Nanocosmetics and Nanomedicines: New Approaches for Skin Care

Nanocosmetics and Nanomedicines Ruy Beck, Silvia Guterres, and Adriana Pohlmann (Eds.) Nanocosmetics and Nanomedicin...

Author: Ruy Beck | Silvia Guterres | Adriana Pohlmann

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Nanocosmetics and Nanomedicines

Ruy Beck, Silvia Guterres, and Adriana Pohlmann (Eds.)

Nanocosmetics and Nanomedicines New Approaches for Skin Care

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Editors Ruy Beck Federal University of Rio Grande do Sul Faculdade de Farmácia Dept. de Produção e Controle de Medicame Av. Ipiranga 2752 90610-000 Porto Alegre Brazil E-mail: [email protected]

Adriana Pohlmann Federal University of Rio Grande do Sul Institut of Chemistry Departamento de Química Orgânica Av. Bento Gonçalves 9500 91501-970 Porto Alegre Brazil Email: [email protected]

Silvia Guterres Federal University of Rio Grande do Sul Faculty of Pharmacy Dept. de Produção e Controle de Medicame Av. Ipiranga 2752 90610-000 Porto Alegre Brazil E-mail: [email protected]

ISBN 978-3-642-19791-8

e-ISBN 978-3-642-19792-5

DOI 10.1007/978-3-642-19792-5 Library of Congress Control Number: 2011923550 c 2011 Springer-Verlag Berlin Heidelberg This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Scientific Publishing Services Pvt. Ltd., Chennai, India. Cover Design: eStudio Calamar S.L. Printed on acid-free paper 987654321 springer.com

Forward

The interaction of particles with biological systems unravels a series of new mechanisms not found for molecules: altered bio-distribution, chemically reactive interfaces, and the combination of solid-state properties and mobility. The tremendous progress and recent advances of nanotechnology has not been accompanied by sufficient studies of nanomaterial toxicity even though they possess unique, completely new properties. One certainty: the toxicity of nanomaterials can neither be extrapolated from the toxicity of bulk materials nor from the toxicity of their constituents in molecular/ionic form. Therefore the research on nanotoxicity is of extremely high scientific, social, and economic value in particular for human applications. As nanomaterial-based products enter the market, there is an urgent need for related research in order to prevent dramatic consequences of any healthoriented issues caused by nanotechnology-driven products. Indeed, the results of research on nanotoxicity have profound significance because the design of nanomaterials used in industry and consumer products should be based on the outcome of such work. This is more urgent in view of the infancy of these studies and that many of the data available on the biological effects of nanomaterials do not always come from studies that can be considered reliable. The responsibilities are enormous not only in social terms but also this research has multi-billion dollar significance for industry and an even greater value for consumers and health care. Therefore one of key questions is to understand the behavior of nanoparticles in biological systems that will certainly opens up new directions for medical treatments and is essential for the development of safe nanotechnology. The widespread use of untested nanomaterials can cause an enormous cost of care for health problems for the word population. In particular, in Brazil the applications and uses of “bio” stands among the most productive and successful area of the Brazilian “nano” initiative in both academia and industry. Indeed, not only in terms of the number and impact of the papers (more than a half of the Brazilian scientific papers in “nano” are from the nano bio area) but also the numbers of commercial products that are already cases of success. With this in mind Pohlmann, Beck and Guterres have ingeniously collected a series of state of the art contributions on an important and hot area on the use of nanomaterials on the use of nanocosmetics and nanomedicines for skin care. The book NANOCOSMETICS AND NANOMEDICINES: New approaches for skin care will certainly stands as the landmark and part of the classical literature on nanomaterials and skin care. Jairton Dupont, Tarragona, 30th January 2011.

Contents

Part I: Fundamentals of Skin Delivery Chapter 1: Transport of Substances and Nanoparticles across the Skin and in Vitro Models to Evaluate Skin Permeation and/or Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renata V. Contri, Luana A. Fiel, Adriana R. Pohlmann, S´ılvia S. Guterres, Ruy C.R. Beck

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Chapter 2: Rheological Behavior of Semisolid Formulations Containing Nanostructured Systems . . . . . . . . . . . . . . . . . . . . . . . . . Marta P. Alves, Renata P. Raﬃn, Solange B. Fagan

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Part II: Nanocarriers for Skin Care and Dermatological Treatments Chapter 3: Polymeric Nanocapsules: Concepts and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fernanda S. Poletto, Ruy C.R. Beck, S´ılvia S. Guterres, Adriana R. Pohlmann Chapter 4: Topical Application of Nanostructures: Solid Lipid, Polymeric and Metallic Nanoparticles . . . . . . . . . . . . . . . . . Nelson Dur´ an, Zaine Teixeira, Priscyla D. Marcato

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Chapter 5: Lipid Nanoparticles as Carriers for Cosmetic Ingredients: The First (SLN) and the Second Generation (NLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Kleber L. Guimar˜ aes, Maria Inˆes R´e Chapter 6: Industrial Production of Polymeric Nanoparticles: Alternatives and Economic Analysis . . . . . . . . . . 123 Luciane F. Trierweiler, Jorge O. Trierweiler

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Contents

Chapter 7: Elastic Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Maria Helena A. Santana, Beatriz Zanchetta Chapter 8: Chitosan as Stabilizer and Carrier of Natural Based Nanostructures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Maria I.Z. Lionzo, Aline C. Dressler, Omar Mertins, Adriana R. Pohlmann, N´ adya P. da Silveira

Part III: Applications of Nanocosmetics and Nanomedicines for Skin Treatments Chapter 9: Performance of Elastic Liposomes for Topical Treatment of Cutaneous Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . 181 Bartira Rossi-Bergmann, Camila A.B. Falc˜ ao, Beatriz Zanchetta, Maria Vit´ oria L. Badra Bentley, Maria Helena Andrade Santana Chapter 10: Druggable Targets for Skin Photoaging: Potential Application of Nanocosmetics and Nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Giselle Z. Justo, S´ılvia M. Shishido, Daisy Machado, Rodrigo A. da Silva, Carmen V. Ferreira Chapter 11: Nanomedicine: Potential Killing of Cancercells Using Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Patricia da Silva Melo, Priscyla D. Marcato, Nelson Dur´ an Chapter 12: Zebrafish as a Suitable Model for Evaluating Nanocosmetics and Nanomedicines . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Carmen V. Ferreira, Maria A. Sartori-da-Silva, Giselle Z. Justo Chapter 13: Nitric Oxide-Releasing Nanomaterials and Skin Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Amedea B. Seabra Chapter 14: Nanocarriers and Cancer Therapy: Approaches to Topical and Transdermal Delivery . . . . . . . . . . . . . . . . . . . . . . . . 269 Juliana M. Marchetti, Marina C. de Souza, Samantha S. Marotta-Oliveira Chapter 15: Nanocarriers to Deliver Photosensitizers in Topical Photodynamic Therapy and Photodiagnostics . . . . . . . 287 Wanessa S.G. Medina, Fab´ıola S.G. Pra¸ca, Aline R.H. Carollo, Maria Vit´ oria L. Badra Bentley

Contents

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Chapter 16: Production of Nanofibers by Electrospinning Technology: Overview and Application in Cosmetics . . . . . . . . . 311 Maria Helena A. Zanin, Natalia N.P. Cerize, Adriano M. de Oliveira Chapter 17: Nanosized and Nanoencapsulated Sunscreens . . . 333 C´ assia B. Detoni, Karina Paese, Ruy C.R. Beck, Adriana R. Pohlmann, S´ılvia S. Guterres About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

Part I

Fundamentals of Skin Delivery

Chapter 1

Transport of Substances and Nanoparticles across the Skin and in Vitro Models to Evaluate Skin Permeation and/or Penetration Renata V. Contri1, Luana A. Fiel1, Adriana R. Pohlmann2, Sílvia S. Guterres1, and Ruy C.R. Beck1 1

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Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brazil [email protected] Universidade Federal do Rio Grande do Sul, Departamento de Química Orgânica, Av. Bento Gonçalves, CP 15003, 91501-970, Porto Alegre, RS, Brazil

Abstract. Nanotechnology can be used to modify the drug permeation/penetration of encapsulated substances, through the manipulation of many different factors, including direct contact with the skin surface and controlled release. In general, nanoparticles cannot cross the skin barrier, which can be explained by the cell cohesion and lipids of the stratum corneum, the outermost skin layer. The device most commonly used to study the transport of substances and nanoparticles across the skin is the Franz vertical diffusion cell, followed by the substance quantification in the receptor fluid or determination of the amount retained in the skin. Microscopy techniques have also been applied in skin penetration or permeation experiments. This chapter will present the fundamental considerations regarding the transport of encapsulated substances and/or nanoparticles across the skin, the experimental models applied in these studies and a review of the main studies reported in the literature in order to allow the reader to gain insight into the current knowledge available in this area.

1.1 Introduction The skin is the largest organ of the human body, presenting a total area of close to 2 m2. It acts as a barrier between the organism and the external environment [1]. Important skin functions include protection against UV radiation, physical and chemical damage and microbiological attack, maintenance of the body temperature and sensorial functions such as pain and temperature [2]. The skin is mainly composed of two layers (epidermis and dermis) besides the subcutaneous tissue [3]. It is composed of a variety of different cells, being considered more complex than the brain regarding this aspect [1]. The epidermis is composed of several lipids including phospholipids, phosphatidylcholine, cholesterol and triglycerides [4]. The main cell types found in the epidermis are keratinocytes, melanocytes, Langerhans cells, and Merkel cells [3]. The epidermis is

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divided into several layers and its outermost layer, the stratum corneum, is responsible for the barrier function of the skin due to its lipophilicity and high cohesion between cells [5, 6]. The stratum corneum is composed of keratinized corneocytes embedded in lipid bilayers [2]. Ceramides, cholesterol and free fatty acids comprise its extracellular lipid compartment [7]. The dermis is the layer next to the subcutaneous tissue and it is composed of collagen, elastin, glycosaminoglycans, and fibroblasts. This layer is highly vascularized besides containing the appendices (sweat glands and pilosebaceous units) and leucocytes, adipocytes and mast cells [3]. Considering the skin anatomy and physiology, some active substances will not provide the desired activity after their cutaneous administration. Nanotechnology can be used to modify the drug permeation/penetration by controlling the release of active substances and increasing the period of permanence on the skin [8, 9] besides ensuring a direct contact with the stratum corneum [10] and skin appendices [11, 12] and protecting the drug against chemical or physical instability [13, 14, 15, 16]. Also, depending on their sizes and structures, some nanostructures can also penetrate across the skin [17]. The aim of this chapter is to discuss the permeation and penetration of nanoencapsulated substances and nanoparticles through the skin. The transport across the skin and the in vitro models and membranes used to evaluate the skin permeation and/or penetration are also reviewed.

1.2 Transport across the Skin Despite the efficient barrier property of the skin, some substances can penetrate across its different layers [18]. The stratum corneum, known as a coherent and compact membrane, is the limiting layer for the penetration process, acting as a passive diffusion barrier [2, 19]. The permeability of some substances through the full-thickness of skin is at least 14 times lower than that of the same substances through the dermis [18]. The integrity of the stratum corneum and the concentration of the applied drug are important aspects that influence the drug penetration profile [19]. The lipids of the stratum corneum, especially ceramides, are important components in terms of its barrier function [2, 20]. There are some passive routes by which a molecule can cross the stratum corneum (Fig. 1.1): intercellular (through solubilization in the extracellular lipids arranged into structured bilayers), transcellular (through the corneocytes and the lipid bilayers) and appendageal (through either the sweat glands or hair follicles) [1]. However, the contribution of the latter route is considered small since the skin appendices occupy 0.1% of the skin surface [21]. The sweat glands are not a common pathway for drugs to pass through the skin due to the tortuous pathway and the ascendant sweat. The hair follicles, on the other hand, are common mechanism of transport for some ions, polyfunctional polar compounds and high molecular weight molecules [5, 22]. It has been verified that the hair follicles, despite their smaller superficial area, play an important role in the permeation of nanoencapsulated substances, since they serve as nanoparticle depots [11, 23]. The impermeability of skin can be a drawback when this route is desirable for the delivery of active substances. Only a small percentage of a substance reaches

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its target when topically applied [1]. The stratum corneum is slightly permeable to both hydrophilic and lipophilic compounds. Thus, the transcellular and intercellular routes are the most important pathways across the skin [19]. The partition coefficient of a substance between the epidermis and its pharmaceutical form is a determining factor in its penetration into the epidermis, which is the determining step of its diffusion rate across the skin [18].

Fig. 1.1 Mechanisms of transport across the skin. (a) intercellular, (b) transcellular and (c) hair-follicles.

1.3 In Vitro Models and Membranes Used to Evaluate Skin Permeation and/or Penetration 1.3.1 In Vitro Diffusion Models The guidelines for dermal absorption of the World Health Organization (WHO) [24] differentiate between the processes of skin permeation and penetration. Penetration is the entry of a substance into a particular layer or structure, whereas permeation is the transport from one layer into a second layer, these being associated with different functions and structures.

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The diffusion cells are the systems normally used to evaluate the drug permeation through the skin [21]. There are vertical or horizontal cells, associated with the drug diffusion, and static or flow-through cells, considering the receptor fluid [24]. Thus, it is important to select a model where the transport is limited only by the skin and not by a stagnated diffusion, which can occur particularly in the case of highly lipophilic substances [5]. Franz (1975) [25] developed a static vertical diffusion cell and verified a good relation between the in vivo and in vitro data for organic substances. The Franz diffusion cell is the most common type of diffusion cell, mainly because of its low cost [2, 5, 26]. Furthermore, it is useful for studying semisolid formulations and is ideal for simulating in vivo performance [24]. In the Franz technique, a membrane (skin) is placed between the diffusion compartments of the cell. The dermis, when present, is turned toward the receptor compartment, which is usually filled with an isotonic saline solution or phosphate buffer containing surfactants or cosolvents in the case of lipophilic substances in order to maintain the sink conditions. The system is maintained under continuous magnetic stirring (Fig. 1.2) [21, 26]. The sample is deposited in the donor compartment on the epidermis. The diffusion rate of the active substance to the receptor compartment is determined by an appropriate and validated analytical method, such as chromatographic or spectroscopic techniques, liquid scintillation counting or other suitable methods [27, 28]. A flow-through cell was developed by Bronaugh and Steward (1985) [29] to automate the collecting of samples from a two-compartment cell. Moreover, this cell facilitates the maintenance of the system viability because the receptor fluid is continuously replaced. The flow-through cell mimics the blood flow through the removal of hydrophobic substances from the skin. However, the amount of substance absorbed and the period necessary for its removal are similar for both the flow-through cell and Franz diffusion cell [26]. Several guidelines [24, 27] recommend static Franz diffusion cells or flowthrough cells for permeation studies. The choice of the cell type must be based on the experimental aims and the substance characteristics, such as theoretical absorption properties. According to the Scientific Committee on Consumer Products guidelines (SCCP, 2006), the static diffusion cell presents the advantage of easy quantification, since the receptor fluid is not replaced continuously, only when the sampling takes place. Recently, the development of equipment based on static permeation cells has presented the possibility of automatic sampling from the receptor fluid, which facilitates the collection and replacement of the medium without the formation of air bubbles. Horizontal diffusion cells, which are also called side-by-side cells, are less common. Here, the cell also comprises two compartments, and the donor and the receptor (usually containing equal volumes) are separated by a membrane [30]. This kind of system is useful for studying mechanisms of diffusion through the skin, such as permeation from one stirred solution into another stirred solution through a membrane [24]. Besides the assay of the active substance in the receptor compartment, the amount of the substance retained on the skin or penetrated into different skin layers is also commonly evaluated using diffusion cells. In order to determine this

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profile, the skin is cleaned after the permeation experiment to remove the excess of formulation. The stratum corneum layers are usually removed by the tape stripping technique where adhesive tapes are placed on the skin surface and the layers are taken by applying constant pressure [21, 31]. In addition, the epidermis and dermis can be separated by heat and force [32, 33]. The amount of substance remaining in each layer is extracted using an appropriate solvent and assayed by a validated analytical method. Studies on the penetration of active substances into different skin layers can also be performed using the alternative Saarbrücken diffusion model [9, 34, 35]. In this system, the skin is placed onto a filter paper wetted with Ringer solution, which is placed into the hole of a Teflon block. The Teflon block with the sample is fitted into the cavity of a Teflon punch and applied to the skin surface. A standard weight is placed on the top of the punch for 2 min to increase the contact between the skin and the formulation. The gap between the two Teflon pieces is then sealed to avoid the loss of water from the skin and the system is placed into a plastic box at 32 ± 1°C. The skin is removed at different time intervals and tape stripping is carried out. After this procedure, the skin is separated into parallel sections using a cryomicrotome and extraction techniques are performed for the quantification of the drug [36].

Fig. 1.2 (a) Horizontal diffusion cell, (b) Vertical diffusion cell and (c) Saarbrucken model.

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1.3.2 Microscopy Techniques Microscopy techniques are interesting tools to visualize the location of the particles or of the active ingredient in the skin tissue after in vitro or in vivo skin permeation or penetration studies. These studies provide information on the penetration pathway or permeation route of substances or particles across the skin [17, 37, 38]. Scanning Electron Microscopy (SEM), Confocal Laser Scanning Microscopy (CLSM), Transmission Electron Microscopy (TEM) and Scanning Transmission Ion Microscopy (STIM) are the microscopic techniques most commonly employed in this type of study [38, 39, 40, 41]. Figure 1.3 shows microscopy images obtained using these different techniques.

Fig. 1.3 (a)* Confocal scanning microscopy images showing fullerene penetration through the skin. All scale bars represent 50 μm. (b)* Transmission electron microscopy image showing that fullerenes are present within the intercellular space of the stratum granulosum cell layer. The scale bar represents 300 nm. (c)** Scanning electron microscopy of Zinc oxide nanoparticles distribution into different areas of in vitro human epidermis. * Reprinted (Adapted) with permission from Rouse et al., 2007. Copyright 2007. American Chemical Society. ** Reprinted (Adapted) with permission from Roberts et al., 2008. Copyright 2008. WILEY-VCH Copyright & Licenses.

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1.3.3 Membranes 1.3.3.1 Types of Membranes Since the skin is an organ with multiple layers, it can be used during in vitro skin permeation/penetration studies with its two basic layers, epidermis and dermis (full-thickness skin) or it can be submitted to treatments that separate its layers supplying different types of membranes (split-thickness skin) to be used for in vitro studies [26, 42, 43]. The choice of the membrane is related to the focus of the study (permeation or penetration). In the full-thickness skin membranes only the subcutaneous tissue is removed [26, 42, 43]. On the other hand, split-thickness skin can be obtained in several ways [26, 42, 43] and the isolation of one specific layer of a skin sample with a controlled thickness can be made using heat and force (heat-separated epidermis), dermatome (dermatomed skin) or enzymatic processes (trypsin-isolated stratum corneum) [44, 45]. Epidermis and stratum corneum membranes are more fragile and some mass balance techniques, such as tape stripping, cannot be applied [27]. Divergences can be found in the literature concerning the effect that these processes can have on the skin characteristics. Some studies have demonstrated that the skin barrier is reestablished with the hydration of these membranes [46, 47]. On the other hand, some reports have claimed that the viability of the skin and the flow through the epidermis can be disturbed by these processes [26, 48, 49]. 1.3.3.2 Sources of Membranes Membranes obtained from diverse sources, such as synthetic [43, 50, 51], vegetable [52], human [53, 33, 43], animal [43, 54] and reconstructed tissues [43, 45, 47] can be evaluated as candidates to predict the transport of substances across the skin and used during in vitro experiments. The advantages and disadvantages of these membrane models need to be investigated regarding their effectiveness to determine the transport of substances across the skin [26]. There is no consensus regarding the standardization of these sources in this type of study. In general, only human, animal or reconstituted skin models should be used as sources of membranes to evaluate the transport of substances across the skin [26, 27]. On the other hand, synthetic membranes are usually recommended and used to study the in vitro release of substances from semisolid or liquid pharmaceutical or cosmetic formulations [43, 55, 56, 57]. 1.3.3.2.1 Human Skin Human skin offers a greater possibility for in vitro-in vivo correlations compared with the other sources of membranes [33, 43, 58, 59, 60]. The samples can be obtained from portions remaining from surgical procedures (plastic surgery or amputation) or autopsies. The most commonly used anatomical regions are abdomen, breast and legs. In the case of skin membranes from a human source, dermatomed skin (200-500 μm) is recommended [27]. To minimize the variability in the results due to differences in the permeation properties of different anatomical regions and volunteers, the anatomical region as well as the sex, race and age of the donors

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should be standardized [90, 92]. However, the availability of human skin sources is limited due to the need to acquire the consent of the volunteer [26]. 1.3.3.2.2 Animal Skin Skin samples obtained from animals allow easily obtainment and standardization of the anatomical region, sex and age of the animal. Thus, the skin of several animal species is frequently used as an alternative to human skin to evaluate the in vitro transport of substances [43]. The use of monkey, rodent, pig and snake skin is reported in the literature. In the evolutionary chain, monkeys represent the species closest to humans. Comparative in vitro-in vivo studies demonstrate that the transport of substances across monkey skin is similar to that across human skin [62, 63, 64]. However, few studies have been carried out using monkey skin for ethical reasons. Studies using rodent skin as a membrane model are more common for the evaluation of the in vitro transport of substances. As sources of membranes, the use of rat, mouse, guinea pig and rabbit skin has been reported. Although it is known that rat skin is around 10 times more permeable than the human skin [27, 64, 65] its use for in vitro experiments is still common in toxicity studies, making in vitro-in vivo correlations possible [64, 66]. Furthermore, a correlation between the skin diffusion coefficients for nortriptyline was observed on comparing Wistar rat skin and human heat-separated epidermis [67]. Considering mouse skin, only full-thickness skin membranes should be used since this type of skin presents a very low thickness [26]. Snake skin has been studied and recommended as a model for the stratum corneum membrane to evaluate in vitro penetration [68, 69]. The stratum corneum membrane is commonly employed to verify the barrier function of the skin, considering it as the main obstacle in the skin penetration of substances [45, 47]. Anatomically, pig skin presents the greatest similarity to human skin [43]. Thus, it is frequently used as a membrane to evaluate the transport of substances through the skin in in vitro studies [43]. Generally, the samples come from the ears or from the abdominal or dorsal regions. Some particularities, such as size and density of hair follicles and the stratum corneum thickness, are indicated as reason for divergences in the results obtained from pig and human skin [34]. The evaluation of the relative permeability of some drugs through membranes obtained from different sources suggests differences in the following order: human < pig < rat < rabbit < mouse [70]. 1.3.3.2.3 Regenerated Skin The extrapolation of results obtained from skin permeation/penetration experiments carried out with animal membranes to the human skin is always questionable given the influence of the inter-species variability. Moreover, there are ethical questions regarding the use of animal skin [71]. The Organization for Economic Cooperation and Development has affirmed that the regenerated human skin models can be used to evaluate the in vitro transport of substances across the skin if there is equivalence [72]. However, this use requires validation of its applicability, which is still under evaluation [73]. The models of regenerated human skin commercially available are: Apligraf®, Epiderm®, SkinEthic® and EpiSkin®. Apligraf®/Graftskin® (Organogénese Incorporation) [74, 75]

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and EpiSkin® (L‘Oréal) are models of two live layers. SkinEthic® (SkinEthic) and Epiderm® (MatTek) [71] are epidermis models. Despite attempts to investigate the appropriateness of using regenerated membranes in in vitro studies on the transport of substances across the skin [45, 73, 76], to date no study has verified that they can act as substitutes for human or animal skin models. These models do not provide a barrier to the transport of substances through the skin [45, 47, 77]. However, regenerated human epidermis models could provide an adequate tool for routine tests, such as the quality control of cosmetic products [73].

1.4 Transport of Nanoparticles and Encapsulated Substances across the Skin The delivery of therapeutic agents without the need for chemical enhancers is desirable to maintain the normal skin barrier function. Treatment with chemical enhancers, such as surfactants and organic solvents, can cause not only a reduction in the barrier function of the skin, but also irritation and damage to the skin [17, 78, 79]. Drug-release nanocarriers, such as liposomes, micelles, polymeric and solid lipid nanoparticles as well as inorganic nanoparticles and sub-micrometric emulsions, have been used as alternatives to chemical enhancers to reduce the damage and increase the permeation of therapeutic agents through the skin [17, 78, 79]. Additionally, controlling the release of therapeutic agents can be achieved with reduced systemic absorption [17, 78, 79]. The transport of nanoparticles and nanoencapsulated substances through the skin is related to the nature and physicochemical properties of the nanoparticles and vehicles, the nature of the substance and the conditions of the skin [78, 79]. Most studies related to inorganic particles are recent (Fig. 1.4) while permeation/penetration studies applying organic (polymeric and lipid) particles have been published since 1999.

Fig. 1.4 Articles which studied the skin penetration/permeation of encapsulated substances or nanoparticles.

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1.4.1 Nanoparticles Based on Organic Compounds Nanoparticles based on organic compounds can be either polymeric or lipid systems, and either vesicular or matrix structures. Such nanoparticles have been proposed for enhancing or controlling the cutaneous penetration of hydrophilic and lipophilic substances. They can be built using a bottom-up or top-down approach. 1.4.1.1 Polymeric Systems Polymer-based nanoparticles are of interest for skin administration due to the controlled release of encapsulated active ingredients, which have to diffuse through the polymeric matrix to permeate the skin. Nanocapsules (See Chapter 3) and nanospheres (See Chapter 4) are the most common types of polymeric nanoparticles studied in terms of their skin permeation (Table 1.1). Polymeric vesicles [80] and dendritic core-multishell nanotransporters [81] have also been reported. Table 1.1 shows an overall view of the reports in the literature on skin permeation/penetration studies employing polymeric nanoparticles. All studies shown in the table are discussed in the following text. In general, polymeric nanoparticles are very stable due to their rigid matrix, and they are able to maintain their structures for long periods of time when topically applied [82, 11]. The contact of the drug with the skin surface is limited by the drug diffusion rate from the nanocarrier [83]. In fact, some reports have shown that polymeric nanoparticles can retard the permeation of encapsulated substances to the receptor compartment or to deep skin layers [9, 54, 84, 85] and increase the amount of drug retained in the stratum corneum in comparison to other skin layers [32, 86]. The amount of drug retained in deep skin layers might increase some time after application [9]. On comparing vesicular and matrical polymeric systems, very similar profiles for the drug penetration into the stratum corneum were observed [33]. Interestingly, an increase in the permeation of active substances through the skin and/or penetration into a specific skin layer following encapsulation in polymeric nanoparticles has also been reported. This has been attributed to the direct contact of the nanoparticles with the stratum corneum due to their high total surface area [31], high thermodynamic activity due to their nanometer scale structure [31, 37], the possibility for their deposition into the hair follicles [31], the surface characteristics [87, 88, 89] and the affinity of the nanoparticle components for certain skin layers [88]. The passive targeting of polymeric nanoparticles to the hair follicles has been reported [11, 83, 90] and the ideal particle size for penetration into the hair follicles was estimated at between 320 and 750 nm [91], although smaller particles also show affinity for the hair follicles [26, 61]. The storage reservoir capacity of the hair follicles is 10 times higher than the storage reservoir capacity of the stratum corneum [23]. Polymeric particles of 320 nm (polydispersion index = 0.06) can be retained in the hair follicles for up to 10 days, being expelled due to sebum production without reaching living cells [11]. Skin furrows may also be the target of polymeric nanoparticles on the skin surface [80, 90, 93].

Poly( -caprolactone), Labrafac hydrophile WL 1219

Poly n-butylcyanoacrylate, benzyl benzoate Polystyrene

Poly( -caprolactone)

Poly(lactic-co-glycolic acid) (PLGA), polystyrene

Poly( -caprolactone) Poly(lactideco-glycolide)

Poly( -caprolactone)

Poly(vinyl alcohol), fatty acids

Poly( -caprolactone)-block-poly(ethylene glycol)

Nanocapsules

Nanoparticles

Nanocapsules

Nanoparticles

Nanocapsules

Nanospheres

Nanocapsules

Nanoparticles

Nanoparticles

Main Components

Nanocapsules

Type*

Minoxidil

Benzophenone-3

Chlorhexidine

Trimethylpsoralen

Octyl methoxycinnamate

-

Octyl methoxycinnamate

-

Indomethacin

Clorhexidine

Active Ingredient

In vitro, full-thickness guinea pig, hairless guinea pig, hairless mouse skin,

In vitro, full-thickness pig skin, Franz diffusion cell,

In vitro, full-thickness and stripped hairless rat skin, Franz diffusion cell

In vitro, full-thickness human skin, Franz diffusion cell

In vitro, full-thickness pig skin, Franz diffusion cell

In vitro, epidermal sheets from mouse skin, confocal laser microscopy

In vitro, full-thickness porcine skin, Franz diffusion cell, confocal microscopy

In vitro, full-thickness porcine skin, Franz diffusion cell, confocal microscopy

In vitro, rat skin, Franz diffusion cell, confocal microscopy

In vitro, full-thickness porcine skin, diffusion cell

Study Type

Table 1.1. Overview of permeation and/or penetration studies applying nanoparticles based on polymeric compounds.

[12]

[85]

[82]

[84]

[32]

[97]

[31]

[90]

[87]

[89]

Reference

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models 13

Latex, polystyrene

Poly(lactideco-glycolide) Poly(lactideco-glycolide)

Nanoparticles

Nanoparticles

Nanoparticles

Nanocapsules

Poly( -caprolactone) and octyl salicylate

Poly( -caprolactone), triglycerides

Poly(lactic-co-glycolic acid)

Nanoparticles

Nanocapsules and nanospheres

Poly(lactide- co -glycolide)

Nanoparticles

Table 1.1 (continued)

In vitro, full-thickness pig skin, laser scanning microscopy In vivo, human skin, tape stripping technique

-

Hinokitiol

In vitro, hairless full-thickness mouse skin, Franz diffusion-cells, confocal microscopy

In vitro, full-thickness and heatseparated human skin, Franz diffusion cell

In vivo, human skin, tape stripping technique, laser scanning microscopy

-

Nimesulide

In vitro, full-thickness human skin, fluorescence microscopy and laser scan microscopy

In vitro, full-thickness human skin, multiphoton microscopy and confocal laser scanning microscopy

In vitro, full-thickness human skin, Saarbrücken penetration model In vitro, heat-separated epidermis, Franz diffusion cell, multiphoton fluorescence imaging

-

-

Flufenamic acid

Franz diffusion cell, confocal microscopy

[37]

[33]

[11]

[23]

[96]

[94]

[9]

14 R.V. Contri et al.

-

Polystyrene and hexadecane

Microcapsules

Poly( -caprolactone)

Nanocapsules

* cited according to the original reference

Poly(lactide)

Nanoparticles

Dendritic core-multishell (CMS) nanotransporters and solid lipid nanoparticles (SLN)

Polyglycerolamine, 1-(2,5dioxopyrrolidin-1-yl)-18-methoxypoly(ethylene glycol)yl octadecanedioate (CMS) and Compritol 888 ATO (SLN)

-

Poly( -caprolactone)–poly(ethylene glycol)-poly( -caprolactone) co-polymer

Polymerosomes

Octylmethoxycinnamate

Octylmethoxycinnamate

In vitro, full-thickness pig skin, Franz diffusion cell

[54]

[86]

[81] In vitro, full-thickness pig skin, Franz diffusion cells, normal and fluorescence light microscopy In vitro, full-thickness pig skin, Franz diffusion cell

[83]

[80]

[92]

[93]

[88]

[98]

In vitro, dermatomed porcine skin, confocal microscopy

In vitro, heat-separated epidermis, Franz diffusion cells, confocal laser microscopy

In vitro, full-thickness porcine skin, Franz diffusion cell, confocal microscopy

Polystyrene and poly-(2 hydroxyethyl methacrylate

Nanoparticles

-

Polystyrene and poly(methyl methacrylate)

Nanoparticles

In vitro, full-thickness pig skin, Franz diffusion cell In vitro, dermatomed porcine skin, Franz diffusion cells, confocal microscopy

Clobetasol propionate

Chitosan and lecithin

Nanoparticles

In vitro, dermatomed pig skin, Franz diffusion cells, scanning and laser electron microscopy

-

-

Dendritic core-multishell (CMS) nanotransporters and solid lipid nanoparticles (SLN)

Polyglycerolamine, 1-(2,5dioxopyrrolidin-1-yl)-18-methoxypoly(ethylene glycol)yl octadecanedioate (CMS) and Compritol 888 ATO (SLN)

Table 1.1 (continued)

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models 15

16

R.V. Contri et al.

The penetration of polymeric nanoparticles into skin layers does not commonly occur under normal skin conditions [37, 83, 90; 94], probably due to the small size of the intercellular space [95]. However, the penetration of poly(nbutylcyanoacrylate) nanocapsules (188 nm) into the epidermis and dermis of full-thickness rat skin has been observed by confocal laser microscopy after cryosectioning of the skin, which also increased the permeation of the encapsulated substance [87]. The type of skin used could have influenced this result since rat skin is more permeable than human skin, as previously mentioned (section 1.3.3.2.2). Hydrophilic vesicles (122 nm) composed of poly(caprolactone)–poly(ethylene glycol)– poly(caprolactone) co-polymer were able to penetrate the epidermis using human epidermis sheets [80]. Accumulation of the vesicles was observed by confocal microscopy in the epidermal layers. The particle sizes mentioned are mean values and the presence of smaller particles due to the polydispersity should be taken into account. In fact, smaller polymeric particles (close to 40 nm) penetrated the full-thickness skin of human [96] and rodent [12] sources, predominantly via the follicular route. Permeation enhancers such as Plurol® (polyglyceryl-6 dioleate), Transcutol® (diethyleneglycol monoethyl ether) and Labrasol® (PEG-8 caprylic/capric glycerides) can promote the nanoparticle penetration across the stratum corneum of mouse skin due to the disruption of the ordered lipid structure [97]. 1.4.1.2 Lipid Systems Nanoparticles based on lipid systems are the most common type of nanoparticles studied for topical application. Solid lipid nanoparticles, nanoemulsions and nanostructured lipid carriers are the main types of matrix nanoparticles while liposomes are the main type of vesicular particles evaluated in permeation studies. Other types cited in the literature include niosomes [99], cubosomes [100, 101], bicellar systems [102], vesicles [103, 104] and nanodispersions [105]. As in the previous table, Table 1.2 provides details of the studies which are discussed herein, regarding the skin permeation/penetration studies performed with lipid nanoparticles. In contrast to the polymeric nanoparticles, they are less stable when applied on the skin surface. After topical application, solid lipid nanoparticles were observed to lose their shape and melt after a period of 2 h [98] due to the interaction between the particle components and skin lipids [81, 98]. This occurrence can reduce the skin barrier function and occlude the skin surface [106, 107], favoring the skin penetration process. Thus, an increase in the permeation to the receptor medium [99, 108, 109, 110, 111, 112, 113, 114] or in the penetration into the skin layers [104, 105, 115, 116, 117, 118, 119] of the active substance is commonly observed when it is encapsulated in different lipid-based nanoparticles, either matrical or vesicular. The lipid components [98, 100, 120] or the surfactant components of nanoparticles can interact with the skin [109, 121]. Moreover, the addition of other components such as ethanol [110, 122] and magnetic nanoparticles [123] to lipid nanoparticles can even enhance the permeation. Besides the enhancement of the permeation/penetration due to encapsulation of substances in lipid nanoparticles, the opposite result has also been reported. This can probably be attributed to an increase in the rigidity of the nanoparticles due to

Tristearin glyceride, stearic acid

Sucrose laurate ester, octaoxyethylene laurate ester

Solid lipid nanoparticles

Elastic and rigid vesicles

Indomethacin

-

Cyclosporin A Indomethacin

Compritol®, Precirol®, Oleic acid, Miglyol® 812

Monoolein, oleic acid

Compritol® 888 ATO, Miglyol®

Solid lipid nanoparticles, nanostructured lipid nanocarrier and nanoemulsion

Hexagonal phase nanodispersion

Nanostructured lipid car-

Octyl methoxycinnamate

Ketorolac

Triptolide

Monooleine

Cubosomes

Octyl methoxycinnamate

Cetyl palmitate, Tego Care 450®

Solid lipid nanoparticles

Nanoemulsion

Vitamin A

Compritol® 888 ATO

Solid lipid nanoparticles Oxybenzone

Vitamin A or E

Lipid nanospheres

Active Ingredient

Main Components

Lecinol, soybean oil

Type*

In vitro, human skin, Franz-type diffusion

In vitro, full-thickness porcine skin, Franz diffusion cell

In vitro, full-thickness pig skin, Franz diffusion cell, fluorescence microscopy

In vitro, human epidermal membranes, Franz diffusion cell In vivo, human skin, tape stripping technique

In vivo, Human skin, tape-stripping technique

In vivo, human skin, tape stripping technique, infrared spectroscopy

In vitro, full-thickness rat skin, Franz diffusion cell

In vivo, human skin, tape stripping technique

In vitro, full-thickness pig skin, Franz diffusion cell

In vitro, full-thickness rat skin, Franz diffusion cell

Study Type

Table 1.2. Overview of permeation and/or penetration studies applying nanoparticles based on lipid compounds.

[59]

[105]

[117]

[100]

[132]

[103]

[112]

[127]

[10]

[121]

Reference

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models 17

Vitamin A palmitate

Compritol® 888 ATO

Solid lipid nanoparticle

Artemisia arborescens essential oil

Triglycerides

Compritol® 888 ATO

Nanoemulsion

Stearic acid

Soya phosphatidylcholine,

Lipid nanoparticles

Ethanolic liposomes

Solid lipid nanoparticles

Nimesulide

Soybean oil

Nanoemulsion

Melatonin

Hinokitiol

Nabumetone

Compritol 888 ATO, Miglyol 812

Nanostructured lipid carriers

Ketorolac

Podophyllotoxin

Tripalmitin

Solid lipid nanoparticles

®

RU58841-myristate

Compritol®, Precirol®

Solid lipid nanoparticles

®

Triptolide

Tristearin glyceride, stearic acid

Solid lipid nanoparticles

In vitro, dermatomed human skin, Franz diffusion cell, confocal microscopy and FT-IR analysis.

In vitro, full-thickness hairless mouse skin, Franz diffusion cell

In vitro, full-thickness pig skin, Franz diffusion cell

In vitro, full-thickness pig skin, Franz diffusion cell

In vitro, full-thickness rat skin, Franz diffusion cell

In vitro, epidermal human membranes, Franz-type diffusion cell

In vitro, full-thickness human skin, Keshary Chien cells

In vitro, full-thickness porcine skin, Franz diffusion cell

In vitro, reconstructed epidermis (SkinEthic), Franz diffusion cell, Fluorescence microscopy

In vitro, full-thickness rat skin, Franz diffusion cell

cell Ascorbyl palmitate

In vitro, full-thickness human skin, Franz diffusion cell

®

812

®

Witepsol E85, Mygliol 812

Solid lipid nanoparticles, nanostructured lipid carriers

riers

Table 1.2 (continued)

[110]

[113]

[129]

[33]

[109]

[126]

[115]

[116]

[134]

[108]

[125]

18 R.V. Contri et al.

Miconazole nitrate -

Compritol® 888 ATO

Soybean phosphatidylcholine

Solid lipid nanoparticle

Ethosomes

Tretinoin

Ketoprofen and naproxen

Compritol® 888 ATO, Miglyol® 812

Lipid nanoparticles

Fruit kernel fats

Corticosterone

Medium-chain triglycerides, tripalmitate, cholesteryl myristate, cholesteryl nonanoate, glycerol monooleate

Lipid nanoparticles

Solid lipid nanoparticles

In vitro, human epidermis, Franz diffusion cell, spectrofluorometry

-

1,2-Dipalmitoyl-sn-glycero-3phosphocholine, 1,2-dioleoylsn-glycero-3-phosphocholine,

Lipossomes

In vitro, human skin scar, confocal microscopy

In vitro, full-thickness human skin, Franz diffusion cell

In vitro, full-thickness rat skin, Franz diffusion cell

In vitro, human epidermal membranes, Franz diffusion cell

In vitro, human heat-separated epidermis trypsin isolated stratum corneum, Franz diffusion cell, Fluorescence light microscopy

In vitro, full-thickness human skin, Franz diffusion cells, confocal microscopy

-

Sabowax CP®, Miglyol® 821

Nanostructured lipid carriers

In vitro, human SC and epidermis, Franz diffusion cell

Ammonium glycyrrhizinate

In vitro, dermatomed human skin, Franz diffusion cell

In vitro, full-thickness rat skin, Franz diffusion cell

In vitro, dermatomed human skin, Franz diffusion cell, confocal microscopy and FT-IR analysis

, -Hexadecyl-bis-(1-aza-18crown-6) (Bolasurfactant)

Tetracaine

Isotretinoin

Precirol® ATO 5

Hexadecane

Methotrexate

Soya phosphatidylcholine,

Niossomes

Nanoemulsions

Solid lipid nanoparticles

Ethanolic liposomes

Table 1.2 (continued)

[122]

[118]

[124]

[128]

[120]

[135]

[107]

[99]

[133]

[119]

[111]

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models 19

* cited according to the original reference

Coenzyme Q10

Precifac® ATO, Miglyol® 812

Nanostructured lipid carrier and nanoemulsion

In vitro, human heat-separated epidermis, Franz diffusion cells

In vitro, dermatomed pig skin, Franz diffusion cells.

Diclofenac diethylamine

Dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine and dihexanoyl phosphatidylcholine

In vitro, hairless mice skin, Franz diffusion cells

In vitro, mouse skin, Franz diffusion cell

In vitro, mouse skin, diffusion cell

In vitro, porcine epidermal membrane, Franz diffusion cell

Berberis koreana extract

Oregonin

Minoxidil

Econazole nitrate

Bicellar systems

Monoolein

Soybean phosphatidylcholine

Elastic liposomes

Cubosomes

Distearyldimethylammonium chloride

Precirol® ATO 5

Cationic vesicles

Solid lipid nanoparticles

Table 1.2 (continued)

[106]

[102]

[101]

[114]

[104]

[131]

20 R.V. Contri et al.

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models

21

the type of lipid used as the raw material in the matrical particles [10, 124, 125] and the association of the drug with the vesicular particles [102]. Interactions between the active substance and the nanoparticle components [59, 126], film forming due to water evaporation [127] and a possible significant adherence to the stratum corneum observed for matrical lipid systems [128, 129] could reduce the release rate and the permeation of encapsulated substances. The particle size seems to play an important role in the permeation of the encapsulated active ingredient. Smaller particles seem to enhance the permeation to a greater extent than larger particles [130, 131, 132]. However, this conclusion is contradictory since it has already been shown that for the same kind of nanoemulsion there was no influence of the emulsion droplet size on the skin penetration of the encapsulated substance when applied to dermatomed human skin [133]. In comparison to nanocapsules, the nanoemulsions present a higher capacity to penetrate and/or permeate the skin, probably due to their flexibility [132] and lack of polymer which has an affinity with the stratum corneum [33]. As in the case of polymeric nanoparticles, an accumulation of lipid nanoparticles in the hair follicles can occur. Solid lipid nanoparticles of around 200 nm were detected in the hair follicles after 24 h of topical application [134]. The penetration of intact particles is not common, since the lipid nanoparticles tend to lose their shape when topically applied [98]. Depending on the nature and size of the particle, it seems possible for the vesicular lipid nanoparticles to penetrate/permeate the skin. The permeation of liposomes of 50, 100 and 200 nm through human epidermal membranes has been demonstrated, although there was a continuous increase in the average particle size in the receptor compartment, probably due to the particle agglomeration [135]. The rigidity of liposomes seems to be related with the skin permeation capacity, especially for larger vesicles [135]. Also, the penetration of elastic vesicles obtained from sucrose laurate ester and octaoxyethylene laurate ester into the deeper stratum corneum layers (close to the junction with the viable epidermis) was observed by means of the human tape stripping technique [103]. It was demonstrated that rigid vesicles, without octaoxyethylene laurate ester, could not penetrate as deeply into the stratum corneum as the elastic vesicles.

1.4.2 Nanoparticles Based on Inorganic Compounds Inorganic nanoparticles are considered nanosized materials. Nanosized materials are built from a top-down approach the nanoparticles being constructed from larger entities. According to their composition and physicochemical properties, these particles have been studied in the biomedical field as nanocarrier systems for targeting and controlling the delivery of active ingredients as well as an imaging device for diagnostic techniques [79]. Nanosized particles of zinc oxide and titanium dioxide, other metallic compounds and fullerenes are the main particles used for these purposes. Considering the cutaneous administration route, inorganic materials are used in a nanostructure form to increase the transport of the active ingredient through the skin, to increase its permanence in the skin or even to improve the cosmetic aspect

22

R.V. Contri et al.

[79, 136]. Regardless of the desired application (transdermal or topical), the transport characteristics of the nanocarrier are related to its dimensions (hydrodynamic diameter and shape - spherical, elliptical, nail-shaped) [78, 136]. Table 1.3 provides an overview of the methodologies used in skin permeation studies which are discussed in this topic. These studies investigated the penetration or permeation capacity and the possible penetration or permeation pathway of nanoparticles based on inorganic compounds proposed for cutaneous application as nanocosmetics or nanomedicines. As can be observed in Table 1.3 there are still few reports in the literature concerning these inorganic nanoparticles compared to organic nanoparticles. These studies have been carried out since 2004. 1.4.2.1 Titanium Dioxide or Zinc Oxide Nanoparticles Titanium dioxide (TiO2) and zinc oxide (ZnO)-based nanoparticles have been studied with a view to their inclusion in sunscreen preparations to reduce the visual opaque effect after the cutaneous application of such preparations compared to the use of bulk forms of these materials (see Chapter 17) [136]. As TiO2 or ZnO are intended to act as physical sunscreens, these materials have to be available on the skin surface to scatter the light. Therefore, the ZnO or TiO2 nanoparticles should not permeate rapidly through the skin. Thus, studies on the permeation capacity, penetration and/or permeation pathways and the location of these particles after topical application have been frequently performed. Several in vitro permeation studies carried out with ZnO or TiO2 nanoparticles showed, despite the size, no permeation of the particles through the skin. According to these results, the particles penetrate the stratum corneum and remain retained in the outermost corneocyte layers [38, 41, 138, 140, 142]. Permeation of the solubilized zinc form seems to be reduced in these studies. Thus, nanoparticle formation is suggested to avoid the presence of zinc in the solubilized form and consequently its permeation [138]. However, a size-dependent permeation was observed when TiO2 nanoparticles were administered in vivo for a long period of time [142]. Particles of 4 nm reached deeper epidermal layers compared to particles of 60 nm, but they did not reach the viable epidermis and dermis. Additionally, these particles were able to permeate through hairless mouse skin in vivo and reach several tissues [142]. However, it is important to consider the source of skin used. As reported in the section 1.3.3, mouse skin seems to be much more permeable than human skin. The main pathways proposed for the penetration of ZnO and TiO2-based nanoparticles are the intercellular route and the hair-follicles [41, 136, 137, 139, 140]. Although one report considered only the passage via the intercellular space as the penetration route for such particles [137], the hair-follicle seems to be an important pathway for particle transport [41, 139]. Another study found 400 μm particles deep in the follicle [139]. Nevertheless, TiO2-based nanoparticles were not found in vital tissue [41, 139]. More details on the use of nanosized materials as sunscreens are given and discussed in Chapter 17.

TiO2

TiO2

TiO2

Titanium dioxide (TiO2) nanoparticles

TiO2 nanoparticles

Rutile titanium dioxide (TiO2) nanoparticles

Gold

Silver

C60

C60

Gold nanoparticles

Silver nanoparticles

Fullerenes

Fullerenes

Fe2O3 or Fe

ZnO

ZnO nanoparticles

Iron oxide (Fe2O3) and iron (Fe) nanoparticles

TiO2

TiO2

TiO2 nanoparticles

ZnO

ZnO nanoparticles

TiO2 nanoparticles

TiO2

Main component

TiO2 nanoparticles

Type

Tape stripping in vivo, pig skin, In vitro, stratum corneum isolated from pig skin. Flow-through diffusion cell. High Performance/Pressure Liquid Chromatography.

In vitro, dermatomed porcine skin. Flow-through diffusion cell. CLSM.

In vitro, intact and damaged human skin. Franz-diffusion cell. Electro thermal atomic absorption spectroscopy (ETAAS). TEM

In vitro, rat skin. Franz-diffusion cell. UV-Vis and X-ray spectroscopy (EDS) and TEM.

In vitro, human full-thickness skin. Vertical cell. ICP-optical emission spectrometry

[145]

[39]

[144]

[40]

[143]

[41]

[142]

In vitro, porcine full-thickness skin. Modified Franz equipment. Flame atomic absorption spectrometry. TEM. In vitro, intact, stripped, and hair-removed skin of Yucatan micropigs. Franzdiffusion cell. ICP-MS.

[141]

[38]

[140].

In vitro, porcine skin, healthy human skin, human skin grafted. TEM and SEM

In vitro, human skin. SEM with X-ray photoelectron spectroscopy. Zinc oxide photoluminescence In vivo

In vitro, healthy and psoriatic skin condition. Particle induced X-ray emission.

[139]

[138]

In vitro, human heat-separated epidermis membranes. Franz-diffusion cell. Inductively coupled plasma-mass spectrometry (ICP-MS). TEM. In vitro, porcine and human skin. Autoradiography.

[137]

Reference

In vivo, pig skin. Ion beam analysis methods.

Study type

Table 1.3 Overview of the methodologies used in permeation and/or penetration studies applying nanoparticles based on inorganic compounds.

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models 23

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R.V. Contri et al.

1.4.2.2 Metallic Nanoparticles Gold, silver, iron and iron oxide nanoparticles have attracted attention in different biomedical segments [78]. Gold nanoparticles [146, 147] as well as iron [148, 149] and iron oxide [148, 150] nanoparticles are interesting photo-thermal agents in hyperthermia and as a tumor imaging device. Silver nanoparticles are widely used in topical formulations due to their antiseptic effect [151]. As nanosized structures, these particles could cross the skin resulting in systemic absorption. Thus, some studies have been carried out, especially in the past 10 years, to determine the penetration capacity of these systems. Gold nanoparticles (mean sizes of 15, 102 and 198 nm) have been shown to permeate through and accumulate in rat skin, and the permeation was found to be size-dependent. Smaller particles demonstrated faster permeation. Additionally, on increasing the particle size, the lag time to particle penetration increases. These findings are related to factors such as the partitioning kinetics, volume fraction in the receiver phase and membrane volume [40]. An increase in the particle diameter increased the amount of metal that permeated through the skin, which could be related to the higher metal loading of the larger particles [40]. An in vitro study demonstrated the capacity of metallic nanoparticles, such as iron and iron oxide nanoparticles, with mean size smaller than 10 nm, to penetrate the skin [143]. This penetration occurs through the hair follicle orifices and the stratum corneum lipid matrix. Although such particles have occasionally been found in viable epidermis, spectroscopy analysis has suggested that these metallic nanoparticles are unable to permeate the skin. The permeation observed was attributed to the particles having extremely small dimensions [143]. In the same way, silver nanoparticles (25 nm) appear to have a low absorption through skin [144]. Silver particles seem to penetrate the stratum corneum reaching, less often, the uppermost layers of the viable epidermis. Since particles with diameters ranging between 7 and 20 nm were mostly found in the infundibulum of the hair follicle and below, the dimensions of nanoparticles can be considered an important parameter in terms of their skin permeation. The low absorption of silver observed in some studies may suggest particle dissolution allowing elemental silver to diffuse through the skin [144]. Therefore, in general, in the case of metallic nanoparticles the penetration into and permeation through the skin is directly related to their mean particle size, smaller particles permeating deeper. This permeation seems to occur mainly through hair orifices and the stratum corneum lipid matrix. 1.4.2.3 Carbon-Based Nanoparticles Carbon nanotubes and fullerenes are carbon-based nanoparticles. These particles are formed of the third most stable form of carbon and differ according their shape [78]. Carbon nanotubes are multi-walled or single-walled hollow tubes while fullerenes are spherical, both with extremely small mean diameters (<100 nm) [17, 78]. To date, no data for carbon nanotubes relating to their penetration into or permeation through the skin have been reported in the literature. Regarding fullerenes, these nanostructures have been proposed for use as rejuvenation

1 Transport of Substances and Nanoparticles across the Skin and in Vitro Models

25

cosmetic products due to their potential anti-oxidant ability [152, 153]. Studies on the anti-oxidant effect were performed using human skin keratinocyte cultures (HaCaT) and showed their capability in terms of reducing the reactive oxygen species levels in the presence of UVB radiation [152]. The mechanical flexion of skin was presented as an important factor influencing fullerene penetration. The particles penetrated the dermis when the skin was previously flexed while no penetration was reported into the dermis of unflexed skin [39]. The penetration degree of fullerenes seems to be dependent on the vehicle type [145]. In vitro and in vivo penetration experiments [145] showed no fullerenes in the receptor medium of a flow-through chamber, using stratum corneum membranes. On the other hand, in vivo experiments show that fullerenes penetrated deeper into the stratum corneum layers when applied with toluene, cyclohexane or chloroform instead of mineral oil. The solvent flux and the super-saturation of fullerenes in the solutions were considered the main factors influencing this difference. Despite the small size of the particles there was a slow penetration rate, which was attributed to the possible occurrence of particle aggregation [145].

1.5 Concluding Remarks Nanotechnology can be applied to control the topical delivery of drugs and active substances. In the case of organic nanocarriers, the transport of nanoencapsulated substances across the skin is determined by several factors, including the physicochemical properties and the nature of the nanocarriers. Those which are capable of interacting with the stratum corneum lipids usually lead to a greater degree of penetration, as commonly observed for lipid nanoparticles, while stable rigid matrixes, such as those of polymeric systems, usually limit the skin permeation of substances. The same parameters (physicochemical properties and the nature of the nanocarriers) are responsible for the transport of the organic and inorganic nanoparticles across the skin, which is most common for smaller nanoparticles. It is important to note that many studies have focused exclusively on investigating the permeation/penetration of nanoencapsulated active substances across the skin, without determining the skin permeation of the entire nanoparticle. On the other hand, in most studies which have evaluated the permeation of nanostructures or nanomaterials, no particle permeation/penetration was observed.

References [1] Hadgraft, J.: Skin, the final frontier. Int. J. Pharm. 224, 1–18 (2001) [2] Degim, T.I.: New tools and approaches for predicting skin permeability. Drug Discov. Today 11, 11–12 (2006) [3] Menon, G.K.: New insights into skin structure: scratching the surface. Adv. Drug. Deliv. Rev. 54(1), 3–17 (2002) [4] Gray, G.M., Yardley, H.J.: Lipid compositions of cells isolated from pig, human, and rat epidermis. J. Lipid Res. 16, 434–440 (1975)

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[114] Kang, M.J., Eum, J.Y., Jeong, M.S., Choi, S.E., Park, S.H., Cho, H.I., Cho, C.S., Seo, S.J., Lee, M.W., Choi, Y.W.: Facilitated Skin Permeation of Oregonin by Elastic Liposomal Formulations and Suppression of Atopic Dermatitis in NC/Nga Mice. Biol. Pharm. Bull. 33(1), 100–106 (2010) [115] Pople, P.V., Singh, K.K.: Development and evaluation of topical formulation containing solid lipid nanoparticles of vitamin. A AAPS Pharm. Sci. Tech. 7(4), 91 (2006) [116] Chen, H., Chang, X., Du, D., Liu, W., Liu, J., Weng, T., Yang, Y., Xu, H., Yang, X.: Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. J. Contr. Release 110, 296–310 (2006) [117] Borgia, S.L., Regehly, M., Sivaramakrishnan, R., Mehnert, W., Korting, H.C., Danker, K., Röder, B., Kramer, K.D., Schäfer-Korting, M.: J. Contr. Release 110, 151–163 (2005) [118] Bhalekar, M.R., Pokharkar, V., Madgulkar, A., Patil, N., Patil, N.: Preparation and Evaluation of Miconazole Nitrate-Loaded Solid Lipid Nanoparticles for Topical Delivery. AAPS Pharm. Sci. Tech. 10(1), 289–296 (2009) [119] Liu, J., Hu, W., Chen, H., Ni, Q., Xu, H., Yang, X.: Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery. Int. J. Pharm. 328, 191–195 (2007) [120] Kuntsche, J., Bunjes, H., Fahr, A., Pappinen, S., Rönkkö, S., Suhonen, M., Urtti, A.: Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model. Int. J. Pharm. 354, 180–195 (2008) [121] Kim, S.Y., Lee, Y.M.: Lipid nanospheres containing vitamin A or vitamin E: Evaluation of their stabilities and in vitro skin permeability. J. Ind. Eng. Chem. 5, 306–313 (1999) [122] He, R., Cui, D.-X., Gao, F.: Preparation of fluorescence ethosomes based on quantum dots and their skin scar penetration properties. Mater. Lett. 63, 1662–1664 (2009) [123] Primo, F.L., Rodrigues, M.M.A., Simioni, A.R., Bentley, M.V.L.B., Morais, P.C., Tedesco, A.C.: In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment. J. Magn. Magn. Mater. 320, e211–e214 (2008) [124] Mandawgade, S.D., Patravale, V.B.: Development of SLNs from natural lipids: Application to topical delivery of tretinoin. Int. J. Pharm. 363, 132–138 (2008) [125] Üner, M., Wissing, S.A., Yener, G., Muller, R.H.: Skin moisturizing effect and skin penetration of ascorbyl palmitate entrapped in Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) incorporated into hydrogel. Pharmazie 60, 751–755 (2005) [126] Puglia, C., Filosa, R., Peduto, A., Caprariis, P., Rizza, L., Bonina, F., Blasi, P.: Evaluation of Alternative Strategies to Optimize Ketorolac Transdermal Delivery. AAPS Pharm. Sci. Tech. 7, E1–E9 (2006) [127] Wissing, S.A., Muller, R.H.: Solid lipid nanoparticles as carrier for susncreens: in vitro release and in vivo skin penetration. J. Cont. Release 81, 225–233 (2002) [128] Puglia, C., Blasi, P., Rizza, L., Schoubben, A., Bonina, F., Rossi, C., Ricci, M.: Lipid nanoparticles for prolonged topical delivery: An in vitro and in vivo investigation. Int. J. Pharm. 357, 295–304 (2008) [129] Lai, F., Sinico, C., De Logu, A., Zaru, M., Müller, R.H., Fadda, A.M.: SLN as a topical delivery system for Artemisia arborescens essential oil: In vitro antiviral activity and skin permeation study. Int. J. Nanomed. 2(3), 419–425 (2007)

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[130] Piao, H., Kamiya, N., Hirata, A., Fujii, T., Goto, M.: A Novel Solid-in-oil Nanosuspension for Transdermal Delivery of Diclofenac Sodium. Pharm. Res. 25(4), 896– 901 (2008) [131] Passerini, N., Gavini, E., Albertini, B., Rassu, G., Di Sabatino, M., Sanna, V., Giunchedi, P., Rodriguez, L.: Evaluation of solid lipid microparticles produced by spray congealing for topical application of econazole nitrate. J. Pharm. Pharmacol. 61(5), 559–567 (2009) [132] Olvera-Martínez, B.I., Zares-Delgadillo, J.C., Calderilla-Fajardo, S.B., VillalobosGarcía, R., Ganem-Quintanar, A., Quintanar-Guerrero, D.: Preparation of polymeric nanocapsules containing octyl methoxycinnamate by the emulsification–diffusion technique: Penetration across the stratum corneum. J. Pharm. Sci. 94, 1552–1559 (2005) [133] Izquierdo, P., Wiechers, J.W., Escribano, E., Garcia-Celma, M.J., Tadros, T.F., Esquena, J., Dederen, J.C., Solans, C.: A study on the influence of emulsion droplet size on the skin penetration of tetracaine. Skin Pharm. Phys. 20(5), 263–270 (2007) [134] Munster, U., Nakamura, C., Haberland, A., Jores, K., Mehnert, W., Rummel, S., Schaller, M., Korting, H.C., Zouboulis, C.C., Blume-Peytavi, U., Schäfer-Korting, M.: Ru 58841-Myristate - Prodrug development for topical treatment of acne and androgenetic alopecia. Pharmazie 60(1), 8–12 (2005) [135] Babu, S., Fan, C., Stepanskiy, L., Uitto, J., Papazoglou, E.: Effect of size at the nanoscale and bilayer rigidity on skin diffusion of liposomes. J. Biomed. Mater. Res. 91(1), 140–148 (2009) [136] Schilling, K., Bradford, B., Castelli, D., Dufour, E., Nash, J.F., Pape, W., Schulte, S., Tooley, I., van den Boschi, J., Schellauf, F.: Human safety review of “nano” titanium dioxide and zinc oxide. Photochem. Photobiol. Sci. 9, 495–509 (2010) [137] Menzel, F., Reinert, T., Vogt, J., Butz, T.: Investigations of percutaneous uptake of ultrafine TiO2 particles at the high energy ion nanoprobe LIPSION. Nucl. Instrum. Methods Phys. Res. B 219-220, 82–86 (2004) [138] Cross, S.E., Innes, B., Roberts, M.S., Tsuzuki, T., Robertson, T.A., McCormick, P.: Human Skin Penetration of Sunscreen Nanoparticles: In-vitro Assessment of a Novel Micronized Zinc Oxide Formulation. Skin Pharmacol. Physiol. 20, 148–154 (2007) [139] Lekki, J., Stachura, Z., Dabros, W., Stachura, J., Menzel, F., Reinert, T., Butz, T., Pallon, J., Gontier, E., Ynsa, M.D., Moretto, P., Kertesz, Z., Szikszai, Z., Kiss, A.Z.: On the follicular pathway of percutaneous uptake of nanoparticles: Ion microscopy and autoradiography studies. Nucl. Instrum. Methods Phys. Res. B 260, 174–177 (2007) [140] Pinheiro, T., Pallon, J., Alves, L.C., Verıssimo, A., Filipe, P., Silva, J.N., Silva, R.: the influence of corneocyte structure on the interpretation of permeation profiles of nanoparticles across skin. Nucl. Instrum. Methods Phys. Res. B 260, 119–123 (2007) [141] Gontier, E., Ynsa, M.D., Biro, T., Hunyadi, J., Kiss, B., Gaspar, K., Pinheiro, T., Silva, J.N., Filipe, P., Stachura, J., Dabros, W., Reinert, T., Butz, T., Moretto, P., Surleve-Bazeille, J.E.: Is there penetration of titania nanoparticles in sunscreens through skin? A comparative electron and ion microscopy study. Nanotoxicology 2(4), 218–231 (2008) [142] Wu, J., Liu, W., Xue, C., Zhou, S., Lan, F., Bi, L., Xub, H., Yang, X., Zeng, F.-D.: Toxicity and penetration of TiO2 nanoparticles in hairless mice and porcine skin after subchronic dermal exposure. Toxicol. Lett. 191, 1–8 (2009)

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[143] Baroli, B., Ennas, M.G., Loffredo, F., Isola, M., Pinna, R., López-Quintela, M.A.: Penetration of metallic nanoparticles in human full-thickness skin. J. Invest. Dermatol. 127, 1701–1712 (2007) [144] Larese, F.F., D’Agostin, F., Crosera, M., Adami, G., Renzi, N., Bovenzi, M., Maina, G.: Human skin penetration of silver nanoparticles through intact and damaged skin. Toxicology 255, 33–37 (2009) [145] Xia, X.R., Monteiro-Riviere, N.A., Riviere, J.E.: Skin penetration and kinetics of pristine fullerenes (C60) topically exposed in industrial organic solvents. Toxicol. Appl. Pharmacol. 242, 29–37 (2010) [146] El-Sayed, I.H., Huang, X., 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. 5(5), 829–834 (2005) [147] Huff, T.B., Tong, L., Zhao, Y., Hansen, M.N., Cheng, J.-X., Wei, A.: Hyperthermic effects of gold nanorods on tumor cells. Nanomedicine 2(1), 125–132 (2007) [148] Baker, I., Zeng, Q., Li, W., Sullivan, C.R.: Heat deposition in iron oxide and iron nanoparticles for localized hyperthermia. J. Appl. Phys. 99, 08H106-08H106-3 (2006) [149] Hadjipanayis, C.G., Bonder, M.J., Balakrishnan, S., Wang, X., Mao, H., Hadjipanayis, G.C.: Metallic iron nanoparticles for MRI contrast enhancement and local hyperthermia. Small 4(11), 1925–1929 (2008) [150] Sonvico, F., Mornet, S., Vasseur, S., Dubernet, C., Jaillard, D., Degrouard, J., Hoebeke, J., Duguet, E., Colombo, P., Couvreur, P.: Folate-Conjugated Iron Oxide Nanoparticles for Solid Tumor Targeting as Potential Specific Magnetic Hyperthermia Mediators: Synthesis, Physicochemical Characterization, and in Vitro Experiments. Bioconjugate Chem. 16, 1181–1188 (2005) [151] Chen, X., Schluesener, H.J.: Nanosilver: A nanoproduct in medical application. Toxicol. Lett. 176, 1–12 (2008) [152] Xiao, L., Takada, H., Maeda, K., Haramoto, M., Miwa, N.: Antioxidant effects of water-soluble fullerene derivatives against ultraviolet ray or peroxylipid through their action of scavenging the reactive oxygen species in human skin keratinocytes. Biomed. Pharmacother. 59, 351–358 (2005) [153] Kaur, I.P., Kapila, M., Agrawal, R.: Role of novel delivery systems in developing topical antioxidants as therapeutics to combat photoageing. Ageing Res. Rev. 6, 271–288 (2007)

Chapter 2 Rheological Behavior of Semisolid Formulations Containing Nanostructured Systems Marta P. Alves, Renata P. Raffin, and Solange B. Fagan Centro Universitário Franciscano, UNIFRA, 97010-032, Santa Maria, RS, Brazil [email protected]

Abstract. This chapter presents an overview of the rheological properties of semisolids before and after the incorporation of nanostructures. Theoretical concepts as well as practical applications are described in order to show the influence of the rheological properties on the physicochemical and biological characteristics of formulations containing nanosystems.

2.1 Introduction Rheological analysis is fundamental in the optimization of topical drug delivery since it is used to predict the in vivo behavior of a material, being indispensable in the development, physicochemical characterization and quality control of dermatological formulations [1]. In particularly, rheology is applied to investigate the viscosity, plasticity, elasticity and deformation of materials under the influence of stress [2,3]. The rheological behavior is specific to each material and is determined by the qualitative and/or quantitative composition of formulations. These rheological properties are associated with important characteristics, such as physicochemical stability, drug delivery properties and applicability of each formulation. In relation to improving the rheological properties, the semisolid forms containing nanocarriers are promising materials that have been widely studied. Given the complexity of nanostructured materials and the recent use of these systems as carriers in topical products, rheological analysis is essential to understanding the interactions between nanostructures, vehicles and/or active compounds. Thus, in the next section, the fundamental concepts of rheology are presented. Also, several examples of the rheological behavior of semisolid formulations containing nanostructured systems, such as liposomes, polymeric nanocapsules and nanospheres, nanoemulsions, and lipid nanoparticles will be explored.

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2.2 Rheology: Fundamental Concepts The rheological characteristics of a product determine several technological aspects, for example, the way in which the product is removed from the packaging [4] or how the product can be spread on the skin. Thus, rheological studies carried out on semisolid systems aim to describe the relation between stress and deformation, mainly in systems for topical application [2,4,5]. The fundamental concepts of rheology are associated with the flow behavior described through the viscosity, elasticity, and plasticity of liquid, solid and semisolid materials under physical deformations. More specifically, viscosity is a measure of the internal resistance of a substance to flow when subjected to tension [4,6]. As a consequence, deformation is elastic if the substance recovers its original shape after the force has been withdrawn, or plastic, if deformation remains [7]. The unit most commonly use for viscosity is the centipoise (1 cP = 0.01 poise) and the corresponding unit in the international system is the millipascal.second (mPa.s) [7]. Similarly, shear rate (γ) corresponds to the variation in speed (dν) between two plains of the liquid as a function of the molecular layer (dr), indicating the rate at which the liquid flows when a specified force is applied [3,4]. The relation between viscosity and shear rate reveals the nature of the rheological behavior, which can be Newtonian or non-Newtonian.

Fig. 2.1 Classification (top) and the relationship between shear stress and shear rate (bottom) for the rheological behavior of fluids.

2 Rheological Behavior of Semisolid Formulations

39

In the case of Newtonian flow, the shear stress (τ) is directly proportional to the shear rate (γ), or τ = ηγ, where η is the viscosity. However, a non-Newtonian fluid does not present a direct linear relationship between shear stress and shear rate, and the interactions among the particles are mainly responsible for the complex rheological performance of these systems. In this case the flow can be viscoelastic, time-dependent (thixotropic or rheopectic) or time-independent (plastic, pseudoplastic or dilatant), as shown schematically in Figure 1.D. Therefore, semisolid materials can be non-Newtonian materials with or without thixotropy or rheopexy. Therefore, non-Newtonian flows can show various behaviors leading to different types of rheograms, such as Bingham (ideal plastic), pseudoplastic described by the power law (shear thinning, shear thickening or dilatant), and pseudoplastic with yield point [2,7]. Subsequently, the representative flow curve, or rheogram, is obtained by plotting shear stress versus shear rate [4]. This can indicate the most appropriate model for a fluid depending on its response to deformation and the experimental data fit for each model [6]. There are several models which may be used to establish the flow index or viscosity (η) in different non-Newtonian models [8], as shown in Table 2.1. Table 2.1 Flow models according to the curves of shear stress (τ) versus shear rate (γ), equations and parameters.

Model

Equation

Bingham

τ = τ 0 + ηγ

(ideal plastic) Casson

Parameter

τo

Yield stress (Pa)

η

Viscosity (Pa.s)

τ 0,5 = τ 00,5 + η 0,5γ 0,5 τo

Shear stress (Pa)

η

Viscosity (Pa.s)

K

Consistency (Pa.sn)

n

Index of flow

τo

Shear stress (Pa)

Bulkley

K

Consistency (Pa.sn)

(yield-pseudoplastic)

n

Index of flow

(plastic) Ostwald

τ = Κγ n

(pseudoplastic) Herschel-

τ = τ 0 + Κγ n

Similarly, thixotropy is important for the percutaneous application of a drug and it represents the difference in the hysteresis loop between the ascending and the descending curves of a rheogram. This behavior occurs when the preparation is subjected to a shear force and its network structure breaks down leading to a

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M.P. Alves, R.P. Raffin, and S.B. Fagan

gradual decrease in viscosity which aids its spreading on the skin. When the shear force is removed, the viscosity recovers slowly and the increased viscosity helps to keep the preparation on the skin [9]. The physicochemical characterization of solid products is used in several studies with the objective of determining the effects over time, at different temperatures, with the incorporation of active substances and of nanocarriers [10-14]. In general, it is observed that polymeric nanoparticles have Newtonian behavior. Lipid nanoparticles can be prepared with different lipid contents and this determines the suspension viscosity. Lipid nanoparticle suspensions can vary from a low viscous Newtonian behavior suspension to a semisolid one containing up to 40% of lipids [15]. Topical formulations are usually semisolids that are easily applied and handled. Aqueous nanoparticle suspensions can be incorporated into different semisolid formulations as hydrophilic bases or to produce lipid nanoparticle gels or creams [16]. The rheological properties of semisolids are very important physical parameters in terms of percutaneous applications [15,17]. Some parameters that should be evaluated are emolience, which is affected by lubricity, and the spreading properties. These two parameters are related to the ability of the formulation to cover a surface and reduce attrition [18]. Different types of nanostructures have been incorporated into semisolids with a view to topical delivery. Each kind of nanostructure has a particular influence on the semisolid rheological behavior, and this is particularly relevant in the case of hydrogels. In general, researchers have evaluated viscosity and the rheological pattern before and after the incorporation of a drug into loaded or unloaded nanoparticles. Solid lipid nanoparticles and nanostructured lipid carriers can be prepared with different solid contents and a great range of viscosities can be achieved. Furthermore, they can be incorporated into hydrogels and creams, which have previously demonstrated stability [19]. Polymeric nanospheres and nanocapsules have a viscosity close to that of water and have also been incorporated into hydrogels and creams [13,14,20]. An increase in viscosity can alter the drug permeation pattern. Thus, in the next section the rheological properties of nanoparticles incorporated into semisolids will be discussed.

2.3 Rheological Properties of Nanoparticles Incorporated into Semisolids Analysis of the rheological properties of nanoparticles incorporated into semisolids for topical drug delivery is essential to determining the technological potential of these systems. Hydrogels can be prepared with natural, semi-synthetic and synthetic polymers [18]. Examples of natural polymers are chitosan and xanthan gum. Chitosan is a

2 Rheological Behavior of Semisolid Formulations

41

cationic polymer which exhibits antimicrobial activity and mucoadhesion [18]. Xanthan gum is a natural polysaccharide used as a food additive and a rheology modifier. Cellulose derivatives are semi-synthetic polymers that do not need neutralization. In addition, they present different degrees of viscosity. Carbomers (Carbopol®) are synthetic polymers that require neutralization in order to gel. Many studies have investigated the incorporation of nanoparticles into different Carbopol gels [16,20-22]. Unloaded solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) prepared with Dynasan® 116 or Dynasan® 116/Mygliol® 812 (70:30) have been incorporated into four different hydrogels [16]. Hydrogels were prepared with 1% xanthan gum, 1.75% hydroethylcelullose (HEC), 0.5% Carbopol® 943 or 1% chitosan. All formulations also contained 5% of glycerol. The incorporation of lipid nanoparticles (SLN and NLC) into xanthan gum hydrogels results in flow curves with plastic characteristics presenting thixotropy. The incorporation of SLN and NLC into HEC, xanthan gum and Carbopol® hydrogels leads to flow curves with plastic characteristics. However, chitosan hydrogels reveal Newtonian behavior with a yield value of approximately zero. It has been observed that SLN have a gel-like structure. The increase in the lipid content of the system leads to different flow characteristics and thixotropy has been observed in systems with higher lipid content. Different drugs have been encapsulated in lipid nanoparticles, which were then incorporated into semi-solids, mainly hydrogels [9,21,24]. Also, in another study Carbopol Ultrez 10® was selected as a gelling agent for valdecoxib-loaded NLC [23]. Rheological studies were carried out using a Brookfield viscosimeter and the viscosity was 85 x 105 cP and the flow index was 0.39, indicating a pseudoplastic flow. Coenzyme Q10-loaded NLC have been prepared through high pressure homogenization and incorporated into 2% xanthan gum hydrogel (2.5% propyleneglycol and 5% glycerin) [24]. The viscoelastic analysis of a NLC gel was performed using a rheometer equipped with a cone-and-plate test geometry. Q10-loaded NLC revealed plastic flow, presenting thixotropy with more elastic than viscous behavior. An increase in the viscosity of a hydrogel containing Q10-loaded NLC was observed during storage (6 months), which can be attributed to the spatial arrangement of the lipid blend in the NLC. Triamcinolone acetonide acetate has been encapsulated in SLN (solid lipid – Precitol ATO® 5) and used to prepare a 0.75% Carbopol ETD® 2020 gel [9]. This SLN gel exhibited better thixotropy than the unloaded gel, which increased slightly with an increase in SLN particle size. Tretinoin-loaded SLN, prepared using the emulsification-solvent diffusion method, have been incorporated in different gels: Xanthan Gum, Carbopol® Ultrez 10, Carbopol® 940 and Carbopol® ETD 2020 [25]. A Brookfield viscosimeter with different spindles was used for the rheological analysis. Carbopol® ETD 2020 led to adequate sensorial characteristics without SLN agglomeration. Xanthan gum yielded gels with low viscosity and tack, and Carbopol® Ultrez 10 and Carbopol®

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M.P. Alves, R.P. Raffin, and S.B. Fagan

940 resulted in the agglomeration of SLN. Carbopol® ETD 2020 gel presented the following rheological parameters: viscosity (at 5 rpm) of 88 x 105 mPa.s, consistency index of 24.05 x 106 dyn/cm2, and flow index of 0.381. NLC containing minoxidil have been incorporated into perfluorocarbon and Carbopol® 940 hydrogels in a concentration of 40% of the colloidal suspension in the gels [21]. Rheological flow patterns were recorded for both formulations. The loaded and unloaded formulations exhibited a pseudoplastic pattern, presenting thixotropy. For the lipid nanoparticles, there was no pattern in rheological behavior on comparing the hydrogels with and without the nanoparticle incorporation. Similarly to the lipid nanoparticles, polymeric nanocapsules and nanospheres have been incorporated into semi-solids and their rheological behavior evaluated before and after the incorporation [14,20,22]. Dexamethasone-loaded poly(εcaprolactone) nanocapsules were prepared by nanoprecipitation and incorporated into Carbopol Ultrez® hydrogel [22]. In order to verify any possible difference in the hydrogel viscosity after the incorporation of nanocapsules, viscosity was assessed using a rotational viscosimeter. There was no significant difference between the hydrogel containing the pure drug and that with drug-loaded nanocapsules. In order to compare different kinds of polymer nanostructures, nanocapsules and nanospheres and a nanoemulsion containing nimesulide were incorporated into 0.2 % Carbopol 940® hydrogels containing 5% sorbitol. The colloidal suspensions were used as the aqueous phase of the semisolids. The gels were evaluated using a rotational viscosimeter, and the data were fitted to conventional flow equations [14]. All gels present non-Newtonian behavior, with the best fit being obtained with the Oswald model, demonstrating a pseudoplastic behavior. No thixotropic phenomenon was observed, and the incorporation of the nanocarrier did not alter the rheological properties of the gels. Liposomes have also been used to prepare semi-solids [12,26,27]. Multilamellar liposomes containing calcein or griseofulvins were incorporated into gels of Carbolpol® 974, hydroxyethylcellulose or a mixture of the two [26]. Rheological measurements were performed in a stress rheometer. The three gels are shearthinning fluids and have a tendency to become Newtonian at asymptotically low shear rates. HEC and the gel mixture showed similar behavior, very different from that of Carbopol. Carbopol presented an elastic solid behavior due to a network of secondary bonds. In contrast, the behavior of the HEC gel and the mixture were closer to that of a viscous liquid. Also, liposomes containing PEG 2000 or stearylamine have been incorporated into Carbopol® 974 hydrogels at two concentrations, 2 and 10 mM. At 2 mM, the liposomes had no influence on the rheological behavior of the gel. However, at 10 mM, the cationic liposomes interacted with the carboxylic groups of the polymer chains and caused an increase in the gel viscosity [12]. Temoporfin liposomes have been evaluated in different Carbopol® 980 concentrations [27]. The results indicated that the more concentrated gels showed an increase in viscosity that reduced the drug penetration into skin. The optimal formulation was obtained with Carbopol at 0.75%.

Minoxidil

None

Coenzyme Q10 Dexamethasone

NLC

SLN NLC

NLC Polymeric nanocapsules

None

Temoporfin

Sterically stabilized and cationic liposomes

Liposome

Polymeric nanocapsules Coenzyme Q10 Polymeric nanocapsules, nano- Nimesulide spheres and nanoemulsion SLN Triamcinolone acetonide acetate NLC Valdecoxib Multilamellar liposomes Calcein and griseofulvin

Drug/Active

Nanoparticle

[9]

Carbopol Ultrez 10® Pseudoplastic Carbopol 974®, Hydroxy- Shear-thinning fluids with a tendency to lethylcellulose become Newtonian at asymptotically low shear rates Carbopol 974P® At 2 mM same as pure gel, i.e., shearthinning behavior (Cross model). At 10 mM cationic liposomes increased gel viscosity Carbopol 980® Plastic flow behavior (Herschel-Bulkley)

Pseudoplastic with thixotropy

[27]

[12]

[23] [26]

[24] [22]

[16]

Carbopol ETD 202®0

Reference [21]

[20] [14]

Rheological behavior

Pseudoplastic with thixotropy Carbopol 940® Perfluorocarbon Carbopol 934®,Chitosan, Plastic with thixotropy, Newtonian, PlasXanthan Gum or Hy- tic or Plastic, respectively droxyethylcellulose 4000 Xanthan gum Plastic Carbopol Ultrez® One point evaluation – no difference with or without NC Carbopol 940® Pseudoplastic Carbopol 940® Pseudoplastic

Semisolid

Table 2.2 Influence on the rheological behavior of each kind of nanostructure incorporated into different semisolids.

2 Rheological Behavior of Semisolid Formulations 43

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In summary, Table 2.2 shows the main rheological behaviors associated with each kind of nanostructure, with different drugs or active compounds incorporated, in different semisolids as presented in this section.

2.4 Conclusions The incorporation of different kinds of nanostructures into semisolids, particularly hydrogels, has been described and the rheological behavior discussed. In each case, depending on the semisolid and on the nanoparticle composition, different patterns were observed. The rheological analysis of each semisolid before and after the incorporation of a certain type of nanoparticle needs to be studied in order to evaluate the viscosity and spreadability for topical administration and the potential for technological application.

References [1] Ramirez, A., Fresno, M.J., Jiménez, M.M., Selles, E.: Rheological study of Carbopol® UltrezTM 10 hydroalcoholic gels, I: Flow and thixotropic behavior as a function of pH and polymer concentration. Die Pharmazie 54, 444 (1999) [2] Radebaugh, G.W.: Rheological and Mechanical Properties of Dispersed Systems. In: Lieberman, J.A., Rieger, M.M., Banker, G.S. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, 2nd edn., New York (1996) [3] Schott, H.: Rheology in Remington: the science and practice of pharmacy. In: Gennaro, A.R. (ed.), 20th edn. University of the Sciences in Philadelphia, Philadelphia (2000) [4] Martin, A.: Rheology in Physical pharmacy: physical chemical principles the pharmaceutical sciences, 4th edn. Lippicott Williams & Wilkins, Philadelphia (1993) [5] Netz, P.A., George, G.O.: Reologia in Fundamentos de físico-química: uma abordagem conceitual para as ciências farmacêuticas. Art. Med., Porto Alegre, RS (2002) [6] Wood, J.H.: Reologia Farmacêutica. In: Lachman, L., Lieberman, H.A., Kang, J.L. (eds.) Teoria e Prática na Indústria Farmacêutica, Fundação Galauste Gulbenkian, Lisboa (2001) [7] Wood, J.H.: Reologia Farmacêutica. In: Lachman, L., Lieberman, H.A., Kang, J.L. (eds.) Teoria e Prática na Indústria Farmacêutica, Fundação Galauste Gulbenkian, Lisboa (2001) [8] Kim, J., Song, J., Lee, E., Park, S.: Rheological properties and microstructures of Carbopol gel network system. Colloid and Polymer Science 281, 614–623 (2003) [9] Liu, W., Hu, M., Liu, M., Xue, C., Xu, H., Yang, X.L.: Investigation of the carbopol gel of solid lipid nanoparticles for the transdermal iontophoretic delivery of triamcinolone acetonide acetate. Int. J. Pharm. 364, 135–141 (2008) [10] Welin-Berger, K., Neelissen, J.A.M., Bergenstahl, B.: The effect of rheological behaviour of a topical anaesthetic formulation on the release and permeation rates of the active compound. Eur. J. Pharm. Sci. 13, 309–318 (2001) [11] Pavelic, Z., Skalko-Basnet, N., Schubert, R.: Liposomal gels for vaginal drug delivery. Int. J. Pharm. 219, 139–149 (2001)

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[12] Boulmedarat, L., Grossiord, J.L., Elias Fattal, E., Bochot, A.: Inflluence of methyl-βcyclodextrin and liposomes on rheological properties of Carbopol 974® NF gels. Int. J. of Pharm. 254, 59–64 (2003) [13] Milão, D., Knorst, M.T., Guterres, S.S.: Hydrophilic gel containing nanocapsules of diclofenac: Development, stability study and physico-chemical characterization. Die Pharmazie 58, 325–329 (2003) [14] Alves, M.P., Pohlmann, A.R., Guterres, S.S.: Semisolid topical formulations containing nimesulide-loaded nanocapsules, nanospheres or nanoemulsion: development and rheological characterization. Die Pharmazie 60, 900–904 (2005) [15] Lippacher, A., Muller, R.H., Mader, K.: Liquid and semisolid SLNe dispersions for topical application: rheological characterization. Eur. J. Pharm. Biopharm. 58, 561– 567 (2004) [16] Souto, E.B., Wissing, S.A., Barbosa, C.M., Muller, R.H.: Evaluation of the physical stability of SLN and NLC before and after incorporation into hydrogel formulations. Eur. J. Pharm. Biopharm. 58, 83–90 (2004) [17] Lippacher, A., Müller, R.H., Mäder, K.: Preparation of semisolid drug carriers for topical application based on solid lipid nanoparticles. Int. J. Pharm. 214, 9–12 (2001) [18] Doktorovova, S., Souto, E.B.: Nanostructured lipid carrier-based hydrogel formulations for drug delivery: a comprehensive study. Expert. Opin. Drug. Del. 6, 165–176 (2009) [19] Pardeike, J., Hommoss, A., Müller, R.H.: Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int. J. Pharm. 366, 170–184 (2009) [20] Terroso, T., Külkamp, I.C., Jornada, D.S., Pohlmann, A.R., Guterres, S.S.: Development of Semi-Solid Cosmetic Formulations Containing Coenzyme Q10-Loaded Nanocapsules Lat. Am. J. Pharm. 28(6), 819–826 (2009) [21] Silva, A.C., Santos, D., Ferreira, D.C., Souto, E.B.: Minoxidil-loaded nanostructured lipid carriers (NLC): characterization and rheological behavior of topical formulations. Die Pharmazie 64, 177–182 (2009) [22] Marchiori, M.L., Lubini, G., Dalla Nora, G., Friedrich, R.B., Fontana, M.C., Ourique, A.F., Bastos, M.O., Rigo, L.A., Silva, C.B., Tedesco, C., Beck, R.C.R.: Hydrogel containing dexamethasone-loaded nanocapsules for cutaneous administration: preparation, characterization, and in vitro drug release study. Drug. Dev. Ind. Pharm. 36, 962–971 (2010) [23] Joshi, M., Patravale, V.: Formulation and Evaluation of Nanostructured Lipid Carrier (NLC)–based Gel of Valdecoxib. Drug Dev. Ind. Pharm. 32, 911–918 (2006) [24] Junyaprasert, V.B., Teeranachaideekul, V., Souto, E.B., Boonme, P., Müller, R.H.: Q10-loaded NLC versus nanoemulsions: Stability, rheology and in vitro skin permeation. Int. J. Pharm. 377, 207–214 (2009) [25] Shah, K.A., Date, A., Joshi, M.D., Patravale, V.B.: Solid lipid nanoparticles (SLN) of tretinoin: Potential in topical delivery. Int. J. Pharm. 345, 163–171 (2007) [26] Mourtas, S., Fotopoulou, S., Duraj, S., Sfika, V., Tsakiroglou, C., Antimisiaris, S.G.: Liposomal drugs dispersed in hydrogels Effect of liposome, drug and gel properties on drug release kinetics. Col. Sur. B: Biointerfaces 55, 212–221 (2007) [27] Dragicevic-Curic, N., Winter, S., Stupar, M., Milic, J., Krajisnik, D., Gitter, B., Fahr, A.: Temoporfin-loaded liposomal gels: Viscoelastic properties and in vitro skin penetration. Int. J. Pharm. 373, 77–84 (2009)

Part II

Nanocarriers for Skin Care and Dermatological Treatments

Chapter 3

Polymeric Nanocapsules: Concepts and Applications Fernanda S. Poletto1, Ruy C.R. Beck2, Sílvia S. Guterres2, and Adriana R. Pohlmann1 1

Universidade Federal do Rio Grande do Sul, Departamento de Química Orgânica, Av. Bento Gonçalves, CP 15003, 91501-970, Porto Alegre, RS, Brazil [email protected] 2 Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brazil

Abstract. This chapter presents an overview of polymeric nanocapsules for dermatological and cosmetic applications, including their preparation methods, physicochemical characterization and models of supramolecular structures. Polymeric nanocapsules are advantageous because of their ability to control the release rate and the penetration/permeation of drugs and active ingredients in the skin. These properties can be modulated through manipulating the qualitative and quantitative compositions of formulations. The chemical nature of raw materials can define the supramolecular structures of the nanocapsule core and surface. In addition, polymeric nanocapsules protect the encapsulated drug or active ingredient from degradation by acting as reservoirs. Aqueous suspensions of polymeric nanocapsules are directly applied on the skin or used as intermediate products for semisolid formulations, such as hydrogels and emulgels. The rheological characteristics of semisolid formulations can be modified by the presence of nanocapsules. Polymeric nanocapsules are valuable devices for skin applications and represent a promising research field in terms of providing products to be explored by industry.

3.1 Introduction In recent decades, polymeric nanocapsules have been studied and proposed for systemic and topical drug delivery [1, 2]. The first academic studies reporting the preparation of nanocapsules date back to the 1980s [3,4]. Conceptually, polymeric nanocapsules are vesicular particles smaller than 1 μm composed of an oily core surrounded by an ultrathin polymeric wall [2]. These devices are stabilized by surfactants and/or steric agents. A monomodal and narrow size distribution is crucial to guarantee their nanotechnological quality [5].

