, 5, 1000–1004. Mitchell, D. T., Lee, S. B., Trofin, L., Li, N., Nevanen, T. K., So¨derlund, H., Martin, C. R. Smart nanotubes for bioseparations and biocatalysis. J. Am. Chem. Soc. 2002, 124, 11 864–11 865. Park, K. H., Chhowalla, M., Iqbal, Z., Sesti, F. Single-walled carbon nanotubes are a new class of ion channel blockers. J. Biol. Chem. 2003, 278, 50 212–50 216. Hu, H., Ni, Y., Montana, V., Haddon, R. C., Parpura, V. Chemically functionalized carbon nanotubes as substrates for neuronal growth. Nano Lett. 2004, 4, 507–511. Matterson, M. P., Haddon, R. C., Rao, A. M. Molecular functionalization of carbon nanotubes and use as substrates for neuronal growth. J. Mol. Neurosci. 2000, 14, 175–182. McKenzie, J. L., Waid, M. C., Shi, R., Webster, T. J. Decreased functions of astrocytes on carbon nanofiber materials. Biomaterials 2004, 25, 1309–1317. Wang, J., Liu, G., Jan, M. R. Ultrasensitive electrical biosensing of proteins and DNA: Carbon-nanotube derived amplification of the recognition and transduction events. J. Am. Chem. Soc. 2004, 126, 3010–3011. Sitharaman, B., Kissell, K. R., Hartman, K. B., Tran, L. A., Baikalov, A., Rusakova, I., Sun, Y.,

26

27

28

29

30

31

32

33 34

Khant, H. A., Ludtke, S. J., Chiu, W., Laus, S., To´th, E´., Helm, L., Merbach, A. E., Wilson, L. J. Superparamagnetic gadonanotubes are high performance MRI contrast agents. Chem. Commun. 2005, 3915–1917. Dwyer, C., Guthold, M., Falvo, M., Washburn, S., Superfine, R., Erie, D. DNA-functionalized single-walled carbon nanotubes. Nanotechnology 2002, 13, 601–604. Cai, H., Cao, X. N., Jiang, Y., He, P. G., Fang, Y. Z. Carbon nanotubeenhanced electrochemical DNA biosensor for DNA hybridization detection. Anal. Bioanal. Chem. 2003, 375, 287–293. Pantarotto, D., Partidos, C. D., Hoebeke, J., Brown, F., Kramer, E., Briand, J.-P., Muller, S., Prato, M., Bianco, A. Immunization with peptide-functionalized carbon nanotubes enhances virus-specific neutralizing antibody responses. Chem. Biol. 2003, 10, 961–966. Chen, R. J., Zhang, Y., Wang, D., Dai, H. J. Noncovalent sidewall functionalization of single-walled carbon nanotubes for proteins immobilization. J. Am. Chem. Soc. 2001, 123, 3838–3839. Shim, M., Shi Kam, N. W., Chen, R. J., Dai, H. J. Functionalization of carbon nanotubes for biocompability and biomolecular recognition. Nano Lett. 2002, 2, 285–288. Dubruel, P., Christiaens, B., Rosseneu, M., Vandekerckhove, J., Grooten, J., Goossens, V., Schacht, E. Buffering properties of cationic polymethacrylates are not the only key to successful gene delivery. Biomacromolecules 2004, 5, 379–388. Shi Kam, N. W., O’Connell, M., Wisdom, J. A., Dai, H. J. Carbon nanotubes as multifunctional biological transporters and nearinfrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11 600–11 605. http://nano.cancer.gov/ http://www.nsf.gov/news/ priority_areas/

279

280

7 Carbon Nanotubes in Cancer Therapy and Diagnosis 35 http://nihroadmap.nih.gov/ 36 http://pubs.acs.org/cen/coverstory/

8148/8148counterpoint.html 37 Sano, M., Kamino, A., Okamura, J.,

38

39

40

41

42

43

44

45

Shinkai, S. Self-organization of PEOgraft-single-walled carbon nanotubes in solutions and Langmuir-Blodgett films. Langmuir 2001, 17, 5125–5128. Banerjee, S., Wong, S. S. Structural characterization, optical properties, and improved solubility of carbon nanotubes functionalized with Wilkinson’s catalyst. J. Am. Chem. Soc. 2002, 124, 8940–8948. Pompeo, F., Resasco, D. E. Water solubilization of single-walled carbon nanotubes by functionalization with glucosamine. Nano Lett. 2002, 2, 369–373. Bahr, J. L., Mickelson, E. T., Bronikowski, M. J., Smalley, R. E., Tour, J. M. Dissolution of small diameter single-wall carbon nanotubes in organic solvents? Chem. Commun. 2001, 1, 193–194. Sun, Y., Wilson, S. R., Schuster, D. I. High dissolution and strong light emission of carbon nanotubes in aromatic amine solvents. J. Am. Chem. Soc. 2001, 123, 5348–5349. Yureli, K., Mitchell, C. A., Krishnamoorti, R. Small-angle neutron scattering from surfactantassisted aqueous dispersions of carbon nanotubes. J. Am. Chem. Soc. 2004, 126, 9902–9903. Matarredona, O., Rhoads, H., Li, Z. R., Harwell, J. H., Balzano, L., Resasco, D. E. Dispersion of singlewalled carbon nanotubes in aqueous solutions of the anionic surfactant NaDDBS. J. Phys. Chem. B 2003, 107, 13 357–13 367. O’Connell, M. J., Bachilo, S. M., Huffman, C. B., Moore, V. C., Strano, M. S., Haroz, E. H., Rialon, K. L., Boul, P. J., Noon, W. H., Kittrell, C., Ma, J., Hauge, R. H., Weisman, R. B., Smalley, R. E. Band gap fluorescence from individual single-walled carbon nanotubes. Science 2002, 297, 593–596. O’Connell, M. J., Boul, P., Ericson, L. M., Huffman, C., Wang, Y.,

46

47

48

49

50

51

52

53

Haroz, E., Kuper, C., Tour, J., Ausman, K. D., Smalley, R. E. Reversible water-solubilization of single-walled carbon nanotubes by polymer wrapping. Chem. Phys. Lett. 2001, 342, 265–271. Rao, R., Lee, J., Lu, Q., Keskar, G., Freedman, K. O., Floyd, W. C., Rao, A. M., Ke, P. C. Single-molecule fluorescence microscopy and Raman spectroscopy studies of RNA bound carbon nanotubes. Appl. Phys. Lett. 2004, 85, 4228–4230. Strano, M. S., Zheng, M., Jagota, A., Onoa, G. B., Heller, D. A., Barone, P. W., Usrey, M. L. Understanding the nature of the DNAassisted separation of single-walled carbon nanotubes using fluorescence and Raman spectroscopy. Nano Lett. 2004, 4, 543–550. Wang, H., Zhou, W., Ho, D. L., Winey, K. I., Fischer, J. E., Glinka, C. J., Hobbie, E. K. Dispersing singlewalled carbon nanotubes with surfactants: A small angle neutron scattering study. Nano Lett. 2004, 4, 1789–1793. Zheng, M., Jagota, A., Semke, E. D., Diner, B. A., McLean, R. S., Lustig, S. R., Richardson, R. E., Tassi, N. G. DNA-assisted dispersion and separation of carbon nanotubes. Nat. Mater. 2003, 2, 338–342. Jelinek, A. and Klocking, H. P. In vitro toxicity of surfactants in U937 cells: Cell membrane integrity and mitochondrial function. Exp. Toxicol. Pathol. 1998, 50, 472–476. Tasis, D., Tagmatarchis, N., Georgakilas, V., Prato, M. Soluble carbon nanotubes. Chem. Eur. J. 2003, 9, 4000–4008. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K., Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., RodriguezMacias, F., Shon, Y.-S., Lee, T. R., Colbert, D. T., Smalley, R. E. Fullerene pipes. Science 1998, 280, 1253–1255. Liu, Z., Shen, Z., Zhu, T., Hou, S., Ying, L., Shi, Z., Gu, Z. Organizing single-walled carbon nanotubes on

References

54

55

56

57

58

59

60

61

62

63

gold using a wet chemical selfassembling technique. Langmuir 2000, 16, 3569–3573. Diao, P., Liu, Z., Wu, B., Nan, X., Zhang, J., Wei, Z. Chemically assembled single-wall carbon nanotubes and their electrochemistry. ChemPhysChem 2002, 3, 898–901. Williams, K. A., Veenhuizen, P. T. M., de la, Torre, B. G., Eritja, R., Dekker, C. Carbon nanotubes with DNA recognition. Nature 2002, 420, 761. Chen, Y., Haddon, R. C., Fang, S., Rao, A. M., Eklund, P. C., Lee, W. H., Dickey, E. C., Grulke, E. A., Pendergrass, J. C., Chavan, A., Haley, B. E., Smalley, R. E. Chemical attachment of organic functional groups to single-walled carbon nanotube material. J. Mater. Res. 1998, 13, 2423–2431. Chen, J., Hamon, M. A., Hu, H., Chen, Y., Rao, A. M., Eklund, P. C., Haddon, R. C. Solution properties of single-walled carbon nanotubes. Science 1998, 282, 95–98. Boul, P. J., Liu, J., Mickelson, E. T., Huffman, C. B., Ericson, L. M., Chiang, I. W., Smith, K. A., Colbert, D. T., Hauge, R. H., Margrave, J. L., Smalley, R. E. Reversible sidewall functionalization of buckytubes. Chem. Phys. Lett. 1999, 310, 367–372. Gu, Z., Peng, H., Hauge, R. H., Smalley, R. E., Margrave, J. L. Cutting single-wall carbon nanotubes through fluorination. Nano Lett. 2002, 2, 1009–1013. Bahr, J. L., Tour, J. M. Covalent chemistry of single-wall carbon nanotubes. J. Mater. Chem. 2002, 12, 1952–1958. Kooi, S. E., Schlecht, U., Burghard, M., Kern, K. Electrochemical modification of single carbon nanotubes. Angew. Chem. 2002, 114, 1409– 1411. Hirsch, A. Funktionalisierung von einwandigen Kohlenstoffnanoro¨hren. Angew. Chem. 2002, 114, 1933–1999. Ahn, C. C., Ye, Y., Ratnakumar, B. V., Witham, C., Bowman, Jr., R. C.,

64

65

66

67

68

69

70

71

72

Maggini, M., Scorrano, G., Prato, M. Addition of azomethine ylides to C60: Synthesis, characterization, and functionalization of fullerene pyrrolidines. J. Am. Chem. Soc. 1993, 115, 9798–9799. Choi, M., Downes, P., Hulman, M., Roth, S., Stepanek, I., Bernier, P., Georgakilas, V., Kordatos, K., Prato, M., Guldi, D. M., Holzinger, M., Hirsch, A. Organic functionalization of carbon nanotubes. J. Am. Chem. Soc. 2002, 124, 760–761. Kordatos, K., Da Ros, T., Bosi, S., Vazquez, E., Bergamin, M., Cusan, C., Pellarini, F., Tomberli, V., Baiti, B., Pantarotto, D., Georgakilas, V., Spalluto, G., Prato, M. Novel versatile fullerene synthons. J. Org. Chem. 2001, 66, 4915–4920. Lu, Q., Freedman, K. O., Rao, R., Huang, G., Lee, J., Larcom, L. L., Rao, A. M., Ke, P. C. Diffusion of carbon nanotubes with singlemolecule fluorescence microscopy. J. Appl. Phys. 2004, 96, 6772–6775. Wu, Y., Hudson, J. S., Lu, Q., Moore, J. M., Mount, A. S., Rao, A. M., Alexov, E., Ke, P. C. Coating single-walled carbon nanotubes with phospholipids. J. Phys. Chem. B 2006, 110, 2475–2478. Lu, Q., Moore, J. M., Huang, G., Mount, A. S., Rao, A. M., Larcom, L. L., Ke, P. C. RNA polymer translocation with single-walled carbon nanotubes. Nano Lett. 2004, 4, 2473–2477. Richard, C., Balavoine, F., Schultz, P., Ebbesen, T. W., Mioskowski, C. Supramolecular self-assembly of lipid derivatives on carbon nanotubes. Science 2003, 300, 775–778. Lichtenberg, D., Goni, F. M., Heerklotz, H. Detergent-resistant membranes should not be identified with membrane rafts. Trends Biochem. Sci. 2005, 30, 430–436. Gao, H., Kong, Y., Cui, D. Detergentresistant membranes should not be identified with membrane rafts. Nano Lett. 2003, 3, 471–473. Zheng, M., Jagota, A., Strano, M. S., Santos, A. P., Barone, P., Chou,

281

282

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

73

74

75

76

77

78

79

80

81

82

83

84

S. G., Diner, B. A., Dresselhaus, M. S., Mclean, R. S., Onoa, G. B., Samsonidze, G. G., Semke, E. D., Usrey, M., Walls, D. J. Structurebased carbon nanotube sorting by sequence-dependent DNA assembly. Science 2003, 302, 1545–1548. Illangasekare, M., Sanchez, G., Nickles, T., Yarus, M. AminoacylRNA synthesis catalyzed by an RNA. Science 1995, 267, 643–647. Lohse, P. A., Szostak, J. W. Ribozyme-catalysed amino-acid transfer reactions. Nature 1996, 381, 442–444. Tarasow, T. M., Tarasow, S. L., Eaton, B. E. RNA-catalysed carboncarbon bond formation. Nature 1997, 389, 54–57. Zhang, B. L., Cech, T. R. Peptide bond formation by in vitro selected ribozymes. Nature 1997, 390, 96–100. Gugliotti, L. A., Feldheim, D. L., Eaton, B. E. RNA-mediated metalmetal bond formation in the synthesis of hexagonal palladium nanoparticles. Science 2004, 304, 850–852. McManus, M. T., Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nat. Rev./Genet. 2002, 3, 737–747. Carter, D. C., Ho, J. X. Structure of serum albumin. Protein Chem. 1994, 45, 153–203. Schultz, G. J., Schindler, H., Schmidt, T. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys. J. 1997, 73, 1073–1080. Ke, P. C., Naumann, C. A. Hindered diffusion in polymer-tethered phospholipid monolayers at the airwater interface: A single molecule fluorescence imaging study. Langmuir 2001, 17, 5076–5081. Ishijima, A., Yanagida, T. Single molecule nanobioscience. Trends Biochem. Sci. 2001, 26, 438–444. Saxton, M. J. Anomalous diffusion due to obstacles – A Monte-Carlo study. Biophys. J. 1994, 66, 394–401. Journet, C., Maser, W. K., Bernier, P., Loiseau, A., dela Chapelle, M. L., Lefrant, S., Denaird, P., Lee, R.,

85

86

87

88

89

90

91

92

93

94

95

Fischer, J. E. Large-scale production of single-walled carbon nanotubes by the electric-arc technique. Nature 1997, 388, 756–758. Hartschuh, A., Pedrosa, H. N., Novotny, L., Krauss, T. D. Simultaneous fluorescence and Raman scattering from single carbon nanotubes. Science 2003, 301, 1354–1356. Bachilo, S. M., Strano, M. S., Kittrell, C., Hauge, R. H., Smalley, R. E., Weisman, R. B. Structureassisted optical spectra of singlewalled carbon nanotubes. Science 2002, 298, 2361–2366. Efron, B., Tibshirani, R. Statistical data analysis in the computer age. Science 1991, 253, 390–395. Doi, M., Edwards, S. The Theory of Polymer Dynamics, Clarendon, Oxford, 1986. Tanford, C. Physical Chemistry of Macromolecules, John Wiley and Sons, New York, 1961. Bustamante, C., Smith, S. B., Liphard, J., Smith, D. E. Single-molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 2000, 10, 279–284. de Gennes, P. G. Scaling Concepts in Polymer Physics, 1979, Cornell UP, Ithaca, New York. Smith, D. E., Perkins, T. T., Chu, S. Self-diffusion of an entangled DNA molecule by reptation. Phys. Rev. Lett. 1995, 75, 4146–4149. Dubruel, P., Christiaens, B., Rosseneu, M., Vandekerckhove, J., Grooten, J., Goossens, V., Schacht, E. Buffering properties of cationic polymethacrylates are not the only key to successful gene delivery. Biomacromolecules 2004, 5, 379–388. Aissaoui, A., Oudrhiri, N., Petit, L., Hauchecorne, M., Kan, E., Sainlos, M., Julia, S., Navarro, J., Vigneron, J. P., Lehn, J. M., Lehn, P. Progress in gene delivery by cationic lipids: Guanidinium-cholesterol-based systems as an example. Curr. Drug Targets 2002, 3, 1–16. Allen, T. M., Cullis, P. R. Drug delivery systems: Entering the main stream. Science 2004, 303, 1818–1822.

References 96 Verma, I., Somia, N. Gene therapy-

97

98

99

100

101

102

103

104

105

promise, problems, and prospects. Nature 1997, 389, 239–242. Pantarotto, D., Briand, J., Prato, M., Bianco, A. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem. Commun. 2004, 1, 16–17. Shi Kam, N. W., Jessop, T. C., Wender, P. A., Dai, H. J. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J. Am. Chem. Soc. 2004, 126, 6850–6851. Salman, H., Zbaida, D., Rabin, Y., Chatenay, D., Elbaum, M. Kinetics and mechanism of DNA uptake into the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7247–7252. Pantarotto, D., Singh, R., McCarthy, D., Erhardt, M., Braind, J.-P., Prato, M., Kostarelos, K., Bianco, A. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew. Chem. 2004, 43, 5242–5246. Liu, Y., Wu, D. C., Zhang, W. D., Jiang, X., He, C. B., Chung, T. S., Goh, S. H., Leong, K. W. Polyethylenimine-grafted multiwalled carbon nanotubes for secure noncovalent immobilization and efficient delivery of DNA. Angew. Chem. 2005, 44, 4782–4785. Wilson, R. W., Bloomfield, V. A. Counterion-induced condensation of deoxyribonucleic acid. A lightscattering study. Biochemistry 1979, 18, 2192–2196. Harpe, A. V., Petersen, H., Li, Y. X., Kissel, T. Characterization of commercially available and synthesized polyethylenimines for gene delivery. J. Controlled Release 2000, 69, 309–322. Shi Kam, N. W., Liu, Z., Dai, H. J. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient Intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 2005, 127, 12 492–12 493. Gillies, E. R., Fre´chet, J. J. M. pHresponsive copolymer assemblies for

106

107

108

109

110

111

112

113

114

controlled release of doxorubicin. Bioconjugate Chem. 2005, 16, 361–368. Shi Kam, N. W., Dai, H. J. Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc. 2005, 127, 6021–6026. Pantarotto, D., Partidos, C. D., Graff, R., Hoebeke, J., Briand, J.-P., Prato, M., Bianco, A. Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides. J. Am. Chem. Soc. 2003, 125, 6160–6164. Georgakilas, V., Tagmatarchis, N., Pantarotto, D., Bianco, A., Briand, J.-P., Prato, M. Amino acid functionalization of water soluble carbon nanotubes. Chem. Commun. 2002, 3050–3051. Nielsen, O., Buchardt, O. Facile synthesis of reagents containing a terminal maleimido ligand linked to an active ester. Synthesis 1991, 819–821. Cai, J., Yang, J., Jones, D. P. Mitochondrial control of apoptosis: The role of cytochrome c. Biochim. Biophys. Acta 1998, 1366, 139–149. Shvedova, A. A., Castranova, V., Kisin, E. R., Schwegler-Berry, D., Murray, A. R., Gandelsman, V. Z., Maynard, A., Baron, P. Exposure to carbon nanotube material; assessment of nanotube cytotoxicity using human keratinocyte cells. J. Toxicol. Environ. Health, Part A 2003, 66, 1909–1926. Warheit, D. B., Laurence, B. R., Reed, K. L., Roach, D. H., Reynolds, G. A. M., Webb, T. R. Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats. Toxicity Sci. 2004, 77, 117–125. Lam, C. W., James, J. T., McCluskey, R., Hunter, R. L. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicity Sci. 2004, 77, 126–134. Sayes, C. M., Fortner, J. D., Guo, W., Lyon, D., Boyd, A. M., Ausman, K. D., Tao, Y. J., Sitharaman, B., Wilson, L. J., Hughes, J. B., West,

283

284

7 Carbon Nanotubes in Cancer Therapy and Diagnosis

115 116

117

118

119

J. L., Colvin, V. L. The differential cytotoxicity of water-soluble fullerenes. Nano Lett. 2004, 4, 1881–1887. http://nanotechweb.org/articles/news/ 4/11/3/1 van der Heijden, A. G., Verhaegh, G., Jansen, C. F., Schalken, J. A., Witjes, J. A. The effect of hyperthermia on mitomycin-C induced cytotoxicity in four human bladder cancer cell lines. J. Urol. 2005, 173, 1375–1380. Raaphorst, G. P., Yang, D. P. The evaluation of thermal cisplatin sensitization in normal and XP human cells using mild hyperthermia at 40 and 41. Anticancer Res. 2005, 25, 2649–2653. Takahashi, I., Emi, Y., Hasuda, S., Kakeji, Y., Maehara, Y., Sugimachi, K. Clinical application of hyperthermia combined with anticancer drugs for the treatment of solid tumors. Surgery, 2002, 131, S78–S84. Westermann, A. M., Jones, E. L., Schem, B. C., van der SteenBanasik, Koper, P., Mella, O., Uitterhoeve, A. L., de Wit, R., van der Velden, J., Berger, C., van der Wilt, C. L., Dahl, O., Prosnitz, L. R., van der Zee, J. First results of triplemodality treatment combining radiotherapy, chemotherapy, and hyperthermia for the treatment of patients with stage IIB, III, and IVA cervical carcinoma. Cancer, 2005, 104, 763–770.

120 Yinghuai, Z., Peng, A. T.,

121

122

123

124

125

Carpenter, K., Maguire, J. A., Hosmane, N. S., Takagaki, M. Substituted carborane-appended watersoluble single-wall carbon nanotubes: New approach to boron neutron capture therapy drug delivery. J. Am. Chem. Soc., 2005, 127, 9875–9880. Willems, A., Gauger, K., Henrichs, C., Harbeck, N. Antibody therapy for breast cancer. Anticancer Res., 2005, 25, 1483–1489. Jazirehi, A. R., Bonavida, B. Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in nonHodgkin’s lymphoma: Implications in chemosensitization and therapeutic intervention. Oncogene, 2005, 24, 2121–2143. Ran, S., He, J., Huang, X., Soares, M., Scothorn, D., Thorpe, P. E. Antitumor effects of a monoclonal antibody that binds anionic phospholipids on the surface of tumor blood vessels in mice. Clin. Cancer Res., 2005, 11, 1551–1562. Huang, X., Bennett, M., Thorpe, P. E. A monoclonal antibody that binds anionic phospholipids on tumor blood vessels enhances the antitumor effect of docetaxel on human breast tumors in mice. Cancer Res., 2005, 65, 4408–4416. Iijima, S. Helical microtubules of graphitic carbon. Nature 1991, 354, 56–58.

285

8

Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis Kiyotaka Shiba 8.1

Introduction

The elimination of cancer-related suffering and death remains an unachieved goal of modern medicine, biology, pharmaceutical science and medical engineering. For this fervent wish to become a reality, what is needed is continued progress, and even ground-breaking innovation, in both the diagnosis of cancer and its treatment. Nanotechnologies have come into the spotlight because of their potential for use as novel diagnostic tools that enable detection of primary cancers at their earliest stages, and in new therapeutic protocols that effectively and selectively exterminate malignant cells. The present chapter introduces the current status of efforts to develop nanomaterials for cancer diagnosis and therapy, mainly by focusing on nonspherical (non-particulate) nanomaterials such as nanotubes, nanowires and nanorods. Because various nanomaterials have been discovered since the first observation of carbon nanotubes, the physicochemical properties of carbonaceous as well as non-carbonaceous nanomaterials are introduced first (Section 8.2). Subsequent sections present potential applications of these molecules in cancer diagnosis (Section 8.3) and treatment (Section 8.4). Progress in the development of spherical nanoparticles for these purposes is reviewed in other chapters of this volume.

8.2

Nanotubes, Nanowires and Nanorods

Nanotubes, nanowires, nanorods, nanotubules, nanoribbons, nanobelts, nanoonion, nanocapsule, graphite cones, nanopolyhedrons and bamboo tubes, among others, are the names that have been given to various, mostly one-dimensional, nanomaterials. Some of them are composed of graphitic tubes (carbon nanomaterials), but others are consist of noncarbonic elements. Some are covalently linked molecules, while others are noncovalently linked assemblages of macromolecules. Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

286

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

Electron micrograph of a single-wall carbon nanotube. (From Ref. [4], with permission.)

Fig. 8.1.

This section an overviews the properties of these nonspherical nanomaterials, giving a clue as to their potentially unparalleled utility in some applications. 8.2.1

Carbon Nanotubes

The discovery of multi-wall carbon nanotubes (MWNTs) by Iijima in 1991 [1] stands as the first of a cascade of brilliant discoveries of various nanomaterials [2, 3]. In 1993, the single-wall carbon nanotube (SWNT), which can be regarded as the simplest form of MWNT, was discovered by Iijima’s and other groups [4, 5]. A SWNT is made up of a single layer of graphite molecules rolled up into a hollow cylinder (Fig. 8.1). Though the diameters of these tubes typically range from 1.0 to 1.5 nm [4], nanotubes with diameters of as little as 0.43 nm have been reported [6–8]. MWNTs are tubes composed of two or more concentrically arranged cylinders (Fig. 8.2). Those consisting of only two cylinders are often referred to as double-wall carbon nanotubes [9]. Carbon nanotubes are synthesized by (a) laser ablation [10], (b) high-pressure CO conversion (HiPCO) [11], or (c) an

Electron micrographs of multi-wall carbon nanotubes. (From Ref. [1], with permission.)

Fig. 8.2.

8.2 Nanotubes, Nanowires and Nanorods

arc-discharge technique [12], and their structures can be dramatically influenced by the conditions under which they are produced, including the metal catalyst used, the temperature, the atmosphere etc. Thus, by applying the appropriate conditions, various SWNT and MWNT derivatives can be obtained, including the nanoonion [13], nanocapsule [14, 15], nanopolyhedron [16], bamboo tube [17], graphite cone [18] and nanohorn [19]. Attractive properties of carbon nanotubes include their high electrical and thermal conductivity, their great tensile strength and their very high elastic modulus [20]. In particular, the electrical properties of nanotubes have received a great deal of attention in nanodevice research. SWNTs can be metallic or semiconducting, depending upon their diameter and chiral angle [21–23], which means that they can act as a molecular wire [24, 25] or as a transistor [26–28]. Indeed, a logic circuit composed of SWNT field-effect transistors (FETs) has been developed [29], and the bottom-up fabrication of such nanoscale block units is expected to break the size limitation that current lithographic technology is encountering. The electrical conductance [30–33] and capacitance [34], the optical properties [35] and the stiffness [36] of nanotubes have all been shown to be altered by surface adsorption of various molecules. In addition, by integrating the sensing abilities of nanotubes into logic circuits, nanoscale biosensors could be designed that simultaneously quantitate different biomolecules (e.g., mRNAs, proteins and metabolic products) and then give a cue for action (e.g., drug release or signal generation) based on programmed logic. The advent of such devices should revolutionize cancer diagnosis and treatment (Section 8.3.1). The strength and stiffness of SWNT or MWNTs [37] have also been explored with the aim of developing novel materials, such as carbon nanotube paper [38, 39], film [40, 41], fibers [42], gel [43], nanotube–collagen composite [44], and nanotube–polyurethane composites [45]. One possible use of these materials is as a scaffold for cell growth [46–49], which may indirectly affect cancer treatment as well as regenerative medicine. 8.2.2

Noncarbon Nanotubes

Graphite-like layered structures can also be formed from elements other than carbon, and an array of noncarbon nanotubes have been fabricated from boron nitride (BN) [50, 51], nickel chloride (NiCl2 ), niobium disulfide (NbS2 ) [52] and molybdenum disulfide (MoS2 ) [53], and even a multielement (boron nitride and carbon) coaxial nanotube [54] has been synthesized. Applications that take advantage of the unique properties of these materials are currently being explored. 8.2.3

Single-wall Carbon Nanohorns

Single-wall nanohorns (SWNHs) were discovered in 1999 and are thus relatively new members of the carbon nanomaterial family [19]. They are spherical struc-

287

288

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

Electron micrograph of single-wall carbon nanohorns. (Courtesy of Professor S. Iijima and Dr. M. Yudasaka.)

Fig. 8.3.

tures with diameters of 80–100 nm, but their constituent parts are graphene nanotubes – i.e., they are spherical aggregates of carbon nanotubes (Fig. 8.3). The constitutive tubes have closed ends with cone-shaped caps (horns) and have diameters of 2–3 nm, which is larger than the typical SWNT. SWNHs have extensive surface areas and multitudes of horn interstices, which enable large numbers of guest molecules to be adsorbed. In addition, as with carbon nanotubes [55, 56], oxidization or acid treatment can be used to create nano-windows in the walls of SWNHs, through which small molecules (e.g., N2 , Ar, C60 etc.) can infiltrate their interior space. Furthermore, the size of the pores can be controlled by appropriately controlling the treatment conditions, enabling the preparation of a series of oxidized SWNHs (oxSWNHs) having distinct molecular sieving effects. Oxidation also introduces functional oxygen groups (e.g., carboxyl and quinine groups) at the pore-edges of oxSWNHs, to which various chemical compounds [e.g., biotin and poly(ethylene glycol)] can be coupled to functionalize the surfaces. In contrast to the synthesis of other carbon nanomaterials, the synthesis of SWNHs (laser ablation of graphite) does not require a metal catalyst. This enables production of extremely pure preparations with no potential for toxicity from contaminating metals. These unique properties are particularly noteworthy because they suggest the feasibility of using SWNHs as carriers in drug delivery systems (see Section 8.4.1). 8.2.4

Nanorods and Nanowires

Whereas nanotubes are hollow cylinders composed of rolled-up single molecular layers, nanorods and nanowires are not hollow. One way to form these structures is to use capillary action to fill the interior space of carbon nanotubes, e.g., with

8.2 Nanotubes, Nanowires and Nanorods

lead [57] or bismuth [55]. Arc discharge in the presence of metals has also been used to fill carbon nanotubes with Y [58], Mn [59], Gd [60], T, Cr, Fe, Cp, Ni, Cu, Zn, Mo, Pd, Sn Ta, W, Dy or Yb [61]. In addition, reacting carbon nanotubes with volatile metal or nonmetal complexes produces carbide (TiC, NbC, Fe3 C, SiC and BC) nanorods [62]. Similarly, reacting Ga2 O vapor with NH3 gas in the presence of carbon nanotubes yields GaN nanorods [63], while analogous reactions have been used to synthesize Si3 N4 nanorods [63] and oriented silicon carbide nanowires [64]. Various other approaches to the fabrication of nanorods (nanowires) that do not require nanotubes have also been developed, yielding GaN nanorods [65, 66], silicon nanowires (SiNW) [67, 68] and indium phosphate nanowires [69]. The nanorods obtained were either single crystal [62, 63, 65–67, 70], polycrystalline [62] or amorphous [62], depending upon the method and materials used [62]. 8.2.5

Self-assembled Nanotubes

Self-assembly from smaller molecular blocks is an alternative approach to synthesizing nanotubular structures. Formation of such structures from lipid molecules was first reported some time ago [71, 72], and has recently been garnering attention as a tool for microfabrication [73–75]. Peptides represent another set of molecules that can be assembled into tubular structures [76, 77]. In contrast to lipid tubes, peptide tubes are nanometer scale structures, the diameters of which can be fine tuned through appropriate design [78]. What is more, casting ionic metal into a peptide nanotube fabricated from dipeptide building blocks has been used to form metal nanowires with diameters of only 20 nm [79] (Fig. 8.4). Block copolymers are also versatile for fabricating various types of microstructures, including tubes [80, 81], vesicles [82] and toroidal structures [83], among others. Lipid, peptide and polymer self-assemblages, along with natural proteinous assemblages, are generally highly biocompatible, so their utility as nano-carriers of drugs, antigens and genes is currently being investigated. They also have been used as templates for inorganic wires fabricated through deposition of various inorganic nanocrystals upon them [84–92].

Silver nanowires were formed by the reduction of silver ions within the peptide nanotubes, followed by enzymatic degradation of the peptide mold. (From Ref. [79], with permission.)

Fig. 8.4.

289

290

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

8.3

Cancer Diagnosis

The recent and rapid progress in genomic medicine has required innovative solutions for high-throughput and parallel identification and/or quantification of large numbers of biomolecules, including mRNA, proteins and metabolites [93, 94]. Microarray technology combined with fluorescence detection would be one of the most popular tools for such high-throughput genomic analysis [95–97]; however, that methodology does not yet have sufficient sensitivity or usability. But by using nanomaterials, a new type of ultrasensitive high-density sensor array could be fabricated. Although such next-generation nanosensors are still in an early phase of development, some elemental technologies have already been shown to work. 8.3.1

Carbon Nanotube-based Detection System

As described in Section 8.2.1, the physicochemical properties of carbon nanotubes can be changed by adsorption of molecules onto their surfaces. The nanotube sensor was first demonstrated as a FET-based gas sensor, in which the electrical resistance of a semiconducting SWNT was altered by exposure to electron-withdrawing NO2 or O2 or to electron-donating NH3 molecules [30, 31, 98]. Since then, SWNTbased FETs have been exploited as biosensors through functionalization of the tube surfaces using various biomolecules. For instance, when the surface was modified with biotin, the electronic properties of the resultant biotin–SWNT were altered upon addition of streptavidin (a protein that strongly binds to biotin) [99]. Similarly, when glucose oxidase was immobilized on the surface of SWNTs, the resultant sensor showed increased conductance upon addition of 0.1 m glucose (a substrate of glucose oxidase), probably reflecting a conformational change in the enzyme [100] (Fig. 8.5). Finally, human U1A RNA splicing factor is an antigen in autoimmune diseases such as systemic lupus erythematosus, and detection of autoantibodies against this protein is a diagnostic criterion for the disease. When U1A protein was immobilized on the surface of SWNTs, the binding of a monoclonal anti-UIA antibody at concentrations of less than 1 nm was detected as an electric signal [33], making this construct suitable for development of an ultrasensitive and label-free diagnostic system.

Enzyme-coated carbon nanotubes as biosensors. Two electrodes connect a semiconducting SWNT with glucose oxidase immobilized on its surfaces. (From Ref. [100], with permission.)

Fig. 8.5.

8.3 Cancer Diagnosis

Carbon nanotube nanoelectrode array. MWNT arrays were grown on Ni spots defined by UV lithography. (From Ref. [104], with permission.)

Fig. 8.6.

RNA, DNA and peptide molecules that bind to specific target substances are termed aptamers [101–103]. They are artificially created using in vitro evolution systems in the order-made manner, and a myriad of aptamers have already been generated against a wide variety of biomacromolecules and inorganic substances. Such aptamers can be used to functionalize the surface of nanotubes to prepare sensors for target substances. For instance, when a DNA aptamer against thrombin [102] was immobilized on the surfaces of SWNTs, the aptamer–SWNT FETs worked as a thrombin sensor. In addition, because aptamers are generally much smaller than antibodies, they could be used to fabricate sensors that are both cheaper and more space-efficient than those fabricated from antibodies. Another type of ultrasensitive DNA/RNA sensor that has been proposed is one in which nanoelectrode arrays composed of MWNTs having one tip modified with oligonucleotides with specifically designed sequences are fabricated on a SiO2 matrix [104] (Fig. 8.6). The hybridization of sub-attomole amounts of single strand DNAs could then be detected as a electronic signal using the Ru(bpy)3 2þ -mediated guanine oxidation method [105]. Such arrays of nanotubes should be easy to construct on silicon tips, thereby providing the opportunity to develop multiplexed detection systems [104]. 8.3.2

Non-carbon Nanotube-based Detection Systems

Though carbon nanotubes are highly promising materials with which to construct nanosensors, they also have some limitations. For instance, existing methods of synthesis produce mixtures of metallic and semiconducting SWNTs, necessitating their efficient separation before defined nanosensors can be constructed. This

291

292

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

makes noncarbonaceous tubes or wires attractive, as most of them require no separation step. For instance, silicon nanowires (SiNWs, Section 8.2.4) [67, 68] are always semiconducting, and their surfaces are easily modified. When the surfaces of boron-doped SiNWs are modified with 3-aminopropyltriethaoxysilane (APTES) and connected to source and drain electrodes, the nanowires function as pHsensing FETs [106]. Similarly, biotin- and calmodulin-immobilized SiNWs function, respectively, as anti-biotin antibodies and Ca 2þ FET sensors. A SiNW-based ultrasensitive DNA sensor has also been developed by functionalizing the surfaces of SiNWs with a PNA (peptide nucleic acid) that recognizes the gene for the cystic fibrosis transmembrane receptor [107]. Here, the conductance of the nanowire increased upon PNA–DNA hybridization. Thus, highly sensitive, real-time nanosensors can be fabricated using SiNW [106]. In addition to SiNW, metal oxide nanowires are also attractive candidates for the fabrication of nanosensors. For instance, In2 O3 NWs with immobilized anti-PSA (prostate-specific antigen) antibodies on their surfaces showed enhanced conductivity upon exposure to PSA [108]. Interestingly, a similar SWNT-based sensor showed suppressed conductivity upon exposure to PSA, enabling complementary detection of PSA antigen with these two devices [108]. 8.3.3

Microcantilevers

Whereas nanotube- or nanowire-based sensors transduce biomolecular recognition into changes in electrical conductance [30, 31] and capacitance [34] or changes in optical properties [35], microcantilevers transduce that recognition into the nanomechanical bending of a cantilever, which can be measured in situ using optical beam detection [109–111]. Microcantilevers are generally made of silicon nitride that is coated on one side with a thin film of gold (Fig. 8.7). They can be constructed using low-cost semiconductor microfabrication processes, thus offering an ideal platform for high-throughput molecular analysis. For instance, a cantilever on which anti-PSA antibody was immobilized could detect free PSA at a concentration of 0.2 ng mL
1 against a background of human serum albumin and plasminogen at concentrations of 1 mg mL
1 . Thus, this technique could compete with

Scanning electron micrograph of a section of silicon nanocantilever. (From Ref. [110], with permission.)

Fig. 8.7.

8.4 Cancer Treatment

ELISA-based diagnoses. Notably, however, although the movement of these cantilevers is on a nanometer scale, a typical microcantilever is 200 mm long, 0.5 mm thick and 20 mm wide, making it difficult to prepare dense arrays of such sensors. 8.3.4

Nano-tag made of Nanorods

Arraying addressable sensors on a miniaturized tip is one approach to constructing a multiplexed detection system. Other approaches include the simultaneous labeling of several biomolecules with distinctive molecular barcodes, which could be produced with the necessary diversity using nanorods [112, 113]. Sequential electrochemical deposition enables construction of combinatorial striping patterns using gold and silver nanorods, which then could be detected and read using light microscopy (Fig. 8.8). Theoretically, 4160 signatures could be generated from 13segment, 6.5-mm-long barcodes.

8.4

Cancer Treatment

While nanotube-based diagnosis systems are still at their early stages of development, nanotube-based approaches to cancer treatment are at the experimental

Fig. 8.8.

Synthesis of multimetal barcodes. (From Ref. [112], with permission.)

293

294

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

stage. By focusing on drug delivery and imaging systems, several ongoing efforts to make the most of the potential of nanotubes in this area will be introduced. 8.4.1

Carriers for Drug Delivery Systems

Although synthetic polymers, lipids and peptides are attracting much attention from those involved in the development of drug delivery systems, new classes of materials constructed from carbon nanotubes may also be suitable for drug, protein and gene delivery. Early observations have shown that fluorescently labeled nanotubes are automatically taken up by cells [114]. This internalization of SWNTs, e.g., by HL60 and Jurkat cells, appears to be mediated by endocytosis, leading to accumulation of SWNTs in the cytoplasm [115]. Moreover, when SWNTs are covalently modified with a peptide from the foot-and-mouth disease virus, they elicit a humoral immune response in mice [116]. Likewise, nanotube-based systems also have been shown to mediate gene delivery in similar fashion [117–119]. In addition to genes and DNA, unmethylated CpG motif [120], which is known to confer nonspecific protection against various intracellular pathogens and siRNAs [121] (small RNAs that interfere with the expression of a target gene), has been linked to SWNTs that were then used to deliver the motif into mammalian cells. SWNHs (Section 8.2.3) are unique among nanomaterials because their constituent parts clearly belong to the nanotube-family, but their overall structure is spherical (Fig. 8.3). The diameters of these spheres, about 80–100 nm, suggests they could show enhanced permeation and retention (EPR) effects [122, 123] and thus could accumulate within solid tumors showing neovascularity. Furthermore, as the synthesis of SWNHs does not require a metal catalyst [124], extremely pure preparations with no contaminating metals can be obtained [125], which would seem to make SWNHs an excellent candidate to be a carrier in a drug delivery system. Indeed, it already has been shown that oxidized SWNHs can adsorb dexamethasone to approximately 200 mg mL
1 and then slowly release the drug in saline buffer [126]. In addition, peptide aptamers [127] could be used to functionalize the surface of SWNHs and prepare composite materials having multifunctionality [128]. 8.4.2

Imaging Agents

New imaging agents that utilize nanomaterials have also been developed, and one using spherical nanoparticles has already shown good results. For example, dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles have been used to image lymph nodes containing micrometastases in patients with prostate cancer [129]. The development of nanotube-based imaging agents is still at a primitive stage, but the intrinsic near-infrared (NIR) fluorescence of SWNTs [130–132] is an attractive property, as tissue and biological fluids are transparent to NIR light [133], perhaps making it possible to use SWNTs as a new class of bioprobe [134, 135] (Fig. 8.9).

8.5 Conclusions

Fluorescence image of cell-engulfed SWNT between 1125 and 1600 nm with excitation at 660 nm. (From Ref. [134], with permission.)

Fig. 8.9.

Multifunctional molecules that can be used for both diagnosis and treatment should be the ultimate objective of nano-medical material development [136]. For instance, spherical multimodal composites composed of gold-coated nanoparticles containing a dielectric silicon core coated with PEG and a cancer-targeting ligand, Her2, have already been developed. Through illumination with NIR, this composite material has been successfully used to reveal microscopic tumors in breast, and then, by increasing the power of the NIR beam (820 nm), the particle’s temperature was increased enough to induce irreversible heat damage to the carcinoma cells [137]. Recently, a similar multifunctional agent was constructed from SWNTs [135]. In that case, SWNTs were functionalized with a folate moiety known to target tumor-associated antigen [138], and were selectively taken up by cells expressing folate receptor. Cells that internalized the SWNT were killed by irradiation with NIR [135].

8.5

Conclusions

Carbon nanotubes and related structures (e.g., nanowires and nanorods) are a recent discovery, and it will take time before materials and protocols that are clinically applicable for the diagnosis and treatment of cancer are developed. Nevertheless, these materials obviously have unique properties, which should soon lead to the development of radically new nanodevices. Although not introduced in this chapter, a family of nanotubes is currently being investigated with the aim of making new devices that could indirectly influence

295

296

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

cancer diagnosis and cancer therapy. These include probe tips for atomic-force microscopy (AFM) [139–141], ion channel blockers [142], and microcantilevers for medical catheter applications [36], among others. Finally, careful attention needs to be paid to the potential toxicity of nanomaterials. So far, studies of their toxicity have produced conflicting results [143–148]. Clearly, much additional study of the metabolic pathway taken by nanomaterials in the body [149] and their potential toxicity will need to be carried out before their clinical use becomes practical.

