Hydrogels for medical and related applications

Hydrogels for Medical and Related Applications In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symp...

Author: Joseph D. Andrade

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Hydrogels for Medical and Related Applications

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Hydrogels for Medical and Related Applications Joseph D . Andrade, EDITOR University of Utah

A symposiu by the Division of Polymer Chemistry, Inc. at the 170th Meeting of the American Chemical Society, Chicago Ill., August 27-28, 1975

ACS

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C.

1976

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

31

Library of Congress

Data

Hydrogels for medical and related applications. (ACS symposium series; 31 ISSN 0097-6156) Includes bibliographical references and index. 1. Colloids in medicine—Congresses. 2. Colloids— Congresses. I. Andrade, Joseph D., 1941II. American Chemical Society. Division of Polymer Chemistry. III. Title. IV. Series: American Chemical Society. ACS symposium series; 31. R857.C66H9 ISBN 0-8412-0338-5

610'.28

76-28170 ACSMC8 31 1-359

Copyright © 1976 American Chemical Society All Rights Reserved. No part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN THE UNITED STATES OF AMERICA

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ACS Symposium Series Robert

F.

Gould,

Editor

Advisory Kenneth B. Bischoff Jeremiah P. Freeman E. Desmond Goddard Jesse C. H. Hwa Philip C. Kearney Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Aaron Wold

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

FOREWORD The ACS S Y M P O S I U SERIES was founded in 1974 to provide a medium for publishin format of the SERIES parallels that of the continuing A D V A N C E S I N C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS S Y M P O S I U M SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PREFACE here is considerable interest and activity in the application of synthetic and biological polymers in medicine. Synthetic polymers are widely used as surgical and dental implants as well as for blood bags, syringes, tubing, etc. Most of the materials used in medicine have properties greatly different from the tissue with which they are interfaced or replacing. Excepting bones, nails, and the outer layers of skin, mammalian tissues are highly aqueous materials, with water contents ranging up to 90% (blood plasma) Hydrogels are three dimensional networks of hydrophilic polymers, generally covalently or ionically cross-linked, which interact with aqueous solutions by swelling to some equilibrium value. Aqueous gel networks can be relatively strong (such as in celluose dialysis membranes) or relatively weak, generally becoming weaker as the water content increases, although such variables as the nature of the cross-linker, polymer network, tacticity, and crystallinity can significantly influence the mechanical behavior. Interest has focused on the utilization of the bulk or the surface properties of hydrogels for biomedical applications. The bulk property of swelling is of particular interest for "swelling implants," i.e., implants which can be implanted in a small dehydrated state via a small incision and which then swell to fill a body cavity and/or to exert a controlled pressure. The swelling of synthetic and natural gels may also help elucidate swelling and osmotic mechanisms in biological tissues. A related bulk property is the permeability of hydrogels for low molecular weight solutes. Solute diffusivity in gels can be used in sustained drug release applications and in the transport of solutes to gelentrapped macromolecules, particularly enzymes immobilized in the gel network. Ion interactions or partitioning within the gels are important in bone interfacing applications. Aqueous gels are relatively subtle systems which equilibrate with and "follow" many environmental changes. The properties of such gels can be highly dependent on cross-linker levels, impurity levels of comonomers, catalyst residues, and stereoregularity. These variables are discussed for hydrophilic methacrylate ester monomers and their gels. These systems receive the most attention in this volume. Monomer XI

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

analysis and purification, free radical initiator effects, and tacticity are discussed for the poly(hydroxyethyl methacrylate) system. The bulk properties of synthetic hydrogels can be widely varied and perhaps tailored to specific end uses. Though the poly (hydroxy ethyl methacrylate ) system is the most widely studied for medical applications, a large repertoire of gel types are available and have been applied to varying degrees in medicine. Questions of long term biostability or intentional biodegradeability are also very important, but are not discussed here. However, a number of fairly basic studies of gel networks and a number of practical applications based on network properties are presented. The surface and interfacial properties of hydrogels are somewhat similar to those of natural biological gels and tissues. A number of analogies or comparisons have been made between the hydrogel/water interface and the living cell/physiologic solution interface. Such interfaces are difficult to stud classical surface chemistry cannot be applied. The effect of the gel/water interface on interfacial fluid dynamics and the use of neutral gel coatings to reduce local fluid movement ( electroosmosis ) in particle electrophoresis experiments are discussed. The interface electrokinetic potential (as measured by the streaming potential) can be varied by use of appropriate comonomer compositions. The wettability of hydrogel surfaces is rather complicated, perhaps because of the mobility of polymer segments in the interfacial zone. Protein adsorption at certain gel interfaces is also discussed and related to interfacial free energy arguments. Aqueous gel surfaces can be used in blood-contact applications, as tubing, catheters, and vascular devices. Such applications depend on the nature of the gel/blood interface. As hydrogels generally lack suitable mechanical properties for vascular device applications, there has been considerable activity in coating or grafting the gels to mechanically suitable supports. In discussing blood compatibility of gels, the application of hydrogels as a substrate for in vitro cell or tissue culture studies should be considered. Data on the blood compatibility of hydrogels is available, particularly for polyacrylamide, and there is considerable interest and activity among medical research scientists and a number of commercial firms in hydrogel-coated catheters, drains, and other conduits. Aqueous gel networks have an important role to play in biomedicine, not only as inert blood conduits and devices, but as biochemically and pharmaceutically active elements. The role of ion and solute partitioning or interactions with gels will prove important for such applications as enzyme entrapment, sustained drug or ion release, and bone induction or xii

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

calcification, as well as for the more popular applications of blood and tissue interfacing. Suitably equilibrated and solute "stocked" gels should prove very important for cell and tissue culture applications. Control of hydration/dehydration phenomena, particularly hysteresis effects, is needed in order to ship and store hydrogel products in a dry state, thus reducing bulk and the possibility of microorganism contamination. This volume is dedicated to the memory of Dr. Willem Prins, a pioneer and leader in the study of gel networks, who died in a boating accident in 1974. I am particularly grateful to Buddy D . Ratner and Allan S. Hoffman for the review paper which begins this volume and to Donald Gregonis for aid in assembling the papers for publication. The participants in the symposium were also the reviewers of the papers. I thank them all for speedy but critical reviews. The secretarial assistance of Terry Smith is gratefully acknowledged University of Utah Salt Lake City, Utah March 5, 1976

JOSEPH

D.