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In the cosmetics and dermatological fields, these colloidal aqueous suspensions increase drug stability [6], delay and control the drug release [7] and increase the drug adhesivity on the skin [8,9]. Nanocapsules can be used to restrict the skin penetration of substances like UV filters [10], or to deliver active ingredients, such as genistein, to the deep layers of skin [11]. The potential dermatological or cosmetic use of nanocapsules was investigated in the early 1990s [12]. The first cosmetic product on the market based on polymeric nanocapsules was launched by the French company L´Oreal in 1995. Since then, new preparation methods have been developed, and useful insights regarding the supramolecular structure of polymeric nanocapsules have been provided [2,13]. The number of articles related to polymeric nanocapsules for cosmetic and dermatological applications significantly increased in the last decade, particularly in the last 5 years, when 75% of the total number of papers was published (Web of Science® Database). The number of patents demonstrates the economic potential of nanocapsules in the cosmetic and dermatological industries (Web of Knowledge® Database). Thus, this chapter is devoted to discussing the general principles of the preparation and characterization of polymeric nanocapsules, as well as some representative formulations for skin applications.

3.2 Models of the Supramolecular Structure of Nanocapsules The first illustrative model proposed for polymeric nanocapsules comprised a vesicle of oil surrounded by an ultrathin polymeric wall [3,4]. However, initially no interaction was considered between the oily core, the external medium and the polymer in this model. In order to investigate this subject in depth, further studies were carried out using different techniques. Nuclear magnetic resonance spectroscopy showed that poly(n-butyl cyanoacrylate) is located at the oil-water interface acting as a nanocapsule wall [14,15]. Furthermore, the core material (triglyceride) and the copolymer emulsifier (Pluronic® F68) showed high mobility in an ethanol/water continuous phase, being temporarily adsorbed on the nanocapsule wall [15]. More recently, the composition of the particle-water interface in aqueous suspensions of nanocapsules was investigated using environment-sensitive fluorescent-labeled poly(methyl methacrylate) [16]. The comparative study of fluorescent dyes either chemically bound to the polymer or dispersed in the core of nanocapsules demonstrated that the polymer interacts at the particle-water interface with both water and oil (triglycerides), while the bulk material did not show swelling in oil or water within 60 days. The polymer swelling test is a simple experiment used to verify whether a vesicular structure containing a polymeric wall can be formed after selecting the materials for the nanocapsule preparation. Swelling experiments carried out with polyesters [poly(epsilon-caprolactone) and poly(D,L-lactide)] have demonstrated that benzyl benzoate is polymer solvent, indicating that nanocarriers prepared using these materials were nanoemulsions rather than nanocapsules [17]. On the other hand, the swelling of poly(epsiloncaprolactone) in octyl methoxycinnamate, a chemical sunscreen, indicated that nanocapsules prepared with these components were vesicles because the polymer was not swollen by the oil [18].

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The nature of the surfactant can also influence the morphological structure of the nanocapsules. Using sodium taurocholate as a surfactant, polyester particles were formed with an acorn morphology, in which a hemisphere of the liquid material of the core (perfluorooctyl bromide) coexisted with a hemisphere of the polymer, whereas using sodium cholate a perfect core-shell structure was obtained. Moreover, the use of poly(vinyl alcohol) as an emulsifier, generated particles presenting both morphologies [19]. Hence, the supramolecular structure of the nanocapsules is related to the nature and the interactions of the raw materials of which the device is composed. Considering the importance of the supramolecular structure in terms of the physicochemical and biological behavior, the composition of the nanocapsules and the resulting structures are discussed in more detail in the following sections.

3.2.1 Core The core in nanocapsules acts as a reservoir for the drug or active ingredient [6,20]. A single chemical substance or a mixture of several can be used as the structural component of the core (Table 3.1). Besides its structural function, this component can have biological activity or effects [21], and different examples of this have been reported in the literature, including nanocapsules containing octyl methoxycinnamate, a UV chemical sunscreen, [22] and turmeric oil, which has properties such as antibacterial, antifungal, antiplatelet, insect repellent, antioxidant, antimutagenic and anticarcinogenic [23]. In general, polymeric nanocapsules have an oily core composed of triglycerides, the liquid active ingredient and, in addition, some examples present a mixture of chemical substances as the core, such as capric/caprylic triglycerides and sorbitan monostearate [24] or vegetable oils and sorbitan monostearate [25] (Table 3.1). In this regard, nanocapsules with sorbitan monostearate and oil as the core have been referred to as lipid-core nanocapsules, which were developed to encapsulate drugs or cosmetic ingredients [5,26,27]. The structural components of the core can dissolve or disperse the drug or active ingredient. Previous studies have reported the encapsulation of active substances in nanocapsules by different mechanisms. In general, nanocapsules have high drug loading capacity compared to polymeric nanospheres as a consequence of the solubility of the drug or active ingredient in the oil core [1]. However, the loading capacity in this case is limited to the saturation concentration of the drug in the organic phase of nanocapsules when the oil is the single structural component of the core. Lipid-core nanocapsules have been developed using not only a liquid lipid (medium-chain triglycerides), but also a solid lipid (sorbitan monostearate) [5,26,27]. Recent studies [5,28] have demonstrated that the release of indomethacin ethyl ester, an anti-inflammatory pro-drug, can be controlled by varying the concentration of the polymer as well as the concentration of caprylic/capric trygliceride or sorbitan monostearate in the core. These new nanocapsules show a different behavior to that of nanocapsules prepared with a single oil as the core. In the latter, the drug release is controlled only by the polymeric wall [29]. The presence of sorbitan monostearate in the core influences the drug release since it increases the non-Newtonian viscosity of the oil

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[5]. As a consequence, the relative permeability of the drug decreases with the increase in the resistance to the substance diffusion.

3.2.2 Surface The nanocapsule surface is directly related to the nature of the polymer and the surfactants used to prepare the device. Synthetic and natural polymers have been used as the wall of nanocapsules (Table 3.1). Ideally, they must be non-toxic and biodegradable. Generally, higher reproducibility and purity can be achieved using synthetic polymers instead natural polymers [30]. The most common surfactants used to stabilize nanocapsules for skin applications are poly(ethylene glycol) derivatives, such as Poloxamer® and Tween®, and phospholipids, such as lecithin. Poly(vinyl alcohol) is also commonly reported [2,9]. Nanocapsules intended for cosmetic and dermatological applications must not cause cutaneous sensitization. Thus, the polymers and surfactant should be selected in order to avoid immune response and to guarantee drug release control. Furthermore, the surfactant or emulsifiers must be carefully selected since they can influence the skin permeation of the substance [31]. The polymeric wall plays an important role in the physicochemical and biological properties of nanocapsules, acting as a barrier to the diffusion [32]. For this reason, the behavior of the drug release from nanocapsules prepared with a single oil as the core is directly related to the characteristics of the wall [29]. Furthermore, the crystallinity of the polymeric wall influences the drug permeability [33]. In addition, active substances can be protected against the photo-degradation induced by UV radiation by encapsulation in nanocapsules, when the wall is comprised not only of semi-crystalline [18] but also amorphous [34] polymers. Nanocapsules can be categorized as ionic or non-ionic according to the presence or absence of formal charge on their surfaces. The presence of charge can increase the hydration forces at the surface of the nanocapsules, favoring repulsion among the particles in suspension and reducing aggregation in clusters [35]. Ionic surfaces can be classified as anionic (negative) or cationic (positive) depending on the chemical nature of polymers and surfactants. Negatively charged polymers are generally formed by acrylic and methacrylic acid, whereas positively charged polymers commonly contain amino quaternary cations as pendant or substituent groups. Chitosan is the most common positively charged polymer used for this purpose [8]. This polysaccharide is derived from chitin by deacetylation, possessing glucosamine groups that protonate under acid conditions. Comparisons between cationic and anionic nanocapsules have demonstrated that the former show affinity for the negatively charged porcine skin surface in contrast to the anionic carriers. In addition, a higher amount of the active substance was delivered from the cationic nanocapsules to the stratum corneum [36]. The cationic nanocapsules can interact more intensely with the skin providing adhesivity [8]. More recently, an amine group was chemically bound to one of the end chains of poly(lactide-coglycolide) to provide acid-base equilibrium by quaternary amino group formation. The functionalized polymer was used to prepare nanocapsules, which showed increased skin penetration of genistein overcoming the barrier function of the

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stratum corneum [11]. Although the drug deeply penetrated into the skin, the nanocapsules remained in the stratum corneum, suggesting a disturbance of the assembled lipid lamellar layers caused by the charge effect which promoted the drug diffusion through the skin layers. Nanocapsules can also have non-ionized surfaces, when hydrophobic or neutral hydrophilic polymers are used. Ionic telechelic groups can influence the nanocapsule surface charge, but this effect decreases for higher molecular weight polymers [37]. For skin applications, the most common hydrophobic polymers used in nanocapsules are polyesters, such as poly(epsilon-caprolactone), poly(D,L-lactide) and their copolymers. Considering the highly hydrophobic character of these polymers, the nanocapsules are stabilized by the use of highly water-soluble surfactants, and thus their fast aggregation in aqueous medium can be avoided via a steric hindrance mechanism. Block-copolymers can be used to introduce hydrophilicity at the nanocapsule surface in order to avoid the use of surfactants. Poly(ethylene glycol) copolymers are the most common raw materials used for this purpose [2,38]. Non-ionic nanocapsules are advantageous because of their greater physicochemical compatibility with other ingredients of dosage forms, increasing the versatility for compounding. Moreover, the release of active substances to the skin is influenced by the degree of hydrophobicity of the polymeric wall. The uptake of Nile Red, a dye used as a model in nanocapsules, was observed to increase as the hydrophobicity of the nanocapsule surface increased [39].

3.3 Nanocapsule Preparation The methods described in the literature to prepare polymeric nanocapsules can be categorized as chemical and physicochemical methods, depending on whether the nanocapsules are generated by in situ polymerization or by interfacial precipitation of the preformed polymer [2]. Both strategies are discussed in detail below.

3.3.1 Chemical Methods Nanocapsules can be prepared through the polymerization of monomers at the surface of droplets in nanoemulsions [3]. The first requirement for this method is to obtain the nanoemulsion template. The template is generally prepared by solvent displacement or high-shear techniques [43]. The polymerization step is carried out under mechanical stirring in the presence of an initiator using specific conditions (pH, temperature, etc). The monomer can be added either into the organic phase before mixing with the aqueous phase or in the external phase after the nanoemulsion preparation. The polymerization rate must be high in order to allow the efficient formation of the polymeric wall around the oily droplet [1,44]. Alkyl cyanoacrylate derivatives are the most common monomers used to prepare nanocapsules by in situ polymerization because they are well tolerated in vivo and the reaction is fast and easily carried out in aqueous medium [44,45]. For nanocapsule preparation, alkyl cyanoacrylate is polymerized through an anionic mechanism using a nucleophile as the initiator. The drug or active

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ingredient can be added before or after the polymerization. Mutual reactivity must be considered between the monomer and the drug when chemical methods are used to prepare nanocapsules [43]. In addition, process variables such as pH and monomer concentration can influence the molecular weight of the polymer [45] affecting the physicochemical characteristics of the formulation. The interfacial polymerization carried out in acid medium can be advantageous over the classical conditions of anionic polymerization to improve the reproducibility of the product characteristics [46]. The advantages associated with the chemical methods include the possibility of synthesizing statistical or block copolymers using simultaneously two different monomers or a monofunctionalized polymer and a cyclic monomer in the reaction medium. A one-step procedure can be carried out combining interfacial polycondensation and spontaneous emulsification. The latter consists of an organic phase containing a lipophilic monomer, oil, and a lipophilic surfactant injected into an aqueous phase containing a hydrophilic monomer and hydrophilic emulsifying agent. The organic solvent and water diffuse into each other forming the dispersed nanodroplets of oil. The monomers react at the oil-water interface, generating the polymeric wall around the oily core [47].

3.3.2 Physicochemical Methods Physicochemical methods involve the interfacial precipitation of preformed polymers. These methods can also be categorized as solvent displacement and high-shear methods. The interfacial deposition of preformed polymers based on the solvent displacement method was first proposed by Fessi and co-workers in 1988 [4,48]. This method constitutes a bottom-up strategy in nanotechnology. In this case, an organic phase containing the oil, the hydrophobic active ingredient, the polymer and a water soluble organic solvent is injected into an aqueous phase containing water and the surfactant under mild stirring. The oily droplets are generated by spontaneous emulsification simultaneously to the precipitation of polymer on the oil-water interface. The main condition required for the nanocapsule formation is the insolubility of the bulk polymer in the inner (oil) and outer (water) phases [4]. In addition, the requirement for spontaneous emulsification is a high miscibility of the organic solvent in water in order to cause supersaturation of the oil in the mixture, leading to its nucleation in small droplets [49,50]. The polymer precipitates onto the oily core due to its reduced solubility in both phases. Physicochemical parameters, such as size and polydispersity, seem to be influenced by the interfacial tension between the oil and the aqueous phase [37]. The interfacial deposition of preformed polymers based on high-shear methods involves mechanical processes consisting of two steps. The first step is the preparation of a miniemulsion, and the second step consists of shearing it to a thin emulsion at the nanoscale diameter with narrow particle size distribution [51]. Thus, emulsification-solvent diffusion is one of the most common high-shear methods. The drug or active ingredient is dissolved in the solvent, partially miscible with water, containing the polymer. This organic phase is emulsified with an aqueous phase containing emulsifying agent in order to obtain an oil-in-water

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(O/W) emulsion. The water and solvent are previously saturated in each other and, generally, rotor/stator devices or ultrasound generators are used to shear the emulsion. The subsequent addition of a second aqueous phase to the O/W emulsion leads to the diffusion of the solvent to the continuous phase resulting in the formation of nanocapsules [52]. The organic solvent is eliminated by dialysis or evaporated under reduced pressure. The chemical composition of the organic phase and the droplet size of the primary emulsion define the final size distribution of the nanocapsules [53]. In this case, the size of the primary emulsion is a function of the mutual diffusion of water and solvent, as well as of the solvent– polymer interaction parameter [54]. Moreover, the control of the primary emulsion size is also dependent on the interface tension gradient. Therefore, the control of the nanocapsule sizes can be achieved by tuning the surface tension of the organic phase through varying the volume fraction of ethanol as the co-solvent in the organic phase [55]. Other high-shear methods used to prepare nanocapsules are salting out [56,57] and emulsification-solvent evaporation [58]. For the former, the precipitation of a polyelectrolyte polymer onto the emulsion droplets is caused by adding salt to the formulation medium. In the case of the latter, the solvent is removed under reduced pressure after the O/W emulsification. Aqueous core-nanocapsules are also prepared by emulsification-solvent evaporation using a multiple emulsion W/O/W. In this case, the organic phase is composed of an organic polymer solution [53,59].

3.4 Methods of Physicochemical Characterization Controlling the particle size and polydispersity is essential to ensure the nanotechnological properties of the nanocapsules. Light scattering and electron microscopy are the most common methods used to determine these parameters (Fig. 3.1). Regarding the light scattering, incident radiation is deflected from its trajectory by the sample. The intensity of the scattered radiation as a function of time, at a fixed scattered angle, provides an insight into the structure of the sample. As nanocapsule diameters are near the visible spectrum of light, laser light scattering is the most common technique used to characterize them [60,61]. Static light scattering involves the measurement of the scattered light intensity at different scattering wave vectors q (Eq.3.1).

q=

4π ⋅ n

λ

⎛θ ⎞ sin ⎜ ⎟ ⎝2⎠

(3.1)

where λ is the wavelength of the incident light, n is the refractive index of the scattering medium and θ is the scattering angle. This method gives parameters such as the radius of gyration (Rg) and second virial coefficient (A2), which characterize, respectively, the particle size determined through its mass distribution around its center of mass and the strength of the interaction between the particles and the medium. Dynamic light scattering (also called photon correlation spectroscopy)

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determines the fluctuations in the intensity of the scattered light. This method provides information regarding the diffusion of the particles in the suspension, and hence gives the hydrodynamic radius (Rh), mean size distribution and the polydispersity index (PI). The mathematical model in dynamic light scattering considers an equivalent sphere. Thus, aggregates present in the formulation can greatly increase the Rh and PI values. In addition, the sample must be sufficiently diluted to avoid decoherence of the incident beam and therefore a lack of accurate information on the particles [62,63]. Recently, formulations were analyzed using equipment based on multiple light scattering theory to circumvent the need for dilution [64]. This is advantageous, because migration and aggregation phenomena as a function of time can be directly monitored in the native sample. In this way, physicochemical instability can be determined earlier than by simply relying on the naked eye [6,65]. In addition, multiple light scattering is an interesting method to analyze the mean particle size of formulations when dilution is not possible [66].

Fig. 3.1 Physicochemical characterization of a polymeric nanocapsule formulation: particle size distribution by (a) laser diffraction, (b) dynamic light scattering (by percentage of light intensity, volume and number), (c) particle tracking analysis (Nanosight®); (d) backscattering scanning profile by Turbiscan® (non-diluted sample); and (e) transmission electron microscopy (bar = 0.2 μm, uranyl acetate stained).

Local properties of individual particles cannot be accurately determined as the light scattering technique characterizes a significant portion of the particle population. However, these properties can be accessed by electron microscopy techniques, such as scanning electron microscopy (SEM) and transmission electron

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microscopy (TEM). The treatment of nanocapsules for SEM analysis involves sample drying and coating with a thin layer of gold or platinum. The sample surface is then scanned by a high-energy beam of electrons. These electrons interact with atoms of the sample producing signals which can provide information on the surface topography, composition, shape and electrical conductivity. Different supramolecular structures of lipophilic surfactants in nanocapsule suspensions (such as vesicles and micelles) as a function of their concentrations have been previously observed by freeze-fracture SEM [37], demonstrating that this technique can be useful in qualitatively evaluating the formulations. SEM can also be used to observe nanocapsules on the skin [67,68] or in a gel matrix before and after storage [69]. However, soft material particles, smaller than 100 nm, may be difficult to resolve by this technique [70]. In contrast to SEM, in TEM a beam of electrons is transmitted through the sample, which is previously dried and stained with contrast agents (such as uranyl acetate and phosphotungstic acid). TEM provides information not only on the size of nanocapsules but also their vesicular structure [37,71] and the polymeric wall and the core [72]. Another technique applied to investigate the nanocapsule structure is atomic force microscopy (AFM), where three-dimensional images of the samples can be achieved [73,74]. Although microscopy techniques are appropriate to analyze individual particles, it is important to note that all microscopic techniques require image treatment of a large number of particles to determine the mean particle size. Nanocapsules may shrink during the drying step, resulting in the underestimation of their diameter [70]. Thus, a combination of at least two methods (such as light scattering and microscopy) to determine nanocapsule size and distribution is recommended. The vesicular structure of nanocapsules can be evidenced using electron microscopy techniques (as previously discussed) or by measuring their density using differential centrifugation [41]. The latter strategy enables the determination of the optimum ratio between the nanocapsule components, when supramolecular structures (such as nanoemulsion and nanospheres) with densities which differ from those of nanocapsules cannot be found [5,37]. This is an important aspect to consider during pre-formulation studies, due to the influence of different nanocarriers on the skin penetration [10,75] (see Chapter 1). The surface characteristics of the nanocapsules can influence not only the physicochemical stability of formulations, but also the behavior of the formulation in the skin. As previously discussed (section 3.2.), bioadhesivity to the stratum corneum is related to the surface charge of the nanocapsules. Although some reports refer to the zeta potential as the surface charge of the particle, this value is the electric potential at the interfacial double layer between the dispersion medium and the stationary layer of fluid attached to the dispersed particle [76]. Zeta potential is usually determined by electrophoretic light scattering. Non-ionic surfactants tend to reduce the absolute zeta potential value, whereas ionic polymers and surfactants increase it. A zeta potential higher than 30 mV (positive or negative) is indicative of adequate stability of the nanocapsules due to charge repulsion.

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However, if the stabilization mechanism is based on steric hindrance, the nanocapsules can have zeta potential values close to zero and still have high stability in suspension [1,77]. In this case, the physical stability can be confirmed by other characterization methods, such as multiple light scattering. The pH values of the bulk [78] and the presence of a drug or active ingredient adsorbed on the nanocapsule surface [79] can influence the zeta potential values. Finally, changes in the zeta potential signal may indicate nanocapsule coating by a specific material [8]. The contact angle and surface tension measurements have also been used to investigate the behavior of nanocapsules in the skin [80]. Quantification methods are chosen according to the molecular structure of the drug or active ingredient. Spectroscopy methods are the most commonly used, particularly those preceded by chromatographic separation. The analytical method used to quantify the active substance must consider nanocapsules as complex matrices [81,82]. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Guideline (ICH) [83] recommends the evaluation of specificity, linearity, range, accuracy, precision, robustness and limits of detection and quantification. Specificity is particularly important to distinguish between the active ingredient and other substances that may be present in the matrix. Since chemical degradation of the active ingredient as a function of time can take place, specificity must also be determined for control samples submitted to thermal and photochemical degradation. Quantification can also be used to determine the chemical stability and, consequently, the shelf life of the formulation. Chemical instability can be caused by other components present in the nanocapsules besides the active ingredient. The acid-base character (pH) can be measured by potentiometry in a predetermined time period. An increase in the acidity indicates hydrolysis of the polymer when carboxylate groups are present in the backbone [8,84]. In the case of nanocapsules prepared with polymethacrylic polymers, a decrease in the pH values is indicative of relaxation of the carboxylic groups at the particle-water interface due to the presence of the drug or as a function of time for drug-free formulations [85].

3.5 Nanocapsules in Skin Formulations: Advantages and Challenges Polymeric nanocapsule suspensions can be directly applied on the skin as a final product or incorporated in semisolid formulations as an ingredient (Table 3.2). Ideally, the nanocapsule suspensions must be of slightly acid pH, which corresponds to the physiological acidity of the skin. After topical application, the nanocapsules form a thin film on the skin with water evanescence. This thin film is related to long-term delivery and the higher storage capacity of the skin in relation to the active ingredient [9,86]. As previously discussed, a positively charged surface can promote bioadhesivity of the nanocapsules to the stratum corneum [11], which can reduce the loss of a substance during application caused by the low viscosity (similar to water) of the suspension.

Surface composition Polymer(s) Surfactant(s)/ emulsifier(s) Poly(caprolactone) Polysorbate 80 Cellulose acetate phthalate Poly(vinyl alcohol) Chitosan and alginate Polysorbate 80 Poly(caprolactone) Cetyltrimethylammonium chloride, sodium lauryl sulfate, gelatin Poly(D,L-lactide) Poly(vinyl alcohol)

Poly(caprolactone) Polysorbate 80 Caprylic/capric triglycerides, sorbitan monostearate Grape seed oil and almond kernel Poly(caprolactone) Polysorbate 80 oil, sorbitan monostearate Retinyl palmitate, sorbitan Poly(D,L-lactide) Poloxamer 188, polysorbate monostearate 80 Caprylic/ Poly(caprolactone) Polysorbate 80, sorbitan capric triglycerides monooleate 1 The encapsulation mechanism of the active ingredient was not considered in this classification.

Isopropylmyristate, octyl methoxycinnamate

Octyl methoxycinnamate Octyl methoxycinnamate Turmeric oil Octyl salicylate

Structural component(s) of the 1 core

Table 3.1 Some representative raw materials used in nanocapsules for cutaneous applications.

[24]

[25] [27] [42]

Benzophenone-3 Retinyl palmitate Dexamethasone

[10]

[40] [41] [23] [31]

Coenzyme Q10

Octyl methoxycinnamate

Octyl methoxycinnamate Octyl methoxycinnamate Turmeric oil Hinokitiol

Active ingredient(s) References

3 Polymeric Nanocapsules: Concepts and Applications 59

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Table 3.2 Examples of final products containing nanocapsules for skin application. Formulation (excipient) Aqueous suspension

Active ingredient

Advantages

Reference

Genistein

[11]

Aqueous suspension

Benzophenone-3

Hydrogel (hydroxyethylcellulose)

Octyl methoxycinnamate

Hydrogel (Carbopol 940®)

Nimesulide

Hydrogel Ultrez 10 NF)

Dexamethasone

Disturbing the tight lamellar layer of the stratum corneum, increasing the diffusion of the active ingredient through the skin. Delaying photodegradation of the active ingredient under UV radiation. Restricting the penetration of the active ingredient into the skin and improving its photostability. Promoting drug penetration into the stratum corneum and/or the layer of viable skin. Potential to promote controlled release of dexamethasone in the treatment of psoriasis. Sustained antimicrobial effect against resident and transient skin flora. Delaying the degradation of the active substance under UVA radiation. Alteration of the rheological behavior, from yieldpseudoplastic to pseudoplastic. Limiting the skin penetration of the active ingredient in viable skin layers.

(Carbopol®

Hydrogel (Sodium salt of carboxymethylcellulose)

Chlorhexidine

Hydrogel (Carbopol 940®)

Benzophenone-3

Hydrogel (Carbopol 940®) prepared using aqueous or spray-dried suspension

Coenzyme Q10

Emulgel (Sepigel® 305)

Octyl methoxycinnamate

[25]

[87]

[75]

[42]

[88]

[68]

[24]

[10]

As semisolid formulations are easier to use, they are preferable to liquid suspensions. Also, the higher viscosity of a gel compared to a nanocapsule suspension has previously been associated with a lower release rate of the nanoencapsulated

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active ingredient in the skin [86]. Hydrophilic gels, such as hydroxyethylcellulose [87] and Carbopol 940® [69,75], are the most common components described to prepare semisolid formulations of nanocapsules. Typically, water is totally or partially replaced by the nanocapsule suspension in these semisolid formulations. A recent study [42] demonstrated that the spreadability factor of hydrogels prepared using Carbopol Ultrez® 10 NF was reduced when dexamethasone loadednanocapsules were incorporated in the matrix. Also, the integrity of the nanocapsules within the hydrogel matrix was demonstrated by TEM and dynamic light scattering. In another study, chlorhexidine-loaded nanocapsules were incorporated into carboxymethyl cellulose gel and their bactericidal activity after application on hands was compared with 60% (v/v) 2-propanol and an ethanol-based gel (Purell®) [88]. The gel containing nanocapsules was as effective as the controls within 30 seconds of application and was superior over long time periods probably due to the high evaporation rate of ethanol. Nanocapsules containing the sunscreen benzophenone-3 were incorporated into hydrophilic gels and the effect of nanoencapsulation of this sunscreen was evaluated [68]. The nanocapsules in a semisolid formulation improved the photostability and the effectiveness of the sunscreen (see chapter 17). In addition, hydrogels did not cause cutaneous sensitization in mice. Hydrophilic gels containing nanocapsules to release nimesulide into the viable layer of the skin were compared with hydrogels containing the pure drug [75]. The gel containing nanocapsules showed higher skin penetration of the drug than those containing other types of nanocarriers (nanospheres and nanoemulsion). Nanocapsules loaded with coenzyme Q10 altered the rheological flow pattern of Carbopol® 940 gels from the yield-pseudoplastic to the pseudoplastic model [24]. In this study, the nanocapsule aqueous suspension was either previously spray-dried or directly used to prepare the hydrogels. The conversion of the suspension into a powder form using lactose as the hydrophilic adjuvant can potentially increase the long-term stability of the nanocapsules. Pseudoplastic behavior was also observed for Carbopol® 940 gels containing nimesulide-loaded nanocapsules [89]. Although hydrophilic gels are the most common vehicles of semisolid formulations containing nanocapsules, other different raw materials can be used. Butyl glucoside, polyacrylamide/C13-14 isoparaffin/laureth-7 (Sepigel® 305) was used to prepare an emulsion gel containing octyl methoxycinnamate-loaded nanocapsules [10]. This formulation improved the safety of the sunscreen reducing its transdermal penetration and its contact with the skin layers without decreasing its efficacy. The incorporation of hinokitiol-loaded nanocapsules into shampoo and hair tonic for hair growth treatment has also been reported [90]. The hair growth effects of both preparations in mice were comparable to those of 3% minoxidil solution used as the positive control. According to the authors, electrostatic interaction between the nanocapsules (positively charged) and skin influenced the hair growth promotion effects.

3.6 Concluding Remarks In this chapter, the use of polymeric nanocapsules for skin applications was discussed. The release rate of drugs or active ingredients can be controlled by loading

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them into nanocapsules. Moreover, nanocapsules act as reservoirs improving the stability of the active substances. Also, the degree of penetration/permeation of a drug or active ingredient into the skin can be modulated according to the polymer and the surfactant used as raw materials. This is of particular interest in relation to UV filters, since the transepidermal delivery must be avoided but increased adhesion to the stratum corneum is desirable since it prolongs the protective effect. Thus, it is important to note that the raw materials used to prepare the polymeric nanocapsules must be carefully selected in order to achieve the desired effect. In this context, a good understanding of the supramolecular structure of nanocapsules is essential, since the release rate of the active substance to the skin is also related to its encapsulation mechanism. Therefore, the physicochemical characterization of these devices must be carried out prior to their use in biological applications. The particle size, polydispersity, zeta potential and drug content are the most important parameters to be evaluated. Light scattering and electron microscopy are the techniques generally reported in the literature to characterize nanocapsules. There are several methods used to prepare polymeric nanocapsules, which can be categorized as chemical and physicochemical methods. The selection of the method depends on the intended design of the supramolecular nanocapsule structure, the nature of the raw materials and the scale of production. Aqueous suspensions can be directly applied to the skin or added to semisolid formulations, usually hydrogels and emulgels because no heating is necessary and the nanocapsule structure is maintained intact in these matrices. Some reports have shown that nanocapsules can modify the rheological properties of semisolid formulations. However, the desired effect was not changed by these modifications. In conclusion, polymeric nanocapsules for skin application are a promising research field in terms of developing valuable devices for industrial application.

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[81] Sartori, T., Seigi Murakami, F., Pinheiro Cruz, A., Machado de Campos, A.: Development and validation of a fast RP-HPLC method for determination of methotrexate entrapment efficiency in polymeric nanocapsules. Journal of Chromatogr. Sci. 46, 505–509 (2008) [82] Fontana, M.C., Bastos, M.O., Beck, R.C.R.: Development and Validation of a Fast RP-HPLC Method for the Determination of Clobetasol Propionate in Topical Nanocapsule Suspensions. J. Chromatogr. Sci. 48, 637–640 (2010) [83] ICH. Validation of analytical procedures: text and methodology Q2(R1) (2005), http://www.ich.org/LOB/media/MEDIA417.pdf [84] Schaffazick, S.R., Guterres, S.S., Pohlmann, A.R., Lucca Freitas, L.: Caracterização e estabilidade físico-química de sistemas poliméricos nanoparticulados para administração de fármacos. Quim Nova 26, 726–737 (2003) [85] Lopes, E.C., Pohlmann, A.R., Bassani, V.L., Guterres, S.S.: Polymeric colloidal systems containing ethionamide: Preparation and Physico-Chemical Characterization. Pharmazie 55, 527–530 (2000) [86] Alvarez-Roman, R., Barre, G., Guy, R.H., Fessi, H.: Biodegradable polymer nanocapsules containing a sunscreen agent: preparation and photoprotection. Eur. J. Pharm. Biopharm. 52, 191–195 (2001) [87] Weiss-Angeli, V., Bourgeois, S., Pelletier, J., Guterres, S.S., Fessi, H., Bolzinger, M.A.: Development of an original method to study drug release from polymeric nanocapsules in the skin. J. Pharm. Pharmacol. 62, 35–45 (2010) [88] Nhung, D.T.T., Freydiere, A.M., Constant, H., Falson, F., Pirot, F.: Sustained antibacterial effect of a hand rub gel incorporating chlorhexdine-loaded nanocapsules (Nanochlorex®). Int. J. Pharm. 334, 166–172 (2007) [89] Alves, M.P., Pohlmann, A.R., Guterres, S.S.: Semisolid topical formulations containing nimesulide-loaded nanocapsules, nanospheres or nanoemulsion: development and rheological characterization. Pharmazie 60, 900–904 (2005) [90] Hwang, S.L., Kim, J.C.: In vivo hair growth promotion effects of cosmetic preparations containing hinokitiol-loaded poly(epsilon-caprolacton) nanocapsules. J. Microencapsul. 25, 351–356 (2010)

Chapter 4

Topical Application of Nanostructures: Solid Lipid, Polymeric and Metallic Nanoparticles Nelson Durán, Zaine Teixeira, and Priscyla D. Marcato Chemistry Institute, Universidade Estadual de Campinas (Unicamp), Brazil [email protected]

Abstract. Nanoparticles are currently widely used in drug delivery systems due to the possibility for sustained release and the protection of labile groups from degradation, and also because they can pass through biological barriers. Topical application of nanostructures represents a promising route mainly for the treatment of skin diseases and even for cosmetic use, such as in anti-aging formulations. Different types of nanostructures, such as solid lipid, polymeric and metallic nanoparticles, have been widely employed. Depending on the characteristics of the nanostructure, drugs or bioactives can be delivery preferentially to different skin layers. In this chapter, we describe the main methods available for the preparation and characterization of these nanostructures. Examples of topical application are described and their main advantages highlighted. These nanostructures offer a feasible approach to modulating bioactive compounds and controlling drug permeation into the skin.

4.1 Introduction Nanostructured carriers for topical applications are currently being widely used due to a number of advantages over conventional formulations. Their main features are sustained release, protection of labile groups from degradation, low toxicity and skin permeation modulation [1,2]. Some mechanisms of permeation through the skin (brick-and-mortar-like structure) are intercellular (through the “mortar”) and others are intracellular (through the “bricks”). Another potential mechanism is via appendices, which is basically through pilosebaceous follicles (1% of the total skin area) or narrow skin pores (transdermal gradient) [3,4]. The physicochemical characteristics of the nanocarriers, such as rigidity, hydrophobicity, size and charge, are crucial to the skin permeation mechanism. More details on the transport of nanoencapsulated substances across the skin are given in Chapter 1.

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Of the different types of nanostructures, polymeric nanoparticles and solid lipid nanoparticles (SLN) have received particular attention in the encapsulation of drugs or active compounds for topical applications. Polymeric nanoparticles can be classified as nanospheres (matrix system) with lower loading capacity or nanocapsules (reservoir system composed of an oily or aqueous core) with higher loading capacity (Fig. 4.1) [1,5]. Polymeric nanoparticles have been investigated since the end of 1980s whereas SLN were developed almost a decade later [6]. The first SLN systems presented some limitations due to their low drug loading and low stability during storage. The development of systems comprising a liquid lipid mixture with solid lipids provided a substitute for SLN and these were named nanostructured lipid carriers (NLC) [7]. More recently, metallic nanoparticles have received attention for topical applications, due to their therapeutic properties, such as the wound healing of silver nanoparticles [8].

Fig. 4.1 Polymeric nanoparticle structures: (a) nanospheres; (b) nanocapsules

In this chapter, the main methods available for the preparation and characterization of polymeric, solid lipid and metallic nanoparticles will be discussed, and recent topical applications described. Further considerations will also be presented.

4.2 Preparation and Characterization of Solid Lipid Nanoparticles (SLN) Solid lipid nanoparticles are prepared with a solid lipid (at room and body temperature), a surfactant and water. In general, the lipids used are triglycerides (e.g. myristyl myristate), partial glycerides (e.g. glyceryl monostearate), fatty acids (e.g. palmitic acid), steroids (e.g. cholesterol) and waxes (e.g. cetyl palmitate). The surfactant is used to efficiently prevent particle agglomeration. Thus, the choice of the best surfactant is a very important factor. Table 4.1 shows the most common ingredients used in the preparation of SLN [9].

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Table 4.1 Lipids and surfactants used in the preparation of SLN.

LIPID Stearic acid

STRUCTURE O OH OH

Glyceryl monostearate

OH O O O

Tristearin

O O O O O

SURFACTANT Poloxamer F68

STRUCTURE CH3 O

O a

O b

OH a

a = 30 unidades b = 75 unidades

Polysorbate 80

There are 4 methods commonly used for the preparation of SLN: hot microemulsion, emulsification and solvent evaporation, solvent diffusion, and high pressure homogenization (HPH). The most important and the most commonly used is the HPH method since it is associated with easy scale-up.

4.2.1 Hot Microemulsion The microemulsion method was developed by Gasco (10) and modified by others authors. The microemulsion is prepared with 10% of melted lipid (e.g. stearic acid), 15% of surfactant (e.g. Polysorbate 20 or 60), more than 10% of cosurfactant (e.g. Poloxamer) and water. Figure 4.2 shows the hot microemulsion process. The last step of this method is the addition of the hot microemulsion to a cold surfactant solution to promote the solidification of the particles [11,12]. The disadvantages of this method are that the excess water needs to be removed and the high surfactant and co-surfactant concentration. The excess water can be removed by ultracentrifugation, lyophilization or dialysis.

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Fig. 4.2 Scheme of SLN preparation by hot microemulsion method

4.2.2 Emulsification and Solvent Evaporation The scheme in Figure 4.3 shows the preparation of SLN by the emulsion and solvent evaporation method.

Fig. 4.3 Scheme of SLN preparation by emulsion and solvent evaporation method

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The advantage of this method is the encapsulation of thermosensitive molecules. However, it does have the disadvantages of residual solvent in the final product and the concomitant production of microparticles [13,14].

4.2.3 Solvent Diffusion Method The diffusion solvent method was firstly developed for the preparation of polymeric nanoparticles. This method is simple and does not require special equipment. However, it is associated with the disadvantages of requiring organic solvents and difficulties related to scale-up. Figure 4.4 shows a scheme for this method [15,16].

Fig. 4.4 Scheme of SLN preparation by solvent diffusion method

4.2.4 High Pressure Homogenization (HPH) Method In the HPH method the dispersion is homogenized at high pressure (500-2000 bar) through a narrow cavity (few micrometers) and accelerated over a short distance at high velocity (~100 km/h). High shear stress and cavitation forces produce submicron particles [11]. Figure 4.5 shows an example of a high-pressure homogenizer used in SLN production.

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Fig. 4.5 Niro-Soavi (Panda 2k) high-pressure homogenizer

Fig. 4.6 Scheme of SLN preparation by hot homogenization method

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HPH can be carried out by two different methods: hot homogenization and cold homogenization. In hot HPH, the whole process is carried out at temperatures above the melting point (Tm) of the lipid. After the emulsion homogenization the dispersion is cooled to form the solid lipid nanoparticles. The scheme in Figure 4.6 shows the hot HPH procedure. In general, 1-3 cycles are sufficient for nanoparticles to form. This method is applied mostly in the case of lipophilic drugs [9,11]. The cold homogenization method is applied to hydrophilic drugs. The scheme in Figure 4.7 shows the cold homogenization method. The solid state of the matrix decreases the migration of the hydrophilic active compound to the aqueous phase, increasing the encapsulation efficiency. Furthermore, in this method it is possible to encapsulated thermosensitive substances due to the very short time in which the drug is exposed to the high temperature.

Fig. 4.7 Scheme of SLN preparation by cold homogenization method

The hot homogenization method produces, in general, nanoparticles below 500 nm, while cold homogenization produces microparticles with larger sizes. However, a size reduction can be obtained by increasing the pressure and using 3-7 cycles in the homogenization [14,17] The advantages of the HPH method (hot or cold) are the easy scale-up and the production of SLN without the use of organic solvents. However, the high pressure can produce the coalescence of the particles and the high temperature used in the process can increase the degradation rate of the drug as well as of the particle [16].

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Solid lipid nanoparticles have been characterized by several techniques including X-ray diffraction (XRD), differential scanning calorimetry (DSC), proton nuclear magnetic resonance (NMR) and microscopy techniques.

4.2.5 X-Ray Diffraction Lipids can present different polymorphic forms. Polymorphism is observed due to the organization of alkane chains into different packing patterns: α (hexagonal), β´ (orthorhombic) and β (triclinic). Hexagonal packing is the most disordered form, orthorhombic packing is less disordered and triclinic packing is the most organized [18,19]. Thus, for the industrial application of SLN it is necessary to control this polymorphism. The polymorphic structure has a direct influence on the encapsulation efficiency and expulsion of the drug during storage. Therefore, its characterization is of great importance. One of the techniques used to characterize the polymorphic structure of SLN is X-ray diffraction (XRD), which determines the length of long and short spacings of the lipid lattice. Furthermore, XRD allows amorphous and crystalline materials to be differentiated and serves to evaluate the influence of constituent oil in long spacings of the nanocrystals of nanostructured lipid carriers (NLC). The association of this technique with differential scanning calorimetry (DSC) is important, since DSC allows amorphous solids and liquids to be differentiated [14,20,21].

4.2.6 Differential Scanning Calorimetry (DSC) The technique of differential scanning calorimetry (DSC) provides information on the physical state and crystallinity of SLN. The degree of crystallinity of a particle is extremely important because it influences the encapsulation efficiency, the release rate and the expulsion of the drug during storage. DSC analysis also provides the fusion temperature and enthalpy of the particles. A high value for the fusion enthalpy suggests a high level of organization in the crystal lattice, because the fusion of a highly organized crystal (perfect crystal) requires more energy to overcome the forces of cohesion in the crystal lattice. Furthermore, it is possible to observe the crystallization behavior of particles, which influences the expulsion of the drug. During storage, the lipid can be converted from the α to the β’ form under certain conditions of temperature, light exposure and water loss from the SLN dispersion [20,22].

4.2.7 Proton Nuclear Magnetic Resonance (NMR) Proton nuclear magnetic resonance (NMR) is an important technique in the characterization of liquid lipid domains within solid lipid nanoparticles. Information on the mobility and arrangement of the molecules of oil can be obtained using the technique of 1H NMR spectroscopy. Through this technique it is possible to verify

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whether a lipid is in the liquid or semi-solid/solid state through the difference in the relaxation time of protons in the liquid state and the semi-solid/solid state. Protons in the liquid state have a narrower signal of higher amplitude than those in the semi-solid/solid state [11,23].

4.2.8 Microscopy Techniques Transmission electron microscopy (TEM) is the most appropriate microscopy technique for obtaining direct images of SLN, allowing the determination of the size (diameter) distribution and determination of the crystal structure, in the case of high-resolution microscopes [15]. However, atomic force microscopy (AFM) has also been used in the characterization of SLN [24].

4.3 Preparation and Characterization of Polymeric Nanoparticles The main methods used for the preparation of polymeric nanoparticles can be divided into two categories: i) polymerization of monomers; and ii) preforming of polymers [25,26]. The former have been less used because undesirable chemical reactions may occur, which may inactivate the active substance and also they require an additional purification step to remove polymerization initiators and residual monomers. Thus, we will focus on the latter which consist of the most commonly employed methods. Many of the most widely used preformed polymers are biodegradable and biocompatible and have been approved by the U.S. Food and Drug Administration (FDA). Examples include poly(D,L-lactide) (PLA), poly(ε-caprolactone) (PCL), poly(D,L-glycolide) (PLG) and poly(lactide-co-gycolide) (PLGA). Another polymer which has been employed in this context is poly(hydroxybutyrate-cohydroxyvalerate) (PHBV), a promising biopolymer for use in drug delivery systems [27]. Polymers that are biocompatible and non-biodegradable, for instance, poly-alkyl-acyanoacrylate, have also been used for topical application. The natural biodegradable and biocompatible polymers chitosan and alginate have also been widely employed in this area [26]. The use of a stabilizer, typically from 0.2 to 2 wt.%, is also necessary because of the increase in the surface tension due to the reduction in size [25,28]. One of the most widely employed polymeric stabilizers is polyvinyl alcohol due to its superior stabilizer capacity, preventing aggregation. However, it is not suitable for intravenous use, limiting its applications. The poloxamers, also known as Pluronics, are widely used due to their lower cytotoxicity. The polysorbates and their esters (Tweens and Spans) are used as non-ionic hydrophilic and lypophilic surfactants. The main methods of preparation based on the use of preformed polymers are: nanoprecipitation, emulsification-solvent evaporation, salting-out, emulsificationsolvent diffusion, and the spontaneous emulsification-solvent diffusion method. Other methods are gelling and self-assembling. The method of choice depends on

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the final desired characteristics of the nanoparticles such as size and polydispersity, and type of polymer, as well as the properties of the drug to be incorporated. Nanoprecipitation is generally the method of choice when both the polymer and drug are soluble in a water-miscible organic solvent. Furthermore, it is a simple, fast, reproducible and low-cost procedure [6,28]. Depending on the chemical components and the preparation steps, the colloidal suspension consists of nanospheres or nanocapsules with the drug dissolved in, entrapped in, adsorbed onto or chemically coupled to the polymer matrix (Fig. 4.1) [1]. In the preparation of nanospheres, the polymer and drug are solubilized in an organic solvent which is miscible with water, such as acetone. Nanoprecipitation is also known as preformed polymer interfacial deposition, when nanocapsules are obtained. In this case, an oil, such as capric/caprylic triglycerides and, optionally, a lipophilic surfactant are also solubilized in the organic phase. The organic phase is added into an aqueous phase and the particles are formed through the rapid diffusion of the organic solvent toward to water. The ingredients that are insoluble in water precipitate forming the nanoparticles. The organic solvent is removed by evaporation or by other extraction methods and the final suspension is concentrated. The average diameters of nanospheres and nanocapsules obtained by this method are typically around 100 nm and 200 nm, respectively [29,30-32]. Both present a low polydispersity index (< 0.2). The variables in this technique are the type of solvent and polymer. Indeed, other variables of the process, such as the volume of phases, the stirring rate, and the type and concentration of stabilizer, in general, do not influence the particle size significantly. In the development of nanocapsules a solubility test should be used to verify that the polymer is insoluble in the oil [33]. Also, the solubility of the drug in the oil should be high, in order to prevent drug ‘leakage’ after preparation. Another commonly-used method is the emulsification-solvent evaporation method in which a water-immiscible solvent is used in the organic phase [34]. The main solvents employed in this technique are dichloromethane and chloroform. This preparation method consists of the addition of an organic phase containing the polymer and drug to an aqueous phase containing the stabilizer. An oil-inwater emulsion is then obtained by emulsification methods such as ultrasound, mechanical stirring, microfluidics or colloidal mills. Particles are formed when the solvent is removed, and each droplet originates one particle. Nanocapsules can also be produced through this method by adding oil to the organic phase. More variables are involved in this process than in nanoprecipitation, including stirring rate, viscosity of the aqueous phase, type of surfactant, solvent evaporation rate and organic/aqueous phase ratio. Size distributions are mainly dependent on the stirring rates used in this method, and other colloidal features, such as Ostwald ripening, significantly affect the process [35]. A modification of this method, the double emulsion water-in-oil-in-water (w/o/w), is generally used to encapsulate hydrosoluble compounds [36]. In this method, the polymer is dissolved in an organic-immiscible solvent with, optionally, a lipophilic surfactant. An aqueous phase containing the active substance is emulsified in the organic phase, obtaining the first emulsion of water-in-oil. This is added to an aqueous phase and the emulsification process is carried out,

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originating the double emulsion water-in-oil-in-water. The organic solvent is removed forming the nanoparticles that entrap the hydrophilic drugs, which are usually larger than particles prepared by the single emulsion methods. Another approach is the use of a multiple emulsion water-in-oil-in-oil, which generally presents better results in the encapsulation of hydrosoluble compounds and a lower polydispersity index than w/o/w. The addition of salts like NaCl to the water phases can avoid the drug migration toward the external aqueous phase, increasing the encapsulation efficiency [37]. The salting-out method is an adaptation of emulsification-solvent evaporation and nanoprecipitation, in which the emulsion is obtained with a polymer solvent that is miscible in water, such as acetone [38]. The aqueous phase is previously obtained with a high concentration of salt or sucrose before the addition of the organic phase. Salts commonly used are calcium chloride, sodium chloride, magnesium acetate, magnesium chloride. The solubility of the organic phase is modified since the water molecules are retained, solubilizing the electrolytes. Instead of the diffusion process, a phase separation is obtained by adding the organic phase to the aqueous phase. After the emulsification process, the nanoparticles are obtained through the addition of pure water. The organic solvent in the drops diffuses toward the water and the polymer is precipitated. The solvent and salting-out agents are commonly removed by tangential filtration. The main variables in this process are the salting-out agent and stabilizer concentration [38,39]. The emulsification-solvent diffusion method consists of a modification of the salting-out method, in which a partially water-miscible solvent such as ethyl acetate is used [40]. Before the emulsification process, saturation of solvent and water is obtained by mixing them and separating the phases. The polymer and active substance are dissolved in the organic solvent saturated with water, while surfactant is dissolved in the aqueous phase. These phases are then emulsified. In the next step, a dilution with water is obtained, in which the organic solvent diffuses out of the droplets and the nanoparticles are formed. The average diameters of the particles are usually between 140 nm and 650 nm [39]. The main variables in this process are stirring rate and type of solvent, which can be adjusted to reduce the particle size and increase the miscibility with water [40]. The method of spontaneous emulsification-solvent diffusion is also an adaptation of the emulsification-solvent evaporation method, in which a water-miscible solvent such as acetone or ethanol is mixed with a water-immiscible solvent (e.g. chloroform) [41]. Diffusion of the miscible solvent toward the water creates an interfacial turbulence between the phases and small-sized droplets are obtained in the emulsification process. Thus, the particles are generally smaller than in the emulsification-solvent evaporation process. The main variable in this technique is the ratio between the organic solvents, a relative increase in the water-miscible solvent leading to a decrease in the particle size. Another method of nanoparticle preparation is the gelling process which involves the addition of a gelling agent or modification of the pH. Most polymers used in this method are soluble in water, for instance, alginate or chitosan. Thus, it is a suitable procedure for the encapsulation of hydrophilic compounds. Alginate

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nanoparticles are obtained through gelation induced by calcium chloride. Chitosan and polylysine are generally used as stabilizers [42]. The particle size in this system is mainly dependent on the molecular weight of the stabilizers and the concentration of alginate. Chitosan nanoparticles are usually obtained by adding polyphosphate ions, such as tri-polyphosphates, and non-ionic stabilizers like Pluronics [43]. The nanoparticle size is a function of the chitosan concentration as well as the pH and the presence of electrolytes. Indeed, drug release can be triggered by variations in the pH or ionic concentration. Suspensions of nanoparticles can be used directly in the final formulation, for instance, in semisolid preparations. A drying step using freeze-drying or spraydrying can be introduced after preparation of the suspensions, increasing the shelf life [29,44]. However, particles can undergo coalescence, and further studies regarding this step need to be carried out. Some stabilizers like PVA and Pluronic F68, for example, avoid coalescence during the freeze-drying process. Alternatively, cryoprotectors can be used, typically in concentrations of 1 to 5% for nanospheres [45] and up to 30% for nanocapsules [29]. Figure 4.8 summarizes these main preparation methods for polymeric nanoparticles using preformed polymers, highlighting the main differences in the processes. More recently, polymeric micelles have also been widely employed due to their superior properties with respect to conventional micellar systems, such as higher drug loading and low critical micellar concentration, leading to a better stability. These can be prepared by conventional methods such as freeze-drying using a water/terc-butanol system, oil-in-water emulsification, dialysis, solvent displacement or simple equilibrium since the mechanism of micelle formation is the self-assembly of amphiphilic or oppositely-charged copolymers in an aqueous phase [46].

Fig. 4.8 Scheme of the main differences in the methods used for the preparation of polymeric nanoparticles

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Nanoparticle systems are generally characterized in terms of their encapsulation efficiency, particle size, zeta potential, stability, morphology, drug-polymer interactions and in vitro release, as well as features associated with skin permeation when intended for topical application.

4.3.1 Encapsulation Efficiency The encapsulation efficiency is usually evaluated by an ultrafiltration/ centrifugation method, in which the solution obtained through filtration (free of particles) is used to quantify the free drug by conventional methods like high performance chromatography (HPLC) or UV-VIS absorbance spectroscopy [47]. The possibility of the presence of precipitated drug must be totally discarded before using this technique. The total drug is considered as the amount of drug added to the formulation or may be also assayed diluting a small amount of suspension in organic solvent. For example, 100 μL of suspension may be diluted in 10 mL of acetonitrile [48]. The encapsulation efficiency, E.E., is calculated by: E.E.= (total drug – free drug)/total drug x 100

(4.1)

4.3.2 Dynamic Light Scattering (DLS) Dynamic light scattering (DLS) is the technique most commonly employed to determine the average diameter and polydispersity index of polymeric nanoparticle systems. This is a fast and reproducible method. Recent advances have made the use of concentrated suspensions (by backscattering analysis) feasible. The charge between the electric double layer of particles (Stern layer) and the diffuse layer, i.e. the zeta potential, is measured by electrophoretic mobility in suspension [1]. The zeta potential measurements are important not only to evaluate the physical stability when the particles are stabilized by charge, but also to predict the interactions of the particles with the biological membranes. Chitosan nanoparticles (positively charged), for instance, are known to be bioadhesive due to the anionic characteristic of biological membranes.

4.3.3 Microscopic Techniques Microscopic techniques like transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) are conventionally used for morphological characterization of nanoparticle systems. TEM has advantages related to the use of suspensions without the need for additional purification to remove stabilizers. However, a staining method, like uranyl acetate staining, is required. Freeze-fracture transmission electron microscopy (FFTEM) has been employed to determine the wall thickness of nanocapsule systems [49].

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4.3.4 In Vitro Drug Release The release of active compounds is dependent on many factors, such as desorption of the adsorbed molecule onto the nanoparticle surface, diffusion through the matrix (nanospheres) or through the polymeric wall (nanocapsules), polymer degradation, molecular weight, crystallinity and a combination of active diffusionpolymer degradation. Generally, burst release is observed when the active compound is not encapsulated in or adsorbed onto the polymeric surface. Firstorder release kinetics is normally observed for nanosphere systems while nanocapsules usually show zero-order release kinetics [2].The main methods of in vitro drug release are the dialysis bag or separation (ultrafiltration/centrifugation). Some authors argue that sink conditions are not perfectly reached in either technique. Thus, usually only the partitioning of the drug is evaluated [47]. An alternative method for investigating the release of the encapsulated molecule from a nanosystem involves model molecules that undergo hydrolysis at the interface between the particle surface and the release medium, in which the product is soluble in the medium. Thus, sink conditions are simulated [50]. The release kinetics of polymeric nanosystems has been investigated using nanocapsules, nanospheres and nanoemulsion systems encapsulating indomethacin ethyl-ester or indomethacin. Indomethacin was only adsorbed on the surface due to its high hydrophilicity while indomethacin ethyl-ester was entrapped [51]. The microdialysis technique is equivalent to ultrafiltration/centrifugation for the evaluation of lipophilic molecules. For example diclofenac has been used as a model molecule to evaluate the release from polymeric nanoparticulate systems [52].

4.3.5 Differential Scanning Calorimetry (DSC) and Other Molecular-Level Methods Differential scanning calorimetry (DSC) can give information at the molecular level, in which thermal events of the nanoparticles are compared with those of the ingredients and physical mixtures of the ingredients. Both the techniques of small angle X-ray scattering (SAXS) [53] and small angle neutron scattering (SANS) can be used for investigations at the molecular level [54]. However, the analysis of the results is generally a difficult task, since the polydispersity index (~0.2) of these nanoparticle systems is considered high for both methods. With regard to more specific characterization for topical applications, Franz diffusion cell experiments using human abdominal or pig ear skin can aid in the determination of the part of the skin in which the drug is held (stratum corneum, epidermis plus epidermis without corneum and/or receptor chamber) using high performance liquid chromatography (HPCL) (for further details see Chapter 1). Confocal laser scanning microscopy (CLSM) is an extremely important tool for the investigation of the skin penetration and permeation, in which labeling is generally provided by chemical reaction with fluorescent probes. A major advantage

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of CLSM is that the tissue can be optically sectioned, thus enabling the depthdependent distribution of the fluorescent probes to be visualized, without tissue fixation and/or sectioning [55].

4.4 Preparation and Characterization of Metallic Nanoparticles Metallic nanoparticles have been widely used for topical application due to their particular properties, such as the wound healing of silver nanoparticles [8]. The metallic nanostructures most commonly used are iron, gold and silver nanoparticles. Of these, silver nanoparticles are without doubt the most commonly employed systems [56]. Thus, we will focus on silver nanoparticles, but procedures used to prepare other metallic nanoparticles are very similar, in which a step involving the reduction of metallic ions is necessary when using most bottom up approaches. The reduction method employing sodium borohydride is that most commonly used to produce silver nanoparticles, and other methods involve the use of citrate, glucose, hydrazine, and ascorbate [57,58]. One strategy for controlling the particle size involves a two-step method in which first strong and then weak reducing agents are used [59,60]. Another procedure utilized to synthesize silver nanoparticles is the solvated metal atom dispersion (SMAD) method [61]. In this method, a metal is co-vaporized with a solvent onto a liquid nitrogen-cooled surface. As the liquid nitrogen is removed, the metal atoms and solvent warm, causing the aggregation of the metal atoms. SMAD can be performed in conjunction with digestive ripening. Nanoparticles resulting from the SMAD method are further refined by heating under inert atmosphere in the presence of selected ligands, which leads to the production of particles within a narrow size range. As a result, monodisperse spherical particles are obtained [62,63]. A recent review of green synthesis methods for the preparation of silver nanoparticles discusses the use of polysaccharides, polyphenols, Tollens reagent, irradiation, biological reduction, and polyoxometalate [48]. In some cases polysaccharides are used and the reduction of silver may be linked to the oxidation of aldehyde groups to carboxylic acid groups [64-67]. Silver nanoparticles can also be synthesized by irradiating silver salt solutions containing reducing and capping agents [68-74]. Biological methods involve the production of silver nanoparticles utilizing bio-extracts from organisms as reducing agents, capping agents or both [75-85]. Such extracts can include proteins, amino acids, polysaccharides, and vitamins [48,86]. Plant extracts have also been used as silver ion reducing agents to produce silver nanoparticles [87-89]. Silver nanoparticles can also be synthesized by several microorganisms such as the bacterial species Bacillus licheniformis and Klebsiella pneumoniae, and fungi such as Verticillium spp., Fusarium oxysporum, Aspergillus flavus, Aspergillus niger [75,84,90-95]. Green synthesis is currently favored over conventional methods [65]. An example of the biosynthesis of silver nanoparticles is described as follows: approximately 10 g of F. oxysporum biomass is placed in a conical flask containing 100 mL of distilled water, kept for 72

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h at 28 oC and then the aqueous solution components are separated off by filtration. In this solution (fungal filtrate) AgNO3 (10−3 mol L-1) is added and the system is kept for several hours at 28 oC. The kinetics of the preparation can be monitored by UV-VIS absorption spectrophotometry at 440 nm [79]. The main characterization methods are UV-VIS absorption, to observe the intensity and shift of the plasmon band, and transmission electron microscopy (TEM) to analyze the morphology. In the example given above, the production of silver nanoparticles can be monitored by UV–VIS at 420 nm. The TEM micrograph shows spherical and homogenous silver nanoparticles with ~2 nm diameter and ~100 nm for the photon correlation spectroscopy (PCS) measurement and with a zeta potential of -26 mV (Fig. 4.9).

Fig. 4.9 TEM image of silver nanoparticles

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4.5 Topical Application of SLN, and Polymeric and Silver Nanoparticles 4.5.1 SLN Application The dermal route is a very attractive route of administration. However, the dermal route is highly selective in relation to drugs that can passively diffuse across the outer layer of skin, the stratum corneum [1]. Thus, different nanocarriers have been studied in order to modulate the penetration/permeation of substances such as SLN through the skin. Over the past five years the use of SLN has gained increasing attention in the cosmetics area. Studies that describe the increase in the chemical stability of labile cosmetic compounds, such as retinol [96], Q10 coenzyme [13] and coenzyme derived from vitamin C [97], using SLN can be found in the literature. Besides increasing the stability of labile compounds, the SLN reduce water loss from the skin and, consequently, increase, its hydration. Moreover, this property smoothes wrinkles and increases the penetration of compounds into specific layers [98]. Other advantages of this carrier in cosmetics are: promoting the sustained release of substances, reducing their systemic absorption and acting as a physical sunscreen [99,100]. Chen and co-workers [24] have investigated the use of SLN in the encapsulation of podophyllotoxin (POD), which inhibits the growth of epithelial cells infected by human papilloma virus (HPV) in the epidermis. It is used as a drug of first-line treatment for vaginal warts. However, this drug when applied topically is absorbed systemically causing severe adverse effects. Therefore, it is necessary to develop a formulation that minimizes its systemic absorption and increases its residence time in the epidermis, which is possible by encapsulating the POD in nanostructures. POD has been encapsulated in SLN prepared by the microemulsion method using lipid tripalmitin. Skin permeation studies were performed in a Franz diffusion cell using pig skin, in which the presence of the drug in the receptor chamber of the Franz cell was not observed after eight hours of application of the POD nanoencapsulated in SLN. This result indicates that the nanoencapsulated drug does not penetrate across the pig skin. Thus, the encapsulation of POD in SLN can prevent systemic absorption of a drug after topical application. Furthermore, a four-fold accumulation of the drug in the pig skin was observed in the case of the POD nanoencapsulated in SLN in relation to the free form. Marcato and co-workers have studied the encapsulation of the sunscreen benzophenone-3 (BZ3) in SLN and polymeric nanoparticles [101]. The interest in developing new sunscreens is increasing due to the harmful effects of UV radiation on the skin, such as erythema and the induction of skin cancer. However, many molecular sunscreens such as BZ3 penetrate into the skin causing photoallergies, phototoxic reactions and skin irritation [102]. The particles were prepared using cetyl palmitate as the lipid and Pluronic F68 as the surfactant by hot high pressure homogenization. The polymeric nanoparticles were prepared using poly(εcaprolactone) (PCL) as the polymer by the nanoprecipitation method. Figure 4.10 shows the AFM image of PCL nanoparticles and SLN. The particles were stable

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for 40 days. The skin permeation of BZ3 encapsulated in PCL nanoparticles decreased more than when the BZ3 was incorporated into SLN. The sun protector factor (SPF) was higher when BZ3 was encapsulated in SLN (SPF 21) than in PCL nanoparticles (SPF 18). In both formulations, the SPF was higher than that of the free sunscreen (SPF 16). This synergistic effect is due to the particle crystallinity. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxic effects in human keratinocytes (HaCaT cells) and BABL/c 3T3 fibroblasts, whereas PCL nanoparticles with BZ3 showed potential phototoxicity in HaCaT cells. Nevertheless, BZ3 free and encapsulated in PCL nanoparticles or in SLN did not produce allergic reactions in mice, indicating that these nanostructures are interesting carriers for sunscreens.

Fig. 4.10 A) AFM topographic image of PCL nanoparticles (scan area 1.5 x 1.5 µm2), B) AFM phase-mode image of SLN (Scan area was 1.25x1.25 µm2)

In another study, the toxicity of SLN prepared with cetyl esters with and without BZ3 was evaluated in a zebrafish embryo model. The zebrafish is emerging as a versatile model system for analyzing a broad spectrum of substances including nanoparticles. In this study, the death of all embryos was observed when BZ3 was tested at concentrations of up to 500 µmol L-1. The BZ3 encapsulated in SLN did not exhibit any toxic effect even at a concentration of 550 µmol L-1. Furthermore, the development of alterations in the zebrafish were not observed, indicating a reduction in the potential for skin damage induced by BZ3 encapsulated in SLN [103]. For further details on the zebrafish model see Chapter 12.

4.5.2 Polymeric Nanoparticles Compounds that have been encapsulated in polymeric nanoparticles vary from cosmetics and drugs to peptides and proteins. Pharmaceutical and cosmetic applications of peptides and proteins have been highlighted in recent years and include cancer, infectious disease, autoimmune disease, AIDS and anti-aging treatments.

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However, the clinical use of peptides and proteins has many challenges [104,105]. The chemical and physical stability of these molecules can be adversely affected by environmental factors that include pH, ionic strength, temperature, high pressure, non-aqueous solvents, metal ions, surfactants, adsorption, stirring and shearing [106]. Furthermore, most of these molecules are hydrophilic and easily biodegradable in the human body [107]. In cosmetic formulations the challenges are the stability of the peptides and their skin penetration. Many peptides are used in anti-aging treatments, but in this case the peptide needs to penetrate through the skin to reach the dermis. However, due to the hydrophilic properties of peptides their penetration into the skin is inhibited [108]. Thus, a nanocarrier system makes feasible the application of peptides and proteins in pharmaceuticals and cosmetics, improving their stability as well as increasing their in vivo activity and skin penetration [82]. For example, glutathione (peptide) was encapsulated in nanoparticles of poly(ε-caprolactone) by the double emulsification method, in which the average size was 315 nm (polydispersity = 0.298), with an encapsulation efficiency of 35% and a slower drug release in comparison to the free drug [37]. Many studies have investigated the feasibility of using polymeric nanoparticles to research skin permeation. In these studies, particle size, lipophilicity, rigidity and charge have been shown to play crucial roles in determining the skin permeation profile [30,109]. Regarding the nanoparticle charge, positive, negative and neutral latex particles of 50 nm, 100 nm, 200 nm and 500 nm have been studied. Only the negatively charged 50 nm and 500 nm particles were able to permeate the skin, as detected in the receptor chamber of Franz diffusion cells [110]. A threshold charge must be reached to allow the adequate repulsion of lipids, which in turn results in temporary channels within the skin, thus leading to the particle permeation. In another study, CLSM images revealed that Nile red dye encapsulated in PCL nanoparticles (average diameter of 250 nm) reached greater depths in the skin when compared to the Nile red in propylene glycol nanoparticles at the same concentration and in a saturated solution [111]. In a subsequent study, fluorescent polystyrene nanoparticles (20 nm and 200 nm) were employed and a preferential particle distribution in the follicular openings was observed instead of deep skin permeation [112]. Based on this study the authors suggested that in the first study the polymeric carriers did not permeate the skin. Rather, the higher dye permeation was possibly related to a uniform release [112]. On using single stained PLGA nanoparticles, transdermal delivery of flufenamic acid, evaluated by CLSM, was improved. Additionally, it has been demonstrated that the carriers had a pH effect, which influenced the ionization state of the drug, thus improving the concentration gradient from the stratum corneum to the dermal side [113]. Nanospheres of poly(L-lactide-co-glycolide) covalently bound to fluorescein and Texas Red as a drug model were investigated by CSLM [114]. No penetration of the particles was found and the fluorescein remained on the skin surfaces, while Texas Red was released and accumulated in the lower stratum corneum to a depth of approximately 20 µm over a period of 5 hours. A comparative study between nimesulide-loaded nanoemulsions, polymeric nanocapsules and nanospheres showed that nanocapsules and nanospheres were better retained in the stratum

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corneum than nanoemulsions. In addition, nanocapsules provided the highest nimesulide concentration in deeper skin layers [115]. Self-assembled vesicles of poly(caprolactone)–poly(ethylene glycol)–poly(caprolactone) copolymer with an average diameter of 122 nm and wall thickness of 25 nm showed deformability by TEM [116]. Permeation studies confirmed that the vesicles pass through the stratum corneum layers. The authors suggested that the deformability of this system increases its permeation through the skin. Retinyl palmitate oil, which is widely used in anti-aging formulations was encapsulated in PLA nanocapsules with an average diameter of 215 nm and also showed deformability by TEM [30]. This system was flexible enough to pass through synthetic pores of smaller diameter and recover a configuration close to the initial (spherical) one and also to reach deeper into the stratum corneum skin layer, that is, approximately 30 μm after 4 h. Nanoparticles of fatty acid conjugated PVA with degrees of substitution of 40% and 80% have been used for benzophenone-3 encapsulation. The system using a high degree of substitution was found to diminish the percutaneous absorption of benzophenone-3 [117]. These findings indicate that most polymeric systems are retained in the stratum corneum and may improve drug release through the skin, which is dependent on the skin absorption characteristics of the drug as well as the drug release properties. Flexibility is a crucial factor in nanoparticle permeation. Systems that are flexible show a deep permeation into the skin layers. Figure 4.11 shows a comparison between polymeric nanospheres and flexible polymeric nanocapsules, which indicates that the capillary adhesion forces in the latter are strong enough to deform particles [30].

Fig. 4.11 Nanoparticle micrographs obtained by TEM of (a) nanospheres of PLA and (b) flexible PLA nanocapsules of retinyl palmitate; (c) proposed mechanism of deformability for this nanocapsule system (adapted from reference 103)

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Indeed, polymeric nanoparticles for topical applications have shown very promising results. According to Guterres and co-workers (2007) [1], polymeric nanoparticles were introduced in the market by L’Oreal in 1995. Subsequently, a lot of new studies and applications in this area have emerged from cosmetics to pharmaceuticals, for instance, the Photoprot® UV filter with FPS 100+++. The main ingredients of this product are polymeric nanocapsules of 240 nm containing buriti oil and vitamin E anti-oxidants, and also avobenzone and octocrylene, as UV filters.

4.5.3 Metallic Nanoparticles Metallic nanoparticles have been used in treatments for several dermal diseases as well as in cosmetics. Undoubtedly, the main kind of nanoparticle employed is silver nanoparticles [56], and in the following we will focus on some examples of applications. A rapid increase in organisms that are resistant to conventionally-used antibiotics/antifungal has been observed. Therefore, there is an inevitable and urgent medical need for the development of new antifungal substances with novel mechanisms of action [118]. In the treatment of onychomycosis (Tinea unguium), which affects over 20% of the world population, nail permeability is one of the important factors for an efficient treatment [119]. The most common treatment is through nail lacquers but this is limited by the low drug permeability. The variable structures of antifungal medications include polyenes (e.g. nystatin) which have both in vitro fungistatic and fungicidal properties; imidazoles (e.g. clotrimazole, tioconazole, econazole, ketoconazole, miconazole, sulconazole, and oxiconazole), which have in vitro fungistatic properties; and allylamines/benzylamines (e.g. naftifine, terbinafine, and butenafine), which have in vitro fungistatic and fungicidal properties [120]. Ciclopirox nail lacquer (8% solution), which has been approved by the US FDA, inhibits the transport of essential elements into the fungal cell, thus disrupting DNA, RNA, and protein synthesis. However, unfortunately, randomized and controlled trials showed that only 1 of 15 patients using the lacquer had a favorable clinical and mycological cured target nail [119]. In this context, considerable efforts have been made in recent years and a new class of antifungal agents (oxaboroles) has recently been described that penetrates the nail more effectively than ciclopirox, achieving impressive levels within and beneath the nail plate [121]. In Europe, an amorolfine lacquer (8% solution) which inhibits the biosynthesis of ergosterol, a component of fungal cell membranes, has been used and this treatment achieved a cure rate of 45-50% [86]. In order to develop appropriate formulations for topical nail application, there is a need for the development of new treatments with better penetrability of the active substance into the nail. One of the new strategies which appears to have great potential is the application of nanobiotechnological techniques. The use of silver nanoparticles is a new approach with a novel mechanism of action on onychomycosis. Thus, a new product containing silver nanoparticles has been developed as a gel [78,92,122]. The clinical response of this gel formulation was evaluated in

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patients. Medication was administered once a day for 120 days. The patients were not allowed to take any other systemic or topical therapy during the course of the study. The study group was formed after voluntary consultation with patients who were between 40 and 70 years of age and suffering from Trychophyton rubrum infection. The diagnosis was confirmed by KOH microscopic examination of a scraping collected from the infected area. Dermatophytes are easily recognized under the microscope by their long branch-like structures [123]. In initial SEM observations of the morphology of the subungual ventral nail plate, elements of fungal hyphae were detected in the toenails of two patients. The thin layers of semihard keratins of the true cuticle, the nail bed horny layer, and the thicker sole horn provide environments which allow fungal elements to thrive. The SEM observation of the cross-sectioned area of the distal toenail in one of the patient illustrates fungal hyphae penetrating the matrix between the keratin folds (Fig. 4.12). Figure 4.12 further exemplifies the pervasive nature of T. rubrum and the difficulty of treatment because of deep penetration into the nail plate. The average size of the fungal hyphae is approximately 2 to 5 μm, with macroaleuriospores reaching 10 μm. Many of the fungi present on the ventral side of the toenail showed evidence of penetration into the corneocytes. The visual aspect after 4 months of treatment in a patient is illustrated in Fig. 4.13.

Fig. 4.12 SEM images of cross-sectioned area of the distal toenail of one of the patients

Fig. 4.13 Silver nanoparticle formulation with local treatment for 4 months: The left toenail corresponds to the right foot and the right toenail correspond to the normal nail of the left foot of the same patients used as the controls

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The patient received ciclopirox for 1.5 years before using the silver nanoparticles formulation. In Figure 4.14, day 0 corresponds to the nails after a period of using ciclopirox continuously. The rapid effect of the silver nanoparticles on the dermatophytes was clear, with no occurrence of fungi at the end of the application. Another application of biogenic silver nanoparticles in healthcare is in treatments for the neglected disease leishmaniasis. It is clear that nanobiotechnology is a promising approach to curing this parasitic disease [75,78,124,125]. A study on BALB/c mice infected with 2 x 106 promastigotes of Leishmania amazonensis GFP in the ear showed that after 10 days of treatment (twice a week for 4 weeks) the wound in the control (mice treated with PBS solution) was large, while the wound of the mice treated with amphotericin B (positive control) exhibited a small lesion (Fig. 4.14).

Fig. 4.14 Photograph of the mice treated with different products: A) PBS, B) chemical nano-Ag, C) biogenic nano-Ag, D) amphotericin B

Furthermore, the same result obtained for amphotericin B was observed when biogenic silver nanoparticles were used, but with a 300-fold lower concentration, demonstrating that this represents a good alternative for leishmaniasis treatment. The chemically synthesized silver nanoparticles also decreased the wound when applied in a concentration 100 times lower than that of amphotericin B. Parasitemia by specific fluorescence units after 42 days of the infection showed the same results (Table 4.2). Table 4.2 Efficiency of silver nanoparticles in murine cutaneous leishnaniosis via intralesional treatment after 40 days of infection

Components Control (PBS) Amphotericin B Silver nanoparticles (biological method) Silver nanoparticles (chemical method)

Concentration (μg kg-1) 0.0 2000.0 6.5

Lesions (mm x 10-2) 100 30 30

FU (fluorescence units) 13500 4000 5000

21.6

50

11000

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4.6 Future Perspectives Nanostructured materials are currently very promising for topical applications, in which nanoparticles can modulate the delivery of active substances and the residence time in the skin. These show advantages not only in the cosmetics area, such as in anti-aging and anti-acne treatments, and hydration and skin care products, but also in the treatment of skin diseases such as skin cancer and vitiligo, and for transdermal delivery of substances. However, there are still some challenges related to the application of these systems to different classes of active substances. It is clear that the most recent advances are in the cosmetics area since the regulatory procedures are faster than those which apply to medicines. Most polymeric nanoparticles and SLN are biodegradable, meaning that they have a very short lifetime when exposed to the natural environment. However, metallic nanoparticles may be stable for long periods. For example, Faraday’s gold colloidal particles were stable for almost a century before being lost in the Second World War. Currently, the main concerns are the safe use of nanoparticles in relation to contamination of the environment or even workers exposure to metallic nanoparticles. Thus, further studies need to be focused on the toxic effects of non-biodegradable nanoparticles and their treatment upon disposal. Acknowledgments. Support from the Brazilian Nanocosmetics Network is acknowledged.

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[58] Marambio-Jones, C., Hoek, E.M.V.: A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 12, 1531–1551 (2010) [59] Schneider, S., Halbig, P., Grau, H., Nickel, U.: Reproducible preparation of silver sols with uniform particle-size for application in surface-enhanced Ramanspectroscopy. Photochem. Photobiol. 60, 605–610 (1994) [60] 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. 211, 122–129 (1999) [61] Stoeva, S., Klabunde, K., Sorensen, C., Dragieva, I.: Gram-scale synthesis of monodisperse gold colloids by the solvated metal atom dispersion method and digestive ripening and their organization into two- and three-dimensional structures. J. Am. Chem. Soc. 124, 2305–2311 (2002) [62] Smetana, A., Klabunde, K., Marchin, G., Sorensen, C.: Biocidal activity of nanocrystalline silver powders and particles. Langmuir. 24, 7457–7464 (2008) [63] Smetana, A., Klabunde, K., Sorensen, C.: Synthesis of spherical silver nanoparticles by digestive ripening, stabilization with various agents, and their 3-D and 2-D superlattice formation. J. Colloid Interface Sci. 284, 521–526 (2005) [64] Batabyal, S., Basu, C., Das, A., Sanyal, G.: Green chemical synthesis of silver nanowires and microfibers using starch. J. Biobased Mater. Bioenergy 1, 143–147 (2007) [65] Panacek, A., Kvitek, L., Prucek, R., Kolar, M., Vecerova, R., Pizurova, N., Sharma, V., Nevecna, T., Zboril, R.: Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. J. Phys. Chem. B 110, 16248–16253 (2006) [66] Singh, M., Sinha, I., Mandal, R.: Role of pH in the green synthesis of silver nanoparticles. Mater. Lett. 63, 425–427 (2009) [67] Venediktov, E., Padokhin, V.: Synthesis of silver nanoclusters in starch aqueous solutions. Russ. J. Appl. Chem. 81, 2040–2042 (2008) [68] Jia, H., Xu, W., An, J., Li, D., Zhao, B.: A simple method to synthesize triangular silver nanoparticles by light irradiation. Spectrochim. Acta A 64, 956–960 (2006) [69] Li, S., Shen, Y., Xie, A., Yu, X., Qiu, L., Zhang, L., Zhang, Q.: Green synthesis of silver nanoparticles using Capsicum annuum L. extract. Green Chem. 9, 852–858 (2007) [70] Long, D., Wu, G., Chen, S.: Preparation of oligochitosan stabilized silver nanoparticles by gamma irradiation. Radiat. Phys. Chem. 76, 1126–1131 (2007) [71] Mahapatra, S., Bogle, K., Dhole, S., Bhoraskar, V.: Synthesis of gold and silver nanoparticles by electron irradiation at 5–15 keV energy. Nanotechnology 18 (2007) [72] Sharma, S., Thakur, M., Deb, M.K.: Preparation of silver nanoparticles by microwave irradiation. Curr. Nanosci. 4, 138–140 (2008) [73] Sharma, S., Thakur, M., Deb, M.: Synthesis of silver nanoparticles using N-1, N-2diphenylbenzamidine by microwave irradiation method. J. Exp. Nanosci. 2, 251–256 (2007) [74] Zeng, H., Zhao, C., Qiu, J., Yang, Y., Chen, G.: Preparation and optical properties of silver nanoparticles induced by a femtosecond laser irradiation. J. Cryst. Growth 300, 519–522 (2007) [75] Durán, N., Marcato, P.D., Ingle, A., Gade, A., Rai, M.: Fungi-mediated synthesis of silver nanoparticles: Characterization processes and applications. In: Rai, M., Kövics, G. (eds.) Progress in Mycology. Scientific Publishers, Jodhpur (2010a)

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[76] Durán, N., Marcato, P.D., Alves, O.L., Da Silva, J.P.S., De Souza, G.I.H., Rodrigues, F.A., Esposito, E.: Ecosystem protection by effluent bioremediation: silver nanoparticles impregnation in a textil fabrics process. J. Nanoparticles Res. 12, 285– 292 (2010b) [77] Durán, N., Marcato, P.D., De Conti, R., Alves, O.L., Costa, F.T.M., Brocchi, M.: Potential use of silver nanoparticles on pathogenic bacteria, their toxicity and possible mechanisms of action. J. Braz. Chem. Soc. 21, 949–959 (2010c) [78] Durán, N., Bergman-Rossi, B., Marcato, P.D., De Conti, R.: Biogenic and chemical silver nanoparticles formulation to treat cutaneous leishmaniasis via intralesional treatment. Brazilian Patent (2010d) (submitted) [79] Durán, N., Marcato, P.D., Souza, G., Alves, O.L., Esposito, E.: Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J. Biomed. Nanotechnol. 3, 203–208 (2007) [80] Durán, N., Marcato, P.D., Alves, O.L., De Souza, G.I.H., Esposito, E.: Mechanistic aspects of biosyntheis of silver nanoparticles by several Fusarium oxysporum strains. J. Nanobiotechnol. 3(8), 1–7 (2005) [81] Li, Y., Kim, Y., Lee, E., Cai, W., Cho, S.: Synthesis of silver nanoparticles by electron irradiation of silver acetate. Nucl. Instrum. Methods B 251, 425–428 (2006) [82] Marcato, P.D., Durán, N.: New aspects of nanopharmaceutical delivery systems. J. Nanosci. Nanotechnol. 8, 2216–2229 (2008) [83] Rai, M., Yadav, A., Gade, A.: A Silver nanoparticles as a new generation of antimicrobials. Biotechnol. Adv. 27, 76–83 (2009) [84] Rai, M., Birla, S., Gupta, I., Ingle, A., Gade, A., Abd-Elsalam, K., Marcato, P.D., Durán, N.: Diversity in synthesis and bioactivity of inorganic nanoparticles: Progress and pitfalls. In: Sekhon, B.S. (ed.) Bioactive Inorganic Nanoparticles, Singapore, Ludhiana (2010) [85] Sanghi, R., Verma, P.: Biomimetic synthesis and characterisation of protein capped silver nanoparticles. Bioresour. Technol. 100, 501–504 (2009) [86] Eby, D., Schaeublin, N., Farrington, K., Hussain, S., Johnson, G.: Lysozyme catalyzes the formation of antimicrobial silver nanoparticles. ACS Nano 3, 984–994 (2009) [87] Kasthuri, J., Veerapandian, S., Rajendiran, N.: Biological synthesis of silver and gold nanoparticles using apiin as reducing agent. Colloids Surf B 68, 55–60 (2009) [88] Shankar, S.S., Ahmad, A., Sastry, M.: Geranium leaf assisted biosynthesis of silver nanoparticles. Biotechnol. Progr. 19, 1627–1631 (2003) [89] Song, J., Kim, B.: Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioproc. Biosyst. Eng. 32, 79–84 (2009) [90] Gade, A., Bonde, P., Ingle, A.P., Marcato, P.D., Durán, N., Rai, M.K.: Exploitation of Aspergillus niger for synthesis of silver nanoparticles. J. Biobases Mat. Bioenerg. 2, 243–247 (2008) [91] Kalishwaralal, K., Deepak, V., Ramkumarpandian, S., Nellaiah, H., Sangiliyandi, G.: Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis. Mater. Lett. 62, 4411–4413 (2008) [92] Marcato, P.D., Durán, N.: Biogenic silver nanoparticles: Applications in medicines and textiles and their health implications. In: Rai, M., Duran, N., Southam, G. (eds.) Metal Nanoparticles in Microbiology. Springer, Germany (2010)

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[93] Mokhtari, N., Daneshpajouh, S., Seyedbagheri, S., Atashdehghan, R., Abdi, K., Sarkar, S., Minaian, S., Shahverdi, H., Shahverdi, A.: Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia: the effects of visible-light irradiation and the liquid mixing process. Mater. Res. Bull. 44, 1415–1421 (2009) [94] Narayanan, K.B., Sakthivel, N.: Biological synthesis of metal nanoparticles by microbes. Advan. Colloid. Interf. Sci. 156, 1–13 (2010) [95] Vigneshwaran, N., Ashtaputre, N., Varadarajan, P., Nachane, R., Paralikar, K., Balasubramanya, R.: Biological synthesis of silver nanoparticles using the fungus Aspergillus flavus. Mater. Lett. 61, 1413–1418 (2007) [96] Jee, J.-P., Lim, S.-J., Park, J.-S., Kim, C.-K.: Stabilization of all-trans retinol by loading lipophilic antioxidants in solid lipid nanoparticles. Eur. J. Pharm. Biopharm. 63, 134–139 (2006) [97] Üner, M., Wissing, S.A., Yener, G., Müller, R.H.: Skin moisturizing effect and skin penetration of ascorbyl palmitate entrapped in solid lipid nanoparticles (SLN) and nanostructures lipid carrier (NCL) incorporated into hydrogel. Phamazie 60, 751– 755 (2005) [98] Souto, E.B., Müller, R.H.: Cosmetic features and applications of lipid nanoparticles (SLN®, NLC®). Int. J. Cosmet. Sci. 30, 157–165 (2008) [99] Attama, A.A., Schicke, B.C., Müller-Goymann, C.C.: Further characterization of theobroma oil beeswax admixtures as lipid matrices for improved drug delivery systems. Eur. J. Pharm. Biopharm. 64, 294–306 (2006) [100] Attama, A.A., Müller-Goymann, C.C.: Effect of beeswax modification on the lipid matrix and solid lipid nanoparticle crystallinity. Colloid Surf A-Physicochem. Eng. Asp. 315, 189–195 (2008) [101] Marcato, P.D., Caverzan, J., Rossi-Bergmann, B., Pinto, E.F., Machado, D., Silva, R.A., Justo, G.Z., Ferreira, C.V., Durán, N.: Nanostructured polymer and lipid carriers for sunscreen. Biological effects and skin permeation. J. Nanosci. Nanotechnol. (2010) (in press) [102] Simeoni, S., Scalia, S., Tursilli, R., Benson, H.: Influence of Cyclodextrin Complexation on the in vitro Human Skin Penetration and Retention of the Sunscreen Agent, Oxybenzone. J. Incl. Phenom. Macrocycl. Chem. 54, 275–282 (2006) [103] Marcato, P.D., Adami, L.F., Caverzan, J., Huber, S.C., Misset, T., den Hertog, J., Ferreira, C.V., Durán, N.: Solid Lipid Nanoparticles as Sunscreen Carrier System and its Toxicity in Zebrafish Embryos. In: Proceeding (615) Nanotechnology and Applications (2008) [104] Lopedota, A., Trapani, A., Cutrignelli, A., Chiarantini, L., Pantucci, E., Curci, R., Manuali, E., Trapani, G.: The use of Eudragit (R) RS 100/cyclodextrin nanoparticles for the transmucosal administration of glutathione. Eur. J. Pharm. Biopharm. 72, 509–520 (2009) [105] Namjoshi, S., Chen, Y., Edwards, J., Benson, H.A.E.: Enhanced transdermal delivery of a dipeptide by dermaportation. Biopolymers 90, 655–662 (2009) [106] Almeida, A.J., Souto, E.: Solid lipid nanoparticles as a drug delivery system for peptides and proteins. Adv. Drug. Deliv. Rev. 59, 478–490 (2007) [107] Ma, J., Feng, P., Ye, C., Wang, Y., Fan, Y.: An improved interfacial coacervation technique to fabricate biodegradable nanocapsules of an aqueous peptide solution from polylactide and its block copolymer with polyethylene glycol. Colloid. Polym. Sci. 279, 387–392 (2001)

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[108] Kasahara, Y.: Applications and prospective problems of peptides for cosmetic. Frag. J. 36, 28–32 (2008) [109] Hoet, P.H.M., Brüske-Hohlfeld, I., Satata, O.V.: Nanoparticles – known and unknown health risks. J. Nanobiotech. 2, 12–26 (2004) [110] Kohli, A.K., Alpar, H.O.: Potential Use of Nanoparticles for Transcutaneous Vaccine Delivery: Effect of Particle Size and Charge. Int. J. Pharm. 275, 13–17 (2004) [111] Alvarez-Román, R., Naik, A., Kalia, Y.N., Guy, R.H., Fessi, H.: Enhancement of topical delivery from biodegradable nanoparticles. Pharm. Res. 21, 1818–1825 (2004b) [112] Alvarez-Román, R., Naik, A., Kalia, Y.N., Guy, R.H., Fessi, H.: Skin penetration and distribution of polymeric nanoparticles. J. Control. Release 99, 53–62 (2004c) [113] Luengo, J., Weiss, B., Schneider, M., Ehlers, A., Stracke, F., Konig, K., Kostka, K.H., Lehr, C.-M., Schaefer, U.F.: Influence of nanoencapsulation on human skin transport of flufenamic acid. Skin Pharmacol. Physiol. 19, 190–197 (2006) [114] Stracke, F., Weiss, B., Lehr, C.-M., König, K., Schaefer, U.F., Schneider, M.: Multiphoton microscopy for the investigation of dermal penetration of nanoparticleborne. J. Invest. Dermatol. 126, 2224–2233 (2006) [115] Alves, M.P., Scarrone, A.L., Santos, M., Pohlmann, A.R., Guterres, S.S.: Human skin penetration and distribution of nimesulide from hydrophilic gels containing nanocarriers. Int. J. Pharm. 341, 215–220 (2007) [116] Rastogi, R., Anand, S., Koul, V.: Flexible polymerosomes—An alternative vehicle for topical delivery. Colloid. Surf B-Biointerfaces 72, 161–166 (2009) [117] Luppi, B., Cerchiara, T., Bigucci, F., Basile, R., Zecchi, V.: Polymeric nanoparticles composed of fatty acids and polyvinylalcohol for topical application of sunscreens. J. Pharm. Pharmacol. 56, 407–411 (2004) [118] Goffeau, A.: Drug resistance: the fight against fungi. Nature 452, 541–542 (2008) [119] Elkeeb, R., AliKhan, A., Elkeeb, L., Hui, X., Maibach, H.I.: Transungual drug delivery: Current status. Int. J. Pharm. 384, 1–8 (2010) [120] Tom, C.M., Kane, M.P.: Management of toenail onychomycosis. Am J. Health Syst. Pharm. 56, 865–871 (1999) [121] Hui, X., Baker, S.J., Wester, R.C., Barbadillo, S., Cashmore, A.K., Sanders, V., Hold, K.M., Akama, T., Zhang, Y.K., Plattner, J.J., Maibach, H.I.: In vitro penetration of a novel oxaborole antifungal (AN2690) into the human nail plate. J. Pharm. Sci. 96, 2622–2631 (2007) [122] Marcato, P.D., Durán, M., Huber, S.C., Rai, M., Melo, P.S., Durán, N.: Biogenic silver nanoparticles and its antifungal activity as a new topical transungual drug delivery. Br. J. Dermatol. (2010) (unpublished) [123] Crissey, J.T.: Common Dermatophyte Infections. A Simple Diagnostic Test and Current Management. Postgraduate Medicine Fev. 191-192, 197–198, 200–205 (1998) [124] Bergman-Rossi, B., Marcato, P.D., De Conti, R., Durán, N.: Efficiency of biogenic and chemical silver nanoparticles in the murine cutaneous leishmaniasis via intralesional treatment. J. Biomed. Nanotechnol. (2010) (unpublished) [125] Marcato, P.D., De Conti, R., Bergmann, B.R., Duran, N.: Silver nanoparticles/Clindamycin: Antileishmanial activity. In: Proc. 7th Bras. MRS Met. (SBPMAT), vol. 7, p. H583 (2008)

Chapter 5

Lipid Nanoparticles as Carriers for Cosmetic Ingredients: The First (SLN) and the Second Generation (NLC) Kleber L.Guimarães1 and Maria Inês Ré1, 2 1

2

Laboratory of Chemical Processes and Particle Technology, São Paulo State Institute for Technological Research, 05508-901 - São Paulo-SP, Brazil Research Center of Albi on Particulate Solids, Energy and Environment (RAPSODEE), Ecole des Mines Albi, Centre RAPSODEE, Campus Jarlard, 81013 - Albi, France [email protected]

Abstract. Colloidal carrier systems like solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were developed with a perspective to meet industrial needs related to a simple technology involving low costs and easiness for scaling-up ensuring the process qualification and validation required for the pharmaceutical and cosmetic industries. Production methods which are suitable for large scale production, innovative applications, recent advances and technological challenges are some of the aspects which are outlined in this chapter. Appropriate analytical techniques suitable for characterizing SLN and NLC are also discussed because of their relevance for product development.