References 1 Iijima, S., Helical microtubules of

2

3

4

5

6

7

8

9

graphitic carbon., Nature 1991, 354, 56–58. Iijima, S., Carbon nanotubes: Past, present, and future., Physica B 2002, 323, 1–5. Tenne, R., Rao, C. N., Inorganic nanotubes., Phil. Trans. R. Soc. London A 2004, 362, 2099–2125. Iijima, S., Ichihashi, T., Single-shell carbon nanotubes of 1-nm diameter., Nature 1993, 363, 603–605. Bethune, D. S., Kiang, C. H., DeVries, M. S., Gorman, G., Savoy, R., Beyers, R., Cobalt-catalysed growth of carbon nanotubes with singleatomic-layer walls., Nature 1993, 363, 605–607. Qin, L.-C., Zhao, X., Hirahara, K., Miyamoto, Y., Ando, Y., Iijima, S., The smallest carbon nanotube., Nature 2000, 408, 50. Wang, N., Tang, Z. K., Li, G. D., Chen, J. S., Single-walled 4 A˚ carbon nanotube arrays., Nature 2000, 408, 50–51. Hayashi, T., Kim, Y. A., Matoba, T., Esaka, M., Nishimura, K., Tsukada, T., Endo, M., Dresselhaus, M. S., Smallest freestanding single-walled carbon nanotube., Nano Lett. 2003, 3, 887–889. Bandow, S., Takizawa, M., Hirahara, K., Yudasaka, M., Iijima, S., Raman scattering study of doublewall carbon nanotubes derived from the chains in single-wall carbon nanotubes., Chem. Phys. Lett. 2001, 337, 48–54.

10 Guo, T., Nikolaev, P., Thess, A.,

11

12

13

14

15

16

Colbert, D. T., Smalley, R. E., Catalytic growth of single-walled nanotubes by laser vaporization., Chem. Phys. Lett. 1995, 243, 49–54. Colomer, J.-F., Stephan, C., Lefrant, S., Tendeloo, G. V., Willems, I., Konya, Z., Fonseca, A., Laurent, C., Nagy, J. B., Large-scale synthesis of single-wall carbon nanotubes by catalytic chemical vapor deposition (CCVD) method., Chem. Phys. Lett. 2000, 317, 83–89. Journet, C., Maser, W. K., Bernier, P., Loiseau, A., Chapelle, M. L. d. l., Lefrant, S., Deniard, P., Lee, R., Fischer, J. E., Large-scale production of single-walled carbon nanotubes by the electric-arc technique., Nature 1997, 388, 756–758. Ugarte, D., Curling and closure of graphitic networks under electronbeam irradiation., Nature 1992, 359, 707–709. Ruoff, R. S., Lorents, D. C., Chan, B., Malhotra, R., Single crystal metals encapsulated in carbon nanoparticles., Science 1993, 259, 346–348. Tomita, M., Saito, Y., Hayashi, T., LaC2 encapsulated in graphite nanoparticle., Jpn. J. Appl. Phys. 1993, 32, L280–L282. Saito, Y., Yoshikawa, T., Inagaki, M., Tomita, M., Hayashi, T., Growth and structure of graphitic tubules and polyhedral particles in arc discharge., Chem. Phys. Lett. 1993, 204, 277–282.

References 17 Saito, Y., Yoshikawa, T., Bamboo-

18

19

20

21

22

23

24

25

26

27

28

shaped carbon tube filled partially with nickel., J. Cryst. Growth 1993, 134, 154–156. Zhang, G., Jiang, X., Wang, E., Tubular graphite cones., Science 2003, 300, 472–474. Iijima, S., Yudasaka, M., Yamada, R., Bandow, S., Suenaga, K., Kokai, F., Takahashi, K., Nano-aggregates of single-walled graphitic carbon nanohorns., Chem. Phys. Lett. 1999, 309, 165–170. Baughman, R. H., Zakhidov, A. A., de Heer, W. A., Carbon nanotubes – The route toward applications., Science 2002, 297, 787–792. Saito, R., Dresselhaus, G., Dresselhaus, M. S., Physical Properties of Carbon Nanotubes, Imperial College Press, London, 1998. Ebbesen, T. W., Lezec, H. J., Hiura, H., Bennett, J. W., Ghaemi, H. F., Thio, T., Electrical conductivity of individual carbon nanotubes., Nature 1996, 382, 54–56. Ouyang, M., Huang, J. L., Cheung, C. L., Lieber, C. M., Energy gaps in ‘‘metallic’’ single-walled carbon nanotubes., Science 2001, 292, 702–705. Tans, S. J., Devoret, M. H., Dai, H., Thess, A., Smalley, R. E., Geerligs, L. J., Dekker, C., Individual singlewall nanotubes as quantum wires., Nature 1997, 386, 474–477. Bockrath, M., Cobden, D. H., McEuen, P. L., Chopra, N. G., Zettl, A., Thess, A., Smalley, R. E., Singleelectron transport in ropes of carbon nanotubes., Science 1997, 275, 1922–1925. Tans, S. J., Verschueren, A. R. M., Dekker, C., Room-temperature transistor based on a single carbon nanotube., Nature 1998, 393, 49–52. Postma, H. W., Teepen, T., Yao, Z., Grifoni, M., Dekker, C., Carbon nanotube single-electron transistors at room temperature., Science 2001, 293, 76–79. Martel, R., Schmidt, T., Shea, H. R., Hertel, T., Avouris, P., Single- and multi-wall carbon nanotube field-effect

29

30

31

32

33

34

35

36

37

38

transistors., Appl. Phys. Lett. 1998, 73, 2447–2449. Bachtold, A., Hadley, P., Nakanishi, T., Dekker, C., Logic circuits with carbon nanotube transistors., Science 2001, 294, 1317–1320. Kong, J., Franklin, N. R., Zhou, C., Chapline, M. G., Peng, S., Cho, K., Dai, H., Nanotube molecular wires as chemical sensors., Science 2000, 287, 622–625. Collins, P. G., Bradley, K., Ishigami, M., Zettl, A., Extreme oxygen sensitivity of electronic properties of carbon nanotubes., Science 2000, 287, 1801–1804. Shim, M., Javey, A., Kam, N. W., Dai, H., Polymer functionalization for airstable n-type carbon nanotube fieldeffect transistors., J. Am. Chem. Soc. 2001, 123, 11 512–11 513. Chen, R. J., Bangsaruntip, S., Drouvalakis, K. A., Kam, N. W. S., Shim, M., Li, Y., Kim, W., Utz, P. J., Dai, H., Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors., Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4984–4989. Snow, E. S., Perkins, F. K., Houser, E. J., Badescu, S. C., Reinecke, T. L., Chemical detection with a singlewalled carbon nanotube capacitor., Science 2005, 307, 1942–1945. Barone, P. W., Baik, S., Heller, D. A., Strano, M. S., Near-infrared optical sensors based on single-walled carbon nanotubes., Nat. Mater. 2005, 4, 86–92. Baughman, R. H., Cui, C., Zakhidov, A. A., Iqbal, Z., Barisci, J. N., Spinks, G. M., Wallace, G. G., Mazzoldi, A., De Rossi, D., Rinzler, A. G., Jaschinski, O., Roth, S., Kertesz, M., Carbon nanotube actuators., Science 1999, 284, 1340–1344. Treacy, M. M. J., Ebbesen, T. W., Gibson, J. M., Exceptionally high Young’s modulus observed for individual carbon nanotubes., Nature 1996, 381, 678–680. Zhang, M., Fang, S., Zakhidov, A. A., Lee, S. B., Aliev, A. E.,

297

298

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

39

40

41

42

43

44

45

46

47

Williams, C. D., Atkinson, K. R., Baughman, R. H., Strong, transparent, multifunctional, carbon nanotube sheets., Science 2005, 309, 1215–1219. Endo, M., Muramatsu, H., Hayashi, T., Kim, Y. A., Terrones, M., Dresselhaus, M. S., ‘Buckypaper’ from coaxial nanotubes., Nature 2005, 433, 476. Sreekumar, T. V., Liu, T., Kumar, S., Single-wall carbon nanotube films., Chem. Mater., 2003, 15, 175–178. Wu, Z., Chen, Z., Du, X., Logan, J. M., Sippel, J., Nikolou, M., Kamaras, K., Reynolds, J. R., Tanner, D. B., Hebard, A. F., Rinzler, A. G., Transparent, conductive carbon nanotube films., Science 2004, 305, 1273–1276. Vigolo, B., Penicaud, A., Coulon, C., Sauder, C., Pailler, R., Journet, C., Bernier, P., Poulin, P., Macroscopic fibers and ribbons of oriented carbon nanotubes., Science 2000, 290, 1331–1334. Fukushima, T., Kosaka, A., Ishimura, Y., Yamamoto, T., Takigawa, T., Ishii, N., Aida, T., Molecular ordering of organic molten salts triggered by single-walled carbon nanotubes., Science 2003, 300, 2072–2074. MacDonald, R. A., Laurenzi, B. F., Viswanathan, G., Ajayan, P. M., Stegemann, J. P., Collagen-carbon nanotube composite materials as scaffolds in tissue engineering., J. Biomed. Mater. Res. A 2005, 74, 489–496. Meng, J., Kong, H., Xu, H. Y., Song, L., Wang, C. Y., Xie, S. S., Improving the blood compatibility of polyurethane using carbon nanotubes as fillers and its implications to cardiovascular surgery., J. Biomed. Mater. Res. A 2005, 74, 208–214. Mattson, M. P., Haddon, R. C., Rao, A. M., Molecular functionalization of carbon nanotubes and use as substrates for neuronal growth., J. Mol. Neurosci. 2000, 14, 175–182. Supronowicz, P. R., Ajayan, P. M., Ullmann, K. R., Arulanandam, B. P.,

48

49

50

51

52

53

54

55

56

Metzger, D. W., Bizios, R., Novel current-conducting composite substrates for exposing osteoblasts to alternating current stimulation., J. Biomed. Mater. Res. 2002, 59, 499–506. Correa-Duarte, M. A., Wagner, N., Rojas-Chapana, J., Morsczeck, C., Thie, M., Giersig, M., Fabrication and biocompatibility of carbon nanotube-based 3D networks as scaffolds for cell seeding and growth., Nano Lett. 2004, 4, 2233–2236. Hu, H. N. Y., Montana, V., Haddon, R. C., Parpura, V., Chemically functionalized carbon nanotubes as substrates for neuronal growth., Nano Lett. 2004, 4, 507–511. Chopra, N. G., Luyken, R., Cherrey, K., Crespi, V., Cohen, M., Louie, S., Zettl, A., Boron-nitride nanotubes., Science 1995, 269, 966–967. Terronesa, M., Hsua, W. K., Terronesb, H., Zhangc, J. P., Ramosb, S., Harea, J. P., Castillob, R., Prassidesa, K., Cheethamc, A. K., Krotoa, H. W., Waltona, D. R. M., Metal particle catalysed production of nanoscale BN structures., Chem. Phys. Lett. 1996, 259, 568–573. Seifert, G., Terronesb, H., Terronesc, M., Frauenheim, T., Novel NbS2 metallic nanotubes., Solid State Commun. 2000, 115, 635–638. Remskar, M., Mrzel, A., Skraba, Z., Jesih, A., Ceh, M., Demsar, J., Stadelmann, P., Levy, F., Mihailovic, D., Self-assembly of subnanometer-diameter single-wall MoS2 nanotubes., Science 2001, 292, 479–481. Zhang, Y., Suenaga, K., Colliex, C., Iijima, S., Coaxial nanocable: Silicon carbide and silicon oxide sheathed with boron nitride and carbon., Science 1998, 281, 973–975. Ajayan, P. M., Ebbesen, T. W., Ichihashi, T., Iijima, S., Tanigaki, K., Hiura, H., Opening carbon nanotubes with oxygen and implications for filling., Nature 1993, 362, 522–524. Tsang, S. C., Chen, Y. K., Harris, P. J. F., Green, M. L. H., A simple

References

57

58

59

60

61

62

63

64

65

66

67

chemical method of opening and filing carbon nanotubes., Nature 1994, 372, 159–162. Ajayan, P. M., Iijima, S., Capillarity induced filling in carbon nanotubes., Nature 1993, 361, 333–334. Seraphin, S., Zhou, D., Jiao, J., Withers, J. C., Loutfy, R., Yttrium carbide in nanotubes., Nature 1993, 362, 503. Ajayan, P. M., Colliex, C., Lambert, J. M., Bernier, P., Barbedette, L., Tence, M., Stephan, O., Growth of manganese filled carbon nanofibers in the vapor phase., Phys. Rev. Lett. 1994, 72, 1722–1725. Subramoney, S. R., Rodney, S., Lorents, D. C., Chan, B., Ripudaman, M., Dyer, M. J., Parvin, K., Magnetic separation of GdC2 encapsulated in carbon nanoparticles., Carbon 1994, 32, 507–513. Guerret-Pie´court, C., Bouar, Y. L., Lolseau, A., Pascard, H., Relation between metal electronic structure and morphology of metal compounds inside carbon nanotubes., Nature 1994, 372, 761–765. Dai, H., Wong, E. W., Lu, Y. Z., Fan, S., Lieber, C. M., Synthesis and characterization of carbide nanorods., Nature 1995, 375, 769–772. Han, W., Fan, S., Li, Q., Hu, Y., Synthesis of gallium nitride nanorods through a carbon nanotube-confined reaction., Science 1997, 277, 1287–1289. Pan, Z., Lai, H.-L., Au, F. C. K., Duan, X., Zhou, W., Shi, W., Wang, N., Lee, C.-S., Wong, N.-B., Lee, S.-T., Xie, S., Oriented silicon carbide nanowires: Synthesis and field emission properties., Adv. Mater. 2000, 12, 1186–1190. Duan, X., Lieber, C. M., Laserassisted catalytic growth of single crystal GaN nanowires., J. Am. Chem. Soc. 2000, 122, 188–189. Goldberger, J., He, R., Zhang, Y., Lee, S., Yan, H., Choi, H. J., Yang, P., Single-crystal gallium nitride nanotubes., Nature 2003, 422, 599–602. Cui, Y., Duan, X., Hu, J., Lieber, C. M., Doping and electrical transport

68

69

70

71

72

73

74

75

76

77

78

in silicon nanowires., J. Phys. Chem. B 2000, 104, 5213–5216. Cui, Y., Lieber, C. M., Functional nanoscale electronic devices assembled using silicon nanowire building blocks., Science 2001, 291, 851–853. Duan, X., Huang, Y., Cui, Y., Wang, J., Lieber, C. M., Indium phosphide nanowires as building blocks for nanoscale electronic and optoelectronic devices., Nature 2001, 409, 66– 69. Zhou, D., Seraphin, S., Production of silicon carbide whiskers from carbon nanoclusters., Chem. Phys. Lett. 1994, 222, 233–238. Yager, P., Schoen, P. E., Formation of tubules by a polymerizable surfactant., Mol. Cryst. Liq. Cryst. 1984, 106, 371–381. Nakashima, N., Asakuma, S., Kunitake, T., Optical microscopic study of helical superstructures of chiral bilayer membranes., J. Am. Chem. Soc. 1985, 107, 509–510. Schnur, J. M., Lipid tubules: A paradigm for molecularly engineered structures., Science 1993, 262, 1669–1676. Karlsson, A., Karlsson, R., Karlsson, M., Cans, A. S., Stromberg, A., Ryttsen, F., Orwar, O., Networks of nanotubes and containers., Nature 2001, 409, 150–152. Karlsson, M., Sott, K., Davidson, M., Cans, A. S., Linderholm, P., Chiu, D., Orwar, O., Formation of geometrically complex lipid nanotubevesicle networks of higher-order topologies., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11 573–11 578. Ghadiri, M. R., Tirrell, D. A., Chemistry at the crossroads., Curr. Opin. Chem. Biol. 2000, 4, 661–662. Matsui, H., Gologan, B., Crystalline glycylglycinebolaamphiphile tubules and their pH-sensitive structural transformation., J. Phys. Chem. B 2000, 104, 3383–3386. Khazanovich, N., Granja, J. R., McRee, D. E., Ronald, A., Milligan, Ghadiri, M. R., Nanoscale tubular

299

300

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

79

80

81

82

83

84

85

86

87

88

ensembles with specified internal diameters. Design of a self-assembled nanotube with a 13 A˚ pore, J. Am. Chem. Soc. 1994, 116, 6011–6012. Reches, M., Gazit, E., Casting metal nanowires within discrete selfassembled peptide nanotubes., Science 2003, 300, 625–627. Yu, K., Zhang, L., Eisenberg, A., Novel morphologies of ‘‘crew-cut’’ aggregates of amphiphilic diblock copolymers in dilute solution., Langmuir 1996, 12, 5980–5984. Raez, J. M., I., Winnik, M. A., Nanotubes from the self-assembly of asymmetric crystalline-coil poly(ferrocenylsilane-siloxane) block copolymers., J. Am. Chem. Soc. 2002, 124, 10 381–10 395. Discher, B. M., Won, Y. Y., Ege, D. S., Lee, J. C., Bates, F. S., Discher, D. E., Hammer, D. A., Polymersomes: Tough vesicles made from diblock copolymers., Science 1999, 284, 1143–1146. Pochan, D. J., Chen, Z., Cui, H., Hales, K., Qi, K., Wooley, K. L., Toroidal triblock copolymer assemblies., Science 2004, 306, 94–97. Cha, J. N., Stucky, G. D., Morse, D. E., Deming, T. J., Biomimetic synthesis of ordered silica structures mediated by block copolypeptides., Nature 2000, 403, 289–292. Lee, S. W., Mao, C., Flynn, C. E., Belcher, A. M., Ordering of quantum dots using genetically engineered viruses., Science 2002, 296, 892–895. Banerjee, I. A., Yu, L., Matsui, H., Cu nanocrystal growth on peptide nanotubes by biomineralization: Size control of Cu nanocrystals by tuning peptide conformation., Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14 678– 14 682. Djalali, R., Chen, Y. F., Matsui, H., Au nanocrystal growth on nanotubes controlled by conformations and charges of sequenced peptide templates., J. Am. Chem. Soc. 2003, 125, 5873–5879. Mao, C., Flynn, C. E., Hayhurst, A., Sweeney, R., Qi, J., Georgiou, G., Iverson, B., Belcher, A. M., Viral

89

90

91

92

93

94

95

96

97

98

assembly of oriented quantum dot nanowires., Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6946–6951. Schembri, M. A., Kjaergaard, K., Klemm, P., Bioaccumulation of heavy metals by fimbrial designer adhesins., FEMS Microbiol. Lett. 1999, 170, 363–371. Mao, C., Solis, D. J., Reiss, B. D., Kottmann, S. T., Sweeney, R. Y., Hayhurst, A., Georgiou, G., Iverson, B., Belcher, A. M., Virusbased toolkit for the directed synthesis of magnetic and semiconducting nanowires., Science 2004, 303, 213–217. Reiss, B. D., Mao, C., Solis, D. J., Ryan, K. S., Thomson, T., Belcher, A. M., Biological routes to metal alloy ferromagnetic nanostructures., Nano Lett. 2004, 4, 1127–1132. Wagner, D. E., Phillips, C. L., Ali, W. M., Nybakken, G. E., Crawford, E. D., Schwab, A. D., Smith, W. F., Fairman, R., Toward the development of peptide nanofilaments and nanoropes as smart materials., Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12 656–12 661. Sander, C., Genomic medicine and the future of health care., Science 2000, 287, 1977–1978. Kitano, H., Cancer as a robust system: Implications for anticancer therapy., Nat. Rev. Cancer 2004, 4, 227–235. Schena, M., Shalon, D., Davis, R. W., Brown, P. O., Quantitative monitoring of gene expression patterns with a complementary DNA microarray., Science 1995, 270, 467–470. DeRisi, J. L., Iyer, V. R., Brown, P. O., Exploring the metabolic and genetic control of gene expression on a genomic scale., Science 1997, 278, 680–686. Lockhart, D. J., Winzeler, E. A., Genomics, gene expression and DNA arrays., Nature 2000, 405, 827– 836. Li, J., Lu, Y., Ye, Q., Cinke, M., Han, J., Meyyappan, M., Carbon nanotube sensors for gas and organic vapor

References

99

100

101

102

103

104

105

106

107

108

detection., Nano Lett. 2003, 3, 929–933. Star, A., Gabriel, J.-C. P., Bradley, K., Gruner, G., Electronic detection of specific protein binding using nanotube FET devices., Nano Lett. 2003, 3, 459–463. Besteman, K. L., Lee, J.-O., Wiertz, F. G. M., Heering, H. A., Dekker, C., Enzyme-coated carbon nanotubes as single-molecule biosensors., Nano Lett. 2003, 3, 727–730. Ellington, A. D., Szostak, J. W., In vitro selection of RNA molecules that bind specific ligands., Nature 1990, 346, 818–822. Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H., Toole, J. J., Selection of single-stranded DNA molecules that bind and inhibit human thrombin., Nature 1992, 355, 564–566. Scott, J. K., Smith, G. P., Searching for peptide ligands with an epitope library., Science 1990, 249, 386–390. Li, J., Ng, H. T., Cassell, A. F., W., Chen, H. Y., Q., Koehne, J., Han, J., Meyyappan, M., Carbon nanotube nanoelectrode array for ultrasensitive DNA detection., Nano Lett. 2003, 3, 597–602. Sistare, M. F., Holmberg, R. C.; Thorp, H. H., Electrochemical studies of polynucleotide binding and oxidation by metal complexes: Effects of scan rate, concentration, and sequence., J. Phys. Chem. B 1999, 103, 10 718–10 728. Cui, Y., Wei, Q., Park, H., Lieber, C. M., Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species., Science 2001, 293, 1289–1292. Hahm, J.-I. Lieber, C. M., Direct ultrasensitive electrical detection of DNA and DNA sequence variations using nanowire nanosensors., Nano Lett. 2004, 4, 51–54. Li, C., Curreli, M., Lin, H., Lei, B., Ishikawa, F. N., Datar, R., Cote, R. J., Thompson, M. E., Zhou, C., Complementary detection of prostatespecific antigen using In2 O3 nanowires and carbon nanotubes.,

109

110

111

112

113

114

115

116

117

J. Am. Chem. Soc. 2005, 127, 12 484– 12 485. Raiteri, R., Nelles, G., Butt, H.-J., Knoll, W., Skladal, P., Sensing of biological substances based on the bending of microfabricated cantilevers., Sens. Actuators B 1999, 61, 213–217. Fritz, J., Baller, M. K., Lang, H. P., Rothuizen, H., Vettiger, P., Meyer, E., Guntherodt, H., Gerber, C., Gimzewski, J. K., Translating biomolecular recognition into nanomechanics., Science 2000, 288, 316–318. Wu, G., Ji, H., Hansen, K., Thundat, T., Datar, R., Cote, R., Hagan, M. F., Chakraborty, A. K., Majumdar, A., Origin of nanomechanical cantilever motion generated from biomolecular interactions., Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560–1564. Nicewarner-Pena, S. R., Freeman, R. G., Reiss, B. D., He, L., Pena, D. J., Walton, I. D., Cromer, R., Keating, C. D., Natan, M. J., Submicrometer metallic barcodes., Science 2001, 294, 137–141. Freeman, R. G., Raju, P. A., Norton, S. M., Walton, I. D., Smith, P. C., He, L., Natan, M. J., Sha, M. Y., Penn, S. G., Use of nanobarcodes particles in bioassays., Methods Mol. Biol. 2005, 303, 73–83. Pantarotto, D., Briand, J. P., Prato, M., Bianco, A., Translocation of bioactive peptides across cell membranes by carbon nanotubes., Chem. Comm. 2004, 16–17. Pantarotto, D., Partidos, C. D., Graff, R., Hoebeke, J., Briand, J. P., Prato, M., Bianco, A., Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides., J. Am. Chem. Soc. 2003, 125, 6160–6164. Kam, N. W. S., Jessop, T. C., Wender, P. A., Dai, H., Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells., J. Am. Chem. Soc. 2004, 126, 6850–6851. Lu, Q., Moore, J. M., Huang, G., Mount, A. S., Rao, A. M., Larcom,

301

302

8 Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis

118

119

120

121

122

123

124

L. L., Ke, P. C., RNA polymer translocation with single-walled carbon nanotubes., Nano Lett. 2004, 4, 2473–2477. Pantarotto, D., Singh, R., McCarthy, D., Erhardt, M., Briand, J. P., Prato, M., Kostarelos, K., Bianco, A., Functionalized carbon nanotubes for plasmid DNA gene delivery., Angew. Chem. Int. Ed. 2004, 43, 5242–5246. Singh, R., Pantarotto, D., McCarthy, D., Chaloin, O., Hoebeke, J., Partidos, C. D., Briand, J. P., Prato, M., Bianco, A., Kostarelos, K., Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: Toward the construction of nanotubebased gene delivery vectors., J. Am. Chem. Soc. 2005, 127, 4388–4396. Bianco, A., Hoebeke, J., Godefroy, S., Chaloin, O., Pantarotto, D., Briand, J. P., Muller, S., Prato, M., Partidos, C. D., Cationic carbon nanotubes bind to CpG oligodeoxynucleotides and enhance their immunostimulatory properties., J. Am. Chem. Soc. 2005, 127, 58–59. Kam, N. W. S., Liu, Z., Dai, H., Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing., J. Am. Chem. Soc. 2005, 127, 12 492–12 493. Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hor, i. K., Tumour vascular permeability and the EPR effect in macromolecular therapeutics., J. Controlled Release 2000, 65, 271–284. Matsumura, Y., Maeda, H., A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS., Cancer Res. 1986, 46, 6387–6392. Kasuya, D., Yudasaka, M., Takahashi, K., Kokai, F., Iijima, S., Selective production of single-wall carbon nanohorn aggregates and their formation mechanism., J. Phys. Chem. B 2002, 106, 4947–4951.

125 Shvedova, A. A., Castranova, V.,

126

127

128

129

130

131

132

Kisin, E. R., Schwegler-Berry, D., Murray, A. R., Gandelsman, V. Z., Maynard, A., Baron, P., Exposure to carbon nanotube material: Assessment of nanotube cytotoxicity using human keratinocyte cells., J. Toxicol. Environ. Health A 2003, 66, 1909–1926. Murakami, T., Ajima, K., Miyawaki, J., Yudasaka, M., Iijima, S., Shiba, K., Drug-loaded carbon nanohorns: Adsorption and release of dexamethasone in vitro., Mol. Pharm. 2004, 1, 399–405. Kase, D., Kulp, J. L., 3rd, Yudasaka, M., Evans, J. S., Iijima, S., Shiba, K., Affinity selection of peptide phage libraries against single-wall carbon nanohorns identifies a peptide aptamer with conformational variability., Langmuir 2004, 20, 8939–8941. Sano, K., Ajima, K., Iwahori, K., Yudasaka, M., Iijima, S., Yamashita, I., Shiba, K., Endowing a ferritin-like cage protein with high affinity and selectivity for certain inorganic materials., Small 2005, 1, 826–832. Harisinghani, M. G., Barentsz, J., Hahn, P. F., Deserno, W. M., Tabatabaei, S., van de Kaa, C. H., de la Rosette, J., Weissleder, R., Noninvasive detection of clinically occult lymph-node metastases in prostate cancer., N. Engl. J. Med. 2003, 348, 2491–2499. O’Connell, M. J., Bachilo, S. M., Huffman, C. B., Moore, V. C., Strano, M. S., Haroz, E. H., Rialon, K. L., Boul, P. J., Noon, W. H., Kittrell, C., Ma, J., Hauge, R. H., Weisman, R. B., Smalley, R. E., Band gap fluorescence from individual single-walled carbon nanotubes., Science 2002, 297, 593–596. Bachilo, S. M., Strano, M. S., Kittrell, C., Hauge, R. H., Smalley, R. E., Weisman, R. B., Structureassigned optical spectra of singlewalled carbon nanotubes., Science 2002, 298, 2361–2366. Hartschuh, A., Pedrosa, H. N., Novotny, L., Krauss, T. D., Simultaneous fluorescence and

References

133

134

135

136

137

138

139

140

Raman scattering from single carbon nanotubes., Science 2003, 301, 1354–1356. Kim, S., Lim, Y. T., Soltesz, E. G., De Grand, A. M., Lee, J., Nakayama, A., Parker, J. A., Mihaljevic, T., Laurence, R. G., Dor, D. M., Cohn, L. H., Bawendi, M. G., Frangioni, J. V., Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping., Nat. Biotechnol. 2004, 22, 93–97. Cherukuri, P., Bachilo, S. M., Litovsky, S. H., Weisman, R. B., Near-infrared fluorescence microscopy of single-walled carbon nanotubes in phagocytic cells., J. Am. Chem. Soc. 2004, 126, 15 638–15 639. Kam, N. W. S., O’connell, M., Wisdom, J. A., Dai, H., Carbon nanotubes as multifunctional biological transporters and nearinfrared agents for selective cancer cell destruction., Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11 600–11 605. Kircher, M. F., Mahmood, U., King, R. S., Weissleder, R., Josephson, L., A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation., Cancer Res. 2003, 63, 8122–8125. Hirsch, L. R., Stafford, R. J., Bankson, J. A., Sershen, S. R., Rivera, B., Price, R. E., Hazle, J. D., Halas, N. J., West, J. L., Nanoshellmediated near-infrared thermal therapy of tumors under magnetic resonance guidance., Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13 549– 13 554. Lu, Y., Sega, E., Leamon, C. P., Low, P. S., Folate receptor-targeted immunotherapy of cancer: Mechanism and therapeutic potential., Adv. Drug Delivery Rev. 2004, 56, 1161–1176. Dai, H., Hafner, J. H., Rinzler, A. G., Colbert, D. T., Smalley, R. E., Nanotubes as nanoprobes in scanning probe microscopy., Nature 1996, 384, 147–150. Wong, S. S., Joselevich, E., Woolley, A. T., Cheung, C. L., Lieber, C. M., Covalently func-

141

142

143

144

145

146

147

148

149

tionalized nanotubes as nanometresized probes in chemistry and biology., Nature 1998, 394, 52–55. Woolley, A. T., Cheung, C. L., Hafner, J. H., Lieber, C. M., Structural biology with carbon nanotube AFM probes., Chem. Biol. 2000, 7, R193–204. Parks, G. A., The isoelectric points of solid oxides, solid hydroxides, and aqueous hydroxo complex systems., Chem. Rev. 1965, 65, 177–198. Warheit, D. B., Laurence, B. R., Reed, K. L., Roach, D. H., Reynolds, G. A., Webb, T. R., Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats., Toxicol. Sci. 2004, 77, 117–125. Lam, C. W., James, J. T., McCluskey, R., Hunter, R. L., Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation., Toxicol. Sci. 2004, 77, 126–134. Huczko, A., Lange, H., Calko, E., Grubek-Jaworska, H., Droszcz, P., Physiological testing of carbon nanotubes: Are they asbestos-like?, Fullerene Sci. Technol. 2001, 9, 251–254. Cui, D., Tian, F., Ozkan, C. S., Wang, M., Gao, H., Effect of single wall carbon nanotubes on human HEK293 cells., Toxicol. Lett. 2005, 155, 73–85. Monteiro-Riviere, N. A., Nemanich, R. J., Inman, A. O., Wang, Y. Y., Riviere, J. E., Multi-walled carbon nanotube interactions with human epidermal keratinocytes., Toxicol. Lett. 2005, 155, 377–384. Manna, S. K., Sarkar, S., Barr, J., Wise, K., Barrera, E. V., Jejelowo, O., Rice-Ficht, A. C., Ramesh, G. T., Single-walled carbon nanotube induces oxidative stress and activates nuclear transcription factor-kB in human keratinocytes., Nano Lett. 2005, 5, 1676–1684. Wang, H., Wang, J., Deng, X., Sun, H., Shi, Z., Gu, Z., Liu, Y., Zhao, Y., Biodistribution of carbon single-wall carbon nanotubes in mice., J. Nanosci. Nanotechnol. 2004, 4, 1019–1024.

303

304

9

Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy Sang Bok Lee and Sang Jun Son 9.1

Introduction

Nanomaterials have been one of the most widely studied and attractive subjects for the last decade in the field of biomedical and biotechnological applications, such as MRI contrasting agent [1], DNA transfection [2], biosensors [3], and drug delivery [4]. These nanoparticles are particularly interesting because many of their properties, such as electronic and optical properties, are quite different from those of bulk material. These new properties have provided new opportunities to develop ideal diagnostic/therapeutic systems and to overcome existing biological barriers such as that only very small amounts of monoclonal antibodies can find their targets in vivo system [5]. For example, liposomes, the simplest biologically friendly form of nanoparticles, are being used to increase drug concentration at tumor sites; their doxorubicin-embedded complex was approved by the FDA 10 years ago for the treatment of Kaposi’s sarcoma, and now they are used in treating breast cancer [6]. While liposomes deal mainly with sizes, not with unique physical properties, for their application, inorganic nanoparticles, such as gold nanoparticles [7], gold nanoshells [8], and quantum dots [9], use the unique electronic and optical properties of nanoscale materials. They are now in commercial use and development as diagnostic and therapeutic tools. All of these nanoparticles can be filled with anticancer drugs and detection agents for the targeted gene therapy and early stage cancer diagnosis [10]. Typically, spherical nanoparticles have been used for most nanobiotechnological applications because they are easy to make. However, spherical nanoparticles still need to be improved in terms of controlling particle sizes, surface functionalization, and environmental compatibility due to their structural limitations when multifunctionality is required, especially on their surfaces. The spherical nanoparticle is not an ideal platform for multifunctional nanomaterials because it has a single surface, so that every surface modification for the multifunctionality takes place at the same surface, which can lead to malfunction or interruption between multifunctional components. Tubular nanoparticles have become highly attractive for multifunctional purposes due to their structural attributes, such as distinctive Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

9.2 Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications

inner and outer surfaces, over conventional spherical nanoparticles. Inner voids can be used for capturing, concentrating, and releasing species ranging in size from large proteins to small molecules because tube dimensions can be easily controlled by template synthesis. Distinctive outer surfaces can be differentially functionalized with environmentally friendly and/or probe molecules to a specific target. These differentially functionalized, or multifunctionalized, nanotubes have enormous potential in biomedical application, including therapeutic tools [11], drug delivery [12], bioseparations, and biocatalysis [13, 14], and have provided a model system to study wetting and diffusion problems in nanoscale containers [15]. In addition to these nanotubes, nanowires or nanorods have also been highlighted with respect to multifunctionality for biotechnological application [16, 17]. Although nanotubes and nanowires offer huge opportunities and might prove to be useful in bio-nanotechnology – most likely cancer technology, such as early detection and targeted drug delivery for cancer tumors – there are as yet few example applications of diagnostic and therapeutic tools. From this viewpoint, the present chapter includes (a) the most recent success story of magnetic nanoparticles, (b) carbon nanotubes, (c) nanotubes and nanowires composed of artificial peptides, and (d) template-synthesized nanotubes, such as silica and magnetic nanotubes, for the various biomedical applications.

9.2

Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications

Magnetic particles have been extensively studied in the field of biomedical and biotechnological applications, including drug delivery, biosensors, chemical and biochemical separation and concentration of trace amount of specific targets, such as bacteria or leukocytes, enzyme encapsulation, and contrast enhancement in magnetic resonance imaging (MRI) [18–20]. Practically, magnetic nanoparticles are ideal candidate materials for the early detection of cancer and targeted drug delivery due to their natural MRI capability, together with the ability to modify the particle surface with targeting moieties [21]. In addition, new synthetic methods have been recently reported to improve control over particle sizes up to 1-nm scale for mass production [22–24]. 9.2.1

MRI and Therapeutic Application of Magnetic Nanoparticles

Recently, a team of Korean scientists have reported that magnetic nanoparticles can be successfully used for cancer diagnosis both in vitro and in vivo [23, 25, 26]. Magnetic nanoparticles can play a key role in magnetic resonance (MR) diagnosis of early state cancer, which requires sensitive and selective detection techniques because of their strong enhancement in MR signal. In addition, nano-scale size is favorable for more specific and active targeting toward cancer cells due to the leaky and small porous structure of such cells. The team, directed by Jinwoo Cheon, re-

305

306

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Schematic illustration of 2,3-dimercaptosuccinic acid (DMSA)-coated water-soluble Fe3 O4 iron oxide (WSIO) nanocrystals and their solubility test (left-hand side) and in vitro cancer diagnosis assisted by WSIO–Herceptin probe conjugate (right-hand side). (From Ref. [26].)

Fig. 9.1.

ported that 9 nm water-soluble Fe3 O4 iron oxide (WSIO) nanoparticles are ideal material for cancer diagnosis. Magnetic Fe3 O4 nanoparticles were synthesized through thermal decomposition of Fe(acac)3 (acac ¼ acetylacetonate) in a hot organic solvent and their surface was covered with 2,3-dimercaptosuccinic acid (DMSA). The carboxylic group of DMSA ligand is coordinated to the metal surface and disulfide crosslinkage between the ligands stabilizes the ligand shell. This ligand increases the solubility in water, and remaining free thiol groups can be used to attach target-specific antibodies (Fig. 9.1). In this report, the DMSA ligand shell was conjugated with the cancer-targeting antibody Herceptin, which has specific binding properties against a HER2/new receptor overexpressed from breast cancer cells. For in vitro study, they examined the binding specificity by treating WSIO-Herceptin probe to a breast cancer cell line, SK-BR-3, and performing an MRI measurement. There was significant darkening of the MR image of the cell line treated with WSIO–Herceptin, while no noticeable difference was observed between the nontreated and WSIO-irrelevant antibody control conjugate-treated cells. The capability of WSIO with multifunctionality for in vivo use was further tested with a set of nude mouse implanted at their proximal thigh region with NIH3T6.7 cell lines overexpressed with HER2/neu cancer markers.

9.2 Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications

Fig. 9.2. In vivo MRI of cancer-targeting events of WSIOirrelevant antibody conjugate (a) and WSIO–Herceptin probe conjugates (b). (From Ref. [25].)

Figure 9.2 shows in vivo MRI of cancer-targeting events of WSIO-antibody conjugates. Color maps of T2-weighted MR images of cancer cell implanted (NIH3T6.7) mice at different temporal points (preinjection, immediate post, 4 h) after the intravenous injection of WSIO-irrelevant antibody control conjugates (a) and WSIO-Herceptin probe conjugates (b) reveals that WSIO–Herceptin conjugates induce an immediate color change in the color-mapped MR signal at the tumor site within 5 min. This implies that the WSIO–Herceptin probe conjugates successfully reach and bind to the target cancer cells at the proximal thigh. In the control experiment with WSIO-irrelevant antibody conjugates, however, no change was observed, confirming minimal nonspecific binding to the tumor site. 9.2.2

Biomedical Diagnostic Application of Magnetic Nanoparticles

Another interesting application of magnetic nanoparticles (MNPs) was reported in Science in 2005 by Kim’s group [27]. They developed a technology called magnetism-based interaction capture (MAGIC) for the detection of molecular interactions in live cells. Combined with confocal microscopy, which has a very narrow focal plane, the induced movement of superparamagnetic nanoparticles was adapted to detect the molecular interaction of interest or to identify molecular targets. As seen in Fig. 9.3, after internalization of MNPs coated with target-specific probes into live cells, application of a magnetic field can induce concentration of MNPs and associated target proteins at the focal plane, and the translocation of the fluorescent signal can be used to determine whether binding events occur in-

307

308

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Schematic of magnetism-base interaction capture (MAGIC) (left-hand side) and fluorescence microscope images showing translocation of MNPs coated with FITC and TAT-HA2 inside cells by the magnetic field (right-hand side). (From Ref. [27].)

Fig. 9.3.

side cells between probe molecule and target proteins. In this study, streptavidinconjugated MNPs were used as generic reagent for the attachment of biotinylated molecules to the nanoprobe, and efficient intracellular uptake of nanoparticles was mediated by transducible fusogenic TAT-HA2 peptide, which has a high density of cationic residues and thus causes an electrostatic interaction with the negatively charged cell surface. To visualize and track the distribution of MNPs within cells, MNPs coated with fluorescein isothiocyanate (FITC) and TAT-HA2 were prepared; when a magnetic field was applied, specific translocation of MNPs was observed inside cells. Furthermore, this translocation was reversible; the MNPs rapidly dif-

9.2 Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications

fused away upon removal of the magnetic field and were redirected when it was reapplied. The authors tested whether the MAGIC principle could be used to detect a specific intracellular target for Asp-Glu-Val-Asp (DEVD), an apoptosis inhibitor known to bind caspase-3. After MNPs modified with DEVD and TAT-HA2 were incubated with HeLa cells transfected with caspase-3 fused to red fluorescent protein (mRFP), the red signal translocated in the direction of the magnetic field. As a systematic target identification (ID) experiment for a bioactive small molecule, a normalized EGFP-tagged cDNA library was expressed in HeLa cells by retroviral transduction, and MNPs coated with FK506 were introduced into these cells to identify the receptors for an immunosuppressant, FK506. They identified 19 positive clones that exhibited specific translocation of EGFP in the direction of the magnetic field, and several known FK506-binding proteins and proteins of unknown function were found in these clones. This MAGIC principle can also be use to probe intercellular signaling processes, such as signal-induced phosphorylation and interaction of the NF-kB/IkB. Enzymes, when immobilized on magnetic nanoparticles (MNPs), can be easily separated from the reaction medium, stored, and reused with consistent results [28]. This system offers a relatively simple technique for separating and reusing enzymes over a longer period than that for both free enzymes alone and enzymes immobilized by physisorption. The most significant advantage of enzyme immobilization on magnetic nanoparticles is their long-term stability, such as reported by Gross’s group using Candida rugosa lipase (E.C.3.1.1.3) immobilized on g-Fe2 O3 magnetic nanoparticles. Lipases are frequently employed enzymes as they are commonly used for the synthesis of enantio-enriched monomers and macromers and for polymerization reactions [29]. MNPs (20 nm) were prepared by sonication of Fe(CO) in decalin and the subsequent annealing of amorphous Fe2 O3 nanoparticles. Acetylated thiophene on MNPs was reacted directly with the enzyme. The enzymatic activity of the immobilized lipase was determined by following the ester cleavage of 4-nitrophenyl butyrate. The activity of Candida rugosa lipase immobilized on MNPs turned out to be lower than that for the free enzyme, but constant activity over one month was seen in the case of lipase immobilized on MNPs. Using bifunctional MNPs, Xu’s group reported an instant and sensitive detection method for pathogens at ultralow concentration [30]. Compared with magnetic microbeads used in biological separation, MNPs promise high performance because of their large surface/volume ratios and easy entry into cells. For fast pathogen detection, FePt nanoparticles modified with vancomycin, a broad spectrum antibiotic, were prepared as a system that combines two kinds of interactions: magnetic dipole interactions that aggregate the MNPs under an applied magnetic field and specific multi-ligand–receptor interactions. Figure 9.4 shows a schematic illustration for the capture of bacteria by vancomycin-conjugated magnetic nanoparticles. Vancomycin was used to detect pathogens because it can bind to the terminal peptide, d-Ala-d-Ala, on the cell wall of a Gram-positive bacterium via hydrogen bonds. After mixing the solution of MNPs modified with vancomycin with a solution of bacterium, a point magnet was used to capture the ‘‘magnetized’’ bacteria and aggregates were analyzed using a microscopic method. The optical and SEM results

309

310

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Illustration of the capture of bacteria by vancomycinconjugated magnetic nanoparticles (A), via a plausible multivalent interaction, and the corresponding control experiment (B). (From Ref. [30].)