ANDRADE

xiii

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1 Synthetic Hydrogels for Biomedical Applications BUDDY D. RATNER and ALLANS.HOFFMAN Departments of Chemical Engineering and Bioengineering, University of Washington, Seattle, Wash. 98195

It is the intention of this paper to review the literature concerning the preparation tions of synthetic hydroge subject is now voluminous and rapidly expanding (e.g. this symposium) it is difficult to produce a completely comprehensive review of the subject. However, a large number of articles will be examined which should give a useful overview of research interests and directions in this growing, multidisciplinary field. This review will be organized as follows: The introduction will discuss general aspects of synthetic hydrogels and point out similarities between the various distinctly different chemical structures which fit into this category. A short section is included within the introduction on problems related to the measurement and description of the biocompatibility of materials. The following six classes of hydrogel materials will then each be treated individually: poly(hydroxyalky1 methacrylates); poly(acrylamide), poly(methacrylamide) and derivatives; poly (N-vinyl-2-pyrrolidone); anionic and cationic hydrogels; polyelectrolyte complexes; and poly(vinyl alcohol). Sections on surface coated hydrogels, the characterization of imbibed water within hydrogels and immobilization or entrapment of biologically active molecules on and within hydrogels for biomedical applications are also included. 1.

Introduction

A. General Aspects of Synthetic Hydrogels. A hydrogel can be defined as a polymeric material which exhibits the ability to swell in water and retain a significant fraction (e.g., > 20%) of water within i t s structure, but which w i l l not dissolve in water. Included in this definition are a wide variety of natural materials of both plant and animal origin, materials prepared by modifying naturally occurring structures, and synthetic polymeric materials. This review article w i l l consider only synthetic hydrogel systems which are being used as, or have 1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR

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p o t e n t i a l f o r use, as b i o m a t e r i a l s . This c o n s t r a i n t i s not i n tended to imply that b i o m a t e r i a l s prepared from n a t u r a l b i o polymers are unimportant or u n i n t e r e s t i n g . Examples of modified n a t u r a l biopolymers which are p r e s e n t l y r e c e i v i n g a t t e n t i o n f o r biomedical a p p l i c a t i o n s i n c l u d e Cuprophan, c r o s s - l i n k e d Dextrans, and c r o s s - l i n k e d , e n z y m a t i c a l l y t r e a t e d c o l l a g e n s . For a review of n a t u r a l t i s s u e s used as b i o m a t e r i a l s see the recent a r t i c l e ly K i r a l y and Nosé ( 1 ) . Hydrogel m a t e r i a l s resemble i n t h e i r p h y s i c a l p r o p e r t i e s l i v i n g t i s s u e more so than any other c l a s s of s y n t h e t i c b i o m a t e r i a l . In p a r t i c u l a r , t h e i r r e l a t i v e l y h i g h water contents and t h e i r s o f t , rubbery consistency give them a s t r o n g , superf i c i a l resemblance to l i v i n g s o f t t i s s u e . Based upon these p r o p e r t i e s a number of advantages, some o b v i o u s l y r e a l and others somewhat s p e c u l a t i v b c i t e d f o hydrogel m a t e r i a l s With respect to the r e a F i r s t , the expanded natur hydroge p e r m e a b i l i t y to s m a l l molecules a l l o w s p o l y m e r i z a t i o n i n i t i a t o r molecules, i n i t i a t o r decomposition products, p o l y m e r i z a t i o n s o l vent molecules and other extraneous m a t e r i a l s to be e f f i c i e n t l y e x t r a c t e d from the g e l network before the hydrogel i s placed i n contact w i t h a l i v i n g system. The i n v i v o l e a c h i n g of a d d i t i v e s used d u r i n g the f a b r i c a t i o n of polymeric m a t e r i a l s has been c i t ed as a cause of inflammation and eventual r e j e c t i o n of implanted b i o m a t e r i a l s £2). Second, the r a t h e r s o f t and rubbery c o n s i s t ency of most hydrogels can c o n t r i b u t e to t h e i r b i o c o m p a t i b i l i t y by minimizing mechanical ( f r i c t i o n a l ) i r r i t a t i o n to surrounding c e l l s and t i s s u e . The most i n t r i g u i n g of the p o t e n t i a l advantages f o r hydrog e l s i s the low i n t e r f a c i a l t e n s i o n which may be e x h i b i t e d between a hydrogel s u r f a c e and an aqueous s o l u t i o n . This low i n t e r f a c i a l t e n s i o n should reduce the tendency of the p r o t e i n s i n body f l u i d s to adsorb and to u n f o l d upon a d s o r p t i o n (3). Minimal p r o t e i n i n t e r a c t i o n may be important f o r the b i o l o g i c a l acceptance of f o r e i g n m a t e r i a l s as the d e n a t u r a t i o n of p r o t e i n s by surfaces may serve as a t r i g g e r mechanism f o r the i n i t i a t i o n of thrombosis or f o r other b i o l o g i c a l r e j e c t i o n mechanisms. The a b i l i t y of s m a l l molecules to d i f f u s e through hydrogels may a l s o be advantageous f o r hydrogel i n v i v o performance. The d i f f u s i o n of important low molecular weight metabolites and ions through the implant and to the surrounding t i s s u e would occur w i t h hydrogels, but not w i t h r e l a t i v e l y hard, impermeable plastics. A number of biomedical a p p l i c a t i o n s f o r hydrogels which have been mentioned i n the l i t e r a t u r e are l i s t e d i n Table I . The wide range of biomedical a p p l i c a t i o n s f o r hydrogels can be a t t r i buted both to t h e i r s a t i s f a c t o r y performance upon i n v i v o i m p l a n t a t i o n i n e i t h e r blood c o n t a c t i n g or t i s s u e c o n t a c t i n g s i t u a t i o n s and to t h e i r a b i l i t y to be f a b r i c a t e d i n t o a wide range of morphologies. The ease w i t h which the p h y s i c a l form