5.1 Introduction Nanotechnology is one of the key technologies of the twenty-first century which wide opens new perspectives for developing science-based solutions for innovative applications. One of these promising applications corresponds to the development of refined cosmetic products whose final performance may be directly impacted by the characteristic nanoscale, thus representing excellent opportunities for both research and business. Nanoparticles as carriers for cosmetic ingredients can be synthesized from different materials. The first generation of lipid nanoparticles named (SLN) was developed at the beginning of the 1990s as an alternative carrier system to emulsions, liposomes and polymeric nanoparticles [1], followed by the nanostructured lipid carriers (NLC), as schematically represented in Fig. 5.1. As a result of intensive work of different research groups, the lipid nanoparticles have been investigated as delivery systems with respect to production, characterization and application [2-7]. The increasing interest in the field of these lipidic structures is demonstrated by a keyword search on “lipid nanoparticles” using the

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Fig. 5.1 Timeline describing the evolution and the development of the lipid nanoparticle concept in direct comparison with the common technologies mainly exploited till the beginning of the 1990’s (adapted and modified from ref. [1]).

WEB OF SCIENCE® database till the date (JUL/2010) as given in Fig. 5.2. Since the last decade, they have been studied intensively for dermal applications, both in pharmaceutical and cosmetic uses.

Fig. 5.2 Increasing trend for the number of scientific publications available in the Web of Science database (up to July 2010) and related to “lipid nanoparticles”.

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Many features of lipid nanoparticles that are advantageous for dermal application of cosmetic and pharmaceutical products have been reported, e.g. occlusive properties, increase in skin hydration, modified release profile, increase of skin penetration associated with a targeting effect and avoidance of systemic uptake. These positive features of lipid nanoparticles leaded to the market introduction of a number of cosmetic products. Examples of cosmetic products currently on the market containing lipid nanoparticles were recently recensed in the literature (see ref [8]).

5.1 Nanocarriers and Skin Interaction Due to their unique size and composition-dependent properties, lipid nanoparticles pose the ability to penetrate through several anatomical barriers providing release of their contents and encouraging applications on the field of controlled drug delivery [9-17]. Small size is a necessary but insufficient characteristic of bionanotechnological products. Such products should also attain and maintain the desirable properties on a relevant time scale. Ideally, they should moreover respond to any change in boundary conditions and thus act as ‘smart’ devices. A perfect nanotechnological product should thus adjust its characteristics promptly and properly to all relevant ambient variations. The primary challenge for developing a successful nanotechnological product for cutaneous administration of cosmetic actives relies on the complex structure of the skin barrier [18]. The challenge is proportional to potential opportunity. Fig. 5.3 illustrates the complexity of the skin consisting of several layers and structures which are organized to represent an excellent biological barrier. Skin anatomy refers to the structure of the skin, which consists of two principal parts: the outer, thinner portion which is called the epidermis and the inner, thicker portion which is known as the dermis. Anatomically, the skin consists of the following basic layers: the stratum corneum (nonviable epidermis), viable epidermis, dermis and subcutaneous tissues. In addition to these structures, there are also several associated appendages: hair follicles, sweat glands, apocrine glands, and nails. The stratum corneum is considered to act as the main barrier for the exchange of substances between the body and the environment because of its composition and structure. Therefore it became the real challenge on active agent delivery into and through the skin. It has been described that solid lipid nanoparticles and nanostructured lipid carriers are able to ensure a high adhesion to the stratum corneum enhancing the amount of active agent which penetrates into the viable skin. Furthermore, for these lipidic particles with a particle size ranging from 200 and 400 nm an occlusive effect has been described on artificial membranes [19].

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Fig. 5.3 Skin (cutis) drawing illustrating the main layers that structure the largest human organ, accounting more than 10% of the body mass. Upper layer: Epidermal layer (Epidermis); Intermediate layer: Dermal layer (Dermis); Bottom layer: Hypodermal layer (Hypodermis). (adapted and modified from ref. [22]).

By reducing the trans-epidermal water loss, an increase of penetration into the skin layers for occlusion sensitive actives has already been reported [20, 21].

5.2 Definitions 5.2.1 Lipid Nanoparticles Despite all positive features and successful application for innovative product design and resultant market introduction, it should be clear the answer for the following question: ‘What exactly are lipid nanoparticles?’ Lipid nanoparticles have a similar structure to nanoemulsions. They are submicron colloidal carriers and their size typically ranges from 40 to 1000 nm. The difference is that the lipid core is in the solid state (see Fig. 5.4). The matrix consists of a single solid lipid or mixtures of lipids. The term lipid includes triglycerides, partial glycerides, fatty acids, steroids and waxes. To stabilize the lipid particle against aggregation, surfactants or polymers are added. Typically, these colloidal carriers with a solid lipid core are composed of 0.1 to 30 wt% solid lipid dispersed in an aqueous medium and if necessary they are stabilized with preferably 0.5 to 5 wt% surfactant. If lipid nanoparticles are intended to be used as a carrier, the active ingredients are dissolved or finely dispersed in the lipid matrix. They may be specially designed for encapsulating hydrophobic active agents once the loading capacity is directly related to the solubility of the active agent in the lipid melt and in the solid matrix originated after crystallization of the lipid upon cooling.

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Fig. 5.4 Schematic representation of (a) nanoemulsions (lipid monolayer enclosing a liquid lipid core) and (b) lipid nanoparticles (lipid monolayer enclosing a solid lipid core).

5.2.2

Solid Lipid Nanoparticles (SLN)

As already mentioned, SLN, the first generation of lipid nanoparticles, is produced by replacing the liquid lipid (oil) of an o/w emulsion by a solid lipid or a blend of solid lipids, i.e. the lipid particle matrix being solid at both room and body temperature [23]. SLN consist of biodegradable physiological lipids or lipidic substances and stabilizers which are generally recognized as safe (GRAS) or have a regulatory accepted status and they are an interesting delivery system. However, there are some potential limitations as the relatively low loading capacity for a number of active substances and potential expulsion of these substances during storage. This is particularly true for SLN prepared from single highly purified lipids which can crystallize in a more or less perfect crystalline lattice. Such a perfect crystalline structure leaves almost none amorphous domains for the incorporation of active substances. Usually, active substances are incorporated between the fatty acid chains, alternatively between lipid layers or in amorphous clusters in crystal imperfections (Fig. 5.5). The lipid nanoparticles’ production methods necessarily comprise at least one step of heating to melt the solid lipid and a consecutive step of controlled cooling which results in the re-crystallization of the lipid matrix. By considering the possibility of polymorphism occurrence, it is common to verify that at least a part of the particles crystallizes in a higher energy modification (α or β'). During storage, these meta-stable structures can transform to the low energy, more ordered β modification [24]. Due to its high degree of order, the number of imperfections in

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Fig. 5.5 Crystalline and amorphous domains concomitantly present in the solid lipid and the active substance incorporation within the formed matrix. During storage it is verified the formation of a continuous “perfect” crystalline structure which results in active ingredient expulsion (adapted and modified from ref. [1]).

the crystal lattice is reduced leading to active ingredient expulsion within a short period of time as schematically represented in Fig. 5.5.

5.2.3

Nanostructured Lipid Carriers (NLC)

A second generation, called nanostructured lipid carriers (NLC), was introduced to overcome the potential difficulties with SLN [25, 26]. In the second generation of lipid nanoparticle technology, the particles are produced using blends of solid and liquid lipids (oils) that induce a melting point depression compared to the pure solid lipid while still being solid at body temperature. In fact, the incorporation of liquid lipids in those structures inhibits the crystallization process by mixing “spatially” different molecules and lefts behind imperfections in the lattice which may accommodate the active ingredient. The solid solution concept is used to produce the lipid particles which are still solid at temperatures up to about 40°C [27]. This kind of approach enables a significant increase related to the loading capacity and also minimizes premature active ingredient expulsion. It is expected, as a result, that the release profile itself is altered because of this novel spatial organization of the ‘building blocks’ that constitute the lipid matrix. Fig. 5.6 illustrates an iconographic model that represents similar (SLN) and dissimilar (NLC) ‘bricks’ that pack together to construct a ‘wall’ without or with a high concentration of defects. The bricks and the wall respectively represent the lipid matrix molecules and the resultant crystalline lattice.

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Fig. 5.6 Formation of an almost perfect crystalline structure in SLN (left) by identically shaped molecules similar to a brick wall. Formation of a solid particle matrix of NLC (right) with many imperfections comparable to building a wall from very differently shaped stones (adapted and modified from ref. [1]).

It can thus be summarized that SLN are particles produced from solid lipids only in contrast to NLC that are particles produced from a blend of solid lipids with a liquid lipid (oil). This results in significant differences regarding the particle matrix structure. NLC possess many imperfections which contribute to increase active loading capacity and minimize or avoid active substance expulsion during storage.

5.3 Preparation of Lipid Nanoparticles The essential prerequisite for entry to the cosmetic market is the availability of large scale production methods at sufficiently low cost that simultaneously meet all the regulatory requirements. Many different techniques for the production of lipid nanoparticles have been described in the literature. Just to point the variety of available methods and their respective variations, some of them are cited here: high pressure homogenization [28, 29], microemulsion technique [13, 30, 31], ultrasonication [32, 33], membrane contractor technique [2, 34], co-flowing micro-channels [35] and several others that should not be considered of less relevance. However, high pressure homogenization technique has so many advantages compared to the most current methods, e.g. easy scale up, avoidance of organic solvents and short production time, that it will be the only method to be further discussed herein. The lipids used may be tryglicerides (tri-stearin), fatty acids (stearic acid, palmitic acid), steroids (cholesterol) and waxes (cetyl palmitate). Various emulsifiers and their combinations to design different hydrophilic-lipophilic balance (HLB) values have been successfully used to stabilize the resulting lipid dispersion.

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Lipid nanoparticles can be produced by either hot or cold high pressure homogenization technique. Fig. 5.7 shows schematically the operational steps of these two available methods. Before continuing to describe the operational procedures related to lipid nanoparticles’ production, it is recommended for the reader to remind the main formulation-based difference between the SLN (first generation) and the NLC (second generation). The active compound to be encapsulated is dissolved or dispersed in melted solid lipid for SLN or in a homogeneous single phase mixture of liquid lipid (oil) and melted solid lipid for NLC.

Fig. 5.7 Production process routine for producing lipid nanoparticles using cold (left) and hot (right) high pressure homogenization (modified from ref. [8]).

In the hot homogenization technique the lipid melt containing the active compound is pre-emulsified in a hot surfactant solution (normally set to 5-10ºC above the melting temperature of the solid lipid or lipid blend) using high speed or high shear stirring. The resultant o/w emulsion is then passed through a high pressure homogenizer applying repeated cycles at a specified pressure condition. The high temperature condition is kept during the entire process and the system is cooled only after finishing the homogenization cycles in order to promote the solidification/recrystallization of the lipid matrix. In the cold homogenization technique the active containing lipid melt is cooled down, just prior to passing through the high pressure homogenizer, resulting in a mass that has to be crushed and ground to obtain lipid microparticles. The micronized lipid particles are then dispersed in a cold surfactant solution yielding a cold pre-suspension which is passed through a high pressure homogenizer

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applying repeated cycles. The number of cycles and working pressure typically selected for the hot homogenization method vary between 2-3 and 500-800 bar. For the cold homogenization method these values commonly lye between 5-10 and 1500 bar [8, 36].

5.4 Characterization of Lipid Nanoparticles An adequate and proper characterization of the lipid nanoparticulate systems is necessary for product design and development (bench-scale production) and also for quality control related to large-scale industrial production. However, physicochemical characterization of lipid nanoparticles is a real challenge due to the colloidal size, complexity and dynamic nature of the delivery system. Some crucial and important parameters to be evaluated are: • • • •

particle size distribution (PSD) physical stability as a function of time degree of crystallinity and lipid modification (polymorphism) morphological aspects

There are several analytical techniques that have been successfully employed to evaluate the respective properties. Although it is not the primary objective of this chapter, some of the fundamental aspects related to these techniques will be briefly discussed in order to open the reader’s mind to the limitations and potentials associated to each of them.

5.4.1 Particle Size Distribution Dynamic Light Scattering (DLS), also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), corresponds to the most powerful technique for routine measurement of sub-micron particle size. This method commonly covers a size range from a few nanometers to about 10 microns for particles in suspension with concentrations ranging from 0.001 to 40%. DLS is a technique used to determine the diffusion coefficient of small particles suspended in a fluid medium. The coefficient is determined by accurately measuring the light scattering intensity of the particles as a function of time. As the particles of interest diffuse within the sample cell due to Brownian motion, an incident beam of laser light illuminates the particles. The particles scatter the light, producing fluctuations in the scattering intensity as a function of time. The scattered light is collected at a chosen angle, and is measured by a highly sensitive detector. Since the diffusion rate of the suspended particles is determined by their size (inertial effects), information about their size is contained in the rate of fluctuation of the scattered light. So, by correlating the fluctuation, it is possible to determine the particle size distribution of the population present. The experimental graphical results are commonly expressed either as a differential or as a cumulative intensity (%) as a function of the particle diameter (nm)

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and this size distribution may be entirely characterized by considering statistical parameters such as the mean diameter and the index of polidispersity. The assessment of information related to particle size distribution may be extremely useful for determining the operational conditions necessary to obtain an intermediate product with the desired properties and also for evaluating the stability kinetics during the shelf-life expected for the product identifying any possible coagulation and aggregation tendency. For example, Jebors et al. [37] demonstrated by using the dynamic light scattering technique that parameters including the organic solvent, amphiphile concentration and the presence of a co-surfactant significantly affect the size of the SLNs obtained. The experimentally determined particle sizes varied between 85 and 215 nm. In contrast, stirring speed and solution viscosity had no effect over particle size distribution. The authors also demonstrated that the SLNs were unstable with respect to freezing-defreezing cycles. The temporal stability was expressed as a ratio of the mean particle diameter of the SLNs after a predetermined period of time in relation to the initial measurement (t=0h).

5.4.2 Zeta Potential Laser Doppler Electrophoresis, also known as electrophoretic light scattering, is the method most popularly used to determine the velocity of the particles suspended in a fluid medium under an applied electric field. This method covers a size range for zeta potential determinations from a few nanometers to about 30 microns with the advantage to handle low volume samples down to 30 μL. Because of the extremely diluted condition required for sample analysis, extreme caution should be taken with any kind of contamination that may affect the results by altering the ionic strength or the pH condition of the system. A common routine for zeta potential determination should take into account a potentiometric titration procedure once the surface charge distribution and resultant magnitude are directly dependent on the pH. In order to determine the speed of the movement of particles, they are irradiated with a laser light and the scattered light emitted from the particles is detected. Since the frequency of the scattered light is shifted from the incident light in proportion to the moving particle’s speed, the electrophoretic mobility of the particles can be measured from the frequency shift (Doppler shift) of the scattered light. When an electric field is applied, the charged particles present in suspension move toward an electrode opposite to their surface charge. Since the speed rate is proportional to the particle’s surface potential, zeta potential (ζ) can be estimated by measuring the velocity of the particles. The assessment of zeta potential may be extremely useful for selecting different additives and determining the efficiency of the resultant stabilization mechanism (except when dealing exclusively with steric stabilization). Sandri et al. [38] observed a significant change in SLNs’ surface charge values as a function of direct interaction with a positively charged polymer (adsorption). The SLNs were

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initially characterized by a negative surface charge potential of about -20 mV and the addition of 1% (w/w) of a cationic polymer implied in a zeta potential of about +30 mV. The magnitude of zeta potential gives an indication of the physical stability of colloidal systems. It directly allows predictions about the storage stability of colloidal dispersions, once in general, particle aggregation is less likely to occur for charged particles (high zeta potential modulus) due to their electrostatic repulsion.

5.4.3 Physical Stability Kinetics The multiple light scattering is a technique which corresponds to a powerful tool that enables quick detection and deep understanding of destabilization phenomena that take place during ageing or shelf-life tests. The technique (multiple light scattering) is used to determine light flux transmitted (T) through and backscattered (BS) from a product. The reading head acquires transmission and backscattering data either at a chosen position on the sample cell or every tens of μm while moving along the cell height. The recorded data is stored as a function of time creating a destabilization phenomena scenario (in terms of particle diameter and concentration) and also enabling the determination of kinetic parameters that might be useful for performance comparison of different samples. This method commonly covers a size range from a few tenths of nanometers to about 1000 microns for particles in suspension with concentrations up to 95 vol.%. Destabilization phenomena (e.g. flocculation, sedimentation, flotation, creaming) necessarily alter the transmitted (T) and backscattered (BS) profiles over the time or over the sample height either because of particle diameter variations or concentration oscillations. As an example it is considered that the top of the sample cell tends to become more turbid when a flotation phenomenon takes place thus increasing the amount of backscattered light in the upper portion of the sample. The opposite is valid for the bottom portion of the sample which clarifies over time thus increasing the transmitted light. Thus, particle size variations due to coalescence or flocculation may be easily monitored through kinetics of particle mean diameter versus time. Particle migration phenomena leading to sedimentation or creaming phenomena can be evaluated by direct measurement of sediment/cream phase thickness. This kind of approach may be directly crossed with zeta potential, particle size distribution and rheological studies to precisely describe the dispersion state enabling the correction of non-optimized formulations or inadequate processing conditions. Gonzalez-Mira et al. [39] demonstrated the applicability of these techniques in an integrated manner to optimize the product development and achieve a NLC formulation with an average diameter of 288 nm and a zeta potential of -29 mV. The stability of the optimized NLC was predicted and confirmed using the multiple light scattering technique by verifying that the transmitted and backscattered profiles didn’t alter over the time.

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5.4.4 Crystal Structure The powder x-ray diffraction method is the most useful of all diffraction methods, which can yield a great deal of structural information about the material under investigation. Essentially the method employs the diffraction of monochromatic xrays by a powder specimen. Powder can mean either real powder bound together or any specimen in polycrystalline form. In powder sample or fine polycrystalline sample there are many small crystalline domains, also called crystallites, randomly oriented so there are always some crystals with planes favorably oriented to yield the diffraction in particular angle determined by Bragg condition. By scanning the sample over a wide range of incident angles, different planes of different crystals will satisfy the reflection condition originating the so called diffractogram (a graphical plot of the diffraction intensity as a function of the measuring angle 2θ). Identification is achieved by directly comparing the x-ray diffraction pattern obtained from an unknown sample with an internationally recognized database containing reference patterns. It is worthy to note that the diffractogram corresponds to the ‘fingerprint’ of a selected substance presenting a respective crystal structure. The assessment of information related to crystal structure is essential for evaluating the occurrence of polymorphic phenomena and also for evaluating the degree of order/disorder achieved by the lipid matrix after the solidification/recrystallization step upon cooling. This kind of information may be extremely useful for determining the optimized re-crystallization conditions (e.g. cooling rate and supercooling state) by selecting the crystal structure of interest based on kinetic and thermodynamic parameters. The distinction between two polymorphic forms may also be evaluated by differential scanning calorimetry (DSC). DSC explores the fact that different polymorphs have different melting points and melting enthalpies. It corresponds to a thermoanalytical technique in which the amount of heat required to increase the temperature of the sample and a reference is measured as a function of time or temperature. The basic principle underlying this technique is that, when the sample undergoes any phase transition, more or less heat will need to flow to it in comparison to the reference to maintain both at the same temperature. Whether less or more heat must flow to the sample depends on whether the process is exothermic (e.g. crystallization) or endothermic (e.g. melting). The result of a DSC experiment is a curve of heat flux versus temperature or versus time. This curve can be used either to determine the enthalpies of transition by directly integrating the peak corresponding to a given transition or to identify the respective correlated temperatures (e.g. melting point). Rahman et al. [40] confirmed by DSC and powder x-ray diffraction studies the non-occurrence of interaction between the components used for the SLN formulation. The starting materials showed sharp melting endothermic peaks at distinct temperatures indicating the crystalline state both for the lipid matrix and the active agent. The physical mixture showed the same peaks of both components while the SLN formulation showed a single characteristic melting point nearby the correspondent for the lipid matrix. This kind of behavior was attributed to the conversion of crystalline active agent into amorphous form in the SLN formulation after processing. This was

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further confirmed by powder x-ray diffraction studies once only crystalline materials promote the occurrence of characteristic crystallographical peaks.

5.4.5 Morphological Aspects High resolution scanning electron microscopy (HRSEM) and high resolution transmission electron microscopy (HRTEM) provide a way to directly observe and characterize nanoparticles. The scanning electron microscope (SEM) is undoubtedly the most widely used of all electron beam instruments because of several factors such as: excellent spatial resolution, relatively straightforward interpretation of the acquired images and the accessibility of associated spectroscopy and diffraction techniques. It provides a powerful tool for morphological examination. With the recent generation of SEM instruments, high quality images can be obtained with magnifications as low as 5x and as high as 1,000,000x. Image resolution of about 0.5 nm can now be achieved in the most recent generation fieldemission-gun SEM (FEG-SEM), clearly rivaling that of a transmission electron microscope (TEM). When an electron beam interacts with a bulk specimen, a variety of electron, photon, phonon, and other signals can be generated. There are three types of electrons that can be emitted from the electron-entrance surface of the specimen: secondary electrons with energies <50 eV, Auger electrons produced by the decay of the excited atoms, and backscattered electrons that have energies close to those of the incident electrons. All these signals can be used to form images or diffraction patterns of the specimen or can be analyzed to provide spectroscopic information. The de-excitation of atoms that are excited by the primary electrons also produces continuous and characteristic X-rays as well as visible light. These signals can be utilized to provide qualitative, semi-quantitative, or quantitative information on the elements or phases present in the regions of interest. All these signals are the product of strong electron-specimen interactions, which depends on the energy of the incident electrons and the nature of the specimen. In a SEM instrument, a fine electron probe, formed by using a strong objective lens to de-magnify a small electron source, is scanned over a specimen in a two-dimensional raster. Signals generated from the specimen are detected, amplified, and used to modulate the brightness of a second electron beam that is scanned synchronously with the SEM electron probe across a cathode-ray-tube (CRT) display. Therefore, a specimen image is mapped onto the CRT display for observation. Due to their composition-dependent characteristics, here expressed by a reduced melting temperature, these lipid nanoparticulate systems are extremely sensitive to temperature modifications which may compromise their integrity. It is known that whenever considering electron microscopy techniques the sample is exposed to heating either on the preparation step or during the image collection because of direct interaction with the accelerated electron beam. By the means of operating the microscope on the environmental mode (low-voltage SEM) it is feasible to collect photomicrographs of the referred sample, which is non-conductive, even without any sample preparation procedures such as sputter-coating which may create artifacts or even compromise the integrity of the colloidal particles.

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The selection of appropriate operating conditions such as a reduced accelerating voltage (< 1 keV) may contribute to preserve the sample characteristics despite the commitment of the image resolution. An alternative approach is to employ an integrated cryogenic stage suitable for freezing the sample thus minimizing any heating decurrent of the Joule effect caused by the interaction with the electron beam. Once the sample is frozen prior and during the experiment it is also possible to collect images without solvent removal that may cause modification changes which influence the particle morphology. The direct observation and characterization of the nanoparticles complement the information of the particle size distribution once the associated shape factor is clearly evidenced. For example, Saupe et al. [41] observed almost spherical particles for SLN and NLC formulations by using the cryo-field emission scanning electron microscopy technique. Any presence of non-entrapped active ingredient, either because of any incompatibility with the lipid matrix or expulsion effect during the re-crystallization steps upon cooling (production process), was also simply evidenced by the presence of outer crystals because of the particular morphological aspect related to lipid nanoparticles.

5.5 Loading Capacity and Active Agent Incorporation Mechanisms By the means of the schematic models displayed in Figs. 5.5 and 5.6 it is easy to understand that both the entrapment efficiency and the loading capacity are directly related to the crystallographical structure of the lipid matrix. Both parameters are of prime importance when evaluating the performance of controlled release applications. As a matter of their relevance, a brief definition of both terms is presented herein. Entrapment efficiency is defined as the percentage of active agent incorporated into the lipid nanoparticles relative to the total active agent added. It specifies the percentage of active agent entrapped within the particles in contrast to free active agent that remains in the dispersion medium. Loading capacity refers to the percentage of active agent incorporated into the lipid nanoparticles relative to the total weight of the lipidic phase (i.e. lipid + active agent). It may be considered a rule that the higher the degree of crystallinity, the lesser the loading capacity [36]. A major drawback of SLN is the frequently low loading capacity which is limited to about 10% of the amount of lipid (leading to about 1% of the final dispersion) to ensure the stability of the system [42]. This is due to the specific nature of the solid lipid matrix and holds true especially with active agents of only moderate lipophilicity. Therefore both the physico-chemical parameters of active agent and lipid have to be considered. The distribution of the active compound within the lipid particle may vary considerably and hence influence on product final performance [9]. Different models of incorporated actives in lipid nanoparticles have already been proposed as presented in Fig. 5.8 and may directly affect some application aspects related to skin permeation and controlled (sustained) release profile.

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Fig. 5.8 Models of incorporated actives in lipid nanoparticles, homogeneous matrix (left), active-free lipid core with active-enriched shell (middle) and active-enriched core with active-free lipid shell (right) (adapted and modified from ref. [8]).

In the case of a homogenous matrix (Fig. 5.8 left) the active agent is molecularly dispersed evenly in the particle matrix. Active agent release takes place by diffusion from the solid lipid matrix and additionally by lipid nanoparticle degradation [9]. For the core-shell structures as depicted in Fig. 5.8 (middle and right) the release profile of the encapsulated active ingredient is directly dependent on partitioning effects between the melted lipid phase and the aqueous surfactant phase during particle production. The reader may incorrectly assume that, once the referred encapsulation technology is mainly applicable to encapsulate hydrophobic actives, the partitioning effect is negligible, but that’s not true. The amount of partitioning will increase with the solubility of the active ingredient in the water phase which means that the temperature and the surfactant concentration will both have a positive effect for the phenomenon [36, 43]. During the cooling step procedure as described for the hot high pressure homogenization (Fig. 5.7) the inverse effect takes place, which means a re-partitioning into the lipid phase. When the recrystallization temperature is reached a crystalline solid lipid core originates limiting the access of the active ingredient and resulting in a preferred concentration in the still liquid outer shell and/or on the surface of the lipid nanoparticles. For the core-shell structure with active enriched shell (Fig. 5.8 middle) this phenomenon can be explained by a lipid precipitation mechanism occurring during particle production. After homogenization each droplet is composed by a mixture of active agent and lipid and is progressively cooled. As a function of a temperature dependence on the lipid solubility, the lipid can precipitate earlier than the active agent to form an active agent-free core or at least a core with reduced active agent content. Reaching the eutectic temperature and composition, lipid and active agent precipitate simultaneously in the outer shell of the particles.

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Active agent enrichment in the shell is also a function of the solubility of the active agent in the aqueous surfactant solution (outer phase) at increased temperature during the production process. The active agent partially leaves the lipid particle and dissolves in the aqueous phase during hot homogenisation. Progressive cooling of the o/w pre-emulsion reduces the active agent solubility in the aqueous phase and, as a consequence, active agent tries to re-partition into the lipid particles leading to enrichment in the particle shell in case the particle core already started to solidify (Fig. 5.9). Such particles are known to lead to a burst release [43]. In contrast, the active agent-enriched core (Fig. 5.8 right) is formed in cases for which cooling of the hot o/w emulsion leads to precipitation of the active agent first. This takes place preferentially for lipid solutions with active agent dissolved at its saturation solubility in the lipid at production temperature. During cooling a super saturation and subsequent active agent precipitation is achieved.

Fig. 5.9 Partitioning effects during the production of lipid nanoparticles by the hot homogenization technique (adapted and modified from refs. [36, 43]).

5.6 Modulation of Active Agent Release As previously indicated in this text, a perfect nanotechnological product should adjust its characteristics promptly and properly as a function of any changes in specific external conditions. NLCs represent a more efficient solution to accommodate the active agent because of their highly disordered lipid structures when compared to the SLNs. By applying an adequate trigger impulse to convert the matrix into a more ordered structure, a desired active agent burst release can be initiated. NLCs of certain structures can be triggered this way; for example, after dermal application the particles will suffer a temperature increase and also a water content loss which might result in an increase of the active agent release.

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5.7 Recent Advances and Advantages of SLN and NLC for Cosmetic Applications 5.7.1 Formulations and Products Both NLC and SLN have many features that are advantageous for dermal applications, thus representing an interesting technology to be exploited for commercial cosmetic products’ development. In general the formulation of topical products (e.g. creams and lotions) is identical for both SLN and NLC. Novel products can be obtained by admixing lipid nanoparticles to existing products, addition of viscosity enhancers to the aqueous phase of SLN/NLC to obtain a gel or the direct production of a final product containing only nanoparticles in a single step process [8]. SLN and NLC possesses some advantages when compared with liposomes (also lipid carriers but without a solid structure) and emulsions, e.g. the protection against chemical degradation of the drug and the modulating capacity of the active compound release.

5.7.2 Encapsulation as a Protective Tool Once the lipid nanoparticles’ technology represents a method for encapsulating active agents it may pose several benefits that contribute to enhance the chemical stability of compounds sensitive to light, oxidation and hydrolysis. Enhancement of chemical stability after incorporation into lipid nanocarriers was proven for many cosmetic actives, e.g. coenzyme Q10 [44, 45], ascorbyl palmitate [46], tocopherol (vitamin E) [44] and retinol (vitamin A) [47-49]. Direct incorporation of active ingredients into the SLN matrix protects them against chemical degradation. Stabilization enhancement was reported for coenzyme Q10 and also for very sensitive retinol. Cosmetic products containing retinol have to be produced applying special and cost-intensive safety conditions concerning light exposition and protective gassing using inert gas. The stabilization of retinol incorporated into SLN offers more cost-effective ways of production and also enables more efficient preservation of the active agent extending the storage period for the product. It may also present several advantages related to controlled release applications. Burst release as well as sustained release has already been reported for SLN and NLC dispersions. Both profiles are of interest when considering dermal application of cosmetic active agents because the former might improve the penetration of the active compounds based on a concentration gradient mechanism and the latter might avoid that high concentrations of irritating compounds enter in contact with the skin over a short period of time. A controlled release mechanism may be exploited to ensure a continuous supply over a prolonged period of time avoiding high concentration peaks. Wissing et al. [50] reported a prolonged release for perfumes incorporated into cosmetic products by using the SLN technology instead of conventional emulsions. Comparing the release of the perfume from an emulsion and SLN, after 6h 100% of the perfume was released from the emulsion but only 75% was released from the SLN.

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5.7.3 The Occlusion Effect Besides the common benefits associated to common encapsulated products, the lipid nanoparticles also present occlusive properties related to film formation on the skin which may result in increased hydration effect (Fig. 5.10).

Fig. 5.10 Film formation associated to the occlusion effect (adapted and modified from ref. [22]).

It was reported by Wissing et al. [21] that the highest occlusion efficiency will be reached by using low temperature melting lipids, highly crystalline and very small particles. Nanoparticles have been found to be 15-folds more occlusive than microparticles [51]. Particles smaller than 400 nm containing at least 35% lipid of high crystallinity have been most effective. Consequently, Souto et al. [52] reported a higher occlusive factor for SLN in comparison to NLC of the same lipid content and justified the obtained results based on the degree of order resultant from the recrystallization process. Comparing NLC with different oil content led to the conclusion that an increase in oil content leads to a decrease of the occlusive factor [5]. Due to reduced water loss caused by occlusion, the skin hydration is increased after dermal application of lipid nanoparticles-containing formulations. Wissing and Müller [53] performed an in vivo study for direct investigation and comparison of skin hydration effects after repetitive application of an innovative o/w cream containing SLN and a conventional one. The study was evaluated during a 28 days period. The SLN containing o/w cream significantly increased the skin hydration in comparison to the conventional cream. Despite the fact that the hydration effect contributes to the healthy appearance of the skin, it also influences directly the percutaneous absorption. For cosmetic applications it is important that the cosmetic active is not systemically absorbed, but it is crucial a certain penetration into the skin for the desired effect to take place. Any increase of the hydration effect results in a facilitation to access the natural pathways through the skin permeability barrier [18].

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The occlusion factor of lipid nanoparticles is directly dependent on several factors: (i) lipid content, (ii) degree of crystallinity of the re-crystallized lipid and also (iii) particle size. The relationships presented here in the sequence assume that two of the three cited parameters are set equal for comparison reasons. As a rule it may be considered that reducing the particle size leads to an increase in particle number and therefore the formed film becomes denser and a higher occlusion factor takes place. The same kind of mechanism is verified by increasing the lipid concentration.

5.7.4 The UV Blocking Effect Complementarily, it was reported by Wissing et al. [21] that SLN can either solely act as a physical UV blocker and also are able to improve the UV protection in combination with organic sunscreens such as 2-hydroxy-4-methoxy benzophenone which allows a reduction of the concentration of the UV absorber. Comparing lipid nanoparticles-containing formulations and conventional emulsions, the amount of organic sunscreen can be reduced by 50% in the SLN formulation while maintaining the protective level of the conventional emulsion [53]. Furthermore, a significant increase in SPF up to about 50 was reported after the encapsulation of inorganic sunscreens such as titanium dioxide into NLC [54].

5.8 Conclusions and Future Perspectives SLN and NLC are very well-tolerated carrier systems, not exclusively designed for, but extremely efficient to assess controlled release of cosmetic and pharmaceutical dermal products. Exploiting the nanotechnology benefits, these carrier systems overcome the fact that the skin is refractive to most molecules, especially hydrophilic ones, despite the existence of trans-barrier pathways. The first lipid nanoparticles-based cosmetic product was introduced to the market only recently in 2005 indicating that the referred technology is only embryonic with respect to its future perspectives. The suitability of this innovative technology has been proven either because just after five years there are already several products available on the market and also because of several patent rights requested and also acquired by different companies during this short period of time. A main aspect that turns the attention to this technology is the fulfillment of key prerequisites related to qualified industrial large scale production with low cost and regulatory requirements’ attendance. A third generation of the referred technology has already been developed to overcome some limitations inherent to the first and second generation. The so called lipid drug conjugates (LDC) are produced by first preparing an insoluble drug-lipid conjugate bulk either by salt formation (e.g. with a fatty acid) or by covalent linking (e.g. to ester or ethers). The obtained LDC is then processed with an aqueous surfactant solution to a nanoparticle formulation using high pressure homogenization. Such matrices may have potential application in site specific targeting of hydrophilic active substances. None application regarding the use of this third generation technology has yet been reported to produce cosmetic products.

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[37] Jebors, S., Leydier, A., Wu, Q., Ghera, B.B., Malbouyre, M., Coleman, A.W.: Solid lipid nanoparticles (SLNs) derived from para-acyl-calix[9]-arene: preparation and stability. J. Microencapsul. 27(7), 561–571 (2010) [38] Sandri, G., Bonferoni, M.C., Gökçe, E.W., Ferrari, F., Rossi, S., Patrini, M., Caramella, C.: Chitosan-associated SLN: in vitro and ex vivo characterization of cyclosporine A loaded ophthalmic systems. J. Microencapsul. 27(8), 735–746 (2010) [39] Gonzalez-Mira, E., Egea, M.A., Garcia, M.L., Souto, E.B.: Design and ocular tolerance of flurbiprofen loaded ultrasound-engineered NLC. Colloids and Surfaces B: Biointerfaces 81, 412–421 (2010) [40] Rahman, Z., Zidan, A.S., Khan, M.A.: Non-destructive methods of characterization of risperidone solid lipid nanoparticles. Eur. J. Pharm. Biopharm. 76, 127–137 (2010) [41] Saupe, A., Gordon, K.C., Rades, T.: Structural investigations on nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers by cryo-field emission scanning electron microscopy and Raman spectroscopy. Int. J. Pharm. 314, 56–62 (2006) [42] Schwarz, C., Mehnert, W.: Solid lipid nanoparticles (SLN) for controlled active agent delivery. II. Active agent incorporation and physicochemical characterization. J. Microencapsul. 16, 205–213 (1999) [43] Müller, R.H., Mehnert, W., Lucks, J.S., Schwarz, C., zur Mühlen, A., Weyhers, H., Freitas, C., Rühl, D.: Solid lipid nanoparticles (SLN) as an alternative colloidal carrier system for controlled drug delivery. Eur. J. Pharm. Biopharm. 41, 62–69 (1995) [44] Dingler, A.: Feste Lipid-Nanopartikel als kolloidale Wirstoffträgersysteme zur dermalen Applikation. Dissertation, Free University of Berlin (1998) [45] Puglia, C., Filosa, R., Peduto, A., de Caprariis, P., Rizza, L., Bonina, F., Blasi, P.: Evaluation of alternative strategies to optimize ketorolac transdermal delivery. AAPS Pharm. Sci. Technol. 7, 1–9 (2006) [46] Teeranachaideekul, V., Müller, R.H., Junyaprasert, V.B.: Encapsulation of ascorbyl palmitate in nanostructured lipid carriers (NLC) - effects of formulation parameters on physicochemical stability. Int. J. Pharm. 340, 198–206 (2007) [47] Jenning, V.: Solid Lipid Nanoparticles (SLN) as a Carrier System for the Dermal Application of Retinol. Dissertation, Free University of Berlin (1999) [48] Jenning, V., Gohla, S.H.: Encapsulation of retinoids in solid lipid nanoparticles (SLN). J. Microencapsul. 18, 149–158 (2001) [49] Jee, J.P., Lim, S.J., Park, J.S., Kim, C.K.: Stabilization of all-trans retinol by loading lipophilic antioxidants in solid lipid nanoparticles. Eur. J. Pharm. Biopharm. 63, 134– 139 (2006) [50] Wissing, S.A., Mäder, K., Müller, R.H.: Solid lipid nanoparticles (SLN) as a novel carrier system offereing prolonged release of perfume Allure (Chanel). In: Int. Symp. Control. Release Bioact. Mater., vol. 27, pp. 311–312 (2000) [51] de Vringer, T.: Topical preparation containing a suspension of solid lipid particles. USA patent 91-200,664 (1999) [52] Souto, E.B., Wissing, S.A., Barbosa, C.M., Müller, R.H.: Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery. Int. J. Pharm. 278, 71–77 (2004) [53] Wissing, S.A., Müller, R.H.: The influence of solid lipid nanoparticles on skin hydration and viscoelasticity—in vivo study. Eur. J. Pharm. Biopharm. 56, 67–72 (2003) [54] Villalobos-Hernandez, J.R., Müller-Goymann, C.C.: Novel nanoparticulate carrier system based on carnauba wax and decyl oleate for the dispersion of inorganic sunscreens in aqueous media. Eur. J. Pharm. Biopharm. 60, 113–122 (2005)

Chapter 6

Industrial Production of Polymeric Nanoparticles: Alternatives and Economic Analysis Luciane F. Trierweiler and Jorge O. Trierweiler Group of Intensification, Modeling, Simulation, Control and Optimization of Processs (GIMSCOP), Departamento de Engenharia Química, Universidade Federal do Rio Grande do Sul. Brazil [email protected]

Abstract. A number of studies in the last 20 years describe the development of several techniques to produce a great variety of nanoparticles applied as drug delivery systems, among them, the nanoprecipitation. However, no study analyzing the profitability of such systems was found in the literature. Therefore, in this work it is analyzed the profitability of a polymeric nanoproduct produced by the method of nanoprecipitation: the bezophenone-3, a sunscreen agent. Based on the laboratory procedures, 3 alternatives were proposed for the industrial production, including a static mixer and a ceramic membrane pre-filtration step. The simplified profitability analysis was done based on a methodology called Bare Module Cost. Its results showed that lowest sale prices were achieved when the particles are produced through the most simplified process and dividing the production in more batches per day. The procedure presented in this chapter is general and can be widely applied to other type of nanoparticles and processes.

6.1 Introduction Polymeric nanoparticles are of especial interest from the pharmaceutical point of view. First, because they are more stable in the gastrointestinal tract than other colloidal carriers and can protect encapsulated drugs from gastrointestinal environment. Secondly, the use of various polymeric materials enable the modulation of physicochemical characteristics (e.g. hydrophobicity, zeta potential), drug release properties, and biological behavior (e.g. targeting, bioadhesion, improved cellular uptake) of nanoparticles. Finally, their submicron size and large specific surface area favor their absorption when compared to larger carriers [1]. In recent years studies indicated that topically applied nanoparticles are primarily restricted to the uppermost stratum corneum layers [2-4]. However, it should be noted that the penetrative abilities of nanoparticles through human skin are complex and are determined by the material properties of the nanoparticle, the size of individual particles, their shape and other physicochemical factors [5].

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One of the methods applied to produce nanoparticles is the nanoprecipitation. This method, first presented in 1989 [6], was largely applied by several authors in the subsequent years [7-10]. It consists of a simple procedure for the preparation of nanocapsules (NC) by interfacial deposition of a preformed, well-defined, and biodegradable polymer following the displacement of a semi-polar solvent miscible with water from a lipophilic solution. As a result, this method yields spherical vesicular nanocapsules, consisting of an oily cavity – where the drug is dissolved – surrounded by a thin wall formed by interfacial deposition of the polymer. When organic and aqueous phases are in contact, the solvent diffuses from the organic phase into the water and carries with it some polymer chains, which are still in solution. Hence, as the solvent diffuses further into the water, the associated polymer chains aggregate forming NC [8]. We did a search through www.scirus.com looking for the following words in the article titles: “profitability and nanoprecipitation” and “economic and nanoprecipitation”. As no article was found, we decided to include in our studies the economic viability of producing of nanocapsules of benzophenone-3 by nanoprecipitation. In order to do that, we first propose three alternatives to the industrial production of dried nanoparticles. After that, the methodology presented by [11] is applied to estimate direct and indirect costs for each alternative, producing 10 and 20 kg/day. Based on the profitability analysis in terms of Net Present Value (NPV) and Discounted Payback Period (DPBP), we established the minimum selling price of each level of production that results in positive NPV at a reasonable DPBP.

6.2 Alternatives Proposal In laboratory scale lipid-core nanocapsules of benzophenone-3 are prepared as following. The organic solution consists of capric/caprylic triglycerides (12.3 mL L1 ), benzophenone-3 (1.11 g L-1), sorbitan monostearate (Span® 60) (2.84 g L-1) and poly(ε-caprolactone) (PCL) (3.70 g L-1) dissolved in acetone. It is poured into an aqueous phase containing polysorbate 80 (Tween® 80) (1.44 g L-1) dissolved in deionized water under moderate stirring and controlled temperature at 40 ºC. Whenever other value is not mentioned, the proportion between aqueous and organic phase was of 2:1. Particles prepared with this procedure usually present mean size less than 300 nm and narrow size distribution[12]. Based on the lab scale process, three alternatives to the industrial production of dried nanocapsules were proposed. In all of them, we suppose that the particles were dried in a spray-drier as suggested by Guterres et al[13]. Dried nanoparticles are interesting as intermediaries in the development of pharmaceutical forms like capsules and tablets of wide industrial application. Because this type of product is usually present in small quantities in pharmaceutical formulations, like cosmetics and cosmeceutics, all alternatives operate in batch mode. In the first alternative, as seen in Figure 6.1, after the nanoprecipitation, the suspension is fed into a batch distillation column where acetone is partially recovered and re-used in the process. This column should operate under reduced pressure since there is a limitation of temperature around 40°C for the processing of

6 Industrial Production of Polymeric Nanoparticles

125

nanoparticles, due to chemical instabilities. One must have in mind that the operation under reduced pressure introduces a series of additional costs involving the vacuum system and use of refrigerant in the condenser.

Fig. 6.1 Nanocapsule Production – Proposal 1 (from now on called P1).

In the second alternative, the nanoprecipitation occurs in a static mixer, as displayed in Figure 6.2. This alternative has the advantage of nanoprecipitation step is continuously done presenting, however, the limitation that the phases preparation would occur still in batch. Note that in this alternative one new equipment and two pumps are added and no other is excluded; that is, the tank where the nanoprecipitation occurred before still exists, because it also serves for the preparation of the aqueous phase, although its size can be reduced and the stirrer can have a reduction in its total power.

Fig. 6.2 Nanocapsule Production – Proposal 2 (from now on called P2).

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In alternative 3, before the distillation, a membrane microfiltration step is added in order to separate the nanoparticles from the stream that will be distilled. Consequently, smaller quantities are processed and the distillation column is able to operate in atmospheric pressure. It must be pointed out that, due to the presence of solvent, the use of polymeric membranes are discarded for this application. Therefore, we propose the use of ceramic membranes. However, the cost of the whole system (membranes, modules, pumps and valves) is still high, around US$ 3,000.00[14]. Additionally, the faster one wants to concentrate the suspension, it will become more expensive due to the need for greater membrane area.

Fig. 6.3 Nanocapsule Production – Proposal 3 (from now on called P3).

In section 6.5 the economic viability of each alternative will be analyzed in terms of Net Present Value (NPV) and Discounted Payback Period (DPBP).

6.3 Definitions 6.3.1 Investment Cost In order to estimate the cost of a project, the purchase price of the main equipments must be known. The most precise estimates are based on supplier’s data, but when it is not available, an alternative is to use cost data of previously purchased equipment. It is still possible to use diagrams relating price and some variable capacity – e.g. volume or area – for the most common equipments. It must be pointed out that any given cost must be adjusted for differences in capacity as well as the year in which the data was generated:

6 Industrial Production of Polymeric Nanoparticles

127

⎛I ⎞ C 2 = C1 ⎜⎜ 2 ⎟⎟ ⎝ I1 ⎠

(6.1)

Where C is the purchase price and I is the cost index (the Chemical Engineering Plant Cost Index (CEPCI), published monthly by the Chemical Engineering Magazine, was used) [15]. The subscript 1 refers to the period in which the price is known and 2 refers to the period where the cost is being estimated. The authors have used the index for January 2009: 539.6. Table 6.1 Parameters used to estimate the installed cost of equipments (to be used with CEPCI of 397) Equipment

k1

k2

k3

C1

C2

C3

B1

B2 Amin Amax

Pmax [barg]

Reboiler (type: kettle)

4.4646 -0.5277 0.3955 0.03881 -0.11272 0.08183 1.63 1.66 10.0 100

Condenser (type: floating head)

4.8306 -0.8509 0.3187 0.03881 -0.11272 0.08183 1.63 1.66 10.0 1000 140

140

Vertical Vessels 3.4974

0.4485

0.1074

-

-

-

2.25 1.82 0.3 520

400

Horizontal Ves3.5565 sels

0.3776

0.0905

-

-

-

1.49 1.52 0.1 628

400

Tower Packing (material: 304 SS)

0.9744

0.0055

-

-

-

3.2999

-

-

0.03 628

-

Source: [11].

In this work Bare Module Cost method was applied because it is widely accepted as the best technique for preliminary cost estimate [11]. The method pro0

vides all costs at the time of purchase ( C P ), whereas the same condition (material and operating pressure). Deviations from standard conditions are considered multiplying factors that depend on the specific type of equipment, system pressure (FP, eqs. 6.4 and 6.5) and construction materials (FM, Table 6.2). Additionally the cost is multiplied by factors (B1 and B2) that synthesize costs with placement, piping, electrical installations, paintings, etc.: C BM = C P0 [B1 + B2 FP FM ]

(6.2)

From May to September 2001, Turton et al.[11] made a price survey with several suppliers. The obtained data were fitted to equation 6.3, resulting in the adjusting parameters of Table 6.1, considering atmospheric pressure and carbon steel. logC p0 = k1 + k2 log10 ( A) + k3 (log10 ( A))

2

(6.3)

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where A is the equipment capacity or the size parameter, e.g., for heat exchangers A is the heat transfer area and for vessels A is the volume. Table 6.2 Material Factors. Equipment

Material

FM

Heat Exchanger, kettle

Cu (shell) / Cu (tube)

1.7

Heat Exchanger, floating head

CS (shell) / Cu (tube)

1.6

Vessels, horizontal or vertical

CS

1

SS

3.6

CS – Carbon Steel; SS – Stainless Steel; Cu – Cooper.

If the equipment is operated in different pressures and/or is constructed with another material, corrections should be made by applying a pressure factor (FP) and/or a material factor (FM). To vessels:

FP,vessels

(P + 1)D + 0.0315 2(850 − 0.6(P + 1)) = 0.0063

(6.4)

where P is the relative pressure and D is the diameter. If FP,vessels < 1 then FP,vessels = 1; if the pressure is lower than -0.5 barg, FP,vessels = 1.25. For other equipments FP is calculated as: log10 FP = C1 + C 2 log10 P + C3 (log10 P )

2

(6.5)

6.3.2 Production Cost It involves direct costs (raw material, waste treatment, utilities, operating labor, maintenance, repairs, supplies, lab costs, patents and royalties), fixed costs (depreciation, local taxes, insurance and royalties) and general costs (administrative costs, distribution, selling, research and development). According to [11] the total production cost before depreciation can be calculated based on five main costs: cost of investment (FCI), labor cost (COL), cost of utilities (CUT), cost of waste treatment (CWT) and cost of raw material (CRM):

COM d = 0.280 FCI + 2.73COL + 1.23(CUT + CWT + C RM )

(6.6)

6.3.3 Basic Concepts Net Present Value (NPV)

It is the algebraic sum of all incomes and outcomes updated based on a discount rate that matches the opportunity cost of an invested capital. Make i the discount rate, n the amortization period and FCLj the value of cash flow at time j:

6 Industrial Production of Polymeric Nanoparticles n

VPL = ∑ j =1

129

FCL j

(6.7)

(1 + i) j

In our analysis, we used i equal to 10% and n equal to 10 years. Discounted Payback Period (DPBP)

It is the time required, after start-up, to retrieve the fixed investment, FCIL, required for the project, with all cash flow discounted back to year zero. Depreciation (d)

Each country has its own laws defining how to apply depreciation. In Brazil, according to the current legislation, goods classified as nuclear reactors, boilers, machinery and mechanical apparatus should be depreciated applying a linear rate of 10% per year for a period of 10 years. Cash Flow

It is calculated as indicated in Table 6.3. Table 6.3 Cash flow calculation (annual calculation, k) Description

Formula COMk + dk

Equation

(incomes – outcomes) *taxes

(Rk-COMk-dk)*t

(16.9)

incomes – outcomes taxes

(Rk-COMk-dk)*(1-t)

(16.10)

Expenses

production cost + depreciation

Taxes Profit after income tax (net profit) Cash flow

net profit + depreciation (Rk-COMk-dk)*(1-t)+dk

(16.8)

(16.11)

6.4 Methodology As explained before, simple costing methods and cost data will be applied to make preliminary estimates of capital and operating costs in order to decide the minimum selling price [US$/kg] which turns profitable each proposed flowsheet. Considering productions of 10 kg/day and 20 kg/day (1 batch or 2 batches of 10 kg each) and based on the method of Bare Module Cost, presented in section 5.3, the estimated investment cost of each flowsheet (P1, P2 and P3) is detailed in Table 6.4. Additional costs are presented in Table 6.5.

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L.F. Trierweiler and J.O. Trierweiler

Table 6.4 Estimated Investment Costs 10 kg, P1

10 kg, P2

10 kg, P3

20 kg, P1

20 kg, P2

20 kg, P3

[US$]

[US$]

[US$]

[US$]

[US$]

[US$]

Condenser

32,243.00

32,243.00

34,579.00

62,177.00

62,177.00

63,821.00

Reboiler

100,620.00

100,620.00

103,131.00 135,607.00

135,607.00 137,632.00

Nanoprecipitation tank

32,600.00

32,600.00

32,600.00

47,200.00

47,200.00

47,200.00

Organic phase preparation tank

19,400.00

19,400.00

19,400.00

26,800.00

26,800.00

26,800.00

Destillation accumulator tank

15,300.00

15,300.00

15,300.00

21,300.00

21,300.00

21,300.00

Water reservoir

35,800.00

35,800.00

35,800.00

54,900.00

54,900.00

54,900.00

Acetone reservoir

24,100.00

24,100.00

24,100.00

35,800.00

35,800.00

35,800.00

Product accumulation tank

40,900.00

40,900.00

40,900.00

63,300.00

63,300.00

63,300.00

Destillation column (tower + packing)

9,501.00

6,971.00

10,581.00

12,079.00

12,079.00

15,313.00

200,000.00

200,000.00

Equipments

Spray-drier Microfiltration membrane

-

Static mixer Total

200,000.00 280,000.00

280,000.00 280,000.00

-

186,387.00

-

-

372,773.00

-

100,000.00

-

-

180,000.00

-

510,564.00

607,934.00

702,777.00 739,163.00

919,163.00 1,118,839.00

Table 6.5 Additional costs. Land

US$ 500,000.00

Taxation Rate

0.42

Discount Rate

0.10

Production Costs

0.18 FCIL + 2.76 COL + 1.21 (CUT + CRM)

Working Capital

0.1 CRM + 0.1 FCIL + 0.1 COL

Operators

2.5 OHD/8; US$ 3,000.00/operator

Supervisors

1 OHD/8; US$ 5,000.00/supervisor

FCIL, fixed-capital investment; COL, cost of operating labor; CUT, cost of utilities; CRM, cost of raw material; OHD, operating hours per day.

6 Industrial Production of Polymeric Nanoparticles

131

6.5 Analysis of Economic Viability This subsection will analyze the results in terms of net present value and return on investment for the three proposed projects, considering daily production of 10 and 20 kg. For each production level was made a sensitivity analysis in terms of fixed investment cost (FCIL) and the purchase price of the most expensive raw material, the polymer (CPCL), where the worst case is calculated in Case 4, accounting for the highest investment as well as the highest price of PCL, as shown in Table 6.6. Table 6.6 Sensitivity analysis. D(FCIL) [%]

D(CPCL) [%]

Base Case

0

0

C1

-5

-5

C2

-5

+10

C3

+20

-5

C4 (worst case)

+20

+10

Cases

C5

0

-5

C6

0

+10

C7

-5

0

C8

+20

0

6.5.4 Production of 10 kg/day Table 6.7 shows the cost of investment (FCIL), working capital (WC), manufacturing cost (COM), operating labor cost (COL) and the cost of raw materials (CRM) for each of the cases analyzed. The sensitivity analysis is done using the values written in Table 6.8. Analyzing Figure 6.4, only selling prices above $1,250.00/kg ensures positive net present value at the end of ten years, both for the base case and the worst case. Moreover, the process (P1) where membrane or static mixer are not used is the most profitable, showing also lower time of return on investment: 3 years in the base case and 4 years in the worst one, as observed in Figure 6.5. Table 6.7 Costs for production of 10 kg/day. Annual Cost [US$] 10 kg/day – P1

10 kg/day – P2

FCIL

510,541.00

702,777.00

10 kg/day – P3 608,007.00

WC

226,717.00

245,941.00

236,464.00

COM

2,692,525.00

2,725,650.00

2,710,069.00

COL

300,000.00

300,000.00

300,000.00

CRM

1,456,637.00

1,456,637.00

1,456,637.00

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L.F. Trierweiler and J.O. Trierweiler

Table 6.8 Sensitivity analysis data when producing 10 kg/day. 10 kg/day – P1

10 kg/day – P2

10 kg/day – P3

FCIL

CPCL

FCIL

CPCL

FCIL

CPCL

Base Case

510,541.00

500.00

702,777.00

500.00

608,007.00

500.00

Case 1

485,013.00

475.00

667,638.00

475.00

577,606.00

475.00

Case 2

485,013.00

550.00

667,638.00

550.00

577,606.00

550.00

Case 3

612,649.00

475.00

843,332.00

475.00

729,608.00

475.00

Case 4

612,649.00

550.00

843,332.00

550.00

729,608.00

550.00

Case 5

510,541.00

475.00

702,777.00

475.00

608,007.00

475.00

Case 6

510,541.00

550.00

702,777.00

550.00

608,007.00

550.00

Case 7

485,013.00

500.00

667,638.00

500.00

577,606.00

500.00

Case 8

612,649.00

500.00

843,332.00

500.00

729,608.00

500.00

Fig. 6.4 Results of NPV sensitivity analysis for the production of 10 kg/day.

Fig. 6.5 Results of DPBP sensitivity analysis for the production of 10 kg/day.

6 Industrial Production of Polymeric Nanoparticles

133

6.5.5 Production of 20 kg/day Table 6.9 shows the cost of investment (FCIL), working capital (CG), manufacturing cost (COM), operating labor cost (COL) and the cost of raw materials (CRM) for each of the proposed flowsheets – P1, P2 and P3 – considering one or two batches. The sensitivity analysis was made as explained before based on the values presented in Table 6.10. Table 6.9 Costs for production of 20 kg/day Annual Cost [US$] FCIL WC COM

20 kg/day – P1

20 kg/dia – P2

20 kg/dia – P3

739,163.00

1,118,839.00

919,162.00

410,243.00

448,211.00

428,243.00

4,940,207.00

5,001,075.00

4,972,607.00

COL

450,000.00

450,000.00

450,000.00

CRM

2,913,274.00

2,913,274.00

2,913,274.00

2 x 10 kg/dia – P1

2 x 10 kg/dia – P2

2 x 10 kg/dia – P3

FCIL

510,541.00

702,777.00

608,007.00

WC

372,381.00

391,605.00

382,128.00

COM

4,465,154.00

4,496,801.00

4,482,698.00

COL

300,000.00

300,000.00

300,000.00

CRM

2,913,274.00

2,913,274.00

2,913,274.00

Analyzing Figures 6.6 and 6.7 it was possible to conclude that: • Selling price of US$ 950.00/kg: for the base case a slightly positive NPV is achieved only when two batches of 10 kg take place in all 3 proposed alternatives. In these cases, the DPBP lies between 3 and 5 years. The production in only one batch is not profitable, resulting in negative NPV. For case 4, the worst case, no project results in positive NPV; • Selling price of US$ 1,000.00/kg: for the base case only the production in one batch using membranes (P2) presents NPV negative and DPBP higher than 10 years; however, for the worst case only the production in 2 batches in plant P1 achieves a positive NPV. For all projects producing in 2 batches, the DPBP is equal or less than 3 years in the base case and between 4 and 6 years in the worst case; • Selling prices of US$ 1,150.00 and US$ 1,200.00/kg: in both cases NPV are positive and DPBPs are no longer than 5 years; • The three projects that consider only one batch proved to be less profitable than those where 2 batches take place.

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L.F. Trierweiler and J.O. Trierweiler

Table 6.10 Sensitivity analysis data when producing 20 kg/day. 20 kg/day – P1

20 kg/day – P2

20 kg/day – P3

FCIL

CPCL

FCIL

CPCL

FCIL

CPCL

base case

739,163.00

500.00

1,118,839.00

500.00

919,163.00

500.00

case 1

702,205.00

475.00

1,062,897.00

475.00

873,205.00

475.00

case 2

702,205.00

550.00

1,062,897.00

550.00

873,205.00

550.00

case 3

886,996.00

475.00

1,342,607.00

475.00

1,102,995.00

475.00

case 4

886,996.00

550.00

1,342,607.00

550.00

1,102,995.00

550.00

case 5

739,163.00

475.00

1,118,839.00

475.00

919,163.00

475.00

case 6

739,163.00

550.00

1,118,839.00

550.00

919,163.00

550.00

case 7

702,205.00

500.00

1,062,897.00

500.00

873,205.00

500.00

case 8

886,996.00

500.00

1,342,607.00

500.00

1,102,995.00

500.00

2 x 10 kg/day – P1

2 x 10 kg/day – P2

2 x 10 kg/day – P3

FCIL

CPCL

FCIL

CPCL

FCIL

CPCL

base case

510,541.00

500.00

702,777.00

500.00

608,007.00

500.00

case 1

485,014.00

475.00

667,638.00

475.00

577,607.00

475.00

case 2

485,014.00

550.00

667,638.00

550.00

577,607.00

550.00

case 3

612,649.00

475.00

843,332.00

475.00

729,608.00

475.00

case 4

612,649.00

550.00

843,332.00

550.00

729,608.00

550.00

case 5

510,541.00

475.00

702,777.00

475.00

608,007.00

475.00

case 6

510,541.00

550.00

702,777.00

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608,007.00

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case 7

485,014.00

500.00

667,638.00

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577,607.00

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612,649.00

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843,332.00

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729,608.00

500.00

Fig. 6.6 Results of NPV sensitivity analysis for the production of 20 kg/day.

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Fig. 6.7 Results of DPBP sensitivity analysis for the production of 20 kg/day.

6.6 Sensitivity Analysis of Sale Price vs. Production The aim of this analysis was to verify what should be the sale price that will ensure the long-term viability of the project even with sales 10, 20 or 30% smaller than planned. The analyzed cases were: (1) production of 10 kg/day; and, (2) production of 20 kg/day, in two batches; both in plant P1. Results presented in Fig. 6.8 indicate that when instead of 10 kg/day, the market demand is only: • 9 kg/day (-10 %), the minimum price that assures positive NPV is US$ 1,300.00/kg, ensuing 4 years of DPBP; • 8 kg/day (-20 %), positive NPV is reached when the price is at least 20% higher than that of nominal production, resulting in DPBP of almost 4 years; • 7 kg/day (-30 %): sale price of US$ 1,660.00 results in slightly positive values of NPV and 4 years of DPBP.

Fig. 6.8 Influence of market demand in the sale price – nominal case: 10 kg/day.

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Results presented in Fig. 6.8 indicate that when instead of 10 kg/day, the market demand is only: • 18 kg/day (-10 %), in this case the same price as the nominal value still assures positive but reduced (0.08) NPV, increasing in 1 year the DPBP, from 2.83 to 3.81; • 16 kg/day (-20 %), positive NPV is reached when the price is at least 10% higher (US$ 1,250.00) than that of nominal production, resulting in DPBP of almost 3 years; • 14 kg/day (-30 %), sale price of US$ 1,330.00 results in slightly positive values of NPV, but more than 10 years of DPBP.

Fig. 6.9 Influence of market demand in the sale price – nominal case: 10 kg/day.

6.7 Conclusions A mandatory step when one intends to industrialize some product is to make a preliminary analysis of profitability, taking into account the main factors that contribute to the investment, production, and fixed costs. Based on this analysis the investor will know [11]: 1. How much money (capital cost) it takes to build a new chemical/pharmaceutical plant; 2. How much money (operating cost) it takes to operate the plant; 3. How to combine items 1 and 2 to provide several distinct types of composite values reflecting process profitability; 4. How to select a “best process” from competing alternatives; 5. How to estimate the economic value of making process changes and modifications to an existing process; 6. How to quantify uncertainty when evaluating the economic potential of a process. Additionally, when projecting an industrial process it is important not to restrict ourselves to reproduce exactly the laboratory procedure; one should take into

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account several technical viable options and analyze their economical potential. For instance, in this work the first step was to analyze how the nanoparticles were prepared in laboratory scale and based on it, we proposed some alternatives for industrial production including or substituting some equipment, like static mixer, batch distillation and membrane process. Hence, based on the economic analysis we were able to carry out the fourth statement above, deciding that the first flowsheet proposal (Figure 6.1) is the one that should be implemented. However, if for safety or quality reasons the particles must present a very small polydispersion index, then for technical reasons, we would recommend implementing the third proposal (Fig. 6.3) because the membrane system can be designed to modulate the particle size distribution by selecting the suitable membranes pore sizes. Finally, from the profitability and sensitivity analysis it was concluded that it is better to divide the daily production in at least two batches instead of one. With this procedure the reduction in the investment cost is reflected in the final price. When 10 kg/day were produced only selling prices above $1,250.00/kg ensures positive net present value at the end of ten years, both for the base case and the worst case. In the case where 20 kg/day were produced the selling price was defined as US$ 1,000.00/kg. With it for the base case only the production in one batch using membranes (P2) presents NPV negative and DPBP higher than 10 years; however, for the worst case only the production in 2 batches in plant P1 achieves a positive NPV. For all projects producing in 2 batches, the DPBP is equal or less than 3 years in the base case and between 4 and 6 years in the worst case. It is important to mention that usually active ingredients in drugs and cosmetics formulations are presented only in small concentration; therefore the price results obtained in this work should not be a limitation for its practical applicability.

References [1] des Rieux, A., Fievez, V., Garinot, M., Schneider, Y.-J., Préat, V.: Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach. Journal of Controlled Release 116(1), 1–27 (2006) [2] Lademann, J., Weigmann, H., Rickmeyer, C., Barthelmes, H., Schaefer, H., Mueller, G., Sterry, W.: Penetration of titanium dioxide microparticles in a sunscreen formulation into the horny layer and the follicular orifice. Skin Pharmacol. Appl. Skin Physiol. 12, 247–256 (1999) [3] Kuntsche, J., Bunjes, H., Fahr, A., Pappinen, S., Rönkkö, S., Suhonen, M., Urtti, A.: Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model. International Journal of Pharmaceutic 354(1-2), 180–195 (2008) [4] Zhang, L.W., Yu, W.W., Colvin, V.L., Monteiro-Riviere, N.A.: Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes. Toxicology and Applied Pharmacology 228(2), 200–211 (2008) [5] Coulman, S.A., Anstey, A., Gateley, C., Morrissey, A., McLoughlin, P., Allender, C., Birchall, J.C.: Microneedle mediated delivery of nanoparticles into human skin. International Journal of Pharmaceutics 366(1-2), 190–200 (2009)

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[6] Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S.: Nanocapsule formation by interfacial polymer deposition following solvent displacement. International Journal of Pharmaceutics 55, R1–R4 (1989) [7] Guterres, S.S., Fessi, H., Barratt, G., Devissaguet, J.P., Puisieux, F.: Poly (DLlactide) nanocapsules containing diclofenac: I. Formulation and stability study. International Journal of Pharmaceutics 113(1), 57–63 (1995) [8] Thioune, O., Fessi, H., Devissaguet, J.P., Puisieux, F.: Preparation of pseudolatex by nanoprecipitation: Influence of the solvent nature on intrinsic viscosity and interaction constant. International Journal of Pharmaceutics 146(2), 233–238 (1997) [9] Govender, T., Stolnik, S., Garnett, M.C., Illum, L., Davis, S.S.: PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. Journal of Controlled Release 57(2), 171–185 (1999) [10] Chorny, M., Fishbein, I., Danenberg, H.D., Golomb, G.: Lipophilic drug loaded nanospheres prepared by nanoprecipitation: effect of formulation variables on size, drug recovery and release kinetics. Journal of Controlled Release 83(3), 389–400 (2002) [11] Turton, R., Bailie, R.C., Whiting, W.B., Shaeiwits, J.A.: Analysis, Synthesis, and Design of Chemical Processes, 2nd edn. Prentice-Hall, New Jersey (2003) [12] Paese, K.: Desenvolvimento tecnológico, estudo da fotoestabilidade e avaliação da permeação cutânea in vitro da benzofenona-3 a partir de nanocápsulas poliméricas incorporadas em diferentes veículos semi-sólidos. UFRGS, Porto Alegre (2008) [13] Guterres, S.S., Muller, C.R., Bassani, V.L., Pohlmann, A.R., Dalla Costa, T.C.T.: Processo de secagem de suspensões coloidais de nanocápsulas e nanoesferas poliméricas por aspersão, PI9906081-7 A2 (1999) [14] Wagner, J.: Membrane Filtration Handbook Practical Tips and Hints. Osmonics, 129 (2001) [15] CEPCI. Chemical Engineering Plant Cost Index. Chemical Engineering 116, 72 (2009)

Chapter 7

Elastic Liposomes Maria Helena A. Santana and Beatriz Zanchetta Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas-UNICAMP, 13083-852, Campinas, SP, Brazil [email protected]

Abstract. Phospholipids type, specific molecules and production processes increase the elastic properties of the membrane, making liposomes useful for the percutaneous transport of drugs and cosmetics. Herein we describe the elastic liposomes state-of-the-art technologies, the main elastic liposome systems, the parameters that quantify the membrane elasticity, the rheology of liposomes flowing down narrow capillaries and the possible mechanisms by which the elastic liposomes transport drugs to the deeper layers of the skin. Future perspectives of the conscientious development of elastic liposomes are envisaged.

7.1 Introduction Nowadays, the cosmetic and pharmaceutical industries face important challenges. Not only will cosmetic formulations substantiate claims of altering the appearance of the skin, but also protect it against the environment aggressiveness, delay the aging process and greatly benefit skin nutrition as well. To meet these demands, modern cosmetics have to demonstrate functionality and physiological benefits [1]. Novel developments in pharmaceutical formulations emphasize the non-invasive routes for drug delivery and, in that sense, the skin offers many advantages over the oral route including avoidance of first-pass metabolism, lower fluctuations in plasma drug levels, targeting of the active ingredient for a local effect and good patient compliance [2]. Additionally, the immunocompetent Langerhans cells in the skin can provide immunization. Therefore, these are exciting times in the area of applications of drugs or cosmetic bioactives through the skin (percutaneous permeation) where the pharmaceutical and cosmetic industries have moved closer together, and in various ways they develop and analyze designed formulations. Some drugs must penetrate until the dermis, in order to reach the blood stream (transdermal permeation), while others must remain at the viable epidermis as observed in cases of parasite treatments, or in cases of immunization where drugs must reach dendritic or Langherhans cells (transcutaneous penetration). On the other hand, the penetration of cosmetic bioactives is desirable, though it must be rigorously controlled, so that it does not reach the dermis, in order to be safe. However, the natural barrier of skin makes it difficult for most free drugs to penetrate into and permeate through it. For this reason, in spite of the many

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advantages offered by the transdermal route over the more conventional oral and parenteral routes, there are still relatively few drugs available in the market for transdermal applications. The barrier properties of skin are attributed primarily to the intercellular lipid bilayers of the stratum corneum. The epidermis is the major element of control. The process for transdermal absorption of compounds is a sequence of deposition onto the stratum corneum, diffusion through the living epidermis and the passage through the upper part of the papillary dermis. From the point of view of penetration of external components, the skin behaves as a mechanical nano-porous barrier perforated by a large number of, probably gap-like or quasi-semicircular, pathways. Figure 7.1 shows a scheme of the cross-section of stratum corneum evidencing dimensions and the intercellular and intracellular routes for transportation of compounds in the skin. Sweat glands and hair follicles were not illustrated due to their low density: sweat glands 0.01% [3] and hair follicles 0.1% [4] of skin surface, on average.

Fig. 7.1 Scheme of the cross-section of stratum corneuum evidencing dimensions and the intercellular and intracellular routes for transportation of compounds in the skin.

Baroli [5] presents a concise and valuable revision involving skin structure, composition, penetration paths through the skin and parameters generally cited to affect skin absorption. The author added conscientious comments on these aspects under the perspective of potentially penetrating nanosized agents. The ingress of foreign agents in the stratum corneum and their further progression toward the viable epidermis is limited by stratum corneum nanoporosity and gradients. Accordingly, most important physicochemical parameters of penetrating agents appear to be dimensions, o/w partition coefficient, and superficial properties. When penetrating agents are molecules, small size (<600 Da) lipophilic and uncharged compounds are the best candidates. For nanometric agents, in healthy individuals, the recommended size is smaller than 5-7 nm to have chances to diffuse intact throughout the fluid portion of the lipidic bilayers. Please see further details on Chapter 1.

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Since the past decades there has been great interest in exploring new techniques to increase drug absorption through skin. A survey of the literature reveals there are hundreds of publications dealing with different approaches for overcoming the low permeability of the skin. These approaches include the use of chemical enhancers, mechanical or electrical treatments for disruption of the skin barrier function such as iontophoresis, electroporation, magnetophoresis, sonophoresis and lipid or polymeric colloidal carrier systems. All of them are based on two strategies: enhancing skin permeability and/or providing a driving force acting on the drug.. Among the carrier systems, vesicular delivery systems have been advantageous and successful for some drugs administered through the percutaneous route. A large variety of raw materials have been used for incorporation or encapsulation of drugs or bioactive cosmetics, aiming to carry them through the layers of the skin. The characteristics of the delivery systems amount to one of the most important factors influencing the efficacy of functional compounds in many industrial products. In order to reach the desirable functionality, delivery vehicles have been designed and constructed demanding the development of new technologies and scalable processes. Besides the conventional attributes of the delivery systems, such as carrying the functional compound to the desired site of action, as to protect it against deactivation and to control the release of the encapsulated compounds, a percutaneous delivery system also must have elasticity to permeate since the stratum corneum until the deepest layers of the skin. Amongst the carriers used in pharmaceutical or cosmetic products, such as niosomes (surfactant vesicles) and polymer nanoparticles, liposomes are the oldest but certainly one of the most successful promoters of percutaneous absorption of drugs. Liposomes or phospholipid vesicles, are conceptually biomimetic model systems. Structurally liposomes are self-assembled colloidal particles, in which an aqueous nucleus is enclosed by one or several concentric phospholipid bilayers. Phospholipids are amphiphilic molecules which in aqueous solution form energyfavorable structures as a result of hydrophilic and hydrophobic interactions. Liposomes also allow surface modifications through the attachment of ligands and bounded or grafted polymers. For historical reasons, when no physical or chemical surface modification is introduced, liposomes are called conventional in order to be distinguished from the surface modified liposomes [6]. Figure 7.2 shows one phospholipid bilayer and various modifications of liposome surface. Elastic liposomes represent a novel concept, which grants to conventional liposomes the capability to deform and flow through capillaries such as the narrow pores of the skin. When elastic vesicles are applied non-occluded to the skin, they permeate through the stratum corneum lipid lamellar regions as a result of the hydration or osmotic force in the skin [7]. It has been found that these deformable fluid vesicles enhance skin permeation and carry the active compound into deeper layers of the skin. Being so, the lag time for permeation is decreased and the amount of active compound is desirably increased [8]. Figure 7.3 shows schematically the ability of elastic liposomes to undergo deformations and to penetrate through the intercellular pores in the skin.

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Fig. 7.2 The phospholipid bilayer and various surface modifications in liposomes

Fig. 7.3 Deformations of elastic liposomes during penetration through the intercellular pores in the skin

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The elastic properties are acquired by modifications in the bilayer and/or at the surface of conventional liposomes. The addition of macromolecules, curved lipids, polymer grafted fatty acids or other molecules that force a twist in the lamellar structure of the vesicles, is capable of creating defects and elasticity in the membrane bilayer. Being flexible, such vesicles were observed to maintain their size before and after pore penetration. They were capable of adapting their shape and volume when passing through the stratum corneum. By contrast, conventional rigid liposomes ruptured during the transport through the stratum corneum and when sufficient pressure was applied during use [8, 9, 10, 11, 12, 13, 14]. This chapter provides a brief revision of state-of-the-art liposomes in percutaneous delivery, describes the main elastic liposome systems, the fundamentals of the membrane elasticity, the characterization of the elastic properties and the mechanisms of liposome permeation in skin. Last but not least, we included future perspectives of the conscientious development of elastic liposomes for pharmaceutical and cosmetic applications.

7.2 State-of-Art Technologies Since their discovery in the 1960’s and the first studies where they were used as a drug delivery system in the 1970’s, liposomes have been utilized as an encapsulation system in a myriad of applications ranging from material science to analytical chemistry, food and medicines. Numerous books and reviews describe the various physicochemical and biological aspects of liposomes, as well as their construction and characterization. This literature has been written by scientists who composed the scientific base of the design and construction of liposomes for the various applications [6, 15, 16]. The potential value of liposomes for topical therapy was first introduced by Mezei and Gulasekharam [17]. After that, the research has been intensified towards further investigation and development of novel lipid vesicles as carriers for the delivery of drugs to skin. The main concern regarding liposome applications on the skin is the topical, percutaneous or transdermal fate of the vesicles. Controversial results using different drugs have led to uncertainties about the real capability of conventional liposomes to act as transdermal carriers. Since the 1990’s it has become evident that conventional liposomes are of little or no value as carriers for transdermal drug delivery, because they do not deeply penetrate skin, but rather remain confined to upper layers of the stratum corneum. Confocal microscopy studies showed that intact liposomes were not able to penetrate into the granular layers of the epidermis [18]. Intensive research led to the introduction of elastic liposomes in the early nineties, as a novel type of liquid-state vesicles characterized by elastic, deformable lipid membranes. This feature enables elastic liposomes to squeeze themselves through intercellular regions of the stratum corneum and permeate through the deepest layers of the skin. Deformable liposomes are composed of phospholipids and an edge activator or other surfactant molecules that give the membrane its characteristic deformability or elasticity. An edge activator is often a single chain surfactant that destabilizes the lipid bilayer of the vesicles and increases the

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deformability of the bilayer by lowering its interfacial tension. An overview of the three decades of research on lipid vesicles for percutaneous drug delivery shows the recent advancements in this field and future perspectives [19]. Benson [20], describe the main types and achievements of elastic vesicles for delivery into and via the skin. The author also summarizes various reported studies involving in vitro skin distribution and permeation from elastic vesicles, as well as other studies evaluating drug delivery of elastic vesicles following topical administration in vivo. The first generation of deformable vesicles, registered as Transfersomes® (IDEA, Munich), was introduced by Cevc and Blume [9]. Those vesicles were composed of phospholipids and sodium cholate or deoxycholate as edge activators. Transfersomes® are known as highly deformable liposomes (also designed as ultradeformable, ultraflexible or elastic). Under non-occluded conditions, elastic liposomes penetrate the intact skin carrying therapeutic concentrations of drugs. An efficiency comparable with subcutaneous administration was also reported for various drugs, such as diclofenac [21], lidocaine, corticosteroids [22], macromolecules such insulin and other peptides [23, 24]. Transfersomes® also could be used for non-invasive transdermal immunization [25]. El Maghraby et al. [26, 27, 28] showed the increased transport of estradiol and 5-fluoracil carried in deformable vesicles through the human skin of cadaver in comparison with the saturated aqueous solution of the drug. The authors also reported the effectiveness of the surfactants Span 80 and Tween 80 as an alternative to sodium cholate in the formulation of deformable liposomes. IDEA, a german biopharmaceutical company founded in 1993, develops and commercializes Transfersomes® based dermatological products. The incorporation of the contraceptive agent, levonorgestrel in proultraflexible lipid vesicles, named protransfersomes, possessed better skin permeation potential, better stability, and higher entrapment efficiency than proliposomal formulation. The composition contained lipid, surfactant (sodium cholate or sodium deoxycholate) alcohol, aqueous phase and drug. This mixture was converted into protransfersomal gel on cooling [29]. Trotta et al. [30, 31, 32] formed flexible vesicles with phosphatidylcholine and dipotassium glycyrrhizinate as the surfactant. Song and Kim [33] used the cationic lipid 1,2-diolyl-3-triethylammoniumpropane (DOTAP) in combination with Tween 20 non-ionic surfactant to form positively charged elastic vesicles aimed to be attracted to the negatively charged skin. They concluded that the positively charged elastic vesicles provided greater skin retention, a finding that has been previously demonstrated with conventional liposomes [34]. Van den Bergh et al. [35, 36, 37, 38] introduced a second generation of elastic vesicles consisting mainly of nonionic surfactants. They were composed of a stabilizing molecule, sacarose lauril ester L-595, a bilayer forming and a destabilizing micelle forming polioxietileno lauril ester, PEG-8-L. These surfactant-based elastic vesicles were shown to be more effective than rigid vesicles in enhancing skin penetration of 3H2O across hairless mouse skin in vitro [39] and also the penetration of various model drugs such as pergolide [40] lidocaine [41] and rotigotine [42] across human skin in vitro.

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Honeywell-Nguyen and Bowstra [13] compared the effectiveness of Transfersomes® and the surfactant-based elastic vesicles as drug delivery systems in skin. They concluded that the degree of intact vesicle permeation across the stratum corneum and the partitioning of intact vesicles into viable epidermis remains to be elucidated in more detail. For the surfactant-based deformable vesicles, the various studies indicated an intact partitioning into the stratum corneum but very limited portioning into the viable epidermis. Although the in vivo studies with Transfersomes® suggested an intact partitioning of vesicles into the viable epidermis and dermis, in vitro studies with Transfersomes® did not indicate an intact partitioning into the receptor phase of the diffusion cell. The authors attribute the inconsistencies in the results to the differences in vesicle composition, vesicle preparation or to differences in skin structure from different species. Ethosomes represent the third generation of elastic lipid carriers, developed by Touitou et al., [43, 44, 45, 46]. The ethosomal system is composed of phospholipid, ethanol and water. Ethosomes have been reported to improve delivery of various drugs to skin. Although the exact process of drug delivery by ethosomes remains a matter of speculation a combination of processes, most likely, contributes to the enhancing effect. As ethanol is a well known permeation enhancer, a synergistic mechanism was suggested among ethanol, vesicles and skin lipids. Ethanol may provide the vesicles with soft flexible characteristics which allow them to more easily penetrate into deeper layers of the skin. It was also proposed that phospholipid vesicles with ethanol have the ability to penetrate into the skin and influence the bilayer structure of the stratum corneum and that may lead to enhancement of drug penetration [47]. Benson [20] cites the Novel Therapeutics Technologies (NTT) as an Israel based company with ethosomes as their core technology.

7.3 Elastic Liposomes Systems Elastic liposomes hold the main structure of the conventional ones, except that the introduction of edge activators or surfactants changes their physico-chemical properties as to add deformability to them. Herein, a brief description of the properties of conventional liposomes supports the description of the elastic liposome properties.

7.3.1 Conventional Liposomes Liposomes or lipid vesicles, are spherical, self-closed structures, composed of curved bilayers which entrap part of the solvent, in which they freely float, into their interior. They may consist of one or several concentric membranes; their size ranges from 20 nanometers (nm) to several dozens micrometers (µm), while the thickness of the membrane is around 4 nm [6]. The precursor phase of liposomes is the symmetric bilayer composed of selfassembled phospholipid molecules with comparable areas of the polar and non-polar portions. Liposomes are formed when, at high concentration, the

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self-assembled molecules form a long-range ordered liquid-crystalline lamellar phase, which diluted in excess water is dispersed in stable colloidal particles. These particles retain the short-range symmetry of their original parent phase. Liposomes can be made entirely from naturally occurring substances, and are therefore biodegradable and non-immunogenic. In some cases the introduction of synthetic lipids is useful for specific characteristics such as stability and charge. Due to their structure, chemical composition and size, all of which can be well controlled by preparation processes, liposomes are eligible for a large range of applications. The most important properties are colloidal size, bilayer phase behavior, mechanical properties, permeability and charge density. The lipid composition, colloidal characteristics, and surface modifications also allow liposomes to have functional properties, such as stability, controlled release of the encapsulated molecules, specific targeting, controlled pH and temperature sensitivity. From the thermodynamic point of view, liposomes are not stable structures, and being so they cannot form themselves spontaneously. In order to produce liposomes, some energy from extrusion, homogenization or sonication, must be dissipated into the system. Helfrich [48] explained the instability of liposomes through the bending elasticity. Symmetric membranes prefer to be flat (spontaneous curvature equal to zero), and energy is required to curve them. Various factors, such as asymmetrical changes by ionization or insertion of molecules like surfactants, change the spontaneous curvature to non-zero values, though the spontaneous formation does not produce stable liposomes for drug encapsulation. Stable liposomes for drug delivery must be in a kinetically trapped and thermodynamically unstable state. As a consequence of that, liposomes maintain their integrity in spite of changes in the environment like dilution or in vivo administration. The vesiculation accumulates an excess of free energy around 10-50 kT (1 kT = 4.11×10-21J at T = 298 K) in the curvature of the liposomes. Subsequently, after formation, liposomes can aggregate, fuse and form larger structures that eventually settle outside the liquid, or may become bioavailable upon vesicle fusion with cell membranes. Phase transition is one of the most important properties of liposomes. It happens when lipid bilayers change from a solid-ordered phase, at low-temperature, to a fluid-disordered phase, above the phase transition temperature. Bilayers can also undergo transitions into different liquid-crystalline phases, such as hexagonal or micellar. The phase transitions can be triggered by physical or chemical factors, resulting in different effects on the liposomes. Thus, transitions from gel to liquidcrystalline phase caused by temperature or ionic strength changes induce liposome leakage. At the liquid-crystalline phase, the use of lipids with low phase transition temperature renders more fluid bilayers which enhance the absorption of some carried drugs in the skin. This is the reason why conventional liposomes are considered promoters of absorption [6].

7.3.2 Elastic Liposomes The elastic liposomes possess the structure and the liquid-crystalline state of the bilayer of the conventional ones. The capability of the membranes to undergo

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deformations remaining their integrity, depends on the lipid composition, on the combination of stabilizing and destabilizing molecules within the lipid membrane. The control of the instability and stability levels, defines the physico-chemical properties and the deformability of the bilayer. In this case, phase transition is also one important property, but the fluidity of the membrane must to be increased by surfactants or edge activators. The combination of the low or high phase temperature lipids as egg or soy lecithins, with the edge activators introduces flexibility in the bilayer packing influencing the fluidity, curvature and the accumulated energy in the elastic liposomes. In terms of deformability, elastic liposomes remember the red blood cells, which must squeeze through tiny capillaries to deliver their payload of oxygen and pick up waste carbon dioxide, functions essential to life. Human red blood cells are discs with a diameter of about eight microns and an area/volume ratio of about 1.73 [49]. When the cells reach vessels such as those in the brain (two microns diameter) they must stretch into a bullet-like shape to squeeze through and then return to their original disc shape upon exiting the vessel. Blood cells must rearrange components of their inner part called cytoskeleton, allowing the cells to become almost liquid-like, in order to squeeze through the narrowest capillaries found in the body. An input of either mechanical energy, such as squeezing or shearing, or chemical energy from ATP is enough to break those bonds and cause the necessary cytoskeleton deformation [50, 51, 52]. Beyond the fluidity of the bilayer, the composition of lipid also influences the interaction between liposomes and the skin. The conventional liposomes prepared from phospholipids were found to have less penetration into skin as compared to liposomes prepared from lipids with composition similar to that of stratum corneum molecules [53] or ceramides [54], which were deposited in the deeper skin layers. Besides the composition, the preparation method of liposomes profoundly affects the capability of deformation and transport across the skin. In general, elastic vesicles are prepared using Bangham´s method similar to conventional liposomes. However, DRVs (Dehydrated-Rydrated Vesicles) were found to be twice as effective as those prepared by reverse evaporation method with respect to the ability to deposit interferons into skin layers [55]. Recently, we could observe that the shear stress imparted by the mechanical stirrers such as ultraturrax or high shear microchannels devices, used to reduce the size of preformed liposomes, influenced the water content and the distance between the bilayers as determined by SAX measurements. That reduction as well as the size of liposomes, was proportional to the level of shear rate introduced in the system. Therefore the shear rate must alter the packing and the mechanical properties of the structure. Investigations of these changes as well as the consequences on the elasticity of the liposome membrane are under way in our group.