Fig. 9.4.

show that MNPs modified with vancomycin selectively bind to most Gram-positive bacteria and vancomycin-resistant enterococci (VRE).

9.3

Carbon Nanotubes

Since Iijima discovered the first carbon nanotubes, using the arc discharge method, in 1991 [31], many nanoparticle-based catalytic methods have been developed and have contributed to the improvement in quality and size control of carbon nanotubes [32, 33]. Typically, carbon nanotubes are one to tens of nanometers in diameter and one to hundreds of micrometers long. Although interest in the application of carbon nanotubes has focused mainly on microelectronic devices [34–38], tremendous public concern has pressurized research into their effect on human health. Scientists have begun this bio-related research with the immobilization of biomolecules on carbon nanotube surfaces [39–42]. 9.3.1

Carbon Nanotubes for Targeted Cancer Cell Death

Hongjie Dai’s group have reported that single-wall carbon nanotubes (SWNTs) can be a good multifunctional biological transporter and near-infrared agent for drug

9.3 Carbon Nanotubes

NIR absorbance of SWNT at 808 nm vs. SWNT concentrations (left-hand side) and schematic of Cy3-DNAfunctionalized SWNT (right-hand side). (From Ref. [43].)

Fig. 9.5.

delivery and cancer therapy [43]. SWNTs prepared from high-pressure CO conversion (Hipco) [44] have strong optical absorbance in the near-infrared (NIR) spectral window to which biological systems are highly transparent [45] and this property can be used for optical stimulation of nanotubes inside living cells to release their cargoes such as DNA and drug molecules or destroy cancer cell selectively. Figure 9.5 shows NIR absorbance of SWNT at 808 nm vs. SWNT concentrations; the molar extinction coefficient of the solubilized SWNTs (molecular mass A 170 kDa, length A 150 nm, diameter A 1.2 nm) measured at l ¼ 808 nm in the NIR was e A 7:9
10 6 m1 cm1 . The high absorbance of SWNTs in the NIR originates from electronic transitions between the first or second van Hove singularities of the nanotubes. This work exploited the high optical absorbance of SWNTs in the 700–1100 nm NIR window transparent to biological systems at a single wavelength by using an 808-nm laser for in vitro radiation [46]. For the DNA transfection experiment, Hipco SWNTs were conjugated with Cy3-DNA by noncovalent adsorption and incubated with HeLa cells at 37  C. As seen in Fig. 9.6, the green color in confocal fluorescence image was observed in the cytoplasm region of HeLa, which implies that DNA–SWNT conjugates were internalized inside the cells, but not the nucleus, with nanotubes as the transporter. Upon irradiating the HeLa cells with NIR after DNA–SWNT uptake, colocalization of fluorescence of Cy3-DNA in the cell nucleus was detected, indicating the release of DNA cargoes from SWNT transporters and nuclear translocation after the laser pulses. Conversely, experiments carried out at 4  C found no uptake of Cy3-DNASWNT conjugates inside cells, suggesting an energy-dependent endocytosis mechanism [47] for the uptake observed at 37  C. For selective cancer cell destruction in vitro, SWNTs were noncovalently functionalized with phospholipids that had a poly(ethylene glycol) (PEG) moiety and folic acid (FA) terminal group and then in-

311

312

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Confocal fluorescence images of HeLa cells showing transporting DNA inside living cells by SWNTs: (a) After incubation with Cy3-DNA-SWNT (green); (b) after nucleus

Fig. 9.6.

staining of HeLa cells by DRAQ5 (red); and (c) after incubation within a Cy3-DNA-SWNT at the low temperature of 4  C and nucleus staining. (From Ref. [43].)

cubated with folate receptor (FR)-overexpressed HeLa cell and normal cells, respectively. FRs are common tumor markers expressed at high levels on the surfaces of various cancer cells and facilitate cellular internalization of folate-containing species by receptor-mediated endocytosis. After NIR irradiation, extensive cell death for the FR-overexpressed HeLa cells was evidenced by drastic cell morphology changes, whereas the normal cells remained intact. The selective destruction of FR-overexpressed cells was a result of selective binding of FA-functionalized SWNTs and FRs on FR-overexpressed cell surfaces and receptor-mediated endocytosis. This shows that SWNTs are molecular transporters or carriers with very high optical absorbance in the NIR regime where biological systems are transparent. Compaired to Au nanoshells employed by Halas, West, and coworkers [48], the SWNTs can be the better NIR absorbing material for cancer cell destruction as lower laser power and shorter radiation times are needed. 9.3.2

Carbon Nanotubes for Detection of Cancer Cell

Carbon nanotubes have been integrated into a biosensor for the detection of prostate cancer by Chongwu Zhou’s group [49]. Figure 9.7 shows, schematically, a device where an active channel made up of nanowires and nanotubes bridges the source/drain electrodes, and the silicon substrate can be used as a gate. They used n-type In2 O3 nanowires (NW) and p-type carbon nanotubes for the complementary detection of prostate specific antigen (PSA), a known oncological marker for the presence of prostate cancer. To detect PSA, they modified the outer surface of NW or SWNT with anti-PSA monoclonal antibody (PSA-AB), a specific ligand for PSA protein. As shown in Fig. 9.7(b), the surface of In2 O3 NW was functionalized with 3-phosphonopropionic acid, which has a COOH group at one end that was used to immobilize PSA-AB by forming amide bonds. The SWNT surface was first functionalized with 1-purenbutanoic acid succinimidyl ester and then treated with the PSA-AB solution (Fig. 9.7c). To investigate the chemical gating effect of PSA on the

9.3 Carbon Nanotubes

(a) Schematic of a nanosensor. (b) Reaction scheme for the modification of In2 O3 NW. (c) Reaction scheme for the modification of SWNT. (From Ref. [49].) Fig. 9.7.

devices, they incubated devices consisting of both individual NWs and individual semiconducting SWNTs in a PBS-buffered solution containing PSA for 15 h. The electrical properties of the devices, including both current–voltage (I-Vds ) and current–gate voltage (I-Vg ) characteristics, were then measured in air before and after the PSA incubation. In this experiment, the authors consistently observed enhanced conductance for NW devices and reduced conductance for SWNT devices after PSA incubation. This complementary response in conductance arises because In2 O3 NWs are n-type and SWNTs are p-type semiconductors. As a result, a chemical gating effect of PSA introduces carriers into In2 O3 NWs, while the PSA binding decreases the carrier concentration in nanotubes, thus reducing conductance. Furthermore, the authors performed real-time PSA detection and succeeded in detecting PSA with a sensitivity of 5 ng mL1 , a level used for the clinical diagnosis of prostate cancer [50]. 9.3.3

Carbon Nanotubes for Targeted Delivery

As new vectors for the delivery of therapeutic molecules, carbon nanotubes (CNTs) have attracted increasing attention due to the ease of translocation across cell membranes and lower toxicity [51, 52]. The use of functionalized carbon nanotubes (f-CNTs) for drug delivery of small molecules and an alternative strategy for the introduction of two different and orthogonal functionalizations to CNTs have been investigated by the group of Alberto Bianco [53]. The orthogonal methodol-

313

314

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Scheme for two different orthogonal CNT modifications. (a) Neat (COCl)2 ; Pht-N(CH2 CH2 O)2 CH2 CH2 NH2 , dry THF, reflux; (b) BocNH(CH2 CH2 O)2 CH2 CH2 NHCH2 COOH/

Fig. 9.8.

(CH2 O)n , DMF, 1258C; (c) hydrated NH2 NH2 , EtOH, reflux; (d) FTIC, DMF; (e) HCl 4 m in dioxane; (f ) Fmoc-AmB, HOBt/EDCHHCl/ DIPEA, DMF; 25% piperidine in DMF. (From Ref. [53].)

ogy allows the selection and control of the attachment of active molecules to sidewalls and tips of the CNTs. Oxidized MWNTs were activated as the acid chlorides and treated with diaminotriethylene glycol (mono-protected as phthalimide) (Fig. 9.8). The Boc-protected amine group was then introduced to the sidewalls of the MWNTs through 1,3-dipolar cycloaddition. The phthalimide group is particularly useful because it is stable to harsh acidic conditions and orthogonal to the Boc group. Thus FITC and amphotericin B (AmB) can be attached to the tips and side-

9.4 Nanotubes and Nanowires Composed of Artificial Peptides

walls, respectively, of the CNTs. Fluorescein and AmB were chosen for tracking the uptake of material and as an antibiotic moiety, the active molecule, respectively. AmB is the most effective antibiotic, but is highly toxic to mammalian cells, originating from the formation of aggregates as a result of its lower solubility in water [54]. From the Human Jurkat lymphoma T cell viability test with either FITC-AmBmodified MWNTs (FITC-AmB-MWNTs) or AmB as control, the authors observed the conjugation of AmB to CNTs remarkably reduced the toxic effects. In addition, maximum fluorescence was observed after only 1 h of incubation, indicating rapid cell uptake of FITC-AmB-MWNTs. Most conjugates were found in cytoplasm and around the nuclear membrane. They excluded endocytosis for the mechanism of cell membrane penetration because NaN3 did not show remarkable influence on the penetration capacity of MWNT; instead, they proposed a spontaneous mechanism: MWNTs behave like nanoneedles and pass through the cell membrane without causing cell death [55]. They supposed the ability of CNTs to internalize AmB rapidly into the cytoplasm of Jurkat cells reduces the possibility of disruption of the cell membrane structure, thus reducing toxicity. They proved that covalent attachment of AmB to the nanotubes not only prevents the aggregation phenomena that the drug typically displays in solution but also that it has little effect on the antifungal activity.

9.4

Nanotubes and Nanowires Composed of Artificial Peptides 9.4.1

Peptide Nanotubes

In 2001, Ghadiri and coworkers reported peptide nanotubes [56–58] that have a new class of antibacterial activities on Gram-positive and/or Gram-negative bacteria [59]. This strategy is based on the self-assembly of ring-shaped cyclic peptide subunits that consist of six or eight alternating d- and l-amino acids. Hydrogen bonding between unit cyclic peptides allows well-defined tubular structures having a uniform internal diameter, which is adjustable by changing the number of amino acid residues in the ring; upon adsorption onto lipid membrane of bacteria, the cyclic peptides can stack to form hollow tubular structures that are open-ended, resulting in an increase in membrane permeability, collapse of transmembrane ion potential, and rapid cell death. Figure 9.9 shows the b-sheet-like, hydrogen-bonded tubular architecture of the self-assembled, eight-residue cyclic d,l-a-peptide, and the modes of membrane permeation accessible to peptide nanotubes. Self-assembly of an eight-residue cyclic d,l-a-peptide subunit is directed by intersubunit backbonebackbone hydrogen bonding to give b-sheet-like, tubular supramolecular structures that are open and hollow. Cyclic d,l-a-peptide nanotubes can display sequencedependent modes of membrane permeation: intramolecular pore, barrel stave, and carpet-like (cyclic peptides are depicted as ring structures). Peptide nanotubes operating through the carpet-like mode of action could have a greater potential for

315

316

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Chemical structure of the cyclic d,l-a-peptides and schematic representation of the mode of interaction of the tube caps in forming heterometric transmembrane channels. (From Ref. [56].)

Fig. 9.9.

membrane discrimination because of their polyvalent display of surface-exposed hydrophilic side chains for potential interactions with various membrane constituents. For antibacterial activity test, a series of six- and eight-residue amphiphilic cyclic d,l-a-peptides was treated against Escherichia coli and methicillin-resistant S. aureus (MRSA), and each peptide was designed to bear at least one basic residue to enhance its target specificity towards negatively charged bacterial membranes. In vitro activity tests revealed that single amino-acid substitution changed membrane selectivity and activity. In addition, the high efficacy observed against lethal MRSA infections in mice indicates that this new class of abiotic structure, the cyclic peptide, and its quick bactericidal action may contribute to limit temporal acquirement of drug-resistant bacteria. 9.4.2

Peptide-Amphiphile Nanofibers

Peptide-amphiphile (PA) nanofibers have been prepared by Stupp’s group using self-assembly [60]. A PA is composed of several peptide residues, which can be customized through the peptide sequence for specific purposes, and an alkyl tail with 16 carbon atoms that pack in the center of the micelle, leaving the peptide segments exposed to the aqueous environment. Figure 9.10(A) shows the chemical structure of one example of PAs used in experiments. Here, the PA was consists of five key structural features. Region 1 is a long alkyl tail that conveys hydrophobic character to the molecule and, when combined with the peptide region, makes the molecule amphiphilic. Region 2 is composed of four consecutive cysteine residues that when oxidized may form disulfide bonds to polymerize the self-assembled structure. Region 3 is a flexible linker region of three glycine residues to provide the hydrophilic head group flexibility from the more rigid crosslinked region. Re-

9.4 Nanotubes and Nanowires Composed of Artificial Peptides

Fig. 9.10. (A) Chemical structure of the peptide amphiphiles (PA). (B) Molecular model of PA. (C) Schematic showing the self-assembly of PA. (From Ref. [60].)

317

318

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

gions 4 and 5 are variable regions and can be modified for specific functionalities. These PAs self-assemble at acidic pH into cylindrical fibers @6–8 nm in diameter and interior cysteine groups are crosslinked through disulfide bonds to yield pHstable nanofibers. Stupp and colleagues have shown many useful applications for these PA nanofibers, with various functional molecules coupled to the peptide residues of PA for specific applications. For example, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA) was conjugated to PA to yield PA nanofibers for the magnetic resonance imaging (MRI) [61]. When the Gd(iii) ion is chelated to the DOTA, Gd(iii) is detoxified and the complex acts as a strong MR agent. A peptide nucleic acid/PA conjugate (PNA-PA) has been prepared to develop isolation tools for the poly A-containing mRNAs and biotin has been incorporated into PA for the high densities of binding sites on their surface [62, 63]. Carbon nanotubes were successfully encapsulated into PA nanofiber without degradation of their structural, electronic, and optical properties [64]. The most interesting application is as a cell differentiator. The authors showed that PA nanofiber composed of the pentapeptide epitope isoleucine-lysine-valine-alanine-valine (IKVAV), which was known to promote neurite sprouting and direct neurite growth, can be used to direct the differentiation of neural progenitor cells largely into neurons while suppressing astrocyte differentiation [65]. Once neural progenitor cells are encapsulated within a three-dimensional network of PA nanofiber it is possible to present to cells the neurite-promoting laminin epitope, which is the peptide element of the PA, at nearly van der Waals density. As a result, the nanofibers can achieve amplification of bioactive epitope presentation to cells.

9.5

Template-synthesized Nanotubes and Nanorods

Carbon nanotubes, self-assembled lipid tubes, and peptide nanotubes have been successfully used in some biomedical applications; however, it is difficult to control their tube diameter or length and some nanotubes such as the lipid tubes must be coated with ceramic or metal to make them rugged enough for biomedical use [66]. Since Martin and coworkers have pioneered a technology, called template synthesis, for preparing monodisperse nanotubes of nearly any size and composed of nearly any material, tubular structures have become highly attractive for multifunctional nanoparticles [67]. The template method is a general approach for preparing nanomaterials that involves the synthesis or deposition of the desired material within the cylindrical and monodisperse pores of a nanopore membrane or other solid surface [68]. Fig. 9.11 shows an alumina template that has a cylindrical nanopore with monodisperse diameters and lengths. Porous alumina template can be obtained by well-established electrochemical anodization on aluminum plate. These pore dimensions can be typically controlled, ranging from 5 to a few hundred nanometers in diameter and from tens of nanometers to hundreds of micrometers in length. Depending on the membrane and synthetic method used, such as chemical or electrochemical deposition of desired material, solid

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.11. Field emission scanning electron micrograph (FESEM) of a home-made alumina template (60-nm in diameter). Top (left-hand side) and cross sectional (right-hand side) images. (Unpublished data prepared by the authors.)

nanowires or hollow nanotubes can be produced. This method has been used to prepare nanowires and nanotubes composed of many types of material, including metals, polymers, semiconductors and carbons. In addition, the template method can be used to prepare composite nanostructures, including concentric tubular composites. Several unique properties make these nanotubes potential candidate for biotechnological applications. First, their inner voids can be filled with large amounts of interesting materials, such as fluorophores for high optical signal, drug molecules to deliver, magnetic materials for in vivo imaging. Second, the distinct outer surfaces can be easily modified with biomolecules. Finally, these nanotubes can be made out of nearly any material. This technology offers tremendous flexibility in making nanotubes with a desired biological property such as biocompatibility or biodegradability [69]. 9.5.1

Differential Functionalization of Nanotube

As another beautiful example of template synthesis, Martin’s group recently reported a very important technology, so-called differential functionalization between the inner and outer surfaces of nanotubes based on a silica nanotube [13]. They used silica nanotubes for proof-of-concept experiments because they are easy to make, readily suspend in aqueous solution, and because silica surfaces can be easily modified using simple silane chemistry with commercially available reagents. The silica walls of nanotubes are completely transparent for UV/Vis light in the range 200–800 nm, allowing it to pass through to the inner materials. Silica nanotubes were synthesized within the pores of nanopore alumina template membranes using a sol–gel method [70]. The authors have developed the following simple procedure for applying different functional groups to the inner versus outer surfaces of these nanotubes (Fig. 9.12). Before being liberated from the template membrane, the inner surface of a nanotube is modified with the first silane. The

319

320

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Schematic of the differential modification procedure. * Membrane surfaces also. (From Ref. [13].)

Fig. 9.12.

outer surfaces of nanotubes are not modified because they are in contact with the membrane template. The template is then dissolved to liberate the nanotubes, affording free outer surfaces that are then modified with a second silane. To demonstrate this differential functionalization, two sets of nanotubes were prepared: (a) with the green fluorescent silane N-(triethoxysilylpropyl)dansylamide attached to inner surfaces and the hydrophobic octadecylsilane (C18) to the outer surfaces and (b) with the blue fluorescent silane triethoxysilylpropylquinineurethane on the inner surfaces and the bare native (hydrophilic) outer surface. Each of these nanotubes were added to a vial containing water and the immiscible organic solvent cyclohexane. The solvents were then mixed and allowed to separate. The green nanotubes with hydrophobic outer surfaces partition into the (upper) cyclohexane phase (Fig. 9.13A), while blue fluorescence is seen only from the aqueous phase with the hydrophilic nanotubes (Fig. 9.11B). When both sets of nanotubes are added to the solvent mixture in the same vial, tubes with the C18 outer surface chemistry partition into cyclohexane and those with the silica outer surface chemistry partition into water (Fig. 9.11C). 9.5.2

Selective Extraction of Drug Molecules

Antibody-functionalized nanotubes can be used to extract selectivity one enantiomer from a racemic mixture. Fab fragments of antibody produced against the drug 4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]-triazol-1-yl-propyl]benzonitrile (FTB,

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.13. Vials containing cyclohexane (upper) and water (lower) under UV light excitation after addition of 10 mg of nanotubes with (A) dansylamide on inner and C18 on outer surfaces and (B) quinineurethane on

inner and no silane on outer surfaces; (C) 10 mg of both A and B nanotubes; 200-nm diameter nanotubes were used. (From Ref. [13].)

Fig. 9.14) [71] were immobilized on both inner and outer surfaces of silica nanotubes by attaching an aldehyde silane to both surfaces of free-standing silica nanotubes liberated from membrane template; the aldehyde group of this silane reacts with free amino groups on the Fab fragment of antibody via Schiff base chemistry. After racemic mixtures of the SR and RS enantiomers of FTB were incubated with Fab-functionalized nanotubes, the tubes were then collected by filtration, and the amounts of two enantiomers in the filtrate solution were measured by a chiral HPLC method. Figure 9.14 shows chromatograms both before (chromatogram I)

Fig. 9.14. Chiral HPLC chromatograms for racemic mixtures of FTB before (I) and after (II, III) extraction with 18 mg mL1 of 200-nm Fab-containing nanotubes. (From Ref. [13].)

321

322

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

and after (chromatogram II) exposure of a solution that contained 20 mm of both enantiomers to the Fab-functionalized nanotubes. They found that 75% of the RS enantiomer and none of the SR enantiomer were removed by the nanotubes and that all of the RS enantiomer was removed (chromatogram III) when the concentration of the racemic mixture was dropped to 10 mm. A control experiment with bare silica nanotubes containing no Fab fragment showed negligible extraction of both enantiomers from a 20 mm solution. Silica nanotubes containing Fab on only the inner surface were prepared by differential functionalization. While nanotubes are still embedded within the pores of template membrane, aminopropyltrimethoxysilane was used to functionalize the inside of the nanotubes with an amine group. After dissolution of the template, the resultant nanotubes were treated with glutaraldehyde, a well-known Schiff base crosslinker, and Fab fragments were immobilized inside the nanotubes through another imine bond between the primary amine of the Fab antibody and the remaining aldehyde group of glutaraldehyde. When a racemic mixture of the drug was incubated with these interior-only Fab-modified nanotubes, 80% of the RS (and none of the SR) enantiomer was extracted, corresponding to almost half the amount extracted by nanotubes with Fab on both their inner and outer surfaces. 9.5.3

Silica Nanotube Carrier for DNA Transfection

The use of silica nanotubes as a biomolecule carrier, such as a DNA transporter, has been explored by Wu and coworkers [72]. They used the template synthesis method to fabricate silica nanotubes (SNT) that can transport DNA molecules to cells. First, silica nanotubes were prepared inside the pores of 200-nm diameter commercial alumina membrane using a sol–gel process involving tetraethyl orthosilicate. The inner silica layer was then modified with 3-aminopropyltriethoxysilane (APTS) to generate the polycationic surface required to hold CdSe/ZnS core–shell quantum dots (QDs) or DNAs through the electrostatic forces. Figure 9.15 shows, briefly, the preparation of fluorescent silica nanotubes and their use for

Schematic illustration of a fluorescent silica nanotube and its application in gene delivery. (From Ref. [72].)

Fig. 9.15.

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.16. Fluorescence microscopy image of COS-7 cells incubated with green QD-silica nanotube after nucleus staining (red) with propidium iodide. (From Ref. [72].)

gene delivery. Before the gene delivery experiment, cultured mammalican cells such as monkey kidney COS-7 cells were treated with green fluorescent silica nanotubes (gfSNTs) to test the cell membrane permeability of silica nanotubes. They found, by confocal microscopy, that the gfSNT entered about 60–70% of the cells by endocytosis and were mostly localized in the cytoplasm (Fig. 9.16). Cytotox-

Fig. 9.17. (A) Bright-field image of COS-7 cells incubated with DNA/silica nanotube complex. Fluorescence image of the same cells (B) showing GFP expression and (C) incubated with GFPDAN and red-fluorescent silica nanotube. (From Ref. [72].)

323

324

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

icity tests on nanotubes in the cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide (MTT) assay, revealed that approximately 80% of the cells were still viable after treatment with gfSNT, indicating that silica nanotubes are not especially toxic under the experimental conditions. For a gene delivery experiment, plasmid DNA was inserted into the nanotube to form a DNA/SNT complex, and the complex was added to COS-7 cells. Cells treated with the DNA/SNT complex showed cytoplasmic GFP expression (Figure 9.17), whereas cells treated with free DNA or red fSNT as a control experiment did not. Although the efficiency of SNT-mediated DNA transfection (ca. 10–20%) is less than that of conventional calcium phosphate (ca. 60–70%), the advantage of this strategy is that SNTs can carry other biomolecules such as RNA or proteins. 9.5.4

DNA Nanotubes

Unlike the previous DNA-functionalized nanotubes [73, 74], Martin’s group have reported DNA nanotubes composed entirely, or predominately, of DNA itself, based on the template synthesis method and DNA hybridization [75]. First, a template membrane was treated with Mallouk’s alternating a,o-diorganophosphonate (a,o-DOP) Zr(iv) to form the outer skin of a DNA nanotube, which guides the growth of a DNA layer [76]. As seen in Fig. 9.18, DNA layers were integrated subsequently into the nanotube from the (a,o-DOP) Zr(iv) skin with the alternating hybridization of the two 15-base segments up to five layers. The nanotubes were then liberated by dissolution of the template in a solution of diluted phosphoric acid and 2 m NaCl at 0  C and collected by centrifugation. A TEM image shows liberated five-layer DNA nanotubes and shorter nanotubes than the alumina template, which is 36 mm thick, indicating that the tubes were broken during membrane dissolution and centrifugation. Because these DNA nanotubes are made of

Scheme of DNA hybridization and TEM image of five tubes with (A) one-layer and (B) three-layer a,o-DOP/Zr(iv) skins. (C) Same as for (A) but after heating to 85  C. (From Ref. [75].) Fig. 9.18.

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.19. Thermal decomposition curves for (A) nanotubes composed of the DNAs in Fig. 9.18. Number of DNA layers: (i) 2, (ii) 3, (iii) 5. Nanotubes were embedded within the pores of

the template. Inset: five-layer tubes liberated from the template. (B) Five-layer nanotubes composed of hybridized 8 (i), 12 (ii) and 15 (iii) base duplexes. (From Ref. [75].)

double-stranded DNA (dsDNA), they can be heated above the melting point at which dsDNA releases the dehybridized ssDNA chains from the tube, which means this strategy can be used to transport DNA. Consequently, DNA release from the DNA-nanotube-containing alumina membranes into buffer solution was investigated, by UV absorbance (260 nm), upon heating from 23 to 85  C. Figure 9.19 shows that there was, initially, no DNA in solution because the nanotubes were sequestered within the pores of template; however, above the melting temperature, the release of ssDNA was detected. The same DNA release experiment carried out for nanotubes liberated from the template membrane gave analogous data. This result shows that a new family of layer-by-layer template-synthesized DNA nanotubes can be used for DNA transport and that their release can be controlled by melting the DNA duplexes. 9.5.5

Nanobarcodes for Multiplexing Diagnosis

In 2001, Keating and Natan’s group exploited metallic nanobarcodes that have alternating metal stripes in a nanorod shape for bioanalysis [16]. Multiplexing and miniaturization is big issues in cancer diagnosis, and in any other bioanalysis. To develop innovative devices that can measure ever-increasing numbers of species from smaller and smaller samples volumes, microarray [77] and encoded microbeads [78], or nanoparticles with distinctive fluorescent signatures [79] have been investigated. Microarrays can encode large numbers of analysis based on position, but slow diffusion of analytes to the surface is an intrinsic problem of planar microarray and the range of analyte concentrations that may be detected at the same time is limited. Although an array of beads in solution, such as microbeads or

325

326

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Fig. 9.20.

Schematic of the synthesis of nanobarcodes. (From Ref. [16].)

nanoparticles, may circumvent the shortcomings of planar microarrays for assay, the number of previously reported bead-based assays that can be performed simultaneously is limited by the number of spectrally distinguishable fluorophores. However, metallic barcodes enable a wide variety of bioanalytical measurements because many thousands of uniquely identifiable particles can be prepared and then used in fluorescence- and mass spectrometry-based assays. Such nanobarcodes have been manufactured electrochemically in the pores of alumina or polycarbonate using the template synthesis method (Fig. 9.20). Striped, cylindrical metal nanoparticles were optically characterized by the pattern of differential reflectivity of adjacent stripes. For example, a metallic nanobarcode composed of Au, Ag, Ni, Pd, and Pt shows different patterns of optical reflectivity, corresponding to their own identity, with respect to the wavelength of illumination light (Fig. 9.21). Figure 9.22 shows that barcoded rods can be used as supports for bioassays, and the selective detection of DNA hybridization on single optically encoded particles was successfully demonstrated for an important first step towards multiplexed hybridization assays. The authors adapted DNA hybridization and immunoassays for use on barcodes. A 24-mer analyte DNA binds to a 12-mer capture DNA immobilized on the surface of a nanobarcode and is later detected by addition of a 12-mer probe DNA that is complementary to the remaining 12 nucleotides and carries a fluores-

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.21. Reflectance optical microscopy images and line profiles for a particle of composition Au-Ni-Pd-Pt with illumination at 430, 520 and 600 nm. (From Ref. [16].)

Fig. 9.22. Sandwich DNA hybridization assay. (i) Fluorescence readout; (ii) shows the rod ID. The analyte (b) was omitted in the controls (iii) and (iv). (From Ref. [16].)

327

328

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

cent tag. Each particle can be seen in both the fluorescence (i) and reflectivity (ii) images. In a control experiment, in which the analyte is omitted, a very low fluorescence background is observed (iii) and particles are visible only in the reflectivity image (iv). 9.5.6

Magnetic Nanotubes

Magnetic nanoparticles have been intensively studied for various biomedical applications, such as early detection of cancer and targeted drug delivery, due to their unique magnetic field-assisted properties like magnetic resonance imaging (MRI) and controlled movement in a magnetic field. Therefore, by combining the attractive magnetic properties with a tubular structure, magnetic nanotubes (MNTs) can be an ideal candidate for multifunctional nanomaterials in biomedical applications, such as targeted drug delivery and MRI. In 2005 Lee’s group reported another interesting nanotube based on the template synthesis method [12]. They showed that MNTs can provide another type of useful tool for biomedical and biotechnological applications, including magnetic-field-assisted bioseparation, biointeraction, drug delivery and MRI. Inner voids of MNTs can be used for capturing, concentrating, and releasing species ranging in size from large proteins to small molecules because the tube dimensions can be easily controlled by template synthesis. Distinctive outer surfaces can be differentially functionalized with environmentally friendly and/or probe molecules for a specific target. The group described the synthesis of MNTs and their uses for magnetic-field-assisted chemical and biochemical separations, immunobinding, and drug delivery. Synthesis and Characterization of Magnetic Nanotubes Silica nanotubes have been synthesized with a layer of magnetite (Fe3 O4 ) nanoparticles on the inner surface of the nanotube using porous alumina film as template (60 and 200 nm pore diameter). To form the layer of magnetite nanoparticles, the silica-nanotube alumina film was dip-coated with a 4:1 mixture solution of FeCl3 (1 m) and FeCl2 (2 m), dried in an Ar stream, and immersed in NH4 OH [80]. For the differential functionalization of MNTs, the inner nanotube surface was treated with octadecyltriethoxysilane (C18) while MNTs were still embedded in the pores of alumina template. Free-standing MNTs were obtained after polishing both sides of the template film mechanically and dissolving the alumina template in a 0.1 m NaOH solution. After the template was dissolved completely, MNTs were collected by filtration. Transmission electron microscopy (TEM) images of MNTs (Fig. 9.23) show clearly the magnetite layers on the inner surface of the nanotubes; bare silica nanotubes have smooth tubular wall surfaces. Room-temperature magnetization curves of MNTs obtained by superconducting quantum interference device (SQUID) showed that both 60- and 200-nm diameter MNTs exhibit superparamagnetic characteristics and their saturation magnetizations are 2.7 and 2.9 emu g1 , respectively. Energy dispersive X-ray (EDX) analysis revealed that these magnetic nanotubes consist mainly of Fe and Si. 9.5.6.1

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.23. Transmission electron micrographs (TEM) of (A, B) 200 and (C, D) 60-nm diameter silica nanotubes without magnetite (A, C) and magnetic nanotubes (MNTs) with magnetite (B, D). (E) Room temperature magnetization curves of MNTs. (From Ref. [12].)

Magnetic Field-assisted Chemical Separation and Biointeraction In an exemplary chemical extraction and separation experiment, MNTs (@10 9 ) functionalized inside with C18 were added to a solution of 1.1 0 -dioctadecyl3,3,3 0 ,3 0 -tetramethylindocarbo-cyanine perchlorate (DiIC18 , 38 mm) in water/ methanol (9:1 v/v, 1 mL). The mixture was then shaken and allowed to stand for 10 min so that the dye molecules could enter the MNTs by the strong hydrophobic interaction. These nanotubes are completely suspendible in aqueous phases (even in pure water) due to their outer hydrophilic silica surfaces. A strong magnet was then used to separate the nanotubes, and thus the DiIC18 molecules, from the solution. Figure 9.24 show that magnetic separation turns an initially red solution, containing the red dye (DiIC18 ), almost transparent and clear. UV/Vis spectroscopy and fluorescence microscopy studies were further performed to show that more than 95% of the DiIC18 was removed from solution and the hydrophobic dye was extracted inside the MNTs. As a control experiment, when MNTs without an inner C18 layer were incubated with DiIC18 , no detectable change was observed. The above results tell us that these MNTs can be used for the extraction, separation, release, and analysis of trace amounts of extremely hydrophobic toxic chemicals, such as polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), in water [81]. MNTs with human IgG inside were prepared and added to a mixed solution (2 mL, pink color) of fluorescein-labeled anti-bovine IgG [green, 0.71 m in 0.1 m phosphate-buffered saline (PBS), pH 7.4] and Cy3-labeled anti-human IgG (red, 0.67 m) to show a magnetic bioseparation using antigen–antibody interaction. BSA-derivatized MNTs (BSA-MNTs) were prepared as well to show a nonspecific 9.5.6.2

329

330

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

A vial containing C-18 modified MNTs before (A) and after (B) magnetic separation for 2 min. (C) A solution containing green FITC-labeled anti-bovine IgG and red Cy3-labeled anti-human IgG after magnetic

Fig. 9.24.

separation with BSA-MNTs (left) and human IgG-MNTs (right). (D) Fluorescence spectra for measuring the amount of remaining red Cy3labeled anti-human IgG in solution. (From Ref. [12].)

biointeraction. The outer surfaces of all the MNTs were functionalized with poly(ethylene glycol) (PEG) silane to prevent nonspecific adsorptions on silica surfaces. After magnetic separation, the solution changes from the original pink to greenish blue only when MNTs with human IgG inside are added, while the solution with BSA-MNTs remains pink. This means that red Cy3-labeled anti-human IgG was separated specifically from the solution by human IgG-MNTs. Fluorescence spectra show that 84% of the Cy3-labeled anti-human IgG was separated by human IgG-MNTs but only 9% by BSA-MNTs. Very interestingly, the magnetic properties of MNTs can be used to facilitate and enhance biointeractions between the outer surfaces of MNTs and a specific target surface. As a potential application, the drug delivery efficiency might be greatly improved by magnetic field-assisted biointeraction when the MNTs carry the drug inside and have probe molecules, such as antibody, on the outer surfaces. In a proofof-concept experiment, onto a anti-rabbit IgG-modified glass slide, 60-nm diameter MNTs with FITC-modified inner surfaces and rabbit IgG-modified outer surfaces were added and incubated for 10 min both with and without a magnetic field applied to the bottom of the glass slide. After washing the unbound nanotubes, the number of bound nanotubes were counted at five different regions of fluorescence microscopy images (Fig. 9.25) and then averaged. A ca. 4.2-fold binding enhancement was observed for the antigen–antibody interactions in the presence of a magnetic field, showing that the efficiency of antigen–antibody interactions can be controlled spatially by means of an external magnetic field. Drug Uptake and Controlled Release MNTs functionalized on their inner surface with an aminosilane (aminopropyltriethoxysilane, APTS) have been prepared for drug delivery experiments. The MNTs were treated with ATPS while still embedded inside the template, which was then dissolved, and the nanotubes were collected by filtration. 5-Fluorouracil (5-FU), 4-nitrophenol, and ibuprofen were then loaded in an organic solvent, such as etha9.5.6.3

9.5 Template-synthesized Nanotubes and Nanorods

Fig. 9.25. Fluorescence microscope images of bound FITCMNTs-Rabbit IgG (60 nm in diameter, 3 mm) following incubation with anti-rabbit IgG-modified glass with and without the application of a magnetic field under the glass substrate. (From Ref. [12].)

nol or hexane, as model drug molecules into the pores of MNTs functionalized with APTS to study the effect of charged hydrogen bonding interactions between the drug and the inner pore surfaces on loading and release. To remove the drug molecules bound onto the outer surface of MNTs, and weakly bound drugs, drugloaded MNTs were washed with PBS (1 mL) and filtered quickly twice, and redispersed in pH 7.4 PBS and incubated at room temperature on a rocking platform. After separation of MNTs from solution using either a strong NdFeB magnet or centrifugation, the amount of released drug was determined by UV/Vis spectroscopy by monitoring changes in absorbance at 264 nm (ibuprofen), 400 nm (4nitrophenol), and 266 nm (5-FU). The percentage release of loaded drug was estimated by measuring the absorbance of drug-released solution after 5 days. UV/Vis spectrophotometry showed that the number of molecules loaded per nanotube was @10 7 for ibuprofen, @10 6 for 4-nitrophenol, and @10 7 for 5-FU. The value for ibuprofen is about twice the monolayer coverage of the MNT inner surface area. Figure 9.26 shows the drug release rate: <10% of ibuprofen was released in 1 h and 80% was released after 24 h. For 5-FU and 4-nitrophenol, however, more than 90% was released in 1 h. Although several factors have to be considered, such as hydrogen bonding, hydrophobic interaction, and solubility, the ionic interaction between

331

332

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

Fig. 9.26. In vitro release of ibuprofen, 4-nitrophenol (4-NO2 Ph), and 5-fluorouracil (5-FU) from MNTs (60 nm diameter, 250 nm long), and the pK a of each drug. (From Ref. [12].)

the inner surface of MNTs and drug molecule is a plausible explanation for this tendency. At pH 7.4, ibuprofen (pK a 4.8) is fully ionized so that Coulomb interaction between negatively-charged ibuprofen and protonated aminopropyl groups on the interior of MNTs contributes considerably to retardation of drug release. With 4-nitrophenol (pK a 7.2) and 5-FU (pK a 8.1), however, the contribution of Coulomb interactions is moderate (4-nitrophenol) or almost negligible (5-FU) because the degree of ionization is less than that of ibuprofen. These results show that the hollow tubular structure of magnetic particles is one of the most promising candidates for various applications, including chemical and biochemical separations and drug delivery, and MNTs with drug-friendly interiors and target-specific exteriors may open up a new field of materials for multifunctional targeted drug delivery.

9.6

Conclusion

We believe that nanotubes or nanowires – with inner chemistry that can be utilized – can be attractive alternatives to spherical nanoparticles – which utilize a single surface – for biomedical and biotechnological applications. Tubular nanoparticles provide more options than spherical ones, especially when multifunctionality is needed because nanotubes have distinctive inner and outer surfaces that can be modified and utilized differentially, enabling them to be equipped with the right function at the right position. The fabrication of nanotubes has been greatly improved by the template synthesis, which affords monodisperse nanotubes with controllable dimensional parameters, such as diameter, length, and wall thickness, together with the differential functionalization technique. However, since the field of

References

nanotubes and nanowires is still in the early stages of biotechnological applications, much fundamental research is needed to solve challenging problems before commercialization of the technology. For example, although nanotubes can accommodate drugs or biomolecules in their inner space, only their inner surface, and not the whole inner volume, is used in chemical bonds or interactions with chemicals. As Martin mentioned in his review [68], capped nanotubes for which the cap can block the nanotube pore and fall off reversibly according to a chemical or biochemical signal might be one way to overcome such biotechnological problems. As shown in magnetic particles used for early-stage cancer diagnosis [26] and magnetism-based interaction capture (MAGIC) [27], or peptide nanotubes used for a new class of antibacterial agent [59], combining the properties of a nanomaterial with an existing technology, such as magnetic resonance (MR) imaging, optical or electronic methods, enables us to exploit a novel concept that can not be attained with the conventional technology. Therefore, by combining properties originating from a tubular structure with existing well-understood technology, nanotubes can open up new opportunities to develop a new class of therapeutic and diagnostic tools and to resolve existing biotechnological problems.

References 1 Perez, J. M., Josephson, L.,

2

3

4

5

Weissleder, R., Viral-induced selfassembly of magnetic nanoparticles allows the detection of viral particles in biological media., J. Am. Chem. Soc., 2003, 125, 10 192–10 193. Radler, J., Koltover, I., Salditt, T., Safinya, C. R., Structure of DNA– cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes., Science, 1997, 275, 810–814. Park, S. J., Taton, T. A., Mirkin, C. A., Array-based electrical detection with nanoparticles probes., Science, 2002, 295, 1503–1506. Murthy, N., Thng, Y. X., Schuck, S., Xu, M. C., Frechet, J. M., A novel strategy for encapsulation and release of proteins: Hydrogels and microgels with acid-labile acetal cross-linkers., J. Am. Chem. Soc., 2002, 124, 12 398–12 399. Li, K. C. P., Pandit, S. D., Guccione, S., Bednarski, M. D., Molecular imaging applications in

6

7

8

9

10

11

nanomedicine., Biomed. Microdevices, 2005, 6, 113–116. Park, J. W., Liposome-based drug delivery in breast cancer treatment., Breast Cancer Res., 2002, 4, 95–99. Cao, Y. W. C., Jin, R. C., Mirkin, C. A., Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection., Science, 2002, 297, 1536–1540. Hirsch, L. R., Halas, N. J., West, J. L., A whole blood immunoassay using gold nanoshells., Anal. Chem., 2003, 75, 2377–2381. Voura, E. B., Jaiswal J. K., Mattoussi, H., Simon, S., Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat. Med. 2004, 10, 993–998. Ferrari, M., Cancer nanotechnology: Opportunities and challenges, Nat. Rev. Cancer, 2005, 5, 161–171. Yinghuai, Z., Peng, A. T., Carpenter, K., Maguire, J. A., Hosmane, N. S., Takagaki, M.,

333

334

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy

12

13

14

15

16

17

18

19

Substituted carborane-appended watersoluble single-wall carbon nanotubes: New approach to boron neutron capture therapy drug delivery., J. Am. Chem. Soc., 2005, 127, 9875–9880. Son, S. J., Reichel, J., He, B., Schuchman, M., Lee, S. B., Magnetic nanotubes for magnetic-field-assisted bioseparation, biointeraction, and drug delivery., J. Am. Chem. Soc., 2005, 127, 7316–7317. Mitchell, D. T., Lee, S. B., Trofin, L., Li, N., Nevanen, T. K., Soderlund, H., Martin, C. R., Smart nanotubes for bioseparations and biocatalysis., J. Am. Chem. Soc. 2002, 124, 11864. Bucak, S., Jones, D. A., Laibinis, P. E., Hatton, T. A., Protein separations using colloidal magnetic nanoparticles., Biotechnol. Prog., 2003, 19, 477. Okamoto, K., Shook, C. J., Bivona, L., Lee, S. B., English, D. S., Direct observation of wetting and diffusion in the hydrophobic interior of silica nanotubes., Nano Lett., 2004, 4, 233–239. Nicewarner-Pena, S. R., Freeman, R. G., Reiss, B. D., He, L., Pena, D. J., Walton, I. D., Cromer, R., Keating, C. D., Natan, M. J., Submicrometer metallic barcodes, Science, 2001, 294, 137–141. Hartgerink, J. D., Beniash, E., Stupp, S. I., Self-assembly and mineralization of peptide-amphiphile nanofibers, Science, 2001, 294, 1684–1687. Kircher, M. F., Mahmood, U., King, R. S., Weissleder, R., Josephson, L., A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation., Cancer Res. 2003, 63, 8122–8125. Kobayashi, H., Choyke, P. L., Brechbiel, M. W., Waldmann, T. A., Lymphatic drainage imaging of breast cancer in mice by micro-magnetic resonance lymphangiography using a nano-size paramagnetic contrast agent., J. Natl. Cancer Inst. 2004, 96, 703–708.