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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3

of a hydrogel can be a l t e r e d a l l o w s the p h y s i c a l p r o p e r t i e s of the hydrogel to be adjusted s p e c i f i c a l l y f o r a g i v e n a p p l i c a t i o n . Hydrogels can o f t e n be prepared i n the form of porous sponges, non-porous g e l s , o p t i c a l l y transparent f i l m s , l i q u i d s which can be subsequently c r o s s l i n k e d t o form g e l s and c o a t i n g s bound by e i t h e r covalent bonds or non-covalent f o r c e s to a s u b s t r a t e p o l y mer m a t e r i a l . I t should be emphasized, however, t h a t a p a r t i c u l a r hydrogel composition s u i t a b l e f o r one b i o m e d i c a l a p p l i c a t i o n may have to be s i g n i f i c a n t l y m o d i f i e d i n composition and form f o r a d i f f e r e n t a p p l i c a t i o n . That i s , the hydrogel system must be matched t o each b i o m e d i c a l use. I n d i v i d u a l c l a s s e s of hydrogel b i o m a t e r i a l s are d i s c u s s e d i n S e c t i o n I I . Table I Potentia Biomedical A p p l i c a t i o n Coatings

"Homogeneous" M a t e r i a l s

Sutures Catheters IUD s Blood Detoxicants Sensors ( e l e c t r o d e s ) Vascular g r a f t s Electrophoresis c e l l s C e l l C u l t u r e Substrates

Enzyme TheraElectrophoresis gels p e u t i c Systems Contact Lenses Artificial A r t i f i c i a l Corneas Organs V i t r e o u s Humor ReplaceDrug D e l i v e r y ments Systems Estrous-Inducers Breast or other S o f t T i s s u e Substitutes Burn Dressings Bone Ingrowth Sponges Dentures Ear Drum Plugs Synthetic Cartilages Hemodialysis Membranes P a r t i c u l a t e C a r r i e r s of Tumor A n t i b o d i e s

f

Devices

Although the presence of imbibed water w i t h i n a polymeric system i s not a guarantee of b i o c o m p a t i b i l i t y , i t i s b e l i e v e d that the r e l a t i v e l y l a r g e f r a c t i o n of water w i t h i n c e r t a i n hydrogel m a t e r i a l s i s i n t r i n s i c a l l y r e l a t e d to t h e i r h i g h b i o c o m p a t i b i l i t y (4), (see S e c t i o n I V ) . However, these h i g h l y hydrated and w a t e r - p l a s t i c i z e d polymer networks are u s u a l l y mechanically weak. Furthermore, the higher the water content of the g e l , the poorer the mechanical p r o p e r t i e s of the g e l become. F o r t u n a t e l y , there are a number of approaches which can be taken to minimize problems due to the poor mechanical p r o p e r t i e s of these g e l s . Probably the s i m p l e s t of these approaches c o n s i s t s of forming the hydrogel over or surrounding a s t r o n g polymeric mesh or woven f a b r i c . Other methods i n v o l v e c o a t i n g a device or m a t e r i a l w i t h a l a y e r of hydrogel. In order t h a t the c o a t i n g