7.3.3 Membrane Elasticity Among the mechanical colloidal properties, elasticity determines functional properties of the vesicles, such as deformation, stability-instability equilibrium and controlled release of the encapsulated molecules. The elasticity of the liposome

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membrane is characterized mainly by three parameters: mean-curvature modulus (or bending rigidity), kc, the Gaussian (or saddle-splay) modulus k c , and the spontaneous (or intrinsic) curvature, c0 (Figure 7.4 a) [56]. The two elastic constants, kc, and k c play very different roles in determining bilayer organization. The magnitude of kc reflects the energy needed to bend the bilayer over from its spontaneous radius of curvature, Ro. k c influences only the topology (and hence the number) of the structures formed, such as transformations between discs and closed spheres or between multiple spheres and cylinders. Both constants have energy units. According to Marsh [56], the starting point for any discussion of bending elasticity constants is the Helfrich [48] expression for the free energy of bending for a membrane (or monolayer) surface of area A (Eq. 7.1).

ΔGC (c , cG ) =

1 2 kc A(c − c0 ) + kc AcG2 2

(7.1)

where kc is the mean-curvature modulus and k c is the Gaussian (or saddle-splay) modulus, the mean curvature is given by 2 G

is given by c

c = c1 + c2 and the Gaussian curvature

= c1 c2, with c1= 1/R1 and c2= 1/R2 being the principal curva-

tures. For cylindrical bending c = c1 and cG=0; for spherical vesicle c/2 = c1= c2 = cG , and for saddle-splay surface c = 0 and c1 = - c2. c0 is the spontaneous (intrinsic) curvature. For a symmetrical bilayer, c0= 0. In the case of a bilayer, the curvature energies of the outer and inner monolayers must be considered. Figure 7.4 b, illustrates the defined curvatures for a monolayer and the geometric parameters which define a curved bilayer vesicle.

Fig. 7.4 (a) Bending of a lipid monolayer with principal curvatures c1= 1/R1 and c2= 1/R2. (b) Geometry of outer (out) and inner (in) monolayers in a curved bilayer vesicle (Marsh, 2006)

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Equation 7.2 gives an expression for the total bending free energy of a symmetrical bilayer:

1 ⎛ ⎞ ΔGc(b ) (c , cG ) = k c( m ) A(c 2 + c 02 ) + 2⎜ k c( m ) − 2k c( m )δ c0 + k c( m ) δ 2 c02 ⎟ A cG2 2 ⎝ ⎠

(7.2)

In the Marsh analysis, the monolayer leaflets of the bilayer are assumed to be uncoupled. i.e., are free to slide over one another. The resultant spontaneous curvature vanish for a symmetrical bilayer, c0 (b) = 0. Therefore, from Eq. 7.1 and 7.2, the elastic constants of the bilayer are twice the size of the monolayer ones. Besides the definitions Marsh [56] also presents experimental values for the elastic constants of lipid monolayers and bilayers for easy reference, as well as he summarizes useful mathematical relations involving the bending moduli and spontaneous curvature. The author also compares data from both bilayers and monolayers, from data obtained through different methodologies and in different laboratories. Although differences of values from different methods and analysis, some relations with the molecular structure could be observed, such as the dependence on the mean curvature modulus, which is the quadratic dependence on acyl chain length, but that does not extend to polyunsaturated lipids. The influence of the polar group is not very strong for phospholipids. Cholesterol, however, does have a very pronounced effect on the mean curvature modulus of bilayers. The effect of chain length and unsaturation on elasticity of lipid bilayers was studied by Rawicz et al. [57]. The authors used micropipette pressurization of giant bilayer vesicles to measure elastic constants of twelve fluid-phase phosphatidylcholine membranes. The main results showed that poly-cis unsaturated chain bilayers are thinner and more flexible than saturated/monounsaturated chain bilayers. The elastic properties of surface modified liposomes by grafted polymer such as the polyethyleneglycol (PEG) change with respect to both the area expansion and the bending of the membrane. There is a direct contribution of the polymer brush to the net membrane elasticity [58]. Marsh [59] investigated the effects of the polymer brush on the membrane stretching and bending. The surface expansion that is induced by the lateral pressure in the brush was considered for a monolayer at the equivalence pressure with fluid lipid bilayers. The calculation indicates that the lateral pressure from the brush region of polymer-grafted lipids induces an appreciable expansion in area of fluid lipid membranes. The area expansion by the polymer brush is a determining feature in the curvature elastic moduli kc and k c only for fluid membranes and short polymers. For longer polymers, the direct contribution from the brush region quickly comes to dominate the curvature moduli, and achieves values several times greater than the intrinsic lipid curvature moduli. The polymer-lipid contribution to the bending modulus is given as a function of the polymer grafting density modulus. For short polymers, around eight monomer units, this contribution never exceeds the bending modulus of the corresponding bare lipid membranes. However, for larger polymer length (PEG 2000-5000 Da) the bending modulus of the bare membrane is exceeded many times, because of the cubic dependence on the number of monomer units. The Gaussian curvature modulus also depends of the polymer properties exactly like the mean curvature modulus does. In the same

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way, the lateral expansion of the membrane by the polymer brush leads to a reduction in the lipid contribution. The spontaneous curvature depends inversely of length of the polymer. The understanding of the molecular origin of the elasticity, described through the above parameters, contributes to a possible tailor-made bilayer organization in order to obtain useful structures for specific applications such as the percutaneous transport of drugs and bioactives.

7.3.4 Mechanisms The various possible mechanisms by which conventional liposomes could improve the delivery of drugs in the skin have been studied and reviewed [60, 61, 62, 63]. Among them, two main mechanisms were considered: 1. Vesicles which can act as drug carrier systems, whereby intact vesicles enter the stratum corneum carrying vesicle-bound drug molecules into the skin; 2.Vesicles which can act as penetration enhancers, whereby vesicle bilayers enter the stratum corneum and subsequently modify the intercellular lipid lamellae. This will facilitate penetration of free drug molecules into and across the stratum corneum. Several experimental results reported in the literature support the penetration enhancing effect (mechanism 2), as the dominant mechanism for drug delivery in the skin from conventional liposomes. For deformable vesicles, the first mechanism was considered by Cevc and Blume [9, 21], Cevc et al. [11]. The authors proposed that the driving force for the vesicle entering the skin is xerophobia (the tendency to avoid dry surrounds). Since the deformable liposomes are flexible, they can spontaneously penetrate the intact skin under the influence of the natural transcutaneous hydration gradient. The deformable vesicles penetrate through the stratum corneum reaching the deeper layers the skin and the blood circulation. However, more recent studies have shown that deformable liposomes only improved skin deposition of the same drugs [27, 28, 64]. Elsayed et al. [18], described the various experiments conducted in order to study the proposed mechanisms, and summarized the various drugs used in skin during in vitro studies. Based on the reviewed studies, Elsayed et al. [18] proposed that both the mechanisms suggested could be supported for deformable liposomes carrying drugs through the skin [8]. In the case of ethosomes, the mechanisms are still not well defined. A combination of processes (ethanol as permeation enhancer) and liposomes as a drug carrier could to be acting. A synergic mechanism was suggested among ethanol, vesicles and skin lipids [44, 46]. However, there are no comparative studies in the literature, regarding ethosomes, deformable and conventional liposomes. Elsayed et al. [18], summarized in vitro studies only with respect to the efficiency and applications of ethosomes as carrier for skin delivery systems. Beside the transport through the skin, the release of drugs from the vesicles is an important step which must also be considered in the mechanisms. The rate and amount of released drugs is a balance between two factors: drug affinity with vesicles, and drug solubility in lipids of the stratum corneum. Poor drug release

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resulted in retention of the drug within vesicles in the stratum corneum and elastic vesicles served as a slow release depot system [65]. Nowadays, due to the large amount of results reported in the literature, there is a better scientific understanding of the mechanisms of drug transport and delivery in the skin. However, due to the complexity of the skin, as well as the large number of variables and properties involved in both mechanisms, there is still discrepancy among the results. Therefore, there are not clear functions yet relating the permeation of drugs carried through the skin with the physico-chemical properties of the carriers, drugs and their interactions, such as size and polydispersity of vesicles, hydrophilicity or lipofilicity of drugs, the changes in elasticity imparted by the drug encapsulation, etc.

7.4 Characterization Methods The permeation of liposomes through the skin is characterized by experimental data and associated mathematical models. The classical permeation of liposome encapsulating drugs through animal skin in the classical experiment of cell Franz, determines the amount of permeated drug. Compared with non-encapsulated drug, the cell Franz experiment characterizes the carrier as a permeation enhancer of the drug, with the capability of a promoter of drug absorption. Associated with the tape stripping technique, both localize the drug retained in the layer of the skin. This is an indirect measurement of the involved elasticity of liposomes. For more detailes, see Chapter 1. The mathematical models used for the description of the transport of free drugs through the skin are empirical or phenomenological. Empirical models are advantageous because they eliminate some imprecision of the phenomenological models caused by uncertainty, allowing better predictions from the practical point of view. [66]. However, phenomenological models are more adequate to represent the transport process aiming to correlate molecular structure and permeability through the skin, to describe the influence of the skin barriers of the chemical agents enhancing the transport the enzymatic action and to allow the prediction of in vivo results from in vitro data. From the physico-chemical point of view, phenomenological models are useful for the evaluation of drugs and to design formulations for the transport through the skin. In general, the phenomenological models for no carried drug transport through the intercellular spaces of the skin, are based on the solution of Fick´s second law for diffusion (Eq. 7.3), which represents the transient diffusion through a infinite semipermeable membrane.

∂C ∂ 2C =D 2 ∂t ∂x

(7.3)

where: C is the drug concentration, D is the diffusivity of drug in the membrane. The boundary conditions are: t = 0, C=0 (initial condition) and x = L, C=0, considering sink conditions. At x=0, C= KC0, due to the partition of drug in the

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membrane, C = KC0. These conditions lead to a solution from Laplace transformate method (Eq. 7.4), in terms of the diffusivity parameter, D.

⎛D 1 2 M = KLC 0 ⎜⎜ 2 t − − 2 6 π L ⎝

∞

∑ n =1

(− 1)n exp⎛ − n

2

D 2 2t ⎞ ⎞ n π ⎟ ⎟⎟ ⎜ 2 ⎝ L ⎠⎠

(7.4)

where M is the mass of permeating solute, D is the drug diffusivity, K the partition coefficient and L the thickness of the membrane. Thus, the prediction of the transport rate of drugs through the skin needs the diffusivity parameter only. Relationships between the diffusivity or permeability and the physico-chemical, properties of the molecules, such as molecular weight or hydrophobicity have been proposed [67, 68]. More rigorous relationships have been obtained from equations for the solvatation energy [69]. Roberts et al [70] examined different in vitro skin diffusion models for percutaneous absorption, in terms of amount penetrating, flux and other useful parameters. The models ranged from the simplest, when a well -stirred formulation (vehicle) of finite volume is applied to the stratum corneum and the solute passes through a receptor sink, to more complexes and realistic models, in which the formulation vehicle is finite, and where the receptor is no longer a sink, and the vehicle cannot be considered well-stirred.

7.4.1 Transdermal Transport of Liposomes The transport of liposomes or carried drugs through the skin is not so much discussed in the literature, compared with the modeling of the free drug permeation, where empirical and phenomenological models have been proposed. Cevc et al [7, 8, 23, 24] associated the transport of aggregates through a semi-permeable barrier with the chemical potential at the both sides of the barrier, which is represented by an osmotic pressure difference. The osmotic pressure is responsible by the movement of the fluid, as well as by the difference between the water activity in the both sides of the barrier. Initially, there is a lag time before permeation, which is necessary for hydration of the vesicle. This time is proportional to the initial excess of water, as well as to the moisture of the application site. The rate of permeation of the vesicles through the skin is influenced by their capability to deform as well as by their capability to exchange water with the external medium. High deformability and hydrophilicity at the surface of the membrane are necessary conditions for the permeation of the aggregate through the semi-permeable barrier [7]. The authors also have written the Eq. 7.5 for the flux of vesicles through the barrier as a function of the transbarrier osmotic pressure difference or the hydrostatic pressure.

{

J v = Aporos [1 − σ v (rv )]Cv Pw − Pv,i (rv )rv2 ΔΠ i

}

(7.5)

where: Aporos is the total area of pores in the permeable medium, σv(rv) is the refractivity coefficient (a measurement of osmotic activity of a given component on

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a barrier), c v is the mean concentration of vesicles, Pw is the barrier permeability to water Pv,i is the barrier penetrability to a vesicle and ΔПi is the transbarrier osmotic gradient . Equation 7.5 reveals that transport flow is controlled by permeantor penetrant-independent pressure difference, in the absence of other free energy contributions. Non-considering small differences in vesicle concentration through the barrier and combining the elastic energy and rupture tension parameters in a new parameter named elasticity, E (Eq. 7.6), Cevc et al [71, 72] proposed an approximated relationship between flux, diameters of vesicles and the pores of the membrane (Eq. 7.7).

J≅E

d p2

(7.6)

d v2

⎛d E ≅ J⎜ v ⎜dp ⎝

⎞ ⎟ ⎟ ⎠

2

(7.7)

where E is the elasticity modulus, J is the flux of vesicles, dv is the diameter of the vesicles and dp is the diameter of the membrane pore. In other models of the same group, they consider the permeability of the porous medium to the flow of liposomes, influenced by the concentration and elastomechanical properties of vesicles, such as the flexibility and compressibility modulus of the lipid bilayer [73]. Bruinsma [74] presented an analytical description of the rheology and shape of axisymmetric vesicles flowing down narrow capillarities. The Helfrich bending free energy was maintained to describe the mechanics of the vesicle surface. The author found that the rheological properties of vesicles in long pores are quite independent of the Helfrich bending energy. However, the kinetics of insertion of vesicles into a pore is certainly dependent on the bending energy. For vesicles with tension, there should be two rheological regimes: at low applied pressure head, the vesicle moves only very slowly and does not obey Darcy´s law of permeation of porous media. With increasing pressure gradients, he found a singular point beyond which the rear of the vesicle becomes tensionless and Darcy´s law is obeyed. This singular point marks a whole sequence of shape transitions of the vesicle, starting from a sphero-cylinder and ending in a bell shape, similar to those reported for red blood cells in physiological literature. The authors modified the classical Darcy´s equation for flowing through ducts, ηV = K(ΔP/ΔL), considering the deformation of vesicle and the presence of a lubricant layer between the surface of vesicle and the wall of the duct, reducing the friction. The proportionality factor between ηV and (ΔP/ΔL) in the modified Darcy´s equation (Eq. 7.8), defines an effective permeability of the porous medium which is influenced by the concentration of vesicles in the pores as well the thickness of the lubricant layer.

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ηV =

R2/ 8 ⎛ ΔP ⎞ ⎜ ⎟ (1 + n RL*/4h* (V )) ⎝ ΔL ⎠

(7.8)

where: L* is the lenght of the sphero-cylinder, n is the number of aggregates divided by the lenght of pore, h* (V) is the thickness of the (velocity dependent) lubrication layer around the vesicle as it flows through the pore and V is the average velocity of fluid in the pore. R is the radius of the sphero-cylinder described by the Equation 7.9.

R = rp − h* (V )

(7.9)

The effective permeability is reduced, as compared to that of an empty pore, by a factor 1/ (1+nRL*/4h* (V)). It depends on the number of vesicles per unit length n inside the pore and is no longer purely geometrical, but the only non-geometrical parameter it will depend on should be the vesicle composition Frisken et al [75] introduced the concept of minimal pressure, from which the flowing is started. (Eq. 7.10).

j = K eff (ΔP − Pmin )

(7.10)

where: j is the flux, K eff is the effective pore permeability, ΔP is the pressure gradient and Pmin is the minimum necessary pressure for the suspension flows through the membrane. Considering the rheological approach of Bruinsma [74] we studied the flow or extrusion of 100 nm diameter egg lecithin liposomes (conventional and surface modified with PEG-8-L), through two stacked polycarbonate membrane, 30 nm pore diameter, under various nitrogen pressures, at 1 mM liposome concentration, using an extrusion cell (T.001- Lipex Biomembranes Inc.) at 25 ºC. The peguilated liposomes were 60:40 lecithin:PEG molar ratio in solution. The analysis of the experimental data aimed to determine a permeability coefficient for the flowing of vesicles through the narrow porous of the synthetic membranes, as an indirect measurement of their deformation capability. Figure 7.5 shows flow rate vs. pressure data for egg lecithin empty liposomes through two stacked polycarbonate membrane, 30 nm pore diameter. At the linear region, the slope of the straight lines determine permeation coefficients to the liposome flow, as 1.17x10-14 m for the conventional liposomes and 4.16x10-14 m for PEG-8-L modified liposomes. Therefore, the addition of PEG-8-L increased four times the flow of liposomes through the narrow pores of the membranes. At 2.5 atm, 17% and 9.4% of the total phosphate was detected in permeated for peguilated and conventional liposomes respectively. No significant difference in the minimum pressure for starting the flow could be observed for both liposomes. These data indicate better deformability and elasticity of peguilated liposomes related to the conventional ones.

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Fig. 7.5 Flow rate vs. applied pressure behavior of 100nm egg lecithin empty liposomes through two stacked 30 nm pore diameter polycarbonate membrane under various applied pressures. At the linear region (a) the slope of the straight lines determine the permeation coefficients to the liposome flow (b). Symbols: (■) water (▲) PEG-8-L surface modified liposomes (60:40 lecithin:PEG molar ratio in solution) (●) conventional liposomes

Figure 7.6 shows TEM images of egg lecithin conventional and peguilated liposomes, before and after liposome permeation at 2.5 atm. The changes in shape are evident, with more elongated structures from peguilated liposomes after permeation. Mean diameter measurements also show significant changes in diameter before and after permeation at various pressures, for conventional and peguilated liposomes (Fig. 7.7). The effect is more pronounced at low pressures (2.5 to 5.0 atm). One hour after extrusion, the size of liposomes increased toward the initial state. These results confirm the Bruinsma rheological analysis. After a lag time, the flow through the porous membranes obeyed Darcy´s law with different permeability coefficients for conventional and peguilated liposomes. The effects of PEG on the deformability of liposomes could be verified, even for short length chains (8 monomers). Compared with cell Franz, this rheological experiment is simpler and “cleaner” assay (because the permeated did not contain other released compounds from the skin) which allows evaluating the vesicles beyond flux and morphology, also performing measurements of mean diameter and polydispersity after extrusion, as well as phospholipid concentration in the fluids retained and permeated through the membrane, as a balance of the total introduced liposomes. Beyond mechanism aspects, the data from the rheological analysis are valuable for the screening of deformable carriers for skin permeation, which could be performed before the cell Franz assay.

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Fig. 7.6 TEM images for 100nm egg lecithin empty liposomes (a,b) conventional and (c,d) PEG8-L surface modified liposomes. (a,c) before (b,d) after permeation (flowing) through two stacked 30 nm pore diameter polycarbonate membranes under 2.5 atm applied pressures The peguilated liposomes are 60:40 lecithin:PEG molar ratio in solution

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Fig. 7.7 Mean diameter of conventional and peguilated (PEG-8-L) liposomes before and after permeation (flowing) through two stacked 30 nm pore diameter polycarbonate membranes under various applied pressures. The peguilated liposomes are 60:40 lecithin:PEG molar ratio in solution

7.5 Final Remarks The evolution of the development of elastic liposomes, the main physico-chemical parameters which influence their elastic behavior and the main proposed mechanisms for skin permeation of empty vesicles and carried drugs were revised Main conclusions from these studies were described in this chapter. The complexity of the skin as a porous biological medium together with the interactions between the vesicle intercellular medium and drug-vesicle, make difficult the analysis of particular results, because it includes liposomes with different types, compositions, carrying different drugs and prepared through different methods. Therefore, some results still appear to be controversial, and the description of the elastic liposome systems still lacks in generalized relations among the performance, physicochemical and mechanical properties of deformable liposomes as a drug carriers for skin permeation. However, we believe that a deeper understanding of the molecular and structural parameters which govern the elasticity of liposomes as well as the rheological behavior under skin permeation will allow in the near future, to predict the capability of lipid and associated surfactants as drug carriers through the skin. These goals will allow the conscious design of liposomes for specific treatments of the skin.

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[51] Parker, K., Winlove, C.: The deformation of spherical vesicles with permeable, constant-area membranes: application to the red blood cell. Biophysical Journal 77, 3096–3107 (1999) [52] Pamplona, D., Calladine, C.: The mechanics of axially symmetric liposomes. Journal of Biomechanical Engineering 115, 149–159 (1993) [53] Egbaria, K., Ramachandran, C., Weiner, N.: Topical Delivery of Ciclosporin: Evaluation of Various Formulations Using in vitro Diffusion Studies in Hairless Mouse Skin. Skin Pharmacology and Physiology 3, 21–28 (1990) [54] Schreier, H., Bouwstra, J.: Liposomes and niosomes as topical drug carriers: dermal and transdermal drug delivery. Journal of Controlled Release 30, 1–15 (1994) [55] Egbaria, K., Ramachandran, C., Kittayanond, D., Weiner, N.: Topical delivery of liposomally encapsulated interferon evaluated by in vitro diffusion studies. Antimicrob. Agents Chemother. 34, 107–110 (1990), doi:10.1128/aac [56] Marsh, D.: Elastic curvature constants of lipid monolayers and bilayers. Chemistry and Physics of Lipids 144, 146–159 (2006) [57] Rawicz, W., Olbrich, K.C., McIntosh, T., Needham, D., Evans, E.: Effect of Chain Length and Unsaturation on Elasticity of Lipid Bilayers 79, 328–339 (2000) [58] Hristova, K., Needham, D.: Phase behavior of a lipid/polymerlipid mixture in aqueous medium. Macromolecules 28, 991–1002 (1995) [59] Marsh, D.: Elastic Constants of Polymer-Grafted Lipid Membranes 81, 2154–2162 (2001) [60] Bouwstra, J.A., Honeywell-Nguyen, P.L.: Skin structure and mode of action of vesicles. Advanced Drug Delivery Reviews 54, S41–S55 (2002) [61] Williams, A.C.: Transdermal and Topical Drug Delivery, 1st edn., London (2003) [62] El Maghraby, G.M.M., Williams, A.C., Barry, B.W.: Can drug-bearing liposomes penetrate intact skin? Journal of Pharmacy and Pharmacology 58, 415–429 (2006) [63] Kirjavainen, M., Urtti, A., Jääskeläinen, I., Marjukka Suhonen, T., Paronen, P., Valjakka-Koskela, R., Kiesvaara, J., Mönkkönen, J.: Interaction of liposomes with human skin in vitro – The influence of lipid composition and structure. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1304, 179–189 (1996) [64] Trotta, M.P.E., Debernardi, F., Gallarate, M.: Elastic liposomes for skin delivery of dipotassium glycyrrhizinate. International Journal of Pharmaceutics 241, 319–327 (2002) [65] Honeywell-Nguyen, P.L., Bouwstra, J.A.: The in vitro transport of pergolide from surfactant-based elastic vesicles through human skin: a suggested mechanism of action. Journal of Controlled Release 86, 145–156 (2003) [66] Yamashita, F., Hashida, M.: Mechanistic and empirical modeling of skin permeation of drugs. Advanced Drug Delivery Reviews 55, 1185–1199 (2003) [67] Flynn, G.: Physicochemical determinants of skin absorption; in principles of route-toroute extrapolation in risk assessment. In: Henry, G.T.R.C.J. (ed.) Principles of Route to-route Extrapolation in Risk Assessment. Elsevier, New York (1990) [68] Potts, R.O., Guy, R.H.: Predicting Skin Permeability. Pharmaceutical Research 9, 663–669 (1992), doi:10.1023/a:1015810312465 [69] Abraham, M., Chadha, H., Mitchell, R.: The factors that influence skin penetration of solutes. Journal of Pharmaceutical Pharmacology 47, 8–16 (1995) [70] Roberts, M., Cross, S., Anissimov, Y.: Mathematical models in percutaneous absorption in Percutaneous Absorption drugs-cosmetics-mechanisms-methodology. In: Bronaugh, R.L., Maibach, H.I. (eds.) Percutaneous Absorption. Marcel Dekker, New York (2005)

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[71] Cevc, G., Blume, G., Schätzlein, A.: Transfersomes-mediated transepidermal delivery improves the regio-specificity and biological activity of corticosteroids in vivo. Journal of Controlled Release 45, 211–226 (1997) [72] Cevc, G., Blume, G.: Hydrocortisone and dexamethasone in very deformable drug carriers have increased biological potency, prolonged effect, and reduced therapeutic dosage. Biochimica et Biophysica Acta (BBA) - Biomembranes 1663, 61–73 (2004) [73] Cevc, G., Schatzlein, A.G., Richardsen, H., Vierl, U.: Overcoming Semipermeable Barriers, Such as the Skin, with Ultradeformable Mixed Lipid Vesicles, Transfersomes, Liposomes, or Mixed Lipid Micelles. Langmuir 19, 10753–10763 (2003) [74] Bruinsma, R.: Rheology and shape transitions of vesicles under capillary flow. Physica A: Statistical and Theoretical Physics 234, 249–270 (1996) [75] Frisken, B.J., Asman, C., Patty, P.J.: Studies of Vesicle Extrusion. Langmuir 16, 928– 933 (2000), doi:10.1021/la9905113

Chapter 8

Chitosan as Stabilizer and Carrier of Natural Based Nanostructures Maria I.Z. Lionzo, Aline C. Dressler, Omar Mertins, Adriana R. Pohlmann, and Nádya P. da Silveira Instituto de Química – Universidade Federal do Rio Grande do Sul. Av. Bento Gonçalves, 9500, CEP 91501-970 C.P. 15003, Porto Alegre – Brazil [email protected], [email protected]

Abstract. The use of nanotechnology in the development of cosmetics can bring new sights to old approaches. Liposomes are classic nanostructured systems largely used because of their ability to entrap hydrophilic and hydrophobic active substances. However, new materials can be obtained by the association of such classical devices with other materials, like the polyelectrolyte chitosan. In this way, chitosan can be used to modulate the surface properties of liposomes. The large surface area makes crucial the knowledge about interfacial phenomena for systems that contain nanoparticles. On the other hand, the macroscopic properties, e.g. rheology, can be modified by the association of chitosan and nanoparticles, affecting sensory attributes. Finally, on the biomedical field it is also possible to produce new devices to be used as support to growth cells employing the same strategy. By analyzing some fundamental aspects of the nanoparticles as well as their carrier it is possible to bring new and better systems based on chitosan.

8.1 Introduction Nanostructured materials can be produced taking advantages of the intrinsic properties of some macromolecules, as the polyelectrolytes. Another way, the association of polyelectrolytes and biomolecules, as verified between natural polymers and certain types of phospholipids, allows the production of hybrid materials with synergistic properties. Polyelectrolytes are defined as macromolecules containing ionizable groups which can form complexes with counter ions or opposite charged molecules [1,2]. Chitosan is a positive charged polyelectrolyte [3] which has been used to stabilize zwitterionic phospholipids systems, as will be presented on section 8.2. Also chitosan, associated to opposite charged polymers, can take part in the formation of micro and nanoparticles, porous matrices [4] and films (section 8.3). In this way, a broad series of materials is available to be used in such association

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(polysaccharides [1,2], proteins [5] as well as synthetic polymers [6]). Another important characteristic of the polyelectrolytes is the ability to form hydrogels [4,7], extending its application on nanocosmetics as a vehicle to carry actives and as a modifier of rheological properties, as can be found in section 8.3. Hence, the aim of this chapter is to provide to chemists, pharmacists and material scientists some new aspects of chitosan applications highlighting on its employ as a versatile component to stabilize and produce technological cosmetics and biomedical materials, overlooking nanocosmetics and nanomedicines.

8.2 Chitosan as Stabilizer of Nanostructures Chitosan (CH) is a polysaccharide composed by poly β(1-4)-linked 2-amino-2deoxy-β-D-glucopyranose and 2-acetamino-2-deoxy-β-D-glucopyranose (Figure 8.1) derived from chitin [3,8,9]. The alkaline deacetylation over 50% of chitin leads to chitosan, which is soluble in acidic medium, pH < 6.0 [10]. This polysaccharide is one of the few cationic polymers from natural source and displays biodegradability, biocompatibility and mucoadhesiveness [3,10]. The properties of chitosan vary as a function of molecular weight and deacetylation degree (DD). It is possible to find molecular weights from 30 to 900 kDa and DD from 70% to 95% [11].

Fig. 8.1 Chemical structure of chitosan.

Several synthesis pathways have led to structures as N-succinilchitosan, N-carboxymethylchitosan and N-carboxybutylchitosan [12]. These new molecules have great appeal in the biomedical area due to its solubility on physiological conditions. Chitosan has been used in the development of nanoparticulated systems by the association with another components [13,14]. Focusing on its association with phospholipids, the interactions involved can be tuned by adsorption, covalent bond and hydrophobic anchoring [15]. In our findings, even on small quantities, chitosan has demonstrated to promote the stabilization of amphoteric phosholipid structures by electrostatic interaction [16,17]. Light scattering (LS) and small angle X-rays scattering (SAXS) results demonstrated that chitosan has the ability to

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aggregate the phosphatidylcholine (PC), in solution, leading to opalescent emulsions [16]. Furthermore, the presence of chitosan induces the organization of lamellar phases. In our pre-formulation studies, pseudo-diagrams (Figure 8.2) representing systems composed by an organic phase (ethyl acetate – EA), phosphatidylcholine and water (W) or aqueous chitosan solution (1 mg/mL) were constructed. By comparing the lamellar microstructure region of the pseudo-diagrams obtained from the systems containing water (Figure 8.2, left) and chitosan solution (Figure 8.2, right), an increase on this region was found when chitosan was present.

Fig. 8.2 Ternary pseudo-diagrams obtained from mixtures of PC/Water (left) and PC/Water/CH solution (right) in ethyl acetate (EA). The structures observed on each region were characterized by visual inspection, dynamic light scattering (DLS) and small angle Xrays scattering (SAXS).

The chitosan properties have been applied also to the improvement of systems such as organogels and liposomes. Organogels are mesophases resulting from the organization of amphiphils on a liquid crystalline phase [18,19]. Liposomes are vesicles formed by the concentric arrangement of one or more phospholipid bilayers surrounding an aqueous core. Their sizes usually vary from 20 nm to 20 μm and they can be obtained from organogels [20,21]. Liposomes are structures with high free energy as compared to the aggregated or precipitated state, requiring the provision of energy to their production [22]. They are stable in the form of closed vesicles for a very long time, but under lateral stress, these fluid membranes can break down, returning to the organogel state [23]. The stability problems include loss of encapsulated molecules, changes on the structure and on the size distribution and molecular degradation [24,25]. Aiming to improve the stability of

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phospholipid vesicles, several approaches have been employed such as the mixture of cationic and anionic surfactants, use of metallic ions, charged peptides, cholesterol, polyethylenglycol, amphiphilic polymers, polysaccharides, among other approaches [26,27,28,29,30]. The association of phospholipids with chitosan has been described by several groups. In our previous studies on this subject [20,31], we found that the reverse phase evaporation technique proved to be a reproducible way to lay out chitosan between the phospholipid bilayers. The steps involving the preparation of the stabilized liposomes are depicted on the Figure 8.3. On the first step (1), the phospholipids are dispersed in a volatile organic solvent, e.g. ethyl acetate or chloroform. Then, an aqueous solution of chitosan is added to the phospholipid dispersion (2). Follow, the mixture is ultrasonicated to produce reverse micelles (3). After, the organic solvent is evaporated, resulting on the precipitation of the reverse micelles and their fusion on the organogel form (4,5). The stabilized CH-loaded liposomes are formed by the organogel re-dispersion in water under shaking (6).

Fig. 8.3 Scheme of the steps to produce chitosomes since the dispersion of the phospholipid in organic solvent (1); addition of chitosan solution (2); emulsification (3); solvent evaporation (4); organogel (5) and CH containing liposomes (6).

The main parameter that can be monitored to estimate the liposome stabilization is related to the phase transition temperature, typical for each phospholipid composition. Because of the anisotropic character of the amphiphils, the transition phases on the organogel can be accompanied by polarized optical microscopy using a heating platform which includes the phase transition temperature [31]. In the case, considering the composition of the liposomes in which 75% were

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represented by distearoyl phosphatidylcholine (DSPC), a saturated lipid, the heating platform was set from 20 to 80 ºC. By analyzing the microscopy results, it was pointed out that chitosan presence led to a more stable organogel phase. The changes on the mesophases (given by the characteristics of textures e.g. Fan, Schlieren, Maltese crosses) caused by the temperature improvement were damped. Also, by LS and SAXS analysis, the impact of the chitosan presence on the particle sizes and on the bilayer repeat distance (d) of liposomes was monitored. The slope on the particle size curve against the temperature accessed by LS experiment was shifted to higher temperatures (from 57 to 64 ºC) when chitosan was present on liposomes. The Bragg peaks reflections resulted from the lamellar structure of liposomes analyzed by SAXS were monitored against the temperature. The peak persistence on the samples containing chitosan was found up to 80 ºC, while without chitosan the peak vanished around 60 ºC, reinforcing the earlier results [31]. Figure 8.4 represents the organization proposed for the multilamellar liposomes stabilized by chitosan, when prepared by means of phase evaporation method. The repeat distance evolution during heating, as well as, the membrane thickness and the bilayer separation have been extensively used as indicatives of the system stability, especially on SAXS studies [32]. The use of theoretical models to fit SAXS spectra obtained from lamellar systems provides parameters that allow the comprehension of the effect of molecules associated to the phospholipids [on going studies].

Fig. 8.4 Representation of multilamellar chitosan containing liposome (left). Details of the lamellar organization (right). Light green indicates the polar region and yellow represents the apolar chains of the phospholipid, dark green represents the chitosan interacting with the polar regions of the phospholipid inside and outside. δ is the membrane thickness; dW is the bilayer separation and d is the repeat distance.

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The different kind of nanoparticles that can be made by associating chitosan and phospholipids depends on several parameters: technique employed, phospholipid composition, concentration of the components, as can be seen on Table 8.1. Table 8.1 Particles prepared by different techniques, using distinct phospholipids (PC) and chitosan (CH) concentrations with respectives sizes and surperficial charges.

Particle type Micelles (PC*) Micelles (PC**) Micelles (PC*-CH) Micelles (PC**-CH)

Technique Emulsification Emulsification

CH conc. Diameter ζ-potencial Ref. ±SD(nm) (mV) (%) -

5±1

-

-

20±5

-

10-4

24±2

-

2.10-1

40±30

-

[16]

Nanogels (PC*)

Emulsification

-

50±30

Nanogels (PC*-CH)

Emulsification

4.10-1

350±150 -

-

206±22

-42±3

[20]

-

190±2

-50±5

***

Liposomes (PC*) Liposomes (PC**)

Reverse phase evaporation

Liposomes (PC*-CH)

2.10-4

212±20

-39±4

[20]

Liposomes (PC*-CH)

-3

290±16

-29±6

[20]

3.10-4

180±13

-16±2

***

3.10-3

214±11

+21±3

***

Liposomes (PC**-CH) Liposomes (PC**-CH)

Reverse phase evaporation

2.10

PC*: phosphatidylcholine 99% with 75% of DSPC(Solae do Brasil S/A). PC**: phosphatidylcholine 95% (Avanti Polar Lipids, Alabaster). ***Not published

The emulsification process can produce micelles and lamellar aggregates (nanogels), which are unstable systems. These kinds of structures suffer a dynamic process of phospholipid exchange between the particles which can lead to precipitation or phase separation [22]. A similar process happens with liposomes, but their closed structures are less susceptible to break. The molecular exchange, observed on liposomes, can take several days until the composition of the vesicles reaches the equilibrium [unpublished results]. During this time, modifications on the particle size and instabilities may occur. The presence of small amounts of chitosan can improve in several times the integrity of phospholipid particles, extending the shelf-life of the preparations. The polyelectrolyte influence prevents the molecular exchange, reducing the coalescence and size alteration. However, the experimentalist must have special attention for the addition of large amounts of chitosan, in order to avoid the occurrence of large size distribution of particles. Light scattering analysis furnishes the sizes of particles composed by phosphatidylcholine and chitosan (Table 8.1). In general, the polymer presence leads to bigger particles as compared to those made only with phospholipids. Besides, the particle sizes are also dependent on the composition of the phospholipid mixture as well as on the characteristics of the polar and apolar moieties. If the lipid

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mixture is rich in anionic amphiphils, e.g. phosphatidic acid, phosphatidylserine or phosphatidylglycerol, the interaction with chitosan is stronger. In relation to the character of the apolar tail, unsaturated chains provide smaller particles than saturated ones. In order to better understand the interaction between phospholipids and chitosan, thermodynamic properties of liposomes and chitosan containing liposomes were obtained by 31P NMR spectroscopy [33]. Van’t Hoff plots where constructed using the equation: ln Δσ = −ΔH o / R(1 / T ) + ΔS o / R , where Δσ corresponds to the chemical shift anisotropy, obtained from the 31P NMR spectra of vesicular systems. In this way, the values of standard enthalpy change and standard entropy change were obtained for both systems: liposomes containing or not chitosan. The resulting enthalpy and entropy values are ΔH◦ =−2.26±0.82 kcal/mol of phospholipid and ΔS◦ =−1.04±1.98 cal/K mol of phospholipid for liposomes (r = 0.90) and ΔH◦ =−2.51±0.63 kcal/mol of phospholipid and ΔS◦ =−1.67±1.99 cal/K mol of phospholipid for liposomes containing chitosan (r = 0.94). Those results showed that the differences of enthalpy and entropy are not significant between liposomes with or without chitosan. However, it may be noted that the overall process is not exactly linear and indeed the lost of linearity is more pronounced for liposomes (r = 0.90). Here we come to two points regarding the structural modification of the systems, which are the phospholipid phase transitions and the fusion processes of the vesicles. As expected, the phosphatidylcholine used in this work cross-over three phase transitions between 25 and 60 ºC: the sub-transition, the pre-transition and the main transition. As previously determined [31], the presence of chitosan slows the phase transition probably dissipating the thermal energy furnished to the system. In this way, the bilayer order/disorder that takes place during the phase transitions is tuned by a kinetic compensation which influences the molecular organization of the phospholipids. As a consequence, chitosan delays the process of phase transition. The difference in linearity of the van’t Hoff plots between liposomes containing or not chitosan corroborates with the fact that the phase transitions are influenced in the same manner by the presence of chitosan. Furthermore, the slight reduction in entropy for chitosomes (chitosan covered liposomes) also suggests that this system needs more energy to cross-over the phase transitions of the bilayer. This result reinforces the idea that chitosan stabilizes the system dissipating thermal energy and hence avoiding the completeness of the main phase transition. Another study [17], using SAXS technique, showed that chitosan presence acts increasing the amount of multilamellar structures in suspension. Employing the width at half height of the first Bragg peak from the SAXS spectra, the number of bilayer repeat distances could be obtained. It was found that the was improved by the increase of chitosan concentration. The mean values varied from 3±1 (liposome) to 12±2 (liposome containing chitosan). By different techniques, it was possible to observe and understand the ability of chitosan to organize the phospholipids. This ability can be applied to the production of stable nanostructured systems. One example using chitosan-coated liposomes to entrap α-hydroxy acids aiming skin renovation showed that the

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encapsulation of the active in the chitosan-coated liposomes sustained the release in comparison with that of the uncoated liposomes [34].

8.3 Chitosan as Carrier of Natural Based Nanostructures Gels are semi-solids formed by a three-dimensional network [35], having prominent role as actives carriers in cosmetic and pharmaceutical industries. It is well known that manipulation of gel properties is important to obtain the desired sensory and textural features for such applications [36]. The use of chitosan in cosmetics has been based on the fact that its topical use conduces to an improvement in wound healing process and because of its antimicrobial activity [37]. Besides, the slightly low pH in which chitosan is soluble is the best to the physiologically conditions of the skin, considering the acid mantle of the integument surface [38]. Chitosan forms gels on aqueous acidic media and the properties of the originated gels can be modulated by nanoparticles addition [39]. The effect caused by the nanoparticles presence depends on the type of interaction between the particle surface and the network of hydrocolloidal molecules. Considering that chitosan gels are cationic, the addition of anionic nanoparticles into the gel is supposed to influence the polymeric chains flow. To evaluate this possibility, liposomes were added into a chitosan gel. Analizing the superficial charge of liposomes by zeta potential measurements, it was found to be between -40 to -50 mV (Table 8.1). After the addition of liposomes to a chitosan gel at 5% wt, the viscosity (η) was improved (Figure 8.5). However, the addition of liposomes into a chitosan gel may lead to the instability of the lamellar structure due to the strong interaction between phospholipids and chitosan. To circumvent this backward, it was proposed the use of chitosan in order to maintain the liposome structure inside the gel and then, to cover the resultant particle with an intermediate polyelectrolyte, i.e. chondroitin sulphate, giving the negative charge necessary to the interaction with chitosan gel, aiming to modulate the viscosity. Chondroitin sulfate (CS) is a negative charged glycosaminoglycan (GAG) with carbonate and sulfate groups [40,41]. To prepare the nanoparticles, phospholipid (POPC) and the chitosan concentrations ranging from 3.10-4 to 3.10-3 %wt, were tested. After the addition of the second polyelectrolyte, the changes of zeta potential values of the composite nanoparticles were monitored. The chitosan containing liposomes have shown zeta potential values ranging from -16±2 mV to +21±3 mV, with the increase of the chitosan concentration. Chondroitin was used to neutralize the positive charges and an excess of it produced negative charges from -11±1 mV to -17±1 mV. This alteration is due to the electrostatic attraction between chondroitin and chitosan, which leads to stable complexes in aqueous media [40,42]. This approach had been well succeeded using other kinds of GAG, as the hyaluronic acid, whose interaction with chitosan on liposomes surface also led to changes on zeta potentials [43]. After the preparation of the mentioned chitosan-chondroitin covered liposomes (CS/CH-liposomes), they were added to the chitosan gels at 5% wt and the viscosity was monitored and compared to that obtained in the presence of liposomes. As

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can be seen on Figure 8.5, the increase in viscosity was higher to the CS/CHliposomes then that caused by the addition of liposomes. The ability of chondroitin to cross-link chitosan explains such effect. However, the simple addition of a chondroitin solution to the chitosan gel showed no difference from the addition of water. This finding exemplifies the benefits of using nanotechnological approaches to traditional methods. The fact that chondroitin is covering the nanoparticles offers a pronounced surface area to the interaction between the polyelectrolytes. Besides, the nanometric size of the particles leads to homogeneous systems.

Fig. 8.5 Viscosity (η) of chitosan gels (2.0%wt) as a function of shear rate (γ) after addition of 5% of chitosan-chondroitin liposomes (T); 5% of liposomes (○); 5% of chondroitin solution (U) and 5% of water (), used as a reference.

Gels containing more than 2% wt of chitosan are expected to have high dynamic viscosities and a cohesive structure which is not desired for some cosmetics [36]. In this way, by the association of nanoparticles it is possible to reduce the chitosan concentration to c.a. 1% wt; however this concentration leads to very fluid gels (η < 1.0 Pa.s). In order to handle the viscosity to intermediary values (5 to 10 Pa.s), the formulator can make use of the addition of different kinds of nanoparticles. Moreover, the nanoparticles addition also improves the sensorial characteristics of the formulations in which they are present [44].

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To produce gels with superior sensorial characteristics, 1% of different nanoelements were added to chitosan gels (1.5%wt) [44]. The nanoelements essayed were i) liposomes made from phosphatidylcholine, ii) wheat nanostarch granules obtained by alkaline leaching and iii) organic liquid crystal molecules [45]. After the incorporation of such particles on the gels, the viscosimetric and spreading properties were evaluated. One commercial formulation made with carbomer was also prepared and essayed as a comparing sample. Figure 8.6 shows the viscosity values from each of the tested gels. The gel containing liposomes, presented viscosity slightly higher than the pure gel, as could be seen previously. Notwithstanding, for those gels containing nanostarch and liquid crystals the mean values of viscosity were lower than the pure gel.

Fig. 8.6 Influence of the addition of water 1%, liposomes 1%, Nanostarch 1% and Liquid crystals 1% on the viscosity (η) of chitosan-glycerol gels (1.5%wt). The viscosity of a commercial formulation of Carbomer gel is 1.5%.

The spreading properties were evaluated by determining the maximum area of spreading [46] of each gel. The results are presented on Figure 8.7. The gels containing liposomes showed the higher spreadability which can be due to the rupture of the vesicles and the formation of lamellar regions that facilitate the sliding of the polymeric chains one each other. To the gels containing liquid crystals, the initial strength applied lead to significant spreading which was gradually

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diminishing with the subsequent increase of the weight. After the addition of 300 g of weight, the profiles of pure chitosan gel and nanostarch and liquid crystal containing gels were similar. Despite the common amphiphilic strucuture of liquid crystals and phospholipids, it was observed a different effect on the viscosity of gels that contained them. That can be due to the organization of the phospholipids on liposomes, while the liquid crystals are molecularly spread on the gel.

Fig. 8.7 Spreading properties of chitosan gels containing just water () and different nanostructures: liposomes (○), nanostarch (Y) and liquid crystals ( ).

⃟

Another application of chitosan is on the production of films to be used on the biomedical field as wound dressings, drug delivery systems and scaffolds for tissue engineering [47,48,49]. Chitosan has been employed as a biomaterial because of the structural similarity to the molecules which forms the extracellular matrix and because of the formation of stable complexes with those molecules [47]. The development of scaffolds can be enriched by the incorporation of nanoparticles on the polymeric matrix. This procedure allows the modulation of mechanical, superficial and functional properties that can improve the cellular viability [50]. Chitosan films may be produced by casting the hydrocolloidal gels. The chitosan films are brittle and their surface is smooth (Figure 8.8A). By adding nanoparticles (CS/CH-liposomes), the resultant film gains a flexible appearance and is possible to observe a rough surface, caused by the presence of the nanoparticles, as can be seen on Figure 8.8B. Among the properties that can be measured to

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characterize films, the contact angle permits to determine the hydrophilicity/hydrophobicity of the surface. To those films with pronounced roughness, the contact angle results seem to be inconsistent. In this way, is necessary to face and compare the findings by differential scanning calorimetry, water sorption and swelling essays. The global analyses showed that particles composed by phosphatidylcholine, chitosan and chondroitin may act as a barrier to the water diffusion into the matrix. It is well known that neither a very hydrophilic nor a very hydrophobic surface favors the cellular attachment [50,51].

Fig. 8.8 Surface of chitosan film (A) and chitosan film containing nanoparticles (B). The particles can be seen on the film surface, modifying their properties.

A set of films with different concentration of nanoparticles was submitted to the cellular adhesion test using mesenchimall stem cells. The results indicated that the chitosan film presented good adhesion. Nevertheless, there was an optimal amount of particles that led to the best film properties aiming cellular adhesion. It seems that an intermediate roughness can facilitate cell adhesion [52].

8.4 Concluding Remarks In this review we have presented some new aspects of chitosan application using systems developed in our research group. Since chitosan is a well-known polysaccharide and it has been applied in different approaches nowadays, challenges in this area are welcome. Our main results are pointing out the importance of chitosan when applied as stabilizer for nanoparticles. The properties of chitosan can be extensively employed in the development of nanoparticles carrier systems, seeking either cosmetic or biomedical applications, in spite of all chitosan systems already known.

References [1] Boddohi, S., Moore, N., Johnson, P.A., Kipper, M.J.: Polysaccharide-Based Polyelectrolyte Complex Nanoparticles from Chitosan, Heparin, and Hyaluronan. Biomacromolecules 10, 1402–1409 (2009)

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[2] Denuziere, A., Ferrier, D., Domard, A.: Chitosan-chondroitin sulfate and chitosanhyaluronate polyelectrolyte complexes Physico-chemical aspects. Carbohydr. Polym. 29, 317–323 (1996) [3] Rinaudo, M.: Chitin and Chitosan: Properties and applications. Prog. Polym. Sci. 31, 603–632 (2006) [4] Berger, J., Reist, M., Mayer, J.M., Felt, O., Gurny, R.: Structure and interactions in chitosan hydrogels formed by complexation or aggregation for biomedical applications. Eur. J. Pharm. Biopharm. 57, 35–52 (2004) [5] Park, W.H.: Insoluble polyelectrolyte complex formed from chitosan and α-keratose: conformational change of α-keratose. Macromol. Chem. Physic 197, 2175–2183 (1996) [6] Lee, J.Y., Nam, S.H., Im, S.Y., Park, Y.J., Lee, Y.M., Seol, Y.J., Chung, C.P., Lee, S.J.: Enhanced bone formation by controlled growth factor delivery from chitosanbased biomaterials. J. Control Release 78, 187–197 (2002) [7] Boucard, N., Viton, C., Agay, D., Mari, E., Roger, T., Chancerelle, Y., Domard, A.: The use of physical hydrogels of chitosan for skin regeneration following thirddegree burns. Biomaterials 28, 3478–3488 (2007) [8] Muzzarelli, R.A.A.: Chitin and its derivatives: New trends of applied research. Carbohydr. Polym. 3(1), 53–75 (1983) [9] Domard, A., Rinaudo, M.: Preparation and Characterization of fully deacetylated chitosan. Int. J. Biol. Macromol. 5, 49–52 [10] Ravi Kumar, N.M.V.: A review of the chitin and chitosan applications. React. Funct. Polym. 46, 1–27 (2000) [11] Canella, K.M.N.C., Garcia, R.B.: Caracterização de quitosana por cromatografia de permeação em gel – influência do método de preparação e do solvente. Quim. Nova 24(1), 13–17 (2001) [12] Sashiwa, H., Shigemasa, Y.: Chemical modification of chitin and chitosan 2: preparation and water soluble property of N-acylated or N-alkylated partially deacetylated chitins. Carbohydr. Polym. 39, 127–138 (1999) [13] Li, W., Yuan, R., Chai, X., Lu, Z., Chen, S., Li, N.J.: Immobilization of horseradish peroxidase on chitosan/silica sol–gel hybrid membranes for the preparation of hydrogen peroxide biosensor. J. Biochem. Biophys. Methods 70(6), 830–837 (2008) [14] Baek, S.-H., Kim, B., Suh, K.-D.: Chitosan particle/multiwall carbon nanotube composites by electrostatic interactions. Colloids Surf A Physicochem. Eng. Asp. 316(13), 292–296 (2008) [15] Sihorkar, V., Vyas, S.P.: Potential of polysaccharide anchored liposomes in drugs delivery. J. Pharm. Pharmaceut. Sci. 4(2), 138–158 (2001) [16] Lionzo, M.I.Z., Mertins, O., Pohlmann, A.R., da Silveira, N.P.: Phospholipid/chitosan self-assemblies analyzed by SAXS and Light Scattering. In: AIP. Conf., vol. 1092, pp. 127–129 (2009), doi:10.1063/1.3086206. [17] Mertins, O., Cardoso, M.B., Pohlmann, A.R., da Silveira, N.P.: Strucutural evaluation of phospholipidic nanovesicles Containing small amounts of chitosan. J. Nanosci. Nanotechnol. 6(8), 2425–2431 (2006) [18] Aliotta, F., Fontanella, M.E., Pieruccini, M., Vasi, C.: Aggregation phenomena in a lecithin-based gel: Transient networks and diffusional dynamics. Phys. Rev. E 59(1), 665–672 (1999) [19] Kumar, R., Katare, O.P.: Lecithin Organogels as a potential Phospholipid-Structured System for Topical Drug Delivery: A Review. AAPS Pharm. Sci. Tech. 6(2), E298– E310 (2005)

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[20] Mertins, O., Sebben, M., Pohlmann, A.R., da Silveira, N.P.: Production of soybean phosphatidylcholine–chitosan nanovesicles by reverse phase evaporation: a step by step study. Chem. Phys. Lip. 138, 29–37 (2005) [21] Mertins, O., da Silveira, N.P., Pohlmann, A.R., Schroder, A.P., Marques, C.M.: Electroformation of giant vesicles from an inverse phase precursor. Biophys. J. 96, 2719– 2726 (2009) [22] Lasic, D.D.: The mechanism of liposome formation. A review. Biochem J. 256, 1–11 (1988) [23] Evans, E., Heinrich, V.: Dynamic strength of fluid membranes Force dynamique des membranes fluides. C. R. Physique. 4, 265–274 (2003) [24] Zana, R.: Dynamics of surfactant self-assemblies: micelles, microemulsions, vesicles and lyotropic phases. Taylor & Francys Group, Boca Raton (2005) [25] Casals, E., Galán, A.M., Escolar, G., Gallardo, M., Estelrich, J.: Physical stability of liposomes bearing hemostatic activity. Chem. Phys. Lip. 125, 139–146 (2003) [26] Lindén, M.V., Wiedmer, S.K., Hakala, S.R.M., Riekkola, M.L.: Stabilization of phosphatidylcholine coatings in capillary electrophoresis by increase in membrane rigidity. J. Chromatogr. A 1051, 61–68 (2004) [27] Volodkin, D., Ball, V., Schaaf, P., Voegel, J.-C., Mohwald, H.: Complexation of phosphocholine liposomes with polylysine. Stabilization by surface coverage versus aggregation. Biochim. Biophys. Acta 1768, 280–290 (2007) [28] Garbuzenko, O., Barenholz, Y., Priev, A.: Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer. Chem. Phys. Lip. 135, 117–129 (2005) [29] Dabholkar, R., Sawant, R.M., Mongayt, D.A., Devarajan, P.V., Torchilin, V.P.: Polyethylene glycol–phosphatidylethanolamine conjugate(PEG–PE)-based mixed micelles: Some properties, loading with paclitaxel, and modulation of P-glycoproteinmediated efflux. Int. J. Pharm. 315, 148–157 (2006) [30] Rizos, A., Tsikalas, I., Tsatsakis, A.M., Shtilman, M.I.: Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer. J. Non-Cryst. Sol. 352(42-49), 4451–4458 (2006) [31] Mertins, O., Lionzo, M.I.Z., Micheletto, Y.M.S., Pohlmann, A.R., da Silveira, N.P.: Chitosan effect on the mesophase behavior of phosphatidylcholine supramolecular systems. Mat. Sci. Eng. C 29, 463–469 (2009) [32] Pabst, G.: Global Properties of Biomimetic Membranes: Perspectives on Molecular Features. Biophis. Rev. Lett. 1, 57–84 (2006) [33] Mertins, O., Schneider, P.H., Pohlmann, A.R., da Silveira, N.P.: Interaction between phospholipids bilayer and chitosan in liposomes investigated by 31P NMR spectroscopy. Colloids Surf. B Biointefaces 75, 249–299 (2010) [34] Phetdee, M., Polnok, A., Viyoch, J.: Development of chitosan-coated liposomes for sustained delivery of tamarind fruit pulp’s extract to the skin. Int. J. Cosmet. Sci. 30(4), 285–295 (2008) [35] Sinko, P.J.: Martin’s:Physical Pharmacy and Pharmaceutical Sciences, 5th edn. Lippincott Williams & Willkins, Philadelphia (2008) [36] Wortel, V.A.L., Verboom, C., Wiechers, J.W., Taelman, M.-C., Leonard, S., Tadros, T.: Linking sensory and rheology characteristics. Cosmet. Toiletries 120(4), 57–66 (2005) [37] Boussens, J.-L.L., Vevey, H.L., Aarau, P.S., Brent, J.-L.V.: Cosmetic preparation containing chitosan. US Patent: 5057542 (1991); Chen, Y.-L., Lee, H., Chan, H.-Y., Sung, L.-Y., Chen, H.-C., Hu, Y.-C.: Biomaterials 28, 2294–2305 (2007)

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[38] Schmid-Wendtner, M.-H., Korting, H.C.: pH and Skin Care. ABW Wissenschaftsverlag GmbH, Berlin (2007) [39] Lee, J.-H., Gustin, J.P., Chen, T., Payne, G.F., Raghavan, S.R.: Vesicle-biopolymer gels: Networks of surfactant vesicles connected by associating biopolymers. Langmuir 21, 26–33 (2005) [40] Denuziere, A., Ferrier, D., Domard, A.: Chitosan-Chondroitin Sulfate and ChitosanHyaluronate Polyelectrolyte Complexes. Physico-chemical aspects Carbohydr. Polym. 29, 317–323 (1996) [41] Chen, Y.-L., Lee, H.P., Chan, H.Y., Sung, L.-Y., Chen, H.-C., Hu, Y.-C.: Composite Chondroitin-6-Sulfate/Dermatan Sulfate/Chitosan Scaffolds for Cartilage Tissue Engineering. Biomaterials 28, 2294–2305 (2007) [42] Piai, J.F., Rubira, A.F., Muniz, E.C.: Self-Assembly Of A Swollen Chitosan/Chondroitin Sulfate Hydrogel By Outward Diffusion of the Chondroitin Sulfate Chains. Acta Biomat. 5, 2601–2609 (2009) [43] Rinaudo, M., Quemeneur, F., Pépin-Donat, B.: Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions. In: Macromol. Symp., vol. 278, pp. 67–79 (2009) [44] Dessler, A.C.: Propriedades reológicas de compósitos sol-gel de quitosana/glicerol/nanoelementos. M.S. Dissertation, Universidade Federal do Rio Grande do Sul (2008) [45] Ritter, O.M.S., da Silveira, N.P., Merlo, A.A.: Synthesis and mesomorphic properties of side chain liquid-crystalline biphenyl-phenyl polyacrylates. J. Braz. Chem. Soc. 17(2), 348–356 (2006) [46] Knorst, M.T.: Desenvolvimento tecnológico e avaliação biológica de formas farmacêuticas plásticas contendo nanocápsulas de diclofenaco. Ph.D. Dissertation, Universidade Federal do Rio Grande do Sul (1991) [47] Kirker, K.R., Luo, Y., Harte Nielson, J.H., Shelby, J., Prestwich, G.D.: Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing. Biomaterials 23, 3661–3671 (2002) [48] Thein-Han, W.W., Saikhun, J., Pholpramroo, C., Misra, R.D.K., Kitiyanant, Y.: Chitosan gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells. Acta Biomat. 5, 3453–3466 (2009) [49] Nair, L.S., Laurencini, C.T.: Biodegradable polymers as biomaterials. Prog. Polym. Sci. 32, 762–798 (2007) [50] Ma, Z., Mao, Z., Gao, C.: Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids. Surf. B Biointerfaces 60, 137–157 (2007) [51] Rodrigues, L.B., Leite, H.F., Yoshida, M.I., Saliba, J.B., Cunha Junior, A.S., Faraco, A.A.G.: In vitro release and characterization of chitosan films as dexamethasone carrier. Int. J. Pharm. 368, 1–6 (2009) [52] Lionzo, M.I.Z.: The influence of polyelectrolytes on phospholipidic structures and its application. Ph.D. Dissertation. Universidade Federal do Rio Grande do Sul (manuscript in preparation)

Part III

Applications of Nanocosmetics and Nanomedicines for Skin Treatments

Chapter 9

Performance of Elastic Liposomes for Topical Treatment of Cutaneous Leishmaniasis Bartira Rossi-Bergmann1, Camila A.B. Falcão1, Beatriz Zanchetta2, Maria Vitória L. Badra Bentley3, and Maria Helena Andrade Santana2 1

Institute of Biophysics, Federal University of Rio de Janeiro –UFRJ, 21.941-902 Rio de Janeiro, RJ, Brazil 2 Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas-UNICAMP, 13083-852, Campinas, SP, Brazil 3 Faculty of Pharmacy, University of São Paulo-USP, Ribeirão Preto, SP, Brazil [email protected]

Abstract . Non-invasive and non-toxic drugs are highly needed for the treatment of several skin diseases. Among those, local topical treatment is more desirable over oral treatment that may produce systemic side-effects. Thickening of the skin is a common feature in chronic skin diseases such as cutaneous leishmaniasis (CL). Nowadays, conventional permeation enhancers that may facilitate the drug passage through the tight stratum corneum skin barrier have been considered obsolete. Thus, appropriate delivery systems capable to carry the drug across the thickened skin are highly needed. This chapter presents an overview on the current therapy of CL and a new approach for topical treatment using conventional and pegylated liposomes as skin carrier systems for an active and highly hydrophobic antileishmanial chalcone.

9.1 Introduction 9.1.1 Cutaneous Leishmaniasis Leishmaniasis is a vector-borne tropical disease that is manifested in a range of clinical forms ranging from the more benign localized cutaneous leishmaniasis (CL) to fatal visceral leishmaniasis. CL is the most common form of the disease, with a global incidence of 1-1.5 million cases in 88 endemic countries [1].The infected sandfly inoculates the parasites into the skin during blood feeding, and these are phagocytosed by local macrophages. Inside the parasitophorus vacuole, the flagellate promastigotes transform into amastigotes that divide by binary fission. After 1-2 weeks the macrophages burst release the new parasites that will

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infect resident or newly arrived macrophages in the dermis. In the majority of cases of CL there is a single self-limiting lesion, but more rarely parasites may infect distant macrophages to produce diffuse CL, mucosal leishmaniasis and even visceral leishmaniasis [2,3]. Parasite species and host immune status are the main factors that will determine the secondary manifestations that develop from the initial skin lesion. CL is characterized by development of an initial skin nodule which eventually ulcerates and grows for as long as a few years. Inflammation with the local production of macrophage attractant chemokines will favor lesion development. The epidermis thickens because of hyperplasia, and the collagen matrix in the dermis is disrupted resulting in necrosis. Epidermal disruption results in discharge to form an ulcer with a central depression and raised border (Figure 9.1 A). In Old World, and less frequently in New World infections, the ulcer may dry and be encrusted. Scanty parasites are found in the necrotic ulcer, whereas abundant parasites are seen in the ulcer border and surrounding inflamed skin (Figure 9.1 B and C). Response to infection is a multifactorial process. Roughly, healing is associated with

Fig 9.1 Cutaneous leishmaniasis lesion development. A) Macroscopic aspect of ulcerated lesion. B) Schematic representation of the location of infected macrophages in the dermis under the still intact skin of an early lesion (left panel). In more advanced stages, viable infected macrophages are found under the ulcer border where the skin is still intact but grossly thickened. Lysed macrophages and scanty parasites are found in the necrotic area of the ulcerated area (right panel). C) Histological appearance of non-infected mouse skin (left) and infected skin showing an abundance of vacuolated infected macrophages in the dermis (arrows). (Photographs kindly provided by Drs Sylvio Celso G. Costa and Celeste S.F. Souza from FIOCRUZ, Brazil).

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the preferential development of Th1-type T cells that produce IFN-γ and TNF-α inflammatory cytokines that activate macrophages to produce nitrogen and oxygen intermediates to kill the intracellular parasites, followed by IL-10-producing regulatory T cells that suppress the effector T-cell-mediated inflammatory response. Finally, recruitment of fibroblasts that deposit fibrin and collagen compose the formation of a healing granuloma [4].

9.1.2 Chemotherapy Treatment of localized CL aims not only to accelerate the healing to avoid disfiguring scars, but also to prevent metastases that lead to the morbid mucosal and diffuse forms caused mainly by L. braziliensis and L. amazonensis. The standard therapy involves daily injections for 20-30 days with of the pentavalent antimonials Pentostam (sodium stibogluconate) or Glucantime (meglumine antimonate). However, a high rate of adverse events, length of treatment, and relapses in up to 25% of cases highlight the limitations of these drugs. Perhaps the biggest challenge now with standard therapy is the increasing resistance to antimonials not only in VL where it has been well documented [5], but also in CL. Although a cure rate of 90 – 100% in CL can be achieved with pentavalent antimonials, a Brazilian study found that cure rates were only 51% and 26% for patients with L. braziliensis and L. guyanensis, respectively [6]. Moreover, patients with heart deficiencies should avoid antimonials due to their cardiotoxic effects. The use of second line drugs, amphotericin B and pentamidine isethionate, and the parenteral formulation of the aminoglycoside paromomycin may be also very toxic [6]. Intravenous liposomal amphotericin B (Ambisome®) although very effective in VL, is not recommended for localized CL as it is much less effective than the free drug [7], and has its use restricted to antimony-unresponsive mucosal leishmaniasis [8]. A study in L. major-infected mice showed that liposomal amphotericin B administered subcutaneously close to the lesion had no significant activity [9]. This is possibly explained by the rapid absorption of the small (60 nm) anionic liposomes to the circulation or to the lower internalization of the drug into the macrophages when in the liposomal form [10]. Attempts to investigate possible new therapies have focused on modestly active oral agents, e.g., antifungal azoles and allopurinol, and local therapy, including cryotherapy, thermotherapy, photodynamic therapy [11], surgery and electrotherapy [12]. Local infiltration (intralesional) with antileishmanial drugs, such as pentavalent antimony [13] and amphotericin B [14] can also enhance healing of the ulcer. A clinical trial against CL in South America with oral miltefosine showed promising results but relatively high toxic doses are needed to be effective. The main limitations of miltefosine are potential for selection of drug resistant parasites and teratogenicity [15]. Thus, the injected and/or systemically administered drugs outlined above are not satisfactory for the treatment of CL, and current topical approaches are described below.

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9.1.3 Topical Treatment of CL Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized CL. The first reports on the topical treatment of CL began almost 30 years ago with the studies of El-On et al on the paromomycin antibiotic showing that a 15% paromomycin sulfate plus 12% methyl benzethonium chloride (MBCl) ointment effected cure of L. major lesions in BALB/c mice [16]. Interestingly, other systemic antileishmanial drugs such as sodium stibogluconate, pentamidine, amphotericin B, and allopurinol have being no significant activity in this CL model. In the same series of experiments, paromomycin in combination with other quaternary ammonium disinfectants or DMSO (a known penetration enhancer), gave significant levels of activity. Two other aminoglycosides, kanamycin and gentamycin in the MBCl formulation also had some activity, but three others, neomycin, amikacin and tobramycin, were not active. The efficacy of topical paromomycin in humans is controversial. Although significant cure rates have been reported in Iran, Guatemala and Ecuador, a placebocontrolled trial in Colombia reported lack of efficacy [17]. This ointment with 12% MBCl is commercially available and marketed in Israel as Leishcutan. However, early studies also reported severe skin irritancy (burning, pruritis and vesicle formation), most probably due to the high concentration of MBCl (12%), a cationic surfactant that is used as a disinfectant at 0.1 to 0.2%. A less toxic formulation of 15% aminosidine with 10% urea in white soft paraffin used twice daily for 14 days is not effective in accelerating cure [18]. A new formulation of 15% paromomycin and 0.5% gentamicin containing different surfactants (WR-279396) also proved less toxic than Leishcutan [19]. Although topical application of amphotericin B in sodium deoxycholate (Fungizone) proved ineffective, topical lipid formulations of amphotericin B such as in colloidal dispersion of cholesterol sulfate (Amphocil®) and in phospholipid (Abelcet®) dispersed in an aqueous solution with ethanol promoted lesion regression in mice infected with L major following 21 days administration [20]. The oral antileishmanial miltefosine did not demonstrate efficacy against CL when used by topical route in conventional cream [21]. In Peru, antimonyresistant CL patients topically treated with a cream containing 5% of the immunostimulant imiquimod greatly improved their response to parenteral Glucantime given as an association therapy for 20 days [22]. Apparently imiquimod cream does not improve Glucantime response in immunocompetent patients, and when applied to CL patients in the Old World produced only a transient cure [23]. Several studies have focused on drugs interfering with sterol biosynthesis. One double-blind trial in Saudi Arabia investigated the clinical efficacy of topical 1% clotrimazole (Canesten®) and 2% miconazole (Daktarin®) with relative improvement of the lesion [24]. More promising experimental studies have been reported with the plant product licochalcone A, which had activity against L. donovani and L. major in experimental models. Two synthetic derivatives of licochalcone A have been further tested. The topical application of an ointment containing two licochalcone synthetic derivatives (50 mg/ml, twice daily for 10 days) in white petroleum caused a

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suppressive effect against lesion growth of L major CL in BALB/c mice, but did not result in a cure [25]. Other chalcones have appeared with potent antileishmanial activity [26], including the nitrated chalcone CH8 used as antileishmanial drug prototype described in this chapter.

9.1.4 Liposomes for Topical Treatment of CL Some lipophylic substances may permeate the normal skin in the presence of permeation enhancers. However, in CL appropriate delivery systems have to be used to carry the drug across both the stratum corneum barrier and the thickened epidermis. Even in more advanced stages of CL where ulcerated lesions have lost the stratum corneum barrier, topical formulations have to permeate both the thickened skin covering the parasite-rich borders and the surrounding areas to reach the intracellular parasites down in the dermis. A range of studies have been reported the use of liposomes to increase antileishmanial drug delivery into and across intact skin. Permeation of amphotericin B loaded in different multilamellar liposomes across isolated rat skin showed that positively charged liposomes promoted a high flux of the drug across the stratum corneum, whereas negatively charged liposomes promoted maximal flux to viable epidermis [27]. New [28] first reported that liposomal formulations of antimonials were active against CL when administered by the i.v. route rather than s.c. into the lesion. This observation was also reported for the liposomal amphotericin B Ambisome [29]. An explanation for that is the reduced internalization of the liposomal formulation by macrophages in relation to the free drug. As described above, one of the most successful formulations for topical treatment of CL is paromomycin in MBCl. In a recent study, paromomycin was loaded at 5% in large unilamellar liposomes composed either by phosphatidylcholine or phosphatidylcholine: cholesterol. Both formulations promoted negligible permeation through intact pig ear skin, perhaps due to their large size (516 nm and 362 nm, respectively). On the other hand, when the stratum corneum was removed from the ear skin by extensive tape stripping to simulate the ulcerated skin as in advanced CL, penetration of drug loaded in liposomes was higher than that of the drug in aqueous solution. That finding was paralled by the in vivo efficacy in L. major-infected mouse where liposomes were applied to the ulcers in hydroxyethylcellulose gel [30]. However, the relevance of that study has to be confirmed, as in ulcerated lesions parasites are more abundant in the dermis of the ulcer border where the epidermis not only is intact but is also thickened. Thus, it seems that delivery systems more effective than conventional liposomes are needed to carry hydrophilic drugs such as paromomycin across the infected skin. In summary, the prepared liposomes have not proved yet an efficient system for optimal topical delivery of the classical antileishmanials such as amphotericin, pentavalent antimonials and paromomycin, which are medium to highly hydrophilic drugs. In this chapter we present the use of flexible or elastic liposomes

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with high deformation capability to increase the topical efficacy of a highly lipophilic antileishmanial chalcone. The elastic properties were provided by two strategies: disturbing the bilayer structure, due to incorporation of the hydrophobic chalcone in 100nm conventional liposomes composed of low phase transition temperature lipid (egg- lecithin), and in addition of that, modifying the surface of liposomes with polyoxiethylene lauryl esther (PEG-8-L).

9.2 Preparation of Elastic Liposomes Loaded with Chalcone CH8 As pointed above, chalcones other than the pioneer licochalcone A have been described with antileishmanial activity. These include the hydrophobic 3-nitro-2’hydroxy-4’,6’-dimethoxychalcone (CH8) that is structurally simpler and more active than licochalcone A [31, Figure 9.2]. The CH8 chalcone has shown much superior antileishmanial activity than the sodium stibogluconate antimonial drug when tested by subcutaneous route in CL caused by L. amazonenesis [32]. However, the CH8 chalcone displayed only a moderate antileishmanial effect when used by the topical route in Lanette cream, despite its lipophylic property and relatively small molecular weight (MW =329). Due to their capability to penetrate the deeper layers across water nano-channels of 30-40 nm in the stratum corneum of non-occluded skin, elastic liposomes such as those covered with polyethylene glycol can squeeze through the pores to reach the dermis (Figure 9.3). The technologies used for production and characterization methods have been described in detail in Chapter 8 of this book. Besides the antileishmanial activity, the hydrophobic nature of the CH8 chalcone also represents a strategy to confer elasticity to egg-lecithin conventional liposomes for topical treatment of CL.

Fig 9.2 Cutaneous leishmaniasis lesion development with amastigote-infected macrophages in the dermis of skin are the target for drug delivery.

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Fig 9.3 Penetration of pegylated elastic liposomes trought the stratum corneum of the skin. Conventional phospholipid or phospholipid / cholesterol liposomes measuring over 40 nm do not permeate through the 40 nm-pores of the stratum corneum and remain on the skin surface. On the other hand, when complexed with polyethylene glycol (PEG) or other appropriate membrane destabilizers, liposomes become more deformable and capable to squeeze through the tight pores of the stratum corneum following the higher water gradient of deeper skin.

9.2.1 CH8- Loaded Conventional Liposomes (CH8-CvL) Lipid vesicles containing egg lecithin were prepared by the conventional Bangham’s method (Chapter 8). Briefly, the egg lecithin was dissolved in methanol/chloroform (1:9 v/v), the organic solvent was evaporated under vacuum in a rotary evaporator, producing a dried lipid film at the bottom of the balloon. The solvent traces were removed by maintaining the lipid film under vacuum during 2 hours. The film was hydrated with HEPES buffer solution (10 Mm), pH 7.4 at 25ºC and 120 rpm, during 1 hour. In a subsequent step, the hydrated vesicles were

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extruded using two stacked polycarbonate membranes (nominal pore size 100 nm) with a high-pressure extruder (Lipex Biomembranes, Vancouver, BC), under 10 Kgf/cm2 nitrogen pressure and 20 consecutive cycles at 25ºC temperature. The CH8 was added to the extruded liposomes by slow dropping of a CH8 ethanolic solution on the preformed dispersion under mild stirring. The incorporation of CH8 in the liposomes was characterized by spectrophotometry, through the absence of the CH8 absorption peak at 337 nm.

9.2.2 CH8- Loaded Pegylated Liposomes (CH8-PL) The incorporation of polyethylene glycol-8 lauryl esther (PEG-8-L) onto preformed CH8- loaded conventional liposomes (Figure 9.4) was carried out by incubation at room temperature, of a 1 mM vesicle dispersion, in a molar proportion 60:40 egg lecithin: PEG-8-L. The incorporation of PEG-8-L was monitored by mean diameter, size distribution and surface tension measurements.

Fig 9.4 Production of elastic liposomes. Anionic conventional liposomes (CvL) are produced by the Bangham’s method, and rehydrated in the presence of CH8 chalcone (CH8-CvL). CH8-CvLs are then pegylated with polyethylene glycol 8-lauryl ether (PEG-8-L) for enhanced elasticity (CH8-PL).

9.3 CH8-Loaded Liposome Characterization 9.3.1 Size Distribution Dynamic light scattering measurements (DLS) were done to determine the mean diameter and size distribution of both CH8-CvL and CH8-PL using a Malvern Autosizer 4700 using a Ne-He laser and measurements at a scattering angle of 90º. The mean diameter and distribution of particle sizes were estimated by CONTIN

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algorithm analysis. For more accurate size characterization, intensity and numberweighted mean diameters and distributions from DLS technique were considered. Although the whole range of diameters is shown in the intensity-weighted distribution (I-distribution), the proportionality to the sixth power of diameter of particle (D) (I α D6) underestimates the small particles, which are only very weakly weighted [33]. The corresponding number-weighted distribution (Ndistribution), converted using Mie theory, is proportional to the first power of diameter (N α D) and determines the real number of particles that yield the observed intensity in each size class [34]. Table 9.1 shows the mean diameter of the liposomal formulations. Table 9.1 Liposome size distribution.

FORMULATION

Mean diameter (nm)

CH8- CvL

76.3 ± 3.8

CvL

60.5 ± 3.8

CH8-PL PL

51.0 ± 4.8 (99,8%) 170.3 ± 19.5 (0,2%) 46.0 ± 8.4 (99,1%) 142.7 ± 37.3 (0,9%)

9.3.2 Morphology Among the mechanical colloidal properties, elasticity determines functional properties of the vesicles, such as deformation, stability-instability equilibrium and controlled release of the encapsulated molecules. Transmission electron microscopy and negative staining method were used to visualize the morphology of the lipid structures of both CH8-CvL and CH8-PL. Carbon-coated 200 mesh copper grids with collodion (parloidin with cellulose acetate) film were used. Each liposome preparation was diluted to 1 mM total lipids and then applied to the carbon grid. After incubation for 5 minutes at room temperature, the excess was blotted. One drop of uranyl acetate (1% w/w in saline solution) was added to the carbon grid and incubated 1 minute at room temperature before the excess was blotted and air-dried. A Carl Zeiss CEM 902 microscope, equipped with a CastaingHenry-Ottensmeyer energy filter was used. The images obtained with a CCD camera (Proscan) showed that the incorporation of CH8 in CvL or PL did not modified the morphology of the liposomes.(Figure 9.5).

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Fig 9.5 Transmission electron micrographs of empty and CH8-loaded conventional ( CvL) and pegylated (PL) liposomes. Size bar: 200nm (left) 100nm (right)

9.3.3 Phospholipids Concentration The phospholipid content in CH8-CvL and CH8-PL was determined by quantification of the total phosphate in the samples, according to methodology developed by [35]. The technique relies on the digestion of phospholipid to inorganic phosphate by sulfuric acid and hydrogen peroxide. The phosphate reacts with the molybdate and the ascorbic acid to form a blue phosphomolybdate complex that can be detected spectrophotometrically at 890 nm. This technique was also used before and after liposome elasticity assay in polycarbonate membranes.

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9.3.4 Elasticity The elasticity of CH8-CvL and CH8-PL was evaluated by their capability to return to their original sizes after forced passage through two stacked 30 nm polycarbonate membranes simulating the pore sizes of the human skin. Single passages were performed at variable pressures: 2.5 (simulating the transepidermal gradient), 5, 12 and 20 atm. The measurements were performed before and after permeation through the membranes. The samples were diluted to 1 Mm concentration, in order to allow the complete permeation along 0.35-1.5 h. The results showed that CH8-CvL and CH8-PL were both deformable and equally elastic.

9.4 Biological Assessments of the CH8-Loaded Liposomes 9.4.1 In Vitro Skin Permeation The capacity of both CH8-CvL and CH8-PL to permeate the isolated pig ear skin after topical application was assessed in vitro using Franz cell [36]. The liposomes were applied onto the intact skin at a concentration of 330 ug/ml in a final volume of 500 ul of HEPES buffer at 37 ºC. Free CH8 incorporated into Lanette cream at a concentration of 6% (w/w) was used as control. After different times ranging from 2 to 12 hours the CH8 content in the receptor solution was quantitated by High Performance Liquid Chromatography (HPLC) to indicate transdermal permeation. The amount of chalcone retained in the stratum corneum (SC) and in the epidermis and dermis (EP + D) was evaluated at 4 and 12 hours after topical application by tape stripping, as described previously [36]. Briefly, the pig ear skin was removed from the Franz cell and the excess of applied drug was cleaned from the upper stratum corneum with running distilled water and pat drying with absorbent paper. The SC was detached from the skin by 13 consecutive tape stripping, and the remaining EP + D was minced in small pieces. The drug retained in the tapes (SC layers) and in the EP + D was extracted with acetonotrile and ultrasound prior to quantitation by HPLC. The results showed that despite the CH8 lipophylicity, no drug was detected in any layer of the skin when formulated in Lanette cream. On the other hand, of the total amount of CH8-CvL and CH8-PL applied, 9.8% and 8.3% detectably entered the skin after 12 hours, respectively. Of these, 3.4% and 2.5% was found in the SC, whereas 6.4% and 5.8% reached the EP+ D, respectively. The similar skin permeation enhancing effect of CH8 loaded in either liposomes, especially to the deeper EP + D may be the result of similar elasticity of CH8-CvL and CH8-PL as measured in polycarbonate membranes. No drug detectably crossed the skin, indicating the absence of transdermal delivery to the general circulation. In a recent study using ultradeformable liposomes measuring 90 nm and loaded with the calcein fluorescent marker, it was suggested that they act as a sustained release system from the skin surface rather than as a carrier system of hydrophylic drugs through the skin [37]. In the present study, the highly hydrophobic CH8 increased the membrane fluidity and elasticity

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of CvL in a manner similar to found with PEG-8-L. The presence of the drug in layers of the skin deeper than the SC may be more a function of the carrier ability of the liposomes rather than its sustained release from the skin surface, since Lanette cream did not promote detectable drug permeation through the skin.

9.4.2 Topical Efficacy against Murine CL Apart from a mild difference in thickness, localized CL lesions in mice resemble human lesions both clinically and histologically. Although liposomes may permeate the ulcer more easily due to the absence of the stratum corneum, it is in the ulcer border and surrounding tissue where the stratum corneum is present that the majority of the infected macrophages reside. Therefore, both CH8-CvL and CH8PL were tested for their efficacy to treat non-ulcerated CL lesions. They were topically applied twice daily for 30 days onto non-ulcerated mouse ears infected with L. amazonensis. Because elastic liposomes expectedly penetrate the skin following the water gradient (Figure 9.3), liposomes in aqueous solution were applied to the skin in a non-occluded way. At the end of the treatment series, it was found that although both liposomal formulations reduced the lesion growth and parasite loads, CH8-CvL was significantly more effective than CH8-PL (Figure 9.6). The fact that both formulations differed in terms of antileishmanial efficacy but not skin permeation suggests that CH8-CvL was better internalized by infected macrophages than CH8-PL. This is substantiated by the demonstration that the presence of PEG-8-L on the liposome surface creates a water shield (stealth liposomes) that prevents their opsonization and internalization by macrophages.

Fig 9.6 Topical treatment of cutaneous leishmaniasis with liposomal CH8 chalcone. BALB/c mice were infected in the ear with Leishmania amazonensis. Animals were treated twice daily for 30 days starting on day 12 of infection with 6.6 µg of CH8 in PBS (CH8 / PBS), in pegylated liposomes (CH8 / PEG) or in conventional liposomes (CH8 / CvL). Controls were vehicles without CH8 (empty line markers and bars). Left panel shows lesion growth, and right panel shows corresponding parasite loads in the lesions measured on day 42 of infection. (Means± SD, n=5).

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9.5 Conclusion So far, the use of liposomes for topical delivery of hydrophilic or weakly hydrophobic antileishmanial drugs such as antimonials, paromomycin and amphotericin B have not produced optimal results in the treatment of CL. However, the incorporation of the hydrophobic chalcone CH8 in the bilayer of small conventional unilamellar liposomes (up to 100 nm) proved to increase liposome membrane elasticity and stratum corneum permeation. Interestingly, this was achieved in the absence of PEG-8L, proving our first strategy in which elasticity may be provided by disturbances in the bilayer structure, due to incorporation of CH8 in egglecithin liposomes. Preclusion of pegylation is of interest not only because the scaling up of process may be simplified and manufacturing costs reduced, but also to prevent potential immunotoxicity by pegylated liposomes [38].

9.6 Future Perspectives Although still in the pre-clinical stage, these results suggest that other potentially active hydrophobic drugs can also enhance liposome elasticity to squeeze through skin pores. The possibility that they may also carry hydrophilic drugs in their aqueous core without reducing membrane elasticity should also be considered as a way to construct a multivalent skin delivery system.

References [1] Shaw, J.: The leishmaniasis - survival and expansion in a changing world. A minireview. Memorias do Instituto Oswaldo Cruz 102, 541–547 (2007) [2] Romero, I., Tellez, J., Suarez, Y., Cardona, M., Figueroa, R., Zelazny, A., Saravia, N.G.: Viability and burden of Leishmania in extralesional sites during human dermal leishmaniasis. PLOS Negleted Tropical Diseases 4, e189 (2010) [3] Aleixo, J.A., Nascimento, E.T., Monteiro, G.R., Fernandes, M.Z., Ramos, A.M.O., Wilson, M.E., Pearson, R.D., Jeronimo, S.M.B.: Atypical american visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy. Transactions of the Royal Society of Tropical Medicine and Hygiene 100, 79–82 (2006) [4] De-Campos, S.N., Souza-Lemos, C., Teva, A., Porrozzi, R., Grimaldi, G.: Systemic and compartmentalised immune responses in a Leishmania braziliensis-macaque model of self-healing cutaneous leishmaniasis. Veterinary Immunology and Immunopathology 137, 149–154 (2010) [5] Maltezou, H.C.: Drug Resistance in Visceral Leishmaniasis. Journal of Biomedicine and Biotechnology 2010, 1–8 (2010) [6] Romero, G.A.S., Guerra, M.V.D., Paes, M.G., Macedo, V.D.: Comparison of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis and L (V.) Guyanensis in Brazil: Therapeutic response to meglumine antimoniate. American Journal of Tropical Medicine and Hygiene 6, 456–465 (2001)

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[7] Gunduz, K., Afsar, S., Ayhan, S., Kandiloglu, A.R., Turel, A., Filiz, E.E., Ok, U.Z.: Recidivans cutaneous leishmaniasis unresponsive to liposomal amphotericin B (AmBisome (R). Journal of the European Academy of Dermatology and Venereology 14, 11–13 (2000) [8] Rabes, T.D., Baquero-Artigao, F., Fernandez, C.G., Miguel, M.J.G., Laguna, R.D.: Treatment of cutaneous leishmaniasis with liposomal amphotericin B. Anales de Pediatria 73, 101–102 (2010) [9] Yardley, V., Croft, S.L.: A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. International Journal of Antimicrobial Agents 13, 243–248 (2000) [10] Legrand, P., Vertut, A., Bolard, J.: Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line. Journal of Antimicrobial Chemotherapy 37, 519–533 (1996) [11] Enk, C.D.: Treatment of cutaneous leishmaniasis with photodynamic therapy. Archives of Dermatology 139, 432–434 (2003) [12] Hejazi, H., Eslami, G., Dalimi, A.: The parasiticidal effect of electricity on Leishmania major, both in vitro and in vivo. Annals of Tropical Medicine and Parasitology 98, 37–42 (2004) [13] Tallab, T.M., Bahamdam, K.A., Mirdad, S., Johargi, H., Mourad, M.M., Ibrahim, K., ElSherbini, A.H., Karkashan, E., Khare, A.K., Jamal, A.: Cutaneous leishmaniasis: schedules for intralesional treatment with sodium stibogluconate. International Journal of Dermatology 35, 594–597 (1996) [14] Ganor, S.: The treatment of leishmaniasis recidivans with local injections of amphotericin B. Dermatology International 6, 141–143 (1967) [15] Velez, I., Lopez, L., Sanchez, X., Mestra, L., Rojas, C., Rodriguez, E.: Efficacy of Miltefosine for the treatment of american cutaneous leishmaniasis. American Journal of Tropical Medicine and Hygiene 83, 351–356 (2010) [16] El-On, J., Jacobs, G.P., Witztum, E., Greenblatt, C.L.: Development of topical treatment for cutaneous leishmaniasis caused by Leishmania major in experimental animals. Antimicrobial Agents and Chemotherapy 26, 745–751 (1984) [17] Kim, D.H., Chung, H.J., Bleys, J., Ghohestani, R.F.: Is paromomycin an effective and safe treatment against cutaneous leishmaniasis? A meta-analysis of 14 randomized controlled trials. PLOS Neglected Tropical Diseases 3, e381 (2009) [18] Ben Salah, A., Zakraoui, H., Zaatour, A., Ftaiti, A., Zaafouri, B., Garraoui, A., Olliaro, P.L., Dellagi, K., Ben Ismail, R.: A randomized, placebo-controlled trial in Tunisia treating cutaneous leishmaniasis with paromomycin ointment. American Journal of Tropical Medicine and Hygiene 53, 162–166 (1995) [19] Soto, J.M., Toledo, J.T., Gutierrez, P., Arboleda, M., Nicholls, R.S., Padilla, J.R., Berman, J.D., English, C.K., Grogl, M.: Treatment of cutaneous leishmaniasis with a topical antileishmanial drug (WR279396): Phase 2 pilot study. American Journal of Tropical Medicine and Hygiene 66, 147–151 (2002) [20] Frankenburg, S., Glick, D., Klaus, S., Barenholz, Y.: Efficacious topical treatment for murine cutaneous leishmaniasis with ethanolic formulations of amphotericin B. Antimicrobial Agents and Chemotherapy 42, 3092–3096 (1998) [21] Sampaio, R.N.R., Lucas, I.C., Takami, H.L.: Inefficacy of the association N-methyl glucamine and topical miltefosine in the treatment of experimental cutaneous leishmaniasis by Leishmania (Leishmania) amazonensis. Journal of Venomous Animals and Toxins Including Tropical Diseases 13, 598–606 (2007)

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[22] Miranda-Verastegui, C., Llanos-Cuentas, A., Arevalo, I., Ward, B.J., Matlashewski, G.: Randomized, double-blind clinical trial of topical imiquimod 5% with parenteral meglumine antimoniate in the treatment of cutaneous leishmaniasis in Peru. Clinical Infectious Diseases 40, 1395–1403 (2005) [23] Seeberger, J., Daoud, S., Pammer, J.: Transient effect of topical treatment of cutaneous leishmaniasis with imiquimod. International Journal of Dermatology 42, 576–579 (2003) [24] Larbi, E.B., Alkhawajah, A., Algindan, Y., Jain, S., Abahusain, A., Alzayer, A.: A randomized, double-blind, clinical-trial of topical clotrimazole versus miconazole for treatment of cutaneous leishmaniasis in the eastern province of saudi-arabia. American Journal of Tropical Medicine and Hygiene 52, 166–168 (1995) [25] Chen, M., Christensen, S.B., Theander, T.G., Kharazmi, A.: Antileishmanial activity of licochalcone a in mice infected with Leishmania-major and in hamsters infected with leishmania-donovani. Antimicrobial Agents and Chemotherapy 38, 1339–1344 (1994) [26] Yunes, R., Chiaradia, L.D., Leal, P.C., Cechinel, V., Torres-Santos, E.C., Falcão, C.B., Rossi-Bergmann, B.: Chalcones as new drug leads against leishmaniasis. In: Richards, R. (ed.) Current Trends in Medicinal Chemistry Ed. Trivandrum Research Trends, ch. 4, pp. 47–56 (2006) [27] Manosroi, A., Kongkaneramit, L., Manosroi, J.: Stability and transdermal absorption of topical amphotericin B liposome formulations. International Journal of Pharmaceutics 270, 279–286 (2004) [28] New, R.R., Chance, M.L.: Treatment of experimental cutaneous leishmaniasis by liposome-entrapped Pentostam. Acta Tropical 37, 253–256 (1980) [29] Yardley, V., Croft, S.L.: A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. International Journal of Antimicrobial Agents 13, 243–248 (2000) [30] Carneiro, G., Santos, D.C.M., Oliveira, M.C., Fernandes, A.P., Ferreira, L.S., Ramaldes, G.A., Nunan, E.A., Ferreira, L.A.M.: Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis. Journal of Liposome Research 20, 16–23 (2010) [31] Chen, M., Christensen, S.B., Theander, T.G., Kharazmi, A.: Antileishmanial activity of licochalcone A in mice infected with Leishmania-major and in hamsters infected with Leishmania-donovani. Antimicrobial Agents and Chemotherapy 38, 1339–1344 (1994) [32] Boeck, P., Falcão, C.A.B., Leal, P.C., Yunes, R.A., Cechinel, V., Torres-Santos, E.C., Rossi-Bergmann, B.: Synthesis of chalcone analogues with increased antileishmanial activity. Bioorganic & Medicinal Chemistry 14, 1538–1545 (2006) [33] Egelhaaf, S.U., Wehrli, E., Müller, M., Adrian, M., Schurtenberger, P.: Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering. Journal of Microscopy 184, 214–228 (1996) [34] Meeren, P.P.A.V., Vanderdeelen, J., Baert, L.: Particle Size Analysis. John Wiley & Sons, New York (1988) [35] Chen, P.S., Totiba, T.Y., Warner, H.: Microdetermination of phosphorus. Analytical Chemistry 28, 1756–1758 (1956)

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Chapter 10

Druggable Targets for Skin Photoaging: Potential Application of Nanocosmetics and Nanomedicine Giselle Z. Justo1, Sílvia M. Shishido2, Daisy Machado2, Rodrigo A. da Silva2, and Carmen V. Ferreira2 1

2

Departamento de Bioquímica (Campus São Paulo) and Departamento de Ciências Biológicas (Campus Diadema), Universidade Federal de São Paulo, 3 de Maio, 100, 04044-020, São Paulo, SP, Brazil [email protected] Laboratory of Bioassays and Signal Transduction, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, s/n, CP 6109, 13083-970, Campinas, SP, Brazil

Abstract. Skin is the organ most exposed to environmental sunlight. Many in vitro and in vivo studies on skin have now unambiguously gathered evidence of the ultraviolet (UV) radiation involvement in the development of human skin pathologies such as sunburn, aging, autoimmunity, immunosuppression and cancer. Thus, the toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The mechanisms by which UV radiation promotes skin damage have been under intense investigation for decades, and much progress has been made in identifying the molecular alterations associated with changes in cellular functions. Furthermore, these studies are providing potential targets for molecular therapies. The aim of this chapter is to summarize the general knowledge available in the field of UV-induced skin damage, with an emphasis on the discussion of molecular markers of cellular senescence and inflammation. Besides the biological consequences of photodamage, this chapter also deals with technologies available for the detection of phototoxicity and characterization of the molecular action of nanostructures, and shows how helpful such approaches can be with a view to improving the photoprotection provided by skin products.