20 Neuwalt, E. A., Varallyay, P., Bago,

21

22

23

24

25

26

27

A. G., Muldoon, L. L., Nesbit, G., Nixon, R., Imaging of iron oxide nanoparticles with MR and light microscopy in patients with malignant brain tumors., Neuropathol. Appl. Neurobiol., 2004, 5, 456–471. Lewin, M., Carlesso, N., Tung, C. H., Tang, X. W., Cory, D., Scadden, D. T., Weissleder, R., Tat peptidederivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 2000, 18, 410–414. Cheon, J., Kang, N.-J., Lee, S.-M., Lee, J.-H., Yoon, J.-H., Oh, S. J., Shape evolution of single-crystalline iron oxide nanocrystals., J. Am. Chem. Soc., 2004, 126, 1950–1951. Song, H.-T., Choi, J.-S., Huh, Y.-M., Kim, S., Jun, Y.-W., Suh, J.-S., Cheon, J., Surface modulation of magnetic nanocrystals in the development of highly efficient magnetic resonance probes for intracellular labeling., J. Am. Chem. Soc., 2005, 127, 9992–9993. Park, J., Lee, E., Hwang, N.-M., Kang, M., Kim, S. C., Hwang, Y., Park, J.-G., Noh, H.-J., Kim, J.-Y., Park, J.-H., Hyeon, T., Onenanometer-scale size-controlled synthesis of monodisperse magnetic iron oxide nanoparticles., Angew. Chem., Int. Ed., 2005, 44, 2872–2877. Huh, Y.-M., Jun, Y.-w., Song, H.-T., Kim, S., Choi, J.-s., Lee, J.-H., Yoon, S., Kim, K.-S., Shin, J.-S., Suh, J.-S., Cheon, J., In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals., J. Am. Chem. Soc. 2005, 127, 12 387–12 391. Jun, Y.-w., Huh, Y.-M., Choi, J.-s., Lee, J.-H., Song, H.-T., Kim, S.-j., Yoon, S., Kim, K.-S., Shin, J.-S., Suh, J.-S., Cheon, J., Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging., J. Am. Chem. Soc. 2005, 127, 5732–5733. Won, J., Kim, M., Yi, Y.-W., Kim, Y. H., Jung, N., Kim, T. K., A magnetic nanoprobe technology for

References

28

29

30

31

32

33

34

35

36

37

detecting molecular interactions in live cells., Science 2005, 309, 121–125. Dyal, A., Loos, K., Noto, M., Chang, S. W., Spagnoli, C., Shafi, K. V. P. M., Ulman, A., Cowman, M., Gross, R. A., Activity of Candida rugosa lipase immobilized on g-Fe2 O3 magnetic nanoparticles., J. Am. Chem. Soc. 2003, 125, 1684–1685. Gross, R. A., Kumar, A., Kalra, B., Polymer synthesis by in vitro enzyme catalysis., Chem. Rev., 2001, 101, 2097–2124. Gu, H., Ho, P.-L., Tsang, K. W. T., Wang, L., Xu, B., Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration., J. Am. Chem. Soc. 2003, 125, 15 702–15 703. Iijima, S., Helical microtubules of graphitic carbon. Nature 1991, 354, 56–58. Ajayan, P. M., Nanotubes from carbon., Chem. Rev., 1999, 99, 1787–1800. Dai, H., Kong, J., Zhou, C., Franklin, N., Tombler, T., Cassell, A., Fan, S., Chapline, M., Controlled chemical routes to nanotube architectures, physics, and devices., J. Phys. Chem. B, 1999, 103, 11 246–11 255. Zhou, C., Kong, J., Yenilmez, E., Dai, H., Modulated chemical doping of individual carbon nanotubes. Science, 2000, 290, 1552–1555. Fan, S., Chapline, M. G., Franklin, N. R., Tombler, T. W., Cassell, A. M., Dai, H., Self-oriented regular arrays of carbon nanotubes and their field emission properties., Science, 1999, 283, 512–514. Kong, Jing, Soh, Hyongsok T., Cassell, Alan M., Quate, Calvin F., Dai, Hongjie, Synthesis of individual single-walled carbon nanotubes on patterned silicon wafers., Nature, 1998, 395, 878–881. Duerkop, T., Kim, B. M., Fuhrer, M. S., Properties and applications of high-mobility semiconducting nanotubes., J. Phys.: Condensed Matter, 2004, 16, R553–R580.

38 Hines, D. R., Mezhenny, S., Breban,

39

40

41

42

43

44

45

M., Williams, E. D., Ballarotto, V. W., Esen, G., Southard, A., Fuhrer, M. S., Nanotransfer printing of organic and carbon nanotube thinfilm transistors on plastic substrates., Appl. Phys. Lett., 2005, 86, 163 101– 163 101. Chen, R. J., Zhang, Y., Wang, D., Dai, H., Noncovalent sidewall functionalization of single-walled carbon nanotubes for protein immobilization., J. Am. Chem. Soc., 2001, 123, 3838–3839. Chen, R. J., Hongjie, D., Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors., Proc. Natl Acad. Sci. U.S.A., 2003, 100, 4984– 4989. Azamian, B. R., Davis, J. J., Coleman, K. S., Bagshaw, C. B., Green, M. L. H., Biochemical single-walled carbon nanotubes., J. Am. Chem. Soc., 2002, 124, 12664–12665. Wong, S. S., Joselevich, E., Woolley, A. T., Cheung, C. L., Lieber, C. M., Covalently functionalized nanotubes as nanometer-size probes in chemistry and biology., Nature, 1998, 394, 52–55. Kam, N. W. S., O’Connell, M., Wisdom, J. A., Dai, H., Carbon nanotubes as multifunctional biological transporters and nearinfrared agents for selective cancer cell destruction., Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11 600–11 605. Nikolaev, P., Bronikowski, M. J., Bradley, R. K., Rohmund, F., Colbert, D. T., Smith, K. A., Smalley, R. E., Gas-phase catalytic growth of single-walled carbon nanotubes from carbon monoxide., Chem. Phys. Lett., 1999, 313, 91–97. O’Connell, M. J., Bachilo, S. M., Huffman, C. B., Moore, V. C., Strano, M. S., Haroz, E. H., Rialon, K. L., Boul, P. J., Noon, W. H., Kittrell, C., Ma, J., Hauge, R. H., Weisman, R. B., Smalley, R. E., Band gap fluorescence from individual single-walled carbon nanotubes., Science, 2002, 297, 593–596.

335

336

9 Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy 46 Koenig, K., Two-photon excited

47

48

49

50

51

52

53

54

lifetime imaging of autofluorescence in cells during UVA and NIR photostress., J. Microsc., 1996, 183, 197–204. Mukherjee, S., Ghosh, R. N., Maxfield, F. R., Endocytosis, Physiol. Rev., 1997, 77, 759–803. Hirsch, L. R., Stafford, R. J., Bankson, J. A., Sershen, S. R., Rivera, B., Price, R. E., Hazle, J. D., Halas, N. J., West, J. L., Nanoshellmediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 13 549– 13 554. Li, C., Curreli, M., Lin, H., Lei, B., Ishikawa, F. N., Datar, R., Cote, R. J., Thompson, M. E., Zhou, C., Complementary detection of prostatespecific antigen using In2 O3 nanowires and carbon nanotubes., J. Am. Chem. Soc., 2005, 127, 12 484–12 585. Li, X., Zhang, Y. P., Kim, H. S., Bae, K. H., Stantz, K. M., Lee, S. J., Jung, C., Jimenez, J. A., Gardner, T. A., Jeng, M. H., Kao, C., Gene Therapy for prostate cancer by controlling adenovirus E1a and E4 gene expression with pses enhancer., Cancer Res., 2005, 65, 1941–1951. Bianco, A., Prato, M., Can carbon nanotubes be considered useful tools for biological applications?, Adv. Mater. 2003, 15, 1765–1768. Bianco, A., Kostarelos, K., Partidos, C. D., Prato, M., Biomedical applications of functionalized carbon nanotubes., Chem. Commun. 2005, 571–577. Wu, W., Wieckowski, S., Pastorin, G., Benincasa, M., Klumpp, C., Briand, J.-P., Gennaro, R., Prato, M., Bianco, A., Targeted delivery of amphotericin B to cells by using functionalized carbon nanotubes., Angew. Chem., Int. Ed. 2005, 44, 6358–6362. Zotchev, S. B., Olyene macrolide antibiotics and their applications in human., Curr. Med. Chem., 2003, 10, 211–223.

55 Cai, D., Mataraza, J. M., Qin, Z.-H.,

56

57

58

59

60

61

62

63

64

Huang, Z., Huang, J., Chiles, T. C., Carnahan, D., Kempa, K., Ren, Z., Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing., Nat. Methods, 2005, 2, 449–454. Bong, D. T., Clark, T. D., Granja, J. R., Ghadiri, M. R., Self-assembling organic nanotubes., Angew. Chem., Int. Ed. 2001, 40, 988–1011. Kim, H. S., Hartgerink, J. D., Ghadiri, M. R., Oriented selfassembly of cyclic peptide nanotubes in lipid membranes., J. Am. Chem. Soc., 1998, 120, 4417–4424. Horne, W. S., Stout, C. D., Ghadiri, M. R., A heterocyclic peptide nanotube., J. Am. Chem. Soc., 2003, 125, 9372–9376. Fernandez-Lopez, S., Kim, H.-S., Choi, E. C., Delgado, M., Granja, J. R., Khasanov, A., Kraehenbuehl, K., Long, G., Weinberger, D. A., Wilcoxen, K. M., Ghadiri, M. R., Antibacterial agents based on the cyclic D,L-a-peptide architecture., Nature 2001, 412, 452–456. Hartgerink, J. D., Beniash, E., Stupp, S. I., Self-assembly and mineralization of peptide-amphiphile nanofibers., Science, 2001, 294, 1684–1688. Bull, S. R., Guler, M. O., Bras, R. E., Meade, T. J., Stupp, S. I., Selfassembled peptide amphiphile nanofibers conjugated to MRI contrast agents., Nano Lett., 2005, 5, 1–4. Guler, M. O., Pokorski, J. K., Appella, D. H., Stupp, S. I., Enhanced oligonucleotide binding to self-assembled nanofibers., Bioconjugate Chem., 2005, 16, 501–503. Guler, M. O., Soukasene, S., Hulvat, J. F., Stupp, S. I., Presentation and recognition of biotin on nanofibers formed by branched peptide amphiphiles., Nano Lett., 2005, 5, 249–252. Arnold, M. S., Guler, M. O., Hersam, M. C., Stupp, S. I., Encapsulation of carbon nanotubes by self-assembling peptide amphiphiles., Langmuir, 2005, 21, 4705–4709.

References 65 Silva, G. A., Czeisler, C., Niece,

66

67

68

69

70

71

72

73

K. L., Beniash, D., Harrington, D. A., Kessler, J. A., Stupp, S. I., Selective differentiation of neural progenitor cells by high–epitope density nanofibers., Science, 2004, 303, 1352–1355. Spector, M. S., Selinger, J. V., Singh, A., Rodriguez, J. M., Price, R. R., Schnur, J. M., Controlling the morphology of chiral lipid tubules., Langmuir, 1998, 14, 3493–3500. Martin, C. R., Nanomaterials: A membrane-based synthetic approach., Science 1994, 266, 1961–1966. Martin, C. R., Kohli, P., The emerging field of nanotube biotechnology., Nat. Rev. Drug Discov. 2003, 2, 29–37. Miller, S. A., Young, V. Y., Martin, C. R., Electroosmotic flow in templateprepared carbon nanotube membranes., J. Am. Chem. Soc. 2001, 123, 12 335–12 342. Steinle, E. D., Mitchell, D. T., Wirtz, M., Lee, S. B., Young, V. Y., Martin, C. R., Ion channel mimetic micropore and nanotube membrane sensors., Anal. Chem. 2002, 74, 2416–2422. Lee, S. B., Mitchell, D. T., Trofin, L., Nevanen, T. K., Soderlund, H., Martin, C. R., Antibody-based bio-nanotube membranes for enantiomeric drug separations., Science 2002, 296, 2198–2200. Chen, C.-C., Liu, Y.-C., Wu, C.-H., Yeh, C.-C., Su, M.-T., Wu, Y.-C., Preparation of fluorescent silica nanotubes and their application in gene delivery, Adv. Mater., 2005, 17, 404–407. Pantarotto, D., Singh, R., McCarthy, D., Erhardt, M., Briand, J.-P., Prato, M., Kostarelos, K., Bianco, A., Functionalized carbon

74

75

76

77

78

79

80

81

nanotubes for plasmid DNA gene delivery., Angew. Chem., Int. Ed. 2004, 43, 5242–5246. Zheng, M., Jagota, A., Strano, M. A., Santos, A. P., Barone, P. S., Chou, G., Diner, B. A., Dresselhaus, M. S., Mclean, R. S., Onoa, G. B., Samsonidze, G. G., Semke, E. D., Usrey, M., Walls, D. J., Structurebased carbon nanotube sorting by sequence-dependent DNA assembly., Science 2003, 302, 1545–1548. Hou, S., Wang, J., Martin, C. R., Template-synthesized DNA nanotubes, J. Am. Chem. Soc., 2005, 127, 8586–8587. Lee, H., Kepley, L. J., Hong, H. G., Akhter, S., Mallouk, T. E., Adsorption of ordered zirconium phosphonate multilayer films on silicon and gold surfaces., J. Phys. Chem. 1988, 92, 2597–2601. Cheung, V. G., Morley, M., Aguilar, F., Massimi, A., Kucherlapati, R., Childs, G., Making and reading microarrays., Nat. Genet., 1999, 21, 15–19. Walt, D. R., Techview: Molecular biology. Bead-based fiber-optic arrays., Science, 2000, 287, 451–452. Han, M., Gao, X., Su, J. Z., Nie, S., Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol., 2001, 19, 631–635. Enzel, P., Adelman, N. B., Beckman, K. J., Campbell, D. J., Ellis, A. B., Lisensky, G. C., Preparation and properties of an aqueous ferrofluid., J. Chem. Educ., 1999, 76, 943–946. Buehler, S. S., Basu, I., Hites, R. A., A comparison of PAH, PCB, and pesticide concentrations in air at two rural sites on lake superior., Environ. Sci. Techol. 2001, 35, 2417–2422.

337

338

10

Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling Li-Shing Huang, Yuh-Yih Chien, Shu-Hua Chen, Po-Chiao Lin, Kai-Yi Wang, Po-Hung Chou, Chun-Cheng Lin, and Yu-Ju Chen 10.1

Introduction

Traditionally, cancer has been considered as a genetic disease; but functionally, it is the altered protein networks and signaling pathways that drive cancer growth, cell survival, tumor invasion, and distant metastasis [1]. Although high-throughput genomic profiling has offered information regarding genomic signature pattern associated with sub-classification of tumor types, these techniques suffer a severe limitation in monitoring the changes in protein levels, which are the actual molecules of action within the cell. In contrast, proteins as the ultimate effectors of gene function directly participate in biological processes to govern cellular growth, differentiation and survival. The change in protein expression patterns in a given disease state should reflect the pathologic processes taking place within the cells. Many studies have shown that the differentially expressed levels of proteins are associated with cancer progression [2, 3]. Protein biomarkers are becoming increasingly important in cancer diagnosis. According to the FDA, biomarkers can be defined as endogenous molecules that are a measurable indicator specifically associated with a particular disease or disease state and are able to differentiate the different physiological states of an individual. More precisely, different types of markers can be used for early detection of disease, for monitoring effects of therapy, for detecting disease recurrence, and for prognosis. Table 10.1 shows a few examples of characteristics of FDA-approved serum tumor markers [4–14]. Although there are only a few types of cancer that can be identified by a single tumor-specific serum marker [15], their expression levels may correlate the progression of cancer with different detection sensitivity and specificity. For example, prostate-specific antigen (PSA) [14, 16] is a specific marker and can be routinely used for screening high-risk population. In contrast, the change in expression pattern of some markers are clinically associated with multi types of cancer (Table 10.1). In the post-genome era, in particular, proteomics opens up new horizons because it promises to accelerate the discovery of new protein disease marker. The completion of the human genome project has catalyzed advances in proteomics to Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

10.2 Fabrication and Biomedical Applications of Nanoparticles

investigate cellular function at the protein level. Particularly, increasingly sophisticated techniques have been rapidly developed for discovering disease biomarkers via large-scale differential profiling [17, 18]. The recognition that every disease induces a specific pattern of change in proteomic microenvironments has important clinical implications for the early detection and progression of disease [19, 20]. Tumors also induce dramatic changes in surrounding stroma, cell–cell contact and angiogenesis. During the changes, plasma, urine, and saliva are readily available samples whose protein content reflects the environment encountered by the blood during its journey through tissues and the circulatory system [21]. Ideally, standard biological sources, such as the above-mentioned body fluids, could be used for identifying biomarkers. A rapid, simple, accurate and inexpensive detection of the relevant marker should be available, together with a measurable and standard baseline as a reference point for diagnosis. However, body fluid-derived proteomes are complex, with a wide range in protein abundance that imposes extreme analytical difficulties for medical studies or clinical diagnoses. Thus, with the advent of a growing number of candidate protein biomarkers for disease diagnosis, it is expected that the number and value of molecular diagnostics, prognosis, and treatment monitoring using protein markers will increase. The development of sensitive techniques with great potential to monitor disease onset is urgently needed for the next phase of cancer diagnosis and monitoring. This chapter overviews a recently developed technique – nanoprobe-based affinity mass spectrometry (NBAMS) – for the characterization of serum protein markers. Section 10.2 summarizes the fabrication of different types of metal nanoparticle and applications in biology and medical science. The principle of mass spectrometry, the detection method in NBAMS, will be described in Section 10.3. Section 10.4 describes the design, workflow, and performance of NBAMS; the advantages of nanoscale probes such as rapid kinetics, specific detection, and high sensitivity are addressed. Section 10.5 reviews the application of NBAMS technology for selected protein pattern analysis in human plasma/blood. Finally, using NBAMS as a multiplexed immunoassay is demonstrated in Section 10.6 for screening of normal individual and cancer patients.

10.2

Fabrication and Biomedical Applications of Nanoparticles 10.2.1

Fabrications and Properties of Nanoparticles

Biomolecule-conjugated nanoparticles (NPs) have been demonstrated to have promising applications in bioanalysis. Among NPs, colloidal gold has attracted the attention of scientists for many years [22, 23]. In general, the synthesis of colloidal gold involves the use of different reducing agents with an aqueous solution of tetrachloroauric acid (HAuCl4 ). By controlling the formation of nuclei and the

339

Biochemical properties

Oncofetal protein, glycoprotein

Mucin identified by monoclonal antibodies Blood group antigen identified by monoclonal antibodies, glycoprotein complex Glycoprotein identified by monoclonal antibodies

Alpha-fetoprotein (AFP)

CA 15-3

CA 19-9

CA 72-4

Colorectal [10] and pancreatic cancer monitoring

Gastrointestinal and ovarian cancer monitoring

48

Breast [6, 7] cancer monitoring

Hepatocellular carcinoma (HCC) and germ-cell (nonseminoma) tumor monitoring and diagnosing

Primary clinical applications

200–1000

300–450

70

Molecular weight (kDa)

Characteristics of some approved serum tumor markers [4, 5].

Protein name

Tab. 10.1.

Breast, gastric, hepatobiliary, hepatocellular, and ovarian [8] cancer Lung and ovarian [8, 9] cancer

37 U mL
1

6 U mL
1

Benign gastrointestinal disease

Pancreatitis and benign gastrointestinal diseases

Benign breast, liver disease

Colorectal, liver, lung, ovarian [8, 9], pancreatic cancer

25 U mL
1

Other related conditions

Pregnancy, hepatitis, and cirrhosis

Other related cancer type

10 ng mL
1

Upper limit of healthy adults

340

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Mucin identified by monoclonal antibodies

Oncofetal antigen, a family of related cellsurface glycoproteins

Hormone, glycoprotein consisting 2 noncovalently bound subunits (a and b) Enzyme, glycoprotein serine protease, total PSA (tPSA) in blood consisting 2 major forms: PSA-ACT (a1anticymotrypsin) complex & free PSA (fPSA)

CA 125

Carcinoembryonic antigen (CEA)

Human chorionic gonadotrophin (hCG), hCG bsubunit (hCGb)

Prostate specific antigen (PSA)

Trophoblastic and testis cancer monitoring

Prostate [12–14] cancer monitoring and diagnosing

28

Colorectal [10], lung, and breast [6, 7] cancer monitoring

Endometrial and ovarian [8, 9, 11] cancer monitoring

36 (hCG)

150–300

200

Gastric, pancreatic, ovarian [8], uterine cancer

Breast, gastrotestinal, lung and ovarian cancer

3 ng mL
1 (5 ng mL
1 for smokers)

5 U L
1 for men and pre-menopausal women; 10 U L
1 for post-menopausal women tPSA: 2.5–6.5 ng mL
1 (by age); fPSA/tPSA ¼ 0:25

Breast, cervical, colorectal, gastrointestinal, lung, pancreatic cancer

35 U mL
1

Benign prostate hyperplasia

Pregnancy, hydatidiform moles, cirrhosis, duodenal ulcer, and inflammatory bowel disease

Benign breast disease, cirhosis, pulmonary emphysema, rectal polyp, ulcerative colitis

Follicular phase of menstrual cycle, early pregnancy, cirrhosis, hepatitis, endometriosis, pericarditis

10.2 Fabrication and Biomedical Applications of Nanoparticles 341

342

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

amount of gold for shell growth, i.e., the conditions of reduction, NPs of variant size could be prepared. For example, small size particles can be formed by using strong reducing agents such as sodium borohydride and white phosphorus [24– 29]. Depending on the reduction conditions, gold nanoparticles (AuNPs) can be synthesized in the size range 0.82–150 nm [30, 31]. Under inert gas conditions, thiol ligands can be assembled on the surface of AuNP by forming the covalent AuaS bond. Besides AuaS formation, the surface of AuNP can also be modified by charge–charge absorption to attach proteins under suitable pH. Antibodyconjugated AuNPs fabricated by charge–charge absorption have been applied in specific cell and tissue labeling that can be visualized under electronic transmission microscopy (TEM) [27]. NPs can also be increased in size by silver enhancement so as to be observed under optical microscopy [32]. In addition, BIA core can be applied to detect the AuNP because the surface plasma resonance (SPR) absorption peaks of gold colloids display in the visible range, 510–550 nm [33]. The aggregation of AuNPs induces a redshift in the SPR peak, which has been applied in many bioassay [34], including DNA–DNA hybridization [35], carbohydrate–protein interactions [36], and carbohydrate–carbohydrate interactions [37]. Recently, gold colloids have been further explored as affinity probes and used with mass spectrometry in biomaterial analysis [38–40]. Besides AuNPs, magnetic nanoparticles (MNPs) have also attracted great attention due to their unique magnetic properties [41–43]. Quantum size effects and the large surface area to volume ratio of MNPs significantly change their magnetic properties and superparamagnetic characteristics [44]. The magnetic properties of iron oxide MNP depend on the particle size [45, 46]. Small particles (<15 nm) show superparamagnetic character, while large particles are ferromagnetic. MNPs of different sizes can be synthesized by changing the reaction parameters, such as the reaction time, temperature, and the concentrations of reagents and stabilizing surfactants. Because of their special properties, MNPs have been utilized in many applications, such as specific cell targeting, virus separation [47], drug delivery [48, 49], magnetic resonance imaging (MRI) [50, 51], and hyperthermia in cancer therapy [52]. MNPs can be fabricated in aqueous or organic solvent conditions. In the aqueous solvent method [53], MNPs are prepared by stirring the mixture of FeCl2 , FeCl3 and NH4 OH (pH adjusting solution) in an ice bath under air. Introduction of air bubbles using a pipette into the mixed solution results in the oxidation of Fe 2þ ions to Fe 3þ , to give Fe3 O4 MNPs. Iron oxide NPs synthesized under organic conditions are better dispersed [54]. In this method, an iron oleic acid metal complex is prepared by the thermal decomposition of pentacarbonyl-iron in the presence of oleic acid at 100  C. Then, iron oxide NPs are generated by ageing the iron complex at 300  C. The size distribution of monodisperse NPs synthesized under these conditions ranges from 4 to 20 nm. When the ratio between pentacarbonyl-iron and oleic acid is changed from 1:2 to 1:3, the particle size is tuned from 7 to 11 nm. In addition to iron oxide MNPs, many alloy MNPs have been synthesized recently. For example, FePt NPs are effective NPs that are easily modified with thiol ligands [55]. The FePt was synthesized by dissolving platinum acetylacetonate and

10.2 Fabrication and Biomedical Applications of Nanoparticles

Fig. 10.1. The Her-2/neu expressing cell was prelabeled with biotinylated antibody and then selectively bound to streptavidin-conjugated SPIO NP, which acted as contrast agent in T2 -weighted MR imaging. (From Ref. [65].)

hexadecane-1,2-diol in dioctyl ether under inert gas and then heating the resulting solution to 100  C. Oleic acid, oleyl amine, and Fe(CO)5 were then added to the reaction mixture, followed by heating at 300  C for 30 min. After cooling the reaction mixture, ethanol was added to precipitate the FePt NPs.

Fig. 10.2. In vivo MR detection of cancertargeting event of WSIO–antibody conjugates (a and b). Color maps of T2-weighted MR images of cancer cell implanted (NIH3T6.7) mice at different temporal points (preinjection, immediate post, 4 h) after the intravenous injection of WSIO-irrelevant antibody control conjugates (a) and WSIO–Herceptin probe

conjugates (b). Whereas no difference is seen in the color-mapped MRI for the control conjugate (a), an immediate (5 min) color change to blue at the tumor site is evident with the probe conjugate (b). (c) T2 versus time after the injection of WSIO–antibody conjugates in (a) and (b) samples. (From Ref. [66].)

343

344

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Semiconductor quantum dots (QDs) are another type of important NPs developed to have many bio-applications, such as medical diagnosis, in vivo cell imaging, and molecular recognition [56]. Not only their good size distribution and uniform shape but also the unique quantum properties makes QDs excellent fluorescent tags. The optical properties of QDs are determined by their sizes. As the particle size decreases, the spacing between two energy levels increases and the wavelength of fluorescence becomes shorter. QDs are synthesized by using semiconductor materials, such as cadmium sulfide, cadmium selenide, cadmium telluride [57], or gallium arsenide [58], under organic or aqueous conditions. QDs with a uniform-size distribution are usually synthesized in organic solvent at high temperature. Trioctylphosphine oxide (TOPO) was first heated followed immediately by injecting the metal precursor (dimethyl cadmium and selenium powder in tributylphosphine) using a syringe. CdSe NPs were then nucleated and the resulting solution became colored. The size of NPs can be modulated by changing the amount of metal precursor and reaction time. QDs so-prepared are hydrophobic due to the surface surfactant layer, TOPO. CdSe NP, one of the most commonly used semiconductor NPs, possesses useful optical properties, including photo-stability, a wide-range excitation, and no red tail in the emission spectrum. Its quantum yield can be increased by coating with a higher band-gap material such as ZnS to form core–shell QDs [59]. Based on these special fluorescent properties, QDs serve as a convenient and effective dye carrier to be monitored either in vitro or in vivo. Recently, DNA encapsulated QD is used as a nanosensor to capture target DNA and the binding signal is amplified by fluorescent resonant energy transfer (FRET) through an external fluorescent tag [60]. Interestingly, various color-coded QDs have been applied in the real-time detection of biomolecule and virus in a microfluidic device [61]. QDs also act as a strong tracing marker in in vivo cell imaging, which facilitates monitoring of the biomolecule distribution [62]. 10.2.2

Metal Nanoparticles in Cancer Diagnosis

Sensitive immunoassay methods are being continuously developed and used in clinical diagnostics to measure specific markers with extremely low concentrations in highly complex biofluids or to detect the premalignant and malignant lesions in early stages. Advances in nanotechnology have facilitated the development of novel ultrasensitive assays for cancer diagnosis in more efficient and economic ways. This section reviews a few iron oxide and gold nanoparticles to illustrate the applications of metal nanoparticles in cancer diagnosis. More information is given in recent reviews [43, 63, 64]. Her-2/neu tyrosine kinase receptor is a 185 kDa protein expressed in approximately 25% of breast cancer cell surface and is an important target in staging and treating breast cancer. Artemov’s laboratory [65] have used a two-step labeling protocol to label the Her-2/neu receptor on a breast cancer cell line (AU-565). As shown in Fig. 10.1, the receptors on cancer cells were first prelabeled with biotiny-

10.2 Fabrication and Biomedical Applications of Nanoparticles

lated humanized mAb (Herceptin, anti Her-2/neu antibody), and then streptavidin superparamagnetic iron oxide microbeads (SPIO-MBs) used as T2 weighted MR contrast agent were selectively bound to the prelabeled receptors. Because the large sizes of SPIO-MBs significantly restricted their delivery and diffusion into cell by passive endocytosis or active transporter system, the particles could remain on the cell surface, facilitating MR imaging. Labeling extracellular receptors is an important advantage for the in vivo application of this method because of the lower probability of modulating cell physiology. A similar approach with the use of nanometer-size magnetic nanoparticles (MNPs) has also been demonstrated in vivo for diagnosis of cancer. Cheon and workers [66] have coated Herceptin on 9-nm Fe3 O4 to give 28 nm sized watersoluble magnetic iron oxide (WSIO)–Herceptin probes. These probes were injected into mice with an implanted fibroblast cell line, NIH3T6.7, which possessed overexpressed Her-2/neu cancer markers. Figure 10.2 showed the T2-weighted MR images obtained at the different temporal points after the intravenous tail injection. No change in the color-mapped MR signal (Fig. 10.2a) and T2 values (Fig. 10.2c) at the tumor site was observed in the control experiment. In contrast, an immediate color change to blue, at the tumor site, became evident within 5 min of the injection of probes. Cheon’s results successfully demonstrated the application of antibody-conjugated MNPs for in vivo MR diagnosis of human cancer cells implanted in live mice. Notably, the T2-weighted MR signal intensity was dependent on the size of WSIO. When the MNPs increased in size, the MR signal intensity decreased [67]. Nanoparticles can also be combined with ELISA assay to develop a more sensitive and efficient assay. Cui and coworkers [68] have fabricated Fe3 O4/Au nanoparticles (GoldMag NPs) with core–shell structure and demonstrated that antibody immobilization efficiency on GoldMag NPs (nanometer scale) was higher than that on Dynal bead (micrometer scale). The anti-HBV (hepatitis B virus) antibodyconjugated GoldMag (anti-HBV GoldMag) was used to detect HBV antigen in blood by ELISA type assay. As shown in Fig. 10.3, the anti-HBV GoldMag NPs were incubated with HBV positive serum first. After a washing step, the HRP (horseradish peroxidase) labeled antibody and its substrate were added into the antibody–nanoparticle complex. By applying an external magnetic field for separation, the liquid phase was measured by UV/Vis spectroscopy. The absorbance at 280 nm showed a 12-fold difference between positive serum and negative serum for HBV antibody. Beside MNPs, AuNPs alone can also be used as contrast reagents for vital optical imaging of precancers and cancers based on their abilities to resonantly scatter visible and near-infrared light. Richards-Kortum and coworkers [69] used bioconjugates of gold nanoparticles (12 nm in diameter) with monoclonal antibodies against epidermal growth factor receptor (EGFR), a transmembrane glycoprotein that is highly overexpressed in epithelial cancers, for molecular specific optical imaging. Because the aggregation of AuNP increased in scattering cross-section per particle, this property produced a large optical contrast between isolated AuNPs and assemblies of AuNPs, and made it suitable for vital optical imaging. Light scat-

345

346

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Schematic of immunoassay using GoldMag NPs for HBV antigen detection. The GoldMag NPs coupled with anti-HBV antibody can capture the HBV antigen by immuno-

Fig. 10.3.

reaction, be isolated from liquid by a magnet, and detected by UV/Vis spectrometry after adding HRP-labeled anti-HBV antibody and HRP substrate. (From Ref. [68].)

tering from the labeled cells was very strong and could be easily observed using low-magnification optics and an inexpensive light source such as a laser pointer. Richards-Kortum’s laboratory has applied anti-EGFR AuNPs to the top of engineered tissue constructs and utilized PVP to help AuNP penetrate the tissue layer and reach the cervical cancer cell layer. Transmittance and reflectance images of engineered tissue constructs labeled with anti-EGFR AuNPs (Fig. 10.4) clearly show the distribution of EGFR expression in living neoplastic cervical tissue. The advantage of using AuNP as contrast reagent is that, as many markers are not uniquely expressed in disease cells but are over- or under-expressed, the scattering from closely spaced aggregates associated with overexpression can magnify the signal difference owing to moderate levels of overexpression. PSA is an important serum marker for the diagnosis and monitoring of prostate cancer. Mirkin and coworkers have developed an ultrasensitive method for detecting PSA using barcode assay [70]. This assay relied on using two particles: a magnetic microparticle for capture target protein and DNA-labeled AuNP for signal amplification. Monoclonal anti-PSA antibody coated magnetic microparticle probes were used to specifically bind target PSA from bio-mediate. The AuNP probes functionalized with hybridized barcode DNA strands and polyclonal antibodies to PSA were used to label microparticle probes through antigen–antibody interaction. Magnetic separation of the complexed probes and PSA was followed by dehybridization of the dsDNA on the AuNP probe to release barcode DNA. Because the AuNP probe carried many DNAs per protein binding event, the binding signal was amplified and PSA could be detected at 30 attomolar concentration. The barcode assay was six orders of magnitude more sensitive than the clinically used ELISA assay for PSA. The same laboratory further discovered that an even more sensitive assay was developed by replacing the AuNPs with polystyrene microparticles, which were loaded with more barcode DNAs to provide a better amplification effect (Fig. 10.5) [71].

10.2 Fabrication and Biomedical Applications of Nanoparticles

Fig. 10.4. Transmittance (A, C, and E) and reflectance (B and D) images of engineered tissue constructs labeled with anti-EGFR/gold conjugated. The tissue constructs consist of densely packed multiple layers of cervical cancer (SiHa) cells. The contrast agents were added on top of the tissue phantoms in 10% PVP solution in PBS (A and B) or in pure PBS (C and D). After incubation for @30 min at room temperature, the phantoms were transversely sectioned with a Krumdieck tissue

Fig. 10.5.

slicer, and the sections were imaged using a Zeiss Leica inverted laser scanning confocal microscope with a 10 (A–D) objective. A small spot on a tissue construct was imaged using a 40 oil immersion objective to show high density of the epithelial cell in the phantom. Reflectance images were obtained with 647 nm excitation. Arrows show the surfaces exposed to the contrast agents. Scale bars are @200 mm (A–D) and @20 mm (E). (From Ref. [69].)

Conventional bio-barcode amplification assay. (From Ref. [71].)

347

348

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

10.3

Principles of Mass Spectrometry

Mass spectrometry is a technique that measures the mass-to-charge ratio (m/z) of molecules. There are three basic components, ion source, mass analyzer and ion detector, in a typical mass spectrometer. Gaseous ions are formed with sample molecules in the ion source and then introduced into the mass analyzer for separation according to their different m/z. Finally, the ions that pass through the mass analyzer are detected by the ion detector and the molecular weight of the analyte can be derived. For the analysis of target protein, mass spectrometry has become the choice of tool for protein characterization. Over the last decade, mass spectrometry has not only advanced technologically but has also greatly expanded in applications to unknown protein identification, post-translational modification and protein quantitation in biological and biomedical research. 10.3.1

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Among various types of mass spectrometers, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become one of the popular tools for protein detection [72, 73]. As shown in Fig. 10.6, the first step involves mixing and co-crystallizing of the sample and excess molar of ‘‘matrix’’ compounds, which are low molecular weight weak organic acids that strongly absorb UV radiation. Even though the exact mechanism of desorption/ionization is not clear, it is generally considered that the matrix absorbs photoenergy from the laser pulse, resulting in energy transfer and desorption/ionization of matrix and

Schematic of matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). Analyte ions are generated by irradiation of laser in the

Fig. 10.6.

MALDI ion source and accelerated into the drift region of TOF mass analyzer. The m/z of the ions are determined according to their arrival time at the detector.

10.3 Principles of Mass Spectrometry

sample molecules from the condensed phase into gaseous phase. Singly and/or doubly charged sample ions are, therefore, formed during this process. The pulsed nature of MALDI is often coupled with analysis with a time-of-flight (TOF) mass analyzer. Ions generated in the MALDI ion source are accelerated electrically into the drift region, a long, straight and electric field-free tube, and drift to the detector at the opposite end. Because ions with different mass are given the same kinetic energy in the ionization region, the lighter and heavier ions will reach the detector at different times. The m/z of an ion can be calculated according to the time, distance of flight and the accelerating voltage. Furthermore, the introduction of peptide mass fingerprints (PMFs) has had a significant impact on unknown protein identification [74]. Based on the properties of the specific enzymatic-hydrolysis of protein and MALDI-TOF MS, protein can be identified simply and rapidly. The protein of interest, which is in many cases purified by gel electrophoresis, is enzymatically or chemically cleaved into a specific set of peptides, and the peptide mixture is analyzed by MALDI-TOF MS. The resulting PMF spectrum is subsequently compared with ‘‘virtual’’ fingerprints obtained by theoretical cleavage of protein sequences stored in databases in a search to identify of possible candidate proteins [75]. Due to its high sensitivity, tolerance to impurities, high speed, and the ability to provide molecule weight information on intact molecule, MALDI-TOF MS has become one of the primary techniques for protein/ peptide profiling and protein identification. 10.3.2

Affinity Mass Spectrometry

Recently, surface-enhanced laser desorption/ionization (SELDI) has evolved rapidly as a new frontier for biomarker discovery and clinical diagnoses based on proteomic pattern analysis [76, 77]. The major difference between SELDI and MALDI is the use of a protein chip array that is composed of a chromatographic surface to retain proteins of interest. SELDI shows diverse applications due its flexibility for surface modification. Despite its advantages of high sensitivity and high throughput, however, the pattern recognition platform, unfortunately, suffers from laboratory-to-laboratory variance due to differences in sample preparation, handling, and analysis software. Many complications may result from suppression effects, a wide dynamic range of protein concentration during simultaneous detection of many proteins in sample. As an alternative to the above approaches, MALDI-TOF MS can be combined with a biologically active probe to rapidly and specifically target, separate, and preconcentrate proteins of interest. This targeted approach, named affinity mass spectrometry, can accelerate research for class-specific proteins or biomarkers. Several analytical affinity capture techniques have been developed for affinity mass spectrometry. The research group of Hutchens and Yip was one of first to demonstrate MS-based affinity capture by immobilization of ‘‘bait’’ DNA on agarose beads for direct MALDI-TOF MS analysis of targeted proteins from complex biofluids [78]. The concept was further tailored by Nelson and coworkers to develop a mass spec-

349

350

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

trometric immunoassay (MSIA) [79]. They used affinity pipette tips (MSIA tips) to selectively retrieve proteins from biological fluids, demonstrating high-throughput quantitative protein analysis as well as screening of heterogeneous glycan structures in plasma proteins [80–82]. Furthermore, ligands and antibodies can also be immobilized or captured on the surface of gold and organic polymer film as probes [83–85]. Other variations of the biologically active probe for affinity mass spectrometry include the assay of direct desorption/ionization on silicon [86, 87] and selfassembled monolayers [88]. Despite the rapid evolution of efficient chip-based or microbead-based assays for biomedical research, protein chip technologies face some technical challenges such as the special requirement of immobilization chemistry and instruments and denaturing/alternating the native bait proteins caused by the chip/bead surface properties. In recent years, nanomaterials have begun to serve as biologically active probes in affinity mass spectrometry and show great potential for application in various biology systems. Chen et al. have used gold nanoparticles immobilized on magnetic particles (Au@magnetic particles) and carbon nanotubes (CNTs) to probe proteins and peptides [38, 89]. Magnetic NPs that conjugated with antibodies were also used to probe pathogenic bacteria by Chen’s group [90]. Chang et al. used high-affinity diamond NPs to extract and preconcentrate proteins from diluted solution and human serum [91]. In addition, Harrison et al. have introduced the C18 functionalized silica NP and aptamer conjugated MNPs as probes [92]. When affinity molecules are coupled to such NPs, they can function as sensitive biosensors that show superior separation capabilities compared with microbeads [93, 94]. There are more advantages of using NPs as ligand carriers [95]. First, there are simple and facile means of anchoring molecules to the nanoscale surface to form versatile and covalently stable conjugates with the capability of specifically interacting with targeted biomolecules. Second, the large surface area-to-volume ratio and the globular shape of an NP allow multivalent and three-dimensional interactions between ligands and receptors without hindrance. Finally, compared with conventional microarray methods using planar solid supports, functionalized NPs can be used as ‘‘water-soluble’’ probes in biological assays in solution. In principle, these ‘‘suspension arrays’’ should improve assay homogeneity and reduce assay time. In our recent report, sugar-conjugated gold NPs were demonstrated as an affinity probe for efficient separation and enrichment of target protein, and then protein identification and epitope mapping in carbohydrate–lectin recognition [40].

10.4

Nanoprobe-based Affinity Mass Spectrometry (NBAMS)

During the past few years, we have focused on developing a versatile, sensitive analytical platform based on surface-engineered NPs and mass spectrometry analysis [96, 97]. The principle of nanoprobe-based affinity mass spectrometry (NBAMS) is to use MNPs as an affinity probe to effectively extract and enrich target protein for direct MALDI-TOF MS detection. The probe, derived form antibody-conjugated

10.4 Nanoprobe-based Affinity Mass Spectrometry (NBAMS)

MNPs, was designed for protein profiling in human plasma. Three serum proteins with different concentrations, C-reactive protein (CRP), serum amyloid A (SAA), and serum amyloid P component (SAP), have been selected as target proteins to demonstrate the general applicability of the method. Both CRP [98, 99] and SAA [100] are exquisitely sensitive systemic markers of acute phase response and present about 1 mg L
1 and 3 mg L
1 in the blood of healthy human, respectively. Overexpression of SAA was also found to be associated with gastric cancer in a recent report [101]. SAP is a biomarker related to Alzheimer’s disease and type 2 diabetes [102], with a concentration about 40 mg L
1 in blood of healthy humans [82, 103]. 10.4.1

Preparation of Nanoprobes and Workflow

Figure 10.7 illustrates the synthesis of antibody-conjugated iron oxide NPs. The iron oxide (Fe3 O4 ) NPs (MNP 1) were obtained by co-precipitation of FeCl2 and

Fig. 10.7. Preparation and characterization of MEG-protected antibody-conjugated MNPs. (From Ref. [97].)