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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remain i n t a c t i t must e i t h e r be used i n a non-solvent environment, be c r o s s l i n k e d , or be i n t i m a t e l y and/or c o v a l e n t l y bound to the support m a t e r i a l . Because of the p o t e n t i a l importance and comp l e x i t y of techniques designed to anchor hydrogel c o a t i n g s , the whole area of hydrogels coated onto other surfaces w i l l be d i s cussed s e p a r a t e l y i n S e c t i o n I I I . F i n a l l y , hydrogels u s u a l l y have a l a r g e number of p o l a r r e a c t a b l e s i t e s on which other molecules may be immobilized by a v a r i e t y of chemical techniques. In a d d i t i o n , b i o l o g i c a l l y a c t i v e molecules can be entrapped w i t h i n the network s t r u c t u r e of c r o s s l i n k e d hydrogels. I m m o b i l i z a t i o n of b i o l o g i c a l l y a c t i v e molecules to and w i t h i n s y n t h e t i c hydrogels w i l l be discussed i n more det a i l i n S e c t i o n V. B. B i o c o m p a t i b i l i t y - O p e r a t i o n a the b i o c o m p a t i b i l i t y o countered i n p r o p e r l y d e f i n i n g the terminology used to d e s c r i b e the response of a l i v i n g system to an implanted f o r e i g n m a t e r i a l . Thus, terms such as "non-thrombogenic", "blood compatible", and "biocompatible" are o f t e n i n d i s c r i m i n a t e l y used to d e s c r i b e a wide range of b i o l o g i c a l responses. Bruck has itemized a number of f a c t o r s which might be d e l e t e r i o u s to the performance of m a t e r i a l s used f o r long-term i n t e r n a l biomedical a p p l i c a t i o n s (5). Based upon h i s d e s c r i p t i o n , the i d e a l b i o m a t e r i a l ( i n terms of b i o l o g i c a l response) could be defined as one which does not cause thrombosis, d e s t r u c t i o n of c e l l u l a r elements, a l t e r a t i o n of plasma p r o t e i n s , d e s t r u c t i o n of enzymes, d e p l e t i o n of e l e c t r o l y t e s , adverse immune responses, damage to adjacent t i s s u e , cancer and/ or t o x i c or a l l e r g i c r e a c t i o n s . No s y n t h e t i c m a t e r i a l developed f u l l y s a t i s f i e s these c r i t e r i a . A l s o , no s i n g l e t e s t method f o r e v a l u a t i n g b i o m a t e r i a l s i s capable of measuring t h i s wide range of f a c t o r s r e l e v a n t to b i o m a t e r i a l response. Based upon the l i m i t a t i o n s imposed by a v a i l a b l e t e s t i n g procedures, d i s c u s s i o n i n t h i s review paper concerning the b i o l o g i c a l performance of b i o m a t e r i a l s w i l l be o r i e n t e d towards the t e s t methods which have been used to evaluate the m a t e r i a l s . With respect to the most commonly used t e s t methods, the f o l l o w i n g comments should a l l o w a more c r i t i c a l reading of t h i s review. Lee White Test (and other r e l a t e d s t a t i c , i n v i t r o coagulat i o n time a s s a y s ) : The Lee White t e s t compares the c o a g u l a t i o n time of r e c a l c i f i e d whole blood i n a t e s t tube made of or coated w i t h the m a t e r i a l to be evaluated w i t h the c o a g u l a t i o n time of blood i n a standard c o n t r o l tube ( u s u a l l y g l a s s ) . V a r i a b l e s which can a f f e c t the r e s u l t s from t h i s t e s t i n c l u d e changing the donor, changes i n the d i e t or medication of the donor, storage time of the blood, venipuncture technique, and v a r i a t i o n s i n the experimental technique used to measure the c l o t t i n g times. The t e s t has a l s o been c r i t i c i z e d because of the l a r g e b l o o d - a i r i n t e r f a c e which i s exposed. The v a l i d i t y of r e s u l t s , p a r t i c u l a r l y as they apply to s i t u a t i o n s i n v o l v i n g contact w i t h f l o w i n g

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5

blood i n an i n v i v o or ex v i v o s i t u a t i o n , have f r e q u e n t l y been questioned. Methods f o r the i n v i t r o e s t i m a t i o n of the blood c o m p a t i b i l i t y of m a t e r i a l s have been c r i t i c a l l y reviewed (6). Vena Cava Ring Test Q) : This t e s t method i n v o l v e s the i m p l a n t a t i o n of s t r e a m l i n e d r i n g s made of or coated w i t h the m a t e r i a l to be evaluated i n t o the vena cava of dogs, u s u a l l y f o r 2 hour and 2 week t e s t p e r i o d s . The f o l l o w i n g terminology has been used to d e s c r i b e the experiment r e s u l t s : "Thrombogenid i s used to designate those m a t e r i a l s (formed i n t o t e s t r i n g s ) which are completely occluded w i t h thrombus a f t e r only 2 hours i m p l a n t a t i o n . "Moderately thromboresistant" i s used to d e s c r i b e m a t e r i a l s which remain patent a f t e r 2 hour i m p l a n t a t i o n but show s i g n i f i c a n t adherent thrombus a f t e r the two week t e s t p e r i o d . The d e s i g n a t i o n " h i g h l y thromboresistant" i s reserved f o r mater i a l s which show l i t t l two week i m p l a n t a t i o n p e r i o d d i f f e r e n t procedure f o r d e s c r i b i n g the r e s u l t s has r e c e n t l y been published ( 8 ) . The vena cava r i n g t e s t does not d i s t i n g u i s h between those m a t e r i a l s which are t r u l y non-thrombogenic and those which cause thrombus formation but are non-thromboadherent. Renal Embolus Ring Test ( 9 ) : This t e s t u t i l i z e s r i n g s f a b r i c a t e d from the m a t e r i a l s to be evaluated implanted i n the canine descending a o r t a j u s t above the r e n a l a r t e r i e s . A cons t r i c t i o n i s made i n the a o r t a below the r e n a l a r t e r i e s to f o r c e a l a r g e f r a c t i o n of the blood f l o w i n g through the t e s t r i n g i n t o the kidneys. A f t e r a p e r i o d of i m p l a n t a t i o n ( u s u a l l y 3-6 days) the r i n g s are examined f o r adherent thrombi and the kidneys are d i s s e c t e d and examined f o r i n f a r c t s presumably caused by thrombi shed from the r i n g s u r f a c e . This t e s t should be a b l e to d i s t i n guish between those m a t e r i a l s which are t r u l y non-thrombogenic and those which are o n l y non-thromboadherent. Almost a l l mater i a l s examined to date have been shown to cause some i n f a r c t damage to the t e s t animal's kidneys. Soft Tissue C o m p a t i b i l i t y Tests: For examining the response of the body to m a t e r i a l s implanted i n s o f t t i s s u e areas (not i n d i r e c t contact w i t h the blood stream) there has been l i t t l e e f f o r t extended towards adopting standardized t e s t procedures. Autian has surmised i n a l i t e r a t u r e review t h a t intramuscular implantat i o n may be the most s e n s i t i v e s i t e f o r e v a l u a t i o n of t h i s t i s s u e response (10). Coleman, King and Andrade have r e c e n t l y d e s c r i b e d a comprehensive p r o t o c o l f o r the e v a l u a t i o n of t i s s u e response to standardized i n t r a m u s c u l a r i m p l a n t a t i o n s (11). However, the b u l k of the papers i n the l i t e r a t u r e do not f o l l o w any p a r t i c u l a r standardized t e s t procedure. The term " t i s s u e compatible", f o r the purposes of t h i s review paper, w i l l be used to d e s c r i b e those m a t e r i a l s which, upon i m p l a n t a t i o n , show a normal acute inflammat i o n r e a c t i o n and then r a p i d l y "heal i n " to a p a s s i v e s t a t e wherei n the implant i s surrounded by a t h i n , uniform f i b r o u s capsule i n which m u l t i n u c l e a t e d g i a n t c e l l s and other inflammatory c e l l s are g e n e r a l l y absent. In v i t r o t e s t s u s i n g c u l t u r e d c e l l s have 1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

a l s o been u t i l i z e d w i t h v a r y i n g degrees o f success t o evaluate the t o x i c i t y and, t o a s m a l l e r e x t e n t , the b i o c o m p a t i b i l i t y o f b i o m e d i c a l polymers. This l i t e r a t u r e has been covered i n a review by A u t i a n (10). II.