10.1 Skin Phototoxicity UV radiation in sunlight (200−400 nm) comprises the wavelengths shorter than those of visible, and is composed of UVC (200−280 nm), UVB (280−320 nm) and UVA (320−400 nm), each of which has distinct biological effects. UVC is effectively blocked from reaching the Earth’s surface by the ozone layer, although

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accidental exposure can occur from man-made radiation sources, such as germicidal lamps. UVA and UVB radiation both reach the Earth’s surface in amounts sufficient to exert a variety of detrimental effects on the skin. Wavelengths in the UVB range of the solar spectrum are mainly absorbed by epidermal cell components (e.g., proteins or DNA), producing inflammation, burns, immunosuppression and eventually skin cancer [1, 2, 3, 4]. UVA radiation penetrates deeply into the skin, reaching the basal layer of the epidermis and even dermal fibroblasts. Although UVA is the predominant component of solar UV radiation to which we are exposed, it was previously considered to be weakly carcinogenic and cause premature skin aging (photoaging). However, it is now generally agreed that UVA is also involved in the development of tumors and in the suppression of immune functions with negative consequences for malignant as well as infectious diseases [2, 5, 6, 7]. The photodamage response has evolved to protect an organism against toxic events that can lead to UV-induced pathologies while promoting the recovery and growth of healthy cells for tissue repair. Therefore, the numbers of repairing cells with low levels of damage required to maintain the skin barrier function must be balanced against the cost of retaining some cells with a low level of mutation [8].

10.1.1 Acute Effects of UV on Skin At the molecular level, acute UV irradiation (a single exposure) can damage many molecules and structures, which may result in changes in cellular functions. DNA is a critical target because its lesions may lead to DNA mutations and, if they are not repaired, they can ultimately induce skin cancer [9]. UV irradiation from 245 to 290 nm is absorbed maximally by DNA [3, 10]. UV irradiation is able to induce mutagenic photoproducts or lesions in DNA mostly by the formation of dimeric photoproducts between adjacent pyrimidines [11, 12]. These dimers are of two main types: cyclobutane dimers (CPDs) between adjacent thymine (T) or cytosine (C) residues, and pyrimidine (6-4) photoproducts between adjacent pyrimidine residues [11, 12]. Overall, these CPDs are produced three times as often as (6-4) photoproducts. Although both lesions are potentially mutagenic, CPDs are considered to be the major contributor to mutations in mammals [12], since (6-4) photoproducts are repaired efficiently compared to CPDs in mammalian cells [13]. In addition, methylation of cytosine has been shown to strongly enhance the formation of dimers at pyrimidine bases when cells are exposed to UVB light and these sites have been implicated as the main sites for p53 gene mutations in human skin tumors [14]. At longer wavelengths (>320 nm), indirect effects mediated by reactive oxygen species (ROS) become more important and induce various types of damage, including DNA breakage, proteinDNA crosslinks and oxidative modifications of nucleic bases, by producing reactive oxygen species such as superoxide anion, singlet oxygen, and hydrogen peroxide via endogenous photosensitizers [15, 16, 17]. One of the best studied lesions is 8-oxo-7,8-dihydroxyguanine, which results from the oxidation of the guanine moiety. This damage has been shown to be pre-mutagenic and probably involved in the photocarcinogenic process initiated by sunlight [18]. UV radiation

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can also activate exogenous molecules (e.g., photosensitizing drugs). Some of these molecules will react directly with DNA (e.g., psoralens), increasing the risk of photocarcinogenesis, whereas others will generate local oxidative stress (e.g., antibiotics and nonsteroidal anti-inflammatory drugs) [10]. In the absence of efficient DNA repair mechanisms, these DNA lesions persist in the genome and may lead to the introduction of deleterious mutations after DNA replication. Depending upon the primary DNA lesion, one or more repair pathways become active such as direct repair, base excision repair (BER), mismatch repair (MMR), double strand break repair, and nucleotide excision repair (NER). The latter is considered to be the major line of defense against carcinogenesis [10]. 10.1.1.1 Role of p53 in the Photodamage Response The major acute effects of UV irradiation on normal human skin comprise sunburn inflammation (erythema), tanning, and local or systemic immunosuppression [3, 8]. The p53 tumor suppressor gene codes for a 53-kDa phosphoprotein involved in gene transcription and control of the cell cycle by coordinating transcriptional control of regulatory genes. Mutations in the p53 gene or its inactivation have been associated with UV-induced skin lesions and cancer [19]. A variety of DNA damaging agents induce high levels of p53 which leads to cell cycle arrest or induces apoptosis in cells with excessive DNA damage. In addition, the p53 protein may directly or indirectly modulate DNA repair [17]. Numerous studies have shown that UV radiation induces the activating phosphorylation of the p53 protein at multiple serine residues, including Ser15 and Ser20 [20] - (Fig. 10.1). Evidence also exists for the involvement of the ataxia telangiectasia mutated (ATM) family of proteins, the mitogen-activated protein kinases (MAPK) p38 kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1 (JNK1) in the phosphorylation of various p53 serine residues in response to UV radiation [21, 22, 23]. The phosphorylation of p53 and/or its negative regulator, the murine double minute 2 (Mdm2) protein, activates p53 through three mechanisms: (1) by disrupting the interaction of p53 and Mdm2, thus stabilizing p53; (2) by enhancing its transcriptional activity; and (3) by promoting the nuclear localization of p53 [17]. Normally, there is little p53 protein in the cell, but in response to UV damage high levels of p53 are induced. The accumulation of activated p53 induces G1 cell cycle arrest, allowing the cellular repair pathway to remove DNA lesions before DNA synthesis and mitosis. In this pathway, p21WAF1/CIP1 competitively forms complexes with cyclin-dependent kinases (CDK), inactivating the cyclin/CDK complexes. However, the overexpression of p21 causes the accumulation of hypophosphorylated Rb protein (pRb) and the sequestration of E2F transcription factor, also leading to cell arrest in the G1 phase [17]. In addition, p21 was shown to directly interfere with DNA synthesis by binding to the proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase δ in eukaryotic cells [19].

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Fig 10.1 Roles of p53 and Bcl-2 family proteins in UV-induced apoptosis and cell survival. Ultraviolet radiation (UVR) may directly interact with DNA, leading to pyrimidine dimer formation and activation of the ataxia telangiectasia mutated (ATM) family proteins. ATR phosphorylates p53 in Ser15, modulating the DNA-binding activity of p53 and enhancing its stability. The tumor suppressor protein p53 transactivates the p21 gene and numerous genes coding for the pro-apoptotic BH3-only members Bid, Noxa and Bax. Phosphorylated p53-induced transcription of p21 causes G1/S cell cycle arrest, allowing the cellular repair pathway to remove DNA lesions before DNA synthesis and mitosis. In contrast, UV at high doses directly activates the death receptors, such as Fas, independent of the ligands, leading to activation of caspase-8, which in turn, activates the executioner caspase 3. In addition, pro-apoptotic Bax and/or Bak can be directly activated by cleaved Bid (tBid), causing mitochondrial outer membrane permeabilization (MOMP) and release of apoptogenic factors. The release of proteins from the intermembrane space triggers the activation of caspase 9. This, in turn, cleaves and activates the executioner caspases, including caspase 3. Antiapoptotic Bcl-2 members sequester the BH3-only proteins, preventing MOMP. Other sensitizing or derepressor proteins, including Noxa and p53, antagonize the antiapoptotic proteins, thus sensitizing cells for death. p53 can also translocate to mitochondria where it directly activates Bax/Bak. Therefore, p53 mediates part of the response of mammalian cells to UVinduced DNA damage, either by stimulating DNA repair or, beyond a certain threshold of DNA damage, by initiating apoptosis. Illustration by Sarah Azoubel Lima.

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Moreover, p53 directly affects two major pathways of DNA repair, NER and BER, by inducing the transcriptional activation of downstream genes. In NER, specifically, p53 transactivates two xeroderma pigmentosum-associated gene products, p48XPE and XPC. The growth arrest and DNA damage 45 (GADD45) stress sensor protein has also been implicated in the suppression of cell growth by binding to UV-damaged DNA and loss of GADD45 results in reduced NER activity [24]. In contrast, when the UV damage is very severe and cannot be repaired, there is an increase in apoptosis. In particular, p53 directly upregulates the expression of pro-apoptotic genes which trigger the release of cytochrome c from mitochondria and caspase activation. Among them are the Bcl-2 family members Bax, p53upregulated modulator of apoptosis (Puma), Noxa, and p53-upregulated apoptosisinducing protein 1 (p53AIP1). These proteins bind to the Bcl-2 protein, antagonizing its antiapoptotic function. Studies in mice have also revealed a concomitant decrease in Bcl-2 protein during apoptosis, followed by epidermal hyperplasia after acute UV exposure, suggesting that UV-induced apoptosis and hyperplasia are closely linked and tightly regulated, and that deregulation of these two events may lead to skin cancer development. p53 may also cause the rapid disruption of mitochondria through upregulation of the death receptor Fas in UV-induced apoptosis. Indeed, induction of apoptotic keratinocytes, sunburn cells, in UV-exposed mouse skin was dependent on the interaction of Fas-Fas ligands (FasL) [17]. Therefore, p53 aids in the DNA repair or the elimination of cells that have excessive DNA damage. In addition, transcription-independent functions have been attributed to the p53 protein. A fraction of p53 can accumulate in mitochondria within cells during apoptosis, but not during p53-independent apoptosis or during cell cycle arrest. Studies have demonstrated that p53 mediates Bax and Bak oligomerization and mitochondrial membrane permeabilization by directly interacting with antiapoptotic Bcl-2 and Bcl-XL, through a mechanism similar to the pro-apoptotic BH3only proteins [25]. Thus, p53 prosurvival functions are transcription dependent, while pro-apoptotic functions can be nuclear or cytosolic (Fig. 10.1) [8]. Other Bcl-2 family members, the BH3-only proteins Bim, Bmf, and Bid, have also been reported to respond to DNA damage-inducing agents. Bid is present at high levels in human epithelial cells and is targeted by p53 for transcriptional upregulation in UV-exposed skin. After dissociation from antiapoptotic Bcl-2 members, Bid can be cleaved and activated in its truncated form, tBid, by caspase 8, JNK-induced protease, or lysosomal cathepsins. tBid can thus activate Bax and Bak and cause mitochondrial outer membrane permeabilization (MOMP), leading to the release of mitochondrial pro-apoptotic contents and subsequent activation of caspase-dependent apoptosis. Another BH3-only protein, Noxa, as well as p53, can bind antiapoptotic Bcl-2 family proteins and release Bid in UV-induced apoptosis (Fig. 10.1) [8, 26]. A role for Bid in the photodamage response has been recently demonstrated in vivo. Bid-deficient mice exhibit profound inhibition of UVB-induced sunburn cell formation and are highly resistant to UV-induced local and systemic immune suppression [27, 28]. Collectively, these data suggest that Bid is an important activator of UV-induced apoptosis.

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10.1.1.2 Inflammation and Melanogenesis Sunburn inflammation (erythema) and tanning (enhanced melanogenesis) are certainly the most well-known responses to UV irradiation. UV-induced melanogenesis, or tanning response, results from the direct effects of UV on melanocytes and from indirect keratinocyte-mediated effects. The skin pigmentation response following UV irradiation comprises two phases: 1) immediate pigment darkening (IPD), which is characterized by an alteration and redistribution of local melanin and has a maximal response immediately after UV exposure; and 2) delayed tanning (DT), which is associated with de novo synthesis of melanin by melanocytes and results from UVB and UVA irradiation. Moreover, delayed tanning from UVB exposure is associated with an increase in the activity and number of melanocytes. While, single exposures induce an increased activity of melanocytes, subsequent doses are required to increase the numbers of melanocytes. Concurrently, melanocyte tyrosinase activity increases, melanocyte dendrites elongate and branch, and melanosome numbers and sizes increase. These events result in accelerated melanin transfer to keratinocytes and, as a consequence, an increase in melanin granules in the epidermis occurs. UVA-initiated tanning has distinct effects that are wavelength-dependent. UVA irradiation between 340 and 400 nm increases melanin density in the basal cell layer, whereas short UVA wavelength radiation (<340 nm) increases the synthesis and transfer of melanized melanosomes to the epidermis [3, 29]. Interestingly, there is now strong evidence that these effects are mediated by direct DNA damage as a consequence of the switching on of specific molecular signaling pathways [10]. The increase in the dose of UV irradiation required to cause sunburn is also associated with hyperplasia of the stratum corneum, epidermis, and dermis. At the same time that UV is activating cell cycle checkpoints and apoptosis, it is also stimulating cell survival mechanisms and inducing cell proliferation, which is evident in the form of epidermal hyperplasia 24–48 h after acute UV exposure. UV triggers these processes by activating receptors to various growth factors and cytokines [3]. In particular, the UV-induced activation of epidermal growth factor receptor (EGFR) mediates the early responses of genes, such as c-fos and c-jun, and the induction of the activator protein-1 (AP-1) transcription factor necessary for the cell cycle progression and proliferation of epidermal cells [30]. The induction of these early-response genes is the result of signaling events triggered by the activation of the stress-activated MAPKs, JNK and p38. In addition, it has been shown that UVB potentiates the signaling pathway mediated by EGFR activation and protein kinase B (Akt) phosphorylation, which promotes cell survival [17, 31]. The clustering and internalization of cell surface receptors for tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 are also induced by UV irradiation. These receptors work together to activate JNK, since EGF, IL-1, and TNF-α produced a synergistic response equal to that caused by UV light. Exposure of skin to solar radiation has been further shown to generate the secretion of IL-1, which displays a broad range of mitogenic and inflammatory activities, including fibroblast proliferation and T-cell activation [17]. In addition, a recent report implicates IL-1β, which is secreted from UV-irradiated keratinocytes, in sunburn-associated inflammation and demonstrates the prominent role of the caspase 1-activating

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NOD-like receptor (NLR) family of proteins in sunburn-associated inflammation [32]. Hence, NLRs are emerging as a crucial link between cell injury and inflammation, in addition to their roles in sensing microbial products in the context of innate immunity [33]. 10.1.1.3 UV-Induced Immunosuppression It is now understood that multiple mechanisms are invoked to orchestrate UVinduced immunosuppression. Differences in UV wavelength, UV dose, duration of UV exposure and genetic factors have revealed that the chief mediators of suppression may differ according to the experimental setting. UV-induced suppression is associated with many types of antigens that appear in conjunction with UV in both murine and human systems. This was first shown using the model for studying UVB-induced immunosuppression, the contact hypersensitivity (CHS) reaction, which is an experimental model of delayed-type hypersensitivity (DTH) using haptens, such as dinitrofluorbenzene and oxazolone, as antigens applied epicutanously to the skin. Many biochemical and molecular factors play a role in orchestrating photodamage responses, including those that lead to tolerance induction. Two types of UV-induced immunosuppression are known to occur, that is, local and systemic. While UV-induced soluble factors mediate the former, the skinspecific antigen presenting cell, the Langerhans cell (LC), plays a major role in abolishing the immune response to antigens applied at the local irradiated site [8]. LCs are located in the suprabasal layer in order to respond to UV stress directly or through activation mediated by apoptotic stimuli and soluble factors produced by surrounding keratinocytes. The fact that UV radiation induces immune tolerance supports the concept that this photodamage response has evolved to protect the skin from autoimmune attack [8]. Recently, evidence indicates that LCs are critical for the generation of regulatory T (Treg) cells. Experiments have established that UV-induced suppression of CHS responses is mediated by T cells, which were able to transfer hapten specific tolerance to naïve mice [34]. Further studies suggest that they represent a specialized subset of skin-specific Treg cells, since they display classic Treg cell markers CD4+CD25+, secrete high levels of the immune system suppressor cytokine IL-10 and express a ligand for dectin-2 receptors on LCs [35]. Recently, Treg cell activation was demonstrated to be dependent on the receptor activator of nuclear factor κB (NFκB), namely RANK, activation on epidermal LCs by interaction with its ligand (RANKL) on keratinocytes [36]. A well characterized consequence of RANK activation of dendritic cells (DCs) is an increase in their survival due to the upregulation of Bcl-XL expression. This mechanism may be operating in UVdamaged LCs to promote their recovery and delay their own demise until they reach the regional draining lymph nodes, where they work as potent stimulators for antigen-specific T cells. Together, these findings are consistent with the idea that LCs are specialized in their response to UV-induced RANK activation, playing a major role in stimulating antigen-specific Treg cells that mediate UVinduced tolerance [8].

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Systemic immunosuppression is mainly caused by the UV-induced release of immunosuppressive mediators by keratinocytes. Several cytokines appear to be involved in systemic immunosuppression, including IL-1, IL-4, IL-6, IL-10, TNFα and transforming growth factor-β (TGF-β) [4]. UV-induced IL-1 from human keratinocytes enhanced the synthesis of prostaglandin E2 (PGE2) which can suppress CHS responses. IL-10, a potent immunosuppressive cytokine is released not only by keratinocytes, but it is also produced by CD11b+ macrophages in human skin [37]. TNF-α is one of several factors involved in UVB-induced immunosuppression, as indicated by the studies on TNFR-deficient mice, in which CHS was suppressed by UVB radiation [38]. Although the interactions between the different cytokines produced in response to UV radiation are not fully elucidated, IL-10 certainly is a major player. IL-10 is a T helper cell type 2 (Th2) cytokine that counteracts the production of T helper cell type 1 (Th1) cytokines, in particular interferon-γ (IFN-γ). UVB-induced IL-10 production by keratinocytes or macrophages shifts the type of immune response from Th1 to Th2, which accounts for the fact that Th1-mediated cellular immune reactions are impaired by UV radiation in mice and human skin [37]. UVB irradiation stimulates antigen-presenting cells to produce a cytokine profile that promotes Th2 cells, and not Th1 cells, which are the effective T cell subset in DTH and CHS responses. Deletion of CD11b+ cells by antibodies makes UV-irradiated mice permissive again for CHS [39]. Thus, CD11b+ cells are included in the complex assembly of players in UVrelated immunosuppression. Although the triggers of UV-induced immunosuppression have not been fully defined, a relationship between apoptosis regulators and immunosuppressive cytokines has been observed. Because DNA is one of the major UV chromophores in the skin, it has been proposed that UV-induced DNA damage may play a crucial role in UV-induced immunosuppression. In fact, data from murine models support the view that DNA damage induces apoptosis which triggers immunosuppression and tumor outgrowth. In addition, studies based on the mixed epidermal cell lymphocyte reaction have indicated that the actions of UVB-induced immunosuppression and pyrimidine dimer induction are comparable [8, 10]. Therefore, it is not surprising that UV-induced DNA damage acts as an important trigger of photodamage responses and affects the skin immune response.

10.1.2 Chronic Effects of UV on Skin Long-term and recurrent exposure to sunlight causes the gradual deterioration of cutaneous structure and function, as a result of cumulative DNA damage resulting from acute DNA injury and chronic inflammation [3]. Chronic effects from repeated exposure to solar UV radiation include specific types of skin cancer, which are associated with cumulative mutations and escape from the immunological surveillance, and a number of deteriorating effects responsible for the majority of the visible signs of skin aging, termed photoaging [40, 41].

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10.1.2.1 Photoaging Chronic exposure to UV radiation in ordinary sunlight is a major factor in the etiology of the progressive undesirable changes in the appearance of skin. Premature aging of the skin is characterized by altered mechanical properties of the dermis with wrinkle formation, laxity, a leathery appearance, increased fragility, blister formation and impaired wound healing. In contrast, intrinsically aged skin is thin with reduced elasticity but reveals a smooth appearance. The major damage of photoaged skin is related to the connective tissue of the dermal compartment with quantitative and qualitative alterations of the extracellular matrix components, such as elastin, glycosaminoglycans and interstitial collagens. For example, collagen type I, which provides tensile strength and stability to the dermis, has been found to be diminished in photoaged skin [42]. Much insight into the mechanisms for these effects at the molecular level has been provided by recent research. Since metalloproteinases (MMPs) are known to degrade collagen and elastin in the skin, it has been suggested that induction of MMPs may be a primary mechanism mediating cutaneous photoaging [43]. In fact, it has been reported that UV radiation upregulated AP-1 and NFκB binding to DNA, which are known stimulators of matrix metalloproteinase [41]. The dermal release of UV radiation-generated ROS probably plays an important additional role in photoaging. Dermal fibroblasts release MMP-1, the major enzyme responsible for collagen 1 digestion, following exposure to both UVA and UVB components of the solar spectrum, although the mechanism of primary cellular damage differs. UVB interacts directly with DNA, the predominant lesions being the CPDs, whereas UVA radiation inflicts damage mostly indirectly through the generation of ROS [44]. In addition, Dong et al. [45] have recently demonstrated that UV-induced DNA damage in keratinocytes of the epidermis is a key event in the processes leading to photoaging, since it not only induces MMP1 gene expression in the irradiated keratinocytes, but also induces the release of soluble mediators that signal fibroblasts of the dermis to release MMP-1. These events in parallel activate collagen digestion, the forerunner to wrinkling. Moreover, as MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes, the significant role of UVB, which deposits most of its energy on the chromatin of the keratinocytes and to a lesser extent in the upper dermis, in photoaging was proposed. Clearly, this work further contributed to replacing the original misconception that ‘UVA is for Aging’ because only UVA penetrates to the dermis, and wrinkles are in the dermis, with the knowledge that UVB-induced DNA damage is an initiator of the events leading to MMP-1 induction [45]. 10.1.2.2 UV-Induced Skin Cancers Skin cancers, i.e., basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma, are some of the most frequently occurring tumors. Currently,

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between 2 and 3 million non-melanoma skin cancers and 132,000 malignant melanomas occur globally each year [46]. UV radiation is recognized as the main etiological agent causing skin cancer, and acts as both a tumor initiator and promoter [47, 48]. While acute UV irradiation causes apoptosis involving p53 and Fas-FasL pathways, chronic UV irradiation results in deregulation of apoptosis, leading to the abnormal proliferation of keratinocytes with DNA damage, acquisition of p53 mutations and loss of Fas-FasL interaction, all of which contribute to the onset of skin cancer [17]. Carcinogenesis by UV radiation often involves the inactivation of one or more tumor suppressor genes or the activation of growth-stimulatory proto-oncogenes. Tumor suppressor genes negatively regulate cell growth and usually require both copies of the gene to be inactivated before loss of control of cell growth occurs. Accumulation of proteins that bind to and sequester tumor suppressor proteins can also make the cell more susceptible to further mutations. Activation of oncogenes is dominant in that a change in only one copy of the gene is required to have an effect. Proto-oncogenes, the normal versions of oncogenes, act to control cell proliferation and differentiation, and constitute three groups: growth factors and their receptors, signal transduction proteins, and nuclear factors [17, 49]. Several genes with important roles in skin carcinogenesis have been extensively studied, including activation of proto-oncogenes, such as Ras, and inactivation of tumor suppressor genes, such as PTCH and INK4a-ARF (CDKN2A) [50]. Non-melanoma skin cancer As discussed earlier, the p53 gene plays an important role in the process of preserving the genome integrity. When it has undergone mutation, the protein functions of p53 can be disrupted, and the ability to manage DNA repair and to engage the apoptosis of highly damaged cells is lost. Thus, UV radiation may be considered a tumor promoter by providing selection pressure for cells carrying a mutated p53 gene. As a consequence, these cells cannot arrest their cell cycle or enter apoptosis even if their genome is heavily damaged [10]. Genetic alterations within the p53 tumor suppressor gene are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific ‘signature’ mutations that can result in oncogenic transformation. There are certain hot spots in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site and, in SCCs and BCCs, a high proportion of these mutations are C to T and CC to TT transitions [3, 49]. FasL, a member of the tumor necrosis factor superfamily, which is preferentially expressed in the basal layer of the skin epidermis, is a key surveillance molecule involved in the elimination of apoptotic (sunburn) cells, but also in the prevention of cell transformation. However, UV light exposure downregulates FasL expression in keratinocytes and melanocytes leading to the loss of its sensor function and increasing the risk of persistent transformed cells. Indeed, a dramatic decrease in apoptotic cells occurs concomitantly with inhibition of FasL expression. This resistance to apoptosis together with epidermal cell proliferation

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represents an adaptive response that apparently serves to sustain a viable stem cell population in the skin. This deregulation of Fas-FasL-mediated signaling has profound consequences in UV-exposed epidermis, including the accumulation of more p53 mutations and suppression of host immunity, events that promote skin cancer outgrowth [17, 51]. Moreover, repeated exposure of skin to UV radiation results in the clonal expansion of initiated p53-mutant cells, suggesting that the selective proliferative advantage of p53-mutant cells may be critical in promoting their clonal expansion [52]. Mutations in the p53 gene alone are not sufficient to cause BCC or SCC, and at the very least, some oncogenic pathway must be activated, such as a mitogenic pathway, which normally starts with the oligomerization and activation of a receptor tyrosine kinase (RTK), such as EGFR. Signal transduction further mediates the activation of transcription factors through a cascade of cytosolic proteins, including Ras. However, the activation of Ras mutations has been reported in a minority of SCCs and BCCs [50]. Mutations in the Hedgehog pathway related genes, especially PTCH, are well known to represent the most significant pathogenic event in BCCs [50]. The PTCH gene is a human homologue of the Patched (Ptc) gene in Drosophila melanogaster. The PTCH1 and 2 proteins are involved in the Hedgehog (Hh) signaling pathway, which is essential for cell growth and differentiation. This pathway is triggered by extracellular Hh (Sonic, Desert and Indian) which couples to PTCH, a serpentine-like receptor woven through the cell membrane. PTCH forms a complex with another serpentine transmembrane protein, Smoothened (Smo). Upon coupling to Hh, PTCH relieves suppression of Smo and engages the intracellular signaling cascade which ultimately releases zinc-finger Gli transcription factors. Retroviral transduction of Sonic Hh into normal human keratinocytes reportedly resulted in the expression of the Gli-target bone morphogenetic protein2B (BMP-2B) and Bcl-2 [53]. The latter protein is commonly expressed in BCCs and is a well-known suppressor of apoptosis [50]. Among the Gli transcription factors, overexpression of Gli2 plays a key role in the marked apoptosis resistance of the BCCs [54]. Recently, Kump et al. [55] also found that high Gli2 expression induced the concomitant high expression of the caspase 8 inhibitor, cFlip (FLICE inhibitory protein), thereby counteracting death ligand-mediated apoptosis. Considering that cFlip and Bcl-2 are highly expressed in BCCs a marked resistance of the tumor to the extrinsic and intrinsic apoptotic pathways is expected as a consequence of high Gli2 expression. Moreover, Gli2 silencing in BCC tissues made the tumor sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death by downregulating cFlip. Since Gli2 silencing downregulates cFlip and Bcl-2, Gli2 was proposed as a key target in tumors with deregulated Hedgehog signaling [55]. A subset of SCCs and BCCs also carries mutations in the INK4a-ARF tumor suppressor gene. The INK4a-ARF locus encodes two independent growth inhibitors and effectors of cellular senescence: the CDK inhibitor p16INK4a and the p53 activator p14ARF (mouse p19Arf). p16INK4a inhibits progression from the G1 to S

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phase of the cell cycle in cells that express pRb. p14ARF mediates G1 and G2 arrest and activates the p53 pathway by binding to human double minute protein 2 (HDM2, mouse Mdm2), thereby blocking HDM2-induced degradation of p53 [17]. Malignant melanoma According to the World Health Organization, malignant melanoma is increasing faster than any other malignancy worldwide, representing a public health problem. Despite the great advances achieved in deciphering the molecular mechanisms involved in melanoma carcinogenesis in the last few years, the formation of malignant melanoma is very complex and may involve mutations in several genes. The role of p53 gene mutations in melanoma is still unclear and the results obtained from tumors are variable. However, the fact that patients affected by the DNA repair deficiency disease, xeroderma pigmentosum, are more prone to melanoma suggests that DNA damage plays a role in the disease. Also, UV-induced pyrimidine dimers in specific tumor suppressor genes or oncogenes are likely to play a role [56]. Moreover, oxidative DNA damage resulting from the photoreaction of melanin, as well as genetic predisposition, should also be considered [10]. Therefore, the likelihood of melanoma is a combination of inherited or constitutional predisposition and exposure to environmental factors, of which exposure to sunlight is the only risk factor proved relevant to melanoma [3]. Some linkage analysis of familial melanomas revealed the presence of a susceptibility gene on chromosome 9p21, and identified the INK4A gene at the locus a few years later. It is estimated that approximately 20% of melanoma families worldwide are linked to P16INK4A mutations. However, the lack of P16 mutations in 9p21-linked families also implicates mutations in the promoter region of the gene and alterations in the methylation pattern as possible causes. Melanoma has also been linked to chromosome 1p36 by linkage and loss of heterozygosity (LOH) studies, and this locus has been implicated in other cancers, suggesting the presence of a new tumor suppressor gene in that locus [3]. The Ras oncogenes have also been implicated in melanoma. More specifically, the N-Ras has been reported in 25–70% cases of melanoma from regularly sunexposed sites. These mutations occur in the vicinity of dipyrimidine sites, the typical UV targets, but they are not dominated by C to T transitions. As mentioned earlier, the Ras proteins function in mitogenic pathways triggered by the activation of an RTK at the cell membrane, such as EGFR. The role of oncogenic Ras in transforming most immortal cell lines and making them tumorigenic upon transplantation into nude mice is well known. However, inactivation of either p16 or p53 was necessary to prevent a G1 arrest of cells expressing oncogenic Ras and induce a tumorigenic state, providing evidence for the cooperation of an RTK mitogenic pathway and dysfuntional INK4A in melanomagenesis [50]. Other studies have also indicated that when keratinocytes or melanocytes are transformed they re-express FasL, allowing the expanding tumor to evade the immune system. Therefore, in addition to Gli2, other genes regulating apoptosis and FasL, which is involved in immune evasion, could be considered as potential targets to prevent tumor formation and growth [54].

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In conclusion, it is clear from these studies that UV radiation adversely affects skin health and appearance via multiple molecular pathways and that research is rapidly gaining ground in terms of understanding the molecular mechanisms involved in DNA damage, and in the determination of the cellular response, that is, to either initiate the cellular repair processes or undergo apoptosis in order to guarantee the skin homeostasis. Certainly, knowledge on the signaling pathways underlying skin pathologies and senescence may help to reveal new candidate targets for the development of future therapies.

10.2 In Vitro Index of Phototoxicity Phototoxicity or photoirritation can be defined as a skin inflammatory reaction, initiated by topical or systemic administration of chemicals such as drugs or cosmetic products, and subsequent exposure to light, particularly to UVA radiation [57, 58, 59, 60]. Depending on the exposure time, the photo-activated molecules can alter biological systems and elicit harmful effects, including phototoxicity (e.g. erythema/edema, pigmentary alterations and visual impairment/ocular damage), photoallergy or photocarcinogenicity [57, 61, 62]. The advances in technology and increase in research promoted by industries has led to the introduction of a huge number of new natural and synthetic compounds into cosmetic formulations [63, 64]. Therefore, during the development of formulations for topical or systemic application which contain chemicals that absorb light in the range of 290-700 nm, the phototoxic potential of the ingredients must be assessed through the use of an appropriate assay [58, 60, 65, 66]. Some in vitro methods have been developed to evaluate the phototoxic potential of chemicals. Basically, in these assays cells (e.g. 3T3) and tissues (reconstructed human epidermis) are employed for screening purposes and/or to define a specific mechanism of phototoxicity [58, 67, 68, 69, 70]. In 1991, ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to develop and validate a protocol for assessing, in vitro, phototoxicity. After validating and confirming the reliability and relevance of the test for predicting phototoxic effects and for identifying phototoxic chemicals, ECVAM and COLIPA elected the 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) as a scientifically validated in vitro test [69, 70]. The basis of this assay is to compare the cytotoxicity of test substances with and without exposure to UV light in fibroblasts [71]. Fig. 10.2 summarizes the main steps of this assay. Cytotoxicity is expressed as a dose-dependent reduction in the growth rate of cells determined by the uptake of the vital dye neutral red (NR) 22 h after treatment [72]. The phototoxic effect is determined by the relative reduction in the viability of cells exposed to an exogenous chemical in the presence and absence of light. The absence of phototoxic potential in a 3T3 NRU phototoxicity assay usually dispenses with the requirement for further phototoxicity testing in vivo [73].

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Fig 10.2 3T3 neutral red (NR) uptake phototoxicity test. For one test chemical it is necessary to prepare two plates: one for determination of cytotoxicity (-UVA) and the other for determination of photocytotoxicity (+UVA). Cells are seeded and incubated for 24 h (5% CO2, 37ºC) or until they reach the middle of a confluent monolayer. Subsequently, the culture medium is removed and cells are incubated with different concentrations of compound in PBS for 1 h, and irradiated or kept in the dark for 50 min. The solutions are then replaced with culture medium and the cells allowed to recover for 22 h in a humidified incubator with 5% CO2, at 37oC. Cell viability is assessed by NR uptake. When possible, the concentration of a test chemical reflecting a 50% inhibition of the cellular NR uptake (IC50) is determined. Using this assay it is possible to predict the potential phototoxic of the compounds, as discussed earlier.

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10.2.1 Photo-Irritation Factor In order to predict the photo-irritation potential the concentration response curves concurrently obtained in the presence and absence of UVA irradiation are compared, and the photo-irritation factor (PIF) is calculated by dividing the medial inhibitory concentration (IC50) of the UVA irradiated plate (+UVA) by the IC50 of the non-irradiated plate (-UVA), according to equation 1.

PIF =

IC50 (−UVA) IC50 (+UVA)

(1)

When no cytotoxicity is observed in the absence of UVA, the PIF is estimated following equation 2, using the highest concentration tested.

PIF =

C max (−UVA) IC50 (+UVA)

(2)

10.3 Molecular Markers of Skin Inflammation, Senescence and Photoaging As discussed above, UV radiation promotes the activation of various signaling pathways involved in skin pathophysiological processes, including inflammation, senescence and photoaging. In this section, we will highlight some molecular markers of the harmful effects of UV radiation in the skin.

10.3.1 Molecular Markers of Inflammation Cytokines Cells in the epidermal and dermal layers produce a range of cytokines, including IL-1, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15 and TNF-α [74]. Morphological and functional changes in epidermal LCs exposed to UV radiation are promoted by TNF-α and IL-1β. Keratinocytes are the major contributors to epidermal cytokine production and the release of cytokines such as IL-1, IL-10 and TNF-α by these cells after UV irradiation has multiple consequences for the migration and functions of inflammatory cells, thus influencing keratinocyte proliferation and differentiation processes, and affecting the production of other cytokines by these cells. In addition, systemic effects on the immune system may also be affected [75]. NFκB Besides cytokines, other proteins that are expressed after UV exposure can be sorted according to their functionality. Among them are proto-oncogene products, including the family members NFκB and AP-1, basic fibroblast growth factor (bFGF) and specific markers of differentiation (keratins) [30, 76]. The transcription factor NFκB is known to exercise functions related to cell differentiation,

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proliferation and survival [77], and is the best studied transcription factor related to cellular response against UV radiation. NFκB is a member of the Rel/NFκB family, which regulates the expression of critical genes for multiple biological processes, including inflammatory reactions, immune responses and apoptosis [78]. The cellular localization of NFκB is strictly controlled by a protein family called inhibitory κB (IκB) proteins [79, 80, 81]. When inactive, NFκB is maintained in the cytoplasm through a strong association with IκBα. Exposure of cells to a variety of extracellular stimuli, such as UV radiation, causes an increase in the production of TNF-α or IL-6, which results in phosphorylation of IκB proteins in Ser32 and Ser36, followed by ubiquitination and their degradation. After this event, NFκB is released and moved to the nucleus where it activates specific gene transcription: pro-inflammatory cytokines, cyclooxygenase (COX) and nitric oxide synthase (NOS) - (Fig. 10.3) [82, 83]. Cyclooxygenase Cycloxygenase (COX) catalyzes the rate-limiting steps of the synthesis of prostaglandins from arachidonic acid. There are two COX isoforms, COX-1 and COX2. Under normal physiological conditions, COX-1 is expressed in most tissues and COX-2 is typically absent in most tissues; however, COX-2 is highly expressed by multiple stimuli [84, 85]. COX-2 is specifically upregulated in the epidermis as a result of chronic exposure to UVB radiation, which occurs via activation of two kinds of MAPKs, ERK1/2 and p38 kinase. Therefore, the level of COX-2 may serve as an early marker of acute skin inflammation [83, 84]. Antioxidant enzymes UV causes depletion of cellular antioxidants and antioxidant enzymes, such as SOD and catalase, promoting an alteration in the cellular redox status [14]. In addition, the pro-inflammatory mediators activated by UV light cause infiltration and activation of neutrophils and other phagocytic cells due to increased permeability of capillaries into the skin. This process results in the generation of reactive oxygen and nitrogen species. In general, the free radical produced will damage the cellular proteins, lipids and carbohydrates, which accumulate in the dermal and epidermal compartments, contributing to photoaging. Proteases Proteases like elastases and cathepsin G released from neutrophils cause further inflammation, and activation of MMPs. Pro-inflammatory cytokines and UVA radiation stimulate dermal fibroblasts to secrete MMPs, which degrade the collagen, fibronectin and gelatin of the dermal extracellular matrix [86]. Repetitive UV exposure provokes an intensive degradation of extracellular matrix components in the dermis by MMPs, which is one of the factors responsible for the wrinkled appearance of skin [87]. The activity of MMPs is regulated on three levels: synthesis, zymogen activation, and proteolytic activity inhibition by proteins called TIMPs (tissue inhibitors of metalloproteinases) [88]. The expression of MMP-1 (colagenase I), MMP-3 and MMP-9 (gelatinase B) is in part responsible for the damage caused to human skin by irradiation with UV light [84, 89]. Thus, an increase in MMP expression is associated with chronological skin aging, as well as

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Fig 10.3 Schematic NFκB signaling pathway involved in the inflammation induced by UV radiation. Nuclear factor κB (NFκB) is formed by p50 and p65 subunits, and its inactive form is maintained in the cytoplasm through a strong association with inhibitory κB (IκB). An extracellular stimulus such as UV radiation results in the IκB phosphorylation by IκB kinase (IκK) proteins and subsequent activation of NFκB through IκB ubiquitination followed by degradation. This allows the release of NFκB, and its translocation into the nucleus to act as a transcription factor of specific genes, for example, COX-2 (COX2; Cyclooxygenase-2), iNOS (NOS2; Inducible Nitric Oxide Synthase), PCNA (Proliferating Cell Nuclear Antigen), and Cyclin D1 (CCND1). Illustration by Sarah Azoubel Lima.

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the deleterious effect of UV radiation [88]. Cathepsins are a family of lysosomal proteases that are abundant in all kind of cells. Cathepsin B plays a key role in changes in the human dermal extracellular matrix, cellular apoptosis, cellular senescence and tumorigenesis in skin [90]. Cathepsin K is a member of the cysteine protease family with strong collagenolytic and elastolytic activities. UV light upregulates cathepsin K expression in fibroblasts in vitro, and this increase is accompanied by increased degradation of elastic fibers. This upregulation was observed to occur to a greater degree with UVA radiation compared with UVB radiation [91].

10.3.2 Molecular Marker of Senescence Cellular senescence occurs when the cell enters a non-replicative state. Human senescent cells display characteristics such as spread morphology, irreversible cell cycle arrest, preferentially, but not exclusively, in G1-S boundary, apoptosis resistance and an increased amount of the most universal senescence molecular biomarker, senescence-associated β-galactosidade (SA-β-Gal) [92]. During senescence some lysosome activities are enhanced, among them, SA-β-Gal activity, which is detectable at pH 6 and allows the identification of senescent cells both in cultures and in mammalian tissues [93].

10.4 Nanocosmetics and Nanomedicine for Overcoming Skin Photoaging Photoaging results from the superimposition of photodamage on intrinsically aged skin, leading to premature skin aging [40]. The skin consists of three layers, the epidermis, the dermis and the subcutis. The outer part is the epidermis, which is composed mainly of keratinocytes distributed in different sub-layers: the stratum basale, stratum spinosum, stratum granulosum, and the most superficial layer, the stratum corneum (SC) [94]. The SC is the primary skin barrier to diffusion and it is filled with a quasi-continuous lipidic matrix [95]. The region below the epidermis, the dermis, consists mainly of a supporting matrix composed by protein fibers, such as collagen, reticulin and elastin, which are produced by fibroblasts. The deepest part of skin, the subcutis, consists of lipocytes and loose connective tissue [94]. Photoaging involves complex alterations in important structural components such as cleavage of fibrillar collagen and disruption of elastin fiber, as well as the production of ROS [40]. Therefore, many cosmetic and cosmeceutical formulations are composed of bioactive species such as antioxidants, alphahydroxy acids, vitamins, retinoids, 2-dimethylaminoethanol (DMAE), and DNA repair enzymes. However, due to the low penetration and/or low stability of these bioactive species, they are commonly associated with nanostructures to enhance their effectiveness. Moreover, the reduced size of nanocarriers allows their interaction with cells and subcellular structures, including macromolecules [96, 97]. Vesicle nanocarriers, such as liposomes, nanosomes, niosomesTM, ethosomes and transfersomes®, are the most commonly used systems in cosmetic

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applications because they may incorporate hydrophobic species, improving the stability and skin tolerance of ingredients, contributing to the safety of cosmetics [95, 98, 99, 100]. Nanoemulsions, nanocapsules and nanospheres have been shown to favor the topical administration of active compounds, mainly due to their increased performance in terms of skin hydration, transparency and sensation to touch [97, 100, 101]. However, important features such as the controlled release of lipophilic substances, increased adhesiveness of the bioactive substance when in contact with the skin, increased compound-loading capacity, and higher stability during storage have been achieved with these nanocarriers [102, 103, 104, 105, 106, 107]. In addition to these systems, fullerenes and their derivatives have been studied as antioxidants and cytoprotective agents in cosmetic formulations, mainly due to their ability to quench ROS [108]. Bioactives Singlet oxygen generated on the surface of the skin can react with free amino acids and oligopeptides, and bind with polysaccharides, ceramides, inter-corneocyte lipids and squalene. These reactions can damage the stratum corneum as well as produce toxic diffusible species that can result in injury to the epidermis and affect the inflammatory response [109]. To counteract these injuries, enzymatic and non-enzymatic antioxidant systems, such as SOD, catalase and glutathione peroxidase are activated. However, these intrinsic antioxidant systems are unable to counterbalance the oxidative attack, and products are required to supply the antioxidant defense [110]. Therefore, many anti-aging cosmetic formulations contain bioactives. Antioxidants The main objective of the use of antioxidants is to revert the depletion of naturally occurring antioxidant reservoirs, and reduce the effects of free radical attack [110]. There are several antioxidants available on the market including coenzyme Q10 (CoQ10), vitamins C and E, and fullerenes. CoQ10, an essential electron carrier in cellular respiration, has been demonstrated to prevent photoaging in topical application. It penetrates into the viable epidermis, reducing the level of oxidation and wrinkle depth, and the detrimental effects of UVA on dermal fibroblasts [111, 112]. The benefits of vitamin C include collagen synthesis and inhibition of MMP-1 in the skin, leading to a reduction in wrinkles. This vitamin can also improve photoprotection due to its antioxidant and anti-inflammatory activities and to its inhibitory action on tyrosinase, which is responsible for depigmenting solar lentigines [113]. One limitation to the topical use of vitamin C is its stability, which is compromised as soon as the product is opened and exposed to air and light. To enhance CoQ10 penetration into skin as well as increase the stability of vitamin C, several nanostructured systems, such as nanostructured lipid carriers (NLC), nanocapsules and nanoemulsions [107, 110, 111, 112] have been researched. Alpha-hydroxy acids Alpha-hydroxy acids, also referred to as fruit acids, cause a desquamation of the outer layers of the skin improving its texture, reducing signs of aging, and

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restoring hydration. Although the action mechanism of AHAs is not completely understood, one hypothesis is that they can reduce the calcium ion concentration in the epidermis and reduce the cell adhesion resulting in desquamation. This reduction in the calcium ion levels can promote cell growth and slow cell differentiation, giving rise to younger looking skin [114, 115]. Retinoids Vitamin A and its derivatives can bind to specific nuclear receptors, and modulate the expression of the genes involved in cellular differentiation and proliferation, normalizing cell keratinization [116, 117]. Moderate cutaneous side effects, especially erythema, desquamation, peeling and burning, as well as increased sensitivity to sunlight, can be avoided with drug delivery system based on nanotechnology [118, 119, 120]. Hyaluronic acid Hyaluronic acid (HA) is a dermal filler and encapsulated low molecular weight HA can be delivered to the dermis where it will be hydrated, thus filling in the wrinkles [121]. DMAE DMAE is an analog of the B vitamin choline and is a precursor of acetylcholine. The benefits of DMAE in dermatology include a potential anti-inflammatory effect and a documented increase in skin firmness with possible improvement in underlying facial muscle tone [122]. The mode of action of DMAE in dermatology is unknown. There have been speculations regarding the possible interference with cholinergic neurotransmission (perhaps analogous with the anti-wrinkle effect of botulinum toxin) and a mild anti-inflammatory effect. Morissette et al. [123] hypothesized that DMAE applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. DNA repair enzymes DNA repair enzymes are one of the new trends in the cosmeceutical field, with promising potential for use in therapy. Draelos et al. [124] tested a moisturizing cream containing the DNA repair enzymes photolyase, UV endonuclease, and 8oxo-guanine glycosylase (OGG1). This pilot study suggested that the topical application of DNA repair enzymes could produce an improvement in subjects with photodamaged skin during a treatment course of 12 weeks. Another DNA repair enzyme, the bacteriophage T4 endonuclease V, is able to substitute the UVdamaged enzyme complex in humans to initiate excision repair. Preclinical studies showed that T4 endonuclease V encapsulated in liposomes (T4N5 liposomes) cross the stratum corneum, reinforcing the DNA repair in the cytoplasm and in the nuclei of keratinocytes in mouse and human skin [120, 125]. Some examples of nanocosmetic products and active ingredients are listed in Table 10.1.

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Table 10.1 Examples of nanocosmetic products and active ingredients on the market. Trade name

Active agent/delivery system

Proposed application

Revitalift

Pro-retinol A/nanosomes Anti-wrinkle

Name of company/ distributor L’oreal

Rénergie Microlift Micro filters (silica and Anti-aging moisturizer protein)/nanoparticles

L’oreal

Advanced Night Repair Protective

Liposome

Skin repair

Estée Lauder

Platinéum

Hydroxyapatite/

Anti-aging

Lancôme

Nanoparticles Radical sponge

Fullerene (C60 nanoparti- Antioxidant and anticles) melanogenesis

Vitamin C60 BioResearch

Biosome DMAE

Liposome/DMAE

Improvement in the skin elasticity

Viafarma

Epiderfill

Nanospheres/low molecular weight hyaluronic acid

Wrinkle filler

Viafarma

Biosome GB

Ginkgo biloba/liposome Antioxidant, vasodilator, stimulator of elastin and collagen production

Viafarma

Collagen Stimula- Stabilized vitamin C se- Stimulation of collagen production tion Factor MAPTM rum/nanocapsules

Cosmetochem

Nano-Lipobelle DN CoQ10, vitamins E and Anti-photoaging and CoQ10 oA C/nanoemulsion metabolic activation

MibelleBiochemistry

NanoVit nc/oA

Sea buckthorn pulp oil, Cell regeneration, protection MibelleBiochemistry vitamin E/nanoemulsion against UV damage

Lipobelle Soyagly- Soy isoflavone aglycone Antioxidant cone (genistein)/liposome

MibelleBiochemistry

10.4.1 Photoprotection Although many formulations to attenuate photodamage have been described, there is a general consensus that prevention is the best way to avoid the undesirable effects of UV exposure. Sun-protection products act by absorbing and/or scattering a large fraction of the incident radiation, thus reducing the number of photons that would be absorbed by endogenous photosensitizers. These molecules can transfer the absorbed energy generating reactive oxygen species, which can induce damage, such as lipid peroxidation and protein oxidation [109, 126]. Sunscreen is effective in decreasing the incidence of actinic keratoses and SCCs [127]. Several methods based on nanoparticulate systems have been used to increase the efficiency and stability of sunscreens and to decrease the skin penetration of the active compounds present in sunscreens. For instance, Wissing and Müller (2001) [128] proposed a sunscreen system based on tocopherol acetate incorporated into solid lipid nanoparticles (SLNs). Perugini et al. [129] proposed the

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encapsulation of cinnamates into nanoparticles of poly-D,L-lactide-co-glycolide, thus increasing their efficacy due to decreased photodegradation [127, 130]. Another approach involving nanostructures is the use of ethosomes to deliver vitamin E into the deeper layers of skin. Encapsulation of vitamin E into ethosomes reportedly enhanced the delivery of vitamin E into the cells located in the deep skin strata [131]. The most common nano-sized materials present in sunscreens are TiO2 and ZnO. Nano-sized TiO2 and ZnO provide superior UV protection by reflecting physically the light and eliminating the unsightly white residues. The inclusion of nanomaterials in day care products has prompted concern regarding the systemic absorption of these particles [94, 127, 132, 133]. An extensive discussion is underway regarding this issue. The Scientific Committee on Consumer Products (SCCP) published an opinion on the safety of nanomaterials in cosmetic products in 2007 [97]. They concluded that based on the current knowledge there is no conclusive evidence for skin penetration into viable tissues by nanoparticles of 20 nm or larger, which are used in metal oxide sunscreens. However, a large number of in vitro and in vivo studies have shown that the nano-sized TiO2 and ZnO remain in the stratum corneum. Although there is no regulation in the United States regarding the testing and labeling standards for sunscreens with nano-TiO2 and ZnO [127], many review articles conclude that inorganic sunscreens with nano- or micro-sized TiO2 and ZnO have a good safety profile [94, 127, 132]. The great concern regarding skin photodamage is reflected in the large number of nanocosmetics and nanomedicines available for overcoming photoaging. In general, the daily use of formulations containing antioxidants, moisturizers and sunscreens in a suitable nanocarrier can avoid the undesirable effects of UV radiation.

10.5 Molecular Action of the Toxicity of Carbon Nanotubes in Skin Cells The application of nanostructures has seen a progressive increase in recent years in different fields. However, from the biological point of view there is little information available on the molecular effects of nanomaterials on the cellular components of skin. In this section we will describe some molecular mechanisms of the toxic effect of carbon nanotubes in some skin cell types. Ding et al. [134] have evaluated in detail the genome profiling of human skin fibroblasts exposed to multi-walled carbon nano-onions (MWCNOs) and multiwalled carbon nanotubes (MWCNTs). These authors observed an increase in cell death by apoptosis/necrosis and a 50% reduction in the proliferation rate of these cells when these nanoparticles were used in a concentrations of 0.6 (MWCNO) and 6 mg/mL (MWCNO). In relation to the genome profiling the treatment of human skin fibroblasts with carbon nanomaterials induced high levels of gene expression changes. MWCNO and MWCNT exposure activates genes that are involved in cellular transport, metabolism, cell cycle regulation, and stress response. Interestingly, it was observed that interferon and p38/ERK-MAPK cascades are pivotal pathway mediators in the induced signal transduction, contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs. ROS generation and activation of NFκB are two events also associated

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with the toxic effect of the single-walled carbon nanotubes (SWCNTs) in human keratinocytes (HaCaT cell line) - [135]. A set of stress responsive genes was differentially expressed in human BJ Foreskin cells treated with 6 μg/mL SWCNTs for 24 h [136]. The heme oxigenase genes (HMOX1 and HMOX2) were highly expressed, suggesting that inducible genes are activated under stress by this material. HMOXs are microsomal enzymes that catalyze the cleavage of the porphyrin to produce biliverdin, free heme iron, and carbon monoxide [137]. HMOX1 is involved in different physiological processes including antioxidative, antiinflammatory, antiproliferative, and antiapoptotic processes. Therefore, the human BJ Foreskin cell response towards SWCNTs might be a protective adaptation to the stress. Interesting, levels of ERCC4, a human gene involved in the nucleotide excision repair pathway, were also increased after SWCNT treatment.

10.6 Final Remarks UV-induced responses are dependent on many factors, such as the cellular target, duration of exposure, dose and type of UV radiation. Insights into the complex mechanisms defining the final outcome of exposure to UV radiation both in vitro and in vivo have increased over recent years, leading to improved formulations based on nanostructured carriers, mainly in the cosmetics field. Conversely, there is a lack of information on the effects, at the molecular level, of nanomaterials on the cellular components of skin. Thus, the optimization of established models for testing nanocarrier systems is only one of the challenges facing dermatotoxicology and the design of new formulations. Overall, optimal use of the existing formulations will require a better understand of the signaling networks which play a role in skin pathologies and senescence. This will certainly open new avenues for the development of novel possibilities for refined and specific future therapies and strategies to manage photoaging and to combat cancer and inflammatory diseases. Acknowledgments. Our research in this field was supported by the governmental agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Rede Nanocosméticos/CNPq.

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[73] Organization for Economic Co-operation and Development (OECD) (2002) OECD Guideline for the testing of chemicals: In vitro 3T3 NRU photoxicity test. Paris: OECD (2002a), Guideline Draft TG 432, http://www.oecd.org/home/ (accessed November 11, 2003) [74] Werth, V.P., Bashir, M.M., Zhang, W.: IL-12 completely blocks ultraviolet-induced secretion of tumor necrosis factor α from cultured skin fibroblasts and keratinocytes. J. Invest Dermatol. 120, 116–122 (2003) [75] Grone, A.: Keratinocytes and cytokines. Vet. Immunol. Immunopathol. 88, 1–12 (2002) [76] Li, D., Turi, T.G., Schuck, A., Freedberg, I.M., Khitrov, G., Blumenberg, M.: Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination. FASEB J. 15, 2533–2535 (2001) [77] Shaulian, E., Karin, M.: AP-1 in cell proliferation and survival. Oncogene 20, 2390– 2400 (2001) [78] Ghosh, S., May, M.J., Kopp, E.B.: NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Ann. Rev. Immunol. 16, 225–260 (1998) [79] Huang, T.T., Miyamoto, S.: Postrepression activation of NFκB requires the aminoterminal nuclear export signal specific to IκBα. Mol. Cell Biol. 21, 4737–4747 (2001) [80] Tam, W.F., Sen, R.: IκB family members function by different mechanisms. J. Biol. Chem. 276, 7701–7704 (2001) [81] Sée, W.A.: Bicalutamide adjuvant to radical prostatectomy. Rev. Urol. 6(2), 20–28 (2004) [82] Karin, M., Ben-Neriah, Y.: Phosphorylation meets ubiquitination: the control of NFκB activity. Annu. Rev. Immunol. 18, 621–663 (2000) [83] Park, J.Y., Pillinger, M.H., Abramson, S.B.: Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin. Immunol. 119, 229–240 (2006) [84] Anggakusuma, Yanti, Hwang, J.K.: Effects of macelignan isolated from Myristica fragrans Houtt. On UVB-induced matrix metalloproteinase-9 and cyclooxygenase-2 in HaCaT cells. J. Dermatol. Sci. 57, 114–122 (2010) [85] Lee, K.M., Lee, K.W., Jung, S.K., Lee, E.J., Heo, Y.S., Bode, A.M., Lubet, R.A., Lee, H.J., Dong, Z.: Kaempferol inhibits UVB-induced COX-2 expression by suppressing Src kinase activity. Biochem. Pharmacol. 80, 2042–2049 (2010) [86] Fagot, D., Asselineau, D., Bernerd, F.: Matrix metalloproteinase-1 production observed after solar-simulated radiation exposure is assumed by dermal fibroblasts but involves a paracrine activation through epidermal keratinocytes. Photochem. Photobiol. 79, 499–505 (2004) [87] Chang, K.C.N., Wang, Y., Oh, I.G., Jenkins, S., Freedman, L.P., Thompson, C.C., Chung, J.H., Nagpal, S.: Estrogen receptor β is a novel therapeutic target for photoaging. Mol. Pharmacol. 77, 744–750 (2010) [88] Rittié, L., Fisher, G.J.: UV-light-induced signal cascades and skin aging. Ageing. Res. Rev. 1, 705–720 (2002) [89] Fisher, G.J., Wang, Z.Q., Datta, S., Varani, J., Kang, S., Voorhees, J.J.: Pathophysiology of premature skin aging induced by ultraviolet light. N. Engl. J. Med. 337, 1419–1428 (1997) [90] Lai, W., Zheng, Y., Ye, Z.Z., Su, X.Y., Wan, M.J., Gong, Z.J., Xie, X.Y., Lui, W.: Changes of cathepsin B in human photoaging skin both in vivo and in vitro. Chin. Med. J (Engl.) 12, 527–531 (2010)

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[91] Condriansky, K.A., Quintanilla-Dieck, M.J., Gan, S., Keady, M., Bhawan, J., Rünger, M.: Intracellular degradation of elastin by cathepsin in skin fibroblast- a possible role in photoaging. Photochem. Photobiol. 85, 1356–1363 (2009) [92] Bernard, D., Gosselin, K., Monte, D., Vercamer, C., Bouali, F., Pourtier, A., Vandenbunder, B., Abbadie, C.: Involvement of Rel/Nuclear Factor-κB transcription factors in keratinocytes senescence. Cancer. Res. 64, 472–481 (2004) [93] Debacq-Chainiaux, F., Erusalimsky, J.D., Campisi, J., Toussaint, O.: Protocols to detect senescence-associated beta-galactosidase (SA-beta-Gal) activity, a biomarker of senescent cells in culture and in vivo. Nat. Protoc. 4, 1798–1806 (2009) [94] Gonzalez, H.: Percutaneous absorption with emphasis on sunscreens. Photochem. Photobiol. Sci. 9, 482–488 (2010) [95] Cevc, G., Vierl, U.: Nanotechnology and the transdermal route: A state of the art review and critical appraisal. J. Control Release 141, 277–299 (2010) [96] Sakamoto, J.H., Smith, B.R., Xie, B., Rokhlin, S.I., Lee, S.C., Ferrari, M.: The molecular analysis of breast cancer utilizing targeted nanoparticle-based ultrasound contrast agents. Technol. Cancer Res. Treat. 4, 627–636 (2005) [97] SCCP - Scientific Committee on Consumer Products, Preliminary opinion on safety of nanomaterials in cosmetic products, 1–56 (2007) [98] Dayan, N., Touitou, E.: Carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs. liposomes. Biomaterials 21, 1879–1885 (2000) [99] Elsayed, M.M.A., Abdallah, O.Y., Naggar, V.F., Khalafallah, N.M.: Lipid vesicles for skin delivery of drugs: Reviewing three decades of research. Int. J. Pharmaceutics 332, 1–16 (2007) [100] Nohynek, G.J., Dufour, E.K., Roberts, M.S.: Nanotechnology, cosmetics and the skin: Is there a health risk? Skin Pharmacol. Physiol. 21, 136–149 (2008) [101] Puglia, C., Rizza, L., Drechsler, M., Bonina, F.: Nanoemulsions as vehicles for topical administration of glycyrrhetic acid: Characterization and in vitro and in vivo evaluation. Drug Delivery 17, 123–129 (2010) [102] Mehnert, W., Mäder, K.: Solid lipid nanoparticles: production, characterization. Adv. Drug Deliv. Rev. 47, 165–196 (2001) [103] Wissing, S.A., Müller, R.H.: The influence of solid lipid nanoparticles on skin hydration and viscoelasticity – in vivo study. Eur. J. Pharm. Biopharm. 56, 67–72 (2003) [104] Guterres, S.S., Alves, M.P., Pohlmann, A.R.: Polymeric nanoparticles. Drug Target Insights 2, 147–157 (2007) [105] Souto, E.B., Müller, R.H.: Cosmetic features and applications of lipid nanoparticles (SLN®, NLC®). Int. J. Cosmetic. Sci. 30, 157–165 (2008) [106] Pardeike, J., Hommoss, A., Müller, R.H.: Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int. J. Pharm. 366, 170–184 (2009) [107] Terroso, T., Külkamp, I.C., Jornada, D.S., Pohlmann, A.R., Guterres, S.S.: Development of semi-solid cosmetic formulations containing coenzyme Q10-loaded nanocapsules. Lat. Am. J. Pharm. 28, 819–826 (2009) [108] McEwen, C.N., McKay, R.G., Larsen, B.S.: C60 as a radical sponge. J. Am. Chem. Soc. 114, 4412–4414 (1992) [109] Giacomoni, P.U., Teta, L., Najdek, L.: Sunscreens: the impervious path from theory to practice. Photochem. Photobiol. Sci. 9, 524–529 (2009) [110] Kaur, I.P., Kapila, M., Agrawal, R.: Role of novel delivery systems in developing topical antioxidants as therapeutics to combat photoageing. Ageing Res. Rev. 6, 271–288 (2007)

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[111] Zhou, H., Yue, Y., Liu, G., Li, Y., Zhang, J., Yan, Z., Duan, M.: Characterization and skin distribution of lecithin-based coenzyme Q10-loaded lipid nanocapsules. Nanoscale Res. Lett. 5, 1561–1569 (2010) [112] Yue, Y., Zhou, H., Liu, G., Li, Y., Yan, Z., Duan, M.: The advantages of a novel CoQ10 delivery system in skin photo-protection. Int. J. Pharm. 392, 57–63 (2010) [113] Burke, K.E.: Interaction of vitamins C and E as better cosmeceuticals. Dermatol. Ther. 20, 314–321 (2007) [114] Wang, X.: A theory for the mechanism of action of the alpha hydroxy acids applied to the skin. Med. Hypotheses 53, 380–382 (1999) [115] Rivers, J.K.: The role of cosmeceuticals in antiaging therapy. Skin Therapy Lett. 13, 5–9 (2008) [116] Sorg, O., Antille, C., Kaya, G., Saurat, J.-H.: Retinoids in cosmeceuticals. Dermatologic Ther. 19, 289–296 (2006) [117] Serri, R., Iorizzo, M.: Cosmeceuticals: focus on topical retinoids in photoaging. Clin. Dermatol. 26, 633–635 (2008) [118] Yamaguchi, Y., Nagasawa, T., Nakamura, N., Takenaga, M., Mizoguchi, M., Kawai, S., Mizushima, Y., Igarashi, R.: Successful treatment of photo-damaged skin of nano-scale at RA particles using a novel transdermal delivery. J. Control Release 104, 29–40 (2005) [119] Shah, K.A., Date, A.A., Joshi, M.D., Patravale, V.B.: Solid lipid nanoparticles (SLN) of tretinoin: potential in topical delivery. Int. J. Pharm. 345, 163–171 (2007) [120] de Leeuw, J., de Vijlder, H.C., Bjerring, P., Neumann, H.A.M.: Liposomes in dermatology today. J. Eur. Acad. Dermatol. Venereol. 23, 505–516 (2009) [121] Viafarma: Revista Fórmulas e Tecnologia 4, 1–11 (2006), http://www.viafarmanet.com.br (accessed August 06, 2010) [122] Grossman, R.: The Role of dimethylaminoethanol in cosmetic dermatology. Am. J. Clin. Dermatol. 6, 39–47 (2005) [123] Morissette, G., Germain, L., Marceau, F.: The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology. Brit. J. Dermatol. 156, 433–439 (2007) [124] Draelos, Z., Yarosh, D., Lang, G., Smiles, K.: Improved photoaging with topical DNA repair enzymes. J. Am. Acad. Dermatol. AB2, P107 (2009) [125] Yarosh, D., Tsimis, J., Yee, V.: Enhancement of DNA repair of UV damage in mouse and human skin by liposomes containing a DNA repair enzyme. J. Soc. Cosmet. Chem. 41, 85–92 (1990) [126] Pazos, M.D.C., Nader, H.B.: Effect of photodynamic therapy on the extracellular matrix and associated compounds. Braz. J. Med. Biol. Res. 40, 1025–1035 (2007) [127] Wang, S.Q., Balagula, Y., Osterwalder, U.: Photoprotection: a review of the current and future technologies. Dermatol. Ther. 23, 31–47 (2010) [128] Wissing, S.A., Muller, R.H.: A novel sunscreen system based on tocopherol acetate incorporated into solid lipid nanoparticles. Int. J. Cosmet. Sci. 23, 233–243 (2001) [129] Perugini, P., Simeoni, S., Scalia, S., Genta, I., Modena, T., Conti, B., Pavanetto, F.: Effect of nanoparticle encapsulation on the photostability of the sunscreen agent, 2ethylhexyl-p-methoxycinnamate. Int. J. Pharm 246, 37–45 (2002) [130] González, S., Fernández-Lorente, M., Gilaberte-Calzada, Y.: The latest on skin photoprotection. Clin. Dermatol. 26, 614–626 (2008) [131] Touitou, E., Godin, B.: Skin nonpenetrating sunscreens for cosmetic and pharmaceutical formulations. Clin. Dermatol. 26, 375–379 (2008)

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Chapter 11

Nanomedicine: Potential Killing of Cancercells Using Nanoparticles Patricia da Silva Melo1,2, Priscyla D. Marcato3, and Nelson Durán3 1

Biological Institute, Universidade Estadual de Campinas (Unicamp), Brazil Veris Faculdades, Campinas, Brazil 3 Chemistry Institute, Universidade Estadual de Campinas (Unicamp), Brazil 2

Abstract. This chapter describes apoptosis as an active process of cellular deconstruction and compares it morphologically with necrosis. In contrast to necrosis, apoptosis involves the regulated action of catabolic enzymes (proteases and nucleases) within the limits of near-to-intact plasma membranes. It also involves the activation of specific caspases that cleave at specific sites after aspartic acid residues. In this context cell death by necrosis and cell death by apoptosis signaling are also discussed in this chapter. One strategy to achieve efficient drug delivery it is to understand the interactions of nanomaterials with the biological environment, targeting cell-surface receptors, drug release, multiple drug administration, stability of therapeutic agents and molecular mechanisms of cell signaling involved in the pathology of several diseases. These aspects are discussed in this chapter mainly in terms of polymeric and silver nanoparticles, in both cases with reference to in vitro and in vivo experiments. The perspectives with regard to the encapsulation of anticancer drugs in nanosystems are also discussed. All of the information available to date indicates that nanomedicine will play a crucial role in cancer treatment.

11.1 General Introduction Due to the increased use of nanoparticles in cosmetic products the risks associated with these particles have to be investigated. Different biological methods can be applied to study the cytotoxicity effect of nanoparticles. In this context, the evaluation of apoptosis and other endpoints (e.g. evaluation of cell cycle progression, cell differentiation and senescence) are essential, mainly in relation to cancer risks. The term “apoptosis” describes an active process of cellular deconstruction which differs morphologically from necrosis. It is a form of conserved cell death that occurs in several physiological and pathological circumstances in multicellular organisms and constitutes a common mechanism of cell replacement, tissue

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remodeling, and removal of damaged cells. Apoptosis is a complex process characterized by cell shrinkage, chromatin condensation, internucleosomal DNA fragmentation, and the formation of “apoptotic bodies” since proliferation and apoptosis are intimately linked [1,2]. Some cell cycle regulators can influence both cell division and programmed cell death: the link between the cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, Ras, PKA, PKC, Bcl-2, cyclins and CK1. The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes including cell death. Several protease families are also implicated in apoptosis, the most prominent being caspases. Caspases are cysteine-containing, aspartic acid-specific proteases which exist as zymogen in the soluble cytoplasm, mitochondrial intermembrane space, and nuclear matrix of virtually all cells. Apoptosis is the subject of intense research due to the susceptibility of tumor cells, including melanoma, which have been found to undergo this kind of cell death in response to anti-tumoral agents [3,4]. This chapter discusses the apoptosis mechanism and the cytotoxicity of polymeric and silver nanoparticles.

11.2 Apoptosis Apoptosis is a morphological phenomenon of programmed cell death, identified by Kerr, Willie and Durrie in 1972 [1]. Visualized by microscopy, the characteristics of the apoptotic cell include chromatin condensation and nuclear fragmentation, plasma membrane blebs and cell shrinkage [2]. Programmed cell death plays a critical role in a wide variety of physiological processes, such as endometrial loss during menstruation and during embryogenesis. It is a mechanism tightly controlled by genetic expressions resulting in changes in the protein expression, protein-protein interactions, and various post-translational modifications, including proteolytic cleavage and phosphorylation due to interactions between the cell and external environment. This process of cell death plays a crucial role in the tissue homeostasis and it is important in some pathologies including cancer. Cancer is usually associated with aberrant cell cycle progression and defective apoptosis induction due to the activation of proto-oncogenes and/or inactivation of the tumor suppressor gene. The evolution of molecular science often provides targets for cancer therapy, since the ability of cancer chemotherapeutic agents to induce cell cycle arrest and apoptosis is an important determinant of their therapeutic response [3, 4].

11.3 Apoptosis Cell Death: Biochemistry and Molecular Aspects Apoptosis plays a regulatory role in the cell proliferation control distinguished from necrosis by unique events including the degradation of chromatin into internucleosomal fragments, a loss of cellular volume, the formation of blebs in the plasma cellular membrane, changes in the mitochondrial membrane potential and phosphatidylserine translocation. Thus, the surface exposure of the phospholipid

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residues (normally on the inner membrane leaflet) allows for the recognition and elimination of apoptotic cells by their healthy neighbors, before the membrane breaks up and organelles and cytosol spill into the intercellular space and elicit inflammatory reactions [5]. In contrast to necrosis, apoptosis involves the regulated action of catabolic enzymes (proteases and nucleases) within the limits of near-tointact plasma membranes. It also involves the activation of specific cysteine proteases (caspases) that cleave at specific sites after aspartic acid residues. Caspases are a unique and closely related set of proteases, so called because of the cysteine at their active site, and the strictly defined four-amino motif (including aspartate at positions 1 and 4) at their target site [6]. Differently to apoptosis, necrosis does not involve any regular DNA and protein degradation pattern and is accompanied by swelling of the entire cytoplasm and of the mitochondrial matrix, which occurs before the membrane ruptures. Cell death by necrosis occurs, generally, in response to severe injury to the cells and is characterized morphologically by cytoplasmatic and mitochondrial swelling, plasmatic membrane rupture and cell content liberation. Consequently, inflammatory response occurs, inducing the injury and cell death of neighboring cells. In cell death by necrosis changes occur in the mitochondrial function, decreasing the ATP production, altering the Na+/K+ pump, triggering cellular swelling due to an increase in the cytosolic Na+. The increase in cytosol Ca2+ induces phospholipase and protease activation, reactive oxygen species (ROS) formation, plasmatic membrane rupture and the migration of signaling macrophages. In contrast to the cell retraction observed in apoptotic cells, in necrosis there is cell swelling due to a loss of membrane permeability. On the other hand, the apoptotic process involves the active participation of affected cells in the self destruction cascade resulting in DNA degradation by endonuclease activation, nuclear disintegration and apoptotic bodies [7]. These are removed from the tissue by macrophages and the signaling occurs through phosphatidylserine externalization designating the cells to be phagocytized. The majority of morphological changes observed in apoptotic cells are due to the activation of caspases. The caspases form a cascade in which they are activated by lethal stimuli arising either at the cell membrane as a result of cytokinereceptor binding, or within the cell, in relation to internally determined signals, often generated in the micro-environment of particular organelles [6].

11.4 Cell Death by Apoptosis Signaling Apoptosis is characterized by multiple molecular events occurring in cascade triggered by an initial event resulting in the collapse of the cellular structure. The majority of signals involved in cell death can be classified as intrinsic or extrinsic pathways. In both categories the caspase cascades end with the cleavage of proteins and in the activation of caspase activated DNase inducing DNA fragmentation. For apoptosis to be initiated by death receptors such as Fas (CD95) and TNF-R1 (tumor necrosis factor receptor), pro-caspase-8 or -10 needs to be present in the complex. However, apoptotic stimuli triggered by chemotherapeutic agents can occur independently of this pathway [8]. In this case, the mitochondrial

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pathway is activated, inducing loss of mitochondrial transmembrane potential, activation of mitochondrial permeability transition (MPT), enhancement of MPT pore opening, and mitochondrial volume enlargement which results in the release of cytochrome c, endonuclease, and apoptosis-inducing factor to the cytosol. When cytochrome c is released to the cytosol it forms a complex called apoptosome, with apoptotic protease activating factor-1 (Apaf-1) and caspase-9. The mitochondrial pathway is frequently activated in response to DNA damage, involving the activation of a pro-apoptotic Bcl-2 family member (Bax, Bid). Proand anti-apoptotic members of the Bcl-2 family control the cytochrome c release from the inner mitochondrial membrane. The anti-apoptotic Bcl-2 family members inhibit the cell death by apoptosis blocking the formation of pores in the mitochondrial membrane and, consequently, inhibiting the cytochrome c release [7, 8]. Multiple stress-inducible molecules, such as p53, JNK (Jun N-terminal kinase), NF-ĸB (necrosis factor) and PKC/MAPK/ERK have an important influence on apoptosis. The MAPK pathway (mitogen-activated protein kinase) includes the subfamilies extracellular signal-regulated kinase (ERK), JNK and p38, being regulated by different extracellular stimuli [9].

11.5 Cell Death and Cancer Cancer involves alterations in the cell cycle control and diminished apoptosis due to the inactivation of genes involved in tumor suppressor and/or activation of proto-oncogenes. These molecular events can be used as targets for chemotherapy [10].The inactivation of programmed cell death is central to the development of cancer. This disabling of apoptotic responses might be a major contributor to treatment resistance and to understanding the main mechanisms for the death of cancer cells [3]. Cancer cells ‘escape’ from recognition and elimination by the host immune system through various strategies. These include the altered expression of proteins and genes involved in cell survival, death and transformation. The inactivation of p53 or mutation in this gene is found in 50% of human cancers, being considered the “guardian of the genome”. The p53 gene is important in terms of tumor suppression because it induces apoptosis mediated by growth arrest and by blocking angiogenesis [10]. Melanoma is the type of skin cancer associated with the highest death toll in the United States and Europe and has been categorized as the most aggressive form of skin cancer. Its incidence has increased worldwide over the last 50 years. The frequency of p53 mutations in melanomaderived specimens is reported to be 5-25% which is relatively low compared with many other cancers, and may be explained by the frequent loss of p14ARF in melanoma through CDKN2A mutations and deletions. p14ARF is a positive regulator of p53, and loss of p14ARF function may have the same effect as the loss of p53 function [11]. In melanoma, p53 protein levels increase with tumorigenesis and tumor development, and despite the presence of functional p53, melanoma is generally regarded as a chemoresistant tumor type. One possible explanation for the development of the resistant phenotype could be the upregulation of p53 antagonists, such as truncated p53 family members [12].

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The MAPK-ERK pathways control the cell proliferation and growth, and have been implicated in the melanoma progression, enhanced cell survival and resistance to apoptosis [13].

11.6 Nanomedicine for the Management of Cancer Cells Nanoparticles can be used as drug delivery systems to transport the drug to the target cell site, to improve the uptake of poorly soluble drugs, and to increase drug bioavailability. Nanosystems with several compositions and biological properties have been extensively investigated for drug and gene delivery applications [14, 15]. To achieve efficient drug delivery it is important to understand the interactions of nanomaterials with the biological environment, targeting cell-surface receptors, drug release, multiple drug administration, stability of therapeutic agents and molecular mechanisms of cell signaling involved in the pathology of several diseases [16].

11.6.1 Polymeric Nanoparticles Polylactic/glycolic acid (PLGA), chitosan, Pluronic® block copolymers and PLGA-based nanoparticles have been used in nanotherapy. Pluronic® block copolymers induce changes in the cell functions, such as alterations in the mitochondrial respiration, the drug efflux transporters expressed in the cell membrane, the multi-drug resistance (MDR), the apoptotic signal transduction and the gene expression. Thus, Pluronic® copolymers can cause considerable sensitization of MDR tumors to various anticancer agents, enhance drug transport across the blood brain and intestinal barriers, and cause transcriptional activation of gene expression [17]. Multi-drug resistance in cancer cells can be acquired after treatment with chemotherapy or can be inherent to the cells, independent of the cell exposure to a substance. The development of MDR inhibits the treatment of cancer patients due to the persistence of the disease in spite of changes in the doses and chemotherapeutic agents used [4]. Multi-functional nanocarriers have been developed to enhance drug delivery and overcome the MDR of anticancer agents in refractory tumors (reported by Jabr-Milane et al., 2008 [4]). The subcellular trafficking is an important determinant of the efficacy of an anticancer agent. In MDR-cells treated with anthracyclines, it has been reported that, in addition to a decrease in the drug accumulation, the intracellular distribution of the drug is changed in epidermoid carcinoma cells [18]. Susa and collaborators [19] observed that the nanoparticle formulation containing doxorubicin is capable of delivering doxorubicin to the nucleus even for the MDR strains (in this situation the drug accumulates primarily in the cytoplasm of drug resistant cells). One probable mechanism associated with the efficacy of nanoparticles against the MDR cells is their ability to overcome efflux pumps such as Pgp in the cell membrane. Moreover several studies suggest that nanoparticle conjugates induce stronger activation of apoptosis-signaling pathways compared with the free drug [20]. Melanoma is associated with deregulation of the MAPK/ERK pathways, which regulate apoptosis and cellular proliferation, respectively. Tran and collaborators

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[21] demonstrated that combining nanoliposomal ceramid with sorafenib synergistically inhibits melanoma survival, decreasing tumor development by doubling apoptosis rates with negligible systemic toxicity. Nanoliposomal ceramide mediates apoptosis primarily by inactivation of Akt, which restores cancer cell apoptotic sensitivity, making them receptive to killing via chemotherapeutic agents such as sorafenib [21].

11.6.2 Silver Nanoparticles 11.6.2.1 In Vitro Experiments It has been reported that silver nanoparticles can induce cytotoxicity in HeLa cells in a lower concentration than silver ions and apoptosis is associated with the cell death. Also, the oxidative stress-related genes, such as ho-1 and mt-2A, were found to be LIP-regulated by silver nanoparticle treatment. Thus, the appropriate concentration of silver nanoparticles must be assessed in order to avoid the induced toxicity [22]. PVP-coated silver nanoparticles and silver ions reportedly induced apoptosis and necrosis in a human monocytic cell line (THP-1) in a dose- and exposure time-dependent manner. The EC50 of silver nanoparticles and silver ions indicated that silver ions were almost 4 times more toxic than silver nanoparticles. After exposure to 5 µg/ml of silver nanoparticles or 1.36 µg/ml of silver ions for 6 h a similar and significant increase in ROS was found to correlate with DNA breakage and high levels of apoptosis and necrosis. These data suggest that silver nanoparticles and silver ions can induce cell death in monocytes through an ROS-mediated apoptotic process (N-acetyl-cysteine prior to exposure to ions or nanoparticles reduces the cell death considerably after 6 h) [8]. Interaction with up to 100 µg/mL of silver nanoparticles did not change the morphology of primary fibroblasts and primary liver cells. The IC50 values for primary fibroblasts and primary liver cells indicated that primary fibroblasts were around 7 times more sensitive than primary liver cells. For the primary fibroblast at half the silver nanoparticle IC50value an enhancement of glutathione and depletion of lipid peroxidation were found. In the case of primary liver cells increased levels of superoxide dismutase and glutathione were observed. The caspase 3 activity assay indicated that the silver nanoparticle concentration required for the onset of apoptosis was much lower than the necrotic concentration. These results indicated that although silver nanoparticles enter the eukaryotic cells (mitochondria and cytoplasm) the antioxidant mechanisms protect the cells from oxidative damage [23]. Silver nanoparticles have been found to reduce the ATP content of the glioblastoma (U251) and in human lung fibroblast cells (IMR-90) resulting in damage to mitochondria and increased production of ROS in a dose-dependent manner. DNA damage was also dose-dependent and more prominent in the cancer cells. The nanoparticle treatment caused cell cycle arrest in the G(2)/M phase possibly due to the repair of damaged DNA and did not show high levels of apoptosis or necrosis. The presence of silver nanoparticles inside the mitochondria and nucleus were

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observed, implicating their direct involvement in the mitochondrial toxicity and DNA damage, probably by involving disruption of the mitochondrial respiratory chain with subsequent ROS production and inhibition of ATP synthesis, which cause DNA damage [24]. In a study with silver nanoparticles and the L929 cell cycle, it was observed that the cells were arrested in the G2M phase and analysis of the ultrastructure of the cells by transmission electronic microscopy showed that silver nanoparticles could be phagocytized. From an investigation into the in vitro cytotoxic mechanism of silver nanoparticles it has been suggested that silver nanoparticles are phagocytized into the cells and then interaction with the mitochondria or nuclei induced apoptosis or necrosis of the cells [25]. The cytotoxicity and genotoxicity of silver nanoparticles have been investigated using the dye exclusion, the comet and the mouse lymphoma thymidine kinase (tk(+/-)) gene mutation (MLA) assays. The IC20 values of silver nanoparticles in L5178Y and BEAS-2B cells were determined and the results of this study indicated that these particles can cause primary DNA damage and cytotoxicity but not mutagenicity in cultured mammalian cells [26]. Silver nanoparticles dispersed in fetal bovine serum showed cytotoxicity to RAW264.7 cells through an increase in the Cl fraction, indicating cellular apoptosis. A decrease in the intracellular glutathione level and increase in NO secretion, TNF-alpha protein and gene levels, and gene expression of the metalloproteinase matrix have been reported. The observation of silver nanoparticles in the cytosol of the activated cells and not in the dead cells was indicative that the silver nanoparticle exerted a cytotoxicty effect through ionized silver ions [27]. Baby hamster kidney (BHK21) and human colon adenocarcinoma (HT29) cell lines have been used in RT-PCR gene expression and flow cytometry analysis to probe the extent of apoptosis and atomic force microscopy to follow the cell membrane topology change induced by silver nanoparticles. The gene expression study revealed that silver nanoparticles induced the p53-mediated apoptotic pathway, indicating its potential as a therapeutic agent for nanomedicinal applications [28]. Silver nanoparticles acting on MLO-Y4 osteocytic and HeLa cervical cancer cells affected the shape and the size of the cells, however, an enhancement of the effect in the presence of etoposide or dexamethasone (chemotherapeutic agents) was observed. Through the caspase 3 behavior a strong interaction between silver nanoparticles and the protein structure of the cell was observed, allowing a high effective action of the apoptotic agent. The authors suggested that this observation may lead to a new class of cancer therapies through the combination of chemotherapeutic agents and nanomaterials [29]. 11.6.2.2 In Vivo Experiments Brain samples from the brain of adult male C57BL/6N mice were removed after 24 h of silver nanoparticle treatment [(i.p.) 100 mg/kg -1000 mg/kg] and dissected into different regions: caudate nucleus, frontal cortex and hippocampus. Total RNA was isolated from the samples and analyzed. The results indicated that the gene expression was affected by the treatment in all samples collected. This result indicates that silver nanoparticles may produce neurotoxicity by generating

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oxidative stress (free radicals) and by altering gene expression, producing apoptosis and neurotoxicity [30]. Mouse embryos (blastocyst stage) in contact with silver nanoparticles exhibited an increase in apoptosis with a decrease in the total cell number and the in vivo implantation of blastocysts was less effective than the control. All of the results indicated that in vitro exposure to silver nanoparticles induces apoptosis and retards early post-implantation development after transfer to host mice. Thus, silver nanoparticles have the potential to induce embryo cytotoxicity, but to a lesser extent than silver ions [31]. Silver hydrocolloids containing 50 µg/ml of nanoparticles were injected into eggs prior to incubation of the eggs and the embryo development was investigated after 48 h and 20 days. During decapitation blood was collected from the neck vein and the serum was obtained by centrifugation. Concentrations of glucose, triacyl-glyceride and cholesterol as VLDL, and activities of asparagine transferase, alanine transferase, and alkaline phosphatase in serum were determined by dry chemistry methods. Immediately after decapitation the livers were frozen in liquid nitrogen and stored at –80°C for future determination of 8-oxo-2’-deoxyguanosine concentration in the liver DNA. There was no negative effect of the nanoparticles on embryo survival, growth and development and morphology. The hydrocolloid did not affect the activity of the enzymes analyzed, the concentrations of glucose, triacylglyceride and cholesterol in blood serum or the genotoxicity measured as the concentration of 8-oxo-2’-deoxyguanosine in the liver DNA [32].

11.7 Future Perspectives Investigations into the regulation of apoptosis and growth arrest initiated by the cellular stress response have provided a detailed insight into fundamental cellular mechanisms with several clinical implications. The encapsulation of anticancer drugs in nanosystems has been proposed as a way to deal with MDR and trigger cell death by apoptosis. In the future it is possible that nanomedicine will play a crucial role in cancer treatment. Acknowledgments. The support from Brazilian Nanocosmetics Network is acknowledged.