351

352

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

FeCl3 under basic conditions and then dispersed in acidic solution [104]. The particle surface was transformed into an amino functionality by the sol–gel process using tetraethyl orthosilicate (TEOS) followed by addition of 3aminopropyltrimethoxysilane (APS) to give MNP 2, which contains amine terminal groups [105]. Transmission electron microscopy (TEM) revealed that the diameter of MNP 1 is in the range of 5–15 nm while the average diameter of the iron oxide core of MNP 2 was 50 nm with a relatively narrow size distribution (Fig. 10.7). Enlargement of the metal core is due to the aggregation of iron oxide during silanization, and the silica shell is visible as a faint circle outside the core. For antibody conjugating, MNP 2 was treated with a crosslinker, bis(N-hydroxysuccinimide ester) (DSS), to crosslink the aminosilane MNPs with antibody [106]. To avoid nonspecific binding on the surface of antibody-conjugated MNPs, the resulting MNPs were protected by a blocking reagent, i.e., terminal aminated methoxy-ethylene glycol (MEG). The final products, MEG-protected antibody-conjugated MNPs, were washed and ready to use for NBAMS. Figure 10.8 shows the workflow of NBAMS. Phosphate-buffered saline (PBS, pH 7.4) diluted plasma sample is incubated with antibody-conjugated MNPs at room temperature. After immunoaffinity capture of antigens, the antigen-captured MNPs are collected by a magnet, and the unbound, non-antigenic components were subsequently removed by a series of washes, abrogating the need for purification and desalting. For subsequent MS analysis, the MNPs were directly mixed with MALDI matrix and directly spotted onto the sample plate for MALDI-TOF MS measurement.

Workflow of nanoprobe-based affinity mass spectrometry (NBAMS). The target antigen is specifically extracted from plasma sample by antibody-conjugated MNPs.

Fig. 10.8.

After separation by magnet, the isolated, antigen-captured MNPs are directly spotted onto a sample plate and analyzed by MALDITOF MS.

10.4 Nanoprobe-based Affinity Mass Spectrometry (NBAMS)

10.4.2

Proof-of-principle Experiment

The feasibility of the NBAMS strategy was first evaluated with a protein pool composed of antigenic proteins (SAP, 20% molar fraction and CRP, 3% molar fraction) and two other ‘‘nonantigenic’’ proteins, myoglobin (Myo, 15%) and enolase (Eno, 62%). The abundance of the targeted antigenic proteins was purposely reduced in mixture to test the extraction efficiency of the targeted protein. As shown in Fig. 10.9(A), the MALDI-TOF mass spectrum of the protein mixture displayed the complexity of the mixture, in which one of the targeted antigens, CRP, was not ob-

Fig. 10.9. MALDI-TOF mass spectra of a protein mixture before (A) and after using (B) anti-CRP-conjugated MNPs, (C) anti-SAPconjugated MNPs, and (D) anti-SAPencapsulated AuNPs to extract a specific protein. The protein solution (60 mL) was composed of 0.5 mm myoglobin (Myo), 0.1 mm

C-reactive protein (CRP), 0.67 mm serum amyloid P component (SAP), and 2.1 mm enolase (Eno). The arrow in (A) indicates the theoretical m/z of CRP. The inset of (C) shows detailed protein expression profiles of wildtype, monosialo-, and asialo-SAP. (From Ref. [96].)

353

354

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

served due to its low abundance (3% molar fraction) and the ion suppression effect [107]. Suppression effects are commonly observed in MALDI-TOF MS due to the presence of salts, buffer, or other more abundant species in complex biological media [108]. The suppression effect can result in reduced signal intensity or even the disappearance of the signal of targeted analyte. Figure 10.9(B) revealed the specificity of the NBAMS methodology, where CRP was extracted and detected with an excellent signal-to-noise ratio of 822. No background peak between m/z 5000 and 50 000 was observed in control experiments before the addition of the protein mixture, showing no ‘‘chemical noise’’ arising from the antibody-conjugated MNPs. The absence of other abundant proteins excluded nonspecific binding arising from electrostatic attraction or hydrogen bonding. The use of nanoprobe-based immunoassay overcomes the suppression effect because the salt and abundant nonantigenic proteins are removed and targeted proteins are selectively concentrated on the MNPs. The strong peak of target protein in mass spectrum without background demonstrates the advantages of nanoprobe-based affinity extraction in providing simultaneous protein isolation, enrichment, and sample desalting without the necessity of additional purification steps. Mass spectrometric detection is also ideal for characterizing post-translational modifications that cannot be predicted from genomic information. The MALDITOF mass spectrum in Fig. 10.9(C) showed a cluster of peaks corresponding to several SAP variants from the affinity extraction using anti-SAP-conjugated MNPs. The expanded view shows that the mass spectrum is dominated by the mass of 25464 G 4 Da, which is consistent with the theoretical value of 25462 Da, as calculated from the known sequence [109]. Accompanying the major peak were two peaks at 25174 and 24881 Da, corresponding to mass shifts of 290 and 583 Da, respectively. Within experimental uncertainty, the shifts can be attributed to the loss of one or two sialic acid residues (mass of each residue: 291 Da) [82]. In addition to MNPs, surface-engineering gold nanoparticles (AuNPs) can also be an affinity probe [40]. The test of AuNP for immunoreaction was performed with 13-nm AuNPs. Antibody-encapsulated AuNPs were prepared as follows. The pH of a gold colloid was adjusted to 8.5 with 0.1 m K2 CO3 , followed by addition of antibody in the solution. The reaction was performed on ice for 60 min and the antibody-encapsulated AuNP was separated by centrifugation. The anti-SAP encapsulated AuNPs were incubated with the same protein pool (Fig. 10.9A). After immunoaffinity extraction, the pellet of AuNP containing targeted antigen was subsequently analyzed by MALDI-TOF MS. Figure 10.9(D) shows the extracted SAP from the mixture; however, signal from the enolase was also observed due to the ‘‘unoccupied sites’’ on AuNP. After further modification with bovine serum albumin on the AuNP surface, the co-extraction of enolase can be diminished (data not shown). In general, antibody–antigen interactions are strong, with dissociation constants (K d ) ranging from 10
7 to 10
11 m. Most antibody–antigen complexes can be dissociated at extreme pH (i.e., pH < 2 or pH > 12). Direct MALDI-TOF MS analysis is performed where particles are placed directly on the MALDI sample plate with addition of matrix. The pH of matrix (SA) solutions is typically less than 2 and thus may serve to elute the antigen bound to the antibody-conjugated MNPs. The direct

10.4 Nanoprobe-based Affinity Mass Spectrometry (NBAMS)

MALDI-TOF MS approach provides rapid and sensitive analysis of affinity-bound analyte, avoiding the potential risk of sample loss. In the linear mode of MALDITOF MS, the mass resolution did not deteriorate when the antigen-bound MNPs were deposited on the MALDI probe. Similarly, a mass accuracy of 0.02% could be routinely obtained by external calibration, comparable to the mass accuracy of conventional MALDI-TOF MS detection. Thus, the ‘‘direct’’ analysis of nanoscale particles does not diminish the performance of the MALDI-TOF MS. To confirm the identity of the captured protein, the MNPs pellet was directly subjected to in situ digestion with trypsin and analyzed by MALDI-TOF MS. A database search of the peptide mass fingerprinting map with the MS-Fit program (http://us.expasy.org/uniprot/P02743) unambiguously matched to serum amyloid P component precursor (Swiss-Prot P02743, see Fig. 10.10). The unique advantage of on-probe protein identification provides potential applications of functionalized MNPs in discovering unknown class-specific proteins.

Fig. 10.10. Assignment of observed peptides from in situ trypsin-digestion of antigen-captured MNPs and their corresponding sequences matched to serum amyloid P component precursor (SAP, P02743 in the SwissProt sequence database). The sequence coverage is 58%. (From Ref. [97].)

355

356

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.11. Effect of incubation time on antibody–antigen recognition using antibodyconjugated MNPs. To investigate the time course of antibody–antigen recognition on MNPs, 1 mL of supernatant was sampled from a 60-mL reaction after different incubation times. The MNPs were conjugated with anti-

SAP (filled squares with solid line) and antiCRP (filled circles with dashed line). After incubation (3–60 min), the quantities of the antigen remaining in the supernatant were detected by MALDI-TOF MS, and the peak intensities were plotted as a function of incubation time. (From Ref. [96].)

10.4.3

Kinetic Study of the Nanoscale Immunoreaction

In traditional immunoassays, the incubation of antibody and antigen is often the rate-limiting step (e.g., 30 min to overnight for conventional ELISA) [110, 111]. The high density of surface antibody should, in principle, speed up antibody– antigen interaction. Figure 10.11 shows the required incubation time of NBAMS approach. The peak intensities, corresponding to unbound antigen (SAP) in solution, decreased dramatically as a function of incubation time over 10 min, at which time free SAP was barely detectable (signal-to-noise ratio was <3). Significantly, the binding of CRP was almost completed in an even shorter incubation time (<3 min). Unlike conventional immunoassays such as ELISA, for which the overall process usually requires at least 4 h, the nanoscale immunoassay can be shortened to within 15–20 min. In addition to high antibody density on MNPs, the rapid immunoreaction may be attributed to the water-soluble MNPs; the ‘‘suspension arrays’’ improves the assay homogeneity and reduces sample handling time. The NBAMS approach directly detected specific antibody-captured antigens by MALDI-TOF MS without using a secondary antibody or a reporter reaction. Thus, this rapid and sensitive approach may be amenable to clinical applications such as high-throughput or population screening. 10.4.4

Detection Limit and Concentration Effect of Nanoprobe-based Immunoassay

The NBAMS assay also reveals high detection sensitivity. Figure 10.12(A–C) shows that the CRP signals decrease progressively with decreasing concentration, ranging

10.4 Nanoprobe-based Affinity Mass Spectrometry (NBAMS)

Fig. 10.12. Mass spectra of CRP extracted from 60 mL of protein solution of (A) 145, (B) 29, and (C) 6 nm, using anti-CRP-conjugated

MNPs; and spectra of SAP extracted from (D) 1.9 mm, (E) 15 nm, (F) 0.6 nm, using anti-SAPconjugated MNPs. (From Ref. [96].)

from 145 to 6 nm, and the current assay detection limit for CRP is 6 nm (Fig. 10.12C). Similarly, Fig. 10.12(D–F) shows the mass spectra of SAP extracted from a series of dilutions of 60 mL PBS, ranging from 1.9 mm to 0.6 nm. Strong peak intensities were observed in all spectra except that for the 0.6 nm solution (signal-tonoise ratio of 3). The better detection limit of SAP compared with CRP may be attributed to the different amounts of antibody on MNPs (the rate of anti-SAP to anti-CRP was 1.4). Theoretically, the assay sensitivity of the NBAMS approach depends on the MALDI-TOF MS detection limit and the efficiency of affinity extraction. Assuming full recovery of all the SAP present in the 60 mL of diluted solution (0.6 nm in Fig. 10.12F), the absolute detection limit is estimated to be 36 fmol, which is comparable to the detection limit by direct deposition of SAP onto the MALDI probe (data not shown). Notably, SAP and CRP levels in sera from healthy individuals were about 1.6 mm and 40 nm, respectively [112, 113]. Considering current progress in the detection limit, the nanoscale immunoassay is capable, with reasonable enrichment, of detecting plasma proteins at the subnanomolar level (0.6 nm). Another advantage of the nanoprobe-based immunoassay is the ability to preconcentrate the antigen from diluted medium to a small volume of MNPs. The concentration effect was demonstrated with a series of solutions with different SAP concentration (8–160 nm). Figure 10.13 shows the MALDI-TOF mass spectra of ex-

357

358

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.13. Mass spectra of SAP extracted from diluted solution using anti-SAP-conjugated MNPs: (A) 160 nm SAP, 50 mL; (B) 40 nm, 200 mL; (C) 16 nm, 500 mL; (D) 8 nm, 1000 mL; (E) 1.6 nm, 1000 mL. The inset of each panel shows the mass spectrum of solution prior to extraction. (From Ref. [96].)

tracted SAP after preconcentration using anti-SAP-conjugated MNPs. By contrast, the SAP peak was barely discernible (or not detected) when the diluted samples were analyzed by conventional MALDI-TOF MS, as shown in the inset of each panel. Similarity, Fig. 10.13(E) shows that as low as 1.6 nm SAP in a 1000 mL solution was detectable with a signal-to-noise ratio of 13, and this concentration was

10.5 Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry

comparable to the detection limit of SAP extracted by MNPs (Fig. 10.12F). All the results demonstrate the MNPs are effective probes that provide simultaneous protein cleaning and concentrating for analyte with very low concentration.

10.5

Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry 10.5.1

Selected Protein Profiling from Human Plasma

Human plasma proteome is the most attractive biological medium containing secreted disease-related markers, holding promise in both disease diagnosis and therapeutic aspects [21]. However, it is a very complex mixture of proteins having a wide and dynamic range of abundance of more than 10 12 . Indeed, 22 highabundance proteins constitute 99% of the protein content in plasma, with the remaining 1% that are of great interest as potential biomarkers [114]. In the NBAMS immunoassay, specificity is one of the key components to analyze the lowabundance proteins. The NBAMS platform has been applied to human plasma to investigate whether the approach can directly detect low-level proteins in plasma. Before immunoaffinity extraction, no protein profile could be obtained from the crude plasma in healthy individual due to the interference of salt and other plasma components (Fig. 10.14A). After a 200-fold dilution of the plasma sample to reduce the salt concentration, the protein profile in Fig. 10.14(B) shows the commonly observed abundant plasma proteins, including human serum albumin (HSA), apolipoprotein A-I (Apo A-I), hemoglobin a-chain (Hb-A), hemoglobin b-chain (Hb-B), and transthyretin (TTR). After immunoaffinity extraction, SAP was detected with concomitant depletion of other proteins of higher concentration (Fig. 10.14C). Similarly, Fig. 10.14(D) shows an apparent peak for CRP, even though the level of this protein is 40-fold lower than that of SAP in healthy individuals. Although the analysis showed minor peaks due to nonspecific binding of other high-abundance plasma proteins, they did not interfere with the unambiguous identification of CRP and SAP by mass spectrometry. 10.5.2

Comparison of Nanoscale and Microscale Immunoassay

To compare the advantages of magnetic nano-size particles with those of magnetic microbeads (MMP), commercially aminated magnetic microbeads (DYNAL BIOTECH, 2.8 mm) were conjugated with anti-SAP antibody to obtain anti-SAP MMP by the same modification process used for MNP. With an equal amount of antibody on the particle surface, the signal intensity and signal-to-noise ratio were dramatically reduced in the mass spectrum of MMP compared with MNP experiments. These results indicate that MNPs afford better affinity extraction of the tar-

359

360

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.14. Comparison of immunoaffinity extraction performance between antibodyconjugated MNPs and magnetic microbeads. Mass spectra showed human plasma (A) without dilution; (B) with 200-fold dilution. High-abundance proteins were so dominant that the signals from SAP or CRP were buried

in the spectrum. Representative mass spectra of (C) for SAP and (D) for CRP were obtained after extraction using antibody-conjugated MNPs. Spectra for antibody-conjugated microbeads are shown in (E) for SAP and (F) for CRP. (From Ref. [96].)

10.5 Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry

get protein, thereby improving the detection. The superior efficiency of MNP could be attributed to its large surface area-to-volume ratio, good reaction homogeneity and fast reaction kinetics in suspension array. Additionally, the mass spectrum of affinity extraction with MNPs preserved good spectral resolution and peak profiles without apparent peak broadening and mass shift. 10.5.3

Suppression of Nonspecific Binding on Magnetic Nanoparticles

Antibody-conjugated MNPs could effectively enhance the target protein signal for MALDI-TOF MS detection. However, nonspecific binding from other non-antigenic protein on the surface of MNPs interferes with the sensitivity and accuracy of MALDI-TOF MS detection. As shown in Fig. 10.15(A), HSA is the major abundant protein present in human plasma and was coextracted by antibody-conjugated MNPs. To avoid nonspecific binding, bovine serum albumin (BSA) was first tested

Fig. 10.15. Mass spectra of nanoscale immunoaffinity extraction of plasma SAP from a 1-mL plasma sample with antiSAP MNPs (A) without blocking, (B) with BSA blocking and (C) with 40 mm MEG blocking. (From Ref. [97].)

361

362

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.16. Concentration effect of MEG on suppression of non-specific binding; the maximum value of target/HSA ratio is normalized as 1.0. (From Ref. [97].)

as a blocking agent to protect the anti-SAP MNPs. However, BSA did not yield good suppression of the HSA peak. Recently, Huang and Zheng [115] have reported that the short molecule, ethylene glycol, is biocompatible material with good resistance to nonspecific binding with biological molecules. The short terminal aminated methoxy-ethylene glycol (MEG) linker was tested as the surface blocking spacer to suppress the serious nonspecific binding effect. Indeed, MEGprotected antibody-conjugated MNPs can significantly reduce non-specific binding during the affinity separation of protein biomarkers in human plasma. By modulating the MEG coupling concentration, the inhibition of nonspecific binding was found to be concentration-dependent (10–100 mm). In Fig. 10.16, three different protein markers, including SAP, CRP, SAA, could be obviously enhanced with the least nonspecific interference from the MEG coupling concentration between 20 and 40 mm. Notably, the amount of antibody on the MNP surface was not affected by the incubation time and the concentration of MEG. Based on above results, moderate surface blocking plays a key role in the enhanced detection specificity of the NBAMS approach. 10.5.4

Enrichment of Target Antigen in Human Plasma

The major advantage of NBAMS methodology is the simultaneous selection and preconcentration of targeted protein onto the MNPs, even in a complex biological medium. The concentration effect of NBAMS in human plasma was evaluated with equal amounts of plasma (1 mL from each subject) diluted 50-, 500-, and

10.5 Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry

Fig. 10.17. Mass spectra of SAP extracted from human plasma (1 mL), undiluted (A), and diluted 50-fold (B), 500-fold (C), or 1000-fold (D), using anti-SAP-conjugated MNPs extraction. Inset of each panel shows the mass spectrum of solution before extraction. (From Ref. [96].)

1000-fold in PBS and analyzed using the nanoprobe-based immunoassay. Figure 10.17 shows the MALDI-TOF mass spectra of plasma SAP extracted from each diluted sample. Incubation of the diluted plasma samples with the antibodyconjugated MNPs resulted in antigen selection and concentration, as demonstrated by the similar mass spectra profiles up to 500-fold dilution (Fig. 10.17C). In the 1000-fold diluted sample (1.8 nm SAP), however, the captured antigen showed significantly lower intensity in the mass spectrum (Fig. 10.17D). This decreased recovery may arise from the incomplete collection of the MNPs from the curved wall of the microcentrifuge tube during the washing steps. Despite the decreased

363

364

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

recovery at 1000-fold dilution, the detection sensitivity in human plasma (estimated to be 1.8 nm) was comparable to the performance of NBAMS in a protein standard (SAP, 0.6 nm in Fig. 10.12F), demonstrating that the nanoscale immunoassay is refractory to the presence of highly abundant nonantigenic proteins, salts, and buffers in plasma. All these results display the general applicability of NBAMS methodology, namely, that concentrations with a 1000-fold difference can be detected successfully. By the good concentration effect, 1 mL of plasma is sufficient to unambiguously identify an antigen of interest. In the NBAMS immunoassay, the rate-limiting step of the total procedure for diseased-related protein analysis by NBAMS is the separation of plasma from blood (30–60 min). To speed up the analysis, the NBAMS approach was performed in a blood sample without prior centrifugation. Figure 10.18(A) shows the profile of the major proteins in human blood, containing Hb-A, Hb-B and HSA. After treatment with anti-SAP- and anti-CRP-conjugated MNPs sequentially, the extracted SAP and CRP was shown in Fig. 10.18(B, C, respectively). Although the highly abundant Hb-A, Hb-B and HSA were coextracted, CRP and SAP were unambiguously identified through mass spectrometric detection within 20 min without interference from the highly abundant contaminates. This demonstrates the feasibility of the NBAMS approach in blood sample analysis and holds promise in speeding up clinical applications. 10.5.5

Plasma Protein Profiling in Normal Individuals and in Patients

The NBAMS approach has been performed with authentic clinical samples plasma (20 mL) from six healthy individuals and four patients with gastric cancer [97]. All sample preparation, washing and on-nanoprobe analysis were performed in parallel batches. CRP and SAP were detected successfully in all the healthy individuals, despite the fact that the levels of a few of them were below the detection limit of ELISA (<0.159 mg L
1 ) [101]. The results in Fig. 10.19 illustrate the measured intensities for CRP and SAP, showing significant upregulation and down-regulation in expression levels, respectively. These observed differences in protein levels are consistent with the literature [116, 117] and the ion intensity measured by the nanoprobe-based assay correlated with the concentration measured by ELISA [101], suggesting that the NBAMS approach shows promise for quantitative protein profiling. Analysis with more clinical samples is required for further evaluation on quantitation accuracy.

10.6

Multiplex Assay

Because of the complex natures of disease, diagnosis using single protein assay often results in insufficient detection specificity. To improve detection specificity and sensitivity to determine disease onset, the utility of multivariant protein

10.6 Multiplex Assay

Fig. 10.18. Mass spectra of a human blood sample (20 mL) from a patient with stroke before (A) and after using (B) antiSAP-conjugated MNPs or (C) anti-CRP-conjugated MNPs extraction.

markers as disease signature present an evolving direction in diagnosis. Developing multiplexed assays that can simultaneously screen multiple protein biomarkers is a rapidly expanding trend in characterizing disease states [118]. For example, Lokshin et al. [119] have developed new profiling technology, LabMAP, which combines the principle of a sandwich immunoassay with fluorescent-bead-based technology to detect tumor markers and cytokines in serum samples. The sensitivity, around 80–90% specificity, of discriminating early-stage ovarian cancer from

365

366

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.19. Screening of SAP and CRP in human plasma from healthy individuals and patients with gastric cancer using nanoprobebased affinity mass spectrometry. The mass

spectrum of CRP (m/z 23 kDa) and SAP (m/z 25 kDa) show significant upregulation and down-regulation, respectively. (From Ref. [97].)

healthy controls was increased from 70 to 80% by using CA 125 alone to 90–100% by using the combination of five markers. In addition to the fluorescence-based immunoassay, Wilson has reported antibody-immobilized electrodes as electrochemical immunosensors. Quantitation can be performed simultaneously for two tumor markers, CEA and AFP, in standard protein solution [120]. Lieber’s group have developed an antibody-labeled silicon-nanowire sensor array for multiplex electrical detection of PSA, PSA-ACT complex, CEA, and mucin-1. This technology allowed highly selective and sensitive detection of 0.9 pg mL
1 of PSA in undiluted serum samples [121]. 10.6.1

Workflow of Multiplexed Assay

In past work, the NBAMS technology has also been successfully demonstrated as a multiplexed immunoassay [97]. Three model proteins, SAA, SAP, and CRP, were still selected as target antigens. Figure 10.20 shows the multiplex immunoassay workflow. Plasma is diluted by PBS, mixed with a mixture of anti-SAA-, antiCRP-, and anti-SAP-MNPs, and incubated at room temperature. In the same way as described previously, MNPs are isolated, washed, and subjected to MALDI-TOF MS analysis after immunoaffinity capture of antigens. This immunoextraction and subsequent analysis can be completed in 1 h.

10.6 Multiplex Assay

Fig. 10.20. Workflow of multiplexed immunoassay. For simultaneous analysis of multiple antigens, various antibody-conjugated MNPs are mixed in the appropriate ratio, and

incubated with plasma sample. After separation by magnet, the isolated, antigencaptured MNPs are directly spotted onto a sample plate and analyzed by MALDI-TOF MS.

10.6.2

Screening for Patient and Healthy Individuals

A total of 18 samples, including nine normal controls and nine gastric cancer patients, have been analyzed by the NBAMS multiplexed immunoassay. Figure 10.21 shows representative results of the multiplex assay for SAA, SAP, and CRP from human plasma. Obvious differences can be observed in the protein profile between the two spectra obtained from healthy control (Fig. 10.21A) and gastric cancer patients (Fig. 10.21B). The measured intensities for both SAA and CRP are considerably higher in the gastric cancer patient sample than in the healthy individual. The relative SAA and CRP intensities are consistent with the concentration measured by ELISA (data not shown), and suggest that our immunoassay shows promise for quantitative protein profiling. The normal and gastric cancer groups can be differentiated with a scatterplot of relative SAA and CRP intensities (Fig. 10.21C). Obviously, the data distribution falls into two clusters in the diagram. A ‘‘virtual diagnosis pattern’’ of the SAA/ SAP and CRP/SAP ratio provides a group separation. The low relative SAA and CRP abundances were observed in plasma from the healthy group, whereas higher ratios were observed for gastric cancer cases. The distribution is consistent with the groups of normal and gastric cancer. It has been increasingly recognized that multivariate parameters characterize disease states better than a single protein assay. By using a mixture of MNPs conjugated with different antibodies, the NBAMS technology demonstrated the capability of simultaneous multiplex protein profiling in complex mixtures like human plasma. Furthermore, this assay provides the speed, high sensitivity, and detection specificity required by disease diagnosis and monitoring. This preliminary data showed the promise for application of rapid screening targeted protein profile for disease diagnosis and monitoring. However, the potential implementation of NBAMS awaits further considerations. Generally, the diagnosis specificity not only depends on the detection specificity but also on statistical specificity of the protein marker. In terms of accuracy and reproducibility of analysis, more samples

367

368

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

Fig. 10.21. Representative protein profiles of human plasma from (A) healthy control (from Ref. [97]) and (B) gastric cancer patient obtained by multiplexed immunoassay. (C) Scatter-plot of relative SAA and CRP intensities shows a differentiation between normal and gastric cancer groups.

References

are required for further evaluation. Further study for quantitative profiling is currently on-going.

10.7

Future Outlook

Surface-conjugated nanoparticles have been shown to act as specific affinity nanoprobes for the multiplexed detection of target proteins in human plasma. Such nanoprobes are envisaged to become excellent tools for the separation and enrichment of low-abundant protein biomarkers. Analysis by MALDI-TOF MS provides unambiguous identification of target antigen. In combination with the ongoing quantitative analysis of plasma protein profiling, this nanoprobe-based immunoassay holds great potential for the early diagnosis of disease, inflammatory events, and eventually cancers. From proof-of-concept experiments to widespread use, as always, the quantification sensitivity, specificity, and reproducibility have to be further evaluated with a large set of clinical specimens. For large-scale analysis, advances in instrumentation will become imperative for the automation of sample preparation, nanoprobe detection, and data analysis. The specificity, speed, and flexibility of the nanoprobe-based affinity assay can easily be adapted for the detection of other class-specific proteins in biological research, clinical proteomics and diagnostics.

Acknowledgments

The authors acknowledge financial support from Academia Sinica Research Project on Nanoscience and Technology and the National Science Council, Taiwan. The authors thank Mr. Li-Li Wen at En Chu Kong Hospital for ELISA analysis, Dr. H.-M. Lin for TEM measurement, and the Institute of Biomedical Sciences, Academia Sinica, Taiwan for the use of MALDI-TOF MS.

References 1 Rosenblatt, K. P., Bryant-

Greenwood, P., Killian, J. K., Mehta, A., Geho, D., Espina, V., Petricoin, E. F., 3rd, Liotta, L. A. Serum proteomics in cancer diagnosis and management, Annu. Rev. Med. 2004, 55, 97–112. 2 Jones, M. B., Krutzsch, H., Shu, H., Zhao, Y., Liotta, L. A., Kohn, E. C., Petricoin, E. F., 3rd Proteomic analysis and identification of new

biomarkers and therapeutic targets for invasive ovarian cancer, Proteomics 2002, 2, 76–84. 3 Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M., Gillespie, J. W., Hu, N., Taylor, P. R., Emmert-Buck, M. R., Liotta, L. A., Petricoin, E. F., 3rd, Zhao, Y. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein

369

370

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

4

5

6

7

8

9

10

11

markers, Mol. Cell. Proteomics 2002, 1, 117–124. Burtis, C. A., Ashwood, E. R., in Tietz Fundamentals of Clinical Chemistry, 5th edn., W. B. Saunders, Philadelphia, 2001, pp. 390–413. [Anon] European Group on Tumour Markers (EGTM), Anticancer Res. 1999, 19, 2785–2820. Soletormos, G., Nielsen, D., Schioler, V., Mouridsen, H., Dombernowsky, P. Monitoring different stages of breast cancer using tumour markers CA 15-3, CEA and TPA, Eur. J. Cancer 2004, 40, 481– 486. Molina, R., Barak, V., van Dalen, A., Duffy, M. J., Einarsson, R., Gion, M., Goike, H., Lamerz, R., Nap, M., Soletormos, G., Stieber, P. Tumor markers in breast cancer – European Group on Tumor Markers recommendations, Tumor Biol. 2005, 26, 281–293. Terry, K. L., Sluss, P. M., Skates, S. J., Mok, S. C., Ye, B., Vitonis, A. F., Cramer, D. W. Blood and urine markers for ovarian cancer: A comprehensive review, Dis. Markers 2004, 20, 53–70. Skates, S. J., Horick, N., Yu, Y., Xu, F. J., Berchuck, A., Havrilesky, L. J., de Bruijn, H. W., van der Zee, A. G., Woolas, R. P., Jacobs, I. J., Zhang, Z., Bast, R. C., Jr. Preoperative sensitivity and specificity for earlystage ovarian cancer when combining cancer antigen CA-125II, CA 15-3, CA 72-4, and macrophage colonystimulating factor using mixtures of multivariate normal distributions, J. Clin. Oncol. 2004, 22, 4059–4066. Duffy, M. J., van Dalen, A., Haglund, C., Hansson, L., Klapdor, R., Lamerz, R., Nilsson, O., Sturgeon, C., Topolcan, O. Clinical utility of biochemical markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines, Eur. J. Cancer 2003, 39, 718–727. Duffy, M. J., Bonfrer, J. M., Kulpa, J., Rustin, G. J., Soletormos, G., Torre, G. C., Tuxen, M. K., Zwirner,

12

13

14

15

16

17

18

M. CA125 in ovarian cancer: European Group on Tumor Markers guidelines for clinical use, Int. J. Gynecol. Cancer 2005, 15, 679–691. Catalona, W. J., Partin, A. W., Slawin, K. M., Brawer, M. K., Flanigan, R. C., Patel, A., Richie, J. P., deKernion, J. B., Walsh, P. C., Scardino, P. T., Lange, P. H., Subong, E. N., Parson, R. E., Gasior, G. H., Loveland, K. G., Southwick, P. C. Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: A prospective multicenter clinical trial, J. Am. Med. Assoc. 1998, 279, 1542–1547. Thakur, V., Singh, P. P., Talwar, M., Mukherjee, U. Utility of free/ total prostate specific antigen (f/t PSA) ratio in diagnosis of prostate carcinoma, Dis. Markers 2003, 19, 287–292. Hernandez, J., Thompson, I. M. Prostate-specific antigen: A review of the validation of the most commonly used cancer biomarker, Cancer 2004, 101, 894–904. Ebert, M. P., Korc, M., Malfertheiner, P., Rocken, C. Advances, challenges, and limitations in serumproteome-based cancer diagnosis, J. Proteome Res. 2006, 5, 19–25. Pavlenko, M., Roos, A. K., Lundqvist, A., Palmborg, A., Miller, A. M., Ozenci, V., Bergman, B., Egevad, L., Hellstrom, M., Kiessling, R., Masucci, G., Wersall, P., Nilsson, S., Pisa, P. A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer, Br. J. Cancer 2004, 91, 688–694. Kantor, A. B., Wang, W., Lin, H., Govindarajan, H., Anderle, M., Perrone, A., Becker, C. Biomarker discovery by comprehensive phenotyping for autoimmune diseases, Clin. Immunol. 2004, 111, 186–195. Petricoin, E., Wulfkuhle, J., Espina, V., Liotta, L. A. Clinical proteomics: Revolutionizing disease

References

19

20 21

22

23

24

25

26

27

28

29

detection and patient tailoring therapy, J. Proteome Res. 2004, 3, 209–217. Liotta, L. A., Kohn, E. C. The microenvironment of the tumourhost interface, Nature 2001, 411, 375– 379. Hanash, S. Disease proteomics, Nature 2003, 422, 226–232. Anderson, N. L., Anderson, N. G. The human plasma proteome: History, character, and diagnostic prospects, Mol. Cell. Proteomics 2002, 1, 845–867. Hayat, M. A. Colloidal Gold: Principles, Methods, and Applications, Academic Press, San Diego, 1989. Daniel, M. C., Astruc, D. Gold nanoparticles: Assembly, supramolecular chemistry, quantum-sizerelated properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 2004, 104, 293–346. Tschopp, J., Podack, E. R., MullerEberhard, H. J. Ultrastructure of the membrane attack complex of complement: Detection of the tetramolecular C9-polymerizing complex C5b-8, Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7474–7478. Faulk, W. P., Taylor, G. M. An immunocolloid method for the electron microscope, Immunochemistry 1971, 8, 1081–1083. Safer, D., Hainfeld, J., Wall, J. S., Reardon, J. E. Biospecific labeling with undecagold: Visualization of the biotin-binding site on avidin, Science 1982, 218, 290–291. Horisberger, M., Rosset, J. Colloidal gold, a useful marker for transmission and scanning electron microscopy, J. Histochem. Cytochem. 1977, 25, 295–305. Birrell, G. B., Hedberg, K. K., Griffith, O. H. Pitfalls of immunogold labeling: Analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy, J. Histochem. Cytochem. 1987, 35, 843–853. Bonnard, C., Papermaster, D. S., Kraehenbuhl, J. P. in Immunolabelling for Electron Microscopy, Polak,

30

31

32

33

34

35

36

37

38

39

J. M., Varndell, I. M. (Eds.), Elsevier, New York, 1984, pp. 95–111. Turkevich, J., Stevenson, P. C., Hillier, J. A study of the nucleation and growth progress in the synthesis of colloidal gold, Discuss. Faraday Soc. 1951, 11, 55–75. Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nat. Phys. Sci. 1973, 241, 20–22. Holgate, C. S., Jackson, P., Cowen, P. N., Bird, C. C. Immunogold-silver staining: New method of immunostaining with enhanced sensitivity, J. Histochem. Cytochem. 1983, 31, 938– 944. Link, S., El-Sayed, M. A. Shape and size dependence of radiative, nonradiative and photothermal properties of gold nanocrystals, Int. Rev. Phys. Chem. 2000, 19, 409–453. Niemeyer, C. M. Nanoparticles, proteins, and nucleic acids: Biotechnology meets materials science, Angew. Chem. Int. Ed. 2001, 40, 4128–4158. Mirkin, C. A., Letsinger, R. L., Mucic, R. C., Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials, Nature 1996, 382, 607–609. Lin, C. C., Yeh, Y. C., Yang, C. Y., Chen, G. F., Chen, Y. C., Wu, Y. C., Chen, C. C. Quantitative analysis of multivalent interactions of carbohydrate-encapsulated gold nanoparticles with concanavalin A, Chem. Commun. 2003, 2920–2921. de La Fuente, J. M., Barrientos, A. G., Rojas, T. C., Rojo, J., Canada, J., Fernandez, A., Penades, S. Gold glyconanoparticles as water-soluble polyvalent models to study carbohydrate interactions, Angew. Chem. Int. Ed. 2001, 40, 2257–2261. Teng, C. H., Ho, K. C., Lin, Y. S., Chen, Y. C. Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis, Anal. Chem. 2004, 76, 4337–4342. Negishi, Y., Nobusada, K., Tsukuda, T. Glutathione-protected gold clusters

371

372

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling

40

41

42

43

44

45

46

47

48

49

revisited: Bridging the gap between gold(I)-thiolate complexes and thiolateprotected gold nanocrystals, J. Am. Chem. Soc. 2005, 127, 5261–5270. Chen, Y. J., Chen, S. H., Chien, Y. Y., Chang, Y. W., Liao, H. K., Chang, C. Y., Jan, M. D., Wang, K. T., Lin, C. C. Carbohydrateencapsulated gold nanoparticles for rapid target-protein identification and binding-epitope mapping, ChemBioChem 2005, 6, 1169–1173. Hafeli, U., in Magnetism in Medicine: A Handbook, Andra, W., Nowak, H. (Eds.), Wiley-VCH Verlag GmbH, Berlin, 1998, p. 15. Weissleder, R., Elizondo, G., Wittenberg, J., Rabito, C. A., Bengele, H. H., Josephson, L. Ultrasmall superparamagnetic iron oxide: Characterization of a new class of contrast agents for MR imaging, Radiology 1990, 175, 489–493. Gupta, A. K., Gupta, M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials 2005, 26, 3995–4021. Goya, G. F., Berquo, T. S., Fonseca, F. C., Morales, M. P. Static and dynamic magnetic properties of spherical magnetite nanoparticles, J. Appl. Phys. 2003, 94, 3520–3528. Bean, C. P., Livingstone, J. D. Superparamagnetism, J. Appl. Phys. 1959, 30, 120S–129S. Lefebure, S., Dubois, E., Cabuil, V., Neveu, S., Massart, R. Monodisperse magnetic nanoparticles: Preparation and dispersion in water and oils, J. Mater. Res. 1998, 13, 2975–2981. Olsvik, O., Popovic, T., Skjerve, E., Cudjoe, K. S., Hornes, E., Ugelstad, J., Uhlen, M. Magnetic separation techniques in diagnostic microbiology, Clin. Microbiol. Rev. 1994, 7, 43–54. Zhang, Y., Kohler, N., Zhang, M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake, Biomaterials 2002, 23, 1553–1561. Widder, K. J., Senyei, A. E., Ranney, D. F. In vitro release of biologically active adriamycin by magnetically

50

51

52

53

54

55

56

57

58

responsive albumin microspheres, Cancer Res. 1980, 40, 3512–3517. Reimer, P., Weissleder, R. Development and experimental use of receptor-specific MR contrast media, Radiologe 1996, 36, 153–163. Pankhurst, Q. A., Connolly, J., Jones, S. K., Dobson, J. Applications of magnetic nanoparticles in biomedicine, J. Phys. D: Appl. Phys. 2003, 36, R167–R181. Luderer, A. A., Borrelli, N. F., Panzarino, J. N., Mansfield, G. R., Hess, D. M., Brown, J. L., Barnett, E. H., Hahn, E. W. Glass-ceramicmediated, magnetic-field-induced localized hyperthermia: Response of a murine mammary carcinoma, Radiat. Res. 1983, 94, 190–198. Schwertmann, U., Cornell, R. M. Iron Oxides in the Laboratory: Preparation and Characterization, Wiley-VCH, New York, 1991. Hyeon, T., Lee, S. S., Park, J., Chung, Y., Na, H. B. Synthesis of highly crystalline and monodisperse maghemite nanocrystallites without a size-selection process, J. Am. Chem. Soc. 2001, 123, 12 798–12 801. Sun, S., Murray, C. B., Weller, D., Folks, L., Moser, A. Monodisperse FePt nanoparticles and ferromagnetic FePt nanocrystal superlattices, Science 2000, 287, 1989–1992. Parak, W. J., Gerion, D., Pellegrino, T., Zanchet, D., Micheel, C., Williams, S. C., Boudreau, R., Gros, M. A. L., Larabell, C. A., Alivisatos, A. P. Biological applications of colloidal nanocrystals, Nanotechnology 2003, 14, R15–R27. Murray, C. B., Norris, D. J., Bawendi, M. G. Synthesis and characterization of nearly monodisperse CdE (E ¼ S; Se; TeÞ semiconductor nanocrystallites, J. Am. Chem. Soc. 1993, 115, 8706–8715. Olshavsky, M. A., Goldstein, A. N., Alivisatos, A. P. Organometallic synthesis of GaAs crystallites exhibiting quantum confinement, J. Am. Chem. Soc. 1990, 112, 9438–9439.

References 59 Seydack, M. Nanoparticle labels in

60

61

62

63

64

65

66

67

68

immunosensing using optical detection methods, Biosens. Bioelectron. 2005, 20, 2454–2469. Dubertret, B. Quantum dots: DNA detectives, Nat. Mater. 2005, 4, 797–798. Agrawal, A., Zhang, C., Byassee, T., Tripp, R. A., Nie, S. Counting single native biomolecules and intact viruses with color-coded nanoparticles, Anal. Chem. 2006, 78, 1061–1070. Bruchez, M., Jr., Moronne, M., Gin, P., Weiss, S., Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013–2016. Ito, A., Shinkai, M., Honda, H., Kobayashi, T. Medical application of functionalized magnetic nanoparticles, J. Biosci. Bioeng. 2005, 100, 1–11. Mornet, S., Vasseur, S., Grasset, F., Duguet, E. Magnetic nanoparticle design for medical diagnosis and therapy, J. Mater. Chem. 2004, 14, 2161–2175. Artemov, D., Mori, N., Okollie, B., Bhujwalla, Z. M. MR molecular imaging of the Her-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles, Mag. Reson. Med. 2003, 49, 403–408. Huh, Y. M., Jun, Y. W., Song, H. T., Kim, S., Choi, J. S., Lee, J. H., Yoon, S., Kim, K. S., Shin, J. S., Suh, J. S., Cheon, J. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals, J. Am. Chem. Soc. 2005, 127, 12 387– 12 391. Jun, Y. W., Huh, Y. M., Choi, J. S., Lee, J. H., Song, H. T., Kim, S., Yoon, S., Kim, K. S., Shin, J. S., Suh, J. S., Cheon, J. Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging, J. Am. Chem. Soc. 2005, 127, 5732–5733. Cui, Y., Wang, Y., Hui, W., Zhang, Z., Xin, X., Chen, C. The synthesis of GoldMag nano-particles and their application for antibody immobilization, Biomed. Microdevices 2005, 7, 153–156.

69 Sokolov, K., Follen, M., Aaron, J.,

70

71

72

73

74

75

76

77

78

79

Pavlova, I., Malpica, A., Lotan, R., Richards-Kortum, R. Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles, Cancer Res. 2003, 63, 1999–2004. Nam, J. M., Thaxton, C. S., Mirkin, C. A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins, Science 2003, 301, 1884–1886. Oh, B.-K., Nam, J.-M., Lee, S. W., Mirkin, C. A. A Fluorophorebased bio-barcode amplification assay for proteins, Small 2006, 2, 103–108. Mann, M., Hendrickson, R. C., Pandey, A. Analysis of proteins and proteomes by mass spectrometry, Annu. Rev. Biochem. 2001, 70, 437–473. Aebersold, R., Mann, M. Mass spectrometry-based proteomics, Nature 2003, 422, 198–207. Henzel, W. J., Watanabe, C., Stults, J. T. Protein identification: The origins of peptide mass fingerprinting, J. Am. Soc. Mass Spectrom. 2003, 14, 931–942. Gevaert, K., Vandekerckhove, J. Protein identification methods in proteomics, Electrophoresis 2000, 21, 1145–1154. Tang, N., Tornatore, P., Weinberger, S. R. Current developments in SELDI affinity technology, Mass Spectrom. Rev. 2004, 23, 34–44. Issaq, H. J., Conrads, T. P., Prieto, D. A., Tirumalai, R., Veenstra, T. D. SELDI-TOF MS for diagnostic proteomics, Anal. Chem. 2003, 75, 148A–155A. Hutchens, T. W., Yip, T. T. New desorption strategies for the massspectrometric analysis of macromolecules, Rapid Commun. Mass Spectrom. 1993, 7, 576–580. Nelson, R. W., Krone, J. R., Bieber, A. L., Williams, P. Mass spectrometric immunoassay, Anal. Chem. 1995, 67, 1153–1158.