B i o m e d i c a l l y Important Hydrogels. Hydrogels may be prepared by v a r i o u s p o l y m e r i z a t i o n t e c h niques o r by conversion of e x i s t i n g polymers. Tablœ I I and I I I l i s t examples o f monomers and polymers used i n p r e p a r i n g these m a t e r i a l s . Although g e n e r a l i z a t i o n s can be made about hydrogels, i t should be apparent from these t a b l e s t h a t t h i s category o f m a t e r i a l s covers a wide range of chemical compositions. There are major d i f f e r e n c e s f o r each type of m a t e r i a l w i t h respect t o s y n t h e s i s , p r o p e r t i e s and b i o c o m p a t i b i l i t y . Therefore, each o f the more important type individually. A. P o l y ( h y d r o x y a l k y 1 m e t h a c r y l a t e s ) . Included i n t h i s class of compounds are p o l y ( 2 - h y d r o x y e t h y l methacrylate) (P-HEMA), p o l y ( g l y c e r y l methacrylate) (P-GMA), and poly(hydroxypropyl methac r y l a t e ) (P-HPMA)* A review a r t i c l e w i t h p a r t i c u l a r em^asis upon h y d r o x y a l k y l methacrylate hydrogels has been p u b l i s h e d (12). P o l y ( 2 - h y d r o x y e t h y l methacrylate) (P-HEMA) hydrogels were f i r s t d e s c r i b e d and s y n t h e s i z e d by Lim and W i c h t e r l e i n the e a r l y I960's (13). Although t h i s polymer was prepared by DuPont s c i e n t i s t s as e a r l y as 1936 (14), they d i d not polymerize the monomer i n the presence of c r o s s l i n k i n g agent i n aqueous s o l v e n t media as did L i m and W i c h t e r l e . To date, P-HEMA hydrogels have probably been among the most w i d e l y s t u d i e d and used of a l l the s y n t h e t i c hydrogel m a t e r i a l s . A d e s c r i p t i o n of the s y n t h e s i s and p r o p e r t i e s o f P-HEMA hydrogels was p u b l i s h e d i n 1965 by Refojo and Yasuda (15). I f HEMA monomer c o n t a i n i n g c r o s s l i n k i n g agent i s polymerized i n the presence o f a good s o l v e n t f o r both monomer and polymer (e.g., ethylene g l y c o l , ethylene glycol-H^O) an o p t i c a l l y transparent (homogeneous) hydrogel i s formed. I f the monomer p l u s c r o s s l i n k i n g agent i s polymerized i n a poor s o l v e n t system f o r the r e s u l t i n g polymer, an opaque, spongy, white (heterogeneous) hydrog e l i s formed. As the HEMA monomer i s an e x c e l l e n t s o l v e n t f o r P-HEMA, i f the c o n c e n t r a t i o n of water (which i s by i t s e l f a nons o l v e n t f o r P-HEMA) i n the water-HEMA mixture i s 43% o r l e s s by weight, a homogeneous g e l w i l l be formed (16)(17). A s o l u b l e P-HEMA polymer which i s s u i t a b l e f o r d i p c o a t i n g a p p l i c a t i o n s can be prepared by p o l y m e r i z i n g t o a low degree o f conversion i n d i l ute e t h a n o l s o l u t i o n monomer from which most o f the contaminating c r o s s - l i n k i n g agent has been removed (18,19). One o f the problems encountered i n the p r e p a r a t i o n o f P-HEMA hydrogels f o r b i o m e d i c a l a p p l i c a t i o n s i s the p u r i t y o f monomer used i n these systems. T y p i c a l i m p u r i t i e s found i n commercial grades o f HEMA monomer are m e t h a c r y l i c a c i d , ethylene g l y c o l

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Synthetic Hydrogels

RATNER AND H O F F M A N

1.

7

T A B L E 3L MONOMERS

CH

HYDROXYALKYL

= C

2

METHACRYLATES

„CH V

USED

IN

HYDROGELS ACIDIC

OR

ANIONIC

3

C02-R

ACRYLIC ACID, DERIVATIVES (R=-H,-CH )

CH

=C-C0 H

CROTONIC

CH - -C=CH-C0 H

2

2

3

R =-CH CH OH, 2

2

-CHo-CH-OH.-CHo-CH-CHo-OH 1 I CH OH

SODIUM

3

ACID

3

STYRENE

CH

SULFONATE

BASIC 2,4

CH

PENTADIENE-I-OL

(R = - H ,

2

X

t

3

VINYL 2

C0 "C H 2

2

4

Ν-R

(R,R' R" = - H , - C H , - C H )

-CH,)

(R,R"=H,-CH -C H , — CH CHOHCH 5

CATIONIC

METHACRYLATE, C H = C

DERIVATIVES

C - C O - N - R R"

2

S O 3 No

2

CH2 =

3

= CH - < w ) -

2

= C H - C H =CH — C H O H

2

AMINOETHYL ACRYLAMIDE DERIVATIVES

OR

(withVAc)