References [1] Kerr, J.F.R., Wyllie, A.H., Durrie, A.R.: Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972) [2] Israels, L.G., Israels, E.D.: Apoptosis. Steam Cells 17, 306–313 (1999) [3] Brown, J.M., Attardi, L.D.: The role of apoptosis in cancer development and treatment response. Nature Rev. Cancer 5, 231–237 (2005) [4] Jabr-Milane, L.S., Van Vlerken, L.E., Yadav, S., Amiji, M.M.: Multi-functional nanocarriers to overcome tumor drug resistance. Cancer Treat. Rev. 34, 592–602 (2008)

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[5] Danial, N.N., Korsmeyer, S.J.: Cell Death: critical control points. Cell 116, 205–219 (2004) [6] Wyllie, A.H.: Where, o Death, is thy sting? A brief review of apoptosis biology. Mol. Neurobiol. 42, 4–9 (2010) [7] Hotchkiss, R.S., Strasser, A., Mcdunn, J.E., Swanson, P.E.: Cell death. N Engl. J. Med. 361, 1570–1583 (2009) [8] Foldbjerg, R., Olesen, P., Hougaard, M., Dang, D.A., Hoffmann, H.J., Autrup, H.: PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Tóxico Lett. 190, 156–162 (2009) [9] Herr, I., Debatin, K.M.: Cellular stress response and apoptosis in cancer therapy. Blood 98, 2603–2614 (2001) [10] Wang, Z., Sun, Y.: Targeting p53 for novel anticancer therapy. Translation Oncol. 3, 1–12 (2010) [11] Kyrgidis, A., Tzellos, T.G., Triaridis, S.: Melanoma: stem cells, sun exposure and hallmarks for carcinogenesis, molecular concepts and future clinical implications. J. Carcinog. 9, 1–15 (2010) [12] Thangasamy, T., Sittadjody, S., Mitchell, G.C., Mendoza, E.E., Radhakrishnan, V.M., Limesand, K.H., Burd, R.: Quercetin abrogates chemoresistance in melanoma cells by modulating •Np73. Np73. BMC Cancer 10, 282–392 (2010) [13] Palmieri, G., Capone, M., Ascierto, M.L., Gentilcore, G., Stroncek, D.F., Casula, M., Sini, M.C., Palla, M., Mozzillo, N., Ascierto, P.A.: Main roads to melanoma. J. Trans. Med. 7, 86–103 (2009) [14] Couvreur, P., Dubernet, C., Puisieux, F.: Controlled drug delivery with nanoparticles: current possibilities and future trends. Eur. J. Pharm. Biopharm. 4, 2–13 (1995) [15] Marcato, P.D., Durán, N.: New Aspects of Nanopharmaceutical Delivery Systems. J. Nanosci. Nanotechnol. 8, 2216–2229 (2008) [16] Suri, S.S., Fenniri, H., Singh, B.: Nanotechnology-based drug delivery systems. J. Occup. Med. Toxicol. 2, 16–22 (2007) [17] Batrakova, E.V., Kabanov, A.V.: Pluronic block copolymers: evolution of drug delivery concept form inert nanocarriers to biological response modifiers. J. Control. Release 130, 98–106 (2008) [18] Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesmann, M.M., Pastan, I.: Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and – sensitive KB cells: effects of verapamil and other drugs. Cancer Res. 46, 5941–5946 (1986) [19] Susa, M., Iyer, A., Ryu, K., Hornicek, F.J., Mankin, H., Amiji, M.M., Duan, Z.: Doxorubicin loaded polymeric nanoparticulate delivery system to overcome drug resistance in osteosarcoma. BMC Cancer 9, 399–411 (2009) [20] Jiang, Z., Chen, B.A., Xia, G.H., Wu, Q., Zhang, Y., Hong, T.Y., Zhang, W., Cheng, J., Gao, F., Liu, L.J., Li, X.M., Wang, X.M.: The reversal effect of magnetic Fe3O4 nanoparticles loaded with cisplatin on SKOV3/DDP ovarian carcinoma cells. Int. J. Nanomedicine 4, 107–114 (2009) [21] Tran, M.A., Smith, C.D., Kester, M., Robertson, G.: Combining nanoliposomal ceramide with sorafenid synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development. Clin. Cancer Res. 14, 3571–3581 (2008) [22] Miura, N., Shinohara, Y.: Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells. Biochem. Biophys. Res. Commun. 390, 733–737 (2009)

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[23] Arora, S., Jain, J., Rajwade, J.M., Paknikar, K.M.: Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol. Appl. Pharm. 236, 310–318 (2009) [24] AshaRani, P.V., Mun, G.L.K., Hande, M.P., Valiyaveettil, S.: Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano. 3, 279–290 (2009) [25] Wei, L.N., Tang, J.L., Zhang, Z.X., Chen, Y.M., Zhou, G., Xi, T.F.: Investigation of the cytotoxicity mechanism of silver nanoparticles in vitro. Biomed. Mat. 5 Special Issue: Sp (4) 044103 (2010) [26] Kim, Y.J., Yang, S.I., Ryu, J.C.: Cytotoxicity and genotoxicity of nano-silver in mammalian cell lines. Mol. cell. Toxicol. 6, 119–125 (2010) [27] Park, E.J., Yi, J., Kim, Y., Choi, K., Park, K.: Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism. Toxicol. In vitro 24, 872–878 (2010) [28] Gopinath, P., Gogoi, S.K., Sanpui, P., Paul, A., Chattopadhyay, A., Ghosh, S.S.: Signaling gene cascade in silver nanoparticle induced apoptosis. Colloids Surf. B-Biointerfaces 77, 240–245 (2010) [29] Mahmood, M., Casciano, D.A., Mocan, T., Iancu, C., Xu, Y., Mocan, L., Iancu, D.T., Dervishi, E., Li, Z.R., Abdalmuhsen, M., Biris, A.R., Ali, N., Howard, P., Biris, A.S.: Cytotoxicity and biological effects of functional nanomaterials delivered to various cell lines. J. Appl. Toxicol. 30, 74–83 (2010) [30] Rahman, M.F., Wang, J., Patterson, T.A., Saini, U.T., Robinson, B.L., Newport, G.D., Murdock, R.C., Schlager, J.J., Hussain, S.M., Ali, S.F.: Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanoparticles. Toxicol. Lett. 187, 15–21 (2009) [31] Li, P.W., Kuo, T.H., Chang, J.H., Yeh, J.M., Chan, W.H.: Induction of cytotoxicity and apoptosis in mouse blastocysts by silver nanoparticles. Toxicol. Lett. 197, 82–87 (2010) [32] Sawosz, E., Grodzik, M., Zielinska, M., Niemiec, T., Olszanka, B., Chwalibog, A.: Nanoparticles of silver do not affect growth, development and DNA oxidative damage in chicken embryos. Arch. Geflügelk 73, 208–213 (2009)

Chapter 12

Zebrafish as a Suitable Model for Evaluating Nanocosmetics and Nanomedicines Carmen V. Ferreira1, Maria A. Sartori-da-Silva2, and Giselle Z. Justo3 1

Laboratory of Bioassays and Signal Transduction, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, s/n, CP 6109, 13083-970, Campinas, SP, Brazil [email protected] 2 Department of Gastroenterology and Hepatology, Erasmus Medical Center, room L-459,Gravendijkwal 230, NL-3015 CE, Rotterdam, The Netherlands 3 Departamento de Bioquímica (Campus São Paulo) and Departamento de Ciências Biológicas (Campus Diadema), Universidade Federal de São Paulo, 3 de Maio, 100, 04044-020, São Paulo, SP, Brazil

Abstract. The assessment of complex in vivo phenotypes using the teleost zebrafish provides an alternative tool to combine detailed toxicological studies with the large-scale screening of chemicals. The zebrafish, Danio rerio, represents a versatile model organism with many molecular, morphological, and physiological similarities to mammals. It is well suited for studies in genetics, embryology, development, and cell biology, and, in recent years, it has become an important vertebrate model for small molecule studies. Several characteristics contribute to the growing interest in this tropical fish for high-throughput screening such as its small size, high reproductive capacity, accessibility for genetic manipulation, optical transparency which allows visual assessment of developing cells and organs, suitability for treatment by water exposure, low maintenance costs, and a large and growing biological database. In the present chapter, we outline the use of the zebrafish in biomaterial nanotoxicity studies and the potential of this model for the phenotype-based screening of skin care products.

12.1 Introduction The rapid increase in the scientific, technological and commercial interest in submicron materials has boosted the fields of nanoscience and nanotechnology. Several products currently on the market contain elements of nanoscience and nanotechnology, and progressively more are entering the active development

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stages. Therefore, both deliberate (medicinal or therapeutic) and inadvertent or uncontrolled (environmental) human exposure to nanomaterials will increase through many different routes [1]. This leads to a requirement for the development of protocols for high throughput screening of bioactivity as well as for assessing predictive toxicity and health hazards. Nanotoxicology is emerging as an important subdiscipline of nanotechnology. It involves studying the interactions of nanostructures with biological systems aiming to elucidate the relationship between the physical and chemical properties of nanostructures, such as size, shape, composition and surface characteristics, and the induction of toxicity [2]. In the past few years, nanotoxicology has focused on in vitro assays, using different cell culture systems. A major shortcoming of this approach is that it does not consider the various levels of physiological reactivity in a whole organism; thus, the data from these studies could be misleading and animal experiments may be required [1, 3]. On the other hand, the characterization of in vivo toxicity is a daunting task due to the complexity of nanomaterials and the lack of protocol standardization, leading to conflicting results and different conclusions regarding their safety. Therefore, the prediction of important aspects of toxicity is compromised. Furthermore, toxicological assessments will require a research effort to develop new standards to study correlations between nanomaterial properties and biological responses, both in vitro and in vivo [1, 4]. It is simple and cost-effective to initially study the toxicity of a nanomaterial using in vitro approaches, such as cell culture systems. However, unfortunately, in vitro assays may have little, if any, correlation to in vivo effects, inhibiting their validation. In addition, the use of small mammalian models for large-scale screening for toxicity and bioavailability may be less economically feasible. Alternatively, Danio rerio, commonly known as the zebrafish (Fig. 12.1), may be considered a well suited vertebrate model to quickly assess the toxicity of submicron materials at low cost [5].

Fig 12.1 Zebrafish. Scale bar, 500 mm. Picture by Vincent Runtuwene (Hubrecht Institute).

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The use of the zebrafish in toxicological studies offers the advantage of being able to integrate the detailed knowledge available on the morphological, biochemical, and physiological characteristics at all stages of early development, and in juveniles and adults of both sexes, with the chemical toxicity results. Also, zebrafish are susceptible to perturbations in the environment, such as exposure to small molecules, including pharmaceuticals in clinical use. Based on these qualities, zebrafish embryos can be considered for the high-throughput phenotype screening of chemicals [6]. In addition, this model offers a promising tool in environmental risk assessment [7]. Thus, chemical screening using the zebrafish may provide new insights into complex biological systems, including target identification and disease modeling, along with the potential to identify novel lead compounds [6]. Furthermore, a recent study has demonstrated that in vivo screening can be used successfully to perform structure-activity relationship (SAR) studies. This validates the zebrafish as an effective model for not only drug discovery but also drug optimization [8]. In the same context, in a comparative study of differentially engineered lead sulfide nanoparticles, Truong et al. [9] have recently shown the effective use of the zebrafish to investigate the effects of nanoparticle surface functionalization and stability for predicting in vivo biological responses.

12.2 Biology of Zebrafish The zebrafish (Fig. 12.1), a popular aquarium fish, is a freshwater cyprinoid teleost which belongs to the order of ray-finned fishes Cypriniformes [10]. It was first described in An Account of the Fishes Found in the River Ganges and its Branches, published by Francis Hamilton in 1822 [11]. The name Danio means ‘of the rice field’ and derives from the Bengali name ‘‘dhani’’. Zebrafish are native to the streams of the South-eastern Himalayan region, being distributed throughout South and Southeast Asia. They are most commonly encountered in slow-moving or standing water bodies, often connected to rice cultivation [11]. Zebrafish are small, rarely exceeding 4 cm (from the tip of the snout to the origin of the caudal fin). It has a fusiform and laterally compressed body shape, with a terminal mouth directed upwards. Males are slender and torpedo shaped while gravid females have a more rounded body shape. The popular name for this fish originates from its color pattern composed of five to seven uniform dark blue longitudinal stripes on the side of the body, extending from behind the operculum into the caudal fin. Males have gold stripes between the blue stripes and tend to have larger anal fins with more yellow coloration. Females have silver stripes instead of gold and adult females exhibit a small genital papilla in front of the anal fin origin. However, the sex of juveniles cannot be properly distinguished without dissection [12, 13]. Domestic strains used in laboratories may exhibit distinguished features. The ‘‘leopard’’ Danio displays a spotted color pattern instead of stripes caused by a

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spontaneous mutation of the wild-type, whereas the ‘‘longfin’’ Danio has a dominant mutation resulting in elongated fins. The TL or Tubingen long-fin, a commonly used wild-type strain, displays both the ‘‘leopard’’ and ‘‘longfin’’ mutations [11]. The approximate generation time for zebrafish is 3-4 months. Fertilization and subsequently embryonic development are external and occur synchronously in large clutches. Females, in the presence of males, can spawn every 2-3 days laying several hundreds of eggs per mating. Spawning activity, in domesticated zebrafish, is influenced by photoperiod and usually commences within the first minute of exposure to light following darkness, continuing for about an hour [14]. Zebrafish eggs are relatively large and embryos are completely transparent during the first 24 h post fertilization (hpf), allowing the visualization of developing organs through the chorion. Embryos develop rapidly and a beating heart and erythrocytes can be seen within 24 h. Precursors of all major vertebrate organs arise within 36 hours, including the brain, eyes, ear and internal organs. The organs are like a minimalist version with far fewer cells compared to higher vertebrates, having an equivalent function in the organism. Within five days, the larva hatches from the egg and is able to swim, displaying food seeking and active avoidance behaviors [15]. During the first three months following hatching the zebrafish growth rate is most rapid and it starts to decrease, approaching zero, by about 18 months. The lifespan in captivity is around 2–3 years although under ideal conditions it may extend to 5 years [16]. The biological characteristics of the zebrafish make it particularly tractable in terms of experimental manipulation and it offers considerable advantages as an animal model system. In the late 1970s, George Streisinger, at the University of Oregon, recognized the virtues of the zebrafish and pioneered its use as a model organism for studies on vertebrate development and gene function [17]. Later, methods for high-efficiency mutagenesis were developed and large-scale genetic screening characterized several mutants and identified numerous genes. The zebrafish genome is now almost completely sequenced. It has approximately 2.0 gigabases divided into 25 chromosomes (1n) (http://www.sanger.ac.uk/Projects/D_rerio/). Although the absolute number of genes is still unknown, a large set of genes has been cloned and conserved coding and regulatory sequences between zebrafish and humans were observed. These findings allow novel insights into the processes of embryonic development and could lead to models for a wide range of human disorders, such as cancer, diabetes, neurodegenerative and cardiovascular diseases [1822]. Furthermore, by 120 hpf zebrafish develop discrete organs and tissues, including brain, heart, liver, pancreas, kidney, intestines, bone, muscles, nerve systems and sensory organs. These organs and tissues have been demonstrated to be similar to their mammalian counterparts at the anatomical, physiological and molecular levels. Therefore, the zebrafish can be considered as an excellent experimental model for predicting the potentiality of a broad spectrum of substances which might affect mammalian metabolism [5, 23, 24].

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12.3 Zebrafish as a Correlative and Predictive Model for Measuring Biomaterial Nanotoxicity Chemical genetics is a method for identifying substances (small molecules, nanomaterials and formulations) that alter the metabolism by targeting metabolic mediators, culminating in the induction or rescue of a specific phenotype [5, 23, 24]. For this purpose, the zebrafish appears as a suitable biological model. Chemical screening using zebrafish has several advantages over similar screening techniques using cell lines: a.

b. c. d.

the cells of a whole organism are in their physiological microenvironment context of cell–cell and cell–extracellular matrix interactions and are nontransformed; the embryo comprises distinct cell types, making it possible to select compounds that have tissue-specific actions; the use of the whole organism provides an indication of the importance of the biotransformation in terms of the final biological effect of certain chemicals; the zebrafish has a high degree of genetic similarity with mammals.

Zebrafish embryos are easily handled since their size is around 1 mm in diameter. Therefore, high throughput chemical genetic screening for substances (small molecules, nanomaterials and formulations) can be easily carried out in 6 to 386well plates (Fig.12.2). The samples to be examined can be directly aliquoted into the embryo medium. Zebrafish embryos absorb small molecules diluted in the surrounding water through their skin and gills. Substances can be administered orally for assays performed after the larvae stage, since zebrafish begin to swallow at 72 hpf. In addition, highly hydrophobic compounds, large molecules and proteins can be injected into the yolk sac, the sinus venosus or the circulation. It is also important to highlight one interesting characteristic of the zebrafish embryo, which is the presence of the chorion that surrounds it during development. Chorion is an acellular envelope made of three intercrossing layers, which allows materials to pass through to the embryo via passive diffusion [25]. The chorion possess pores or channels ca. 0.5–0.7 μm in diameter [26], meaning that the size of common nanoparticles, including quantum dots, nano-sized metals, metal oxide particles, carbon nanotubes and polymeric nanoparticles, should not preclude passage through the chorion. After treatment for a specified period, for instance up to 72 h, analysis of the mortality and morphological defects (phenotypic changes) is performed (Fig. 12.2b), in which the central nervous system, cardiovascular system, neural crest and ear are examined [27, 28]. In the context of nanomaterials, zebrafish embryos have been employed to examine the toxicity potential of a huge spectrum of nano-based materials such as precious metal nanoparticles, carbon nanotubes and polymers. Some of these materials have being used in potential nanopharmaceutical formulations.

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Fig 12.2 Compound screening experiment. (a) untreated embryo at 24 h post fertilization (hpf); (b) 24 hpf abnormal embryo after treatment with toxic compound; (c) embryos placed in 6-well plate under toxicity screen treatment. Picture by Vincent Runtuwene (Hubrecht Institute).

Henry et al. [29] performed a very elegant study, in which they evaluated the alteration in survival and gene expression of larval zebrafish after exposure to aggregates of fullerene C60-water or fullerene C60-tetrahydrofuran. These authors observed the survival of larval zebrafish placed in C60-tetrahydrofuran and tetrahydrofuran-water but not in C60-water. Regarding gene expression, a change was observed when the zebrafish were exposed to C60-tetrahydrofuran whereas no change was observed on exposure to tetrahydrofuran-water. Under both conditions the genes involved in controlling oxidative damage were significantly upregulated. Importantly, analysis of C60-tetrahydrofuran and tetrahydrofuran-water by gas chromatography followed by mass spectrometry revealed the presence of tetrahydrofuran oxidation products such as γ-butyrolactone and tetrahydro-2-furanol. This finding indicates that the effects observed in tetrahydrofuran treatments may be due to γ-butyrolactone toxicity. Heiden et al. [28] examined the developmental toxicity of low generation G3.5 and G4 polyamidoamine dendrimers, as well as Arg-Gly-Asp-conjugated forms, using the zebrafish embryo as a model. These authors observed that G4 dendrimers, which have amino functional groups, were toxic and attenuated the growth and development of zebrafish embryos at sublethal concentrations. On the other hand, G3.5 dendrimers, with carboxylic acid terminal functional groups and Arg-Gly-Asp-conjugated G3.5, did not display any toxic activity toward zebrafish embryos. Arg-Gly-Asp-conjugated G4 dendrimers were less potent in causing embryo toxicity than G4 dendrimers. Another important finding was the fact that embryos exposed to G4 dendrimers survived longer when they were dechorionated prior to exposure. According to Heiden et al. [28] the positive charge of the G4 dendrimer might interact with the negative charge of the chorion, which could be responsible for concentrating the dendrimer around the embryo, thus increasing the effective dose.

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In recent years, both single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) have been utilized as nanocarriers for drug delivery. The safety of carbon nanotubes (CNT) is still in debate due to the lack of information regarding detailed toxicity. Studies have implicated size (aggregation) as a primary source for potential toxicity in vivo [4]. In this respect, Cheng et al. [30] evaluated the biodistribution and the acute and long-term effects of a single injection of functionalized MWCNTs in zebrafish embryos. The translocation of the MWCNTs around the zebrafish body was monitored for up to 92 h. The injected fluorescent-labeled MWCNTs (in the 1-cell stage) were allocated to all blastoderm cells of the embryos through proliferation, and were distinctively excluded from the yolk cell. When introduced into the circulation system fluorescent-labeled MWCNTs moved to the compartments and were subsequently eliminated by the body 96 h after administration. It was also observed that treated zebrafish embryos generated immune response by accumulating circulating white blood cells in the trunk region. Interestingly, the larvae of the second generation had lower survival rates as compared to the control sample, suggesting a negative effect on the reproductive potential.

12.4 Screening of Skin Care Products Based on Zebrafish Development The zebrafish presents some metabolic characteristics that make it a good model for in vivo testing of products for skin care. For instance, melanin in zebrafish can be identified within 24 h of development [31], and the melanogenic regulation by compounds can be easily examined [32]. Some well-known melanogenic inhibitors (1-phenyl-2-thiourea, arbutin, kojic acid, 2-mercaptobenzothiazole) and two other small molecule compounds (haginin and YT16i) have been evaluated as potential modulators of melanogenesis in zebrafish [32]. These compounds produced inhibitory effects on the pigmentation of zebrafish, which is probably due to their potential to inhibit tyrosinase activity. In relation to the toxicity analysis, the compound YT16i caused strong malformations and also affected the cardiac function. These findings provided some evidence that the zebrafish is a useful tool in the discovery of novel hypo- and hyperpigmenting agents. It has been shown that the treatment of cultured normal human melanocytes (NHMC) and zebrafish with NAD(P)H:quinone oxidoreductase-1 (NQO1) inhibitors, ES936 and dicoumarol, significantly decreased melanogenesis, which highlights the importance of this enzyme for pigmentation [33]. Metal and metal oxide nanomaterials comprise a large segment of the growing nanotechnology market. Several metals, such as gold, silver, copper, nickel, cobalt, zinc, and titanium, are used in nanoparticle preparations. Metal oxide nanoparticles, such as nanoscale zinc oxide (nZnO), titanium dioxide (nTiO2), and alumina (nAl2O3), are widely used as sunscreens and pigments in many formulations, cosmetics and antibacterial ointments. Zhu et al. (2008) [34] revealed that both nZnO and bulk ZnO delay the hatching rate and the development of zebrafish

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embryos and larvae. Furthermore, tissue ulceration, pericardial edema, and body arcuation were visible, which are indicative of embryo malformation. Interestingly, Griffitt et al. (2009) [35] examined the effects of nanoparticle composition and dissolution on zebrafish gill tissue. Following toxic (nanocopper or nanosilver) or nontoxic (nTiO2) nanometal exposure, global gene expression analysis demonstrated that several genes involved in ribosomal function were affected by nTiO2 exposure indicating the possibility that, although not toxic within the time period studied, longer exposure to nTiO2 might cause toxicity in zebrafish gills. These findings suggest that longer-term studies are necessary in order to carry out a risk assessment of nanomaterials in aquatic organisms. Oxidative stress induced by free radical formation seems to play a major role in the molecular mechanism of in vivo nanotoxicity [4, 36]. In excess, free radicals cause damage to biological components through the oxidation of lipids, proteins, and DNA. Moreover, oxidative species may upregulate redox-sensitive transcription factors, such as nuclear factor κB (NFκB) and activator protein-1 (AP-1), as well as activate stress kinases involved in inflammation [4]. Singlet oxygen generated on the surface of the skin can damage the stratum corneum as well as produce toxic diffusible species, which results in injury to the epidermis and inflammatory response [37]. Free radicals can be generated from several sources including phagocytosis of foreign material, insufficient concentrations of antioxidants, Fenton reaction in the presence of transition metals, and environmental factors [4]. Due to the limited capacity of the intrinsic cellular enzymatic and non-enzymatic antioxidant systems to counteract oxidative stress, several bioactives have been studied for their ability to supply the antioxidant reservoir. Fullerene (C60) has attracted interest for application in a variety of products due to its unique physicochemical and optical properties. In contrast to the powerful antioxidant property demonstrated for this nanomaterial, a few reports have claimed a strong oxidative potential through photoactivation. It has been proposed that oxidative damage in exposed organisms is a consequence of the generation of highly reactive singlet oxygen in the process of light absorption and energy transfer to triplet oxygen [38]. In order to gain information on the oxidative potential of C60, Usenko et al. [39] addressed the effects of light, chemical supplementation and depletion of glutathione (GSH) on C60-induced oxidative stress responses in zebrafish embryos. In the presence of the glutathione precursor N-acetylcysteine (NAC) and reduced light, C60 exposure led to a significantly decreased toxicity. Conversely, an increased sensitivity to C60 was observed when GSH inhibitors, buthionine sulfoximine and diethyl maleate, were co-administered. Co-exposure of C60, or its less toxic hydroxylated derivative C60(OH)(24) [40], with increased concentrations of hydrogen peroxide resulted in a dose-dependent mortality. Furthermore, microarray analysis indicated alterations in the expression of several stress response genes, including glutathione-S-transferase, glutamate cysteine ligase, ferritin, alpha-tocopherol transport protein and heat shock protein 70, at 36 and 48 hpf, suggesting that C60 elicits oxidative stress in this model [39]. Interestingly, the same authors have previously shown that at a high concentration (200 μg/L) C60 significantly increased malformations, pericardial edema, and mortality. In contrast, exposure to concentrations of C60(OH)(24) an order of magnitude

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higher caused less pronounced effects. Additionally, necrosis and apoptosis were observed in zebrafish embryos exposed to C60, whilst C60(OH)(24)-exposed embryos did not present signs of apoptosis, corroborating the lower toxicity of the hydroxylated derivative observed in other models [40]. Many compounds have been shown to provide protective effects against reactive oxygen species-induced injury in skin, including natural products such as isoflavones [41]. For instance, topical treatment of human and mouse skin with epigallocatechin before UVB exposure significantly reduces UVB-induced erythema development, hydrogen peroxide production and leukocyte infiltration [42, 43]. Wang et al. [44] reported that UVB light affected the zebrafish pelvic fin, the toxicity of which was prevented by EGCG. This study also indicated the potential of the zebrafish model for use in the screening of new compounds, such as small chemicals which are derivatives of EGCG or other dietary agents for sun protection, for stability and toxicity [44]. In the same context, we have recently demonstrated that retinyl palmitate polymeric nanocapsules toxicity in the zebrafish model is dependent on the stability of the particle in water. The results revealed that free retinyl palmitate at 60 μmol/L caused typical signs of toxicity, such as edema, blood accumulation and altered body axial curvature at 48 hpf. However, at 60 μmol/L, retinyl palmitate polymeric nanocapsules of poly(D,L-lactide) and Pluronic F68 without a stabilizing agent caused 100% mortality at 24 hpf, whilst no effects were observed for nanoparticles of poly(D,L-lactide) and Pluronic F68, even at concentrations 20 times higher than those of retinyl palmitate polymeric nanocapsules [45]. This study emphasizes the applicability of the zebrafish model in the optimization of nanoparticle formulations based on environmental factors and their constituent properties. Even though fullerenes and their derivatives as well as retinyl palmitate polymeric nanoparticles have been studied as agents for topical application [45, 46, 47, 48], a multitude of opportunities for their application in the future might be expected. Thus, assessment of the potential toxicity in vivo is desirable. In addition, the growing use of nanotechnologies requires careful assessment of unexpected toxicities and biological interactions if introduced or absorbed into the systemic circulation. In this regard, the embryonic zebrafish model fulfills the requirements for cheap and rapid assessment of nanomaterial toxicity in vivo.

12.5 Future Perspectives Over the past decade, the zebrafish has emerged as a valuable organism for developmental studies due to a combination of advantageous features which resemble invertebrate biology. These features also make it a valuable model for other purposes, including assessment of in vivo toxicity of nanoscale materials, as outlined in the above sections. It also provides unique research opportunities for disease modeling, drug discovery (particularly to identify lead compounds that modulate specific biological processes), and regenerative medicine.

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Interestingly, despite the growing literature on in vivo chemical screening in the zebrafish, this organism has received little attention as a platform for aging research [49]. In particular, this fish species is very appropriate for the investigation of basic processes implicated in aging, such as insulin signaling and oxidative stress, as well as for the molecular characterization of the mechanisms of action of nano-based anti-aging products. In a recent review, Eimon et al. [50] highlighted the potential of the zebrafish to become a powerful in vivo model for studying cell death, since it offers genetic tractability coupled with the potential for analysis during early development and is suitable for high throughput experimental approaches. Importantly, a diversity of apoptosis-related genes comparable to mammals is found in the zebrafish and the biochemical pathways involved in apoptosis induction are functionally analogous, although further studies are necessary to clarify the roles played by mitochondria and mitochondrial factors in zebrafish apoptosis. Nevertheless, the zebrafish may provide advantages for the study of apoptotic cell death occurring not only during embryonic development but also in response to several stressor stimuli, such as chemicals [50]. The similarity of the effects observed for many small molecules in zebrafish and humans reinforces the perspective of expanding the use of this model system to the screening of nanocarriers and nano-sized materials designed to induce or inhibit apoptosis in different clinical sets. Another potential area of research would be the identification of complex signaling pathways governing biological process, such as cell proliferation, differentiation and growth, which are under tight control but might be dramatically altered by the growing use of nanotechnologies in vivo. Indeed, in the last few years, proteomics has been applied to investigate the physiological aspects of zebrafish biology and the effect of contaminants on its development [51]. The adaptation of conventional assays combined with new technical approaches will certainly integrate fish proteomics with other ‘‘omics’’, creating future challenges in nanothecnology research and suggesting the continued use of zebrafish for screening and toxicity evaluation. Although a full validation of the zebrafish model for the assessment of detailed mechanisms of action and safety will require the evaluation of a large number of samples from diverse classes, the zebrafish is undoubtedly appropriate for the high throughput screening of nanoscale compounds. We are thus hopeful that the use of novel techniques will make the zebrafish a prominent model in nanotechnology research in the forthcoming years. Acknowledgments. Our research in this field was supported by the government agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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[19] Amatruda, J.F., Patton, E.E.: Genetic models of cancer in zebrafish. Int. Rev. Cell. Mol. Biol. 271, 1–34 (2008) [20] Kinkel, M.D., Prince, V.E.: On the diabetic menu: zebrafish as a model for pancreas development and function. Bioessays 31, 139–152 (2009) [21] Chico, T.J., Milo, M., Crossman, D.C.: The genetics of cardiovascular disease: new insights from emerging approaches. J. Pathol. 220, 186–197 (2010) [22] Sager, J., Bai, Q., Burton, E.A.: Transgenic zebrafish models of neurodegenerative diseases. Brain. Struct. Funct. 214, 285–302 (2010) [23] den Hertog, J.: Chemical genetics: Drug screens in Zebrafish. Biosci. Rep. 25, 289– 297 (2005) [24] McGrath, P., Li, C.Q.: Zebrafish: a predictive model for assessing drug-induced toxicity. Drug. Discov. Today 13, 394–401 (2008) [25] Berghmans, S., Butler, P., Goldsmith, P., Waldron, G., Gardner, I., Golder, Z., Richards, F.M., Kimber, G., Roach, A., Alderton, W., Fleming, A.: Zebrafish based assays for the assessment of cardiac, visual and gut function-potential safety screens for early drug discovery. J. Pharmacol. Toxicol. Methods 58, 59–68 (2008) [26] Rawson, D.M., Zhang, T., Kalicharan, D., Jongebleod, W.L.: Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasmamembrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of structural and functional relationships with respect to cytoprotectant penetration. Aquac. Res. 31, 325–336 (2000) [27] Peterson, R.T., Link, B.A., Dowling, J.E., Schreiber, S.L.: Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. USA 97, 12965–12969 (2000) [28] Heiden, T., Dengler, E., Kao, W., Heideman, W., Peterson, R.: Developmental toxicity of lowgeneration PAMAM dendrimers in zebrafish. Toxicol. Appl. Pharmacol. 225, 70–79 (2007) [29] Henry, T.B., Menn, F.M., Fleming, J.T., Wilgus, J., Compton, R.N., Sayler, G.S.: Attribuiting effects of aqueous C60 nanoaggregates to tetrahydrofuran decomposition products in larval zebrafish by assessment of gene. Environ. Health Perspect. 115, 1059–1065 (2007) [30] Cheng, J., Chan, C.M., Veca, L.M., Poon, W.L., Chan, P.K., Qu, L., Sun, Y.P., Cheng, S.H.: Acute and long-term effects after single loading of functionalized multiwalled carbon nanotubes into zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 235(2), 216–225 (2009) [31] Sinha, R.P., Häder, D.P.: UV-induced DNA damage and repair: a review. Photochem. Photobiol. Sci. 1, 225–236 (2002) [32] Choi, T.Y., Kim, J.H., Ko, D.H., Kim, C.H., Hwang, J.S., Ahn, S., Kim, S.Y., Kim, C.D., Lee, J.H., Yoon, T.J.: Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pigment. Cell. Res. 20, 120–127 (2007) [33] Choi, T.Y., Sohn, K.C., Kim, J.H., Kim, S.M., Kim, C.H., Hwang, J.S., Lee, J.H., Kim, C.D., Yoon, T.J.: Impact of NAD(P)H:quinone oxidoreductase-1 on pigmentation. J. Invest. Dermatol. 130, 784–792 (2010) [34] Zhu, X., Zhu, L., Duan, Z., Qi, R., Li, Y., Lang, Y.: Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early developmental stage. J. Environ. Sci. Health A: Toxicol. Hazard Subst. Environ. Eng. 43, 278–284 (2008)

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[35] Griffitt, R.J., Hyndman, K., Denslow, N.D., Barber, D.S.: Comparison of molecular and histological changes in zebrafish gills exposed to metallic nanoparticles. Toxicol. Sci. 107, 404–415 (2009) [36] Lanone, S., Boczkowski, J.: Biomedical applications and potential health risks of nanomaterials: molecular mechanisms. Curr. Mol. Med. 6, 651–663 (2006) [37] Giacomoni, P.U., Teta, L., Najdek, L.: Sunscreens: the impervious path from theory to practice. Photochem. Photobiol. Sci. 9, 524–529 (2010) [38] Arbogast, J.W., Darmanyan, A.P., Foote, C.S., Rubin, Y., Diederich, F.N., Alvarez, M.M., Anz, S.J., Whetten, R.L.: Photophysical properties of C60. J. Phys. Chem. 95, 11–12 (1991) [39] Usenko, C.Y., Harper, S.L., Tanguay, R.L.: Fullerene C60 exposure elicits an oxidative stress response in embryonic zebrafish. Toxicol. Appl. Pharmacol. 229, 44–55 (2008) [40] Usenko, C.Y., Harper, S.L., Tanguay, R.L.: In vivo evaluation of carbon fullerene toxicity using embryonic zebrafish. Carbon. N Y 45, 1891–1898 (2007) [41] Lin, J.Y., Tournas, J.A., Burch, J.A., Monteiro-Riviere, N.A., Zielinski, J.: Topical isoflavones provide effective photoprotection to skin. Photodermatol. Photoimmunol. Photomed. 24, 61–66 (2008) [42] Katiyar, S.K., Elmets, C.A.: Green tea polyphenolic antioxidants and skin photoprotection. Int. J. Oncol. 18, 1307–1313 (2001) [43] Katiyar, S.K., Mukhtar, H.: Green tea polyphenol (−)-epigallocatechin-3-gallate treatment to mouse skin prevents UVB-induced infiltration of leukocytes, depletion of antigen-presenting cells, and oxidative stress. J. Leukoc. Biol. 69, 719–726 (2001) [44] Wang, Y.H., Wen, C.C., Yang, Z.S., Cheng, C.C., Tsai, J.N., Ku, C.C., Wu, H.J., Chen, Y.H.: Development of a whole-organism model to screen new compounds for sun protection. Biotechnol. NY 11, 419–429 (2009) [45] Durán, N., Justo, G.Z., Ferreira, C.V., Silva, R.A., Machado, D., Shishido, S.M., Teixeira, Z., Den Hertog, J.: Cellular and molecular approaches to in vitro and in vivo assessment of preliminary toxicological and biological activities of nanomaterials. Nanotoxicology 2 (S1),S82 (2008) [46] McEwen, C.N., McKay, R.G., Larsen, B.S.: C60 as a radical sponge. J. Am. Chem. Soc. 114, 4412–4414 (1992) [47] Lens, M.: Use of fullerenes in cosmetics. Recent Pat. Biotech. 3, 118–123 (2009) [48] Teixeira, Z., Zanchetta, B., Melo, B.A., Oliveira, L.L., Santana, M.H., ParedesGamero, E.J., Justo, G.Z., Nader, H.B., Guterres, S.S., Durán, N.: Retinyl palmitate flexible polymeric nanocapsules: Characterization and permeation studies. Colloids Surf. B Biointerfaces 81, 374–380 (2010) [49] Gerhard, G.S.: Small laboratory fish as models for aging research. Ageing Res. Rev. 6, 64–72 (2007) [50] Eimon, P.M., Ashke, A.: The zebrafish as a model organism for the study of apoptosis. Apoptosis 15, 331–349 (2010) [51] Forné, I., Abián, J., Cerdà, J.: Fish proteome analysis: Model organisms and nonsequenced species. Proteomics 10, 858–872 (2010)

Chapter 13

Nitric Oxide-Releasing Nanomaterials and Skin Care Amedea B. Seabra Chemistry Institute, Universidade Estadual de Campinas, UNICAMP, CP 6154, 13083-970, Campinas, SP, Brazil [email protected]

Abstract. The gaseous free radical nitric oxide (NO) is a key endogenous found molecule involved in several physiological and pathophysiological processes in different organs and tissues. NO is synthesized by several cell types in the human skin, where regulates diverse homeostatic functions, such as the control of dermal blood flow, the promotion of wound healing, the skin pigmentation, and the antiaging effects. Due to its unique chemistry structure and biological versatility, NO is also an important mediator of diverse human skin diseases, and plays a defense role against skin pathogens. Therefore, there is an explosive interest on the development of biological friendly vehicles for topical NO release in several dermatological applications. In this scenario, the preparation of NO-releasing nanomaterials, as controllable NO dispenser, is a promising strategy for diverse applications in human skin, as discussed in this chapter.

13.1 Nitric Oxide and Skin: Current Status Nitric oxide (NO) is an endogenous found molecule that has diverse physiological functions, including vasodilation of blood vessels [1, 2], neurotransmission [3], inhibition of platelet adhesion and aggregation [4], apoptosis [5], antioxidant actions [6], antitumour and antimicrobial effects [7, 8] and promotion of wound healing [9, 10]. Considered merely as an environmental pollutant, it was only at the late of 1980s that this small free radical was shown to be a key molecule in the vascular system [11]. At the present moment, NO is one of the most studied molecules and a great volume of biomedical research has established NO as a fascinating biologically active free radical and a key regulator in many organs

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and tissues of the human body, including the skin [12, 13]. In this context, NO is considered a keyword for a diverse scientific community with a special relevance for dermatologists. Figure 13.1 shows the large increase in the number of publications that contain the expression “nitric oxide” in the topic, according to the Web of Science®. It can be observed that the great interest of the scientific community by NO was started in the 90s and it is still relatively high until nowadays.

Fig 13.1 Number of published papers with the expression “nitric oxide” from 1988 to 2009. Data obtained from Web of Science®.

In vivo, the nitric oxide synthase (NOS) enzymes produce NO through the oxidation of L-arginine to L-citrulline [14]. The three NOS isoforms are situated in different intracellular location, and are the products of different genes. There are two constitutive isoforms: eNOS present in endothelial cells, and nNOS first identified in the peripheral and central nervous systems [15]. Both eNOS and nNOS are calcium-dependent isoforms, which produce low amounts of NO (picomolar to nanomolar range) for short periods mainly for homeostatic functions [16, 17]. In contrast, iNOS is a calcium-independent inducible isoform, greatly involved in inflammation and pathological conditions, which produces high amounts of NO (milimolar range) upon induction by a variety of stimuli, such as cytokines [18] (Figure 13.2).

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Fig 13.2 Endogenous synthesis of NO by constitutes eNOS and nNOS and inducible iNOS isoforms from oxidation of L-arginine to L-citrulline. In human skin, NO can have regulatory functions or cytotoxic effects, as listed in the picture.

During the past years, the physiologic and pathophysiologic roles of NO in the skin have been the focus of an increasing attention, and this research field remains as active as ever. The human skin is a site for NO production since all three NOS isoforms are expressed in human skin cells, such as melanocytes, adipocytes, endothelial cells, macrophages, neutrophils, fibroblasts, keratinocytes and Langerhans cells [12, 19, 20]. For example, it has been reported that ultraviolet radiation and cytokines induce NO synthesis by iNOS in keratinocytes [21], eNOS has been identified in fibroblasts, and nNOS in keratinocytes and melanocytes [19, 20]. As the skin has a constant exposure to a variety of environmental challenges, such as chemicals irritants, mechanical trauma, UV radiation, and infectious microorganisms, it is a site of significant immunologic activity. Consequently, a variety of immune reactions in the skin involves NO. As inducible isoform, iNOS is expressed following stimulation with inflammatory cytokines, such as interferon γ (IFNγ), interleukins (IL-1β, IL-2, IL-6, IL-8), tumour necrosis factor (TNFα) and UV radiation, generating significantly great and sustained amounts of NO [12]. This scenario is mostly observed in skin pathologies, such as acute and chronic skin diseases [22]. Contrarily, eNOS and nNOS have homeostatic roles in the skin such as limiting UV-induced epidermal apoptosis, and maintaining dermal blood flow [23, 19, 20]. Thus, depending on the nature of the NOS isoform, NO can be involved in physiologic or pathophysiologic responses in human skin (Figure 2).

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13.2 The Role of NO in Physiologic Responses in the Skin The chemical nature of NO makes it a key player in the bioregulation of important physiologic processes in the skin. NO is lipophilic, it has a small size, it is devoid of charge and diffuses easily in the biological medium crossing most physiologic barriers [24]. Indeed, NO moves rapidly from cell to cell, independently of membranes receptor and channels, along a concentration gradient [25]. Consequently, NO has a relative high diffusion rate which is ca. 1.4-fold higher than O2 or CO and a great ability to penetrate cell membranes [26]. As highly diffusible molecule, NO maintain skin homeostasis by regulating physiologic responses in the skin, such as dermal circulation, ultraviolet-mediated melanogenesis and skin pigmentation, delay the onset of wrinkles and the maintenance of the protective barrier against microorganisms [19, 20, 27]. Moreover, diverse studies have reported that NO is able to accelerate and promote wound healing in acute and chronic wounds, being considered one of the key molecules involved in wound repair process [9, 10]. Thus, due to the multifaceted physiologic roles of NO in human skin, several NO donors are currently being used in various types of experimental studies and there is a great interest on the development of NO-releasing biomaterials for dermatological applications [28, 13].

13.3 NO and Skin Diseases The skin has the largest surface area in the body and acts as protective barrier for the internal organs. As highly metabolic tissue, the skin is a biological layer responsible to defend mechanically and biochemically human body against diverse environmental insults [29]. Moreover, the skin has the ability to indicate endogenous pathologic conditions such as artherosclerosis, AIDS, diabetes mellitus, mental stress, and aging [30 -33]. It has extensively reported that NO is a key mediator in cytotoxic events in host defense against diverse pathogens, such as bacteria, candida, leihsmania and fungi [34, 8, 35]. High amounts of NO (mili molar range) are release by iNOS, upon stimulation with cytokines and tumor necrosis factor - α, under pathological conditions by different cell types, especially by macrophages [36]. The high amount of NO released has bacterial, viricidal, leishmanicidal and tumoricidal activities [37]. It has been reported that NO exerts its cytotoxic activity by diverse mechanisms, such as the inhibition of DNA replication of the pathogen, the prevention of mitochondrial respiration by interference with enzymes of the electron transport chain of the pathogen, and the S-nitrosation of cysteine residues of vital enzymes of the pathogen [38, 8]. As lipophilic molecule, NO diffuses easily through the pathogen membranes and interacts with intracellular targets, causing toxic effects. The highly oxidant peroxynitrite (ONOO-) can be formed inside pathogens’ cells by the reaction of NO with superoxide anion radical (O2·-) (Eq. 1), which is present in oxidative stress process and is one of the products of bacterial cellular respiration. NO· + O2·- → ONOO-

(13.1)

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Peroxynitrite causes severe biological damages, including lipid membrane peroxidation [28], attack of metabolic heme-containing enzymes forming nitrosyl compounds, DNA, and iron-sulfur cluster of important enzymes, impairing skin pathogen metabolism and growth [39]. Moreover, S-nitrosothiols (RSNOs), which are NO donors due to the homolytic cleavage of S-N bond (Eq. 2), have been shown to have leishmanicidal actions [8]. RS-NO → RS· + NO·

(13.2)

One of the proposed mechanisms by which the NO-donors S-nitrosothiols (RSNOs) have leishmanicidal effects is based on the bimolecular reaction of the intact RSNO molecule with parasite proteins [8]. In particular, RSNOs are able to transfer the NO group to a vital parasite target: cysteine protease enzymes, through a chemical reaction namely trans-S-nitrosation. This enzyme modification might impair the parasite survival. Therefore, NO is directly involved in many skin diseases infected with bacteria, and protozoa. Moreover, NO is involved in skin disorders such as psoriasis, skin cancer, among others. Psoriasis is a chronic skin disease characterized by an inflammatory dermatosis due to the presence of dermal hyperproliferative keratinocytes [40]. Clinically, patients with psoriasis have red or white scaly skin lesions with a thickened epidermis [41]. Skin lesions present keratinocyte hyperproliferation and T cells infiltrated, which secrete gamma interferon (γ-IFN) for the maintenance of the psoriatic phenotype. Psoriatic skin lesions contain elevated iNOS and decreased eNOS expressions, mainly in the dermis, in comparison to healthy normal skin, indicating the importance of iNOS in the mediation of cutaneous inflammation [23]. Recently, it has been observed that the endogenous concentration of arginine (the substrate for NO production by NOS) is reduced in the psoriactic lesions leading to a relative decrease in NO production [42]. Thus, dermatological treatment of psoriasis with NO donors might have the potential therapeutic effect to reduce the size of psoriatic plaques. In addition to psoriasis, dermatological NO donors have a great potential to treat nonhealing chronic wounds, especially those infected with bacteria. The presence of a significant bacterial burden in the wound bed impairs the wound repair [43]. Most of the wound bed infected with bacterial burden is composed of a complex and organized network of bacteria namely bacterial biofilm [44]. Clinically, it is difficult to treat these infected wounds with conventional antibiotics and consequently, the wound healing process is impaired and delayed [45]. Pure NO, in the gas form, has been successfully used in the dermatological treatment of human nonhealing chronic venous ulcer covered with bacterial biofilm [46]. Notably, the concentration of gaseous NO required to kill bacteria (200 ppm) was found to be nontoxic to human dermal fibroblasts [47]. However, in practice, the use of a simply gaseous NO is not the most appropriated strategy for dermatological applications, since a complete absence of oxygen is required to avoid NO oxidation to nitrogen dioxide. Moreover, a sustained and controlled NO release directly to the target site in the skin is desired. Therefore, topical applications of NO-releasing vehicles capable to release therapeutically amounts of NO for a prolonged time are considered a promising approach to treat nonhealing infected wounds [13, 48].

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Indeed, a variety of NO donors, such as S-nitrosothiols, NO-releasing zeolites, diazeniumdiolate (NONOate), and sodium nitroprusside (SNP) have been studied in vivo as therapeutic agents for various clinical conditions. Recently, it has been reported the preparation of polymeric NO-donor comprised by a solid hydrophobic film of poly(nitrosated polyester)/poly(methyl methacrylate) (PNPE/PMMA) that acts as sustained NO donor for more than 24h [34]. The sustained and controlled NO released from this polymeric material exerted a potent dose- and time- dependent antimicrobial effects against Staphylococcus aureus and Pseudomonas aeruginosa strains, opportunistic pathogens found in burn and chronic wound infections. NO released from this biomaterial reduced the viability of these multidrug-resistant bacteria by more than 98% after 12 h of incubation, and may have either inhibited bacterial biofilm formation or elicited biofilm dispersal or detachment [34]. Similarly, it has been reported the preparation of NO-releasing silica nanoparticles modified with the NO donor diazeniumdiolates (NONOates) [28]. The NOreleasing nanoparticles showed not only a more efficient antimicrobial effect against Pseudomonas aeruginosa compared to small molecule of NO donors [28], but also the ability to kill microbial cells within established biofilms [49]. Taken together, these results show that exogenous NO has potent significant antibacterial properties that can be beneficial in reducing bacterial burden in infected wound in burn injuries or non-healing ulcers. Thus, the development of formulations that release therapeutically amounts of NO in topical applications for long periods of time is highly under the interested of scientists belonging to the chemical, materials science and biochemical communities.

13.4 NO and Wound Healing Successful wound healing process involves numerous of orchestrated, overlapping and complex physiological responses, which includes: clot formation, inflammation, re-epithelialization, angiogenesis, granulation tissue formation, wound contraction, scar formation and tissue remodelling [50]. These highly interconnected and overlap events require the presence and interaction of many cell types, such as inflammatory cells, fibroblasts, keratinocytes and endothelial cells, plus the involvement of growth factors and enzymes [51]. Briefly, the injury is characterized by a disruption of blood vessels and extravasation of blood constituents, which produces the formation of a provisional extracellular matrix. The inflammatory phase is characterized by platelets aggregation at the wound site, which releases various growth factors and cytokines, increasing the permeability of nearby blood vessels. This response recruits inflammatory cells, such as neutrophils and macrophages, and also fibroblasts and endothelial cells to the wound site [52]. All these stimuli lead to the next phase, the granulation tissue formation, characterized by the differentiation of many cells types into myofibroblasts, which produce the extracellular matrix and generate the mechanical force to close the wound site [53]. The granulation tissue formation phase is mainly characterized by the re-epithelialization by proliferation and migration of epithelial cells [54]. In the final phase, there is a replacement of granulation tissue by a scar tissue, due to the degradation of extracellular matrix components by matrix metalloproteinases (MMPs) [55].

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NO participates in diverse physiologic events involved with wound healing process, including, angiogenesis (formation of new microvessels), chemoattractant of cytokines, monocytes and neutrophils, control of collagen synthesis and deposition, cellular proliferation, and apoptosis [9, 10, 26, 56]. As NO is involved in wound repair process, it is expected that a reduction in the levels of NO production in the wound site might impair and delay normal wound healing process. In fact, it has been reported a decrease in NO production in the wound bed, caused by gene knockout or pharmacological inhibition, that resulted in impairment of wound healing as evidenced by increased closure time, lower wound breaking strength and decreased collagen deposition [57]. Conversely, supplementation with L-arginine, the substrate for NO synthase, increases closure of wounds [58]. Diabetic rats show low NO levels, and delayed healing in wounds, and this can be partially reversed by L-arginine supplementation and also by exogenous NO donor [56]. Therefore, an improvement of wound healing process is expected to be achieved due topical application of NO-releasing materials. For this dermatological application, it is desirable to develop a material that releases therapeutical amounts of NO, for prolonged time, the material should be appropriate for wound healing and be convenient to apply in the clinical setting without deleterious side effects. A promising NO-releasing formulation for dermatologic purpose was prepared by incorporating the NO donor S-nitrosoglutathione (GSNO) in a hydrogel of poly(ethylene oxide) 99 -poly(propylene oxide) 65 -poly(ethylene oxide) 99 (PEO-PPO-PEO, Pluronic F-127) [59, 1, 2, 9, 10]. Pluronic F-127 is used as a vehicle in several biomedical applications. Initially, the thermal and photochemical NO release profile from GSNO-containing Pluronic hydrogel was characterized [59]. In subsequent assays, this NO-releasing hydrogel was topically applied on the forearm of healthy human subjects and on the foot sole skin of streptozotocininduced diabetic rats, with impaired microcirculation. In both applications, it was observed an increase in the dermal blood flow, indicating that NO released from this material has the ability to exert its biological effects [1, 2]. Indeed, cutaneous microdialysis showed a correlation between an increase in dermal blood flow and a diffusion of NO through the dermis of human subjects, followed topical application of GSNO-containing hydrogel [2]. Finally, the ability of this GSNOcontaning Pluronic hydrogel to promote and accelerate rat excisional wound healing was investigated [9,10]. It has been reported that daily topical applications of the hydrogel during the early phases of rat cutaneous wound repair accelerates wound closure and re-epithelialization and affects granulation tissue organization [9]. In a sequent study, the effects of topical application of GSNO-containing hydrogel in different phases of rat excisional cutaneous wound healing were investigated. It was observed that NO is important in all phases wound repair (coagulation, inflammation, proliferation and remodeling), but if applied on both inflammatory and proliferative phases, the improvement in wound healing was found to be more impressive [10]. Moreover, the effect of topical application of GSNO-releasing hydrogel on the healing of ischemic rat’s wounds (flap model of impaired wound) was evaluated [60]. The results showed that this formulation has the ability to treat chronic wounds.

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Chronic, nonhealing skin wounds are normally sign observed in uncontrolled diabetes, which are associated with an impaired micro and macrocirculation leading to a poor blood supply to the wound site, infection and callus formation, frequently associated with a significant morbidity [61]. This scenario is coupled to low endogenous NO synthesis, leading to a decrease in collagen deposition and wound-breaking strength [50]. In fact, diabetic human skin fibroblasts produce less NO than normal human skin fibroblasts [62]. Addition of the NO donor Snitrosoglutathione significantly increase the rate of fibroblasts proliferation compared to untreated normal fibroblasts [62], suggesting that NO donors might aid chronic wound healing. These studies suggest that topical application of NO-donor biomaterials at acute and chronic wounds have the ability to promote and accelerate tissue repair. Ideally, the NO-donors biomaterials should have half-lives able to produce a long and sustained NO release. The ability of these NO-donors biomaterials to simply and effectively deliver NO in a clinical situation coupled with the vasodilatory, antibacterial and wound healing properties of NO, make them attractive candidates for the treatment of acute and diabetic ulcers.

13.5 Nanotechnology and NO in Skin Treatments Due to the involvement of NO in many physiologic and pathophysiologic responses in human skin, as discussed above, there is an increasing interesting on the development of NO-releasing biomaterials for dermatological purposes. These biomaterials should be biocompatible, preferably biodegradable, release controlled and sustained amounts of NO, at the target site, and with non-toxic side products formation. Different approaches have been used to control the NO release in a specific site, including polymeric solid films, hydrogels and nanoparticles [63, 59, 13]. Many different classes of NO donors, such as S-nitrosothiols, diazeniumdiolates (NONOates), and ruthenium derivatives, have been prepared and applied in biological systems [13, 48]. These NO donors can be incorporated into polymeric matrices, such as polyethylene glycol, poly(vinyl alcohol), chitosan, or chemically linked to a polymeric backbone, such as polynitrosated polyester, as previous reported [64], in order to mimic the endogenous NO production directly at the target site. One of the first reports of topical applications of NO-generating formulations on human skin was published almost a decade ago [65]. In this work, a dermatological formulation composed by 3 % salicylic acid and 3% nitrite in aqueous cream, capable to generate NO, was topically applied on patients with tinea pedis. The antimicrobial effects of this cream were confirmed by a clinical improvement and mycological cure after one month of daily treatment [65]. The disadvantage of this strategy is the indirect NO generation in situ (nitrite is reduced to NO in acidified cream) and by the fact that this formulation does not involve nanomaterials for NO delivery. Thus, the beneficial effect of NO on the skin can be increased by using a topical formulation able to release free NO in a sustained fashion. This approach might be achieved by using NO-releasing nanoparticules, as described below.

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A very promise strategy that has recently been used to obtain NO-releasing vehicles for biomedical applications is based on the combination of NO donors with nanostructured materials [13]. The preparation of NO-releasing nanomaterials for dermatological applications is a recent development, and an important feature of such donors is the inert nature of the carrier molecule or vehicle. Pharmaceutical nanoparticle properties and drug delivery prolife from these NO-releasing nanomaterials have been widely studied in order to increase their solubility, stability, and their specificity targets [13, 28, 48]. Several works describe the preparation of different scaffolds for NO-releasing nanomaterials for biomedical applications, including dermatologic therapies. In particular, it has been reported the bacterial effects of diazeniumdiolate-modified aminoalkoxysilanes nanoparticles against P. aeruginosa [28]. The great advantage of this formulation is that in vitro cytotoxicity experiments conducted with L929 mouse fibroblasts confirmed that NO-releasing nanoparticles are nontoxic to mammalian fibroblast cells at concentrations capable of killing P. aeruginosa [28]. In contrast, small NO donors molecues, such the diazeniumdiolates, when administrated at bactericidal concentrations exhibit a significant toxicity to fribroblasts culture. Thus, NO-releasing nanoparticles might be use topically to cover bacteria infected wounds in patients [28]. Additionally, zeolites can be use as vehicle to carry and delivery NO in dermatological applications. The advantage of using zeolites nanoparticles is based on the fact that the vehicle can be considered inert material, since zeolites per se produces little inflammation [66]. As a class of crystalline aluminosilicate micropourous, zeolites contains an inorganic three-dimensional network of channels, rings, and cages, composed of linked tetrahedra built from an open network composed of AlO4 and SiO4 [66]. Zeolites has a natural affinity for NO, since the open structure of the framework allows ion exchange, reversible dehydration, and the adsorption of small molecules, such as NO [67]. Moreover, zeolites are a good storage nanoporous material, since NO can bound within the framework and be released from this stable storage material in biologically relevant amounts upon contact with water. In other words, gaseous NO can be stored into zeolites and released upon hydration, such as after its application on the skin surface. For example, it has already been reported that the topical application of NO-releasing zeolites on human skin increased dermal blood flow, illustrating the ability of this nanomaterial to deliver bioactive NO on human skin [68, 69]. The increased dermal blood flow, measured by Laser-Doppler, correlated directly with the concentration of NO delivered transepidermally, as measured by cutaneous microdialysis [68]. Thus, the dermatological use of NO-releasing zeolites might find applications such as to increase dermal blood flow, to promote wound healing, and to treat dermatological infections cause by microorganisms. The lipophilicity of NO and its neutral charge facilitates its free diffusion through aqueous solutions and across skin membranes, thus pure NO released from zeolites can diffuse across the epidermis to the dermis to exert its biological effects, such as the vasodilation. Moreover, the amount and kinetics of NO release from this nanomaterial can be adjusted for the desired level, which depends on the specific clinical requirements. Indeed, the rate of NO release from zeolites can be tailored by altering the type

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and number of metal cations in the structure, allowing NO delivery patterns to be modulated [70]. NO-releasing zeolites can be considered an effective nanomaterial in human skin without dramatic pro-inflammatory effects. In an alternative approach, the tripeptide glutathione (GSH) was incorporated into alginate/chitosan nanoparticles [71]. The encapsulated thiol (GSH) was nitrosated in the presence of sodium nitrite forming the NO donor Snitrosoglutathione (GSNO) in the alginate/chitosan nanoparticles. It was found that the encapsulation of this NO donor in alginate/chitosan nanoparticles increases the GSNO stability, compared to non-encapsulated GSNO [71], illustrating that nanoparticles have the ability to control the rate of NO release from GSNO. Cytotoxicity studies demonstrated that free GSNO was not cytotoxic to fibroblast V79 cells in the lysosomal (Neutral red) and slightly cytotoxic to mitochondrial (MTT) assays (20-25% around to 16 μM concentrations). However, the cytotoxicity characterization of nanoparticles containing GSNO showed that this material was completely non cytotoxic to cellular viability. These results show that GSNO containing alginate/chitosan nanoparticles have a great potential to used in dermatological applications, since it slowly releases NO and it has no cytotoxicity. Moreover, it has been reported the preparation of NO-releasing platform using nanotechnology based in a silane hydrogel containing chitosan [72]. Topical or intrademal applications of NO-releasing silane/chitosan hydrogel on bacterial abscesses in mice were found to be effective against methicillin-resistant staphylococcus. Clinically, bacterial abscesses are difficult to treat and can lead to complications such as sepsis, tissue damage and death. The dermatological application of NO-releasing silane/chitosan nanoparticle was responsible to improve skin architecture and to reduce the infected area [72]. As liphophilic and small molecule, NO can diffuse easily into the abscess and have its beneficial effect such as the reduction of bacterial burden, the attraction of immune cells to the affected area, the promotion of local vasodilation, and the increase of vascular permeability [24]. This example shows that NO-releasing nanoparticles can be use in the treatment of cutaneous infected wounds where NO induces a protective immune response. However, an important aspect of these NO-releasing nanomaterials for dermatologic applications is the lack of a complete and detailed characterization of their toxicity in vivo. In order to propose the use of NO-releasing nanomaterials for topical applications, the clearance of the nanomaterial and NO donor after the release of NO in biological medium have to be characterized. Some of NO-releasing nanomaterials contain metals in their structure, which might cause toxicity in vivo.

13.6 NO and Skin Pigmentation Enhancers UV radiation is the main physiological stimulus for human skin pigmentation. Upon UVA and UVB radiation, melanocytes, localized within the epidermalmelanin unit, synthesize and release melanin to the surrounding keratinocytes, which produce paracrine factors affecting melanocyte proliferation, dendricity, and melanin synthesis, leading to skin pigmentation [73 ]. It has been reported that

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NO is involved in the skin pigmentation process, since UVA and UVB radiation stimulate human keratinocytes NOS to produce NO and released it to the surrounding. NO produced by UV-irradiated keratinocytes plays an important role in the paracrine mediation of UV-induced melanogenesis. In addition, NO may also contributes to skin erythema in response to UV radiation, by increasing dermal blood flow in humans. It has already been reported that the incubation of human epidermal melanocytes with the NO donor sodium nitroprusside (200 μM) increased 3.5-fold melanin production [73]. The involvement of NO in skin pigmentation is also evidenced when keratinocytes are co-cultured with melanocytes and exposed to UVA and UVB radiation. NO is produced in keratinocytes and released to the medium, where up-regulates cyclic guanosine monophosphate (cGMP), actives tyronise in melanocytes resulting in melanogenesis. Thus, current evidence indicates that NO production in keratinocytes and melanocytes is required for UVB-induced melanogenesis [73]. In another work, the importance of NO in skin pigmentation was evidenced by suppressing endogenous NO production in animal model. The specific NOS inhibitor N- (G)-nitro-L- arginine methyl ester (L-name) was topically applied on the back of guinea pigs before and after UV-irradiation exposure. It was observed that the presence of an NO inhibitor lead to suppression of UV-induced skin pigmentation, indicating that NO is required for skin pigmentation [74]. On a purely cosmetic level, these results suggest that dermatological applications of NO donors may be use as a novel tanning agent. Moreover, the endogenous pigmentation provides a broad wavelength to protect skin against cancer and DNA damage caused by solar radiation [75]. Thus, it can be hypothesized that topical application of skin pigmentation stimulators, such as NO donors, before sun exposure has the potential to reduce photodamage and skin cancer. In this scenario, topical formulations that release NO might be use for tanning and at the same time be able to prevent skin cancer.

13.7 Future Directions and Challenges in Dermatological Therapeutics Due to the importance of NO in cutaneous biology there is an increasing interest on the design of NO-releasing formulations for dermatological purposes. The role of NO in skin physiology and pathophysiology has become considerably investigated only in the last 10-15 years. It should be noted that consider the skin as a target organ for pharmacological studies and therapeutic interventions for NOreleasing formulations is an extremely exciting field of study. In this scenario, topical applications of NO donors have diverse applications on human skin, such as the treatment of skin diseases, the promotion and acceleration of wound healing, as discussed in this chapter. Besides these medical uses, topical application of NO-releasing biomaterials can find purely cosmetic and aesthetic applications, such as skin tanning, fight against wrinkles and promotion of hair growth [76, 27, 76]. For example, NO donors have the ability to increase collagen type I synthesis

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in human fibroblasts [27]. Increased collagen production is a well known allied to delay skin aging, since alterations of dermal matrix contributes to the formation of wrinkles. Facial cream containing NO donors might find cosmetic applications to avoid and delay wrinkles formation. Thus, there is a great interest on both medical and purely cosmetic and aesthetic of applications of NO donors. Although NO donors are no yet commercially available for dermatologic applications, it is expected to have these compounds in the dermatopharmacologic market in the near future. However, key questions might be addressed such as what NO donor is more convenient for a specific dermatological application, and how it should be administrated. In this aspect, the use of NO-releasing nanomaterials has been proved to be an extremely promising strategy [13]. The ideal NO-releasing nanomaterial for dermatological applications should be able to load the desired amounts of NO, release NO for a sustained and controlled manner at the specific target, and be biocompatible, preferentially biodegradable. Despite so many challenges, this is an exciting time to be engaged in the development of new NO-releasing nanomaterials for clinical skin research.

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Chapter 14

Nanocarriers and Cancer Therapy: Approaches to Topical and Transdermal Delivery Juliana M. Marchetti, Marina C. de Souza, and Samantha S. Marotta-Oliveira Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil [email protected]

Abstract. The main goals of nanotechnology in cancer are to develop safer and more effective diagnostics and therapeutics. Nanotechnology can bring advantages to drug delivery, overcoming the limitations of conventional formulations. Drugs encapsulated in targeted nanocarriers are promising for the improvement of efficacy and safety of not only currently available drugs, but also certain chemical or biological compounds that were not previously used due to toxic effects or because they were not able to be administered. Drug delivery across the skin is an extremely attractive route due to the possibility of targeting skin diseases (topical), for achieving systemic effects (transdermal administration), providing patient convenience, and avoiding first-pass hepatic metabolism. However, this route still remains a challenge due the highly organised stratum corneum structure. Several strategies have been studied to optimize topical and transdermal drug delivery, including physical techniques, such as eletroporation and iontophoresis, and nanotechnology-based drug delivery systems. This work discusses nanotechnology evolution and the use of several nanotechnology strategies to increase skin penetration and permeation in the improvement of cancer treatment.

14.1 Nanotechnology in Healthcare: Evolution Recently, the term nanotechnology has become popular and has shown current trends in science and technology. The term nanotechnology refers not to a specific area, but to a variety of disciplines ranging from the basic science of materials to applications in healthcare. Nanotechnology has contributed greatly to biology, biotechnology, electrical engineering, mechanics, chemistry, materials, mathematics, and chemistry, among many other fields [1]. Nanotechnology is now a major focus of research activities, development and innovation in all industrialised countries. In 2000, a government agency was created in the U.S., the National Nanotechnology Initiative (NNI), which was responsible for financing the research and development, infrastructure, education and commercialisation of nanotechnology. In 2005, the resources invested were

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around 1.05 billion dollars and have continued to increase [2]. There are already some industrial products based on nanotechnology, and their number increases rapidly every day. It is estimated that from 2010 to 2015, the world market for materials, industrial products, and processes based on nanotechnology will be a trillion dollars, according to the United States National Nanotechnology Initiative (US-NNI) [3]. In the past two decades, researchers involved in the development of pharmaceuticals reported that nanotechnology was a fundamental step in drug development, and a wide range of drug delivery systems were designed based on this technology [4, 5]. As evidence, there has been a progressive increase in the number of commercially available nanotechnology-based drugs for the treatment of many diseases [6, 7]. In the present century, it is also one of the key technologies for atomic and molecular engineering, which is potentially able to change the features of human society further than any conventional technology [8, 9]. Throughout the history of medicine, alternative methods for drug delivery have been needed. Fifty years ago, a polymer–drug conjugate (polyvinylpyrrolidone– mescaline) was synthesised and is considered the first nanoparticle therapeutic [6] [10, 11]. In the mid-1960s, lipid vesicles were among the first drug delivery systems using nanotechnology, acknowledged later as liposomes [12]. These two classes of nanocarriers represent most of the marketed nanoparticle therapeutics and continue being extensively investigated presently [6, 8]. Subsequently, a variety of other biomaterials for drug delivery were developed. In the 1970s, there were conceptualised targeted drug conjugates, expounding the key principles that underpin much of the current thinking. Albumin-based nanoparticles, reported in 1972, were the precursor to the first protein-based nanoparticles to receive regulatory approval: albumin-bound paclitaxel (Abraxane®, Abraxis BioScience, AstraZeneca), which was approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic breast cancer in 2005 [6]. However, the first nanoparticle therapeutic, approved by the FDA in the 1983, was a combination of cyclosporine and Cremophor® EL (polyoxyethylated castor oil, which is capable of solubilising extremely lipophilic drugs through the formation of micelles), marketed as Sandimmune® by Novartis. Cremophor® is also used in the preparation of the cytotoxic anticancer drug paclitaxel (Taxol®, Bristol-Myers Squibb). The first controlled-release polymer composition (polymer-based nanocarriers), an implantable form of goserelin acetate (a synthetic analogue of luteinising hormone releasing hormone), marketed as Zoladex® by AstraZeneca, was approved by the FDA in 1989 for the treatment of certain types of prostate and breast cancers. This controlled-release polymer–drug composed of small polymer rods, also known as ‘drug depots’, allow a drug or other molecule of interest to be trapped inside a polymer matrix and released over an extended period of time as it slowly diffuses out of the polymer matrix. Langer’s laboratory pioneered the use of these types of materials, beginning with non-degradable polymer matrices in their early work and leading to therapeutics such as a biodegradable depot form of the cytotoxic drug carmustine (Gliadel®, Eisai), which was approved by the FDA for the treatment of brain cancer in 1996 [6].

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The first long circulating liposome, or “stealth liposome”, was described in 1987. Subsequently, the use of polyethylene glycol (PEG) to increase circulation times for liposomes and polymeric nanoparticles (1990 and 1994, respectively) allowed the development and approval of Doxil® (doxorubicin liposome), for the treatment of AIDS-associated Kaposi’s Sarcoma (1995) [6, 8]. Several other liposome-based therapeutics have been approved by the FDA for the treatment of disease, including liposomal amphotericin B (AmBisome®, Gilead) for fungal infections and liposomal morphine (DepoDur®, Pacira Pharmaceuticals) for postsurgical analgesia. As a result, there are over two dozen nanotechnology-based therapeutic products that have been approved for clinical use up to 2009 [13]. Nanotechnology is an area relatively new to science and its application in medicine is promising. The advances made so far suggest that nanotechnology will have a great impact on the prevention, diagnosis, and treatment of diseases [5, 14, 15].

14.2 Technological Advances: Nanotechnology and Cancer The term cancer is generically used to represent more than 100 tumour types, which include malignant tumours from different regions of the body. It is defined by a rapid and disordered growth and spreading of abnormal cells [16], which can be formed due to external factors (e.g., smoking, chemicals, and infections) or internal factors (e.g., inherited metabolism mutations, hormones, and immune conditions), and replicate at a higher rate than healthy cells, placing strain on nutrient supply and metabolic waste product elimination. Tumour cells will displace healthy cells until the tumour reaches a diffusion-limited maximum size. To grow beyond this size, the tumour must recruit blood vessels to provide the necessary nutrients to fuel continued expansion. Cells at the outer edge of the tumour mass have the best access to nutrients, while cells on the inside that rely on diffusion to deliver nutrients and eliminate waste products die, creating a necrotic core. Thus, tumours exhibit densely vascularised regions to acquire an adequate supply of nutrients for rapid growth and necrotic regions or haemorrhages [17, 18]. Since 2003, malignant neoplasms have been classified as the second leading cause of human death, representing almost 17% of known deaths. According to the World Cancer Report (2008) of the International Agency for Research on Cancer (IARC/WHO), the global impact of cancer more than doubled in the past 30 years. In the last decade, researches have focused on using distinctive characteristics of tumour cells and the vasculature around these cells to deliver chemotherapeutic drugs, gene therapy, imaging devices, and other active agents directly and selectively into cancerous tissues. These studies have mainly been concentrated on conjugated polymers, liposomes, micelles, dendrimers and polymeric nanoparticles [19]. Although many new drugs that are potential candidates for cancer treatment have been developed successfully by the pharmaceutical industry, cancer still remains a leading cause of death and is a severe threat to human health. Anticancer drugs also show serious side effects, such as high toxicity for normal cells [18]. This limits the dose or cumulative doses that can be administered to patients,

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which can then lead to relapse of the tumour and the development of drug resistance. The main challenges for worldwide researchers are therefore the enhancement of cancer treatment efficacy and reduction of the severe side effects that restrict the use of antineoplasic drugs. The development of new drug molecules that can act specifically on tumour tissue is a strategy that has been used to achieve this goal [20]. However, new drug development is a process that requires large investments and spans a massive amount of work. On average, over 20% of revenues from currently marketed pharmaceuticals (about US$ 40 billion) are invested in developing new medicines. According to the Tufts Centre for the Study of Drug Development (USA), it takes about 10-15 years and $ 450-900 million until the new drug can be launched. In addition, for each 10,000 new molecules developed, only one becomes a marketed drug. New drug delivery systems, however, can be developed for 20% of this value and in half the time, which allows pharmaceutical companies to maximise the return on their investments quickly. Additionally, these systems give a second life to old drugs, with better efficacy and patient compliance [21]. As such, this research has developed new forms of administration and absorption for drugs, as well as ways to increase its action time, maximise the efficacy and minimise side effects. Many medicines containing drug nanocarrier systems are currently in the early stages of clinical trials. These systems are very promising as chemotherapeutic agent vehicles, as they present several advantages over conventional formulations. These advantages include reduction of side effects, increased therapeutic index, easier administration and increased patient treatment compliance [5, 20, 22, 23]. Moreover, nanocarriers are also able to increase the blood circulation time of drugs, allowing a reduction in the dose required to achieve therapeutic levels over an extended period of time, and delivering the drug directly to the tumour site, reducing the toxicity and healthy tissue interactions [5, 24–27]. Furthermore, the formulation of a drug in a nanoparticulate system can lead to a reduced renal and hepatic clearance and decreased immune system recognition, changing the pharmacokinetic properties and biodistribution of the drug. An example of a nanocarrier system already in development is XyotaxTM, currently being tested in clinical phase III. This product consists of an acidic polyglutamic-paclitaxel conjugate. Currently Paclitaxel (Taxol®), an anticancer drug approved since 1992, is one of the first drugs of choice for the treatment of metastatic breast and ovarian carcinomas at advanced stages [29]. Its conventional formulation causes a high rate of hypersensitivity due to the high concentrations of polyethoxylated castor oil and ethanol need to solubilise the drug. The nanostructured system increased the drug solubility in water (> 20 mg/kg) and showed significantly higher therapeutic efficacy with reduced levels of side effects compared to a conventional formulation of paclitaxel in phase II clinical trials [30]. Nanocarriers can also improve drug stability, allowing the development of potentially effective new chemical entities that have stalled during preclinical or clinical development because of suboptimal pharmacokinetic or biochemical properties. Thus, nanocarriers may also facilitate the development of multifunctional systems for targeted drug delivery [31, 32], combined therapies [33], or

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systems for simultaneous therapeutic and diagnostic applications. Furthermore, various nanosystems, as a result of their small size, accumulate at higher concentrations than conventional drugs at specific areas of the body [34]. Moreover, the association of increased vascular permeability and a damaged lymphatic drainage system lead to a higher permeability and retention of nanosystems in tumours and inflamed tissues [4, 35, 36]. In the 1980s, a ground-breaking discovery was made while investigating a poly(styrene-co-maleic acid) conjugated to the cytotoxic drug neocarzinostatin [36, 37]. Compared to free neocarzinostatin, a significantly enhanced accumulation of the conjugated system at the tumour site was observed, which the authors ascribed to the unique structural features of the tumour vasculature. This phenomenon became known as the enhanced permeability and retention (EPR) effect. The enhanced permeability allows macromolecules to escape circulation due to the inherent leakiness of the underdeveloped tumour vasculature. In addition, the lack of an efficient lymphatic system leads to retention of those macromolecules in the tumour bed. Typically, to capitalize on the EPR effect, a drug carrier must be the size range from approximately 10 nm to 100 nm. Entities smaller than 10 nm are rapidly cleared by the kidneys or through extravasation, and entities larger (~100–200 nm) are cleared by the reticulo-endothelial system. Polymer–drug conjugates are particularly well suited to take advantage of the EPR effect because the molecular mass of the polymer can be easily altered, allowing the effective size of the construct to be tuned systematically [5]. The tumour tissue vasculature is highly heterogeneous and encompasses areas from vascular necrosis to extensively vascularised areas, ensuring an adequate supply of oxygen and nutrients for tumour growth [38]. The transport of macromolecules across tumour microvasculature may occur through open interendothelial junctions or transendothelial channels. The pore cut-off size of these transport pathways in various models has been estimated to be in the range of <1 µm, and in vivo measurement of extravasation of liposomes into tumour xenografts suggested a cut-off size in the range of 400 nm. In general, particle extravasation is inversely proportional to size, so smaller particles (< 200 nm size) would be most effective for extravasation of the tumour microvasculature [39]. The tumour lymphatic system is also abnormal, resulting in fluid retention in tumours and high interstitial pressure with an outward convective interstitial fluid flow [39]. This property is thought to promote tumour cell intravasation, resulting in tumour metastasis and blockage of nanocarrier extravasation from microvasculature into the tumour interstitium. However, the lack of an intact lymphatic system also results in the retention of the nanocarriers in the tumour interstitium, since these particles are not readily cleared. When taken together, the leaky microvasculature and the lack of an intact lymphatic system results in an enhanced permeation and retention (EPR) effect and “passive” cancer targeting through the accumulation of nanocarriers in the tumoral area at a higher concentration than in the plasma or other tissues [35, 39]. The release of drugs from nanocarriers in this case then results in a relatively higher intratumoural drug concentration, which translates into enhanced tumour cytotoxicity. These nanocarriers may be further modified for “active” cancer targeting by functionalising the surface of

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nanocarriers with ligands such as antibodies, aptamers, peptides, or small molecules that recognize tumour-specific or tumour-associated antigens in the tumour microenvironment. When nanocarriers are targeted to the extracellular portion of transmembrane tumour antigens, they may be specifically taken up by cancer cells through receptor mediated endocytosis [40, 41]. The specific targeting, intracellular uptake, and regulated therapeutic delivery of a payload are properties that are derived from rational design of nanocarriers. The application of nanotechnology to cancer therapy, including the development of “smart” nanoparticles, is indeed an exciting and promising area of investigation. In the following sections, we review some of the most important breakthrough nanotechnology platforms for cancer therapeutic applications aiming topic and transdermal uses [39]. The principal goals of cancer nanotechnology are to develop safer, yet more effective, diagnostic and therapeutic tools for the fight against cancer. Drug encapsulated and targeted nanoparticles are a promising class of therapeutics with the potential to improve efficacy and safety of currently available drugs. Recent improvements in engineering of nanotechnology platforms, including polymeric nanoparticles, liposomes, dendrimers, and polynucleotide nanoparticles, have led to the development of a variety of new nanoscale platforms, such as quantum dots, nanoshells, gold nanoparticles, paramagnetic nanoparticles, and carbon nanotubes [42]. There is also an increasing number of targeting molecular platforms that have emerged for use in the engineering of nanocarriers. These include antibody and antibody fragments, peptides, aptamers, small molecules, and carbohydrates. The intrinsic properties of nanocarriers, including the density of ligands on their surface, might affect the treatment efficacy and should be optimised. Targets in the vasculature are easily accessible for bioconjugates to be internalised, but physical and physiological barriers in solid tumours might limit the diffusion of therapeutic nanocarriers into target cells. Each class of nanoparticles might be useful for a particular application based on the specific physiological environment and clinical requirements. Hence, multifunctional nanocarriers are now emerging to combine radiotherapy and chemotherapy, with or without imaging properties. To move these technologies forward, novel approaches are needed to accelerate the discovery process, such as synthesis and screening automation. Future areas of nanotechnology innovation for cancer treatment include the development or optimisation of novel bioresponsive and self-regulated delivery systems. These are truly exciting times and, with continual support, medicine will continue to be a major beneficiary of nanotechnology for years to come [39]. Nanotechnology also is progressing rapidly in the field of in vivo imaging and therapeutics. This very fast progress will have important implications for management of cancer in the near future. Nanocarriers have been used for a wide variety of applications, from the detection of apoptosis through magnetic resonance imaging (MRI), to sentinel lymph node mapping, to photothermal ablation of tumours. Previously developed nanoscale platforms, such as liposomes and micelles, have also been modified and improved.

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14.3 New Strategies for Topical and Transdermal Treatment of Cancer Among the various routes of administration, the skin and mucous membranes have been widely studied. Drug administration in the skin is extremely attractive, not only due to the possibility of targeting skin diseases (topical), but also for obtaining systemic effects (transdermal administration). In addition, it provides patient compliance and avoids first-pass hepatic metabolism. Usually, topical administration is a challenge in pharmaceutics. It is well known that the delivery of several drugs in and across the skin is a difficult task due to the barrier function of the skin, provided by the highly organized structure of the stratum corneum (SC) [43, 44]. Please, see Chapter 1. To enable the successful therapeutic application of hydrophilic molecules, which the stratum corneum is a major barrier to in cutaneous delivery, a suitable release system is required. Lipid nanoparticles, for example, may improve epidermal penetration because lipid carriers attach themselves to the skin surface, allowing lipid exchange between the outermost layers of the SC and the carrier [45]. Several compounds and techniques have been studied to increase drug penetration in the skin, including penetration enhancers, physical techniques like eletroporation and iontophoresis, and drug delivery systems [43, 44]. In this context, nanoscale carrier systems were already evaluated in several studies for skin interactions and their impact on drug penetration [46], as previously discussed in Chapter 1. Innovation in drug delivery has made possible the use of certain chemical or biological compounds that were not previously used due to toxic side effects or lack of effective administered [47]. As such, several strategies can be employed in order to optimise topical and transdermal drug delivery. As an example, drug targeting can be employed to deliver chemotherapeutic agents directly inside the tumour, reducing side effects [48]. Moreover, there are many strategies to increase skin penetration and permeation, including chemical and physical methods, as discussed below.

14.3.1 Target Delivery The main objectives of target delivery are to reduce side effects and improve the efficacy of therapeutics. One of the advantages of nanoscale drug delivery systems is their ability to deliver drugs directly to their site of action (e.g., targeting tumoral tissues) [49]. These systems can also be employed to target very inaccessible sites, like the brain, once they are permeable to biological barriers [50]. These systems are able to penetrate the tumour mass due to the “leaky” nature of the tumour microvasculature, with contains pores varying from 100 to 1000 nm in diameter. The vasculature of health tissues, on the other hand, presents tight intercellular junctions of less than 10 nm. Therefore, tumour tissues can be targeted by the construction of systems with diameters larger than the intercellular gaps of health tissues but smaller than the pores of the tumour microvasculature [48]. Due

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to their high surface area, nanocarriers can also improve the pharmacokinetics and biodistribution of drugs [39]. Several metals, such as gold (Au), silver (Ag) and cooper (Cu), as well as inorganic materials, such as silica or alumina, have been used to produce nanoparticles with excellent optical and photoelectric properties [51]. Gold nanoparticles, besides being very good contrast agents, are very effective in photodermal cancer therapy since they can efficiently convert strongly absorbed light into localized heat [52]. Furthermore, they are very good vectors for gene delivery [53]. Micellar systems, nanoscopic core-shell structures created spontaneously by amphiphilic molecular assembly, like co-polymers and phospholipids, have been extensively used to increase the solubility of poorly-water soluble drugs. This type of delivery system can also be designed for drug delivery through the skin. As an example, Sibata and co-workers [54] incorporated zinc (II) phthalocyanine (ZnPc), a classical photosensitiser used in photodynamic therapy (PDT), into a phospholipid micellar system. The system provided better drug stability and enhanced the fluorescence quantum yield and lifetime of the triplet excited state over conventional formulations. These results demonstrated that this type of system is promising for use in PDT and photodiagnostics. Another system containing ZnPc incorporated in a magnetic nanoemulsion was developed by Primo et al. [55] for targeted delivery of a drug in the treatment of skin cancer. The in vitro assays demonstrated that the presence of magnetic nanoparticles significantly increased the drug amount in the deeper layers of the skin, making it a promising strategy for potentiating the effects of ZnPc.

14.3.2 Chemical and Formulation Enhancers of Skin Penetration and Permeation Several compounds can be used to enhance drug delivery into or across the skin. According to Shah [56], these compounds can increase the drug diffusivity in the skin, promote a reversible fluidisation of the lipids present in the stratum corneum, optimise the thermodynamic activity of the drug in its vehicle or alter the partition coefficient of the drug, favouring its release from the formulation. Some of these compounds are essential oils and terpenes, thiomenthol derivatives, oxazolidinones, iminosulfuranes and azone analogues [57]. Lipid nanocarriers have been used successfully for drug delivery in the skin. Liposomes are one of these nanocarriers, being used to administer drugs by both systemic and topical routes [58]. Studies have demonstrated that the inclusion of some organic solvents, like ethanol, in these lipid structures can further enhance skin permeability. In 1997, Touitou et al. [59] developed a structure named ethosome that matched this type of lipid nanostructure. Further studies demonstrated that this system was more efficient than conventional liposomes and hydroalcoholic solutions in the topical delivery of drugs to the skin, increasing both delivered amount and depth [60]. Fang et al. [58] compared the skin delivery of 5-aminolevulinic acid (ALA) incorporated in conventional liposomes versus ethosomes. Results obtained by confocal laser scanning microscopy from the in vitro

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skin penetration study showed that ethanol significantly increased the deposition of the drug in the skin. As suggested by Touitou [61] ethanol promoted a fluidising effect of the ethosomal lipids and lipid bilayer of the stratum corneum, favouring skin penetration of drugs. Invasomes are another variation of liposomal systems composed of phospholipids, ethanol and terpenes. Studies carried out by differential scanning calorimetry (DSC) and X-ray diffraction showed that terpenes are able to increase drug permeation by the disruption of the lipid packaging of the stratum corneum and/or disturbing the stacking of the bilayers [62, 63]. In work performed by DragicevicCuric et al. [64] temoporfin-loaded invasomes were submitted to in vitro skin penetration studies and compared to conventional liposomes and ethosomes. The results showed that the invasomes promoted an increase in skin penetration when compared to conventional liposomes and even to ethosomes. As described previously [65, 66], ethanol and terpenes show a synergistic effect on skin penetration. Drugs that are under investigation for topical application using lipid nanoparticles nowadays include, for instance, glucocorticoids, retinoids, non-steroidal antiinflammatory drugs, COX-2 inhibitors, antimycotics, and anticancers. It was shown that it is possible to enhance the percutaneous absorption with lipid nanoparticles. These nanocarriers may even allow drug targeting to the skin or even to its substructures. Thus they have potential to improve the benefit/risk ratio of topical drug therapy [67].

14.3.3 Physical Methods: Iontophoresis, Sonophoresis, and Electroporation 14.3.3.1 Iontophoresis Iontophoresis is a non-invasive technique that uses a low intensity electric current to promote drug delivery across biological membranes into the bloodstream [68]. The electric current, supplied from a battery, is distributed using positive (anode) and negative (cathode) electrodes through an electrolytic solution that flows on the skin in the direction of the blood flow [69, 70]. The device is put on the skin and an electronic controller starts the passage of the electric current between the electrodes. The drug is then repelled from the reservoir directly into the skin and bloodstream [71, 72]. In May 2006, the FDA approved the Ionsys® device (Ortho McNeil, Inc.), the first patient controlled iontophoretic device for transdermal delivery of fentanyl, an opioid analgesic drug used for the treatment of chronic pain [73]. The device, containing a fentanyl hydrochloride (HCl) - loaded hydrogel formulation, precisely releases a dose equivalent of 40.0 μg over 10 minutes to 24 hours for a maximum of 80 doses. This device is composed of a plastic top containing a 3 V lithium battery and a second plastic housing that contains two hydrogel reservoirs (one, the anode, loaded with the formulation containing fentanyl HCl, and the other, the cathode, loaded with a placebo formulation), as well as a polyisobutylene skin adhesive. The hydrogel formulation is protected by a siliconised plastic layer that must be removed before placement on the skin [74].

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Iontophoresis has been used in association with nanostructured formulations to improve drug delivery in (topical) and across (transdermal) the skin. Lidocaine hydrochloride (a hydrophilic drug) skin penetration was evaluated in a study where a microemulsion-based formulation was topically applied, followed by the application of a short-term iontophoretic current. It was shown that the combined use of a microemulsion and iontophoresis promoted an increase in drug concentrations in epidermis and dermis compared with the application of the microemulsion alone, besides the lag time had been reduced, favouring the accumulation of the drug in the skin [75]. Liu et al. investigated the influence of iontophoresis in the trandermal delivery of triamcinolone acetonide acetate (used as a model drug), formulated in a solid lipid nanoparticles hydrogel. The study showed the electric field application enhanced expressively the drug amount in the skin compared with the application of the hydrogel formulation alone. It was also evaluated the influence of particle size in drug skin penetration and one could see that the smaller the particles the higher the drug amount in the skin [76]. 14.3.3.2 Sonophoresis Sonophoresis is a physical technique to improve transdermal drug delivery using ultrasound energy [77]. Studies have suggested that the enhancement of drug delivery in the skin by sonophoresis is due to alterations of the skin barrier properties, but how this process occurred was not clear until now [78]. Nowadays, this technique is still limited to topical applications, specifically to increase the delivery of anti-inflammatory [76] and local anaesthetic drugs [79], but new applications, such as gene and drug delivery for the treatment of solid tumours, has been investigated [80]. Ultrasound (US) has also been applied in association with chemical enhancers to improve transdermal penetration of drugs [81]. The effects of US on biological tissues can be divided into thermal and nonthermal effects. The thermal effects are related to the absorption of acoustic energy by the tissues and fluids [82]. The non-thermal effects include cavitating bubbles and other effects, like radiation torque and pressure and acoustic streaming. The effect of particular relevance to drug delivery is the degree that the fluid or particle motion increases in the convection currents, increasing the drug transport toward or into the cells [83]. Harada et al. investigated the effect of the never before tried association of US and nanoparticles of titanium dioxide – TiO2 (a potential sensitizer by photodynamic therapy), over melanoma cells in in vitro and in vivo experiments. In this study, activation of TiO2 was induced by US irradiation. The in vitro studies demonstrated that the application of ultrasound in the presence of TiO2 enhanced the percentage of apoptotic cells in 2.73 times compared with untreated cells. In vivo experiments carried out in mice showed the treatments with TiO2 or US alone were not able to reduce tumor growth compared with the untreated group. But, the association of TiO2 and US lead to a significant (P<0.05) inhibition of tumour growth when compared with the control group [84].