373

374

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling 80 Tubbs, K. A., Nedelkov, D., Nelson,

81

82

83

84

85

86

87

88

89

R. W. Detection and quantification of beta-2-microglobulin using mass spectrometric immunoassay, Anal. Biochem. 2001, 289, 26–35. Nedelkov, D., Tubbs, K. A., Niederkofler, E. E., Kiernan, U. A., Nelson, R. W. High-throughput comprehensive analysis of human plasma proteins: A step toward population proteomics, Anal. Chem. 2004, 76, 1733–1737. Kiernan, U. A., Nedelkov, D., Tubbs, K. A., Niederkofler, E. E., Nelson, R. W. Proteomic characterization of novel serum amyloid P component variants from human plasma and urine, Proteomics 2004, 4, 1825–1829. Brockman, A. H., Orlando, R. Probe-immobilized affinity chromatography/mass spectrometry, Anal. Chem. 1995, 67, 4581–4585. Brockman, A. H., Orlando, R. New immobilization chemistry for probe affinity mass spectrometry, Rapid Commun. Mass Spectrom. 1996, 10, 1688–1692. Ching, J., Voivodov, K. I., Hutchens, T. W. Polymers as surface-based tethers with photolytic triggers enabling laser-induced release/desorption of covalently bound molecules, Bioconjugate Chem. 1996, 7, 525–528. Wei, J., Buriak, J. M., Siuzdak, G. Desorption-ionization mass spectrometry on porous silicon, Nature 1999, 399, 243–246. Zou, H., Zhang, Q., Guo, Z., Guo, B., Zhang, Q., Chen, X. A mass spectrometry based direct-binding assay for screening binding partners of proteins, Angew. Chem. Int. Ed. 2002, 41, 646–648. Su, J., Mrksich, M. Using mass spectrometry to characterize selfassembled monolayers presenting peptides, proteins, and carbohydrates, Angew. Chem. Int. Ed. 2002, 41, 4715–4718. Chen, W. Y., Wang, L. S., Chiu, H. T., Chen, Y. C., Lee, C. Y. Carbon nanotubes as affinity probes for

90

91

92

93

94

95

96

97

98

peptides and proteins in MALDI MS analysis, J. Am. Soc. Mass Spectrom. 2004, 15, 1629–1635. Ho, K. C., Tsai, P. J., Lin, Y. S., Chen, Y. C. Using biofunctionalized nanoparticles to probe pathogenic bacteria, Anal. Chem. 2004, 76, 7162–7168. Kong, X. L., Huang, L. C., Hsu, C. M., Chen, W. H., Han, C. C., Chang, H. C. High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis, Anal. Chem. 2005, 77, 259–265. Turney, K., Drake, T. J., Smith, J. E., Tan, W., Harrison, W. W. Functionalized nanoparticles for liquid atmospheric pressure matrix-assisted laser desorption/ionization peptide analysis, Rapid Commun. Mass Spectrom. 2004, 18, 2367–2374. Gu, H. W., Ho, P. L., Tsang, K. W. T., Wang, L., Xu, B. Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration, J. Am. Chem. Soc. 2003, 125, 15 702–15 703. Xu, C. J., Xu, K. M., Gu, H. W., Zhong, X. F., Guo, Z. H., Zheng, R. K., Zhang, X. X., Xu, B. Nitrilotriacetic acid-modified magnetic nanoparticles as a general agent to bind histidine-tagged proteins, J. Am. Chem. Soc. 2004, 126, 3392–3393. Hamley, I. W. Nanotechnology with soft materials, Angew. Chem. Int. Ed. 2003, 42, 1692–1712. Chou, P. H., Chen, S. H., Liao, H. K., Lin, P. C., Her, G. R., Lai, A. C., Chen, J. H., Lin, C. C., Chen, Y. J. Nanoprobe-based affinity mass spectrometry for selected protein profiling in human plasma, Anal. Chem. 2005, 77, 5990–5997. Lin, P. C., Chou, P. H., Chen, S. H., Liao, H. K., Wang, K. Y., Chen, Y. J., Lin, C. C. Ethylene glycol-protected magnetic nanoparticles for a multiplexed immunoassay in human plasma, Small 2006, 2, 485–489. Berton, G., Cordiano, R., Palmieri, R., Pianca, S., Pagliara, V., Palatini, P. C-reactive protein in

References

99

100

101

102

103

104

105

acute myocardial infarction: Association with heart failure, Am. Heart J. 2003, 145, 1094–1101. Kuhn, E., Wu, J., Karl, J., Liao, H., Zolg, W., Guild, B. Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards, Proteomics 2004, 4, 1175–1186. Uhlar, C. M., Whitehead, A. S. Serum amyloid A, the major vertebrate acute-phase reactant, Eur. J. Biochem. 1999, 265, 501–523. Juan, H. F., Chen, J. H., Hsu, W. T., Huang, S. C., Chen, S. T., Yi-Chung Lin, J., Chang, Y. W., Chiang, C. Y., Wen, L. L., Chan, D. C., Liu, Y. C., Chen, Y. J. Identification of tumorassociated plasma biomarkers using proteomic techniques: From mouse to human, Proteomics 2004, 4, 2766– 2775. Pepys, M. B., Herbert, J., Hutchinson, W. L., Tennent, G. A., Lachmann, H. J., Gallimore, J. R., Lovat, L. B., Bartfai, T., Alanine, A., Hertel, C., Hoffmann, T., JakobRoetne, R., Norcross, R. D., Kemp, J. A., Yamamura, K., Suzuki, M., Taylor, G. W., Murray, S., Thompson, D., Purvis, A., Kolstoe, S., Wood, S. P., Hawkins, P. N. Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis, Nature 2002, 417, 254–259. Chen, S.-H. Affinity Mass Spectrometry for Targeted Proteomics. Master’s Thesis, Department of Chemistry, National Taiwan University, Taipei, Taiwan, 2005. Kang, Y. S., Risbud, S., Rabolt, J. F., Stroeve, P. Synthesis and characterization of nanometer-size Fe3 O4 and gamma-Fe2 O3 particles, Chem. Mater. 1996, 8, 2209–2211. Liu, X. Q., Ma, Z. Y., Xing, J. M., Liu, H. Z. Preparation and characterization of amino-silane modified superparamagnetic silica nanospheres, J. Magn. Magn. Mater. 2004, 270, 1–6.

106 Perez, J. M., Simeone, F. J., Saeki,

107

108

109

110

111

112

113

114

Y., Josephson, L., Weissleder, R. Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media, J. Am. Chem. Soc. 2003, 125, 10 192–10 193. Krause, E., Wenschuh, H., Jungblut, P. R. The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins, Anal. Chem. 1999, 71, 4160–4165. Knochenmuss, R., Zenobi, R. MALDI ionization: The role of inplume processes, Chem. Rev. 2003, 103, 441–452. Pepys, M. B., Rademacher, T. W., Amatayakul-Chantler, S., Williams, P., Noble, G. E., Hutchinson, W. L., Hawkins, P. N., Nelson, S. R., Gallimore, J. R., Herbert, J., Hutton, T., Dwek, R. A. Human serum amyloid P component is an invariant constituent of amyloid deposits and has a uniquely homogeneous glycostructure, Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5602–5606. Fu, J. Y., Muller, D. Simple, rapid enzyme-linked immunosorbent assay (ELISA) for the determination of rat osteocalcin, Calcif. Tissue Int. 1999, 64, 229–233. Wang, X., Chen, F., Wan, P. J., Huang, G. Development of monoclonal antibody-based enzymelinked immunosorbent assay for gossypol analysis in cottonseed meals, J. Agric. Food Chem. 2004, 52, 7793– 7797. Pepys, M. B., Hirschfield, G. M. C-reactive protein: A critical update, J. Clin. Invest. 2003, 111, 1805–1812. Pepys, M. B., Dash, A. C., Markham, R. E., Thomas, H. C., Williams, B. D., Petrie, A. Comparative clinical study of protein SAP (amyloid P component) and C-reactive protein in serum, Clin. Exp. Immunol. 1978, 32, 119–124. Tirumalai, R. S., Chan, K. C., Prieto, D. A., Issaq, H. J., Conrads,

375

376

10 Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling T. P., Veenstra, T. D. Characterization of the low molecular weight human serum proteome, Mol. Cell. Proteomics 2003, 2, 1096– 1103. 115 Zheng, M., Huang, X. Nanoparticles comprising a mixed monolayer for specific bindings with biomolecules, J. Am. Chem. Soc. 2004, 126, 12 047–12 054. 116 Jang, J. S., Cho, H. Y., Lee, Y. J., Ha, W. S., Kim, H. W. The differential proteome profile of stomach cancer: Identification of the biomarker candidates, Oncol. Res. 2004, 14, 491–499. 117 Solakidi, S., Dessypris, A., Stathopoulos, G. P., Androulakis, G., Sekeris, C. E. Trypsin inhibitor; Kazal pancreatic; Tumor markers; biological; carcinoembryonic antigen; C-reactive protein; a1-antitrypsin; gastrointestinal neoplasms, Clin. Biochem. 2004, 37, 56–60.

118 Kingsmore, S. F., Patel, D. D.

Multiplexed protein profiling on antibody-based microarrays by rolling circle amplification, Curr. Opin. Biotechnol. 2003, 14, 74–81. 119 Gorelik, E., Landsittel, D. P., Marrangoni, A. M., Modugno, F., Velikokhatnaya, L., Winans, M. T., Bigbee, W. L., Herberman, R. B., Lokshin, A. E. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer, Cancer Epidemiol. Biomarkers Prev. 2005, 14, 981–987. 120 Wilson, M. S. Electrochemical immunosensors for the simultaneous detection of two tumor markers, Anal. Chem. 2005, 77, 1496–1502. 121 Zheng, G., Patolsky, F., Cui, Y., Wang, W. U., Lieber, C. M. Multiplexed electrical detection of cancer markers with nanowire sensor arrays, Nat. Biotechnol. 2005, 23, 1294–1301.

377

11

Nanotechnological Approaches to Cancer Diagnosis: Imaging and Quantification of Pericellular Proteolytic Activity Thomas Ludwig 11.1

Introduction

Most cancer related deaths are not caused by the growth of a tumor in its primary location. Metastasis with subsequent local tissue invasion accounts for more than 90% of all lethal outcomes in cancer (reviewed in Refs. [1, 2]). Despite great efforts to improve our understanding of the metastatic cascade (Figs. 11.1 and 11.2) and the accumulation of detailed knowledge about single aspects of this process, comparably little has resulted in patient benefits so far (reviewed in Ref. [3]). This chapter outlines reasons for this dilemma by analyzing the challenges that result from local, nanoscale processes for in vitro and in vivo diagnostics. It combines, for the first time, a thorough review of the biological basis of proteolytic activity as the prototype of a highly regulated and spatially restricted process in cancer with a critical analysis of conventional state of the art methods and how the application of nanotechnology creates unique opportunities to further both our understanding and treatment of malignancies. The mechanisms that confine and concentrate protease activity in the pericellular microenvironment of cancer cells are prerequisites of tumor cell invasion and key factors in the regulation of tumor microecology. This chapter starts with a detailed description of these mechanisms with a special emphasis on the technical problems related to them and how this is connected to the obvious gaps in our knowledge regarding their regulation. With a reference to the biological background, the next section of the chapter provides the reader with information about the principles and disadvantages of conventional state of the art methods applied in this field. The current imperfections of most methods offer unique opportunities for innovative nanotechnological approaches. As one recent example, atomic-force microscopy is presented as a technique for the nanoscale imaging and quantification of proteolytic activity in the pericellular microenvironment of cancer cells in vitro. In general, techniques that enable real-time, high resolution imaging or precise quantification of local proteolytic activity in vitro and in vivo remain major chal-

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

378

11 Nanotechnological Approaches to Cancer Diagnosis

Metastasis: The formation of metastasis is a complex and highly dynamic process. It is characterized by the spreading of tumor cells through vessels (2 and 4), lymphatic ducts (3) or cavities (1) to form

Fig. 11.1.

new colonies away from their origin. It is undoubtedly a topic of high clinical relevance as it accounts for over 90% of all lethal outcomes in cancer.

lenges. These methods will play an important role in cancer diagnosis, the understanding of basic principles in metastasis, the identification of new therapeutical and targets and, hence drug design in the near future. The immediate need for these methods has been demonstrated by the results from clinical protease inhibitor trials. Although extensive preclinical data supported the inhibition of protease activity as a promising strategy for cancer therapy, clinical trials with various protease inhibitors, i.e., matrix metalloproteinaseinhibitors (MMPIs), did not bring the expected breakthrough in cancer treatment [4, 5] (reviewed in Refs. [6–9]). It might be too early to abandon the concept of protease inhibition in cancer therapy in general, as the design of these clinical trials must be considered. MMPIs had to compete, for example, against cytotoxic drugs in late-stage cancer patients. More recent insights into protease function in cancer development and angiogenesis indicate that protease inhibition is unlikely to provide considerable patient benefits in this setting, since proteases are crucial for

11.1 Introduction

Fig. 11.2. Cancer cell invasion. Metastasis depends essentially on the ability of cancer cells to invade intact tissue structures (2), and to overcome physiological barriers such as basement membranes upon intra- (3) and extravasation (4). In skin cancer, as depicted here, the breach of the epidermal basement membrane (1) by cancer cells is a defining step with far reaching consequences in terms

of treatment and prognosis of the patient. The upper layer of the skin is free of lymph and blood vessels. No metastasis can be found until the basement membrane has been crossed by the cancer cells. Patients can therefore be healed by total excision of the tumor and no further treatment such as chemotherapy is necessary at this stage.

establishing new metastasis and may not be necessary to maintain them. Animal studies support this position as MMPI treatment was most effective in early stages of cancer [10]. It seems therefore reasonable to initiate new trials that employ protease inhibition early in cancer treatment or in addition to established treatments like surgery and radiation. Nonetheless, the principle drawback in the clinical application and evaluation of protease inhibitors is the significant gap in our understanding of the complex regulation mechanisms of local proteolytic activity due to methodological obstacles. In addition, there is a serious lack of reliable techniques and parameters to assess protease inhibitor effectiveness in vivo. None of the patient trials proved so far that the administered drug dose was sufficient to target local proteolytic activity in situ. Surrogate markers, such as serum gelatinase activity, have proven unreliable for tumor screening purposes and were, therefore, inappropriate for monitoring substance response. These results from clinical trials illustrate the necessity to develop

379

380

11 Nanotechnological Approaches to Cancer Diagnosis

functional methods to test drug efficacy by the quantification of local proteolytic activity.

11.2

Quantification of Local Proteolytic Activity – an Objective

Cancer metastasis is a complex, highly dynamic multistep process that includes:







Cell detachment from the primary tumor mass, migration within and intrusion into surrounding tissue structures and ECM, degradation of physiological barriers such as endothelial basement membranes, penetration into the vascular lumen (intravasation), distribution by the blood stream and survival in the circulation, adhesion to distant endothelia and extravasation.

and, finally, establishment of a colony in a new environment distant from the cancer cells origin (Figs. 11.1 and 11.2) [11] (reviewed in Refs. [12–14]). The expression of proteases and the development of proteolytic activity is a prerequisite for tumor cells to achieve these aims. Regardless of their diverse etiology, the function of proteases in tumor cell spreading constitutes a final common pathway of all invasive malignancies. In agreement, high concentrations and activities of proteases have been found more often in and around cancers than in normal, benign or premalignant tissues. Most proteases are produced as inactive proenzymes that undergo proteolytic cleavage for their activation. Once activated, their activity is modified by several cellular mechanisms and biochemical factors such as protein trafficking and endogenous inhibitors. The excessive post-translational modification of protease activity is responsible for the critical difference between the concentration of proteases and their activity. This limits the application of many conventional methods, which cannot reliably distinguish between the active or inactive protease. Proteolytic activity in cancer cell invasion is a very local phenomenon. Although a protease can be widely expressed throughout a tumor, it may, for example, only be active at the leading edge in a small subset of cells at the tumor–stroma interface. Specific mechanisms localize and concentrate protease activity in the pericellular microenvironment of cells. This spatial restriction challenges the maximum achievable resolution of common in situ imaging techniques and in vitro diagnostics. Until now, no method has proven sensitive enough to employ proteolytic activity in cancer for serum diagnostics and screening purposes. General considerations can be applied to evaluate the quality of diagnostic tools (reviewed in Ref. [15]). For example, numerous methods and assays have been described for the investigation of enzymatic activity in vitro as well as in vivo. Despite their heterogeneity in terms of strategy and aims, they can be grouped considering aspects of their primarily targeted parameters. These are qualitative or quantitative evaluation of the local, or total, concentration or activity of one or several enzymes

11.2 Quantification of Local Proteolytic Activity – an Objective

Fig. 11.3. Evaluation parameters of assay methods. Several criteria can be applied to assess diagnostic tools such as protease assays. Most proteases are secreted as inactive proenzymes, and enzymatic activity is additionally regulated by endogenous inhibitors. Proteolytic activity is usually restricted to the pericellular microenvironment of cancer and stromal cells, and may only be present in a small fraction of a sample. Assays detecting protease concentrations (top lefthand side box, at the back) give, therefore, only indirect clues to functionally relevant local proteolytic activity (bottom right-hand side box, at the front). Usually, several complementary methods have to be employed

in combination to understand results in the context of proteolytic orchestration. As an example, western blot analysis of cell lysates can determine the overall concentration of a single protease at a time, but is usually not capable of determining the active fraction of this enzyme. The presence of a broad variety of proteases and inhibitors, in the same spot or separate compartments, gives rise to a complex interplay that cannot be assessed by this method. In situ zymography is able to localize proteolytic activity, but it must be considered that the activity observed with a specific substrate may not be of particular functional relevance for the investigated microenvironment.

(Fig. 11.3). The usefulness of an assay depends as much on its sensitivity and specificity as it does on its compatibility for a particular question and application. In most cases, a combination of complementary techniques is required to get an oversight of the functional relevance of a result. As an example, the most valuable functional information is usually derived from the quantitative evaluation of local enzymatic activity (lower, right-hand box in Fig. 11.3); it is also important to get additional information about the spatial distribution and overall concentration, i.e., of a protease in a specimen (upper, left-hand box in Fig. 11.3). The functional context is derived from the knowledge of the quantitative relationship between concentration and activity and the total to local ratio. Hence, the aim must be to develop dynamic high-resolution imaging techniques or methods that enable precise quantification of local activities in vivo. Investigation of the function of proteases and their utilization for diagnostic purposes in cancer faces two major obstacles – post-translational regulation and spatial restriction.

381

382

11 Nanotechnological Approaches to Cancer Diagnosis

11.2.1

Regulation of Protease Activity

Protease activity is tightly regulated at the:


transcriptional, translational and
post-translational level.


The transcription of MMPs (matrix metalloproteinases) can, for example, be up- or down-regulated by various stimuli such as extracellular matrix proteins, phorbol esters and cell stress [16–19] (reviewed in Refs. [20, 21]). It has been demonstrated for MMP-9 that expression can be increased post-transcriptionally through an enhancement of translational efficiency [22]. Most methodological problems are related to the post-translational regulation of proteases, due to the crucial difference between concentration and activity (Fig. 11.3). The activity of proteases is post-translationally regulated by diverse, principle mechanisms:








Secretion activation inactivation endogenous protease inhibitors glycosylation oligomerization and protein trafficking.

Secretion Secretion of most proteases seems to be constitutive, which means that they are secreted once they were synthesized. Strong indicators suggest that, at least in inflammatory cells, MMPs can be stored in vesicles and secreted upon stimulation [23]. Although described for cancer cells, aspects of the regulation of this rapid response to environmental stimuli remain vague [24]. 11.2.1.1

Activation As most proteases are secreted as latent enzymes, their activation becomes a critical control point. Extracellular activation of proteases can be initiated by other already active proteolytic enzymes. This is usually obtained by the removal of a peptide that helps to maintain latency. For instance, MMPs are activated by a mechanism generally known as the ‘‘cysteine switch’’ [25, 26]. This model suggests that a cysteine coordinates the active-site zinc atom in the latent enzyme. All identified modes of MMP activation lead to a dissociation of this cysteine residue, which is accompanied by exposure of the active site. Few proteases – among them MMP-11, MMP-13, and MT-MMPs (membrane type-MMPs) – are activated intracellularly, which makes these proteases important 11.2.1.2

11.2 Quantification of Local Proteolytic Activity – an Objective

pacemakers of proteolytic cascades by subsequent activation of other proteases [27–30]. The molecular basis of this intracellular activation is the presence of a specific motif between the propeptide and catalytic domain of these proteases that is recognized by proprotein convertases that process secretory precursor proteins in the trans-Golgi network. Inactivation Proteases can be inactivated by proteolysis, protease inhibitors and removal from the extracellular space or plasma membrane. Although similar in principle, the proteolytic processing of proteases leading to their inactivation is far less understood than the proteolytic processing leading to their activation. In addition to their irreversible inactivation, limited proteolysis of MMPs can also modify their distribution, substrate specifity and ability to be inhibited. Cleavage of a sequence close to the transmembrane domain of MT-MMPs can generate, for example, soluble subspecies of these proteases [31–34]. Although these shed proteases are catalytically active and may now act as soluble proteases, they have lost most of their invasion promoting activity and effects on cell function [35, 36]. Obviously, protease action must occur in the immediate cellular surrounding to contribute significantly to the regulation of the pericellular microenvironment. MT-1MMP cannot only undergo autocatalytic shedding. Overexpression can lead, furthermore, to an autocatalytic inactivation that results in a truncated membrane-bound remnant that has lost its catalytic domain [31, 37, 38]. Autocatalytic inactivation could serve as a self-limiting mechanism to down-regulate excessive proteolytic activity. The proteolytic processing of many proteases varies, depending on the prevalence of the activity of other proteases. Summarizing, the presence and interaction of many different representatives of proteases from distinct families, that have 11.2.1.3

1. different substrate affinities, concentrations and actual states of activity, that are 2. in addition capable of rapidly activating and inactivating themselves and each other and 3. coexist in a tiny space of only several nm 3 with 4. a no less complex and dynamic mixture of substrates (Fig. 11.7 below) leads to a complex and fragile balanced composite that determines the functional relevant net proteolytic activity for this micro-compartment. Endogenous Protease Inhibitors Protease activity is modified on the post-translational level by various endogenous inhibitors. For example, the activity of MMPs is tightly regulated by tissue inhibitors of metalloproteases (TIMPs). To date, four structurally related TIMPs have been identified. The upregulation of TIMPs has been shown to attenuate the invasive behavior of several tumors, whereas reduction in TIMP-levels by means of antisense RNA can confer tumorigenicity to non-invasive cells [39]. 11.2.1.4

383

384

11 Nanotechnological Approaches to Cancer Diagnosis

TIMPs are clearly multifunctional proteins [40, 41]. TIMP-1 was isolated from fibroblasts and initially characterized to inhibit collagenase activity [42]. Soon after its discovery, TIMP-1 turned out to be identical to a known growth factor that was described to have erythroid-potentiating activity in previous studies [43]. Besides their MMP-inhibiting properties and erythroid-stimulating activity, other growth promoting activities of TIMPs have been reported [44]. Paradoxically, the presence of endogenous protease inhibitors can be of significant importance for the activation of some proteases. TIMP-2 was shown to be required for efficient proMMP-2 activation by MT1-MMP both in vivo and in vitro [37, 45, 46]. Besides being inhibited by TIMPs, MMP activity is regulated by the recently identified membrane-bound MMP-inhibitor RECK (reversion-inducing cysteinerich protein with Kazal motifs) and other abundant inhibitors such as a2 macroglobulin [47]. Glycosylation Glycosylation has recently been discovered as an alternative way of protease activity regulation. Data provide evidence that the glycosylation of proteases may regulate their substrate targeting. In MT-1MMP, O-glycosylation seems to determine whether the protease can degrade ECM and activate MMP-2 or can only cleave ECM components [48]. This might have drastic consequences on local proteolytic activity, as MT-1MMP is considered to be one of the key activators of proteolytic cascades. Technically, this aspect has far reaching consequences. It limits the use of simple indicator probes of proteolytic activity, as subtle but functionally crucial differences in specifity of a protease can easily be overseen. Glycosylation is not only relevant for the ‘‘fine tuning’’ of proteases. Moreover, it is important for the suppression of tumor cell invasion that is mediated by the membrane-bound inhibitor RECK [49]. N-Glycosylation of RECK acts through multiple mechanisms such as suppressing MMP-9 secretion and inhibiting MMP2 activation, which appear to be regulated independently by the glycosylation at different sites [49]. 11.2.1.5

Oligomerization Multimeric complexes of membrane-bound proteases facilitate their autocatalytic processing and the activation of other proteases. The homo-oligomerization of MT1-MMP is stimulated by at least three of its domains and takes part in the regulation of its activity and turnover [50–52]. The formation of homo-oligomers of MT1-MMP turns out to be of particular relevance for the activation of proMMP-2 [50–52]. It has been hypothesized that other regulatory proteins may be involved in this by facilitating protease clustering [53]. These findings reveal that the oligomerization of proteases further determines the ‘‘functional activity’’ that a protease can unfold in a given context and demonstrate that the nanoscale surrounding of a single protease can determine its actual function. 11.2.1.6

11.2 Quantification of Local Proteolytic Activity – an Objective

Protein Trafficking The internalization of membrane-bound proteases provides cells with an effective way to regulate the proteolytic activity on their surface. Most studies regarding this mechanism have been carried out for MT1-MMP. Endocytosis of this prominent protease can be mediated by at least two distinct pathways – clathrin-coated pits and caveolae [54–57]. Caveolae are not only involved in the internalization of MT1MMP in cancer cells, but are an issue in the regulation of MT1-MMP in endothelial cells as well [55]. The rapid internalization of MT1-MMP via clathrin-coated pits is dynamin mediated and depends on the cytoplasmic tail of the protease [51, 54, 58–60]. Once internalized, MT1-MMP can be recycled to the surface [54, 61]. The rapid trafficking of MT1-MMP has the potential to regulate MMP-2 activation and thus represents an extra path of proteolytic cascade initiation [24]. Interestingly, internalization and dynamic turnover of proteases are necessary to maintain their proper function [62]. Besides internalization, trafficking deep within the cancer cells can participate in the regulation of events on the cell surface. For instance, internalized MT1-MMP can be transported to CD63-positive lysosomes for its degradation [63]. The tetraspanin CD63, a well investigated resident of late endosomal and lysosomal membranes, promotes thereby the internalization, lysosomal targeting and proteolysis of the protease [63]. Despite proliferating knowledge about the trafficking of proteases, it is still illdefined as to how it affects local proteolytic activity quantitatively. It is, for example, still not known whether specific sub-fractions of proteases, which means active, inactivated or inhibitor complexed proteases, are internalized and how this might be regulated. Conceivably, the internalization route might differ for these subtypes of a protease and could ultimately determine whether a protease is degraded or recycled. One reason for this gap in our current understanding has been the already mentioned, namely, inherent problems in the nanoscale quantification of complex local processes – here proteolytic activity. 11.2.1.7

11.2.2

Mechanisms of Confining Proteolytic Activity

An important concept of tumor cell invasion is that cells do not indiscriminately release proteases (Fig. 11.4). Several specific mechanisms that confine and concentrate protease activity in the pericellular microenvironment and cellular subdomains of tumor cells have been identified (reviewed in Refs. [15, 20, 51, 64– 69]). These mechanisms are prerequisites of tumor cell invasion and key factors in the regulation of tumor microecology and consist of


Expression of membrane anchored proteases; the binding of soluble proteases by membrane-bound receptors;
local activation of soluble proteases by membrane-associated activators;


385

386

11 Nanotechnological Approaches to Cancer Diagnosis

Detection of local proteolytic activity by fluorescence-labeled gelatin. In cancer cells, proteolytic activity is a locally restricted phenomenon. It can be detected and visualized by surface-bound indicator substrates. (a) Tumor cells (1) were seeded on coverslips (4) coated with fluorescence-labeled gelatin (3). Proteolytic matrix cleavage (2) causes focal

Fig. 11.4.

loss of fluorescence. (b) Glioblastoma and astrocytoma cells from primary cultures were fixed 48 h after seeding them on fluorescencelabeled gelatin (green). Dark spots in the gelatin coating reflect areas of proteolytic activity. Cells were labeled by indirect immunofluorescence for MMP-2 using Cy3conjugated secondary antibodies (red).




the presence of cell surface receptors for protease activating enzymes; the regulated localization of receptors and proteases to distinct subcellular domains (i.e., invadopodia);
specific binding of latent proteases by ECM components;
induction of proteases expression at the tumor–stroma interface.


Membrane-type Matrix Metalloproteinases The most obvious mediators of cell surface bound proteolytic activity are undoubtedly the transmembrane- and GPI-anchored membrane type (MT) matrix metalloproteinases (MMPs). The first MT-MMP (MT1-MMP) was discovered by Hiroshi Sato and colleagues in 1994, and was thought to act primarily as a membranebound activator of the gelatinase MMP-2 [70]. Besides its crucial role in activation of soluble proteases, MT1-MMP itself is now credited for its important contribu11.2.2.1

11.2 Quantification of Local Proteolytic Activity – an Objective

tion to ECM macromolecule cleavage and its broad-spectrum proteolytic capacities [71]. To date, six MT-MMPs have been identified that are linked to the plasma membrane by a transmembrane domain or a glycosylphosphatidyl inositol (GPI) anchor. The importance of localized proteolytic activity is demonstrated by the observation that expression of either wild-type MT1- or MT2-MMP confers invasiveness to COS-1 cells, whereas the expression of truncated, soluble forms of these transmembrane domain proteases has no invasion promoting effect [36]. Expression of MT1-MMP in cancer cells amplifies their metastatic potential, and constitutively expressing cell lines are generally more invasive than non-expressing cell lines [51, 72, 73]. Besides the pivotal role of MT1-MMP in tumor cell invasion, expression of this protease appears to be of critical importance for a diversity of physiological functions. It is, for example, crucial for the fibrin-invasive activity of endothelial cells during angiogenesis and wound healing [74, 75]. MT1-MMP deficient mice present the most severe phenotype observed in MMP deficient mice so far. Although these mice are born with no grossly abnormal features, the loss of MT1-MMP interferes with normal postnatal development. MT1-MMP deficient mice develop various bone and joint disorders, like dwarfism, osteopenia and arthritis, which suggest a severe impairment of the remodeling of collagen-rich tissues [69, 76]. Cell Surface Receptors for Protease Binding Non-MT-MMPs are bound to the membrane or localized to invadopodia by specific interactions with integrins or other cell surface receptors. For example, MMP-2 is bound by the a v b 3 integrin [77], and CD44 mediates docking of MMP-9 to the plasma membrane [78]. Impressively, CD147 (extracellular matrix metalloproteinase inducer, EMMPRIN) is capable of both induction of MMP expression in stromal cells and subsequent binding of these proteases [79, 80]. Besides MMPs, accumulating evidence indicates the responsibility of serine proteases for the metastatic phenotype of many tumors. The urokinase-type plasminogen activator (uPA) drives the plasmin cascade system, and its expression is elevated in glioblastomas and correlates with malignant progression of astrocytomas [81]. The uPA receptor (uPAR) can tie uPA to the surface of tumor cells. Results indicate that only cell-bound, not soluble, uPA can effectively generate plasmin for the activation of gelatinases (MMP-2 and MMP-9) [82]. 11.2.2.2

ECM Binding of Proteases Physiologically, binding of MMPs to ECM components can prevent loss of the secreted enzyme via diffusion, provide a reservoir of latent enzyme at the target site for rapid activation and might facilitate cellular regulation of local proteolytic activity. Several specific interactions of latent MMPs and ECM proteins on cells or in the extracellular space have been described. For example, heparan sulfate proteoglycans, common components of the ECM, can bind proMMP-7 [83], and 11.2.2.3

387

388

11 Nanotechnological Approaches to Cancer Diagnosis

proMMP-9 can form a complex with a1 and a2 chains of collagen IV on cells [84, 85]. Cellular Microdomains During cancer cell invasion, membrane-bound proteases are not randomly distributed on the cell’s surface. Cancer cells can accumulate proteases in specialized regions such as the leading edge or invadopodia – a term derived from ‘‘invasive pseudopodia’’. These subcellular domains have proven to be ‘‘hotspots’’ of ECM degradation during cancer cell invasion. MT1-MMP is concentrated at the lamellipodium through interaction with CD44, which links the protease, furthermore, directly to F-actin [86]. CD44 also recruits MMP-9 to this subcellular localization [87]. At invadopodia, particularly MMP-2, MMP-9 and MT1-MMP can be found. The clustering of active MMP-2 at invadopodia is directly mediated by the a v b 3 integrin [88]. The cytoplasmic tail of MT1-MMP has been identified to regulate its docking to invadopodia and has been suggested to be a prerequisite for the subsequent focal ECM degradation by this protease [89]. Lipid rafts have been discussed in connection with the organization of membrane proteases in cellular microdomains. Interestingly, the aberrant and persistent inclusion into caveolin-enriched lipid rafts limits the tumorigenic function of MT1-MMP in malignant cells [90]. Although cell membranes are certainly not homogeneous mixtures of lipids and proteins, almost all aspects of lipid rafts – their size, composition and turnover – remain controversial (reviewed in Ref. [91]). Despite this, there is a broad consent about their overall biological relevance (reviewed in Ref. [92]). Most aspects of the dispute around the concept of these cellular microdomains are clearly size and resolution related methodical difficulties. Nanotechniques like AFM (atomic-force microscopy) have proven useful for the investigation of lipid rafts in artificial lipid bilayers in vitro, but direct investigation in cells is far more difficult [93]. One important function of microdomains in cancer cell invasion could be the partitioning of proteases into distinct cellular localizations, which then serve as platforms for the regulation of local proteolytic activity via protein oligomerization and trafficking. The restriction of proteolytic activity to small subcellular regions can increase the effectiveness and economy of local ECM degradation, but further complicates the detection of these activities. 11.2.2.4

The Tumor–Host Conspiracy The local host tissue is a constant and active participant during tumor development and metastasis. The extracellular matrix and stromal cells form a complex microenvironment that can limit or even unwillingly facilitate tumor cell invasion (reviewed in Ref. [94]). A striking example of tumor–stroma cooperation is the contribution of matrix-degrading enzymes by the host itself. For example, MMP-11 (stromelysin-3) was discovered in stromal cells of breast carcinomas [95], before its production by tumor cells was described. Most interesting findings originate from studies in MMP knockout mice (Section 11.2.3.3). 11.2.2.5

11.2 Quantification of Local Proteolytic Activity – an Objective

Cancer cells can harbor the ability to induce the production of proteases in stromal cells by factors such as EMMPRIN [79, 80, 96]. In agreement with this, expression levels of matrix degrading enzymes are usually strongly elevated at the invasive front of the tumor–stroma interface [97]. 11.2.3

Local Proteolytic Activity Regulates Complex Cellular Functions

Proteolytic cleavage of extracellular matrix (ECM) is a potent regulator of many physiological and pathological events [20, 65, 98–100]. It affects fundamental processes such as cell growth, differentiation, apoptosis, and migration. The mechanisms that localize and concentrate protease activity in the pericellular microenvironment of cells are prerequisites for processes like angiogenesis, bone development and inflammation. The role of local proteolytic activity in tumor cell invasion and all these other processes is not restricted to the function of breaking down the ECM by means of a path clearing process [101]. Local Proteolytic Activity in Cancer Cell Migration To overcome barriers, cells must be able to move actively to their new location. Several direct and indirect effects of proteolytic activity on tumor cell migration can be observed. The ability of cells to migrate on Laminin-5 is positively correlated with the expression of plasma membrane bound MT1-MMP [102]. The same MMP confers the ability to migrate on myelin substrates to transfected 3T3 fibroblasts [103]. Other results indicate that MT1-MMP plays an important role in glioma cell spreading and migration on white matter [103]. For cancer cell migration in 2D systems, there is virtually no need to cleave the ECM as a barrier. Despite this, a synthetic MMP-inhibitor, tissue inhibitor of metalloproteinases-1 and -2, and the COOH-terminal hemopexin-like domain of MMP-2 inhibit the migration of adenocarcinoma cells on gelatin matrix-coated coverslips [104]. The specific extinction of proteins by small interfering RNAs (siRNAs) has been utilized to target the expression of MMP-9 in a Ewing’s sarcoma cell line, constitutively expressing this protein [105]. Silencing of MMP-9 resulted in a migratoryadhesive switch, marked by decreased spreading on extracellular matrix coatings and inhibition of migration towards fibronectin [105]. As migratory behavior was unchanged by gelatinase inhibitors, the authors conclude that the migrationpromoting activity of proMMP-9 may be independent of its proteolytic activity [105]. 11.2.3.1

Local Proteolytic Activity and Cell Signaling It has been suggested that all cancers acquire the same capabilities during their development [1]. These include 11.2.3.2




limitless replicative potential, sustained angiogenesis,
tissue invasion and metastasis,


389

390

11 Nanotechnological Approaches to Cancer Diagnosis


insensitivity to growth inhibiting signals, self-sufficiency in growth signals, and
the ability to evade apoptosis-inducing signals.


All of these aspects seem to be affected by local proteolytic activity, if not entirely dependent upon it, as local proteolysis during tumor cell invasion is not restricted to cleavage of ECM components [20, 65]. Several other molecules have been identified as important targets of tumor cell associated proteases. Among these are chemokines and their receptors, adhesion molecules, clotting factors and proteinase inhibitors. For example, the association of MMP-7 with CD44v3 regulates heparin-binding epidermal growth factor (HB-EGF) precursor processing [106]. Subsequent HB-EGF binding to its receptor (ErbB4) might regulate other cellular events, such as cell survival [106]. Fas ligand cleavage by the same protease [107] also has a significant effect on tumor cell survival and is implied in protecting tumor cells from chemotherapeutic drug cytotoxicity [108]. Furthermore, most of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases are membrane bound and involved in cleavage of diverse surface associated molecules, such as TNF-a [109–111]. Functional Insights from Matrix-metalloprotease Deficient Mice Mice, deficient for single MMPs, helped to shed light on functions of these proteases in physiology and disease. The putative central physiological role of several MMPs in vivo, which was deduced from their involvement in pathological processes and their abundance during development, proved to be wrong. Many knockout mice, which are the MMP-2 [112], MMP-3 [113], MMP-7 [114], MMP-9 [115], MMP-11 [116], MMP-12 [117] and MMP-20 [118] deficient mice, display no dramatic phenotype (reviewed in Ref. [69]). It must be considered that MMPs have broadly overlapping substrate specifities. Hence, the lack of dramatic phenotypes in these mice probably demonstrates the redundance of vital mechanisms, rather than their uniqueness. Obviously, only a few MMPs are indispensable. Given the importance of MTMMPs for local proteolytic activity and their potential role as triggers of proteolytic cascades, unsurprisingly, MT1-MMP deficient mice bring along the most severe phenotype observed in MMP deficient mice so far. Although these mice are born with no grossly abnormal features, the loss of MT1-MMP is obviously incompatible with normal postnatal development. These mice develop various bone and joint disorders, like dwarfism, osteopenia and arthritis [69, 76]. Despite the absence of lethal phenotypes, mouse models uncovered specific physiological functions of single MMPs. For example, MMP-20 (enamelysin) null mice develop a profound tooth phenotype [118]. MMP-20 is a tooth-specific protease that is only expressed during the early through middle stages of enamel development [118]. Its substrates are enamel specific matrix proteins that are also expressed during this developmental time period [118]. Although MMP deficient mice might not be considered a major breakthrough for proving the overall importance of all MMPs in development, they made a major 11.2.3.3

11.3 Evaluation of Classical Methods for Quantification of Net Proteolytic Activity

contribution to the understanding of host-derived MMPs for tumor cell invasion and metastasis. MMP-2 and MMP-9 deficient mice develop normally, but in these mice the number of metastatic colonies formed by intravenously injected B16-BL6 melanoma or Lewis lung carcinoma cells is dramatically decreased [112, 119, 120]. MMP-9 knockout mice show a decreased incidence of invasive tumors, but bone marrow transplantation reconstitutes many properties of carcinogenesis in these mice [121]. Mice heterozygous for the ApcMin allele (Min/þ) are susceptible to benign intestinal tumors [114]. In this mouse model, MMP-7 ablation by gene targeting and homologous recombination resulted in a reduction of tumor frequency and diameter [114]. Similarly, anthracene derivative induced tumorigenesis is reduced in MMP-11 (stromelysin-3) null mice, and fibroblasts from these mice fail to support metastasis formation by breast cancer cells (MCF-7) upon co-implantation in a nude mice model [116]. Macrophages secrete a selection of proteases, but macrophages lacking MMP-12 (macrophage metalloelastase) from the corresponding null mice are unable to penetrate reconstituted basement membranes in vitro and in vivo [117].

11.3

Evaluation of Classical Methods for Quantification of Net Proteolytic Activity

Classic biochemical methods can be employed to investigate the expression of enzymes and their inhibitors on the transcriptional and translational level. Enzyme expression can be quantified in cell and tissue extracts by real-time reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis, whereas in situ hybridization is useful to localize mRNA expression in tissue sections. ELISA, Western blot, immunocyto- and histochemistry are useful to evaluate the concentration or distribution of any given protein, but it is not possible to deduce enzymatic activity from its mere concentration in a tissue section or extract (Fig. 11.3) (reviewed in Ref. [15]). As previously stated, most proteases, such as MMPs, are produced as inactive proenzymes and their activity is additionally regulated by natural inhibitors [21]. Most antibodies for immunohistochemistry cannot distinguish between the activated, inactivated or pro-form of a protease [122]. In general, antibodies recognize structural epitopes, not function. Thus, RT-PCR, in situ hybridization and immunological methods may be suitable to localize or quantify the expression and production of proteases and their inhibitors, but do not indicate the resulting matrix degrading activity. Various chromogenic, fluorogenic and radioactive assays have been developed to detect and quantify protease activity in tissue extracts [123–125]. Additionally, antibodies can recognize neo-epitopes, e.g., on collagens or aggregacan, that are generated by their proteolytic cleavage. Several ELISA assays exploit the formation of these specific neo-epitopes as an indicator for proteolytic activity (‘‘substrate cleav-

391

392

11 Nanotechnological Approaches to Cancer Diagnosis

age ELISAs’’) [126, 127]. The biological function of cryptic fragments and neoepitopes has been reviewed elsewhere [128, 129]. Gel enzymography – also referred as substrate zymography – [130] and reverse enzymography [42] offer a way to analyze the activities of MMPs and TIMPs in complex biological samples. Enzymography can detect both the proenzyme and the active MMP. In short, zymography involves gel SDS electrophoresis of samples under nonreducing conditions. Protease substrates such as gelatin or casein are incorporated in the gel and retained during electrophoresis. After SDS is removed from the gel by washing, MMPs refold, regain their activity and digest the substrate incorporated in the gel. By staining the gel, usually with Coomassie Blue, proteolytic activity is evident as an unstained band at the corresponding weight of the protease. One advantage of gel enzymography is the activation of proenzymes after electrophoresis, which enables convenient quantification of the ratio of pro-MMPs to overall activated MMPs. It is, however, impossible to assess net MMP activity in situ with this technique, as MMP-TIMP complexes are separated upon electrophoresis. As a modification of zymography, reverse zymography was developed to detect protease inhibitors such as TIMPs [42]. After electrophoresis and washing, the gel is either incubated in conditioned media, containing soluble proteases, or proteases were added to the gel before polymerization. The activity of protease inhibitors is visualized by Coomassie Blue staining. The result is a dark band, consisting of partially undegraded substrate protected from proteolysis in the presence of the inhibitor. Summarizing, the above assays can determine net MMP activity, but there are numerous limitations regarding the quantification of the endogenous and local balance of specific matrix degrading activity and inhibition in situ. Most assays cannot distinguish between different enzymes, as most ECM-cleaving proteases have broadly overlapping substrates. Preparation of sample extracts precludes, furthermore, the localization of enzyme activity and real-time quantification. Also, tissue homogenization leads to artificial interactions of inhibitors and proteases that were located within different compartments in tissues and cells. Functionally important, high local proteolytic activity that is only present in a small proportion of a tissue section (i.e., tumor–stroma interface) might be underestimated or not be recognized after dilution in the entire tissue extract upon homogenization (Fig. 11.3, total versus local activity).