2

4

9

CH

CH

3

—Ν — VINYL

P Y R R O L I DON Ε

C H = CH- Ν 2

HYDROPHOBIC ACRYLICS

ETHYLENEGLYCOL DIMETHACRYLATE DERIVATIVES

CHj) = C ^

CH = C 2

,

3

4

9

C=CH

Ο

3

(R =-CH ,-C H ,

Rv

1

CO

(USED A S COMONOMERS) (R=-H,-CH ) -OCH ,-CN,-OCH CH OCH 3

2

2

(CH CH 6) 2

2

X

3

CH =CH

METHYLENE-BISACRYLAMIDE

CH=CH

2

CO

CO

I

NH ^CHp

I

n2

Table I I I EXAMPLES OF CONVERTED POLYMERS USED AS HYDROGELS -f-CH -CH-)-

-fCH -CH*

2

2

Ο

OH

C=0 CH

3

-f C H - C H = CH-CH -)" 2

4 CH -CH-CH-CH 4OH OH

2

2

2

POLYELECTROLYTE COMPLEX

-iCh^-ÇHiCN

·•

-fCH ~CH-) CH 2

fCH —CH4—(CH -CH-)C= 0 C0 H NH 2

2

2

2

C H

2

=C

C°

H3

+

C0 C H OH 2

2

-<-CH -CH-} 2

+~ MIXTURE OF 2

4

(J OCN-R-NCO + HO-f C H — C H — Ο ή ^ - Η — H N R - N H - C ^ O - f - C ^ - O ^ - O ^ ] 2

2

CO

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2

1

8

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

d i m e t h a c r y l a t e , and ethylene g l y c o l . The monomer can be p u r i f i e d by a complex procedure i n v o l v i n g e x t r a c t i n g w i t h hexane, d i s t i l l a t i o n under vacuum and treatment w i t h alumina (18,20). Even a f t e r t h i s p u r i f i c a t i o n , low l e v e l s (^0.01 - 0.1%) of i m p u r i t i e s remain i n the monomer. These i m p u r i t i e s could have long-term e f f e c t s on the b i o c o m p a t i b i l i t y o f P-HEMA g e l s . I t has been suggested that l a c k o f r e p r o d u c i b i l i t y i n the measurement of the thrombogenicity o f P-HEMA m a t e r i a l s i n i n v i v o t e s t s might be due to as yet u n i d e n t i f i e d i m p u r i t i e s i n HEMA monomer (21). A p a r t i c u l a r l y advantageous property o f P-HEMA hydrogels f o r biomedical a p p l i c a t i o n s i s t h e i r h i g h degree o f chemical s t a b i l i t y . P-HEMA g e l s were found t o be r e s i s t a n t t o a c i d h y d r o l y s i s and r e a c t i o n w i t h amines (22). I n another study using a r e l a t e d c r o s s l i n k e d polymer, p o l y ( d i e t h y l e n e g l y c o l m e t h a c r y l a t e ) , a l k a l i n e h y d r o l y s i s wa only a t h i g h pH and a t i n n e u t r a l o r near n e u t r a l aqueous s o l u t i o n s are a l s o r e l a t i v e l y temperature s t a b l e and can be steam s t e r i l i z e d w i t h no apparent damage t o the m a t e r i a l s (24). The s w e l l i n g behavior o f P-HEMA g e l s i n v a r i o u s s o l v e n t s has been s t u d i e d i n great d e t a i l . In water the water content o f homogeneous P-HEMA g e l s i s remarkably i n s e n s i t i v e t o the concentration of c r o s s l i n k e r i n the p o l y m e r i z i n g mixture i n the 0-4 mole % c r o s s l i n k e r range (15,25). The water content o f heterogeneous P-HEMA g e l s s t r o n g l y depends upon the amount o f water (polymer non-solvent) present d u r i n g the p o l y m e r i z a t i o n r e a c t i o n (15). Tables which compare the r e l a t i v e degree of s w e l l i n g o f P-HEMA g e l s i n a wide v a r i e t y o f organic s o l v e n t s have been prepared by two authors (16,^2). The s w e l l i n g behavior o f t h i s g e l i n a v a r i e t y o f aqueous s o l u t i o n s has a l s o been described i n a number of s t u d i e s (25-29). Theories r e l a t i n g the s w e l l i n g o f P-HEMA gels to both hydrophobic and hydrogen bonding i n t e r a c t i o n s w i t h i n the g e l m a t r i x have been proposed (25 26>30>31). The s w e l l i n g o f P-HEMA g e l s has a l s o been s t u d i e d w i t h respect t o the fundamental equations d e s c r i b i n g the s w e l l i n g o f c r o s s l i n k e d networks (32). The mechanical p r o p e r t i e s o f P-HEMA hydrogels have r e c e i v e d some a t t e n t i o n (33). These g e l s are p a r t i c u l a r l y s u i t e d f o r s t u d i e s on the fundamental aspects o f rubber e l a s t i c i t y , as the t o p o l o g i c a l aspects o f the c r o s s l i n k e d networks can be e a s i l y v a r i e d by changing the c r o s s l i n k i n g agent, c r o s s l i n k i n g agent c o n c e n t r a t i o n , t o t a l monomer c o n c e n t r a t i o n o r p o l y m e r i z a t i o n s o l v e n t . A l s o , co-monomers are e a s i l y i n c o r p o r a t e d i n t o the system. This l i t e r a t u r e has r e c e n t l y been reviewed by Janacek (33). Biomedical s t u d i e s u t i l i z i n g P-HEMA hydrogels are numerous (34-64). Many of these are l i s t e d i n Table IV. Fundamental s t u d i e s on the h e a l i n g o f d i s k s o f P-HEMA subcutaneously implanted i n r a t s and p i g s were performed p r i m a r i l y by researchers at the I n s t i t u t e o f Macromolecular Chemistry, Prague (31-36). Homogeneous P-HEMA and heterogeneous P-HEMA m a t e r i a l s w i t h varying 5