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14.3.3.3 Electroporation The application of powerful electric pulses for a short period promotes cellular membrane disorganization, which can be employed as a tool to improve skin drug delivery (electroporation) [85, 86]. Electroporation of cells is accomplished using electrical pulses varying from 0.1 to 1 kV cm-1 for 100 µs [87]. This method has been studied especially for the introduction of DNA and anticancer drugs into cells [88]. Studies showed that exposure to short intense electrical pulses increased the in vitro cytotoxic activity of some chemotherapeutic agents [89]. The strong but reversible alteration in skin permeability makes electroporation an interesting tool to deliver macromolecules (up to at least 40 kDa). This technique could also be used for various topical treatments [90]. Electroporation has been used frequently to potentiate the cytotoxic effects of anticancer drugs in a technique named electrochemotherapy. This application is especially interesting for hydrophilic and non-permeable cytotoxic drugs, like bleomycin and cisplatin. Furthermore, electrochemotherapy has been studied to treat metastatic nodules of solid tumours in the skin and subcutaneous tissues [91]. The association of electroporation and nanocarriers has been studied to treat different kinds of malignant tumours, as can be seen in the work conducted by Xu et al. where a novel technique combining electroporation and the conjugation of TiO2 nanoparticles with monoclonal antibodies was evaluated. The objectives were to increase photokilling selectivity and efficiency of photoexcited TiO2 due to the conjugation with the monoclonal antibodies and to accelerate the delivery of TiO2 nanoparticles with the application of electroporation. These objectives were achieved successfully in experiments conducted with human LoVo cancer cells, showing that is a promising method than can be extensively explored to treat many different kinds of tumours, just changing the type of antibody [92].

14.3.4 Microneedles Microneedles are composed of a matrix with microprojections containing drugs or vaccines and applied to the skin in order to facilitate transdermal delivery of the active substances. The release mechanism in this case is not based on diffusion, as in other transdermal devices, but in the transitory mechanical rupture of the skin layers to reach the target site [93]. These systems are similar to traditional needles, but manufactured on a microscale. Microneedles must be long enough to penetrate the stratum corneum, but not so long as to reach nerve endings, reducing the risk of infection. Another advantage is that no professional staff is required for the insertion of microneedles [94]. The active substances on microneedles may be delivered by four different mechanisms: skin puncture by a matrix composed of solid microneedles followed by the application of a formulation containing the drug in the pre-treated area; insertion of solid microneedles recovered by the drug and their further removal; introduction of biodegradable microneedles into the skin for dissolution and subsequent release of the drug; and injection of active substances through hollow microneedles [95].

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Improvement of the intradermal delivery of ALA (a protoporphyrin XI (PpXI) precursor) with a silicon microneedle array was tried by Donnelly et al. [96] for potentiating topical skin cancer treatments. In this study, full thickness murine skin was firstly punctured by the microneedles and then an ALA-loaded bioadhesive patch was put over the pre-treated area. The ALA skin penetration test showed that pre-treatment with the microneedles lead to a significant increase in transdermal delivery compared to intact skin. In the same study, the influence of pre-treatment with microneedles in the in vivo production of PpXI in murine cells was also evaluated. Furthermore, the authors observed an increase in the PpXI in normal murine skin after microneedle puncture. The optimization of the in vitro skin permeation of docetaxel, a high molecular weight and poorly-water soluble anticancer drug, across rat and porcine skin with elastic liposomes and microneedles was investigated. The study showed that elastic liposomes were able to increase the drug skin permeation compared to conventional liposomes independent of pre-treatment with microneedles. However, it was also observed that the microneedles promoted an increase in transdermal flux for all formulations (conventional and elastic liposomes) and decreased the lag time from the elastic liposomes by approximately 70%, suggesting that the application of elastic liposome and microneedle pre-treatment could be useful for improving the skin permeation of high molecular weight and poorly-water soluble compounds [97]. At the time of publication, there were a few microneedle devices available on the market, such as Macroflux® (Zozano, Pharma) and Nanoject® (Debiotech). The Macroflux® technology was developed to deliver peptides, proteins, high and low molecular weight molecules and vaccinations by the transdermal route. This system is composed of titanium microneedles placed in a patch that can be used to deliver drugs by releasing then into the viable layers of the skin and favouring its absorption (http://www.zosanopharma.com/). The Nanoject® system employs MEMS technology (microelectro mechanical system). This system allows a high level of reproducibility in microneedle manufacturing at a low cost, being applicable to intradermal and hypodermic drug delivery, diagnostic, collect of biological fluids, and immunization. It is built from metal microneedles with side holes that facilitate drug perforation and prevents coring of cutaneous tissue. Furthermore, the needles have a circular tip, which was designed to cause the least possible damage to the skin and facilitate healing (http://debiotech.com/products/msys/uneedle.html).

14.4 Final Considerations Binomial nanoscience / nanotechnology is currently one of the most debated topics in the scientific and technological community. The multidisciplinary nature of the subject contributes significantly to the expansion of this new horizon of science. Projects in several areas have started leaving research labs and moving to industry. This trend is slowly turning into a nanotechnology-based industry, indispensable to any branch of science or technology. The impact will be felt more quickly in areas of cosmetics and pharmaceuticals, as nanocoated pharmaceuticals - or pharmaceuticals engineered on the nanometric scale - promise to reach

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inaccessible points and act more effectively. One cannot state the exact number of companies dedicated to nanotechnology, but in developed countries it is difficult to find a high tech company, which is not involved in this activity nowadays. Over the next four decades, science will move closer to more perfectly mimicking life. Viruses, which behave like natural nanomachines that trick the immune system, are a great source of inspiration. For example, 60 U.S. companies are all developing an intelligent molecular system capable of navigating into the bloodstream and recognising specific proteins released by HIV. Another great possibility is the use of nanoparticles to repair neurons damaged by trauma, stroke or neurodegenerative diseases like Parkinson's. Nanotechnology is beginning to fulfil its potentials with products being made commercially available to people. The fight against cancer has also been gaining reinforcements from nanotechnology. Nanocarriers have been widely researched to improve the treatment of all kinds of cancer, including skin malignant tumours. The topical and transdermal routes may be some of the most important ones to deliver anticancer drugs.

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Chapter 15

Nanocarriers to Deliver Photosensitizers in Topical Photodynamic Therapy and Photodiagnostics Wanessa S.G. Medina, Fabíola S.G. Praça, Aline R.H. Carollo, and Maria Vitória L. Badra Bentley Faculdade de Ciências Farmacêuticas de Ribeirão Preto, University of São Paulo, Brazil [email protected]

Abstract. Topical photodynamic therapy is used for the prevention and treatment of non-melanoma skin cancer. Until recently, clinically approved indications have been restricted to superficial basal cell carcinoma and nodular, actinic keratoses and, since 2006, Bowen’s disease. Most photosensitizers are relatively hydrophobic and will be attracted to membranes, but even the exception molecules, having substituents that render them watersoluble, bind to membranes because of their hydrophobic ring systems. The primary function of the skin, however, is to protect the body from unwanted influences from the environment. This protection is provided primarily by the stratum corneum, which consists of corneocytes surrounded by lipid regions. The major limitation of PDT is the poor penetration of photosensitizers through biological barriers, like the skin. Over the past 10 years, a considerable number of studies have therefore been conducted on the development of different strategies to overcome these difficulties, including nanocarriers to delivery photosensitizers and their precursors, nanoemulsions, liposomes, ethosomes, invasomes, liquid crystals and magnetic nanoparticles, among others.

15.1 Topical Photodynamic Therapy 15.1.1

General Considerations

Photodynamic therapy (PDT) is an emerging technology for the important therapeutic options in management of neoplastic and non-neoplastic diseases [1] and has been investigated in the treatment of patients with either carcinoma in situ or superficial cancers who are unable to tolerate or who refuse surgical resection. Photodynamic therapy is based on the administration of a photosensitizing drug and its selective retention in the malignant tissue. Most photosensitizers (PSs) are relatively hydrophobic and will be atracted to membranes, but even the exception molecules, having substituents that render them watersoluble, bind to membranes because of their hydrophobic ring systems.

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Photodynamic therapy is based on the administration of light-sensitive drugs or photosensitizers and its selective retention in the malignant tissue. Subsequent activation by exposure of a photosensitizing agent to light at a wavelength matching the absorption spectrum leads to a photophysical reaction resulting at the cell death due to the production of free radicals and/or reactive oxygen species, especially oxygen singlets (1O2) [2,3] (Figure 15.1).

Fig. 15.1 Schematic representation of Photodynamic Therapy for skin cancer

In this process, when light is absorbed, the energy of the absorbed photons leads to the absorbing molecule to be electronically excited. This kinetic energy may be converted into heat by the collision of the excited molecule with surrounding molecules through radiationless decay. Alternatively, it may be reemitted as fluorescence. The electronic energy levels between the transitions occur by absorption of ultraviolet-visible light (λ= 200 to 700 nm) and the photosensitizers absorb in the visible spectral regions below 700 nm, where light penetration into the skin is only a few millimeters, thus clinically limiting PDT to treating surface and relatively superficial lesions. The physical processes of luminescence are best described with the aid of Figure 15.2. The electronic states are represented by the triplet states, T1 and T2, and the singlet states, S0 to S2. Excitation begins in the ground state. These are two "magnetically" different excited and energized states that giving rise as a quantum mechanical consequence of electron spin. In each electronic state, the molecule has its electrons arranged in a different combination of the available orbits and spin orientations-the latter distinguishing the singlet and triplet states. Singlet oxygen is also highly reactive and likely to engage in irreversible chemical reactions with a susceptible binder or luminophore. Fortunately, direct photoexcitation of oxygen to the triplet state from the ground state is essentially forbidden. The oxygen molecule is unusual in that its ground state is triplet and it has a pair of excited singlet states at only 1.0 eV above the ground state. T1 can initiate photochemical reactions directly, arise to the reactive free radicals, or by energy transfer to the ground-state oxygen molecule (1O2) to yield excited singlet state oxygen molecules 1O2. This excitation to lead to 1O2 requires at least 20 kcal/mol, which places limits on the wavelength of absorption of the PS, which usually raises it to one of its lowest-energy singlet states [4].

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If the energetics is appropriate, photooxidative reactions may occur by excited singlet oxygen molecules mediation. This photodynamic mechanism of cytotoxicity is generally accepted for most PSs [5].

Fig. 15.2 Energy level diagram of photodynamic therapy for a typical type II PS and oxygen. The sensitizer in its ground state (S0) absorbs a photon of light and is excited to its first singlet state (S1). It spontaneously decays to its excited triplet state (T1) via intersystem crossing. From T1, energy is transferred to ground state molecular oxygen (3O2), creating reactive singlet oxygen (1O2). Adapted from Ryan et al., 2009 [6].

Mitochondria is considered an important target for anticancer therapy due to their crucial role in photodynamic cell death from the release of pro-apoptotic proteins, such as cytochrome C. In addition, apoptosis induction by PSs primarily localized to the mitochondria is an extremely rapid process [4]. Moreover, one important attribute of successful PSs is their ability to damage mitochondria, the most sensitive targets for directly triggering PDT-induced apoptosis. For example, PSs that target mitochondria kill cells more efficiently than those targeted to the plasma membrane or lysosomes. For those PSs which do not localize in mitochondria, mitochondrial damage may still be the event which ultimately leads to cell death because mitochondria are the central converging point for signals, irrespective of where they initiated [2,7]. In the tissues and cells, such as complex environments, the subcellular localization of the PS and their chemistry is important for effective photochemistry to occur. The lifetime of singlet oxygen is very dependent on its environment and can vary over many orders of magnitude [8]. However, the most unstable of these, singlet molecular oxygen, will not migrate more than a fraction of a micron from the site of formation and the energy transfer reactions involving 1O2 require close proximity of the target and sensitizer. Therefore, the cellular structures close to both a high oxygen concentration and a high sensitizer will be more effectively damaged upon illumination. Despite its advantages over current treatments, PDT is yet to gain general clinical acceptance [1].

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Skin Cancer and Non-neoplastic Disorders

The last decade has seen a breakthrough in the study of photodynamic reactions, which have led to different clinical trials aimed at developing other indications for dermatological PDT. However, these treatments still require longer trial periods and larger numbers of participants to ascertain the treatment protocols. Among the new indications for PDT, multiple inflammatory dermatoses, cancer, infectious hair removal, rejuvenation and vascular lesions are all included. The use of topical PDT for these indications may be useful given the advantages that this technique is noninvasive, has high specificity to the target tissue, is well tolerated, can treat multiple lesions in one session, has no cumulative toxicity, allowing multiple treatments, and good cosmetic results [9]. Topical photodynamic therapy is used for the prevention and treatment of nonmelanoma skin cancer. Until recently, clinically approved indications have been restricted to superficial basal cell carcinoma and nodular, actinic keratoses and, since 2006, Bowen’s disease. However, the range of indications is continuously expanding [9]. For example, many non-neoplastic dermatological diseases, like localized scleroderma, acne vulgaris, psoriasis, acne, common warts, cutaneous T cell lymphoma and viral warts, also have therapeutic benefits from PDT. Currently, the only Food and Drug Administration (FDA) approved indication for 5ALA (5-aminolevulinic acid) PDT and MAL (methyl ester of 5-ALA) PDT is for the treatment of actinic keratoses. The off-label and alternative uses include treatment of basal cell carcinoma (BCC), photo-aging, acne vulgaris, Bowen’s disease and hidradenitis suppurativa [10]. MAL also induces photosensitizing porphyrins, but does not induce strong generalized cutaneous photosensitization [10]. Due to the easy accessibility of skin to light activation, incoherent lamps and LED arrays are both suitable for PDT. The production of reactive oxygen intermediates, like singlet oxygen, depends on the applied light dose, as well as the concentration and localization of the PS in the diseased tissue. Once irradiated, cytotoxic effects resulting in tumor destruction or immunomodulatory effects improving inflammatory skin conditions are induced in the diseased tissue [11]. Clinically, rather rapid tumor destruction results upon PDT. In addition, as most PSs do not accumulate into connective tissues, one can achieve healing with minimal scar tissue generation. This combination of relatively selective and rapid tumor destruction with excellent cosmetic outcome has therefore continued to push PDT to the forefront of treatment for cutaneous lesions [12]. Furthermore, numerous publications have demonstrated the effectiveness of PDT for the treatment of other cutaneous malignancies and non-oncologic indications as well (Table 15.1) [9].

15.1.3

Photosensitizers and Precursors

PSs are chemical compounds with a chromophore that, when it is absorbed by cancer cells and exposed to light of specific wavelengths, becomes active and generates reactive oxygen species (ROS). It is these ROS that are commonly used

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Table 15.1 Diseases, photosensitizer drugs and carriers

DISEASES Acne

PHOTOSENSITIZER DRUGS AND PRO-DRUGS 5-ALA Methylene blue 5-ALA and derivatives

Non-melanoma skin cancer

5-ALA 5-ALA and M-ALA Zinc phtalocyanine tetrasulphonate (ZnPcSO4)

Neoplastic skin lesion

5-ALA and derivatives

Nasopharyngeal carci5-ALA noma and oral leukoplakia Psoriasis

Zinc phtalocyanine (ZnPc)

Cutaneous skin neoplasias

Phenothiazinium

CARRIER Liposomes[13] Liposomes[14] Cubic phase based monoolein and water[15] Monoolein and propylene glycol[16] Nanostructured lipid cubic phase[17] Lamellar and cubic liquid crystalline phases[18] Sensitive adhesive matrix[6] Mucoadhesive thermoresponsive polymeric gel[19] Aqueous-based topical gel[20] Liposomes AND nanoparticles[21]

for light-induced cytotoxicity in photodynamic therapy. In addition, PSs in PDT are the vessels that allow for the transfer and translation of light energy into type II chemical reactions. The reactive end products of this pathway result in a rapid cyto- and vasculotoxicity, which are essential in photodynamic therapy [22]. A large number of photosensitizing agents have been tested in vivo and in vitro in PDT experiments, but very few have shown desirable properties. For this reason, recent studies have focused on the development and efficacy of new PSs [23,24]. Prerequisites for an ideal sensitizer include chemical purity, a high tumor to normal tissue ratio, chemical and physical stability, rapid accumulation in tumor tissue, activation at therapeutic wavelengths with optimal tissue penetration, and rapid clearance from the body [6,23]. The main classes of PSs are porphyrin derivatives, their precursor (the 5-ALA, phthalocyanines, and chlorins). They exhibit different photochemical and photophysical properties in their mechanisms of action and light activation, so each one is combined with a specific type of light, in a specific type of administration and dose to kill cancer cells [25]. 15.1.3.1 Porphyrins The first family of photosensitizer discovered was based on hematoporphyrin (Hp) and its derivatives. They were the first systematically studied PSs used for clinical

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PDT and are currently the most extensively used PSs. Photofrin® (Axcan Pharma) is a combination of monomers, dimers, and oligomers derived from chemical manipulation of hematoporphyrin, which has the longest clinical history and patient track record in PDT [22,26]. Further substitutions and changes to the porphyrin ring structure resulted in additional PSs of interest. One example is a commercially available product based on verteporfin, a highly photoactive benzoporphyrin derivative of porphyrin. Visudyne® (Novartis Pharmaceuticals), indicated for the treatment of patients with predominantly classical subfoveal choroidal neovascularization (CNV) due to macular degeneration, pathologic myopia and ocular histoplasmosis syndrome [27]. Other examples are meso-tetra(3hydroxyphenyl)porphine (m-THPP) and meso-tetra(para-sulfophenyl)-porphin (TPPS4), but both can cause toxicity if used systemically. However, TPPS4 has potential for the topical treatment of skin tumors, where no neurotoxicity was found upon systemic administration [28]. 15.1.3.2 5-ALA and Derivatives 5-ALA is a small (167.6 Daltons), water-soluble pro-drug that is a naturally occurring precursor in the biosynthetic pathway of heme [26]. Its topical application represents the most commonly used PDT technique in dermatology. Administration of excess exogenous 5-ALA avoids the negative feedback control that heme exerts over its biosynthetic pathway. Due to the limited capacity of ferrochelatase to convert protoporphirin IX (PpIX) into heme, the presence of excess exogenous 5-ALA in cells induces accumulation of PpIX [23]. The hydrophilic nature of 5-ALA, as evident from its low octanol-water partition coefficient of 0.03 [29,30], does impair permeation because stratum corneum, the outermost layer of the skin, presents an essentially hydrophobic barrier to the permeation of exogenously applied agents [31,32]. This low penetration capacity of this highly hydrophilic molecule into deeper skin layers limits its use in PDT [33]. Therefore, many authors have sought to increase 5-ALA penetration. Steluti et al. (2005) used formulations containing glycerol monooleate in propylene glycol solutions with 5-ALA. According to them, this formulation significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In addition, in vivo studies also showed increased PpIX accumulation in mouse hairless skin [34]. Another alternative to solve the penetration problem of 5-ALA is the use of its ester derivatives: methyl-, butyl-, hexyl-, and octyl-5-ALA. Studies comparing the permeation/retention of 5-ALA and its esters across the skin showed that the more lipophilic forms had increased permeation and were more retained in viable epidermis and dermis than 5-ALA [35]. Another study showed that, in normal tissues, 5-ALA derivatives generated PpIX more slowly than 5-ALA, suggesting that they were less rapidly taken up and/or converted to free 5-ALA. However, the biological stock of PpIX may be enhanced over long exposure periods, especially in the case of 5-ALA-methyl ester or 5-ALA-hexyl ester [36]. The log P of octyl ester is nearly four orders of magnitude more lipophilic than 5-ALA and shows a strong affinity for the stratum corneum, which may diminish its bioavailability. This information shows that a pro-drug with very high lipophilicity does not guarantee therapeutic success [39].

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Two products based on 5-ALA are available for use in topical PDT: Levulan® (DUSA Pharmaceuticals, Inc.), a topical solution of 5-ALA (20%) for actinic keratoses [38] and Metvix® (Galderma/PhotoCure ASA), a cream containing 16% methyl aminolevulinate for topical therapy of pre-cancerous and skin cancer lesions, such as actinic keratoses, basal cell carcinomas and Bowen's disease [39]. 15.1.3.3 Chlorins Chlorins are reduced porphyrins with a strong absorption band in the 640–700 nm range, which can be altered by chelated metal ions[40]. One example of a chlorins is chlorin e-6, (Ce6), sometimes termed phytochlorin, which is derived from chlorophyll A. HPPH (Photochlor) is a chlorin-based PS that is highly active at 665 nm and is intravenously introduced with minimal toxicity [22]. Metatetra(hydroxyphenyl)chlorin (m-THPC), which is a second-generation PS with potent antitumor efficacy, has been approved for human use in Europe. Foscan® Temoporfin (m-THPC) (Biolitec Pharma, Ltd.) is indicated for palliative treatment of advanced head and neck squamous cell carcinoma [41]. Furthermore, benzoporphyrin derivative monoacid ring A (BPD-MA) has been found to be effective in treating basal cell and squamous cell carcinomas [42]. A study using chlorin p6 (CP6), a chlorophyll A derivative, assessed its pharmacokinetics and tumor response to PDT in hamster cheek pouch model. In this study, CP6 was administered either intraperitoneally or topically [43]. According to the authors, for the tumors tested (size >8 mm), topical application was more effective than intraperitoneally administration. In addition, the level of CP6 in the tumor and surrounding tissues was seen to decrease within 24 h of administration, becoming undetectable at longer time intervals (>72 h) [43]. In another study using chlorin e6, authors conducted a clinical assessment of PDT for metastatic pigmented melanoma. The drug was intravenously injected, and patients were exposed to light between 1 h and 24 h post-injection. It was observed that tissues subjected to PDT with chlorine e6 showed complete regression of the melanoma, with no recurrence during the study period [44]. 15.1.3.4 Phthalocyanines Phthalocyanines are second-generation PSs compounds that are highly colored, generally lipophilic, contain a diamagnetic metal ion, have shown high photodynamic efficiency in the treatment of animal tumors, and reduced phototoxic side effects [23,45]. In addition, these structures are active in the range of 650 nm to 850 nm, have structures similar to porphyrins and most are hydrophobic, requiring delivery agents for clinical use (such as a liposomal preparation) [15]. Phthalocyanines may be linked to a variety of metals, such as aluminum [46] and zinc [47]. A specific phthalocyanine, the Zn(II)-2(3), 9(10), 16(17), 23(24)-tetrakis-(4oxy-N-methylpiperidinyl)phthalocyanine (Zn(II) phthalocyanine), has been formulated in an azone-containing gel for topical administration in the dorsal skin of Balb/c mice. From the results, it appeared the PS localized in the epidermal layers [48]. In another study, three Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse

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embryo fibroblasts and HaCaT human keratinocytes). The authors showed that tetra-substituted phthalocyanines had more affinity for cells then mono-substituted ones and, as a consequence, the tetra-substituted phthalocyanines exhibited a higher phototoxicity against all three cell lines [49]. Beyond those cited above, other PSs, like texaphyrins (water soluble synthetic expanded porphyrins) [50], hypericin (an active plant pigment from Hypericum) [51], methylene blue (a phenothiazinium compound, originally used as an histological dye) [52] and Nile blue (a benzophenoxazine dye )[53] have all been suggested as PSs for photodynamic therapy.

17.2 Skin as Administration Site for Topical PDT 17.2.1 Skin Structure, Barrier and Permeability PDT has been applied to almost every type of superficial non-melanoma skin cancer and numerous benign skin disorders (in particular, basal cell carcinoma, actinic keratoses and squamous cell carcinoma) using the skin as the site of administration for topical PDT [26,37,54,55,56,57]. The primary function of the skin, however, is to protect the body from unwanted influences from the environment. This protection is provided primarily by the stratum corneum, which consists of corneocytes surrounded by lipid regions. As most drugs applied to the skin permeate along the lipid domains, the lipid organization is considered to be very important for skin barrier function [58,59,60]. In the stratum corneum, the lipids in the lamellar phases form predominantly crystalline lateral phases and two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover, a subpopulation of lipids likely forms a liquid phase [61,62]. Diseased skin is often characterized by an altered lipid composition and organization and a reduced barrier function [54,62,63,64]. Underlying the stratum corneum is the viable epidermis (50–100 μm thick), which is responsible for generating the stratum corneum. The viable epidermis consists of various layers, such as the stratum basale, the stratum spinosum and the stratum granulosum. In addition, the epidermis is a dynamic, constantly self-renewing tissue in which a loss of the cells from the surface of the stratum corneum (desquamation) is balanced by cell growth in the lower epidermis. There is an extremely large variability in rates of dermal absorption between different chemicals [60,64]. In general, more lipophilic photosensitizers are absorbed more readily, as the stratum corneum provides a formidable barrier to hydrophilic compounds [58,65]. Because skin permeation is so important, different non-invasive approaches have been used to monitor the skin barrier physical properties in vivo [59,66]. The quantification of parameters, such as transepidermal water loss, stratum corneum hydration, and skin surface acidity, is essential for the integral evaluation of the epidermal barrier status. However, the permeability barrier status can be modified by different external and internal factors, such as climate, physical stressors, and a number of skin disorders [64,67,68].

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Neoplastic lesions are an epithelial tumor that originates in the epidermis, in squamous mucosa or areas of squamous metaplasia [69]. Macroscopically, the tumor is often elevated, fungating, or may be ulcerated with irregular borders [55]. Microscopically, tumor cells destroy the basement membrane and form sheets or compact masses that invade the subjacent connective tissue (dermis). In general, skin cancers are characterized by an increase in keratinocyte proliferation, a decrease in thickness of the stratum granulosum and an increase in stratum spinosum layers [46]. The reduced barrier function, which is correlated with signs of scaling, enables increased percutaneous absorption of topically applied compounds [70]. The plaques are largely devoid of intracellular lipid, reducing the convoluted lipid pathway to the dermoepidermal junction. The disordered stratum corneum and disrupted epithelial barriers offered by many neoplastic lesions allows enhanced penetration of some delivery systems containing photosensitizers due to poor continuity in intracellular lipid structures [65]. This further improves the selectivity of photosensitizers accumulated into the skin and explains the successful employment of topical PDT to skin cancer [71]. Most primary cutaneous lesions are small and superficial. With topical PDT, the lesion often disappears with no wound issues. As the majority of true squamous cell cancers will be deeper than 2 mm, topical PDT alone may result in high local failure rates. However, superficial lesions of less than 2 mm depth will respond well [72].

17.2.2 Drug Delivery Systems in Topical PDT Topical delivery systems are placed on the skin to deliver drugs to the local tissue directly under the application site or within tissues under and around the site of application. Additionally, as little as 1% and usually no more that 15 % of the drug in a dermatological formulation is bioavailable from the topical formulation. An optimal therapy therefore calls for appropriate pharmaceutical formulations that bring aimed therapeutic outcomes and the lowest levels of adverse effects. Suitable carrier and delivery systems for drugs, such as PS, are dependent on a simple but effective strategy to realize high selectivity, high efficacy and low risk of adverse events [73]. The major limitation of topical PDT is the poor penetration of PSs through biological barriers, like the skin. Over the past 10 years, a considerable number of studies has therefore been conducted on the development of different strategies to overcome these difficulties, including nanocarriers to delivery PSs and their precursors [74,75], nanoemulsions [76], liposomes, ethosomes, invasomes [77], liquid crystals [57,78] and magnetic nanoparticles [79], among others. 17.2.2.1 Polymeric Nanoparticles Several PSs have been encapsulated in polymeric nanoparticles in order to reach specific sites of action, minimize collateral effects, like sensitivity to the sun, and to prevent aggregation of PSs, which impedes PS molecules from being activated by light. The PSs most commonly studied with polymeric particles were mTHPP

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(5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin) [80,81], PpIX [82], hematoporphyrin [83], phthalocyanines [84], and verteporfin [85]. For the synthesis of nanoparticles, biodegradable and nonbiodegradable materials can both be used. The main advantages of biodegradable particles, like PLA (poly(D,L-lactide), and its copolymer with glycolic acid, PLGA ((poly(d,l-lactideco-glycolide)), are their high drug loading, possibility for controlling drug release, and existence of a large variety of materials and manufacturing processes [86]. Non-biodegradable nanoparticles, like ceramic and polyacrylamide, have a different role in PDT because of their inability to degrade and release drugs in controlled amounts [87]. 17.2.2.2 Nanoemulsions The effective formulations for drugs efficacy can be severely limited by poor solubility or instability in the vehicle. Nanoemulsion drug delivery system is one of the most promising technologies, which is currently being applied to enhance the bioavailability and solubility of lipophilic drugs. The nanosized droplets lead to an enormous increase in interfacial areas and influence the transport properties of the drug [88]. Nanoemulsions are prepared by spontaneous emulsification (titration method), which can simply be the blending of oil, water, surfactant and cosurfactant, in the right proportion, with mild agitation, then the order of mixing of the components is generally considered not to be critical. Though nanoemulsification is a spontaneous process, the time required for these systems to reach equilibrium can be long, although the small driving forces. Nanoemulsions have been employed in pharmaceutical compositions as topical medicines for the treatment of dermatological diseases such as neurodermatitis, psoriasis, keratosis, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, Bowen’s disease, vulvar intraepithelial neoplasia (VIN) and nodular or subcutaneous cancers [89]. Nanoemulsions have also attracted considerable attention in recent years for application in personal care products as vehicles for the controlled delivery of active ingredients into particular skin layers [89]. The higher solubilization capacity and thermodynamic stability that nanoemulsions demonstrated, offers advantages over, suspensions and emulsions (unstable dispersions), because they can be manufactured with little energy input and have long shelf lives [90]. With nanoemulsions, the ratio of adsorbed layer thickness to droplet radius is large; therefore, the steric repulsion is strong, preventing any flocculation in the system [91,92]. The resulting nanoemulsion is stable because of the small droplet size and the absence of interfacial energy that results from the use of large amounts of mechanical and thermal energy when the emulsion is formed [89,93]. For example, Primo et al. (2007) demonstrated the preparation of, basically, an oil/water (O/W) based nanoemulsification of an aqueous phase system containing a non-ionic surfactant (Poloxamer 188) and magnetic compounds [93]. 17.2.2.3 Vesicular Systems Liposomes are considered the first generation of novel drug delivery systems and, for this reason, have been widely studied. They are characterized as microscopic

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vesicles and may contain one or more concentric spheres composed of lipid bilayers, that are separated by aqueous or buffer compartments [94]. During the continuous improvement of the efficacy and safety of PDT, liposomes, with their high loading capacity and flexibility in accommodation of PSs with variable physicochemical properties, came into focus as a valuable carrier and delivery system [95]. As a result, many different PSs were encapsulated in liposomes to increase their delivery to cells. One of the most studied was 5-ALA, which was enclosed in liposomes having a lipid composition (ceramide, cholesterol, palmitic acid and colesteryl sulfate) similar to the mammalian stratum corneum. This liposomal system presented a lower permeation through mouse skin, but a significant (p<0.05) increase in the 5-ALA retention in epidermis without SC plus dermis was observed when compared to an aqueous solution [96]. Another author worked with two types of liposomes, glycerol dilaulate and phosphatidylcholine, to enhance 5-ALA topical delivery and examined the resulting 5-ALA-induced protoporphyrin (PpIX) expression in rat pilosebaceous units throughout the hair cycle. According to the results, both liposomal 5-ALA formulations increased PpIX expression in rat dorsal skin and pilosebaceous units when compared with free 5-ALA. However, iontophoresis combined with liposomal 5-ALA reduced the expression intensity of PpIX in hair bulbs despite achieving deeper and wider expression of PpIX throughout the transfollicular pathway [97]. Besides 5-ALA, its derivatives have also been carried by liposomes. Di Venosa and colleagues (2008) designed liposomes of different compositions to improve delivery of 5-ALA and its esterified derivatives, 5-ALA-hexyl ester and 5-ALAundecanoylester. Egg yolk phosphatidyl choline, phosphatidic acid and phosphatidyl glycerol were employed in the preparation of the liposomes, with different resulting entrapment percentages for 5-ALA [98]. Other PSs can also enjoy the advantages of liposomal formulations for drug delivery. Chloroaluminum-phthalocyanine encapsulated in liposomes has been evaluated in oral carcinoma cells, as well as genotoxicity and cytotoxicity of the encapsulated PS in both the dark and under irradiation using a 670 nm laser [99]. Ethanol-containing liposomes loaded with temoporfin (mTHPC) were developed to investigate their enhanced skin penetration. According to the authors, liposomes without ethanol delivered the lowest amount of mTHPC into the skin, while liposomes containing 20% ethanol showed the highest penetration enhancement [100]. Liposomal formulation can be used in PDT for treatment of diseases other than cancer. Fadel and colleagues (2009) have proposed a liposomal delivery method for methylene blue (MB) for selective treatment of acne vulgaris. They used phosphatidylcholine from soybeans and cholesterol to produce the vesicles. Their studies demonstrated that MB loaded in liposomes and formulated in hydrogel could be delivered into sebaceous glands and rendered capable of being targeted by a diode laser and selectively damaged [101]. New generations of vesicles based on liposomes are being developed for enhanced delivery to, and through, skin. One of these novel vesicle carriers is ethosomes, which are malleable vesicles composed primarily of phospholipids, ethanol (in a relatively high concentration) and water. Different reports show a promising future for ethosomes in more effective transdermal delivery of various agents

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[102]. 5-ALA was the first drug tested with this new vesicle when Fang and coworkers (2008) compared 5-ALA encapsulated in liposomes with that in ethosomes for skin delivery in photodynamic therapy. The results showed that ethosomes were smaller than liposomes, had good entrapment efficiency, were stable upon storage and had higher penetration ability than liposomes [103]. The same group also studied the topical delivery of 5-ALA-encapsulated ethosomes formed from phosphatidylethanolamine as the lipid component in a hyperproliferative murine model. They found that the application of ethosomes produced either a significant increase in 5-ALA cumulative amounts (5 to 26-fold) or an increase in PpIX intensity (3.64-fold) both in normal and hyperproliferative murine skin compared to an aqueous solution of 5-ALA [104]. Another type of new vesicle is the flexible liposome (flexosome). They are selfregulating, lipid-based drug carriers containing biocompatible membrane softeners in the liposomal bilayer for targeted and non-invasive delivery of agents into and through the skin [105]. For example, Dragicevic-Curic and coworkers (2010) used neutral, anionic and cationic flexosomes in order to increase the topical delivery of temoporfin (mTHPC) [106]. Neutral and cationic flexosomes appeared to be physically stable during storage at 4 °C for 9 months in contrast to anionic flexosomes. In addition, cationic vesicles delivered the highest mTHPC-amount to the SC and deeper skin layers [107]. The last new vesicles of interest are the invasomes, which contain, in addition to phospholipids, terpenes (cineole, citral and d-limonene) and ethanol as penetration enhancers, which make the vesicles deformable [108]. For PDT use, they have been tested for enhancement of the skin delivery of mTHPC. In 2008, mTHPC-loaded invasomes were developed, characterized and investigated for in vitro percutaneous penetration of mTHPC into abdominal human skin using Franz diffusion cells [107]. The photodynamic efficacy of the vesicles was then investigated after their topical application on the skin of mice bearing subcutaneously implanted human colorectal tumor HT29 and photoirradiation. The results showed that groups of mice treated with mTHPC-invasomes prior to photoirradiation showed a significantly smaller (p<0.05) increases in tumor size compared to control groups (mice without any treatment or only photoirradiated) [108]. In 2009, the same group tested a new series of mTHPC-loaded invasomes by varying the ratio between d-limonene, citral and cineole in the standard terpene mixture. These new mTHPC-loaded invasomes were characterized and investigated for stability and in vitro penetration of mTHPC into abdominal human skin using Franz diffusion cells. The authors observed that invasomes containing just 1% (w/v) cineole reached the highest penetration enhancement of mTHPC [109]. 17.2.2.4 Liquid Crystal The state of matter whose properties are intermediate between a crystalline solid and an isotropic liquid is called liquid crystal or the mesomorphic phase. This phase has some characteristics of solid crystal, as evidenced by x-ray diffraction, but also some of the disorder of liquids, characterized by the ability to flow. In fact, molecules in the liquid crystalline phase present both structural order and mobility [110,111]. Various types of molecules can form liquid crystalline phases,

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but all of them have an elongated shape and a polar group. Such molecules tend to align themselves parallel to each other and are held together by van der Waals forces. Changes in the environment cause the molecules to rearrange themselves, keeping some degree of order, to form the mesomorphic phase [112,113]. The formation of mesophases can be brought about in two different ways: thermally, so that the formed mesophase is called a thermotropic phase, or by the addition of solvents, forming lyotropic liquid crystals. Thermotropic liquid crystals are formed by heating the system to a temperature above which the crystal lattice is no longer stable [114]. The type of liquid crystalline phase formed depends on the solvent content, the temperature of the system and on the molecular shape of the amphiphiles, phospholipids and polar lipis. In general, polar lipids, such as glyceryl mooleate or monoolein, exhibit different phases when exposed to water. Monoolein is a mixture of glycerides of oleic acid and other fatty acids, consisting mainly of monooleate [115,116,117]. Among the mesophases exhibited by monoolein, hexagonal, lamellar and cubic phases have all been described, as well as a nanodispersion (Figure 3). The lamellar phase has a long-range order in one direction and displays anisotropic properties. Its structure consists of linear bilayers separated by an aqueous phase [118]. Additionally, the hexagonal phase can be described as a bidimensional lattice composed of infinite water rods. In aqueous solvents, structures having a hydrocarbon core surrounded by an interfacial layer of hydrated polar groups is formed, an arrangement known as hexagonal phase I. Reverse structures are also possible where water rods separated by lipid layers are formed (hexagonal phase II). This phase is anisotropic and stiffer in consistency than the lamellar phase [111,112,117]. Furthermore, the cubic phase can be described as a very viscous, isotropic and transparent gel, physically stable upon contact with excess water [117]. Its structure consists of curved lipid bilayers extending in three dimensions and separated by water channels, whose diameters are about 5 nm when the system is fully hydrated. The proposal of the cubic phase as an ideal candidate for drug delivery purposes is based on its unique structure and physicochemical characteristics. The structure, with a three-dimensional bicontinuous lipid bilayer separating two congruent networks of water channels, allows for the incorporation of both hydrophilic and hydrophobic molecules because they do not disrupt the structure of the mesophase [118]. The ability of controlling the release of drugs, as well as its excellent biocompatibility, make liquid crystals of monoolein particularly attractive as vehicles for topical and transdermal delivery of drugs. In the last few years, liquid crystalline phases based on monoolein and water have been receiving a lot of attention as potential delivery matrices for topical delivery of pro-drugs and PSs in PDT of skin cancer [119]. Therefore, the incorporation of photosensitizers into liquid crystalline phases might be investigated in order to guarantee the integrity of the mesophase and consequently, to study the release characteristics of the system. As such, the penetration of 5-ALA using formulations containing monoolein was evaluated as an interesting and useful pharmaceutical formulation by Regilene et al. (2005) [17]. They proposed to increase 5-ALA penetration and showed that propylene glycol solutions containing different concentrations of monoolein

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significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin models. Turchiello et al. (2003) investigated the potential of the cubic phase as a topical delivery system for pro-drugs used in PDT of cutaneous diseases for the first time [57]. The pro-drug 5-ALA and its ester derivatives (hexylester, octylester, and decylester), a classical second-generation PS (m-THPC) and also the endogenous photosensitizing agent PpIX were incorporated into a gel cubic phase (monoolein/water; 70:30 w/w) and their subsequent physicochemical properties were investigated. It was shown that 5-ALA and its ester derivatives remained stable in the cubic phase gel formulation over the 30-day period of investigation. Indeed, the PSs m-THPC and PpIX were shown to maintain stability for considerably longer periods of time: 90 and 126 days, respectively. Furthermore, no sign of bacterial contamination was observed in the gel cubic phase after a follow-up time of 6 months. In vivo work carried out on patients observed enhanced PpIX production following application of a monoolein cubic phase gel formulation of 5ALA containing 2% w/w of 5-ALA compared to application of the traditional 5ALA cream formulation, which contains 20% w/w of 5-ALA [57]. In the same way, Bender et al. (2005) investigated the efficiency of PpIX production using nanostructured lipid cubic phase systems as drug delivery vehicles for 5-ALA and m-5-ALA [78]. They showed that PpIX formation from topical application of 5ALA and m-5-ALA could be significantly increased by using nanostructured lipid cubic systems instead of standard ointments, such as unguentum M. Garcia et al. (2004) investigated the ability of the lamellar and cubic liquid crystalline phases to increase the penetration of the hydrophilic photosensitizer ZnPC by in vitro and in vivo studies of penetration and retention in skin using porcine ear skin and hairless mouse as model membranes. Cubic and lamellar phases provided the highest drug retention in epidermis plus dermis for both in vitro and in vivo studies when hydroxyethyl cellulose was used as the control formulation [18]. 17.2.2.5 Magnetic Nanoparticles The history of magnetism dates back to earlier than 600 B.C., but it is only in the twentieth century that scientists have begun to understand it and develop technologies based on this understanding [121]. Curiously, the first systematic study of magnetism using scientific methods was made not by a physicist, but by a physician, William Gilbert (1540–1603), the father of electrical and magnetic science. Gilbert's principal work was his treatise on magnetism, entitled De magnete, magneticisque corporibus, et de magno magnete tellure (London, 1600). Currently, in view of recent needs for miniaturization and high efficiency of electrical equipment, there has been an increasing demand for upgrading of permanent magnet materials. A permanent magnet is one made from a material that stays magnetized. Materials that can be magnetized, which are also the ones that are strongly attracted to a magnet, are called ferromagnetic [122]. These include iron, nickel, cobalt, some rare earth metals and their alloys, and some naturally occurring minerals, such as lodestone. Permanent magnets are made from "hard"

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Fig. 15.3 Representative microscopic characterization of liquid crystalline phases and a nanodispersion: A and B illustrate hexagonal and lamellar phases visualized by polarized light microscopy, respectively; C and D illustrate nanoparticles of the hexagonal liquid crystalline phase visualized by cryo-transmission electronic microscopy (adapted from Lopes et al., 2006) [120].

ferromagnetic materials that are designed to stay magnetized, while "soft" ferromagnetic materials, like soft iron, are attracted to a magnet but do not tend to stay magnetized. Magnetic nanoparticles, composed of a magnetic (e.g., iron oxide) core and a biocompatible polymeric shell (e.g., dextran), are an effective drug delivery system [123]. The particles encapsulate drugs and can be targeted to a desired treatment location by externally localized magnetic steering. As a result of enhanced permeability and retention, macromolecules and nanoparticles are able to extravasate into the interstitium through the hyper-permeable vasculature of most hyperplastic tissues. Moreover, deficient lymphatic drainage retards the clearance of the macromolecular structures, rendering further selectivity enhancement [124]. Systemic toxicity is thus minimized, and local therapeutic effects are increased. Increases in the local drug dose at the treatment target are also achieved. More importantly, the magnetic nanoparticles have other favorable features, such as MRI visibility for MRI imaging and nanoparticle tracking [125]. Wet chemical routes to magnetic nanoparticles are simpler, more tractable and more efficient with appreciable control over size, composition and sometimes even the shape of the nanoparticles. Iron oxides (either Fe3O4 or g-Fe2O3) can be

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synthesized through the co-precipitation of Fe2+ and Fe3+ aqueous salt solutions through the addition of a base [126]. The control of size, shape and composition of nanoparticles therefore, depends on the type of salts used (e.g., chlorides, sulfates, nitrates, perchlorates, etc), the Fe2+ to Fe3+ ratio, the pH and the ionic strength of the media. The magnetite is conventionally prepared by adding a base to an aqueous mixture of Fe2+ and Fe3+ chloride at a 1:2 molar ratio. Several authors have reported magnetic iron oxide nanoparticles coated with silic a[127]. An advantage of having a surface enriched in silica is the presence of surface silanol groups that can easily react with alcohols and silane coupling agents [128]. Various biological molecules, such as antibodies, proteins and targeting ligands, may also be bound to the surfaces of the nanoparticles by chemical coupling via amide or ester bonds to make the particles target specific. This modification is exciting, as the possibilities for targeting protein coatings are almost limitless [129,130]. Another possibility is liposomes [73,131], which have the ability to encapsulate a large number of magnetic nanoparticles and deliver them together to a target site, avoiding dilution. Combining a therapeutic agent in the payload further enhances the multifunctionality of these delivery vehicles. Primo et al. (2008) recently demonstrated the nanoencapsulation of zinc phtalocyanineZnPc in a magnetic nanoemulsion for use in PDT trials for cancer treatment [79]. The entrapment of ZnPc showed favorable properties that were promising for future applications in PDT, and the in vitro assays showed that the amount of the ZnPc in the deep layers of the skin was significantly higher in the presence of magnetic nanoparticles, making its topical application in skin cancer PDT possible.

17.3 Future Directives Liquid crystalline lipid-water phases with an inverse structure (inverse hexagonal phase and the cubic phases) can co-exist in equilibrium with an excess of water. Studies on their dispersion properties have shown that they can transform into colloidal dispersions that are kinetically stable [132]. The preparation of such dispersions and some of their structural features have been receiving a lot of attention recently as delivery systems in medicine [133]. Similar particles with periodicity in the micrometer range occur in biology as a conformation alternative in cell membrane assemblies. Dispersions of cubic and hexagonal lipid-water phases have been used for the development of controlled release formulations of biologically active agents in the field of drug delivery and may be a new topical delivery system for use in PDT of skin cancer [134,135]. For photodynamic therapy, these nanodispersed systems can be applied to the delivery of photosensitizer drugs and pro-drugs into the skin. The nanosized structures and the enhanced penetration of this preparation can increase the accumulation of photosensitizer in the deeper skin layers, where the cancer and non-neoplastic diseases occur. Advances in PDT have been the result of the synthesis of new PSs and also combination of PSs. In this aspect, nanocarriers will provide higher bioavailability in skin and mucous membranes and target delivery into cancerous tissues.

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[87] Chatterjee, D.K., Fong, L.S., Zhang, Y.: Nanoparticles in photodynamic therapy: An emerging paradigm. Adv. Drug Deliv. Rev. 60, 1627–1637 (2008) [88] Ghosh, P.K., Murthy, R.S.R.: Microemulsions: a potential drug delivery system. Curr. Drug Deliv. 3, 167–180 (2006) [89] Guglielmini, G.: Nanostructured novel carrier for topical application. Clin. Dermatol. 26, 341–346 (2008) [90] Shafiq-Un-Nabi, S., Shakeel, F., Talegaonkar, S., Ali, J., Baboota, S., Ahuja, A., Khar, R.K., Ali, M.: Formulation Development and Optimization Using Nanoemulsion Technique: A Technical Note. AAPS Pharm. Sci. Tech. 8(2), Article 28 (2007) [91] Dahms, G.: 22nd International Federation of Societies of Cosmetic Chemists Congress. Edinburgh Oral Proceedings Keynote Lecture, pp. 519–529 (2002) [92] Tadros, T.: Presented at: 21st International Federation of Societies of Cosmetic Chemists Congress. Berlin Oral Proceedings, pp. 442–458 (2000) [93] Primo, F.L., Michieleto, L., Rodrigues, M.A.M., Macaroff, P.P., Morais, P.C., Lacava, Z.G.M., Bentley, M.V.L.B., Tedesco, A.C.: Magnetic nanoemulsions as drug delivery system for Foscans: Skin permeation and retention in vitro assays for topical application in photodynamic therapy (PDT) of skin cancer. J. Magn. Magn. Mater. 311, 354–357 (2007) [94] Gulati, M., Grover, M., Singh, S., Singh, M.: Lipophilic drug derivatives in liposomes. Int. J. Pharm. 165, 129–168 (1998) [95] Derycke, A.S.L., De Witte, P.A.M.: Liposomes for photodynamic therapy. Adv. Drug Deliv. Rev. 56, 17–30 (2004) [96] Pierre, M.B.R., Tedesco, A.C., Marchetti, J.M., Bentley, M.V.L.B.: Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study. BMC Dermatol. 1, 5 (2001), doi:10.1186/1471-5945-1-5. [97] Han, I., Jun, M.S., Kim, S.K., Kim, M., Kim, J.C.: Expression pattern and intensity of protoporphyrin IX induced by liposomal 5-aminolevulinic acid in rat pilosebaceous unit throughout hair cycle. Arch. Dermatol. Res. 297, 210–217 (2005) [98] Di Venosa, G., Hermida, L., Batlle, A., Fukuda, H., Defain, M.V., Mamone, L., Rodriguez, L., Macrobert, A., Casas, A.: Characterization of liposomes containing aminolevulinic acid and derived esters. J. Photochem. Photobiol. B 92, 1–9 (2008) [99] Tapajós, E.C.C., Longo, J.P., Simioni, A.R., Lacava, Z.G.M., Santos, M.F.M.A., Morais, P.C., Azedesco, A.C., Azevedo, R.B.: In vitro photodynamic therapy on human oral keratinocytes using chloroaluminum-phthalocyanine. Oral Oncol. 44, 1073–1079 (2008) [100] Dragicevic-Curic, N., Scheglmann, D., Albrecht, V., Fahr, A.: Characterization, stability and in vitro skin penetration studies. Colloids and Surfaces B Biointerfaces 74, 114–122 (2009) [101] Fadel, M., Salah, M., Samy, N., Mona, S.: Liposomal methylene blue hydrogel for selective photodynamic therapy of acne vulgaris. J. Drugs Dermatol. 8, 983–990 (2009) [102] Rajashree, H., Harshal, G., Suneeta, S., Sujata, L., Vilasrao, K.: Ethosomes and its Applications in Transdermal Drug Delivery. Curr. Drug Ther. 4, 92–100 (2009) [103] Fang, Y., Tsai, Y., Wu, P., Huang, Y.: Comparison of 5-aminolevulinic acidencapsulated liposome versus ethosome for skin delivery for photodynamic therapy. Int. J. Pharm. 356, 144–152 (2008)

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[104] Fang, Y.P., Huang, Y.B., Wu, P.C., Tsai, Y.H.: Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior. Eur. J. Pharm. Biopharm. 73, 391–398 (2009) [105] Song, Y., Kim, C.: Topical delivery of low-molecular-weight heparin with surfacecharged flexible liposomes. Biomaterials 27, 271–280 (2006) [106] Dragicevic-Curic, N., Gräfe, S., Gitter, B., Winter, S., Fahr, A.: Surface charged temoporfin-loaded flexible vesicles: In vitro skin penetration studies and stability. Int. J. Pharm. 384, 100–108 (2010) [107] Dragicevic-Curic, N., Scheglmann, D., Albrecht, V., Fahr, A.: Temoporfin-loaded invasomes: development, characterization and in vitro skin penetration studies. J. Control Release 127, 59–69 (2008) [108] Dragicevic-Curic, N., Gräfe, S., Albrecht, V., Fahr, A.: Topical application of temoporfin-loaded invasomes for photodynamic therapy of subcutaneously implanted tumours in mice: A pilot study. J. Photochem. Photobiol. B 91, 41–50 (2008) [109] Dragicevic-Curic, N., Scheglmann, D., Albrecht, V., Fahr, A.: Development of different temoporfin-loaded invasomes-novel nanocarriers of temoporfin: characterization, stability and in vitro skin penetration studies. Colloids Surf B Biointerfaces 70, 198–206 (2009) [110] Singh, S.: Phase transitions in liquid crystals. Phys. Rep. 324, 107–269 (2000) [111] Tyle, P.: Liquid Crystals and their applications in drug delivery. In: Rosoff, M. (ed.) Controlled Release of Drugs: Polymer and Aggregate Systems, ch. 4, pp. 125–162. VCH Publishers, New York (1989) [112] Alfons, K., Sven, E.M.: Drug Compatibility with the Sponge Phases Formed in Monoolein, Water, and Propylene Glycol or Poly(ethylene glycol). J. Pharm. Sci. 87, 1527–1530 (1998) [113] Carr, M.G., Corish, J., Corrigan, O.I.: Drug delivery from a liquid crystalline base across Visking and human stratum corneum. Int. J. Pharm. 157, 35–42 (1997) [114] Ganem-Quintanar, A., Quintanar-Guerrero, D., Buri, P.: Monoolein: a review of the pharmaceutical applications. Drug Dev. Ind. Pharm. 26, 809–820 (2000) [115] Caboi, F., Amico, G.S., Pitzalis, P., Monduzzi, M., Nylander, T., Larsson, K.: Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein:water system. I. Phase behavior. Chem. Phys. Lipids 109, 47–62 (2001) [116] Clogston, J., Rathman, J., Tomasko, D., Walker, H., Caffrey, M.: Phase behavior of a monoacylglycerol (Myverol 18-99K):water system. Chem. Phys. Lipids 107, 191– 220 (2000) [117] Chang, C.M., Bodmeier, R.: Low viscosity monoglyceride-based drug delivery systems transforming into a highly viscous cubic phase. Int. J. Pharm. 173, 51–60 (1998) [118] Shah, J.C., Sadhale, Y., Chilukuri, M.D.: Cubic phase gels as drug delivery systems. Adv. Drug. Del. Rev. 47, 229–250 (2001) [119] Bender, J., Ericson, M.B., Merclin, N., Iani, V., Rosen, A., Engstrom, M.J.: Lipid cubic phases for improved topical drug delivery in photodynamic therapy. J. Control Release 106, 350–360 (2005) [120] Lopes, L.B., Ferreira, D.A., Paula, D., Garcia, M.T.J., Thomazini, J.A., Fantini, M.C., Bentley, M.V.L.B.: Reverse hexagonal phase nanodispersion of monoolein and oleic acid for topical delivery of peptides: in vitro and in vivo skin permeation of cyclosporin A. Pharm. Res. 23, 1332–1342 (2006)

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[121] Babincova, M., Babinec, P.: Magnetic Drug Delivery And Targeting:Principles And Applications. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 243–250 (2009) [122] Morrish, A.H.: Physical Principles of Magnetism. IEEE Press, New York (2001) [123] Jain, T.K., Morales, M.A., Sahoo, S.K., Leslie-Pelecky, D.L., Labhasetwar, V.: Ionoxide nanoparticles for sustained delivery of anticancer agents. Mol. Pharmacol. 2, 194–205 (2005) [124] Chertok, B., Moffat, B.A., David, A.E., Yu, F., Bergemann, C., Ross, B.D.: Iron oxide nanoparticles as a drug delivery vehicle for MRI monitores magnetic targeting of brain tumors. Biomaterials 29, 487–496 (2008) [125] Lu, Z.R., Ye, F., Vaidya, A.: Polymer Platforms for Drug Delivery and Biomedical Imaging. J. Control Release 122, 269–277 (2007) [126] Schwertmann, U., Cornell, R.M.: Iron oxides in the laboratory: preparation and characterization. VCH, Weinheim (1991) [127] Gupta, A.K., Gupta, M.: Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26, 3995–4021 (2005) [128] Berry, C.C., Curtis, A.S.G.: Functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D Appl. Phys. 36, R198–R206 (2003) [129] Sajja, H.K., East, M.P., Mao, H., Wang, Y.A., Nie, S., Yang, L.: Development of multifunctional nanoparticles for targeted drug delivery and noninvasive imaging of therapeutic effect. Curr. Drug Discov. Technol. 6, 43–51 (2009) [130] Kikumori, T., Kobayashi, T., Sawaki, M., Imai, T.: Anti-cancer effect of hyperthermia on breast cancer by magnetite nanoparticleloaded anti-HER2 immunoliposomes. Breast. Cancer. Res. Treat. 113, 435–441 (2009) [131] Babincova, M., Babinec, P.: Possibility of magnetic targeting of drugs using magnetoliposomes. Pharmazie 50, 828–829 (1995) [132] Yamashita, J., Shiono, M., Hato, M.: New lipid family that forms inverted cubic phases in equilibrium with excess water: molecular structure-aqueous phase structure relationship for lipids with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17tetramethyloctadecanoyl chains. J. Phys. Chem. B 112, 12286–12296 (2008) [133] Lee, E.S., Oh, Y.T., Youn, Y.S., Nam, M., Park, B., Yun, J., Kim, J.H., Song, H.T., Oh, K.T.: Binary mixing of micelles using Pluronics for a nanosized drug delivery system. Colloids Surf B Biointerfaces (2011), doi:10.1016/j.colsurfb.2010.08.033 [134] Ferreira, D.A., Bentley, M.V.L.B., Karlsson, G., Edwards, K.: Cryo-TEM investigation of phase behaviour and aggregate structure in dilute dispersions of monoolein and oleic. Int. J. Pharm. 310, 203–212 (2006) [135] Lopes, L.B., Spereta, F.F.F., Bentley, M.V.L.B.: Enhancement of skin penetration of vitamin K using monoolein-based liquid crystalline systems. Eur. J. Pharm. Sci. 32, 209–215 (2007)

Chapter 16

Production of Nanofibers by Electrospinning Technology: Overview and Application in Cosmetics Maria Helena A. Zanin, Natalia N.P. Cerize, and Adriano M. de Oliveira Laboratory of Chemical Processes and Particle Technology, São Paulo State Institute for Technological Research, 05508-901, São Paulo, SP, Brazil [email protected]

Abstract. This chapter relates to the techniques to produce polymer nanofibers and specifically discusses one of those techniques which has been recently investigated, namely Electrospinning. An overview of this technology is described, considering the work trend based on nanofiber and electrospinning publication in scientific paper and patents. Moreover, issues regarding the main process control parameters, applications of polymer nanofibers and the appropriate characterization of these nanofibers or mats are presented. Applications in cosmetic field are outlined, as well as the future perspective of the electrospinning technology in cosmetic application.

16.1 Overview 16.1.1 Introduction In the introduction of this chapter it is essential to define the term nanofiber. The original word “fiber” or “fibre” has its origin from the latin word “fibra” and the nano term comes from the definition that has been discussed abundantly. It refers to the physical quantity regarding to the scale of a billionth of a meter: nanometer. In a broad sense, fibers with diameter less than 1 micron are referred to as nanofibers, and can be called nanostructured material if filled with nanoparticles to form composite nanofibers [1]. When the polymer fiber has its diameter reduced to the nanoscale, the surface area per unit mass is larger [2] (on the order of 100 m2/g for a nanofiber of approximately 100 nm of diameter) and permits easier addition of surface functionalities [3]. Due to these prominent properties, the nanofibers became a noteworthy material for many important applications that have been increasing in recent years in diversified areas. The literature presents different processing techniques to obtain polymer nanofibers [4] such as drawing [5], template synthesis [6], phase separation [7], selfassembly [8] and electrospinning [9]. The drawing process is similar to dry

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spinning, one-by-one fabrication of very long single fibers using citrate molecules. Each fiber is pulled out from a microdroplet or from a micropipette, during the solvent evaporation. However this technique requires a material with pronounced viscoelastic behavior to undergo strong deformations while being cohesive enough to support the stresses developed during pulling. The final section of fibers is very dependent on the drawing rate, the cooling or evaporation rate and also on precise composition of the material [4, 5]. The process is simple and requires a SiO2 surface, a micropipette and a micromanipulator. It is a laboratory scale process in which the nanofibers are produced one by one. The template synthesis method is based on the synthesis of the desired material within the porous of a nanoporous membrane. The membranes employed have cylindrical pores with uniform diameter where each pore is seen as a beaker and the nanostructure of desired material is electrochemically or chemically synthesized by oxidative polymerization. The membrane materials most used for this synthesis are the polycarbonates; however a number of other materials can be used. The process is simple and requires standard laboratory equipment. It is a laboratory scale process limited to the conversion of specific polymers directly to nanofiber structures. The phase separation technique is based on thermodynamic demixing of a homogeneous polymersolvent solution into a polymer-rich phase and a polymer poor phase, usually either by exposure of the solution to another immiscible solvent or by cooling of the solution below a bimodal solubility curve. This method has been used in the preparation of porous polymer membranes for years [7] and the process consists of five basic steps to obtain, for example, a nanofibrous foam material: polymer dissolution, phase separation and gelation, solvent extraction from the gel with the water, freezing and then freeze drying with vacuum [10]. The process is simple and requires a refrigerator, a freeze dryer and also a standard laboratory. Self assembly is one of the few practical strategies for making ensembles of nanostructured material. It is based on spontaneous organization of individual components into an ordered and stable structure with association of organic molecules through non-covalent interactions. Nevertheless this method is limited to some molecules such as diblock copolymers, triblock copolymer, triblocks from peptideamphiphile and dendrimers [4]. Electrospinning, the last mentioned technique, is a process that creates fibers through an electrically charged jet of a polymer solution or polymer melt, in which the diameter of fibers can vary from a few microns to nanometers. The basic configuration of electrospinning, illustrated by figure 16.1, consists of a syringe pump, a metallic fiber collector, a capillary with a metal tip to hold the polymer solution and a direct current voltage supply. During the process there is a creation of an electric field between a grounded fiber collector and a positively charged capillary field with a polymer solution. The polymer solution jet is achieved when the electrostatic charge is larger than the surface tension of the polymeric solution at the capillary tip. The fibers are collected as a mat of fibers on the surface of a grounded cylinder (fiber collector) (1, 4, 9, 11). Electrospinning technique, on the other hand, has the advantage of being not only laboratory but also an industrial scale technique. Furthermore, there is the possibility of using different polymers according to the application. Many universities and technological institutions have dedicated their researches to the

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Fig. 16.1 Illustration of a monoaxial electrospinning apparatus.

development of nanofibers as mat for different applications and the majority of the work is on the laboratory scale. Discussing the entire range of nanofiber production process is beyond the scope of this chapter. Instead we will highlight a few important parameters that govern the nanofiber formation with relevant examples from the literature reinforcing the main applications of these vehicles.

16.1.2 History of Nanofibers Obtained by Electrospinning The process known as electrospinning processing is an old technique and its first descriptions were presented in the patents US 692631 [12] and US 705691 [13]. Later, Formhals brought a contribution to the development of this technique with his works, described in several patents in the 1930s and 1940s [14, 15, 16]. In the same century, 1969, Geoffrey Ingram Taylor contributed with his work to the electrospinning technique by developing a mathematical model of the cone shape formed by the fluid droplet under the effect of an electric field, which is known up to now as Taylor’s cone [17]. In the beginning, this technique was known as electrostatic spray or electrostatic spinning and subsequently in the 1990s, was renamed as electrospinning [18]. The Figure 16.2 presents a statistical analysis of scientific publications in nanofibers area employing a known scientific database: Web of Science. In this statistical search were found publications referred as technical-scientific articles and patents. The period evaluated was limited from January 1997 to September 2010. The literature search was performed in two different ways: covering the term "nanofiber" (broader and more generic way) and comprising term "nanofiber" associated with the term "electrospinning" (a more focused search), both for

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articles and patents. It was observed that the rise of technical-scientific publications related to nanofibers was followed by a significant increase in patents, which could be regarded to an evidence of the commercial viability of the technology. Regarding the production process, the technique of electrospinning also showed a significant increase in publications in the last decade, which also demonstrates the importance of this process for the nanofibers production area.

Fig. 16.2 The graph shows the significant increase in the number of scientific papers and patents published since 1997 regarding the polymeric electrospun fiber (accessed on 21.09.2010). * Data reported in the 2010 year refers to the January to September months.

16.2 Preparation of Nanofiber by Electrospinning Technology As mentioned before, the nanofibers can be obtained by different technological paths, each having its advantages and disadvantages. However, as noted in the statistics of publications on nanofibers, electrospinning technique has been highlighted in recent years because of its versatility for adaptation to both laboratory and industrial scale processes and for using different polymer families. The electrospinning method consists in an electric voltage, usually up 30 kV applied at the tip of capillary/needle which contains the polymer solution with a specific surface tension and the ground fiber collector in order to facilitate the ejection of the charged jet toward the fiber collector, following which a pendent droplet of the polymer at the tip of capillary that becomes electrified and induces charge accumulation on the surface of droplet. This droplet deforms into a cone shape (Taylor’s cone). Such deformation is attributed to two reasons: a) electrostatic repulsion between the surface charges of the droplet, and b) Coulombic force exerted by the external electric field applied. The polymeric solution jet

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moves toward the fiber collector with a simultaneous evaporation of the solvent molecules, leading to the deposition of a mat of nanofibers on the collector surface [3, 18]. This system is known as a simple nozzle or monoaxial, as can be seen in Figure 16.1, due to the fact that it employs only one capillary containing the polymer solution to be electrospun. However, there is a more recent configuration of electrospinning known as coaxial configuration [19, 20]. This involves two coaxial capillaries which permit simultaneous electrospinning of two polymer solution into core-shell structured nanofiber. In this configuration there are two separate polymer solutions or aqueous flow through to different capillaries, which are coaxial with a smaller capillary inside a larger capillary. The concept is similar to the monoaxial electrospinning which the electrostatic force induces a high charging potential shearing the coreshell droplet into polymeric fibers. The greatest advantage of coaxial electrospinning is its versatility in the type (hydrophilic or hydrophobic) and size (ranging from 100nm to 300μm) of fibers it can produce. This configuration of electrospinning has been considered as a good alternative to encapsulate active agent that is insoluble in organic solvent and may suffer loss of bioactivity when dispersed in the polymer solution on drug delivery applications [20, 21]. Moreover, there are other electrospinning configurations, such as mixing and multiple electrospinning, core shelled electrospinning, blow assisted electrospinning [22] and the industrial electrospinning machine that might represent a huge contribution to the process scaling and bring a technological challenge to the innovative applications [23].

16.2.1 Polymers Used in the Electrospinning Process The raw materials commonly used in industrial production of conventional fibers, fabrics and non-wovens, are polymers of natural origin first and, secondly synthetic polymers. Reasons identified by several industries for this preference of polymeric materials are: practicality, moldability, flexibility, lightness, durability and chemical and physico-chemical stabilities. In this context, considering the possibility of obtaining fibers at nanoscale, polymers have been chosen as the main candidates for this development, in line with historical developments and the fact that polymeric materials are the most exploited for the production of nanofibers nowadays. The types of polymer amenable to electrospinning are numerous and individual material properties must be considered depending on its applications. The electrospinning process may be modified to yield electrospun fiber with the desired morphology and properties. For example, in applications as wound dressing, the polymer selection is limited to that which is soluble in a skin- or tissue-compatible solvent [24]. The polymer can be dissolved in a suitable solvent and electrospun or it can be directly electrospun from a melt. In this case the presence of organic solvent is eliminated, which is excellent for scaled-up processes. However the melting polymer demands a processing at elevated temperature, whereas the polymer solution can be electrospun at room temperature [20]. The polymer family used in nanofibers production includes the homopolymer and copolymer of natural and synthetic polymers as well as the blends of both polymers.

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The electrospinning process based on natural polymers has been utilized to generate non woven mats for biomedical applications due to biocompatibility and low immunogenicity compared to the synthetic polymers among other applications. Nevertheless these raw materials present a limitation as properties vary from batch to batch [25]. The most frequently used natural polymers are chitosan, collagen, gelatin, casein, hyaluronic acids, cellulose acetate, silk protein, chitin, fibrinogen [22, 26, 27, 28, 29]. Synthetic polymers can be tailored according to the properties desired to the application and also permits a good uniformity. The typical synthetic polymer used in biomedicine and extended to drug delivery applications including the hydrophobic biodegradable polyesters, such as poly(latic acid-co-glycolic acid) (PLGA), polylactide (PLA) and poly (ε-caprolactone) (PCL). Also the hydrophilic biodegradable polymers, such as polyvinylpyrrolidone (PVP), poly(vinil alcohol) (PVA) and poly (ethylene oxide) (PEO), have been employed in biomedical application [28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39]. Some applications demand a structure with properties of two or more polymers which can be achieved by polymerization of two different polymers (copolymer such as PLGA, a random copolymer of glycolide (G) and lactide (L) represents a classical polymer that is well studied and described in the literature, lactide and ε-caprolactone (PLLA-CL), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(p-dioxanoneco-L-Lactide)-block-poly(ethylene glycol) (PPDO/PLLA-b-PEG) [1, 33] or by physical mixture of two or more polymers (blend) [27, 28, 32, 37, 40, 41, 42, 43, 44]. The blends open the possibility of combining different polymer families as natural and synthetic polymers without a chemical reaction between the polymers. In order to evaluate the potential polymers for use in electrospinning and their applications, Table 16.1 was built by compiling various scientific works that demonstrate the versatility of polymer nanofibers.

16.2.2 Electrospinning Process Control Parameters Although the electrospinning process to obtain the nanofibers is considered relatively simple, many parameters can affect the fiber characteristic and should be controlled. These parameters include [3, 9, 45]: type of polymer and polymer solution properties (molecular weight, viscosity, conductivity, surface tension); process parameters (electric potential at the capillary/voltage, flow rate, distance between the capillary and the fiber collector, capillary design and geometry and collector composition); ambient parameters: temperature and humidity. The manipulation of one or more these parameters can determine the morphology and the structures of the fibers according to the characteristics required in the application. For the readers interested in knowing more details about these parameters, we recommend to see some articles that present a good compilation about them [3, 18, 21, 22, 46]. However, evaluating the interdependence of these parameters in electrospinning processes is still very complex.

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Briefly, the parameters evaluated by the literature are described below: Solution polymer parameters affect the formation of the nanofibers, causing defects in the fibers in the form of beads and junction by their low concentration or viscosity, low conductivity and also by their reduction of molecular weight. The beads are illustrated in figure 16.3. The fiber diameter increases with the increase of the concentration/viscosity of the polymer solution and higher conductivity yields smaller fibers in general, except for the process using the polymers polyacrilic acid (PAA) and polyamide-6. The surface tension effect on the morphology of nanofiber and size of electrospun fiber have been investigated, however there is no conclusive results on this issue yet.

Fig. 16.3 SEM image of electrospun PVP-Eudragit ® RS100 nanofibers with beads.

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Process parameters, such as low flow rate and high voltage affect the fiber diameter, yielding fibers with small diameters. Moreover, high voltage can conduct the formation of beads and the high flow rate delays the drying of the fiber until it reaches the collector. The distance between the capillary and the fiber collector affects the fiber drying and requires a minimum distance to give the fibers sufficient time to dry before achieving the fiber collector. The geometry and composition of the collector affect the surface of fiber, where the porous collector produces more porous fiber and metallic collector smooth fibers. The design of the capillary can also produce hollow fibers by the coaxial design. Ambient parameters play a role in the properties of the polymeric solution and consequently affect the morphology of the nanofibers. For example, solution viscosity decreases with increasing temperature, and this consequently reduces fiber diameter. Also the humidity results in the appearance of circular pores on the fibers.

16.3 Characterization of Electrospun Nanofibers During the development of a new material it is crucial to clearly outline information as to how relevant process parameters may be controlled, thus facilitating the material development process. In the case of cutting edge products involving nanotechnology, the necessity of those characterization tools is even more needful requiring the use of reliable and accurate techniques that allow monitor the formation of new material, as well as its characteristic, associated with the process parameters. These techniques should yield information with high accuracy and precision by allowing manipulation in the nanometer scale, and thus success on final product. It must be borne in the mind that the basic properties of nanofibrous membranes are morphology, molecular structure and mechanical properties. The morphological characteristics of a single fiber such as average fiber diameter, pores on the fiber surface as well as the morphology of nanofibrous membrane (such as porosity and beads) are basic properties of the nanofibers. The molecular structure of a nanofiber bears influence on the optical, thermal and mechanical behavior of the nanofibrous membranes [1]. The mechanical properties can be evaluated by investigating the material resistance characteristics. The main techniques employed to investigate the nanofibers characteristics are Electron Microscopy to determine the characteristics of single fiber diameter and morphology (presence of beads, the surface porosity, etc); Atomic Force Microscopy – AFM and BET measurement to provide information about pore size, surface area and porosity; Differential Scanning Calorimetry – DSC, to determine thermal behavior; X-Ray Diffraction to identify the crystalline structure of nanofibers membranes and loaded active agents; Dynamic Mechanical Analysis – DMA to evaluate the mechanical properties. Among all the techniques employed to characterize nanofibers, Scanning Electron Microscopy (SEM) is an unanimous and noteworthy tool due to the fact that it provides information on the micro/nanometer scale with high resolution such as fiber diameter, porosity surface, mat orientation or non woven morphology.

DCM/DMF -

Erythromycin

Lisozime

DCM

Eth/Ac

Chl

PCL

PLA

HPMCP

Blends

Swelling

Collector-needle dist: 30 cm

Conductivity Mechanical Properties

Collector-needle dist: 10cm Polymer/Drug: 9/1

Voltage: 15 kV Collector-needle dist: 12cm

PCL/PLGA-b-PEO

In vitro Release

DSC

SEM, LSCM

Surface tension

Voltage: 10 kV

Flow rate: 1.2 mL/h

SEM, Viscosity

EDX

Flow rate: 5 mL/h

Voltage: 10-17 kV

SEM, TEM

OM

Voltage: 31 kV

Flow rate: 0,5-1,5 mL/h

SEM

Characterization

Flow rate: 0,5 mL/h

Process Conditions

PLA/PLGA-b-PEO

-

DMF

PVP

Drug

Solvent

Polymer

Table 16.1 Process conditions, characterization and applications of nanofibers in current scientific works

Drug Delivery

Drug Delivery

Tissue Engineering

Microgels

Application

32

26

31

30

Ref

16 Production of Nanofibers by Electrospinning Technology 319

Chl

DCM

TFA/DCM

DMF/DMSO

Eth

PU

PLGA

Blend

Chi/PLA

PVP

Table 16.1 (continued)

Antibacterial Activity SEM XR TEM

Collector-needle dist: 11cm Flow rate: 2 mL/h Voltage: 15 kV Collector-needle dist: 20cm

Fe3O4

Nanoparticles

Magnetic separation, Drug Delivery and Magnetic sensors

Flexible magnetic membranes,

Applications

Flow rate: 1,3 mL/h

Viscosity

Voltage: 10-40 kV

Cytotoxicity Test

LSCM, XR, DSC

Drug Delivery

Biomedical Application

Wound-dressing

Voltage: 5-12 kV

SEM, In vitro Release

Swelling

Voltage: 30 kV Collector-needle dist: 17cm

SEM

Flow rate: 2,1 mL/h

SEM, XR

Paclitaxel

-

34

27

33

47

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DMF

PU

Chl

PCL, PLA

PLGA, PVA

Diclofenac sodium and Human Serum Albumin

Eth:Water

PMMBH

FTIR-ATR,

Collector-needle dist: 20-30cm In vitro Release

Voltage: 16-26 kV

SEM, CM

In vitro Release

Voltage: 20 kV Collector-needle dist: 20-30 cm

SEM

SEM

In vitro Release

Flow rate: 1 mL/h

Collector-needle dist: 10cm

SEM, DSC

In vitro Release

Collector-needle dist: 20cm Voltage: 16 kV

SEM, FTIR

Voltage: 10 kV

Metoclopramide hy- Flow rate: 0.5-12.5 mL/h drochloride

Rolipram

Itraconazole and ketanserin

Methylene blue

Blend PLA/PLGA

DMAc

Water

PAA/ PAH

Table 16.1 (continued)

35

50

Drug Delivery

Drug Delivery

40

49

Drug Delivery

Drug Delivery

Drug Delivery; Biomedi- 48 cal Application

16 Production of Nanofibers by Electrospinning Technology 321

CM:DMF

DMF

Formic Acid: Raspberry ketone Water

PLA

PLGA

Blend

PVA/Gelatin

m-Cresol

Nylon-6

Carmofur

THF: DMF

PU

Dexamethasone

Chl/DMF

PCL

Table 16.1 (continued)

In vitro Release

Collector-needle dist: 20cm

XR,

Collector-needle dist: 10cm

Voltage: 30 kV

Drug Delivery

Textile and Filters

Drug Delivery

SEM, Conductivity, Vis- Drug Delivery cosity, Mechanical Properties, In vitro Release

Collector-needle dist: 10-25 cm In vitro Release

Voltage: 10-30 kV

Flow rate: 5 mL/h

SEM, DSC

Mechanical Properties

Voltage: 27 kV Collector-needle dist: 12-13cm

FTIR-ATR

Flow rate Ny: 0.2 mL/h

SEM, TEM

FTIR-ATR,

Voltage: 9.5 kV

Flow rate PU: 2 mL/h

SEM, DSC

Flow rate: 1 mL/h

28

36

52

51

322 M.H.A. Zanin, N.N.P. Cerize, and A.M. de Oliveira

PVP

PVA/PLGA

Blend

PCL/PLA

Ketoprofen

Water or Ac Haloperidol

Eth

Flow rate: 6-15 mL/h

SEM, FTIR DSC, In vitro Release

Voltage: 15 kV Collector-needle dist: 15cm

In vitro Release

SEM, FTIR

In vitro Release

Surface Tension

Flow rate: 2 mL/h

Collector-needle dist: 15cm

Voltage: 15 kV

Flow rate: 2 mL/h

Collector-needle dist: 19,4cm

Tetracycline Ampho- Voltage: 20 kV tericin

Chlorotetracycline

SEM

SEM, Viscosity

Chl

-

Blend

Ketoprofen In vitro Release

Chl

Blend PU/PCL

PU, PCL,

Table 16.1 (continued)

Drug Delivery

Drug Delivery

Drug Delivery

Drug Delivery

38

37

42

41

16 Production of Nanofibers by Electrospinning Technology 323

Chl

Eth

PLA-b-PEG

PVP

PSM/Cellulose

Ibuprofen

Doxorubicin

Collector-needle dist: 12cm

XR, FTIR

Voltage: 15 kV

In vitro Release

Collector-needle dist: 24cm

SEM, DSC

CM

Voltage: 48 kV

Flow rate: 2 mL/h

SEM

Viscosity, Swelling

Flow rate: 3.6 – 5.4 mL/h

Collector-needle dist: 16cm

Voltage: 14 kV

Flow rate: 0.6 mL/h

Blend SEM, FTIR

Density, Porosity

Voltage: 14-16 kV

PLGA-b-PEG-NH2

DMAc:Ac

SEM, LSCM, FTIR

Lysozyme

Flow rate: 1.2 mL/h

Blend PCL/

DMF/THF

Table 16.1 (continued) 43

Drug Delivery

Drug Delivery

39

53

Biomedical Applications 44

Drug Delivery

324 M.H.A. Zanin, N.N.P. Cerize, and A.M. de Oliveira

DMAc/Ac

Vitamins A and E

SEM In vitro Release Mechanical Properties

Flow rate: 1 mL/h Voltage: 17.5 kV Collector-needle dist: 15cm

Drug Delivery

29

Ac: Acetone; ATR: Spectroscopy Attenuated Total Reflectance; Chi: Chitosan; Chl: Chloroform; CM: Confocal Microscopy; Collector-needle dist: Collector-needle distance; DCM: Dichloromethane; DMAc: N,N-dimethylacetamide; DMF: Dimethylformamide; DMSO: Dimethylsulfoxide; DSC: Differential Scanning Calorimetry; EDX: Energy Dispersive X-ray Analysis; Eth: Ethanol; FTIR: Fourier Transform Infrared Spectroscopy; HPMCP: Hydroxylpropyl methyl cellulose phthalate; LSCM: Laser Scanning Confocal Microscopy; OM: Optical Microscopy; PAA: poly(acrilic acid); PAH: Poly (allylamine hydrochloride); PCL: Polycaprolactam; PEG: Poly (ethylene glycol); PLA: Polylactide; PLGA: Poly(lactic acid-co-glycolic acid); PMMBH: Poly(maleic anhydride-alt-2-methoxyethyl vinyl ether) n-butyl hemiester; PSM: Poly[styrene-co-(maleic sodium anhydride)]; PU: Polyurethanes; PVA: Polyvinylalcohol; PVP: Polyvinylpyrrolidone; SEM: Scanning Electron Microscopy; TEM: Transmission Electron Microscopy; TFA: Trifluoroacetic acid; THF: Tetrahydrofuran; XR: X-ray Analysis.

Cellulose Acetate

Table 16.1 (continued)

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New materials that can be used as filters, scaffolds and drug delivery require a suitable selection of tools for their characterization taking into consideration the influence of process parameters in the final product and focusing on development of a streamlined process. More specific applications require individual techniques such as swelling in case of hydrogels, for systems which employ more than one polymer as is the case of coaxial electrospinning, tools for distinguishing various materials and for defining the nanofiber morphology, for example, core-shell structures (confocal microscopy and Transmission Electron Microscopy). And finally, the characterization of drug delivery nanofibrous system using in vitro release studies, measurement of encapsulation efficiency, dyes for confocal scanning laser microscopy, when it involves more elaborated procedures as the encapsulation of different active agents in diversified areas (pharmaceuticals, cosmetics, veterinary, agrochemical, among others). An overview of the techniques used for characterization of nanofibers can be found in Table 16.1. Note that SEM is the most used technique requiring an easy sample preparation. DSC is always present due to be very sensitive and widely applicable.

16.4 Application of Polymer Nanofibers A combination of three main aspects of nanofibrous membranes: 1) size of fiber, 2) highly orientated molecular structure and 3) high surface area, give rise to applications in filtration, cellular matrices, catalyst substrates, ultra-strong composites, bioreactors, functional textiles, drug encapsulation, wound dressings and stent manufacture [54]. Although the first applications of nanofibers are related to unit processes, such as construction of ultrafiltration membranes, currently the main applications of nanofibers are related to biomedical research, such as tissue engineering and controlled drug release. In the field of tissue engineering the nanofibers can be used to build different tissues such as vasculature, bone, neural and tendon / ligament [20]. Concerning drug delivery application, including controlled release systems, the nanofibers provide opportunity for encapsulation of different active agents such as antibiotics, anticancer drugs, proteins, and DNA. Table 16.1 presents some scientific works that present loaded-nanofibers containing different active agents obtained for drug delivery purpose. Recently, our group successfully demonstrated use of electrospinning of nanofibers in odontological and cosmetic applications. The nanofibers studied were obtained by using PVP, PVA, Eudragit® RS100 and blends of these polymers. The Figure 16.4 shows the morphology and fiber diameter of nanofibrous membranes of PVP/ Eudragit® RS100 blends which diameters vary from 150 to 300 nm.

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Fig. 16.4 SEM image of PVP/ Eudragit® RS100 electrospun nanofiber.

16.5 Application of Polymeric Nanofibers in Cosmetics In spite of the increase in the number of electrospun polymer nanonofiber publications in the recent years, there is relatively scarce work, including scientific papers

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and patents, in the cosmetic field regarding the use of electrospun nanofibers. The development of nanofibers in this field have been focused on skin treatment applications, such as care mask, skin healing and skin cleansing, with active agents (cosmetics) with controlled release in some cases. The cosmetic application tends to be included in the biomedicine application, which includes the drug delivery system employing the active agents used in cosmetics, body care supplements [29, 55]. Therefore, the cosmetic and drug delivery are closely related areas. Comparative studies of Taepaiboon and coworks [29] of spun fiber mats and cast film loaded with vitamin E (α tocoferol) and vitamin A (retinoic acid) presented a gradual and monotonous increase in the cumulative release of vitamins over the test periods (24 h for Vitamin E loaded nanofibers and 6 h to the vitamin loaded ones) for the most samples, while the corresponding cast films presented a burst release of the vitamins. The polymer used was cellulose acetate (CA) solubilized in a mixture of acetone/N,N-dimethylacetamide 2:1 v/v. The loading of vitamin E and Vitamin A were 5 and 0.5 wt%, respectively, based on the weight of CA%. The electrospun nanofibers provide the advantage of increasing drug release when compared to cast-films due to the increased surface area [56]. An overview of some publications in drug delivery with different polymer families is depicted in the Table 16. 1. In terms of patents, one of them belongs to Seiler et al. [57] which describes an application of nanofiber matrices formed by electrospinning process, containing an active agent, incorporated in the fibers, to cosmetic application or other application as drug delivery and growth of cells. The invention comprises the using of hyperbranched polymers (with a hydrophobic core having polyester units and hydrophobic end groups) and active agents typically used in cosmetic preparation such as colorants, emollients, sunscreens, exfoliants, antioxidants, humectants and nutrients. The other three patents [9, 58, 60] relate to the skin care (cosmetic sheets).

16.6 Future Perspectives of Nanofibers by Electrospinning in Cosmetic Application The nanofibers as mat to vehicle active agent to cosmetic applications are still not much used, however there is a high growth potential in this field due to the large surface area per unit mass (on the order of 100 m2/g for a nanofiber of approximately 100 nm of diameter) and the possibility of industrial processing. Moreover, it amenable to the processing of different polymers such as natural, synthetic and blends, according to their solubility or melting point. It is important to point out that although most of the researches in the field of polymeric nanofiber by electrospinning consider the technique simple, costeffective and also easily scalable from laboratory to commercial production, only a limited number of companies have commercially implemented electrospun fibers. One strategy for implementing this technology to the cosmetic area is to use as model systems that have already been developed in the biomedical/drug delivery field and adapting it to the requirements of the cosmetic application. One of

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the challenges in development of nanofibers will be to demonstrate the advantages of drug delivery systems based on nanofibers compared to nanoparticulated systems. These investigations have the potential to open opportunities for the development of laboratory scale processes which could be later scaled up, taking into consideration the different electrospinning machine configurations commercially available. Acknowledgments. The authors acknowledge the financial support by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to the infrastructure to the nanofiber studies.