11.3.1

Functional Detection of Local Proteolytic Activity by In Situ Zymography

In situ zymography (ISZ) overcomes some of the limitations of conventional zymography and other biochemical assays. It enables both localization and estimation of net MMP activity in tissue sections, and preserves sample histology.

11.3 Evaluation of Classical Methods for Quantification of Net Proteolytic Activity

The method has undergone innumerable modifications and adaptations, but the basic principle remains unchanged. In short, ISZ can be seen as an adaptation of zymography to frozen tissue sections. Sections are placed on a surface coated with a protease substrate (e.g. gelatin on microscope slides). After incubation and washing, uncleaved substrate is detected by protein staining of the coating. Proteolytic activity results in unstained spots. The most commonly used techniques today are either photographic emulsion-based or fluorescence-labeled substrate-based ISZ [122, 125]. Photographic emulsion ISZ utilizes the effect of gelatin to release a thiol group containing propeptide upon cleavage. This thiol group induces a structural change in colloidal silver, which results in a change of color. In fluorescencelabeled substrate-based ISZ, thin coatings of fluorescence-labeled substrates indicate proteolytic activity by local loss of fluorescence after proteolytic cleavage. ISZ has almost exclusively been used for the localization of MMP activities, especially the gelatinases (MMP-2 and MMP-9), although it can be adjusted to virtually any protease group. Most studies that employ ISZ used complementary techniques to provide accurate insights into the function of a specific protease. Several of these studies confirmed that proteolytic activity was not restricted to the tumor cells, but could also be found in the stroma surrounding the tumor [131, 132], stressing once more the regulatory function of tumor–host interaction for invasion and metastasis. Although ISZ and related techniques can localize even subtle increases of proteolytic activity and enable one to assess the balance between activated proteases and inhibitors, only three to four levels of proteolytic activity can be classified for quantification purposes [131, 133]. It can be concluded that ISZ is a semiquantitative method for the detection of local proteolytic activity and acts as a complement to, but does not replace, gel zymography and other biochemical assay methods (Fig. 11.3). However, the major disadvantage of ISZ is the restriction of its application to excised specimen after invasive procedures, which greatly limits its application for any screening purpose or early diagnosis of cancer. 11.3.2

Tumor Cell Invasion Assays

The ability of cancer cells to invade the surrounding stromal microenvironment and breach formerly restrictive basement membrane boundaries is a defining step in tumor progression and a hallmark in the development of the metastatic phenotype (reviewed in Ref. [1]). Invasion is a complex, multifactorial event, that requires directed migration and proteolytic activity to degrade extracellular matrix barriers (reviewed in Refs. [134–136]). Diverse in vitro assays have been developed to simulate in vivo conditions and understand key regulators of this process. Most experimental setups are variations of a modified Boyden chamber assay, introduced in 1987 by Albini and colleagues [137]. The original assay, published by Boyden 1962 for the analysis of leukocyte chemotaxis, is based on a chamber of two mediumfilled compartments, separated by a microfilter membrane [138]. In general, cells

393

394

11 Nanotechnological Approaches to Cancer Diagnosis

migrate from the upper medium compartment through the pores of the filter membrane to the lower medium compartment, which contains chemotactic agents. At the end of the assay the number of cells that have migrated to the lower side of the membrane is determined by fixation and staining of the membrane. For tumor cell invasion assays, the filter membrane is coated with an ECM preparation – most commonly matrigel – to resemble the basement membrane. NIH 3T3 conditioned media or FCS is usually used as a chemoattractant to stimulate directed migration [139–141]. In all these assays, invasion is not only dependent on local proteolytic activity, but also on matrix composition [142, 143], the ability of the cells to adhere and detach from this matrix [144–146], and the resulting and intrinsic ability of these cells to migrate [147] (reviewed in Refs. [135, 148, 149]). Electrical Resistance Breakdown Assay In addition to protease substrate based assay systems, local proteolytic activity can be quantified by an innovative, electrophysiological and indicator cell based assay. The electrical resistance breakdown assay offers a complex but stable cell based model system for the dynamic quantification of tumor cell-associated local proteolytic activity, cancer cell invasiveness and the investigation of basic mechanisms in this process. The system can be used for large and even heterogeneous tumor cell populations from primary tumor cultures and cell lines. Setups, application and the theoretical background of this assay have been described elsewhere in detail [150–155]. 11.3.2.1

11.3.3

In vivo Detection of Proteolytic Activity

The early detection of small primary tumors is still the most promising method to improve cancer survival rates and represents the working basis for extensive screening programs of common tumors. Several high-affinity substances targeted against tumor associated markers or angiogenesis have been used to detect solid tumors in animal models and clinical trials. The targeting of tumors by functional imaging with sufficient sensitivity and specificity is a diagnostic challenge. The limiting factor is the tumor-to-background ratio, which results from the lack of specific processes exclusively found in tumors. There has been a major drive to design biocompatible probes for in vivo detection and imaging of cancer cells. The rationale of this approach is based on the specific binding of easily detectable molecules to proteins that are predominantly expressed by tumor cells. The development of protease activated near-infrared fluorescent (NIRF) probes has been a huge step towards the functional imaging of tumors. The increased and localized proteolytic activity in invading cancer cells is exploited by these small, synthetic substrates. Optically quenched NIRF probes generate a strong NIRF signal after enzymatic cleavage, and have proved capable of detecting and imaging differential protease expression in nude mice in vivo. For example, protease

11.3 Evaluation of Classical Methods for Quantification of Net Proteolytic Activity

specific NIRF probes were designed to be either activated by cathepsin-B [156] or cathepsin-D [157]. Tissue studies of breast cancer patients link aggressive tumor behavior and high expression levels of these proteases [158, 159]. In principle, NIRF probes seem to have the potential to evolve to a functional method for protease activity monitoring in clinical applications and MMPI trials. The feasibility of this approach has already been demonstrated by direct imaging of MMP-2 activity in response to an MMPI (prinomastat) in a nude mice model [160]. Despite the achievements of NIRF probes in detecting proteolytic activity, small synthetic substrates do not resemble complex protease–ECM interactions. The cleavage of a synthetic substrate by a single protease or protease group does not imply functional relevance of this activity for the heterogeneous ECM micromilieu that surrounds a tumor cell. At present, it is not possible with this technique to relate the detected proteolytic activity to the net ability of living tumor cells to digest specific, local ECM compositions (Fig. 11.7 below) in situ. 11.3.4

Multiphoton Microscopy and Second-harmonic Generation

Multiphoton microscopy (MPM) has become a preferred fluorescence imaging technique for in vivo studies, due to its ability to image processes deep within living tissues and its extremely low out of focal plane excitation. In standard fluorescence microscopy, a single photon causes excitation of a fluorescent dye. Multiphoton microscopy (MPM) is based on the simultaneous absorption of multiple low energy photons to evoke an electronic transition that is equivalent to the absorption of one single high-energy photon. For example, the simultaneous absorption of two red photons (two-photon excited fluorescence) can cause an excitation that is usually caused by a single photon in the range of UV-light. Multiphoton excitation occurs only at the beam focus. Without using dyes, intrinsic signals of molecules can be utilized for coherent nonlinear photon microscopic techniques, such as second-harmonic generation (SHG) imaging microscopy. Processes like SHG are based on photon scattering rather than photon absorption. With SHG, the first order hyperpolarizability of a sample enables two incident photons of a given wavelength to scatter coherently and produce a photon of half the wavelength (twice the energy). In fluorescence microscopy a proportion of the excitation energy is lost during relaxation of the excited state. Other than this, SHG involves neither excitation of molecules nor absorption of energy. Hence, it shows no photobleaching and theoretically no phototoxicity. SHG does not need exogenous dyes, but is confined to sources that lack a center of symmetry. Higher-ordered structures in biological specimen such as myosin, collagen, and microtubules fulfill this requirement. MPM and SHG are noninvasive techniques that can be used to image cancer cell induced ECM remodeling, as they permit three-dimensional (3D) reconstruction and investigation of tissues [161], ECM morphology [162], cell metabolism [163] and tumors [164] in vivo.

395

396

11 Nanotechnological Approaches to Cancer Diagnosis

The full theory of SHG and multiphoton microscopy and their application in biological systems are beyond the scope of this chapter, but have been thoroughly reviewed elsewhere [163, 165–169]. 11.3.5

In vitro Detection of Local Proteolytic Activity by Labeled Substrates

Although detection of proteolytic activity on the subcellular level is not feasible by in situ zymography, the thin fluorescence labeled substrate coatings can be utilized to detect and image local proteolytic activity in the microenvironment of cells under tissue culture conditions (Fig. 11.4). The method was developed to detect focal, proteolytic ECM degradation below cells [170], and facilitated functional investigation of the regulation of proteolytic activity at invadopodia. It was used, for example, to demonstrate that invadopodial docking of MT-MMP1 is dependent on its transmembrane and cytoplasmic domain [89]. Overexpression of MT-MMP1 without invadopodial localization failed in these experiments to initiate ECM degradation [89]. A similar experimental setup proved the ability of integrins to direct proteases to sites of invasion, which gave further insights in the connection between integrins, adhesion and proteolysis [171]. Furthermore, fluorescence labeled protease substrates were used to localize local matrix degradation in the microenvironment of melanoma cells in a combined setup of fluorescence microscopy and atomic-force microscopy [172]. Instead of detecting proteolysis by loss of fluorescence, intramolecularly quenched substrates can be used. Cleavage causes an increase of fluorescence emission in these substrates. Results from functional imaging of proteolytic activity in living cells with quenched substrates supported the previously suggested functional importance of intracellular protease activity for matrix degradation [173]. Studies with DQ-collagen IV confirmed that proteolysis of ECM proteins occurs, for example, intracellularly in glioma [174] and breast cancer cells [175]. In addition to the described applications, ‘‘quenched substrates’’ offer crucial advantages for imaging of proteolytic activity within 3D matrix systems [176].

11.4

Novel Approaches to Local Proteolytic Activity

Atomic-force microscopy offers an interesting and novel approach for highresolution imaging and nanoscale quantification of local proteolytic activity in the microenvironment of live cancer cells [172]. By moving a fine tip across the sample, the AFM detects height changes and produces a 3D image of surface topography (Fig. 11.5). The atomic-force microscope (AFM) was developed by Binnig and coworkers [177], and evolved from the scanning tunneling microscope (STM) [178]. The possibility of scanning native biological samples (i.e., live cells) under physiological conditions – that means in most cases in liquid at 37  C – opened up a

11.4 Novel Approaches to Local Proteolytic Activity

Fig. 11.5. Schematic setup for atomic-force microscopy. AFM utilizes the deflection of a thin silicon nitride spring with a fine probe at its end to reconstruct a 3D topographical map of sample surfaces (here native rat tail tendon in fluid). A laser beam is focused at the end of the triangular spring. Deflection of the spring is registered by a photo detector (hair cross on left-hand side). A feedback loop couples the

photo detector to a piezo tube at the other end of the silicon spring (not shown). The corresponding height is calculated from the physical constants of the piezo-crystal and the voltage that must be applied to it to bring the laser beam back into the center of the photo detector. The AFM tip is moved line wise (horizontal arrow) across the sample to collect x, y and z data.

whole new perspective for biological, medical and biomaterial research. STM was the first instrument to generate real-space images of surfaces with atomic resolution, but was initially unable to image in fluids or scan non-conducting biological samples [179]. Since that time, AFM has become a rapidly emerging technique in biomedical research (e.g. Fig. 11.6). A wide range of biological samples have been successfully studied under native conditions with the AFM, ranging from proteins incorporated in artificial [180] and native lipid bilayers [181], to whole cells, their mechanical properties, subcellular structures and dynamic changes [151, 182–185]. Nanoscale images of, and structural insights into, the ECM have been obtained by AFM. It was used for example to investigate the interaction of basement membrane macromolecules [186], the surface ultrastructure of collagen fibrils and their association in human cornea and sclera [187], the assembly mechanism of fibrous long spacing collagen [188], the substructure of native, hydrated rat tail tendon ECM [189] and imaging of collagen III polymerization in solution [190]. Among

397

398

11 Nanotechnological Approaches to Cancer Diagnosis

Nanoscale imaging of local proteolytic activity by atomic-force microscopy. The image displays the leading edge of a melanoma cell on a gelatin-coated coverslip (principles shown in Figs. 11.4 and 11.5). By stepwise reduction of the scanned matrix area, the AFM tip is directed to the pericellular microenvironment of the tumor

Fig. 11.6.

cell (right) and distant control areas (left). Images of pericellular gelatin (right) illustrate the decrease in size and frequency of larger protein structures compared to intact matrix (arrows in left-hand image). These morphometric alterations can automatically be quantified with the AFM software.

various other applications, AFM was used to examine the structure of single collagen XVI molecules on mica [191]. AFM of normal human skin basement membrane preparations [192] displays the structural diversity of different fibrillar networks and the close meshed, underlying laminin-collagen IV net. Imaging of ECM compositions illustrates the high spatial resolution of the AFM in biological samples (Fig. 11.7). The ability of the AFM to image ECM-components with high resolution has been exploited in an investigation of protease–substrate interactions in vitro. For example, Sun et al. have revealed by AFM the existence of several non-specific

11.4 Novel Approaches to Local Proteolytic Activity

Fig. 11.7. 3D reconstruction of AFM images of normal human skin preparations, enriched in basement membranes. The images depict the molecular diversity and complexity of native ECM compositions. (a) Larger scale

magnification; (b) collagen-IV/laminin-V network with typical collagen IV conformations. Arrows: NC-1 domains of collagen IV. (c) Macromolecules such as fibrillin can be identified by their structure.

binding sites for MMP-8 on single type II collagen helices, but only one specific cleavage site for this enzyme [193]. Lin and colleagues demonstrated AFM imaging of real-time proteolysis of single collagen I molecules in fluid [194]. AFM was used alone and in combination with fluorescence microscopy for imaging of local proteolytic activity in the pericellular microenvironment of cancer cells (Figs. 11.2 and 11.4) [172]. Amelanotic melanoma cells were seeded on thin coatings of fluorescence-labeled gelatin or collagen IV (setup comparable to Fig. 11.4). Local loss of fluorescence due to ECM protein cleavage was used for fluorescence microscopic detection of proteolysis and localizing the AFM tip, and served as a positive control. Additional experiments proved that fluorescence microscopy

399

400

11 Nanotechnological Approaches to Cancer Diagnosis

is not a prerequisite for this approach, as AFM imaging of pericellular matrix is sufficient to detect proteolysis on a subcellular level. As the AFM acquires primarily x-, y- and z-data during scanning, it is very easy to calculate and analyze differences in height and volume of subnanometer structures. AFM depicted significant differences in average height, volume and molecular weight distribution of pericellular matrix proteins between the microenvironment of invasive cancer cells and distant control areas [172]. By treating it as the segment of a sphere, the molecular weight of a protein imaged by AFM can be calculated from the molecular volume of the structure. In these experiments, the AFM registered significant changes in average matrix volume as little as 0.003 fl mm2 and decreases in average height of less than 0.5 nm. By focusing on the complex composition of ECM, the possibility of time lapse AFM of primary tumor cell cultures on native human basement membranes is currently being evaluated (Fig. 11.7). In summary, the AFM can image and quantify nanoscale ECM alterations in the microenvironment of cancer cells. Considering that direct measurement of local proteolytic activity and high-resolution imaging of ECM remodeling have proven difficult, this method is particularly advantageous as it enables nanoscale resolution and functional investigations on the subcellular level. The advantages of AFM as a rapidly evolving nanotechnique for functional high resolution imaging under physiological conditions are striking, as it can provide functional data in addition to 3D nanoscale images of local phenomena. Current advances in making the AFM a high-resolution tool for simultaneous topography and specific functional recognition imaging of single molecules (‘‘molecular recognition’’) like integrins or cellular microdomains such as lipid rafts opens up interesting perspectives [93, 195]. Furthermore, physical parameters such as cell traction, elasticity of membrane compartments and ECM compositions or the binding forces of ligand–receptor complexes can be quantified with nanoscale resolution and integrated in the molecular recognition maps.

11.5

Conclusions and Perspectives

The most devastating process in cancer is metastasis, as it represents the milestone of an irreversible stage of progression that inevitably determines disease prognosis. Regardless of the advancement of therapies for cancer that has already spread, the early diagnosis and removal of the malignancy is still the most promising strategy. The limitations of conventional state of the art methods offer unique opportunities for innovative, nanotechnology based approaches. The development of improved methods that enable real-time high-resolution imaging and quantification of local enzymatic activities will play a major role in the design of new drugs and the understanding of basic principles in tumor cell invasion. These methods themselves harbor the potential to contribute directly to patient benefits due to earlier diagnosis of malignancies and functional monitoring following clinical interventions.

References

Acknowledgments

As it is sometimes not possible to cite all relevant articles on a subject, several references have been used as examples to illustrate major principles. I thank Uwe Hansen for the generous supply with various matrix proteins, Sylvia Puttman and Volker Senner for their cooperation and contribution of primary brain tumor cells, and Helga Bertram and Stephan Kusick for their excellent technical assistance in tissue culture and Atomic Force Microscopy. I thank Joerg Hinnerwisch, Mark Lal, Hans Oberleithner, Val Prasad and Michael J. Morton for revising and improving the original manuscript and for interesting discussions. Excerpts of this chapter have been previously published as a review [15]. This work was supported by the Deutsche Forschungsgemeinschaft (LU 854/2-1 and LU 854/3-1) and ‘‘Innovative Medizinische Forschung’’ (LU 110343) of the University of Mu¨nster.

Abbreviations

AFM ECM EMMPRIN HSPG ISZ MMP MT-MMP MMPI MPM NIRF RECK SHG TIMP uPA

Atomic-force microscope Extracellular matrix Extracellular matrix metalloproteinase inducer Heparan sulfate proteoglycan In situ zymography Matrix metalloproteinase Membrane type-matrix metalloproteinase Matrix metalloproteinase inhibitor Multi-photon microscopy Near-infrared fluorescence Reversion-inducing cysteine-rich protein with Kazal motifs Second-harmonic generation Tissue inhibitor of matrix metalloproteinases Urokinase-type plasminogen activator

References 1 Hanahan D, Weinberg RA. The

hallmarks of cancer. Cell 100, 57–70, 2000. 2 Sporn MB. The war on cancer: A review. Ann. New York Acad Sci. 833, 137–146, 1997. 3 Sporn MB. The war on cancer. Lancet 347, 1377–1381, 1996. 4 Wang X, Fu X, Brown PD, Crimmin MJ, Hoffman RM. Matrix metalloproteinase inhibitor BB-94

(batimastat) inhibits human colon tumor growth and spread in a patientlike orthotopic model in nude mice. Cancer Res. 54, 4726–4728, 1994. 5 Brown PD. Matrix metalloproteinase inhibitors: A novel class of anticancer agents. Adv. Enzyme Regul. 35, 293–301, 1995. 6 Baker AH, Edwards DR, Murphy G. Metalloproteinase inhibitors: Biological actions and therapeutic

401

402

11 Nanotechnological Approaches to Cancer Diagnosis

7

8

9

10

11

12

13

14

15

16

17

opportunities. J. Cell Sci. 115, 3719–3727, 2002. Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: A tail of a frog that became a prince. Nat. Rev. Mol. Cell Biol. 3, 207–214, 2002. Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: Trials and tribulations. Science 295, 2387–2392, 2002. Overall CM, Lopez-Otin C. Strategies for mmp inhibition in cancer: Innovations for the post-trial era. Nat. Rev. Cancer 2, 657–672, 2002. Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 284, 808–812, 1999. Al-Mehdi AB, Tozawa K, Fisher AB, Shientag L, Lee A, Muschel RJ. Intravascular origin of metastasis from the proliferation of endotheliumattached tumor cells: A new model for metastasis [In Process Citation]. Nat. Med. 6, 100–102, 2000. Bogenrieder T, Herlyn M. Axis of evil: Molecular mechanisms of cancer metastasis. Oncogene 22, 6524–6536, 2003. Murphy PM. Chemokines and the molecular basis of cancer metastasis. N. Engl. J. Med. 345, 833–835, 2001. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat. Rev. Cancer 2, 563–572, 2002. Ludwig T. Local proteolytic activity in tumor cell invasion and metastasis. Bioessays 27, 1181–1191, 2005. Shrivastava A, Radziejewski C, Campbell E, Kovac L, McGlynn M, Ryan TE, Davis S, Goldfarb MP, Glass DJ, Lemke G, Yancopoulos GD. An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors. Mol. Cell. 1, 25–34, 1997. Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol. Cell. 1, 13–23, 1997.

18 Kheradmand F, Werner E, Tremble

19

20

21

22

23

24

25

26

P, Symons M, Werb Z. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science 280, 898–902, 1998. Faisal Khan KM, Laurie GW, McCaffrey TA, Falcone DJ. Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression. J. Biol. Chem. 277, 13 778– 13 786, 2002. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463–516, 2001. Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T. Regulation of matrix metalloproteinases: An overview. Mol. Cell Biochem. 253, 269–285, 2003. Fahling M, Steege A, Perlewitz A, Nafz B, Mrowka R, Persson PB, Thiele BJ. Role of nucleolin in posttranscriptional control of MMP-9 expression. Biochim. Biophys. Acta 1731, 32–40, 2005. Raza SL, Nehring LC, Shapiro SD, Cornelius LA. Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases. J. Biol. Chem. 275, 41 243–41 250, 2000. Zucker S, Hymowitz M, Conner CE, DiYanni EA, Cao J. Rapid trafficking of membrane type 1-matrix metalloproteinase to the cell surface regulates progelatinase a activation. Lab. Invest. 82, 1673–1684, 2002. Van Wart HE, Birkedal-Hansen H. The cysteine switch: A principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. U.S.A. 87, 5578–5582, 1990. Springman EB, Angleton EL, Birkedal-Hansen H, Van Wart HE. Multiple modes of activation of latent human fibroblast collagenase: Evidence for the role of a Cys73 activesite zinc complex in latency and a ‘‘cysteine switch’’ mechanism for

References

27

28

29

30

31

32

33

34

35

36

activation. Proc. Natl. Acad. Sci. U.S.A. 87, 364–368, 1990. Yana I, Weiss SJ. Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases. Mol. Biol. Cell. 11, 2387– 2401, 2000. Pei D, Weiss SJ. Furin-dependent intracellular activation of the human stromelysin-3 zymogen. Nature 375, 244–247, 1995. Kang T, Nagase H, Pei D. Activation of membrane-type matrix metalloproteinase 3 zymogen by the proprotein convertase furin in the trans-Golgi network. Cancer Res. 62, 675–681, 2002. Illman SA, Keski-Oja J, Parks WC, Lohi J. The mouse matrix metalloproteinase, epilysin (MMP-28), is alternatively spliced and processed by a furin-like proprotein convertase. Biochem. J. 375, 191–197, 2003. Osenkowski P, Toth M, Fridman R. Processing, shedding, and endocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP). J. Cell Physiol. 200, 2–10, 2004. Itoh Y, Seiki M. MT1-MMP: An enzyme with multidimensional regulation. Trends Biochem. Sci. 29, 285–289, 2004. Hernandez-Barrantes S, Bernardo M, Toth M, Fridman R. Regulation of membrane type-matrix metalloproteinases. Semin. Cancer Biol. 12, 131–138, 2002. Toth M, Osenkowski P, Hesek D, Brown S, Meroueh S, Sakr W, Mobashery S, Fridman R. Cleavage at the stem region releases an active ectodomain of the membrane type 1 matrix metalloproteinase. Biochem. J. 387, 497–506, 2005. Cao J, Sato H, Takino T, Seiki M. The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J. Biol. Chem. 270, 801–805, 1995. Hotary K, Allen E, Punturieri A, Yana I, Weiss SJ. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen

37

38

39

40

41

42

43

44

matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J. Cell Biol. 149, 1309–1323, 2000. Lehti K, Lohi J, Valtanen H, KeskiOja J. Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface. Biochem. J. 334(Pt 2), 345–353, 1998. Toth M, Hernandez-Barrantes S, Osenkowski P, Bernardo MM, Gervasi DC, Shimura Y, Meroueh O, Kotra LP, Galvez BG, Arroyo AG, Mobashery S, Fridman R. Complex pattern of membrane type 1 matrix metalloproteinase shedding. Regulation by autocatalytic cells surface inactivation of active enzyme. J. Biol. Chem. 277, 26 340–26 350, 2002. Khokha R, Waterhouse P, Yagel S, Lala PK, Overall CM, Norton G, Denhardt DT. Antisense RNAinduced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells. Science 243, 947–950, 1989. Jiang Y, Goldberg ID, Shi YE. Complex roles of tissue inhibitors of metalloproteinases in cancer. Oncogene 21, 2245–2252, 2002. Murphy G, Willenbrock F. Tissue inhibitors of matrix metalloendopeptidases. Methods Enzymol. 248, 496–510, 1995. Welgus HG, Stricklin GP, Eisen AZ, Bauer EA, Cooney RV, Jeffrey JJ. A specific inhibitor of vertebrate collagenase produced by human skin fibroblasts. J. Biol. Chem. 254, 1938–1943, 1979. Docherty AJ, Lyons A, Smith BJ, Wright EM, Stephens PE, Harris TJ, Murphy G, Reynolds JJ. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 318, 66–69, 1985. Hayakawa T, Yamashita K, Tanzawa K, Uchijima E, Iwata K. Growthpromoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells. A possible new growth factor in serum. FEBS Lett. 298, 29–32, 1992.

403

404

11 Nanotechnological Approaches to Cancer Diagnosis 45 Butler GS, Butler MJ, Atkinson SJ,

52 Itoh Y, Takamura A, Ito N, Maru Y,

Will H, Tamura T, van Westrum SS, Crabbe T, Clements J, d’Ortho MP, Murphy G. The TIMP2 membrane type 1 metalloproteinase ‘‘receptor’’ regulates the concentration and efficient activation of progelatinase A. A kinetic study. J. Biol. Chem. 273, 871–880, 1998. Wang Z, Juttermann R, Soloway PD. TIMP-2 is required for efficient activation of proMMP-2 in vivo. J. Biol. Chem. 275, 26 411–26 415, 2000. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, Nishimura S, Imamura Y, Kitayama H, Alexander DB, Ide C, Horan TP, Arakawa T, Yoshida H, Nishikawa S, Itoh Y, Seiki M, Itohara S, Takahashi C, Noda M. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell 107, 789–800, 2001. Wu YI, Munshi HG, Sen R, Snipas SJ, Salvesen GS, Fridman R, Stack MS. Glycosylation broadens the substrate profile of membrane type 1 matrix metalloproteinase. J. Biol. Chem. 279, 8278–8289, 2004. Simizu S, Takagi S, Tamura Y, Osada H. RECK-mediated suppression of tumor cell invasion is regulated by glycosylation in human tumor cell lines. Cancer Res. 65, 7455–7461, 2005. Rozanov DV, Deryugina EI, Ratnikov BI, Monosov EZ, Marchenko GN, Quigley JP, Strongin AY. Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells. J. Biol. Chem. 276, 25 705–25 714, 2001. Seiki M, Koshikawa N, Yana I. Role of pericellular proteolysis by membrane-type 1 matrix metalloproteinase in cancer invasion and angiogenesis. Cancer Metastasis Rev. 22, 129–143, 2003.

Sato H, Suenaga N, Aoki T, Seiki M. Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J. 20, 4782–4793, 2001. Itoh Y, Seiki M. MT1-MMP: A potent modifier of pericellular microenvironment. J. Cell Physiol. 206, 1–8, 2006. Remacle A, Murphy G, Roghi C. Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface. J. Cell Sci. 116, 3905–3916, 2003. Galvez BG, Matias-Roman S, YanezMo M, Vicente-Manzanares M, Sanchez-Madrid F, Arroyo AG. Caveolae are a novel pathway for membrane-type 1 matrix metalloproteinase traffic in human endothelial cells. Mol. Biol. Cell. 15, 678–687, 2004. Annabi B, Lachambre M, BousquetGagnon N, Page M, Gingras D, Beliveau R. Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains. Biochem. J. 353, 547–553, 2001. Williams TM, Medina F, Badano I, Hazan RB, Hutchinson J, Muller WJ, Chopra NG, Scherer PE, Pestell RG, Lisanti MP. Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo. Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion. J. Biol. Chem. 279, 51 630– 51 646, 2004. Remacle AG, Rozanov DV, Baciu PC, Chekanov AV, Golubkov VS, Strongin AY. The transmembrane domain is essential for the microtubular trafficking of membrane type1 matrix metalloproteinase (MT1MMP). J. Cell Sci. 118, 4975–4984, 2005. Lehti K, Valtanen H, Wickstrom S, Lohi J, Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic

46

47

48

49

50

51

53

54

55

56

57

58

59

References

60

61

62

63

64

65

66

67

68

69

70

domain. J. Biol. Chem. 275, 15 006– 15 013, 2000. Jiang A, Lehti K, Wang X, Weiss SJ, Keski-Oja J, Pei D. Regulation of membrane-type matrix metalloproteinase 1 activity by dynaminmediated endocytosis. Proc. Natl. Acad. Sci. U.S.A. 98, 13 693–13 698, 2001. Wang X, Ma D, Keski-Oja J, Pei D. Co-recycling of MT1-MMP and MT3MMP through the trans-Golgi network. Identification of DKV582 as a recycling signal. J. Biol. Chem. 279, 9331–9336, 2004. Uekita T, Itoh Y, Yana I, Ohno H, Seiki M. Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity. J. Cell Biol. 155, 1345–1356, 2001. Takino T, Miyamori H, Kawaguchi N, Uekita T, Seiki M, Sato H. Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase. Biochem. Biophys. Res. Commun. 304, 160–166, 2003. Werb Z. ECM and cell surface proteolysis: Regulating cellular ecology. Cell 91, 439–442, 1997. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer 2, 161–174, 2002. Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Semin. Cancer Biol. 10, 415–433, 2000. Yana I, Seiki M. MT-MMPs play pivotal roles in cancer dissemination. Clin. Exp. Metastasis 19, 209–215, 2002. Ellerbroek SM, Stack MS. Membrane associated matrix metalloproteinases in metastasis. Bioessays 21, 940–949, 1999. Holmbeck K, Bianco P, Yamada S, Birkedal-Hansen H. MT1-MMP: A tethered collagenase. J. Cell Physiol. 200, 11–19, 2004. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 61–65, 1994.

71 Ohuchi E, Imai K, Fujii Y, Sato H,

72

73

74

75

76

77

78

Seiki M, Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem. 272, 2446–2451, 1997. Seiki M. Membrane-type matrix metalloproteinases. APMIS 107, 137–143, 1999. Tsunezuka Y, Kinoh H, Takino T, Watanabe Y, Okada Y, Shinagawa A, Sato H, Seiki M. Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay. Cancer Res. 56, 5678–5683, 1996. Lafleur MA, Handsley MM, Knauper V, Murphy G, Edwards DR. Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs). J. Cell Sci. 115, 3427–3438, 2002. Collen A, Hanemaaijer R, Lupu F, Quax PH, van Lent N, Grimbergen J, Peters E, Koolwijk P, van Hinsbergh VW. Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix. Blood 101, 1810–1817, 2003. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, BirkedalHansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99, 81–92, 1999. Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 85, 683–693, 1996. Yu Q, Stamenkovic I. Cell surfacelocalized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev. 14, 163–176, 2000.

405

406

11 Nanotechnological Approaches to Cancer Diagnosis 79 Sun J, Hemler ME. Regulation of

80

81

82

83

84

85

86

87

MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions. Cancer Res. 61, 2276–2281, 2001. Guo H, Li R, Zucker S, Toole BP. EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface. Cancer Res. 60, 888–891, 2000. Yamamoto M, Sawaya R, Mohanam S, Bindal AK, Bruner JM, Oka K, Rao VH, Tomonaga M, Nicolson GL, Rao JS. Expression and localization of urokinase-type plasminogen activator in human astrocytomas in vivo. Cancer Res. 54, 3656–3661, 1994. Festuccia C, Dolo V, Guerra F, Violini S, Muzi P, Pavan A, Bologna M. Plasminogen activator system modulates invasive capacity and proliferation in prostatic tumor cells. Clin. Exp. Metastasis 16, 513–528, 1998. Yu WH, Woessner JF, Jr. Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). J. Biol. Chem. 275, 4183–4191, 2000. Olson MW, Toth M, Gervasi DC, Sado Y, Ninomiya Y, Fridman R. High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV. J. Biol. Chem. 273, 10 672–10 681, 1998. Toth M, Sado Y, Ninomiya Y, Fridman R. Biosynthesis of alpha2(IV) and alpha1(IV) chains of collagen IV and interactions with matrix metalloproteinase-9. J. Cell Physiol. 180, 131–139, 1999. Mori H, Tomari T, Koshikawa N, Kajita M, Itoh Y, Sato H, Tojo H, Yana I, Seiki M. CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain. EMBO J. 21, 3949–3959, 2002. Bourguignon LY, Gunja-Smith Z, Iida N, Zhu HB, Young LJ, Muller

88

89

90

91

92

93

94

95

96

WJ, Cardiff RD. CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. J. Cell Physiol. 176, 206–215, 1998. Deryugina EI, Ratnikov B, Monosov E, Postnova TI, DiScipio R, Smith JW, Strongin AY. MT1MMP initiates activation of pro-MMP2 and integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells. Exp. Cell Res. 263, 209–223, 2001. Nakahara H, Howard L, Thompson EW, Sato H, Seiki M, Yeh Y, Chen WT. Transmembrane/cytoplasmic domain-mediated membrane type 1matrix metalloprotease docking to invadopodia is required for cell invasion. Proc. Natl. Acad. Sci. U.S.A. 94, 7959–7964, 1997. Rozanov DV, Deryugina EI, Monosov EZ, Marchenko ND, Strongin AY. Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of membrane type-1 matrix metalloproteinase in malignant cells. Exp. Cell Res. 293, 81–95, 2004. Lai EC. Lipid rafts make for slippery platforms. J. Cell Biol. 162, 365–370, 2003. Edidin M. The state of lipid rafts: From model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283, 2003. Henderson RM, Edwardson JM, Geisse NA, Saslowsky DE. Lipid rafts: Feeling is believing. News Physiol. Sci. 19, 39–43, 2004. Mueller MM, Fusenig NE. Friends or foes – bipolar effects of the tumour stroma in cancer. Nat. Rev. Cancer 4, 839–849, 2004. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer OL, Chenard MP, Rio MC, Chambon P. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348, 699–704, 1990. Tang Y, Kesavan P, Nakada MT, Yan L. Tumor-stroma interaction: Positive

References

97

98

99

100

101

102

103

104

feedback regulation of extracellular matrix metalloproteinase inducer (EMMPRIN) expression and matrix metalloproteinase-dependent generation of soluble EMMPRIN. Mol. Cancer Res. 2, 73–80, 2004. Hofmann UB, Eggert AA, Blass K, Brocker EB, Becker JC. Expression of matrix metalloproteinases in the microenvironment of spontaneous and experimental melanoma metastases reflects the requirements for tumor formation. Cancer Res. 63, 8221–8225, 2003. Stamenkovic I. Extracellular matrix remodelling: The role of matrix metalloproteinases. J. Pathol. 200, 448–464, 2003. Chang C, Werb Z. The many faces of metalloproteases: Cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 11, S37–S43, 2001. Gingras D, Bousquet-Gagnon N, Langlois S, Lachambre MP, Annabi B, Beliveau R. Activation of the extracellular signal-regulated protein kinase (ERK) cascade by membranetype-1 matrix metalloproteinase (MT1MMP). FEBS Lett. 507, 231–236, 2001. McCawley LJ, Matrisian LM. Matrix metalloproteinases: They’re not just for matrix anymore! Curr. Opin. Cell Biol. 13, 534–540, 2001. Koshikawa N, Giannelli G, Cirulli V, Miyazaki K, Quaranta V. Role of cell surface metalloprotease MT1MMP in epithelial cell migration over laminin-5. J. Cell Biol. 148, 615–624, 2000. Belien AT, Paganetti PA, Schwab ME. Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter. J. Cell Biol. 144, 373–384, 1999. Nabeshima K, Inoue T, Shimao Y, Okada Y, Itoh Y, Seiki M, Koono M. Front-cell-specific expression of membrane-type 1 matrix metalloproteinase and gelatinase A during cohort migration of colon carcinoma cells induced by hepatocyte growth factor/scatter factor. Cancer Res. 60, 3364–3369, 2000.

105 Sanceau J, Truchet S, Bauvois B.

106

107

108

109

110

111

112

Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing’s sarcoma cells. J. Biol. Chem. 278, 36 537–36 546, 2003. Yu WH, Woessner JF, Jr., McNeish JD, Stamenkovic I. CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev. 16, 307–323, 2002. Powell WC, Fingleton B, Wilson CL, Boothby M, Matrisian LM. The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr. Biol. 9, 1441–1447, 1999. Mitsiades N, Yu WH, Poulaki V, Tsokos M, Stamenkovic I. Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity. Cancer Res. 61, 577–581, 2001. Schlondorff J, Blobel CP. Metalloprotease-disintegrins: Modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J. Cell Sci. 112(Pt 21), 3603–3617, 1999. Moss ML, Lambert MH. Shedding of membrane proteins by ADAM family proteases. Essays Biochem. 38, 141– 153, 2002. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385, 729–733, 1997. Itoh T, Ikeda T, Gomi H, Nakao S, Suzuki T, Itohara S. Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J. Biol. Chem. 272, 22 389–22 392, 1997.

407

408

11 Nanotechnological Approaches to Cancer Diagnosis 113 Mudgett JS, Hutchinson NI,

114

115

116

117

118

119

120

Chartrain NA, Forsyth AJ, McDonnell J, Singer II, Bayne EK, Flanagan J, Kawka D, Shen CF, Stevens K, Chen H, Trumbauer M, Visco DM. Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction. Arthritis Rheum. 41, 110–121, 1998. Wilson CL, Heppner KJ, Labosky PA, Hogan BL, Matrisian LM. Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc. Natl. Acad. Sci. U.S.A. 94, 1402–1407, 1997. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411–422, 1998. Masson R, Lefebvre O, Noel A, Fahime ME, Chenard MP, Wendling C, Kebers F, LeMeur M, Dierich A, Foidart JM, Basset P, Rio MC. In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy. J. Cell Biol. 140, 1535–1541, 1998. Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD. Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc. Natl. Acad. Sci. U.S.A. 93, 3942–3946, 1996. Caterina JJ, Skobe Z, Shi J, Ding Y, Simmer JP, Birkedal-Hansen H, Bartlett JD. Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype. J. Biol. Chem. 277, 49 598– 49 604, 2002. Itoh T, Tanioka M, Matsuda H, Nishimoto H, Yoshioka T, Suzuki R, Uehira M. Experimental metastasis is suppressed in MMP-9deficient mice. Clin. Exp. Metastasis 17, 177–181, 1999. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. Reduced angiogenesis and tumor

121

122

123

124

125

126

127

128

129

130

progression in gelatinase A-deficient mice. Cancer Res. 58, 1048–1051, 1998. Coussens LM, Tinkle CL, Hanahan D, Werb Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103, 481–490, 2000. Yan SJ, Blomme EA. In situ zymography: A molecular pathology technique to localize endogenous protease activity in tissue sections. Vet Pathol. 40, 227–236, 2003. Catterall JB, Cawston TE. Assays of matrix metalloproteinases (MMPs) and MMP inhibitors: Bioassays and immunoassays applicable to cell culture medium, serum, and synovial fluid. Methods Mol. Biol. 225, 353–364, 2003. Lopez-Otin C, Overall CM. Protease degradomics: A new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519, 2002. Frederiks WM, Mook OR. Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: Review and protocols. J. Histochem. Cytochem. 52, 711–722, 2004. Billinghurst RC, Ionescu M, Poole AR. Immunoassay for collagenasemediated cleavage of types I and II collagens. Methods Mol. Biol. 151, 457–472, 2001. Fosang AJ, Last K, Jackson DC, Brown L. Antibodies to MMP-cleaved aggrecan. Methods Mol. Biol. 151, 425–449, 2001. Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr. Opin. Cell Biol. 16, 558–564, 2004. Schenk S, Quaranta V. Tales from the crypt[ic] sites of the extracellular matrix. Trends Cell Biol. 13, 366–375, 2003. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196–202, 1980.