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

RATNER AND H O F F M A N

9

Synthetic Hydrogek

degrees o f p o r o s i t y were examined. I n g e n e r a l , most o f implants were w e l l t o l e r a t e d and d i d not provoke unfavorable r e a c t i o n . However, P-HEMA g e l s which were extremely macroporous (those prepared w i t h greater than 70% water i n the p o l y m e r i z a t i o n mixture) demonstrated poor mechanical p r o p e r t i e s , and c e r t a i n u n d e s i r a b l e h e a l i n g c h a r a c t e r i s t i c s i n c l u d i n g c a l c i f i c a t i o n a t the margins o r center o f the implant. I t was t h e r e f o r e concluded t h a t homogeneous o r microporous heterogeneous P-HEMA g e l s might be more suitable f o r implant purposes. Table IV Biomedical Studies U t i l i z i n g P-HEMA Hydrogels Description E v a l u a t i o n o f Tissue Respons Antibiotic Delivery: From Coated Suture From Coated U r e t h r a l Catheter In Otolaryngology Anti-tumor Drug D e l i v e r y Device Coated I n t r a u t e r i n e Device L i v e r Resection Blood C o m p a t i b i l i t y Coated Sutures Corneal Surgery Soft Contact Lens Ureter P r o s t h e s i s Breast Augmentation Latex Spheres f o r C e l l Surface Studies Hemodialysis and Hemoperfusion Ligament P r o s t h e s i s Bone Formation i n P-HEMA Sponges

Reference

(41,42) (41.43,44) (45) (46) (47) (48) (41,49-52) (41,42,50) (53) (54,55) (56) (57) (58) (59-62) (63) (40,64)

Further problems were noted w i t h the h e a l i n g o f porous P-HEMA sponge m a t e r i a l s by Winter and Simpson (40). They found that pieces o f P-HEMA sponge implanted f o r 62 days i n the s k i n o f young p i g s showed evidence of woven bone formation. The e f f e c t was found t o be r e a d i l y r e p r o d u c i b l e . The h e a l i n g process f o r these porous m a t e r i a l s i s unusual i n that the sponges are appare n t l y w e l l t o l e r a t e d f o r the f i r s t month o f i m p l a n t a t i o n . However, from 31 days onwards m u l t i n u c l e a t e g i a n t c e l l s are found i n the implant (64). Rubin and M a r s h a l l attempted t o u t i l i z e the observed bone formation i n implanted spongy P-HEMA t o anchor i n t o the femur and t i b i a a k n i t t e d Dacron-porous P-HEMA a n t e r i o r c r u c i a t e ligament p r o s t h e s i s (63). F i x a t i o n i n t o the bone was not achieved although bone formation was noted i n p a r t s o f the polymer. Due t o the poor mechanical p r o p e r t i e s o f P-HEMA g e l s , there have always been experimental d i f f i c u l t i e s i n e v a l u a t i n g the blood c o m p a t i b i l i t y o f these m a t e r i a l s by accepted i n v i v o t e s t i n g

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

techniques (e.g., the vena cava r i n g t e s t o r the r e n a l embolus r i n g t e s t ) . However, the blood c o m p a t i b i l i t y o f P-HEMA was observed i n a few s t u d i e s e i t h e r i n v i t r o , o r , u s i n g somewhat l e s s c o n v e n t i o n a l techniques, i n v i v o (41, 49-52). I t was found, i n a l l cases, t o be a r e l a t i v e l y "non-thrombogenic" (or a t l e a s t non-thromboadherent) m a t e r i a l , although the i n v i v o t e s t s t h a t were used do not a l l o w a systematic grading o f thrombogenicity, and do not compare the performance o f P-HEMA t o a number o f other commonly used m a t e r i a l s . More systematic s t u d i e s o f the blood c o m p a t i b i l i t y of P-HEMA are discussed i n the s e c t i o n on s u r f a c e coated hydrogels. The primary c l i n i c a l use f o r P-HEMA hydrogels has been f o r f l e x i b l e , h y d r o p h i l i c contact l e n s e s . This i s a l s o the o n l y a p p l i c a t i o n which has r e c e i v e d U.S. Food and Drug A d m i n i s t r a t i o n approval ( s p e c i f i c a l l y f o S o f l e n s f o r the c o r r e c t i o Lambert poly(HEMA-co-ethylene d i m e t h a c r y l a t e - c o - m e t h a c r y l i c a c i d co-g-Povidone) Soft-con as a t h e r a p e u t i c 'bandage"). The propert i e s and a p p l i c a t i o n s f o r such contact lenses have been reviewed (54). Studies d e a l i n g s p e c i f i c a l l y w i t h oxygen p e r m e a b i l i t y through hydrogel contact l e n s e s (65), and h y d r a t i o n and l i n e a r expansion o f h y d r o p h i l i c contact Tenses (66) have been p u b l i s h e d . In another study methyl methacrylate contact l e n s e s and P-HEMA contact lenses were observed w i t h respect t o t h e i r e f f e c t upon the growth o f c o r n e a l e p i t h e l i a l t i s s u e i n c u l t u r e . The t o l e r ance o f the t i s s u e t o continuous exposure t o the contact lenses was found t o be s i g n i f i c a n t l y higher f o r the h y d r o p h i l i c lenses A number o f problems have been noted w i t h hydrophilic contact l e n s e s . These i n c l u d e low v i s u a l a c u i t y as compared t o hard l e n s e s , accumulation o f m a t e r i a l on and w i t h i n the l e n s e s , suscept i b i l i t y t o mechanical damage, low p e r m e a b i l i t y t o oxygen, ( r e v e r s i b l e ) changes i n the shape and r e f r a c t i v e index o f the l e n s e s , and the need f o r frequent s t e r i l i z a t i o n (54*67)· The p r i n c i p l e advantages o f these hydrogel lenses are t h a t they are easy t o f i t , w e l l t o l e r a t e d and can be used f o r t h e r a p e u t i c purposes other than simple r e f r a c t i v e c o r r e c t i o n . Concerning the degree t o which these lenses are t o l e r a t e d , i t has been r e c e n t l y reported that the Bausch and Lomb Soflens can be worn c o n t i n u o u s l y 10 days w i t h few or no problems (6£) . However, another study r e p o r t s that i t i s unadvisable t o wear s o f t contact lenses c o n t i n u o u s l y f o r long periods o f time due t o the development o f c o r n e a l edema (68). Other hydroxyalky1 methacrylates have not r e c e i v e d n e a r l y as much a t t e n t i o n as P-HEMA. Poly(hydroxypropyl methacrylate) hydrog e l s c o n t a i n i n g a mixture o f the two isomers can be r e a d i l y p r e pared (25). Although the water content of these hydrogels i s f a i r l y low (^25%) they demonstrate e x c e l l e n t mechanical properties. P o l y ( g l y c e r y l methacrylate) hydrogels e x h i b i t a c o n s i d e r a b l y higher e q u i l i b r i u m water content than P-HEMA g e l s (28,60-71). Therefore i t has o f t e n been thought t h a t t h i s m a t e r i a l might show