References [1] Ramakrishna, S., Fujihara, K., Teo, W.E., Lim, T.C., Ma, Z.: An introduction to electrospinning and nanofibers. World Scientific Publishing Co. Pte. Ltd., Singapore (2005) [2] Gibson, P., Schreuder-Gibson, H., Rivin, D.: Transport properties of porous membranes based on electrospun nanofibers. Colloids and Surf A: Physicochem. and Eng. Aspects 187-188, 469–481 (2001) [3] Huang, Z.M., Zhang, Y.Z., Kotaki, M., Ramakrishna, S.: A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos. Sci. Technol. 63, 2223–2253 (2003) [4] Jayaraman, K., Kotaki, M., Zhang, Y., Mo, X., Ramakrishna, S.: Recent Advances in Polymer Nanofibers. J. Nanosci. Nanotechnol. 4, 52–65 (2004) [5] Ondarçuhu, T., Joachim, C.: Drawing a single nanofibre over hundreds of microns. Europhys. Lett. 42(2), 215–220 (1998) [6] Martin, C.R.: Membrane-Based Synthesis of Nanomaterials. Chem. Mater. 8, 1739– 1746 (1996) [7] Smith, L.A., Ma, P.X.: Nano-fibrous scaffolds for tissue engineering. Colloids and Surfaces Biointerfaces 39, 125–131 (2004) [8] Whitesides, G.M., Grzybowski, B.: Self-Assembly at All Scales. Sci. 295, 2418– 2421 (2002) [9] Doshi, J., Reneker, D.H.: Electrospinning Process and Applications of Electrospun Fibers. J. Electrost. 35, 151–160 (1995) [10] Ma, P.X., Zhang, R.: Synthetic nano-scale fibrous extracellular matrix. J. Biomed. Mater Res. 46, 60–72 (1999) [11] Barnes, C.P., Sell, S.A., Boland, E.D., Simpson, D.G., Bowlin, G.L.: Nanofiber technology: Designing the next generation of tissue engineering scaffolds. Adv. Drug Deliv. Rev. 59, 1413–1433 (2007) [12] Cooley, J.F.: Patent US 692631 (1902) [13] Morton, W.J.: Patent US705691 (1902) [14] Formhals, A.: Patent US 1975504 (1934) [15] Formhals, A.: Patent US 2077373 (1937) [16] Formhals, A.: Patent US 2187306 (1940) [17] Taylor, G.I.: Electrically Driven Jets. Proceedings of the Royal Society of London Series A: Mathematical and Physical Sciences 313, 453–475 (1969)

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[18] Murugan, R., Ramakrishna, S.: Design Strategies of Tissue Engineering Scaffolds with Controlled Fiber Orientation. Tissue Eng. 13, 1845–1866 (2007) [19] Jiang, H., Hu, Y., Li, Y., Zhao, P., Zhu, K., Chen, W.: A facile technique to prepare biodegradable coaxial electrospun nanofibers for controlled release of bioactive agents. J. Control Release 108, 237–243 (2005) [20] Sill, T.J., Recum, H.A.: Electrospinning: Applications in drug delivery and tissue engineering. Biomater 29, 1989–2006 (2008) [21] Chakraborty, S., Liao, I., Adler, A., Leong, K.W.: Electrohydrodynamics: A facile technique to fabricate drug delivery systems. Adv. Drug Deliv. Rev. 61, 1043–1054 (2009) [22] Bhardwaj, N., Kundu, S.C.: Electrospinning: A fascinating fiber fabrication technique. Biotechnol. Adv. 28, 325–347 (2010) [23] Petrik, S., Maly, M.: Production nozzle-less electrospinning nanofiber technology, http://www.elmarco.cz/upload/soubory/dokumenty/ 66-1-1-mrs-fall-boston-09.pdf (acessed September 16, 2010) [24] Smith, D.J., Reneker, D.H., McManus, A.T., Schreuder-Gibson, H.L., Mello, C., Sennet, M.S.: Patent US 6753454 (2004) [25] Liang, D., Hsiao, B.S., Chu, B.: Functional electrospun nanofibrous scaffolds for biomedical applications. Adv. Drug. Deliv. 5, 1392–1412 (2007) [26] Wang, M., Wang, L., Huang, Y.: Electrospun hydroxypropyl methyl cellulose phthalate (HPMCP)/Erythromycin fibers for targeted release in intestine. J. of Appl Polym Science 160, 2177–2184 (2007) [27] Ignatova, M., Manolova, N., Markova, N., Rashkov, I.: Electrospun Non-Woven Nanofibrous Hybrid Mats Based on Chitosan and PLA for Wound-Dressing. Appl. Macromol. Biosci. 9, 102–111 (2009) [28] Yang, D., Li, Y., Nie, J.: Preparation of gelatin/PVA nanofibers and their potential application in controlled release of drugs. Carbohydrate Polym. 69, 538–543 (2007) [29] Taepaiboon, P., Rungsardthong, U., Supaphol, P.: Vitamin-loaded electrospun cellulose acetate nanofiber mats as transdermal and dermal therapeutic agents of vitamin A acid and vitamin E. Eur. J. Pharm. Biopharm. 67, 387–397 (2007) [30] Diaz, J.E., Barrero, A., Marquez, M., Fernandez-Nieves, A., Loscertales, I.G.: Adsorption properties of microgel-PVP composite nanofibers made by electrospinning. Macromol. Rapid. Commun. 31, 183–189 (2010) [31] Xie, J., MacEwan, M.R., Willerth, S.M., Li, X., Moran, D.W., Sakiyama-Elbert, S.E., Xia, Y.: Condutive core-sheath nanofibers and their potential application in neural tissue engineering. Advanced Functional Mat. 19, 2312–2318 (2009) [32] Kim, T.G., Lee, D.S., Park, T.G.: Controlled protein release from electrospun biodegradable fiber mesh composed of poly( -caprolactone) and poly(ethylene oxide). International Journal of Pharmaceutics – Pharmaceutical Nanotechnol 338, 276–283 (2007) [33] Xie, J., Wang, C.H.: Electrospun Micro- and Nanofibers for Sustained Delivery of Paclitaxel to Treat C6 Glioma in Vitro. Pharm Research 23(8), 1817–1826 (2006) [34] Wang, H., Tang, H., He, J., Wang, Q.: Fabrication of aligned ferrite nanofibers by magnetic-field-assisted electrospinning coupled with oxygen plasma treatment. Mater Research Bulletin 44, 1676–1680 (2009) [35] Tiwari, S.K., Tzezana, R., Zussman, E., Venkatraman, S.S.: Optimizing partitioncontrolled drug release from electrospun core–shell fibers. Int. J. Pharm. 392, 209– 217 (2010)

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[36] Ren, J., Liu, W., Zhu, J., Gu, S.: Preparation and Characterization of Electrospun, Biodegradable Membranes. J. Appl. Polym. Sci. 109, 3390–3397 (2008) [37] Fathi-Azarbayjani, A., Chan, S.Y.: Single and Multi-Layered Nanofibers for Rapid and Controlled Drug Delivery. Chem. Pharm. Bull. 58(2), 143–146 (2010) [38] Yu, D.G., Branford-White, C., Shen, X.X., Zhang, X.F., Zhu, L.M.: Solid Dispersions of Ketoprofen in Drug-Loaded Electrospun Nanofibers. J. Dispersion Sci. Technol. 31, 902–908 (2010) [39] Yu, D.G., Zhang, X.F., Shen, X.X., Brandford-White, C., Zhu, L.M.: Ultrafine ibuprofen-loaded polyvinylpyrrolidone fibermats using electrospinning. Polym. Int. 58, 1010–1013 (2009) [40] Zhu, Y., Wang, A., Shen, W., Patel, S., Zhang, R., Young, W.L., Li, S.: Nanofibrous Patches for Spinal Cord Regeneration. Adv. Funct. Mater. 20, 1433–1440 (2010) [41] Kenawy, E., Abdel-Hay, F.I., El-Newehy, M.H., Wnek, G.E.: Processing of polymer nanofibers through electrospinning as drug delivery systems. Mater. Chem. Phys. 113, 296–302 (2009) [42] Buschle-Diller, G., Cooper, J., Xie, Z., Wu, Y., Waldrup, J., Ren, X.: Release of antibiotics from electrospun bicomponent fibers. Cellul. 14, 553–562 (2007) [43] Kim, T.G., Park, T.G.: Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules. Biotechnol. Prog. 22, 1108–1113 (2006) [44] Cao, S., Hu, B., Liu, H.: Synthesis of pH-responsive crosslinked poly[styrene-co(maleic sodium anhydride)] and cellulose composite hydrogel nanofibers by electrospinning. Polym. Int. 58, 545–551 (2009) [45] Chronakis, I.S.: Novel nanocomposites and nanoceramics based on polymer nanofibers using electrospinning process – A review. J. Mater Process. Technol. 167, 283– 293 (2005) [46] Pham, Q., Sharma, U., Mikos, A.G.: Electrospinning of Polymeric Nanofibers for Tissue Engineering Applications: A Review. Tissue Eng. 12, 1197–1211 (2006) [47] Shah, P.N., Manthe, R.L., Lopina, S.T., Yun, Y.H.: Electrospinning of L-tyrosine polyurethanes for potential biomedical applications. Polym. 50, 2281–2289 (2009) [48] Chunder, A., Sarkar, S., Yu, Y., Zhai, L.: Fabrication of ultrathin polyelectrolyte fibers and their controlled release properties. Colloids and Surfaces B: Biointerfaces 58, 172–179 (2007) [49] Verreck, G., Chun, I., Rosenblatt, J., Peeters, J., Dijck, A.V., Mensch, J., Noppe, M., Brewster, M.E.: Incorporation of drugs in an amorphous state into electrospun nanofibers composed of a water-insoluble,nonbiodegradable polymer. J. Control Release 92, 349–360 (2003) [50] Piras, A.M., Chiellini, F., Chiellini, E., Nikkola, L., Ashammakhi, N.: New Multicomponent Bioerodible Electrospun Nanofibers for Dual-controlled Drug Release. J. Bioact. Compat. Polym. 23, 423–443 (2010) [51] Martins, A., Duarte, A.R.C., Faria, S., Marques, A.P., Reis, R.L., Neves, N.M.: Osteogenic induction of hBMSCs by electrospun scaffolds with dexamethasone release functionality. Biomater 31, 5875–5885 (2010) [52] Han, X., Huang, Z., He, C., Liu, L.: Preparation and Characterization of Core–Shell Structured Nanofibers by Coaxial Electrospinning. High Performance Polym. 19, 147–159 (2007) [53] Xu, X., Chen, X., Ma, P., Wang, X., Jing, X.: The release behavior of doxorubicin hydrochloride from medicated fibers prepared by emulsion-electrospinning. Eur. J. Pharm. Biopharm. 70, 165–170 (2008)

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[54] Stanger, J., Tucker, N., Staiger, M.: Electrospinning Rapra Review Reports. Smithers Rapra Technology 16, 1–206 (2005) [55] Ramakrishna, S., Fujihara, K., Teo, W.E., Yong, T., Ma, Z., Ramaseshan, R.: Electrospun nanofibers: solving global issues. Mater. Today 9, 40–49 (2005) [56] Agarwal, S., Wendorff, J.H., Greiner, A.: Use of electrospinning technique for biomedical applications. Polym. 49, 5603–5621 (2008) [57] Seiler, M., et al.: Patent WO2009133059 (2009) [58] Kim, Y.H., et al.: Patent. KR 2007-78821 (2009) [59] Tajima, T., Kusamoto: Patent EP 2020455 (2009) [60] Hasui, T., Takeda, M.: Patent JP2009097121 (2009)

Chapter 17

Nanosized and Nanoencapsulated Sunscreens Cássia B. Detoni1, Karina Paese1, Ruy C.R. Beck1, Adriana R. Pohlmann2, and Sílvia S. Guterres1 1

2

Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000, Porto Alegre, RS, Brazil [email protected] Departamento de Química Orgânica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, CP 15003, 91501-970, Porto Alegre, RS, Brazil

Abstract. Nanotechnology represents an important complement to traditional chromophore-based UV filters. Nanosized metal oxides are widely used due to their broad protection spectrum and reduced skin irritation. These particles are already present in most commercial sunscreen lotions even though their photoreactivity has been highlighted as a potential cause of cytotoxic effects in human skin cells (e.g. fibroblasts, epithelial cells). However, this cytotoxicity is not likely to represent a significant health risk to consumers as several skin penetration assays have shown that the photoreactive particles penetrate at most to the stratum spinosum of the epidermis. Nanoencapsulation of traditional organic UV filters is a more recent approach to improve skin retention, photostability and the UV blocking ability of the free molecules. All of these improvements have been confirmed with different scientific assays for different particles, especially polymeric nanocapsules and solid lipid nanoparticles. These technological advantages offered by nanometric particles for sun protection formulations have made them commercially important. In inventories from non-governmental organizations there are approximately 30 commercial sunscreen products listed as containing nanoparticles.

17.1 Introduction Sun protection has become a major concern and the efficacy and efficiency of sunscreens are being challenged. The development of new formulations based on nanotechnology has been shown to be a promising approach to skin administration. Today, nanotechnology plays an important role in overcoming traditional drawbacks associated with sunscreens by improving their photostability, skin retention, sun protection factor (SPF) and spectrum of protection. Sunscreen formulations work by combining organic and inorganic filters. Inorganic ingredients like zinc oxide (ZnO) or titanium dioxide (TiO2) reflect, absorb and scatter ultraviolet (UV) radiation. Organic chromophore-containing molecules, such as octyl methoxycinnamate (OMC) or benzophenone-3 (BZ3), absorb UV radiation. Regarding the application of nanotechnology to sunscreen formulations, there are two distinct

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strategies. The first considers the nanoencapsulation of organic filters in carriers prepared with biodegradable materials which act as devices to control their release and delivery to the skin, and also block the UV radiation themselves. The second strategy is based on the incorporation of nanosized filters into the formulations, i.e., they are not nanocarriers of complex architecture but nanoparticles prepared directly by nanosizing the bulk material of the sunscreen. The efficacy of sunscreen formulations is directly dependent on the affinity that the different sun filters have with the skin surface, the stratum corneum [1]. The use of nanoparticulate carriers, such as nanocapsules, increases considerably the time of permanence of substances topically applied on the skin [2, 3], a finding also observed for organic sun filters, such as OMC [4, 5]. Another benefit of nanoencapsulated sunscreens is the increase in photoprotection [6] due to the synergic effect of the absorption of UV radiation by the organic sunscreens and the reflection and scattering of this radiation by the nanosized particles [7]. The efficiency of the sunscreen formulations is also dependent on the photostability of the organic filters used [1]. The improvement in the photostability of chromophores when encapsulated in nanocarriers has been demonstrated for different substances under UVA radiation [5, 8, 9]. Mineral UV filters have been extensively used in attempts to raise the SPF of sunscreen formulations, but they form a visible pigmented layer on the skin. To circumvent this inconvenience and improve the acceptance of these formulations, micronized oxides are now obtained in the nanometric scale. When applied on the skin these nanosized particles form a thin transparent layer [10]. They have been commercialized in sunscreen lotions since 1990 and great safety concerns are being raised by society in general and the scientific community. Since their mechanisms and efficiency are well established and they have a consolidated position in the commercial arena, research on these particles is currently focused on evaluating their toxicity after topical application and UV exposure. This chapter presents an overview of the new insights and data generated over the last few years on the dermatological applications of nanotechnology in photoprotection. We attribute the term nanosized to all nanoparticles that do not have an organic chromophore in their constitution and are capable of blocking UV radiation. All organic chromophore molecules loaded into submicrometric particles are referred to here in as nanoencapsulated UV filters.

17.2 Nanosized UV Filters Nanosized UV filters are particles capable of scattering, reflecting, and/or absorbing solar radiation. These UV filters are usually nanometric and consist of different materials, which can be organic or inorganic. The use of these particles in sunscreens is advantageous because of their: i) ability to increase the SPF; ii) broad spectrum of UV protection; and iii) reduced potential irritancy [11]. TiO2 and ZnO account for most particulate sun filters used in sunscreens. In 1989 the first sunscreen containing nano-TiO2 was commercialized, while ZnO was introduced in 1991 [12]. The use of TiO2 and ZnO in their nanosized forms in place of their bulk forms solved the problem of the opaque film formed after the

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use of traditional sunscreens. Nowadays, in order to have cosmetic acceptability, the particle size of metal-oxide sunscreens needs to be around 20–50 nanometers (nm). Particle sizes of TiO2 ranging from 200 to 500 nm reflect more visible light, and when applied on the skin present the disadvantage of being opaque [13]. The term physical UV filters was initially attributed to these sunscreens because their first known mechanism of sun blocking was through the physical phenomena of reflection and scattering. However, inorganic UV filters also absorb considerable UV radiation [13]. The combination of absorption, scattering and reflection leads to protection across the whole UVA/UVB spectrum (290–400 nm). The absorption and scattering of each system will be determined by the intrinsic refractive index, the size of the particles, the dispersion in the emulsion base, and the film thickness [12]. UV-transparent nanoparticulate materials, such as polymers and lipids, may also have the physical sun blocking properties of reflection and scattering [7, 9]. An association of these nano- or microsized particles with an inorganic physical filter or organic sunscreen leads to a synergic sun screening effect. Both of these strategies will be further discussed later in this chapter (sections 17.2.3 and 17.3.3).

17.2.1 TiO2 and ZnO-Induced Cytotoxicity and Genotoxicity The use of nanomaterials continues to grow rapidly in numerous areas, such as cosmetics and pharmaceutical products. Evaluating carefully the toxicity and safeness of these nanomaterials is of extreme importance [14]. Raw materials, such as ZnO and TiO2, have their toxicity tested in relation to many administration routes, however, here we will focus on their topical application as a nano-sunscreen agent (Table 17.1). In addition to being an effective sun block, TiO2 is also known for its photocatalytic activity. TiO2 as microparticles is generally inert, but in the form of nanoparticles under irradiation it is a strong oxidizing agent, capable of interacting with a great deal of biological molecules, presenting antibiotic activity. This process is used in photocatalysis in which TiO2 nanoparticles are used for the removal of organic material, such as pollutants from soil, aqueous and atmospheric ecosystems [15, 16, 17]. ZnO is also associated with health concerns. The absorbed UV radiation can promote free radical formation and lead to a consequent genotoxicity. The photocatalytic properties of these materials thus raise health concerns which need to be considered by consumers, manufacturers, governments agencies and research scientists [18]. TiO2 has three different polymorphs, anatase, rutile and brookite, in addition to an amorphous phase. Only the polymorphs anatase and rutile are present in sunscreen lotions and in heterogeneous photocatalysts. Rutile and anatase are tetragonal crystals and in both cases, within a unit cell, each Ti atom is coordinated to six oxygen atoms. Compared to rutile TiO2, the O-Ti-O bonds in anatase are more susceptible to deformations or buckling. Buckling reduces the crystal symmetry and results in a larger unit cell dimension. Anatase is more effective as a photocatalyst when compared to the rutile form [19]. Through electronic microscopy

[24]

[23]

[18]

[21]

[14]

[17]

[16]

[15]

Reference

165, 30 or 50-70 nm, re- Transmission electron microscopy, DNA damage potential in human epidermal cells. dynamic light scattering and Brunspectively auer-Emmett-Teller ZnO, SiO2, single19.6 nm, 20.2 nm, diameter: Evaluation of the cytotoxicity, oxidative stress and walled carbon nano- 8 nm length: < 5 μm, 12.3, genotoxicity induced by four different nanomaterials. tubes and carbon black nm, respectively Cytotoxicity of human skin fibroblasts exposed to TiO2 10-20, 20-100, 20-40, and Scanning electron microscopy UVA light. 20,20,20 nm, respectively

ZnO

-

Cultured skin cell apoptosis and activation of the DNA-damage-response pathway under UVA exposure.

Cytotoxicity on human skin fibroblasts.

Cytotoxicity on human dermal fibroblasts.

Cytotoxicity induced by TiO2–UVA treatment.

Effect of TiO2 particles on UV light induced pyrimidine dimer formation.

Study design

[22]

9 nm

TiO2

X-ray diffraction

-

X-ray diffraction

-

Method of particle size determination -

Effect on cellular functions of human skin – keratinocytes, sebocytes, melanocytes and fibroblasts

Samples varied from 11 – 30 nm

TiO2

3-5 nm

TiO2

50-70 nm and < 150 nm

15,17,30,37, 85,90,93,120 and 400 nm

TiO2

ZnO and TiO2

-

Particle size

TiO2

Material

Table 17.1 Publications on skin-related TiO2 and ZnO toxicity studies

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analysis, it is possible to observe that, while anatase nanoparticles generally aggregate into spherical equidimensional grains, rutile nanocrystals tend to arrange more compactly into larger, striated structures (Figure 17.1). These clustered crystals are considerably more intergrown than anatase crystals [17, 20].

Fig. 17.1 Transmission electron micrographs of (A) anatase, (B) anatase:rutile (60:40) and (C) rutile crystals in water (figure obtained from [17]).

The photocatalytic properties of nanosized TiO2 have been the focus of many research studies in recent years in order to evaluate the potential toxicity of these particles when applied as sunscreens and exposed to UV radiation [24]. Since the catalytic activity of TiO2 is photo-dependent, the cytotoxicity and genotoxicity of these particles will also depend on the level of irradiation, which can vary according to time of exposure or power of the light source [16, 24]. TiO2 can produce hydroxyl radicals, hydrogen peroxide and molecular oxygen when exposed to UV radiation. The production of reactive oxygen species (ROS) has been associated with a variety of nanomaterials. ROS can result in oxidative stress, inflammation and consequent protein, DNA and membrane damage, leading to cell death. This increase in ROS is one of the most probable mechanisms of cytotoxicity [16, 18]. The formation of hydroxyl radicals is dependent on the concentration of the anatase form and on the intensity of the UV radiation. The anatase form leads to a greater formation of hydroxyl radial than the rutile and amorphous forms. Cell viability reduces significantly after UVA irradiation. Anatase crystals present cytotoxicity even without being irradiated by UVA light, while rutile does not modify the cell viability significantly. Thus, the cytotoxicity is strongly associated with the concentration of the anatase form, and weakly associated with the intensity of the UVA radiation. It is possible that the different crystalline forms produce OH radicals through different pathways and that the radicals produced by rutile crystals are less stable than those produced by the anatase form, thus being less toxic [16]. Size is a very important parameter in nanoparticle characterization when considering biological applications. This parameter may influence the cytotoxicity, since smaller sizes lead to larger surface areas and consequently greater reactivity. Different sized TiO2 particles have been compared: the amorphous TiO2 (17 nm),

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four rutile types [anatase mixtures containing rutile as the major component (37, 93, 400 and 85 nm)] and mixtures containing anatase as the major component (15, 30, 30, 90 and 120 nm). The size of the particles was found to influence the formation of hydroxyl radicals. It was observed that for anatase TiO2 30 nm particles generated the most OH radicals in cell cultures exposed to UVA radiation while for the rutile form 90 nm particles were the most photoreactive [16]. Various methods are used to measure particle size and it is important to bear in mind that different measurement techniques are not directly comparable [25]. The oxidative stress has been evaluated using human epidermal cell cultures exposed to ZnO. In this study, the size of ZnO particles was determined by transmission electron microscopy (TEM), dynamic light scattering (see Chapter 3) and the Brunauer-Emmett-Teller (BET) data provided by the supplier. The results for the particle size were TEM 30 nm, dynamic light scattering 165 nm and BET 50-70 nm. As can be noted, the different methods used to determine particle size may result in very different diameters (Figure 17.2). In toxicological assays, dynamic light scattering is the most important technique because it determines the particle diameter under conditions closest to that of a biological assay [18].

Fig.17.2 Schematic representation of the analytical techniques used by Sharma et al. 2009 to measure nanoparticles and their results.

Decreases in catalase, glutathione, and superoxide dismutase levels, which directly compensate for the ROS formation, indicate a condition of oxidative stress in the cells. Oxidative stress is usually initiated by an imbalance between the free radical formation and the antioxidant defense system. An increase in peroxide formation has been observed after exposure to ZnO particles, indicating lipid

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peroxidation, which can lead to membrane damage initiated by the release of lactate dehydrogenase [18]. The cellular internalization of nanosized particles is also an important factor for cyto- and genotoxicity. Titanium anatase particles of 9 nm at a concentration of 15 µg ⁄cm2, visualized by phase contrast microscopy, have been found to reach the cytoplasm of fibroblasts and melanocytes after 24 hours. However, interestingly, there was no cellular uptake of TiO2 by keratinocytes or sebocytes [22]. Hybrid TiO2/p-amino benzoic acid nanoparticles were also detected by confocal microscopy in the cytoplasm of monocytes but not in their nuclei. The internalization was via macropinocytosis and not the receptor [26]. The nanoparticle material is another important parameter for cytoxicity and oxidative stress. Carbon black nanoparticles (diameter 12.3 nm), single-walled carbon nanotubes (diameter 8 nm, length 5 nm), silicon dioxide (20.2 nm) and ZnO (19.6 nm), with the diameters determined by TEM, have been compared. Fibroblasts were incubated with different concentrations of nanoparticles (5, 10, 20, 50 and 100 µg/mL) for 24 hours in order to determine the cytotoxicity and oxidative effects. ZnO particles presented the highest level of oxidative effects on the cells, which caused a reduction in the glutathione concentration and the enzymatic activity of superoxide dismutase, as well as an increase in malondialdehyde. Also, the ZnO particles showed greatest cytotoxicity in doses of 10 µg/mL or higher. There was no difference in the membrane damage caused by the different materials. Since all particles caused similar degrees of membrane damage, identified by the raised lactate dehydrogenase levels, and ZnO presented the highest level of ROS generation, the acute cytotoxicity is not caused by membrane damage and may be caused by internalized ZnO particles. Considering that ZnO and SiO2 crystalline particles showed similar shape and size, the main distinct factor which leads to the greater toxicity of ZnO particles is the particle chemical composition. On the other hand, single-walled carbon nanotubes were less cytotoxic than ZnO but caused greater DNA damage. The damage caused to the DNA when the cells are exposed to single-walled carbon nanotubes may be associated with a mechanical mechanism rather than oxidative effects. The single-walled carbon nanotubes can penetrate the cell nucleus through nucleopores and destroy the DNA double helix. However, even with this result, it is not verified that the different genotoxicity levels of nanoparticles is primarily due to particle shape rather than chemical composition, since there are still technical difficulties associated with evaluating the intracellular translocation of nanoparticles [23]. Particle concentration and time of exposure also have to be taken into account when evaluating toxicity. Different concentrations of ZnO particles suspended in ultrapure water; (0.008, 0.08, 0.8, 5.0, 8.0 and 20.0 µg/mL) were studied in a comparative cytotoxicity assay. The cells were exposed to ZnO particles for 48 hours and the cell viability was evaluated after 3, 6, 24 and 48 hours. The cytotoxicity generated by ZnO application on human epidermal cells was found to be time- and dose-dependent, exhibiting a positive toxic effect above 5 μg/mL after exposure to ZnO for more than 6 hours. For higher concentrations (20 μg/mL), the cytotoxicity was evident after 3 hours. The toxicity of ZnO nanoparticles was also noted through morphological alterations in the cells after 6 hours with a

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concentration of 8 μg/mL. DNA damage was also observed. This genotoxicity, possibly related to oxidative stress, could lead to apoptosis or cause interference in normal cellular functions leading to cell death [18]. The role of the particle composition in the in vitro cytotoxicity of TiO2 and ZnO on human skin fibroblasts without UV irradiation was verified in 2007. The IC50 (50% inhibitory concentration) and total lethal concentration indicated that after 4 hours of exposure to both particles only slightly adverse effects were observed while after 24 hours substantial cytotoxic effects occurred. The ZnO total lethal concentration after 24 hours was around 290 ppm and for TiO2 it was around 15,000 ppm [14]. The cytotoxicity of TiO2 in anatase and rutile forms in human dermal fibroblast cell cultures and in pulmonary carcinoma cells after UVA irradiation (356 nm) has been compared. Anatase nanoparticles were found to be more cytotoxic at high concentrations (1500 μg/mL) than the rutile crystals. Specifically, the anatase nanoparticles presented a LC50 of 3.6 µg/mL and the rutile of 550 µg/mL. In addition, the anatase particles reduced mitochondrial activity (or cell viability) and raised the interleucine-8 level (inflammatory response). The cytotoxicity of the anatase crystalline form is related to the higher levels of reactive oxygen species formation [17]. The ex vivo production of ROS under different conditions of exposure (dark or illuminated) is also greater when anatase is used in place of rutile TiO2. The crystalline forms differ substantially in terms of the surface chemistry of the particle with respect to the formation of ROS [17], indicating the importance of surface modifications such as those which occur through coating and ion doping. The crystalline form has a great influence on the metal oxide photoreactivity. The anatase form of TiO2 is much more reactive than the rutile form. Therefore, one of the strategies available to prevent free radical formation is the use of predominantly the rutile crystalline form. However, the prevention of free radical formation and consequent damage to skin cells should also include surface modifications with inert coatings or doping with specific ions. The coatings include silicon oxides (silica), silicones and organosilanes, and those based on aluminum oxides (alumina), and doping can be carried out with manganese ions [21, 24]. Nanosized UV-blocker coatings are based on forming a physical barrier layer, such as silica or alumina, which is deposited around the UV-absorbing particle (TiO2 and ZnO). This acts by trapping photogenerated electrons within the particle and preventing surface access leading to free radical formation. Ion doping is based on the incorporation of a number of dopant ions into the titanium nanoparticles at a low concentration (i.e. less than 1 %) [27]. Few research studies have evaluated the skin-related cytotoxicity of coated or doped metal oxides. Two studies have confirmed the importance of these factors in terms of the potential toxicity. One of these was performed using ZnO and TiO2 in different anatase:rutile ratios with different surface modifications [21] and the other was preformed with different commercial TiO2 particles [24]. Potential toxicity may be assayed by comparing the photoreactivity of the particles. Oxidation of organic materials by hydroxyl radicals from illuminated metal oxides can be easily determined by monitoring the oxidation of a test molecule.

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One study used methylene blue as a liable molecule and verified that a mixture of anatase and rutile with an organosilane coating is more photocatalytically active when compared to ZnO and pure rutile with different coatings. Considering all of the samples analyzed, 100 % rutile particles with an aluminum coating or manganese doping were the least photoactive. Under certain conditions the simultaneous presence of ZnO and TiO2 enhances the photoactivity of TiO2 [21]. In a more recent study, a similar photobleaching assay with the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical was used. Only anatase or anatase/rutile mixtures presented significant photoreactivity confirming that 100 % rutile TiO2 particles are not photocatalytic. Mixtures of anatase/rutile coated with organic compounds, as used by the authors (TEGO Sun®), did not show a reduction in the photoactivity. All particles assayed in this study had diameters close to 20 nm, except one sample which varied from 20 to 100 nm. Even so, the authors did not evaluate the effect of particle size on the assays. In this assay, no statistical difference between the rutile crystals with different types of coating and the irradiated control sample (without the presence of TiO2 particles) was observed [24]. Biologically relevant molecules such as deoxyribose, which constitutes part of the DNA structure, may also be used in photoreactivity assays. The degradation of deoxyribose by TiO2 nanoparticles was evaluated with a colorimetric method based on reaction of the degradation products with thiobarbituric acid (TBA). Manganese-doped rutile did not show any statistical difference from the control (absence of particles) while aluminum and dimethicone-coated rutile particles showed some reactivity and anatase particles were the most reactive [24]. The in vitro photoreactivity assay provides a good preliminary evaluation, but comparisons between different material particles and different molecules are not possible because, as photocatalysts, metal oxides may be associated with many pathways [28]. Assays using cultures of different skin cells (human epithelial and fibroblast cells) may offer a simple and reliable way to evaluate the safety of nanosized filters by determining cellular damage by apoptosis [21, 24] and DNAdamage [21]. Monochromatic 365 nm UVA irradiation caused signs of apoptosis, with condensed and fragmented nuclei in cultured cells after 75 min of irradiation. UVA alone induced 10–30% apoptotic cells in the total cell population. The morphological changes in the nuclei were attributed to a caspase-linked pathway [21]. The TiO2 may present a toxic or protective effect depending on the characteristics of the particles. The protective property of TiO2 has been extensively proven in the last decade with optical experiments and SPF assays, but in vitro cellular studies that confirm the protective efficacy of these particles are scarce. Uncoated rutile/anatase crystal mixtures, and organosilane and dimethicone-coated TiO2 particles have shown destructive behavior in in vitro assays with human cells [17, 24], whereas the rutile crystal form, either alumina-coated or manganese-doped, is protective against UV radiation [15, 21]. In an in vitro toxicity assay with epidermal cells the most toxic particles increased the number of apoptotic cells by up to 90%, and this dropped to 1% with the most protective particles, which is similar to natural apoptosis in the absence of UVA.

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It is known that epithelial cells, when exposed to UV radiation, form pyrimidine dimers which are usually repaired, but when a great number of dimmers are formed the repair might not occur effectively, leading to DNA damage. If the DNA damage occurs at an important gene, such as the tumor suppressing genes, this damage could lead to skin cancer. The formation of pyrimidine dimers through an in vitro assay, using bladder cancer cell cultures and an in vivo assay using hairless mice, was evaluated. TiO2 anatase nanoparticles were suspended in buffer, applied to the cell cultures or mice and exposed to irradiation at 254 nm (UVC) and 360 nm (UVA). After UVC radiation an increase in the number of pyrimidine dimmers was verified, which was not observed with UVA radiation. On the other hand, with the application of TiO2 in cells, prior to UVC irradiation, the pyrimidine dimer formation was suppressed. The reduction in the dimer formation was proportional to the quantity of TiO2 applied. Evaluating the in vivo pyrimidine dimer formation after UVC irradiation, the authors observed that the presence of TiO2 reduced the number of dimers formed, as in the in vitro assay. UVA radiation also had no effect on dimer formation in the in vivo assay. This reduction in the dimmer formation indicates a protective property of TiO2 particles because these particles absorb radiation before it affects the cells, reducing the UV-induced damage. The formation of agglomerates reduces the UV light absorption decreasing the protective effect promoted by TiO2. Pyrimidine dimmer repair occurs 24 to 48 hours after the UV radiation exposure has ceased and the presence of TiO2 did not affect this repair, since it occurs at the cell nuclei and most likely the particles did not enter the cells [15]. Cellular damage evaluation using the colorimetric 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide assay, which evaluates the presence of intracellular dehydrogenases of viable living cells, leading to the formation of purple formazan crystals, is a commonly used technique. The cell viability decreases to around 60% after 30 min of UVA irradiation, in the absence of particles, and reduces to approximately 30% after 60 min. Anatase and rutile TiO2 nanoparticles coated with alumina and dimethicone, respectively, cause a slight but significant decrease in the cell viability before UV irradiation [24]. In contrast to the observations of Rampaul and co-workers (2007), there were no significant differences (toxic or protective) in the presence of any sample tested compared to the irradiated control, although the pure anatase sample was again found to be slightly more cytotoxic [21]. The photoreactivity of TiO2 also leads to the possibility of genotoxicity, as previously noted. Consequently, an assay for DNA damage can aggregate important data to the toxicity profile of a product. DNA damage can be indirectly evaluated by observing the DNA damage response of histone phosphorylation. In the presence of UVA irradiation and rutile doped with manganese, the histone phosphorylation was slightly elevated when compared to only UVA irradiation. However, the activation of this pathway with UVA exposure in the presence of anatase/rutile particles coated with organosilane is around five times greater than with UVA irradiation in the absence of particles [21]. Observing directly the DNA damage, using the comet assay, particles coated with alumina, uncoated or doped with manganese exhibited toxic effects even

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when not exposed to UVA light. Since, the manganese-doped particles did not decrease the cell viability under UVA irradiation it is possible that the manganesedoped particles cause DNA damage through a different pathway, which does not lead to cellular death. After UVA exposure the manganese-doped particles did not show any difference from the irradiated control, but the uncoated and anatase particles resulted in greater DNA damage than the control [21]. Since the toxicity of photoactivated TiO2 is associated with the generation of ROS, as seen earlier in this section, the production of ROS within human skin fibroblasts incubated in the presence of TiO2 particles with different coatings and exposed to UVA has also been studied. Uncoated particles and particles coated with organic materials (containing 80% anatase) were found to be more damaging to DNA than all of the rutile-coated particles, and they both induced a remarkably significant increase in intracellular ROS compared to the irradiated control. In the presence of the other TiO2 samples there was no significant change in ROS production, except for the manganese-doped particles for which a decrease in ROS production was observed [24]. In fact, manganese doping has been shown to lead to an increase in the UVA/UVB absorption ratio of titanium, a reduction in the free radical generation rates by over 90 %, and free radical scavenging behavior. Manganese-doped particles (50–100 nm) were compared to octylsilylated TiO2 (20–100 nm) and a 75 % anatase / 25 % rutile crystal structure, and a silica-alumina-coated 100 % rutile titania of similar size. Besides being less photoreactive than the other particles, manganese doping results in an increased absorbance in the UVA region of the UV spectrum and an absorbance tail in the visible region compared to the undoped material. This effect results in an increase in the wavelength of maximum absorption and the average UVA/UVB absorbance ratio of manganese-doped titania compared with other samples. The wavelength of maximum absorption of the doped material is 385 nm, compared with 383 nm for the silica-alumina-coated material and 380 nm for the octylsilylated material. The UVA/UVB average absorption ratio is 1.05 for manganese-doped titania, 0.78 for octylsilylated and 0.87 for silica-alumina-coated titania [29]. Most dopants (Al, Mo, Fe, among others) significantly enhance the generation of active species and DNA damage. With respect to undoped material, iron and chromium have little effect, while chromium, manganese and vanadium show significant benefits over undoped material [27]. Additionally, researchers have concluded that the use of the anatase crystalline form and uncoated TiO2 in sunscreens are the best options. Data suggest that TiO2 particles coated with alumina or doped with manganese are not cyto- or genotoxic to human skin cells [21, 24].

17.2.2 Skin Penetration In general, the toxic effect after exposure to the above-mentioned insoluble nanoparticles (ZnO and TiO2) occurs with an increase in ROS, causing an oxidative stress situation, which induces damage to the membrane, proteins and DNA with consequent cell death. To cause an increase in ROS at the viable cell level, the nanoparticles must penetrate and permeate the skin. Many studies have been

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carried out with the objective of establishing the degree of penetration of TiO2 and ZnO nanoparticles into the skin. Cutaneous penetration of ZnO nanoparticles, without coating and with a mean diameter of 80 nm, and of TiO2 in needle form (30-60 nm x 10 nm) coated with silica and methicone or only methicone, was evaluated using an in vitro technique with pig skin as the membrane. All nanoparticles were previously incorporated in oil in water (O/W) emulsions and the electron microscopy images of the emulsions indicated that the particles were distributed in the aqueous phase, either isolated or agglomerated. The elements Zn and Ti were analyzed by atomic spectroscopy in the different skin layers. Both the ZnO and TiO2 particles with coatings remained in the stratum corneum and did not penetrate into the deeper layers of the skin [30]. In the same context, a study evaluated the penetration of ZnO nanoparticles coated with silicone with sizes ranging from 26 to 30 nm (transmission electron microscopy, X-ray, dynamic light scattering, BET) using Franz diffusion cells and transmission electron microscopy (TEM). Zinc was quantified through inductively coupled plasma-mass spectrometry. A tendency toward larger concentrations of Zn2+ in the receptor chamber was verified, but the total absorbed was less than 0.03% of the amount applied, a value considered not statistically significant due to the amount of Zn normally present in the skin. When analyzed by TEM, it was observed that the ZnO nanoparticles remained at the external surface of the stratum corneum and no particles were detected in the lower layers of the stratum corneum or in the viable epidermis. Considering that the particles were not visualized in the deeper layers of the stratum corneum and that there is a tendency to observe higher levels of zinc in the receptor chamber, it is possible to conclude that some particles dissolved permitting the diffusion of elementary zinc through the membrane [31]. The penetration of TiO2 from a commercially available sunscreen product containing micronized TiO2 in a hydrophobic emulsion (Anthelios XL SPF 60, La Roche Posay, France) was tested on human skin xenografts. Intact human skin xenografts were treated in vivo with TiO2 emulsion for 24 h using an occlusive bandage. In this assay, TiO2 nanoparticles did not reach the ‘living’ cell layers, remaining in the stratum corneum in the human skin xenografts [22]. Even though many researchers have confirmed that ZnO and TiO2 nanoparticles do not penetrate the stratum corneum, this result is not conclusive. In similar assays some authors have detected TiO2 in the stratum granulosum (Figure 17.3) [32]. Nanosized TiO2 characterized by TEM and presenting lance-like shape (45–150 nm long and 17–35 nm wide) was studied in different formulations to evaluate skin penetration. Among other formulations, a commercially available product, Eucerin® Micropigment Cream 15 (5% TiO2 concentration) produced by the Beiersdorf Company, was evaluated. The formulation was applied on disinfected pig skin using allergy-testing plasters (1 cm2) and fixed with covering plasters. The different skin layers and their components were identified by element images and density distributions with spatially resolved ion beam analysis. The TiO2 present in the commercial formulation penetrated the stratum corneum and reached the stratum granulosum (the first layer of viable cells in the epidermis) in the first

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8 hours of exposure. Even after 48 hours, the particles did not penetrate to deeper layers [32]. In another in vivo study, using human foreskin grafts transplanted into mice, nanoparticles incorporated in an emulsion (Anthelios XL F60, La RochePosay, France) also reached the stratum granulosum [33]. TiO2 particles were found to be in contact with “live” cells, which may suffer cellular and DNA damage in this layer of the skin.

Fig. 17.3 Schematic representation of the skin penetration of nanosized particles according to research articles published in the last 5 years.

In another study, uncoated particles and dimethicone/methicone copolymercoated particles (5 % by weight) in a sunscreen formulation were topically applied on live minipigs. The epidermis of the minipigs treated with sunscreens containing TiO2 showed elevated titanium levels. Titanium levels and isolated TiO2 particles were also detected in the dermis of the abdominal and neck regions of the minipigs. However, there was no pattern of distribution suggesting that the particles present in the deeper layers of the skin could be caused by contamination during the tissue sectioning from the epidermis (location of application) to the dermis. A few isolated particles represent only a small fraction of the total amount of TiO2 applied and nanosized TiO2 in sunscreens do not show significant penetration into intact skin [34]. There is no evidence to date of ZnO or TiO2 particles from sunscreen lotions penetrating into layers deeper than the stratum granulosum. Based on the currently available scientific evidence, these particles do not reach dermal layers or the systemic circulation (see Chapter 1). The above findings clearly demonstrate that the

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first two layers of the epidermis function as an effective barrier against nanosized UV filters in intact skin.

17.2.3 Novel nanosized UV filters Besides reducing the photoreactivity, the coating of inorganic sunscreen particles is being explored as a novel strategy to enhance the SPF. The use of carnauba wax combined with decyl oleate in the form of solid lipid nanoparticles to encapsulate TiO2 has been proposed. These innovative particles enhanced the UV attenuation of the inorganic sunscreen, as demonstrated through an improvement in the intrinsic SPF of the substances. The improved UV protection is possibly due to an increase in the number of solid species in the nano dimension, and also the higher UV absorption due to the compounds present in the complex carnauba wax mixture. Furthermore, these new vehicles provide a positive effect on the viscosity of the systems giving the formulations a better fixation. Additionally, higher erythemal UVA protection factors were obtained after the crystals were covered with carnauba wax:decyl oleate or dispersed within wax:oil nanoparticles [35]. The use of organic materials as physical filters is also being explored, with a view to associating them with organic sunscreens in the future. Using the absorption properties to predict the blocking efficacy, one study verified that nanostructured lipid carriers blocked UV radiation to different extents. Carnauba wax nanostructured lipid carriers were found to be better UV blockers than glyceyl dibehenate (Compritol 888). This might be a result of the lipid crystallinity or of the absorptive properties of carnauba wax components [36]. The association of a polymer and TiO2 has also been suggested as a UV filter. TiO2-loaded nanocapsules with a chitosan shell were introduced as a synergetic combination of organic–inorganic hybrid spheres. Nanocapsules with an eicosapentaenoic acid core containing TiO2 were also produced. The main objective of this association was also to enhance the sun protection ability of TiO2. The irradiation wavelength absorption range was broadened by the incorporation of eicosapentaenoic acid into the nanocapsules. The nanocapsules exhibited a high UV protection rate of up to 95 % for both UVA and UVB, as did the uncoated particles [37].

17.3 Nanoencapsulated UV Filters Nanostructured carrier systems have been extensively studied for oral and parenteral routes of drug administration and they are also useful for the release of different drugs and active ingredients through the skin [38]. These systems modulate the transepidermal diffusion and can alter the pharmacokinetics and distribution through the skin [39]. Nanostructures have advantages that make them promising vehicles for cosmetic application. Some of these advantages are the protection of liable substances against chemical degradation, the possibility of controlling the release of the active components, the occlusive ability of the particles, and the potential synergic

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effect with chemical sunscreens increasing the SPF [40]. These carrier systems present a reduced size, which increases the contact between the active substance and the stratum corneum. Furthermore they facilitate the inclusion of active substances in dermatological products [2, 3]. To ensure their efficiency, sunscreens must penetrate the stratum corneum and remain predominantly in the upper layers promoting UV protection [41, 42]. The amount of sunscreen inside the stratum corneum can be directly associated with the SPF. Nanoparticles are being proposed as a topical vehicle, especially for sunscreens, with a view to prolonging the residence time of the sunscreens in the stratum corneum. The sunscreen efficacy requires that it adheres to the skin as a protecting film, presenting a high affinity with the stratum corneum. Therefore, the vehicle in which the active substance is incorporated ideally needs to dissolve the sunscreen and thus remain in contact with the skin [1, 6]. The many advantages of using nanocarriers have been demonstrated for different kinds of structures (Table 17.2 and 17.3). The verification that nanoencapsulation improves sunscreens, and the possible mechanisms involved, will be discussed in this section.

17.3.1 Skin Penetration Organic sunscreens must be retained in the upper part of the skin, the stratum corneum, where the chromophores absorb UV radiation and prevent skin damage. Nanotechnology has been applied with the purpose of maintaining the active molecules at the most efficient location. The region of the nanoparticle where the molecules are located is another important aspect, because this has a direct influence on the substance release profile. Substances need to be internally encapsulated, at least partially, to aggregate the benefits of skin retention brought by nanotechnology [1, 6, 51]. In vitro release experiments are useful to characterize this behavior. The release profile of nanocapsules containing OMC incorporated in a gel was compared to that of a formulation containing non-encapsulated OMC, showing similar behavior. This phenomenon can be explained by the highly hydrophobic nature of the polymer and OMC as well as the crystallinity of the polymer used [poly(ε-caprolactone)] which contributed to the reduction in the OMC diffusion to the receptor compartment. The low diffusion suggests that OMC is encapsulated and not adsorbed on the external surface of the nanocapsules [6]. On the other hand, the retention of OMC in the stratum corneum of pig skin, through in vitro tape stripping, demonstrated that the amount of the organic sunscreen retained in this layer was 3 to 4 times higher when nanocapsulated in poly(εcaprolactone) particles than the non encapsulated form. After 6 hours of experiment the sunscreen was not detected in the receptor compartment [4]. The influence of encapsulation on the in vitro percutaneous absorption of OMC was also studied, comparing the penetration of free and encapsulated OMC in O/W and W/O emulsions [1]. Corroborating data previously published, the nanocapsules containing OMC presented less penetration through the stratum corneum when compared to the emulsions containing the free active substance [4]. These

Poly(İ-caprolactone)

Poly(İ-caprolactone)

Polyvinyl alcohol and fatty acids Cellulose acetate phthalate Cellulose acetate phthalate Poly(İ-caprolactone)

Nanocapsules

Nanocapsules

Nanocapsules

Nanoparticles

Poly(İ-caprolactone)

Poly(İ-caprolactone)

Poly(lactic acid)

Nanocapsules

Nanocapsules

Nanocapsules

and

and

Poly-D,L-lactide-coglycolide Poly(İ-caprolactone)

Nanoparticles

Nanocapsules nanoemulsion Nanocapsules nanoemulsion Nanocapsules

Main component Poly(İ-caprolactone)

Nanoparticle type Nanocapsules

OMC

OMC

BZ3

OMC + quercetin

OMC

OMC

BZ3

OMC

OMC

trans-2-ethylhexyl-pmethoxy-cinnamate OMC

Encapsulated substance OMC

Reduce skin penetration of OMC and the encapsulated substance distribution in the skin. Evaluate the transdermal permeation of OMC and the retention of the substance in the different layers of the skin. Improve OMC photostability by nanoencapsulation with different surfactants, concentrations of polymer and organic phase volume. Evaluate percutaneous permeation of nanocapsules with different proportions of polyvinyl alcohol:fatty acids. Optimize the preparation of nanocapsules by emulsificationdiffusion and evaluate the in vivo distribution. Evaluate the in vivo penetration of OMC encapsulated in nanocapsules and nanoemulsion. Evaluate OMC and OMC/quercetin nanocapsules photostability. Increase photostability, in vitro efficacy and evaluate in vivo allergenic effect. Evaluate the distribution profile of nanoencapsulated OMC in the skin. Reduce skin penetration of OMC.

Objective Evaluate the in vitro release profile of the encapsulated sunscreen included in a hydrogel and reduce UV erythema induction (in vivo). Improve the organic sunscreen photostability.

Table 17.2 Characteristics of formulations containing polymer nanoencapsulated UV filters and their objective.

[49]

[48]

[9]

[5]

[47]

[46]

[45]

[44]

[1]

[4]

[43]

Reference [6]

348 C.B. Detoni et al.

BZ3

Cetylpalmitate

Stearic acid

Hydrogenated soya phosphatidylcholine and Tristearin

Solid lipid nanoparticles

Cetylpalmitate

BZ3

trans-2-ethylhexyl-p-methoxycinnamate Bisethylhexyloxyphenolmethoxyphenyltriazine OMC and diethyltoluamide

BZ3

Cetylpalmitate

Calixarene and solid lipid

Encapsulated substance Vitamin E

Main Component Cetylpalmitate

Solid lipid nanoparticles

Solid lipid nanoparticles Solid lipid nanoparticles

Nanoparticle Solid lipid nanoparticles Solid lipid nanoparticles Solid lipid nanoparticles

Evaluate the percutaneous absorption of coencapsulated OMC and diethyltoluamide (sunscreen and insect repellent). Evaluate the UV absorptive effects of the sunscreen and penetration of the model sunscreen BZ3 with and without carriers.

Promoting photochemical stability.

Objective Evaluate UV-blocking ability of solid lipid nanoparticles containing Vitamin E. Raise sunscreen skin retention evaluated by in vitro release and in vivo skin penetration. Confirm the synergic effect of SLN and a conventional chemical sunscreen through UVblocking ability. Photostabilization of chromophores.

Table 17.3 Characteristics of formulations containing lipid nanoencapsulated UV filters and their objective.

[54]

[53]

[8]

[52]

[40]

[51]

Reference [50]

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results highlight the importance of nanoencapsulation in terms of the cutaneous penetration of this sunscreen. The formation of a protective film on the skin contributes to the differentiated skin penetration. Even taking these findings into account, there is a question that remains unanswered. Does the sunscreen penetrate after being released from the nanoparticles or still encapsulated in nanoparticles? A confocal microscopy study showed the distribution of nanocapsules containing the stain Nile red in skin compared to the administration of the pure substance. A better distribution of the stain was observed when encapsulated in nanoparticles, but the authors affirm that the micrographs did not allow them to define whether the distributed Nile red was originally nanoencapsulated or free. In general, to date, studies have indicated that the particles are retained and that the substance penetrates after the release [4]. In order to determine the precise location in the skin of OMC released from polymeric nanocapsules incorporated in a hydroxyethylcellulose gel, a novel extraction technique has been proposed. Two protocols were used. In the first protocol OMC was extracted from the skin using a conventional organic solvent, acetonitrile, which dissolves the nanocapsule structure and OMC. In the second protocol OMC was extracted with isopropyl myristate, which dissolves the sunscreen but not the polymer. The amounts of OMC quantified in the lower layers of the skin using the two protocols were similar. On the other hand, on the cutaneous surface there was a significant difference between the amount of total (dissolved nanocapsules) and free OMC quantified, indicating that the nanocapsules accumulate on the surface of the skin and release the active substance which may penetrate. This effect was attributed to the particle size and the lipophilic nature of the polymer, poly(ε-caprolactone), both factors contributing to the retention of nanocapsules at the surface of the skin [48]. A similar study was performed with OMC encapsulated in nanoparticles of poly(lactic acid). Nanoencapsulated OMC remained mostly on the skin surface (80 %) and the quantities that reach the viable epidermis were three times lower than the unencapsulated substance incorporated in an emulgel. This result indicates a possible sustained release of the sunscreen from the nanoparticles [49]. Some researchers study how the design of the particles influences the percutaneous absorption of the encapsulated substance. The method of preparation of the nanocapsules and their skin penetration were evaluated. OMC nanocapsules were obtained through the emulsification-diffusion technique with diameters of between 400 and 615 nm and nanoemulsions of 160 nm. The skin penetration of these carriers was analyzed through in vivo tape stripping using a conventional nanoemulsion as the control. The incorporation of the organic sunscreen into the nanoemulsion enhanced its skin penetration when compared to OMC incorporated into nanocapsules or conventional emulsions. The increased skin penetration of OMC in nanoemulsions was related to their size and flexibility [46]. Another factor influencing the skin penetration is the chemical nature of the polymer used to formulate the nanoparticles. Different physical-chemical and functional properties can be obtained when changing the polymer [3]. In this context, Luppi and co-workers (2004) studied the permeation, using Franz cell diffusion, of nanoencapsulated BZ3 with the polymer polyvinyl alcohol combined with

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different fatty acids with two degrees of substitution (40 and 80 %). This study aimed to optimize a formulation that reduces the percutaneous absorption of BZ3 through the skin and into the systemic circulation. The nature and molar ratio influenced the particle size and the ability of BZ3 to interact with the skin. The nanoparticles obtained with the polyvinyl alcohol with a low degree of substitution (40 %) showed a higher amount of BZ3 located in the epidermis, while nanoparticles with a high degree of substitution seemed to prevent the percutaneous absorption of BZ3 [45]. Besides polymeric capsules, solid lipid nanoparticles may also enhance sunscreen retention on the skin surface. The release and percutaneous absorption of sunscreens encapsulated in solid lipid nanoparticles may also be controlled by changing the particle preparation method or components. On comparing the release mechanism of formulations containing BZ3 in the shell with that of formulations containing BZ3 in the core, a burst release was observed for solid lipid nanoparticles with BZ3 in the shell. For formulations with BZ3 in the core a sustained release was observed [51] and these formulations thus represent a system suitable for sunscreen encapsulation. However, both of these solid lipid nanocapsule models present lower BZ3 release rates when compared to conventional emulsions. The penetration was assayed by in vivo tape stripping. The penetration rate for the formulation containing BZ3 in the core was 1.7 % (solid lipid nanoparticles) while for the corresponding emulsion it was 3.4 %. Solid lipid nanoparticles promoted a 50 % decrease in the skin penetration. Thus, it was suggested that solid lipid nanoparticle formulations form a film on the skin upon water evaporation, as reported for polymeric nanocapsules [4, 51]. The in vitro permeation across the skin of BZ3-loaded solid lipid nanoparticles of hydrogenated soya phosphatidylcholine and tristearin showed that the solid lipid nanoparticles in cream exhibited fast permeation (4.79 %) in the first hour followed by a steady permeation with 23.6 % of the BZ3 permeated by the end of a 24 h period [54]. The presence of BZ3 on the surface of solid lipid nanoparticles was responsible for the fast permeation followed by the steady permeation, which might be due to diffusion of the drug from the core of the solid lipid nanoparticle. The retention of solid lipid nanoparticles in the skin layers has also been evaluated using a hydrophobic fluorescent probe, Rh 6G. The cream containing solid lipid nanoparticles shows bright fluorescence intensity in the stratum corneum. The unencapsulated Rh 6G shows bright fluorescence in the viable epidermis due to the formation of a uniform thin film by the nanoparticles, as suggested by many authors. The skin permeation of co-encapsulated OMC and an insect repellent, diethyltoluamide, in solid lipid nanoparticles was evaluated with the Franz diffusion cell assay using human stratum corneum and epidermis as the membrane. The flux (µg/cm2 per h) of OMC penetration in the solid lipid nanoparticle was much lower than that of the OMC in an emulsion also containing both active substances [53]. Inorganic materials may also serve as chemical sunscreen carriers. Sunscreens incorporated in the interlayer region of anionic clay particles have shown increased photostability and decreased release from the formulation. The authors intercalated 2-phenyl-1H-benzimidazole-5- sulfonic acid in MgAl and ZnAl hydrotalcite (HTlc).

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The percentage of free 2-phenyl-1H-benzimidazole-5-sulfonic acid released from the cream was more than 35 % after 15 min and reached 100 % after 5 hours. The results for both intercalated product formulations were 10 % after 15 min and 90 % after 8 hours [55]. A burst release followed by sustained release of the encapsulated sunscreen agent may be seen as a benefit. The fast release improves the initial saturation of the stratum corneum and promotes a faster onset of action, while a sustained release maintains a constant level of sunscreen at the skin surface [54]. The advantages of the organic sunscreen retention in the upper layers of the skin range from the reduction of undesired effects of potential phototoxic and photoallergic interactions and systemic effects to the enhancement of the protection efficacy. Nanotechnology has been used to add this characteristic to different organic sunscreens and has also shown the ability to alter the penetration and release profiles according to the design and material. The lower degree of penetration of organic sunscreens promoted by nanoencapsulation may be a result of the formation of a film on the surface of the skin. The fraction of sunscreen found in the deeper skin layers is probably not encapsulated in the nanocapsules, which remained at the surface of the skin [48].

17.3.2 Photostability To ensure a constant efficacy during the exposure period, organic sunscreens should not undergo chemical alterations when irradiated with solar light. The photostability of a sunscreen is related to the maintenance of the photoprotection capacity after the UV radiation is absorbed. Organic sunscreens are less stable than physical filters due to their mechanism of action, which is dependent on the absorption of UV radiation and conversion of the molecule from the fundamental state to its excited state. The excited molecule returns to its fundamental state, dissipating the absorbed energy through different mechanisms such as heat, light, fluorescence or the transfer of energy to another molecule. The photostability of a UV filter is strictly associated with its capacity to release absorbed energy without undergoing structural changes. If the excited molecule does not dissipate the absorbed energy it may undergo structural rearrangements or fragmentation [43, 56]. A reduction in the absorption capacity due to the instability of organic sunscreens results in an increase in the radiation which directly affects the skin [57, 58, 59]. According to some articles, the topical administration of nanocarriers containing organic sunscreens can offer advantages in comparison to the conventional products by enhancing the photostability of the active substance [5, 9, 8] Nanoparticles of poly-D,L-lactide-co-glycolide were used to improve trans-2ethylhexyl-p-methoxy-cinnamate photostability when exposed to UVA and UVB radiation for 4 hours. The photodegradation of the organic sunscreen was reduced when it was encapsulated by nanoparticles. The degree of degradation was 35.3 % for the nanoparticles and 52.3 % for the non encapsulated sunscreen [43]. The influence of nanoencapsulation on the photostability of sunscreens was also comparatively studied for polymeric nanocapsules of poly(ε-caprolactone) containing OMC, quercetin and different lipophilic surfactants in the oily core

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(sorbitan monosterate and phospholipids). As controls, a methanolic solution of OMC and a methanol/water suspension of OMC were also prepared. All samples were exposed to UVA radiation and the OMC was quantified at certain intervals. The capacity of the nanocarries, in specific nanocapsules, to partially protect the sunscreen from photodegradation was confirmed by the observed difference between the samples. After 12 days of exposure the nanocapsules presented a recovered amount of OMC of 17 to 35 % while the methanolic solution presented values below 1 % [5]. This photostabilizing property of the nanocarriers may be attributed to the scattering and reflecting properties of the particles. Hydrogels containing BZ3 in nanocapsules of poly(ε-caprolactone) present higher chemical stability than gels containing the free substance. After 13 hours of UVA exposure the formulation containing the nanoencapsulated BZ3 had a recovery of 56 % while only 29 % of the initial BZ3 concentration was recovered from the gel containing the free substance [9]. Lipid-based nanostructures also share the photostabilizing property with polymeric nanoparticles. Bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), a photochemically unstable sunscreen, has been encapsulated with cetyl palmitate in solid lipid nanoparticles and evaluated in terms of the stability of the free and encapsulated forms under UV radiation. In the photochemical stability study, after UV radiation the residual free BEMT content gradually decreased to around 19 % whereas the residual content of BEMT encapsulated in the solid lipid nanoparticle decreased to around 8 %, confirming the potential stabilizing ability of nanoencapsulation [8]. The association of a solid lipid nanoparticle with a hydrophilic molecule/chromophore complex also showed the photostabilization of chromophores. A calixarene and trans-2-ethylhexyl-4-methoxy-cinnamate complex was included in solid lipid nanoparticles. The combination of the components (calixarene and trans-2-ethylhexyl-4-methoxy-cinnamate) during the preparation of solid lipid nanoparticles controlled the photochemistry and eliminated the photodimerization. The calixarene/trans-2-ethylhexyl-4-methoxy-cinnamate crystal and the calixarene/trans-2-ethylhexyl-4-methoxy-cinnamate-loaded solid lipid nanoparticles were exposed to UV radiation and analyzed, being 40 % cis isomer and 60 % trans isomer, with no dimers being detected. However, on increasing the trans-2ethylhexyl-4-methoxy-cinnamate concentration dimers were detected, confirming that at lower concentrations the trans-2-ethylhexyl-4-methoxy-cinnamate is isolated and the reaction between molecules is prevented [52]. The association of chomophores with ZnAl and MgAl can also lead to an improvement in the photostability. The absorption of the free sunscreen decreases faster when exposed to 297 nm radiation than both of the intercalated compounds using fluorescence as the analytical method. After 2 hours of irradiation, the fluorescence of the ZnAl–sunscreen system reduces to around 15 % while that of the MgAl–sunscreen system reduces to around 22 %. These results indicate that the intercalated compounds are more photostable than the free sunscreen and that the ZnAl–HTlc matrix offers a more efficient protection of the chromophore [55].

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Another successful organic–inorganic ultraviolet active hybrid material association was prepared using a sol-gel process, BZ3 derivatives and tetraethylorthosilicate. Samples with BZ3 and the derivative maintained 79 and 80 % of their UV absorption ability, respectively, after irradiation over the entire UV spectrum. When this derivative is located on the surface of the particles, the UV filter photostability increases and 89 to 93 % of the UV absorption ability, respectively, is maintained after irradiation [60]. The capacity of nanoparticles to photostabilize chomophores may be a result of the isolation of the molecule from contact with other reactive molecules (including those of the chromophore) in the formulation or of the reflective and scattering properties of the particles, which prevent radiation from affecting the molecule. Probably, the combination of the two mechanisms of chemical stabilization leads to the effect observed in research studies.

17.3.3 UV-Blocking Ability Some assays evaluate directly the efficacy of the association of an organic sunscreen with nanoparticles through the UV absorption of the formulation or even UV-induced erythema inhibition. Gels containing OMC-loaded polymeric nanoparticles showed a better protection against UV irradiation than gels containing the unencapsulated sunscreen. When comparing the protection against UV radiation-induced erythema, the formulation containing nanocapsules demonstrated a significantly higher protection ability than the non-encapsulated OMC gel. The better efficacy of the encapsulated OMC indicates that the sunscreen adhered to the skin, forming a protective film [6]. The in vitro sun blocking efficacy (Sun-to-See®-assay) of tocopherol acetateloaded solid lipid nanoparticles has also been evaluated. Solid lipid nanoparticles absorbed 100 % more than the reference emulsion. Tocopherol acetate-loaded solid lipid nanoparticles presented better UV-blocking properties than empty solid lipid nanoparticles and a tocopherol acetate-containing emulsion. Confirming the blocking properties of solid lipid nanoparticle, the absorption of the placebo solid lipid nanoparticle formulation exceeds even that of the tocopherol acetatecontaining emulsion. The incorporation of tocopherol acetate as a chromophore into both formulations leads to an increase in absorption [50]. The authors continued their investigation confirming the synergic effect of solid lipid nanoparticles and a conventional organic sunscreen [40] (Figure 17.4). Again, the solid lipid nanoparticle formulations were distinctly more effective than the corresponding emulsions. The concentration of the molecular sunscreen (BZ3) can be decreased by 50 % while maintaining the absorption level. Similarly to solid lipid nanoparticles, the UV filter-loaded polymeric nanocapsules also scatter and absorb UV light. On analyzing the UV absorption spectrum of free and encapsulated BZ3 incorporated into a hydrogel it is possible to observe the synergism [9].

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Fig. 17.4 Diagram showing the UV blocking mechanism (scattering + absorbing) obtained with nanoencapsulated UV filters.

The in vivo SPF is the most reliable assay for determining the inhibition of UVB radiation-induced erythema. Albino rats have been used in the SPF assay to show that a cream base containing unloaded solid lipid nanoparticles presented better protection against UV radiation when compared to the pure cream base because, as previously reported, solid lipid nanoparticles can act as a physical barrier to UV radiation. The SPF obtained for solid lipid nanoparticles in a cream base formulation was found to be three times higher when compared with the cream base containing a similar amount of BZ3. This may be due to the synergistic effect of solid lipid nanoparticles which also act as physical sunscreens [54]. In conclusion, nanoencapsulated sunscreens have higher SPF values than the free sunscreen and the unloaded polymeric and lipid nanocarriers. This confirms a synergic effect of the chemical and physical mechanisms of UV protection.

17.4 Nanoparticles in Sunscreens Today: The Commercial Arena Nanotechnology is already a common feature in the sunscreen market, mostly related to TiO2 and ZnO nanosized particles, and to a lesser extent nanoencapsulated substances. It is difficult to find and analyze the nano-products currently on the Table 17.4 TiO2 nanoparticles available on the market.

Product Eusolex® T-2000 Eusolex® T-AVO Eusolex® T-S Eusolex® T-ECO Eusolex® T Eusolex® T-AQUA Eusolex® T-Oleo

Nanostructure used Ultra-fine rutile-base TiO2 with amphiphilic surface. Ultra-fine rutile-base TiO2 coated with silicon dioxide. Ultra-fine TiO2 coated with stearic-acid. Simethicone-coated ultra-fine TiO2. Ultra-fine TiO2 (anatase modification). Ultra-fine TiO2 (rutile modification). Silica-coated TiO2.

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Table 17.5 Commercially available products containing nanoparticles with sun blocking properties. Product Red Vine Year Round Hair Sun Protection Lancome Soleil Soft-Touch Moisturising Sun Lotion Revlon Skinlights Instant Skin Brightener Lotion All Day Suncare

SPF -

Nanostructure used Nanoparticles

15

Vitamin nanocapsules

15

Micronized topaz powder, micronized rose quartz powder Patented nanotechnology Micronized zinc oxide Micronized ZnO (ZCOTE®) Micronized zinc oxide Microfine TiO2-doped manganese (OPTISOL™) Micronized ZnO (ZCOTE®) and micronized TiO2 (TCote®) Micronized ZnO (ZCOTE®) Micronized ZnO (ZCOTE®) Micronized ZnO (ZCOTE®) Micronized ZnO (ZinClear™) Micronized ZnO (ZCOTE®) Nanoparticle TiO2 surface-treated Micronized zinc and micronized TiO2 Nanocapsules containing avobenzone and octocrylene (NANOPHOTON®)

15 and 30

BioMedic Pigment Shield Dermatone

18 20

Powder sunscreen Optisol™ Sun Defence

20 25

Sensitive Skin Sunscreen

25

Advanced Protection

30

Q-SunShade™

30

Blue Lizard® Regular

30

Rosacea Care Sunscreen “30” Sheer physical UV defense

30 50

SunVex® IM50

50

Cotz

58

Photoprot

100

Company Responsible Korres Natural Products Ltd. L'Oreal

Revlon

Kara Vita La Roche-Posay Dermatone Innovative skincare Oxonica® Ltd.

Applied Therapeutics™

Skin Rx Solutions International Cosmeceuticals, Inc. Crown Laboratories, Inc. Rosacea Care SkinCeuticals® SunVex® Fallene BIOLAB Sanus Farmacêutica

market, because until 2010 there was no mandatory nano-product labeling. However, some inventories, surveys and studies confirm that such products are available in great numbers. In March 2006 the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for Scholars displayed publicly the first searchable

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inventory of consumer products based on nanotechnology, finding over thirty (30) nano-sunscreens on store shelves of the United States and the United Kingdom by the year 2010 [61, 62]. Inorganic nanoparticles, such as TiO2 and ZnO, are the most common products of nanotechnology present in the market. Most companies acquire these nanosized particles as raw materials. As an example, pharmaceutical companies are responsible for many forms of nanosized TiO2 available as raw materials for sunscreen formulations (Table 17.4). The Australian Therapeutic Goods Administration (TGA) concluded in February 2006 that there are approximately 400 nano-sunscreen products containing manufactured nanoparticles of ZnO or TiO2 currently available on the market. Furthermore, 70 % of TiO2 sunscreens and 30 % of zinc sunscreens contained these ingredients in the nanometric form. In May 2006 the organization Friends of the Earth released a report: “Nanomaterials, sunscreens and cosmetics: Small ingredients, big risks" where 22 commercial sunscreen lotions produced by companies such as Christian Dior and Boots are listed [63]. In the United States, a non-profit organization, Environmental Working Group (EWG), also released an inventory of consumer products involving nanotechnology, including 27 sunscreens [64]. Some examples of commercially available products are listed in Table 17.5.

17.5 Conclusions Nanosized metal oxides and nanoencapsulated UV filters have brought many technological advantages to sunscreen lotions. Nanosized particles are widely used to broaden the protection spectrum and increase the SPF by reflecting, scattering and absorbing UV radiation. These metal oxide particles are mainly present as TiO2 and ZnO, which have been components of commercial sunscreen lotions since 1990. Since these particles are known for their photoreactivity reasonable concern is raised by the consumers, manufacturers and the scientific community. The photoreactivity of these particles is potentially a cause of cytotoxic effects in human skin cells (e.g. fibroblasts, epithelial cells). The penetration of these particles into the skin will, therefore, determine their toxicity. It is very important that these particles do not reach the deeper layers of the skin where their cytotoxicity can lead to damage in viable cells. However, several skin penetration assays have shown that TiO2 and ZnO particles penetrate at most to the stratum spinosum of the epidermis. Nanoencapsulation of traditional organic UV filters is a more recent technological approach to improving the skin retention, photostability and UV blocking ability of the free molecules. Nanoencapsulation enhances the retention of different organic sunscreens in the upper layers of the skin and alters the penetration and release profiles of the active molecule according to the design and material of the particle. The lower penetration of organic sunscreens promoted by nanoencapsulation may be a result of the formation of a film on the surface of the skin. The photostabilization of active molecules presented by nanoencapsulation may be a result of the isolation of the molecule from contact with other reactive molecules (including other identical molecules) in the formulation and of the reflective and

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scattering properties of the particles, which prevents radiation from affecting the molecule. In addition, the nanoencapsulation of chromophores leads to a synergic effect of the chemical and physical mechanisms of UV protection. All of these technological advantages brought by nanosized and nanoencapsulated sun protection formulations have made them important in the commercial arena, and these particles are currently commercialized as raw materials and are present in many final products in various countries. Inventories of commercially available sunscreen products containing nanoparticles have been published by non-governmental organizations and the number of products is close to 27-30 sunscreens.

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[12] Murphy, G.M.: Sunblocks: Mechanisms of action. Photodermatol. Photoimmunol. Photomed. 15, 34–36 (1999) [13] Serpone, N., Dondi, D., Albini, A.: Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products. Inorganica. Chim. Acta. 360(3), 794– 802 (2007) [14] Dechsakulthorn, F., Hayes, A., Bakand, S., Joen, L., Winder, C.: In vitro cytotoxicity assessment of selected nanoparticles using human skin fibroblasts. AATEX 14, 397– 400 (2007) [15] Kubota, Y., Niwa, C., Ohnuma, T., Ohko, Y., Tatsuma, T., Mori, T., Fujishima, A.: Protective effect of TiO2 particles on UV light induced pyrimidine dimer formation. J. Photochem. Photobiol. A Chem. 141, 225–230 (2001) [16] Uchino, T., Tokunaga, H., Ando, M., Utsumi, H.: Quantitative determination of OH radical generation and its cytotoxicity induced by TiO2-UVA treatment. Toxicol. In Vitro 16, 629–635 (2002) [17] Sayes, C.M., Wahi, R., Kurian, P.A., Liu, Y., West, J.L., Ausman, K.D., Warheit, D.B., Colvin, V.L.: Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol. Sci. 92(1), 174–185 (2006) [18] Sharma, V., Shukla, R., Saxena, N., Parmar, D., Das, M., Dhawan, A.: DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicol. Lett. 185, 211–218 (2009) [19] Liang, Y., Gan, S., Chambers, S.A., Altman, E.I.: Surface structure of anatase TiO2(001): Reconstruction, atomic steps, and domains. Phys. Rev. B 63, 235402(1)– 235402(7) (2001) [20] Matthews, A.: The crystallization of anatase and rutile from amorphous titanium dioxide under hydrothermal conditions. Am. Mineral. 61, 419–424 (1976) [21] Rampaul, A., Parkin, I.P., Cramer, L.P.: Damaging and protective properties of inorganic components of sunscreens applied to cultured human skin cells. J. Photochem. Photobiol. A Chem. 191, 138–148 (2007) [22] Kiss, B., Bíró, T., Czifra, G., Tóth, B.I., Kértesz, Z., Szikszai, Z., Kiss, Á.Z., Juhász, I., Zouboulis, C.C., Hunyadi, J.: Investigation of micronized titanium dioxide penetration in human skin xenografts and its effect on cellular functions of human skinderived cells. Exp. Dermatol. 17, 659–667 (2008) [23] Yang, H., Liu, C., Yang, D., Zhang, H., Xi, Z.: Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. J. Appl. Toxicol. 29, 69–78 (2009) [24] Tiano, L., Armeni, T., Venditti, E., Barucca, G., Mincarelli, L., Damiani, E.: Modified TiO2 particles differentially affect human skin fibroblasts exposed to UVA light. Free Radical. Bio. Med. 49, 408–415 (2010) [25] Schilling, K., Bradford, B., Castelli, D., Dufour, E., Nash, F., Pape, W., Schulte, S., Tooley, I., van den Boschi, J., Schellauf, F.: Human safety review of “nano” titanium dioxide and zinc oxide. Photochem. Photobiol. Sci. 9, 495–509 (2010) [26] Migdal, C., Rahal, R., Rubod, A., Callejon, S., Colomb, E., Atrux-Tallau, N., Haftek, M., Vincent, C., Serres, M., Daniele, S.: Internalisation of hybrid titanium dioxide/para-aminobenzoic acid nanoparticles in human dendritic cells did not induce toxicity and changes in their functions. Toxicol. Lett. (2010), doi:10.1016/j.toxlet.2010.07.017

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About the Editors

Adriana Raffin Pohlmann teaches Organic Chemistry at the Federal University of Rio Grande do Sul (UFRGS), Brazil, since 1990, and nowadays she is Associate Professor II at the Department of Organic Chemistry in the Institute of Chemistry (UFRGS). She received her PhD in Therapeutic Chemistry at the University of Paris V, France, in 1997. In 1998, she received the Roussel-Uclaf award for her Dissertation and a Laureate Diploma from the College of Pharmaceutical and Biological Sciences, University of Paris V, France. She was Head of the Department of Organic Chemistry (1999-2001), First Director of the Center of Nanoscience and Nanotechnology at UFRGS (2006), member of the Committee of the Postgraduate Program in Chemistry (2001-2003; 2009-2011) and Vice-Dean of the Institute of Chemistry (20032007). Her main research is focused on the organic chemistry applied to drug nanocarriers, including polymeric nanocapsules and nanospheres, with the view of understanding and controlling their sizes, shape, surface and physico-chemical properties. She advises Graduate and Post-Graduate students in Chemistry and Pharmaceutical Sciences. She is a National Council for Scientific and Technological Development (CNPq/Brazil) researcher developing works as leader of the group: Micro- and nanoparticles for therapeutics. She was the Vice-Director of one of the Brazilian National Nanotechnology Network (Nanocosmetics Network), and Coordinator of a collaborative IBSA project between India, Brazil and South Africa both supported by the Brazilian Ministry of Science and Technology. She has published more than 100 research papers, 1 book and 5 book chapters, and 4 patents. She is member of the Editorial Board of the Journal of Biomedical Nanotechnology. She is Ad-hoc reviewer for more than 20 Scientific Journals and for National and State Agencies to support scientific research. She is a member of the Brazilian Association of Pharmaceutical Scientists (ABCF) and the Brazilian Chemical Society (SBQ). Sílvia Stanisçuaski Guterres is a Professor of Pharmaceutical Technology in the College of Pharmacy at the Federal University of Rio Grande do Sul, Brazil, since 1989. She received her PhD in Pharmaceutical Nanotechnology from the University of Paris XI, France, in 1995 and nowadays she is an Associate II Professor, teaching pharmaceutical technology and cosmetology for undergraduate pharmacy students at College of Pharmacy and graduate students at the Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF/UFRGS – UFRGS Pharmaceutical Sciences Graduate Program). She

364

About the Editors

have advising more than 35 graduate students (master and Ph.D. levels) from 1997 to 2010. She is a National Council for Scientific and Technological Development (CNPq/Brazil) researcher and leader of the research group entitled Nanostrucutured Systems for Drug Administration. Her main research is focused on the development, physicochemical characterization and biological applications of innovative nanocarriers aiming at drug delivery via oral, cutaneous and parenteral routes. She is the Director of one of the Brazilian National Nanotechnology Network (Nanocosmetics Network), supported by the Brazilian Ministry of Science and Technology. She is the Coordinator of a collaborative project between France (Université de Paris-Sud, Professor Elias Fattal) and Brazil (UFRGS) for Nanotechnolgy (CAPES/COFECUB 540-2005) supported by the Brazilian Ministry of Education. She has published more than 100 research papers and 6 book chapters. She is a member of the Brazilian Chemical Society (SBQ), the Center of Nanoscience and Nanotechnology (CNANO/UFRGS) and the Brazilian Association of Pharmaceutical Sciences (ABCF). She is member of the Editorial Board of the Journal of Drug Science and Technology. Ruy Carlos Ruver Beck is a Professor of Pharmaceutical Technology in the College of Pharmacy at the Federal University of Rio Grande do Sul, Brazil, since 2010. He received his PhD in Pharmaceutical Sciences from the Federal University of Rio Grande do Sul, Brazil, in 2005, with a collaborative period in the Saarland University, Germany (Departament of Biopharmacy and Pharmaceutical Technology). From 2005 to 2010 he was a Adjunt Professor II at the Federal University of Santa Maria, Brazil. Nowadays, he is an Adjunt III Professor, teaching pharmaceutical technology and homeophaty for undergraduate pharmacy students at College of Pharmacy and graduate students at the Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF/UFRGS – UFRGS Pharmaceutical Sciences Graduate Program). He is a researcher of the research group entitled Nanostrucutured Systems for Drug Administration. His main research is focused on the development, physicochemical characterization and biological applications of innovative nanocarriers aiming at drug delivery via oral, ocular, cutaneous and parenteral routes. He is a member of one of the Brazilian National Nanotechnology Network (Nanocosmetics Network), supported by the Brazilian Ministry of Science and Technology. He has published more than 30 research papers. He is a member of the Center of Nanoscience and Nanotechnology (CNANO/UFRGS) and the Brazilian Association of Pharmaceutical Sciences (ABCF). He is Ad-hoc reviewer for more than 15 Scientific Journals and for State Agencies to support scientific research.

Subject Index

acne, 290, 297 aging, 253, 256, 264 alumina, 245 and Scanning Transmission Ion Microscopy, 8 antibiotics, 326 anticancer, 326 antileishmanial, 181, 183–186, 192, 193 antioxidants, 212, 214, 215, 218, 328 antitumour, 253 apoptosis, 199–202, 204, 206–209, 212, 214, 218, 229–236, 253, 255, 259, 289 atomic force microscopy, 57 backscattering analysis, 81 basal cell carcinoma, 205 biodegradable, 260, 264 biofilm, 257–258 cancer, 229–230, 232–236, 242, 257, 263, 269–271, 273–276, 279–281 carbon nanotubes, 24, 218, 243, 245 Carbopol, 41–42, 61 carcinoma, 287, 290–291, 293–294, 296–297 caspases, 229–231 cationic nanocapsules, 52 cationic polymers, 164 chalcone, 181, 185–186, 188, 191–193 chemical enhancers, 11, 141 chitosan, 80–81, 163–174, 233 chitosan nanoparticles, 262 Cholesterol, 149 chomophores, 204, 290, 353, 354

clinical trials, 272 cold homogenization, 75, 108 comet assay, 342 Confocal Laser Scanning Microscopy, 8 confocal microscopy, 339, 350 controlled release, 3 core-shell, 115 cosmetics, 9, 11, 21, 25, 50, 85–86, 89, 92, 101–103, 107, 117–119, 124, 137, 139, 141, 143, 163–164, 170–171, 209, 214–215, 218, 229, 311, 326–328, 335, 357 cream, 184, 186, 191, 351–352, 355 cytokines, 202, 204, 211–212 cytotoxic, 255–256, 262, 270, 273, 279, 290 cytotoxicity, 229, 234–236, 289, 291, 297, 333, 335, 337, 339–340, 357 deformable, 187, 191 Deformable liposomes, 143 depreciation, 128–129 dermatological, 253, 256–257, 259–264 dermatology, 292 dermis, 3–4, 6–7, 9, 16, 22, 25, 103 dialysis bag, 82 differential scanning calorimetry, 76, 112, 174 DMAE, 214, 216, 217 DNA damage, 199–202, 204–206, 208–209 dried nanoparticles, 124 drug delivery, 315–316, 326, 328

366 drug delivery systems, 269–270, 272, 275 dynamic light scattering, 81, 109, 188, 338, 344 elastic liposomes, 185, 192 elasticity, 139, 141, 143, 146–151, 153–154, 157, 186, 188–191, 193 electron microscopy, 55–57, 62, 77, 81, 84, 113, 114, 189, 318, 326, 338, 344 electroporation, 141, 277, 279 electrospinning, 311, 313–316, 326, 328 electrospun, 314–315, 317–318, 327–328 eletroporation, 269, 275 emulgel, 350 emulsification-solvent diffusion, 77, 79 emulsification-solvent evaporation, 77–79 encapsulation efficiency, 75–76, 79, 81, 87 enhancers, 275–276, 278 environment, 229–231, 233 epidermis, 3, 103 erythema, 354–355 ethosomes, 145, 150, 214, 218, 276–277, 287, 295, 297 fibroblasts, 255, 257–258, 260–261, 264, 333, 339–340, 343, 357 fixed costs, 128, 136 flexosomes, 298 flowsheets, 133 flow-through cell, 6 Franz, 3 Franz diffusion cell, 6 fullerenes, 8, 21, 24–25, 247 full-thickness skin, 9 functionalization, 241 gelling process, 79 gels, 170–173 genotoxicity, 335, 337, 339–340, 342 glutathione peroxidase, 215 gold nanoparticles, 24, 274

Subject Index hair follicles, 4, 10, 12, 21, 140 healthcare, 269 heat-separated epidermis, 9 high pressure homogenization, 71, 85, 107–108, 115, 119 Horizontal diffusion cells, 6 hot homogenization, 74–75, 108–109, 116 Human skin, 9 Hyaluronic acid, 216 hydrodynamic radius, 56 hydrogels, 40–41, 259–260, 262 hypersensitivity, 203 immunosuppression, 197–199, 203–204 in situ polymerization, 53 In vitro diffusion models, 5 in vitro models, 3–4 in vitro-in vivo studies, 10 in vivo screening, 241 industrial application, 124 industrial process, 136 industrial production, 123–124, 137 inflammation, 197–199, 202, 204, 211–213 inorganic nanoparticles, 11, 21–22, 25 inorganic sunscreen, 346 interfacial deposition, 54 interfacial precipitation, 53–54 invasomes, 277, 287, 295, 298 investment, 128–129, 131, 133, 136–137 iontophoresis, 141, 269, 275, 277–278, 297 iron oxide nanoparticles, 24 keratinocytes, 255, 257–258, 262–263, 294 lab scale process, 124 leishmaniasis, 181–183, 186, 192, 256 leishmanicidal, 256–257 licochalcone, 184, 186 Light scattering, 55, 62 lipid nanoparticles, 101–105, 107–109, 114–119, 275 lipid particles, 106, 108, 116 lipid peroxidation, 234 Lipid vesicles, 187

Subject Index lipid-core nanocapsules, 51, 124 liposomal, 183, 185, 189, 192 liposomes, 11, 16, 21, 42, 101, 117, 139, 141–147, 149–157, 163, 165– 173, 181, 183, 185–187, 191, 214, 216, 270–271, 273–274, 276–277, 280, 287, 295, 297, 302 liquid crystals, 172–173, 287, 295, 299 liquid-crystalline phase, 146 loading capacity, 51, 104–107, 114 magnetic nanoparticles, 287, 295, 301–302 mammalian, 240, 242 mechanical properties, 318 melanin, 202, 208 melanocytes, 255, 262–263 melanogenesis, 202, 217, 256, 263 melanoma, 205–206, 208, 232–233 Membranes, 9 Metallic nanoparticles, 83, 89 metalloproteinases, 258 micelles, 11 microdialysis, 259, 261 microemulsion, 71–72, 85, 107 microneedles, 279–280 microparticles, 335 monoolein, 291, 299–300 morphology, 315–318, 326 mucosal leishmaniasis, 182 multiple light scattering, 111 nanocapsule surface, 52–53, 58 nanocapsules, 12, 37, 40, 42, 49–53, 55, 57–58, 61–62, 70, 78, 80, 82, 87–89, 333–334, 346–347, 350–354, 356 nanoemulsions, 16, 42, 50, 53, 276, 287, 295–296, 350 nanoencapsulation, 334, 347, 350, 352–353, 357 nanofibers, 311–319, 326–329 nanomaterials, 218–219, 229, 233, 235 nanomedicine, 229, 236 nanoparticles, 217–218, 311 nanopharmaceutical, 243 nanoprecipitation, 77–79, 85, 123–125 nanoshells, 274 Nanosized materials, 21 nanosomes, 214, 217

367 nanospheres, 12, 37, 40, 42, 70, 78, 80, 82, 87–88 Nanostructured carriers, 69 nanostructured lipid carriers, 16, 40, 41, 101, 103, 106, 346 nanotoxicology, 240 nanotubes, 339

necrosis, 229–232, 234–235 neurotoxicity, 292 Newtonian, 38 Nitric oxide, 253 non-Newtonian, 38 octanol-water, 292 organic sunscreens, 334, 352, 357 organogels, 165 Oxidative stress, 246 p53, 198–201, 206–208 particle size, 21, 103, 109–111, 114, 119 pegylated, 181, 187–188, 190, 192–193 penetration, 3–5, 7–12, 16, 21–22, 24–25, 42, 82, 85, 87, 90, 287–288, 291–292, 295, 297–300, 302 permeation, 3, 69, 81–82, 85–88, 139, 141, 143–145, 150–157, 181, 185, 191–193 pharmaceuticals, 241 pharmacokinetics, 346 phase transition, 146, 166, 169 phosphatidylcholine, 165, 167–169, 172, 174, 297 phospholipid vesicles, 166 phospholipids, 276–277 photoaging, 198, 204–205, 211–212, 214–215, 217–219 photocarcinogenesis, 199 photochemistry, 289 photodamage, 197–199, 201, 203–204, 214, 217–218 photodegradation, 352–353 photodynamic, 287, 289–291, 293–294, 298, 302 Photodynamic therapy, 287–288 photo-irritation factor, 211 photoprotection, 334, 352 photoreactivity, 333, 340–342, 346, 357

368 photosensitizers, 287–288, 294–295, 299 photostability, 333–334, 351–354, 357 phototoxic, 293 phototoxicity, 197, 209–210, 294 pig skin, 10 Pluronic, 259 polyelectrolytes, 163, 171 polyesters, 316

polyethyleneglycol, 149 Polymer–drug conjugates, 273 polymeric nanoparticles, 12, 16, 21, 70, 73, 77, 80, 85–87, 89, 92, 101, 123, 271, 274, 295 polymeric wall, 49–54, 57 porphyrin, 291–292, 296 potentiometry, 58 preformed polymers, 77, 80 Proteases, 212 psoriasis, 257, 290, 296 purchase price, 126–127, 131 quantum dots, 243, 274 reactive oxygen species, 337, 340 regenerated human skin, 10 rheological, 153–155, 157 Rheological analysis, 37 rheological properties, 37, 40, 42 Saarbrücken diffusion model, 7 SAXS, 82 Scanning Electron Microscopy, 8 semisolids, 37, 40, 42, 44 silica nanoparticles, 258 silver nanoparticles, 24, 70, 83–85, 89, 91, 229–230, 234–236 skin cancer, 287–291, 293–295, 299, 302 skin diffusion models, 152 small angle X-rays scattering, 164–165 solid lipid nanoparticles, 11, 16, 21, 40–41, 70, 75–76, 101, 103, 333, 346, 351, 353–355 sonophoresis, 141, 278 sorbitan monostearate, 51

Subject Index split-thickness skin, 9 squamous cell carcinoma, 294, 296 stability, 261–262, 291, 296, 298, 300 stabilizer, 163–164, 174 stratum corneum, 4, 103, 246, 287, 292, 294–295, 297 sunscreens, 217–218, 328, 333–335, 337, 343, 345–347, 351–352, 355, 357–358 superoxide dismutase, 338–339 surfactant, 51–52, 54, 62 sustained release, 350–352 synthetic polymers, 315–316 systemic effects, 352 tape stripping, 7, 9, 21 thixotropy, 39 titanium dioxide, 22, 245, 333 topical application, 58 toxicity, 240–241, 243–248 transdermal, 139–140, 143–144 transdermal delivery, 269, 277, 279–280 transepidermal water loss, 294 transfersomes, 144–145, 214 Transmission Electron Microscopy, 8 tumour necrosis factor, 255 ultradeformable liposomes, 191 UV radiation, 197–199, 203–207, 209, 211–214, 218–219, 255, 262 UVA, 197, 202, 205, 209–212, 215 UVB, 197–198, 201–205, 212, 214 UV-blocking, 354 UVC, 197 visceral leishmaniasis, 181 viscosity, 37, 51, 58, 60, 316–318 wounds, 253, 256–263 x-ray diffraction, 76, 112 zebrafish, 86, 239–248 zeolites, 258, 261 zeta potential, 57, 62, 81, 84, 110–111 zinc oxide, 21–22, 245, 333, 356

Author Index

Alves, Marta P.

37

Badra Bentley, Maria Vit´ oria L. Beck, Ruy C.R. 3, 49, 333 Bentley, Maria Vit´ oria L. Badra Carollo, Aline R.H. 287 Cerize, Natalia N.P. 311 Contri, Renata V. 3 da Silva, Rodrigo A. 197 da Silva Melo, Patricia 229 da Silveira, N´ adya P. 163 de Oliveira, Adriano M. 311 de Souza, Marina C. 269 Detoni, C´ assia B. 333 Dressler, Aline C. 163 Dur´ an, Nelson 69, 229 Fagan, Solange B. 37 Falc˜ ao, Camila A.B. 181 Ferreira, Carmen V. 197, 239 Fiel, Luana A. 3 Guimar˜ aes, Kleber L. 101 Guterres, S´ılvia S. 3, 49, 333 Justo, Giselle Z. Lionzo, Maria I.Z.

181 287

Machado, Daisy 197 Marcato, Priscyla D. 69, 229 Marchetti, Juliana M. 269 Marotta-Oliveira, Samantha S. Medina, Wanessa S.G. 287 Mertins, Omar 163

Paese, Karina 333 Pohlmann, Adriana R. 3, 49, 163, 333 Poletto, Fernanda S. 49 Pra¸ca, Fab´ıola S.G. 287 Raﬃn, Renata P. 37 R´e, Maria Inˆes 101 Rossi-Bergmann, Bartira

181

Santana, Maria Helena Andrade 181 Sartori-da-Silva, Maria A. 239 Seabra, Amedea B. 253 Shishido, S´ılvia M. 197 Teixeira, Zaine 69 Trierweiler, Jorge O. 123 Trierweiler, Luciane F. 123

197, 239 163

269

Zanchetta, Beatriz 139, 181 Zanin, Maria Helena A. 311

139,