References 131 Minami R, Tsunoda H, Iijima T,

132

133

134

135

136

137

138

139

140

141

Yoshikawa H, Nemori R, Noguchi M. Early acquisition of gelatinolytic activity in carcinogenesis of the uterine cervix. Mod. Pathol. 16, 1164–1170, 2003. Di Nezza LA, Misajon A, Zhang J, Jobling T, Quinn MA, Ostor AG, Nie G, Lopata A, Salamonsen LA. Presence of active gelatinases in endometrial carcinoma and correlation of matrix metalloproteinase expression with increasing tumor grade and invasion. Cancer 94, 1466–1475, 2002. Kawamura K, Kamiya N, Suyama T, Shimbo M, Oosumi N, Suzuki H, Ueda T, Tobe T, Igarashi T, Ito H, Ishikura H. In situ gelatinolytic activity correlates with tumor progression and prognosis in patients with bladder cancer. J. Urol. 172, 1480–1484, 2004. Curran S, Murray GI. Matrix metalloproteinases in tumour invasion and metastasis. J. Pathol. 189, 300–308, 1999. Friedl P, Hegerfeldt Y, Tusch M. Collective cell migration in morphogenesis and cancer. Int. J. Dev. Biol. 48, 441–449, 2004. Murphy G, Gavrilovic J. Proteolysis and cell migration: Creating a path? Curr. Opin. Cell Biol. 11, 614–621, 1999. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 47, 3239–3245, 1987. Boyden S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115, 453–466, 1962. Chen HC. Boyden chamber assay. Methods Mol. Biol. 294, 15–22, 2004. Shaw LM. Tumor cell invasion assays. Methods Mol. Biol. 294, 97–106, 2004. Benbow U, Orndorff KA, Givan AL. Invasion assays and matrix metalloproteinases. Quantification of cellular invasion using propidium iodide

142

143

144

145

146

147

148

149

150

labeling and confocal laser scanning microscopy. Methods Mol. Biol. 151, 485–494, 2001. Shang M, Koshikawa N, Schenk S, Quaranta V. The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration. J. Biol. Chem. 276, 33 045– 33 053, 2001. Kuratomi Y, Nomizu M, Tanaka K, Ponce ML, Komiyama S, Kleinman HK, Yamada Y. Laminin gamma 1 chain peptide, C-16 (KAFDITYVRLKF), promotes migration, MMP-9 secretion, and pulmonary metastasis of B16-F10 mouse melanoma cells. Br. J. Cancer 86, 1169–1173, 2002. Ray JM, Stetler-Stevenson WG. Gelatinase A activity directly modulates melanoma cell adhesion and spreading. EMBO J. 14, 908–917, 1995. Yan L, Moses MA, Huang S, Ingber DE. Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells. J. Cell Sci. 2000. Kucik DF, Wu C. Cell-adhesion assays. Methods Mol. Biol. 294, 43–54, 2004. Sieuwerts AM, Klijn JG, Foekens JA. Assessment of the invasive potential of human gynecological tumor cell lines with the in vitro Boyden chamber assay: Influences of the ability of cells to migrate through the filter membrane. Clin. Exp. Metastasis 15, 53–62, 1997. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2, 91–100, 2002. Bussemakers MJ, Schalken JA. The role of cell adhesion molecules and proteases in tumor invasion and metastasis. World J. Urol. 14, 151–156, 1996. Ludwig T, Puttmann S, Bertram H, Tatenhorst L, Paulus W, Oberleithner H, Senner V. Functional measurement of local proteolytic activity in living cells of

409

410

11 Nanotechnological Approaches to Cancer Diagnosis

151

152

153

154

155

156

157

158

159

invasive and non-invasive tumors. J. Cell Physiol. 202, 690–697, 2005. Ludwig T, Ossig R, Graessel S, Wilhelmi M, Oberleithner H, Schneider SW. The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion. Am. J. Physiol. Renal. Physiol. 283, F319–F327, 2002. Schnaeker EM, Ossig R, Ludwig T, Dreier R, Oberleithner H, Wilhelmi M, Schneider SW. Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: Prerequisite in human melanoma cell invasion. Cancer Res. 64, 8924–8931, 2004. Schneider SW, Ludwig T, Tatenhorst L, Braune S, Oberleithner H, Senner V, Paulus W. Glioblastoma cells release factors that disrupt blood-brain barrier features. Acta Neuropathol. (Berl) 107, 272–276, 2004. Zak J, Schneider SW, Eue I, Ludwig T, Oberleithner H. High-resistance MDCK-C7 monolayers used for measuring invasive potency of tumour cells. Pflugers Arch. 440, 179–183, 2000. Ludwig T, Fakih S, Krebs B, Oberleithner H. Platinum complex cytotoxicity tested by the electrical resistance breakdown assay. Cell Physiol. Biochem. 14, 425–430, 2004. Bremer C, Tung CH, Bogdanov A, Jr., Weissleder R. Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes. Radiology 222, 814–818, 2002. Tung CH, Mahmood U, Bredow S, Weissleder R. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 60, 4953–4958, 2000. Poole AR, Tiltman KJ, Recklies AD, Stoker TA. Differences in secretion of the proteinase cathepsin B at the edges of human breast carcinomas and fibroadenomas. Nature 273, 545–547, 1978. Thorpe SM, Rochefort H, Garcia M, Freiss G, Christensen IJ, Khalaf

160

161

162

163

164

165

166

167

168

S, Paolucci F, Pau B, Rasmussen BB, Rose C. Association between high concentrations of Mr 52,000 cathepsin D and poor prognosis in primary human breast cancer. Cancer Res. 49, 6008–6014, 1989. Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743–748, 2001. Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. U.S.A. 100, 7075–7080, 2003. Brown E, McKee T, DiTomaso E, Pluen A, Seed B, Boucher Y, Jain RK. Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat. Med. 9, 796–800, 2003. Richards-Kortum R, Sevick-Muraca E. Quantitative optical spectroscopy for tissue diagnosis. Annu. Rev. Phys. Chem. 47, 555–606, 1996. Wang W, Wyckoff JB, Frohlich VC, Oleynikov Y, Huttelmaier S, Zavadil J, Cermak L, Bottinger EP, Singer RH, White JG, Segall JE, Condeelis JS. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res. 62, 6278–6288, 2002. Friedl P. Dynamic imaging of cellular interactions with extracellular matrix. Histochem. Cell Biol. 122, 183–190, 2004. Mohler W, Millard AC, Campagnola PJ. Second harmonic generation imaging of endogenous structural proteins. Methods 29, 97–109, 2003. Moreaux L, Sandre O, Charpak S, Blanchard-Desce M, Mertz J. Coherent scattering in multi-harmonic light microscopy. Biophys. J. 80, 1568–1574, 2001. Williams RM, Zipfel WR, Webb WW. Multiphoton microscopy in biological research. Curr. Opin. Chem. Biol. 5, 603–608, 2001.

References 169 Campagnola PJ, Loew LM. Second-

170

171

172

173

174

175

176

177

178

harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat. Biotechnol. 21, 1356–1360, 2003. Monsky WL, Lin CY, Aoyama A, Kelly T, Akiyama SK, Mueller SC, Chen WT. A potential marker protease of invasiveness, seprase, is localized on invadopodia of human malignant melanoma cells. Cancer Res. 54, 5702–5710, 1994. Mueller SC, Ghersi G, Akiyama SK, Sang QX, Howard L, PineiroSanchez M, Nakahara H, Yeh Y, Chen WT. A novel protease-docking function of integrin at invadopodia. J. Biol. Chem. 274, 24 947–24 952, 1999. Kusick S, Bertram H, Oberleithner H, Ludwig T. Nanoscale imaging and quantification of local proteolytic activity. J. Cell Physiol., 204, 767–774, 2005. Szpaderska AM, Frankfater A. An intracellular form of cathepsin B contributes to invasiveness in cancer. Cancer Res. 61, 3493–3500, 2001. Sameni M, Dosescu J, Sloane BF. Imaging proteolysis by living human glioma cells. Biol. Chem. 382, 785–788, 2001. Sameni M, Moin K, Sloane BF. Imaging proteolysis by living human breast cancer cells. Neoplasia 2, 496–504, 2000. Horino K, Kindezelskii AL, Elner VM, Hughes BA, Petty HR. Tumor cell invasion of model 3-dimensional matrices: Demonstration of migratory pathways, collagen disruption, and intercellular cooperation. FASEB J. 15, 932–939, 2001. Binnig G, Quate CF, Gerber Ch. Atomic force microscope. Phys. Rev. Lett. 56, 930–933, 1986. Baro AM, Miranda R, Alaman J, Garcia N, Binnig G, Rohrer H, Gerber C, Carrascosa JL. Determination of surface topography of biological specimens at high resolution by scanning tunnelling microscopy. Nature 315, 253–254, 1985.

179 Binnig G, Roher H, Gerber Ch,

180

181

182

183

184

185

186

187

188

189

Weibel E. Surface studies by scanning tunneling microscopy. Phys. Rev. Lett. 49, 57–61, 1982. Muller DJ, Engel A, Matthey U, Meier T, Dimroth P, Suda K. Observing membrane protein diffusion at subnanometer resolution. J. Mol. Biol. 327, 925–930, 2003. Schillers H, Danker T, Madeja M, Oberleithner H. Plasma membrane protein clusters appear in CFTRexpressing Xenopus laevis oocytes after cAMP stimulation. J. Membr. Biol. 180, 205–212, 2001. Oberleithner H, Ludwig T, Riethmuller C, Hillebrand U, Albermann L, Schafer C, Shahin V, Schillers H. Human endothelium: Target for aldosterone. Hypertension 43, 952–956, 2004. Ludwig T, Oberleithner H. Platinum complex toxicity in cultured renal epithelia. Cell Physiol. Biochem. 14, 431–440, 2004. Engel A, Muller DJ. Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7, 715–718, 2000. Santos NC, Castanho MA. An overview of the biophysical applications of atomic force microscopy. Biophys. Chem. 107, 133–149, 2004. Chen CH, Hansma HG. Basement membrane macromolecules: Insights from atomic force microscopy. J. Struct. Biol. 131, 44–55, 2000. Miyagawa A, Kobayashi M, Fujita Y, Hamdy O, Hirano K, Nakamura M, Miyake Y. Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy. Cornea 20, 651–656, 2001. Paige MF, Rainey JK, Goh MC. A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy. Micron 32, 341–353, 2001. Raspanti M, Congiu T, Guizzardi S. Tapping-mode atomic force

411

412

11 Nanotechnological Approaches to Cancer Diagnosis microscopy in fluid of hydrated extracellular matrix. Matrix Biol. 20, 601–604, 2001. 190 Taatjes DJ, Quinn AS, Bovill EG. Imaging of collagen type III in fluid by atomic force microscopy. Microsc. Res. Tech. 44, 347–352, 1999. 191 Kassner A, Tiedemann K, Notbohm H, Ludwig T, Morgelin M, Reinhardt DP, Chu ML, Bruckner P, Grassel S. Molecular structure and interaction of recombinant human type XVI collagen. J. Mol. Biol. 339, 835–853, 2004. 192 Kassner A, Hansen U, Miosge N, Reinhardt DP, Aigner T, Bruckner-Tuderman L, Bruckner P, Grassel S. Discrete integration of collagen XVI into tissue-specific

collagen fibrils or beaded microfibrils. Matrix Biol. 22, 131–143, 2003. 193 Sun HB, Smith GN, Jr., Hasty KA, Yokota H. Atomic force microscopybased detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices. Anal. Biochem. 283, 153–158, 2000. 194 Lin H, Clegg DO, Lal R. Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope. Biochemistry 38, 9956– 9963, 1999. 195 Stroh C, Wang H, Bash R, Ashcroft B, Nelson J, Gruber H, Lohr D, Lindsay SM, Hinterdorfer P. Single-molecule recognition imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 101, 12 503–12 507, 2004.

413

Index 2-in-1 system, gold nanoparticles 97–98

a AbVF see VEGF antibody-2C3 active targeting 65, 124, 185 – folate receptors 66 – lectins 65 affinity mass spectrometry – nanoprobe-based 338–369 – principles 349 AFM see atomic-force microscopy agonists, LHRH 192 AMI-121 (Ferumoxsil) 150–151 AMI-227 (Combidex) 151 AMI-25 (Feridex) 151 AMI25 iron particles 184 aminated particles, bioconjugation 64 amine-containing silane compound (APTS) 62 amine-reactive fluorescein isothiocyanate 62 aminopropyltriethoxysilane (APTS) 330–331 amorphous silica nanoparticles 61–65 angiogenesis – and cancer 88–96 – blocked extracellular matrix breakdown 91 – molecular MRI 159 Annexin V-FITC 267–268 antibodies 27 – monoclonal 26–27 antibody–gold conjugate 61 antibody-2C3 97–98 antibody-functionalized nanotubes 320 antibody-mediated tumor targeting 66 anti-carcinoembryonic antigen (CEA) antibody 163–164 APO-BrdU TUNEL assay 245–247 apoptosis – cyt-c-induced 267–268 – molecular MRI 160

APTS see aminopropyltriethoxysilane artificial peptides, nanotubes 315–317 assay methods 381 atomic-force microscopy (AFM), proteolytic activity detection 396–400 Au NP see gold nanoparticles avidin–biotin binding, bioconjugation 65 avidin–gold conjugate 61

b barcodes – multimetal 293 – nano 325–327 bFGF, activity 94 bifunctional dendritic clusters 30 bioactivity, dendrimers 20–21 bio-barcode amplification assay 347 biocompatibility – dendrimers 22 – gold nanoparticles 99–104 bioconjugation, gold nanoparticles 60, 64–65 – Qdot 57 biodistribution studies 28 biointeraction, magnetic field-assisted 329 biomarkers, targeting 25–27 biomolecule-conjugated nanoparticles 339 biosensors, SWNTs 269, 312–313 blood analysis, nanoprobe-based affinity mass spectrometry 359–364 BNCT see boron neutron capture therapy BOM see bubbles-overlapping mode (BOM) boron neutron capture therapy (BNCT) 29 bovine serum albumin (BSA) 238, 248–252, 361–362 breast cancer cells (MCF7) 253–258 breast cancer markers 68 BSA see bovine serum albumin BSA-MNTs 329–330 bubbles-overlapping mode (BOM) 109

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

414

Index cellular functions, local proteolytic activity 389–390 cellular microdomains, proteolytic activity 388 c cellular uptake, of nanoparticles 188 C60 273 charged-coupled device (CCD) technology 48 CACO-2 cells 245–246 chemical separation, magnetic field-assisted cadmium selenide (CdSe) 52 329 cadmium sulfide (CdS), crystalline 52 chemoselective ligation reaction 32 cancer chemoselective targeting, drugloaded – chemotherapeutic approaches 179 dendrimers 32 – conventional approaches to detection 177– chemotherapeutic approaches, cancer 179 178 cis-platin 88 – multidrug resistance 179 CLIO see crosslinked iron oxides – statistics 175–176 CLIO–antibody conjugates 160 cancer cell death, targeted 310–311 cancer cell migration, local proteolytic activity coated nanoparticles 181 colon cancer (CACO-2) 245–246 389 Combidex (AMI-227) 151 cancer detection, nanotechnology 106–108 computed tomography (CT) scanning 47 cancer diagnosis confining proteolytic activity, mechanisms – carbon nanotubes 290–293 385–388 – nanotechnological approaches 377–400 confocal fluorescence images, MCF7 cells cancer imaging 44–72 256–257 – magnetic resonance nanoparticle probes conjugation strategies, polymeric 17–18 147–168 contrast agents – molecular MRI 163–165 – magnetic nanoparticle 150–156 – techniques 46–48 – nanoparticle-based 53–64 cancer marker protein profiling, nanoprobe– nanoparticles-based 52 based 338–369 – non-hydrolytically synthesized 154–156 Candida rugosa lipase 309 – optical 50 carbohydrate epitopes, metabolically– silica- or dextran-coated iron oxide 150– engineered 31 151 carbon nanotubes (CNTs) 232–277, 285–295, – SPION 204 310–314 controlled drug release, magnetic nanotubes – cancer diagnosis 290–293 330–331 – cancer treatment 293–295 conventional synthetic polymers, comparison – differential functionalization 319–320 to dendrimers 5 – drug delivery systems 294 convergent synthesis, of dendrimers 11 – enzyme-coated 290 core–shell nanoparticle design 54 – functionalized 313–315 COS-7 cells 323 – monodisperse 318 covalent conjugation strategies, dendrimers carboxylated particles, bioconjugation 64 17–19 carcinoembryonic antigen (CEA) 163–164 C-reactive protein (CRP) 353–354, 356–358, cardiac infarct 158–159 363–364 cascade reactions, dendrimer synthesis 10– crosslinked iron oxide (CLIO) particles 127– 11 128 CCD see charged-coupled device CRP see C-reactive protein CDDP 184 crystalline cadmium sulfide (CdS) 52 CdSe NP 343 CT see computed tomography CEA see carcinoembryonic antigen Cy3-DNA functionalized SWNT 311–312 cell signaling, local proteolytic activity 389 Cy3-DNA-SWNT complexes, NIR laser light cell surface receptors, for protease binding excitation 271 386 cyanogen bromide chemistry, bioconjugation cell tracking, iron oxides 128 65 cell-labeling, nanoparticle-based 55 buckyball C60 273 building blocks, dendrimers 12–13

Index cyclic peptides 316 cysteine switch 382 cyt-c-induced apoptosis 267–268 cytochrome c 267–268 cytotoxicity, SWNTs 272–275

d

DABCYL (4,40 -dimethylaminophenylazobenzoic acid) 108 deformability, of dendrimers 8 dendrimer-based cancer-targeted drug delivery 18 dendrimer-based drug delivery 9 dendrimers 1–33 – basic properties and applications 3–9 – bioactivity 20–21 – biocompatibility 22 – building blocks 12–13 – cascade reactions 10–11 – cascade synthesis 11 – chemoselective targeting 32 – comparison to conventional synthetic polymers 5 – comparison to proteins 6–7 – convergent synthesis 11 – covalent conjugation strategies 17–19 – deformability 8 – divergent synthesis 11 – drug delivery 15–23 – encapsulation of guest molecules 16–17 – flexibility 8 – gene delivery 16 – generations 3–5 – heterogeneously-functionalized 13–14 – immunogenicity 22 – internal cavities 16 – kidney filtered 21 – labeled 28 – magneto 152–153 – multifunctional 25–27 – oligosaccharide coatings 14 – paramagnetic 133 – paramagnetic polyamidoamine 133 – passive accumulation 24 – self-immolative 20 – surface modification 14 – synthesis 10–14 – toxicity 22–23 – vaccines 19 – water solubility 22 dendritic clusters, bifunctional 30 dendritic PEI 259–260 dextran crosslinked iron oxide (CLIO) particles 127–128

dextran-coated iron oxide contrast agents 150–151 dextran-coated MIONs 188 dextran-coated USPIOs 294 DHLA see dihydroxylipoic acid diethyl tetraminepentaacetic acid (DTPA) 187 differential functionalization, of nanotubes 319–320 diffuse optical tomography (DOT) 50 dihydroxylipoic acid (DHLA)-capped quantum dots 67 2,3-dimercaptosuccinic acid (DMSA) 156– 157 2,3-dimercaptosuccinic acid (DMSA)-coated water-soluble Fe3 O4 iron oxide (WSIO) nanocrystals 306 4,40 -dimethylaminophenylazobenzoic acid (DABCYL) 108 disulfide bonding, bioconjugation 65 divergent synthesis, of dendrimers 11 DMSA see 2,3-dimercaptosuccinic acid DNA, single-stranded DNA hybridization 326–327 DNA microarray analysis 100 DNA nanotubes 324–325 DNA–dendrimer conjugates 31 DOT see diffuse optical tomography DOTA see 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid drug delivery – dendrimer-based 9 – dendrimer-based cancer-targeted 18 – dendrimers 15–23 – nanoparticles as vehicles 181–188 – SWNT transporters 252–269 – targeted 313–314 – to tumors 179 drug delivery systems, carbon nanotubes 294 drug molecules, selective extraction 320–321 drug release, controlled 330–331 drug uptake, magnetic nanotubes 330–331 DTPA see diethyl tetraminepentaacetic acid dye-doped nanoparticles 52 dye-doped silica nanoparticles – cancer imaging 69 – contrast agents 61–64

e ECM see extra cellular matrix ECM binding, of proteases 387 ECM-components, atomic-force microscopy (AFM) 397–400 EFGR see epidermal growth factor receptor

415

416

Index EGF 94, activity 94 electrical resistance breakdown assay 394 electronic structure, semiconducting SWNTs 270 ELISA see enzyme linked immunosorbent assay encapsulation, of guest molecules 16–17 endocytosis, receptor-mediated 197–198 endogenous protease inhibitors 383 endosomal rupture 267 endothelial proliferation, inhibition 90 engineered transferrin receptor (ETR) 163 enhanced permeability and retention (EPR) effect 24–25, 125 – passice targeting 66 enzyme linked immunosorbent assay (ELISA) 264–265, 345–346, 356, 364, 391 enzyme-coated carbon nanotubes 290 epidermal growth factor receptor (EFGR) 107 epidermal growth factor receptor (EGFR) 345–346 EPR see enhanced permeability and retention extra cellular matrix (ECM) components – AFM 396–400 – angiogenesis 89

folate, targeting 26 folate receptor (FR) – active targeting 66 – HeLa cells 312 foot and mouth disease virus (FMDV), peptides 264–265 fragment binding antigen see Fab FTP see 4-[3-(4-fluorophenyl)-2-hydroxy-1[1,2,4]-triazol-1-yl-propyl]benzonitrile fullerenes, paramagnetic 132 functionalized carbon nanotubes 313–315 – antibody 320 – differentially 319–320 – see also single-wall nanotubes (SWNT) functionalized SWNT, Cy3-DNA 311–312

g

gadolinium chelates 187 gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) 184, 187 Gd-DTPA see gadoliniumdiethylenetriaminepentaacetic acid gene delivery – dendrimers 16 – SWNT transporters 252–269 gene expression, molecular MRI 161–162 gene transfection, SWNT transporters 258– 262 f generations, of dendrimers 3–5 Fab (fragment binding antigen) conjugates GFP see green fluorescent proteins 158, 320–322 gfSNTs see green fluorescent silica FePt NP 342 nanotubes Feridex (AMI-25) 151 glycosylation 14 ferrofluids 187 – protease activity 384 Ferumoxsil (AMI-121) 150–151 glycosylation abnormalities 31 FET see field-effect transistor GnRH see gonadotropin releasing hormone field-effect transistors (FETs), SWNT 287, – see also ligand luteinizing hormone releasing 290–291 hormone FITC see fluorescein isothiocyanate gold conjugates 96–98 FITC-AmB modified MWNTs 315 gold nanoparticles (Au NPs) 86–110, 342– FITC-doped silica nanoparticles 69 346, 354 flexibility, of dendrimers 8 – anti-angiogenic properties 91 fluorescein isothiocyanate (FITC) 308 – biocompatibility 99–104 – amine-reactive 62 – bioconjugation 64–65 fluorescence microscopy, single-molecule – cancer imaging 68 248–250, 253 fluorescence-mediated molecular tomography – cellular adhesion effects 99 – conjugates 60–61 (FMT) 49–50 – contrast agents 57–60 fluorescent quantum dots 52 4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]-triazol- – internalization 101 – in-vivo effects on angiogenesis 95 1-yl-propyl]benzonitrile (FTB) 321 – local biological effects 99 5-Fluorouracil (5-FU) 330–331 – near-normal culture conditions 103–104 FMDV see foot and mouth disease virus – serum-free conditions 101–102 FMT see fluorescence-mediated molecular – synthesis 58–59 tomography

Index – synthetic approaches 105–106 – systemic and remote effects 99 – transcription pattern 101–104 – VEGF165 92–95 gold precursors 58 gold radioisotopes 96 gold–TNF conjugates 96 gonadotropin releasing hormone (GnRH) see ligand luteinizing hormone releasing hormone GPBR see G-protein binding receptor G-protein binding receptor (GPBR) 193– 196 green fluorescent proteins (GFP) 49, 51 green fluorescent silica nanotubes (gfSNTs) 323–324 guest molecules, encapsulation 16–17

i

ibuprofen 330–331 IKVAV see isoleucine-lysine-valine-alaninevaline imaging – of pericellular proteolytic activity 377–400 – optical 44–72 – with dendrimers 28 imaging agents, cancer treatment 294 immunoassay – nanoprobe-based 356–358 – nanoscale 359–360 immunocytochemistry techniques, metastase detection 178 immunotherapy 26–27 implant, effects by host 100 iMQC see intermolecular multiple-quantum coherence In2 O3 nanowires 312–313 h half-cylinder wrapping 243–245 induced toxicity 23 HBV see hepatitis B virus infarct, molecular MRI 158 HEK 293 cells 198 inflammation, molecular MRI 158 HeLa cells 259, 311–312 a v b 3 -integrin, MRI 130 heparin sepharose 93 intermolecular multiple-quantum coherence hepatitis B virus (HBV) antibody 345–346 (iMQC) 209–210 HER2 68 internal cavities, dendrimers 16 Her-2/neu receptor 343–344 internalization, gold nanoparticles 101 Herceptin conjugates, WSIO 164–167 invasion assays, tumor cell 393–394 heterogeneously-functionalized dendrimers iron oxide contrast agents, silica- or dextran13–14 coated 150–151 HL60 cells 267 iron oxide MNPs 342, 344 host, effects on the implant 100 iron oxide nanoparticles 187 hot solution-phase mediated Qdot synthesis – cell tracking 128 55 – ligand-directed targeting 127 HSA see human serum albumin – molecular MRI 157–165 human breast cancer – non-hydrolytically synthesized 154–156 – LHRH-SPION uptake 206–207 – poly(ethylene glycol) (PEG)-ylated 155 – MDA-MB-435 67 – siloxane–poly(ethylene glycol) (PEG) coated human embryonic cells see HEK 293 cells 156 human IgG 329–330 – superparamagnetic 203–209 human mammary epithelial tumor (MDA-MB- – ultrasmall superparamagnetic 294 231) 67 – water-soluble 156–157 human plasma, enrichment of target antigen isoleucine-lysine-valine-alanine-valine (IKVAV) 362–363 318 human plasma analysis, nanoprobe-based ISZ see in situ zymography affinity mass spectrometry 359–364 human serum albumin (HSA) 361–362 k HUVEC cells 93–94, 160 kidney, dendrimer filter 21 – gold nanoparticles 101–104 – migration 95 l – transcription pattern 101–104 labeled dendrimers 28 hydrochloroauric acid 58 labeled substrates, proteolytic activity detection hyperthermia, LHRH-bound magnetic 396 nanoparticles 202 lectin–gold conjugate 61

417

418

Index lectins, active targeting 65 LHRH see luteinizing hormone releasing hormone LHRH conjugated magnetic nanoparticles 174–211 LHRH receptors 175, 189–200 – cancer types 176 LHRH-bound magnetic nanoparticles 201– 209 LHRH-SPION nanoparticles 201 – subcellular distribution 208 LHRH-SPION uptake, receptor-targeted 205–209 LHRH-SPION-hecate, destruction of metastases 203 ligand-directed targeting, iron oxides 127 ligation reaction, chemoselective 32 lipid rafts 388 liposomes – magneto 152–153 – paramagnetic 131 local proteolytic activity 380–390 lock and key function 7 LPA see lysophosphatidic acid LPC see lysophosphatidylcholine LPG see lysophosphatidylglycerol luteinizing hormone releasing hormone (LHRH) 174–211 – agonists 192 – amino acid sequences 191 – analogs 192 – function–signal transduction pathways 194–196 – ligand 189–200 lysophosphatidic acid (LPA) 241 lysophosphatidylcholine (LPC) 241–247 lysophosphatidylglycerol (LPG) 241–245 lysophospholipids, single-wall nanotubes (SWNT) 239–246 lytic peptides, LHRH-bound magnetic nanoparticles 203

m macrophage (THP-1) cells 245–246 MAGIC see magnetism-base interaction capture magnetic field-assisted chemical separation 329 magnetic microbeads (MMP) 359–360 magnetic nanoparticles (MNP) 342–344 – biomedical diagnostic applications 307– 309 – contrast agents 150–156 – for biomedical applications 305–309

– – – – –

LHRH conjugated 174–211 LHRH-bound 201–209 suppression of nonspecific binding 361 vancomycin-conjugated 309–310 MEG-protected antibody-conjugated 350– 351 magnetic nanotubes (MNT) 328–332 magnetic resonance (MR) 47, 148 magnetic resonance imaging (MRI) 121– 138 – higher resolution 188 – magnetic nanoparticles 305–306 – molecular 157–165 – nanoparticle probes 147–168 – nanoparticles 186–187 – SPION contrast agents 204 – targeting mechanisms 124–125 magnetism-base interaction capture (MAGIC) 308–309 magnetodendrimers 152–153 magnetoferritin 152 magnetoliposomes 152–153 MALDI-TOF MS see matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometric immunoassay (MSIA) 349–350 mass spectrometry – nanoprobe-based 338–369 – principles 348–349 – time-of-flight 348 matrix breakdown, extracellular 91 matrix metalloproteinase 2 (MMP-2) 49 matrix metalloproteinase inhibitors (MMPIs) 378–379 matrix metalloproteinases (MMPs) 382–396 – membrane-type 386 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS), principles 348 matrix-metalloprotease deficient mice 390 MCF7 cells 253–258, 274–275 MCF-7 cells 202 MDA-MB-231 67 MDA-MB-435 67 MDA-MB-435S.luc cancers 206–209 MEG see methoxy-ethylene glycol MEG-protected antibody-conjugated MNPs 362 membrane-type matrix metalloproteinases (MT-MMPs) 386–391, 396 metabolically-engineered carbohydrate epitopes 31 metal nanoparticles 344–347

Index metalloproteinases, membrane-type 386 metastase detection 177–178 – nanoparticles 186–188 metastasis, formation 377–379 methicillin-resistant S. aureus (MRSA) 316 methoxy-ethylene glycol (MEG) 350–351 microarray analysis, DNA 100 microbeads, SPIO 345 microcantilevers, cancer diagnosis 292 microemulsion (W/O) method, reverse 62 microscale immunoassay, comparison to nanoscale immunoassay 359–360 MION see monocrystalline iron oxide nanoparticles mix-and-match strategy, bifunctional dendritic clusters 30 MMP see matrix metalloproteinase MMP-2 see matrix metalloproteinase 2 MMP-9 389 MMPI see matrix metalloproteinase inhibitor MNT see magnetic nanotubes molecular MRI, iron oxides 157–165 molecular recognition 7 monoclonal antibodies, targeting 26–27 monocrystalline iron oxide nanoparticles (MION) 127, 206 – dextran-coated 188 monodisperse nanotubes 318 MR see magnetic resonance MRI see magnetic resonance imaging MRI imaging agents 28 MRSA see methicillin-resistant S. aureus MT-MMPs see membrane-type matrix metalloproteinases multi-CRAZED sequence 209–210 multidentate phosphine oxide ligands 155 multidrug resistance, cancer 179 multifunctional dendrimers 25–27 multifunctional nanoparticles 52 multifunctional nanotubes 304–332 multimetal barcodes 293 multiphoton microscopy, proteolytic activity detection 395 multiphoton microscopy (MPM) 395 multiplex assay 364–369 – screening for patient and healthy individuals 367–368 – workflow 366 multiplexing diagnosis, nanobarcodes 325– 327 multi-wall nanotubes (MWNT) 286–287 – functionalized 314–315 – PEI 258–260 MWNTs see multi-wall nanotubes

n nanobarcodes, for multiplexing diagnosis 325–327 nanocantilevers 285–295 nanocrystals 306 nano-devices, dendrimers 15 nanogold see gold nanoparticles nanoparticle design, core–shell 54 nanoparticle-based cell-labeling 55 nanoparticle-based contrast agents 53–64 nanoparticle-based optical contrast agents 70 nanoparticles – as vehicles for drug delivery and diagnosis 181–188 – biomolecule-conjugated 339 – classification 135 – coated 181 – dye-doped 52 – fabrication and biomedical applications 339–347 – iron oxide 187 – LHRH conjugated magnetic 174–211 – LHRH-bound magnetic 201–209 – LHRH-SPION 201 – magnetic 305–309, 361 – metallic 344–347 – metastase detection 186–188 – MRI 305–306 – multifunctional 52 – non-hydrolytically synthesized iron oxide 154–156 – optical imaging 44–72 – paramagnetic 128–133 – perfluorocarbon 129–130 – phosphor 70 – poly(ethylene glycol) (PEG)-ylated iron oxide 155 – polymer 134 – siloxane–poly(ethylene glycol) (PEG) coated iron oxide 156 – superparamagnetic 126–128 – tumor detection 186–188 – ultrasmall superparamagnetic iron oxide 294 – vancomycin-conjugated magnetic 309–310 – water-soluble iron oxide 156–157 nanoparticles-based contrast agents 52 nanoprobe-based affinity mass spectrometry (NBAMS) – for cancer marker protein profiling 338– 369 – kinetic study of the nanoscale immunoreaction 356 – proof-of-principle experiment 353–355 – workflow 351–352

419

420

Index nanoprobe-based immunoassay 356–358 nanorods 285–295 – template-synthesized 318–332 nanoscale immunoassay, comparison to microscale immunoassay 359–360 nanoscale immunoreaction, kinetic study 356 nanosensors, SWNT 312–313 nanotechnology, cancer detection 106–108 Nanotechnology for Cancer Diagnosis and Therapy initiative 233 nanotube carriers, silica 322 nanotubes 285–295 – carbon 285–295 – differential functionalization 319 – DNA 323–324 – magnetic 328–332 – multifunctional 304–332 – noncarbon 287 – paramagnetic 133 – self-assembled 289 – template-synthesized 318–332 nanowires 285–295 – multifunctional 304–332 nanowires (NW), In2 O3 312–313 NBAMS see nanoprobe-based affinity mass spectrometry nearinfrared (NIR) light 48 near-infrared fluorescent (NIRF) probes 394–395 net proteolytic activity, classical methods for quantification 391–396 NHS see N-hydroxysuccinimide N-hydroxysuccinimide (NHS) 235 NIR see nearinfrared NIR laser light excitation, Cy3-DNA-SWNT complexes 271 4-nitrophenol 330–331 NMR see nuclear magnetic resonance noncarbon nanotubes 287 – cancer diagnosis 291 non-hydrolytically synthesized iron oxide nanoparticles 154–156 non-invasive techniques, metastase detection 178 nonspecific binding, on magnetic nanoparticles 361 NP see nanoparticles nuclear magnetic resonance (NMR) 122 – see also magnetic resonance

o oligomerization, protease activity 384 oligosaccharide coatings, of dendrimers 14

opsonization 181 optical contrast agents 50 – nanoparticle-based 70 optical imaging – contrast agents 50 – of cancer 44–72

p PA see peptide-amphiphile PAMAM see poly(amidoamine) paramagnetic nanoparticles 128–133 paramagnetic polyamidoamine dendrimers 133 passive targeting 66, 124, 183–184 PEG see poly(ethylene glycol) PEI see polyethylenimine peptide coated quantum dots 67 peptide nanotubes 315 peptide nucleic acids (PNA) 235 peptide-amphiphile (PA) nanofibers 316–317 peptides, artificial 315–317 perfluorocarbon (PFC) nanoparticles 129– 130 pericellular proteolytic activity, nanotechnological approaches 377–400 PET see positron emission tomography PFC see perfluorocarbon phagokinetic track assay, quantum dot 67 phosphine oxide ligands, multidentate 155 phospholipids (PL) 261–262 – single-chained 239 phosphor nanoparticles, up-converting 70 phosphotyrosine phosphatase (PTP) 196 PL see phospholipids plasma protein profiling, in normal individuals and in patients 364 PL-PEG-SWNT complexes 261–262 PNA see peptide nucleic acids poly(amidoamine) (PAMAM) 3–7 – chemical composition 11 – induced toxicity 23 – paramagnetic dendrimers 133 – uptake by target cell 21–22 poly(ethylene glycol) (PEG) poly(ethylene glycol) (PEG)-ylated iron oxide nanoparticles 155 poly(rU) RNA 239, 248–258 polyethylenimine (PEI), dendritic 259–260 polymer nanoparticles 134 polymers – conjugation strategies 17–18 – conventional synthetic 5 positron emission tomography (PET) 47 pro-drugs

Index – covalently-delivered 19 – encapsulated 17 proof-of-principle experiment, affinity mass spectrometry 353–355 prostate specific antigen (PSA) 312–313 prostate-specific antigen (PSA) 109, 292, 346 protease activity, regulation 382–385 protease assays 381 protease binding – cell surface receptors 386 – ECM 387 protease inhibitors, endogenous 383 protein A–gold conjugates 60 protein delivery, SWNTs 266–268 protein profiling – human plasma analysis 359 – nanoprobe-based 338–369 protein trafficking 385 proteins, comparison to dendrimers 6–7 proteolytic activity – AFM 396–400 – classical methods for quantification 391– 396 – confining 385–388 – in vitro detection 396 – in vivo detection 394–396 – local 380–390 – pericellular 377–400 PSA see prostate-specific antigen PT see thiol-derivatized PEG PTP see phosphotyrosine phosphatase PT-PCR 103–104

q Qdot synthesis – hot solution-phase mediated 55 – reverse-micelle mediated 56 quantum dots (Qdots) 133 – bioconjugation 57 – cancer imaging 66–67 – contrast agents 53–56 – fluorescent 52 – peptide coated 67 – phagokinetic track assay 67 – semiconductor 344 – surface passivation 56 – TOP/TOPO-capped 56

r radioisotope labeling assay 255–258 radioisotopes, gold 96 receptor-mediated endocytosis 197–198 reflectance fluorescence imaging 49 relaxation times, MRI 123, 148

RES see reticuloendothelial system reticuloendothelial system (RES) 126, 154 reverse microemulsion (W/O) method, dyedoped silica nanoparticles 62 reverse transcriptase polymerase chain reaction (PT-PCR) 103–104 reverse-micelle mediated Qdot synthesis 56 RNA – HUVEC cells 103 – poly(rU) 239, 248–252 – small interfering 248 RNA interference, SWNT transporters 261– 262 RNA polymers, solubilization of SWNTs 239 RNA translocation, SWNT transporters 253–257 RT-PCR 391 – metastase detection 178 rule-of-five guidelines 15

s SAP see serum amyloid P second-harmonic generation (SHG) – imaging microscopy 395–396 – proteolytic activity detection 395 seed mediated route, gold nanoparticle synthesis 59 SELDI see surface-enhanced laser desorption/ionization self-assembled nanotubes 289 self-immolative dendrimers 20 semiconducting SWNTs, electronic structure 270 semiconductor quantum dots 344 sensors see biosensors SERS see surface-enhanced Raman scattering serum amyloid P (SAP) 351, 353, 356–358, 363–364 serum tumor markers 340–341 shell-crosslinked nanoparticles 135 SHG see second-harmonic generation sialic acid engineering 32 silica nanoparticles, dye-doped 61–64, 69 – FITC-doped 69 silica nanotube carriers, for DNA transfection 322 silica nanotubes, green fluorescent 323–324 silica-coated iron oxide contrast agents 150– 151 silicon dioxide nanoparticles 61–65 silicon nanowires (SiNWs) 292 siloxane–poly(ethylene glycol) (PEG) coated iron oxide nanoparticles 156

421

422

Index silver nanowires 289 single-chained phospholipids 239 single-mismatch detection, of cancer 107 single-molecule fluorescence microscopy (SMFM) 248–250, 253 single-photon emission CT (SPECT) 48 single-stranded DNA (ssDNA) 238–239 single-wall nanohorns (SWNHs) 287 single-wall nanotubes (SWNT) 232–277 – biocompatibility 233–246 – biomolecular complexes 247–251 – biosensing 269 – cancer treatment 269–277 – covalent modifications 236 – Cy3-DNA functionalized 311–312 – cytotoxicity 272–275 – functionalization 235–237, 311–312 – gene and drug delivery 252–269 – lysophospholipids 239–246 – nanosensors 312–313 – noncovalent modifications 237–246 – oxidation 235 – protein delivery 266–268 – semiconducting SWNTs 270 – solubility 234–246 – water-soluble 237–239 SiNWs see silicon nanowires siRNA see small interfering RNA small interfering RNAs (siRNAs) 248, 261– 263 SMFM see single-molecule fluorescence microscopy solubilization of SWNTs 237–247 SPECT see single-photon emission CT SPIO see superparamagnetic iron oxides SPIO microbeads (SPIO-MBs) 345 SPIO–C2 conjugates 161 SPION see superparamagnetic iron oxide nanoparticles SPION contrast agents, targeted delivery 204 SPR see surface plasmon resonance ssDNA see single-stranded DNA Starburst clusters 3 Stober’s method, dye-doped silica nanoparticles 62 streptavidin–gold conjugate 61 subcellular distribution, of LHRH-SPIONs 208 superparamagnetic iron oxide nanoparticles (SPION) 201 – LHRH 203–209 superparamagnetic iron oxides (SPIO) 126

superparamagnetic nanoparticles 126–128 surface modification, dendrimers 14 surface plasmon resonance (SPR) 58 surface-enhanced laser desorption/ionization (SELDI) 349 surface-enhanced Raman scattering (SERS) 108 surfactants, solubilization of SWNTs 244 SWNHs see single-wall nanohorns SWNT see single-wall nanotubes SWNT field-effect transistors (FETs) 287, 290–291 SWNT transporters, gene and drug delivery 252–269 SWNT–biomolecular complexes, diffusion 247–251 SWNT-BSA complex 248–252 SWNT-Cy3-DNA complexes, NIR laser light excitation 271 SWNT-LPC complex 241–247 SWNT-LPG complex 241–245 SWNT-PL-PEG complexes 261–262, 272 SWNT-poly(rU) complex 248–258

t TAAs see tumor associated antigens target antigen, human plasma 362–363 target cells 21 targeted cancer cell death, carbon nanotubes 310–311 targeted delivery, of nanoparticles 188 targeted drug delivery, carbon nanotubes 313–314 targeting, active 65, 185 – chemoselective 32 – ligand-directed 127 – of metabolically-engineered carbohydrate epitopes 31 – of specific biomarkers 25–27 – passive 66, 183–184 – tumor cells 182–185 targeting mechanisms, MRI 124–125 TAT-HA2 peptide 308 template-synthesized nanotubes and nanorods 318–332 TEOS see tetraethylorthosilicate 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA) 318 tetrachloroauric acid (HAuCl4 ) 339 tetraethylorthosilicate (TEOS) 62 tetramethylorthosilicate (TMOS) 62 thiol-derivatized PEG (PT) 97 THP-1 cells 245–246

Index time-of-flight (TOF) mass spectrometry, matrixassisted laser desorption/ionization 348 TIMPs see tissue inhibitors of metalloproteases tissue inhibitors of metalloproteases (TIMPs) 383–384 TMOS see tetramethylorthosilicate TNF see tumor necrosis factor TOF see time-of-flight TOP/TOPO-capped Qdots 56 TOPO see trioctylphosphine oxide transcription, global pattern 101–104 trioctylphosphine oxide (TOPO) 344 tumor associated antigens (TAAs) 26 tumor cell invasion assays 393–394 tumor detection, nanoparticles 186–188 tumor markers 340–341 tumor morphology 180 tumor necrosis factor (TNF) 97 tumor targeting 182–185 – antibody-mediated 66 tumor–host conspiracy 388

u U1A antigen-Tween conjugates 269 ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles 126 – dextran-coated 294 ultrasonography (US) 48 up-converting phosphor nanoparticles 70 urokinase-type plasminogen activator (uPA) 387 US see ultrasonography USPIO see ultrasmall superparamagnetic iron oxide

v vaccine delivery, SWNTs 263–265 vaccines, dendrimer conjugates 19 vancomycin-conjugated magnetic nanoparticles 309–310 vascular endothelial growth factor (VEGF) 89, 159–160 VEGF see vascular endothelial growth factor VEGF antibody-2C3 (AbVF) 97–98 VEGF121, activity 94 VEGF165 – activity 94 – inactivation 92 – signaling events 94–95

w W/O see water-in-oil (W/O) water-in-oil (W/O) microemulsion 63 water-soluble Fe3 O4 iron oxide (WSIO) nanocrystals, 2,3-dimercaptosuccinic acid (DMSA)-coated 306 water-soluble iron oxide nanoparticles (WSIO) 156–157 water-soluble SWNTs 237–239 whole blood analysis, nanoprobe-based affinity mass spectrometry 359–364 WSIO see water-soluble iron oxide nanoparticles WSIO–antibody conjugates 343 WSIO–Herceptin conjugates 164–167, 307

z zinc selenide (ZnSe) 52 zinc sulfide (ZnS) 52 in situ zymography (ISZ) 392–393

423