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

RATNER AND H O F F M A N

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11

a higher degree of b i o c o m p a t i b i l i t y than P-HEMA. This has not been described i n the l i t e r a t u r e to date. I t has been suggested that dehydrated P-GMA might be swollen i n s i t u i n cases where i t i s necessary to r e p l a c e the v i t r o u s humor of the eye (70). Β· P o l y ( A c r y l a m i d e ) , Poly(Methacrylamide) and D e r i v a t i v e s . Gels of poly(acrylamide) (PAAm) and of some N - s u b s t i t u t e d d e r i v a t i v e s of PAAm can be r e a d i l y prepared by the f r e e r a d i c a l p o l y m e r i z a t i o n of an aqueous s o l u t i o n of acrylamide c o n t a i n i n g a s m a l l f r a c t i o n of c r o s s l i n k i n g agent ( o f t e n N, N-methylenebisacrylamide). The g e l s formed are o p t i c a l l y t r a n s p a r e n t , mechanically weak, and can have extremely h i g h water contents (> 95%). The water content i s dependent upon the per cent c r o s s l i n k e r i n the system, u n l i k e the homogeneous P-HEMA g e l system. Another technique was r e c e n t l y p r i m a r i l y of polyacrylamid ( a c r y l o n i t r i l e ) and p o l y ( a c r y l i c a c i d ) (72). This technique i n v o l v e s p o l y m e r i z i n g a c r y l o n i t r i l e i n concentrated aqueous z i n c c h l o r i d e and then p a r t i a l l y h y d r o l y z i n g the r e s u l t i n g g e l . The high water contents which are a t t a i n a b l e w i t h acrylamide g e l s prepared by s o l u t i o n p o l y m e r i z a t i o n or by h y d r o l y s i s make them a t t r a c t i v e f o r b i o m e d i c a l a p p l i c a t i o n s (see S e c t i o n I V ) . A number of s t u d i e s have been c a r r i e d out on the h y d r o l y s i s of acrylamide and methacrylamide polymers (73-75). Hydrolysis occurs at s i g n i f i c a n t r a t e s at elevated temperatures i f the polymers are i n a c i d i c or b a s i c s o l u t i o n s . Thus, polyamide g e l s should be r e l a t i v e l y s t a b l e under c o n d i t i o n s of p h y s i o l o g i c a l pH and temperature. However, problems might occur a t steam auto c l a v e temperatures and t h e r e f o r e t h i s procedure might be c o n t r a indicated. The t i s s u e c o m p a t i b i l i t y of p o l y ( N - s u b s t i t u t e d acrylamides) was i n v e s t i g a t e d by Kopecek, et a l . (76). N - s u b s t i t u t e d a c r y l a mides were used f o r t h i s study because of t h e i r s u p e r i o r hydrol y t i c s t a b i l i t y compared to p o l y ( a c r y l a m i d e ) . I t was found that d i s c s of p o l y ( N , N - d i e t h y l a c r y l a m i d e ) , p o l y ( N - a c r y l y l morpholine), p o l y ( N - e t h y l acrylamide) and a l s o the methacrylamide d e r i v a t i v e , poly[N-(2-hydroxypropyl) methacrylamide], implanted subcutaneously i n r a t s were w e l l t o l e r a t e d by the animals and d i d not provoke unfavorable r e a c t i o n . The long term b i o l o g i c a l i n t e r a c t i o n of these hydrogels w i t h the t e s t animals was described as being s i m i l a r to t h a t observed w i t h implanted P-HEMA m a t e r i a l s , which were a l s o i n v e s t i g a t e d by t h i s group. The thrombogenicity of a number of PAAm g e l s was i n v e s t i g a t e d i n v i t r o u s i n g the Lee-White c o a g u l a t i o n time t e s t (52). Where the c o a g u l a t i o n time of f r e s h blood samples i n g l a s s tubes was approximately 12 minutes, blood i n PAAm tubes prepared u s i n g s i n g l y r e c r y s t a l l i z e d acrylamide monomer showed a c l o t t i n g time of ^45 minutes. When g e l s formed from t r i p l y r e c r y s t a l l i z e d acrylamide monomer were t e s t e d they showed c l o t t i n g times i n excess of 24 hours. The d e l e t e r i o u s e f f e c t s of incomplete removal -

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of i n i t i a t o r by-products on the thrombogenicity o f acrylamide m a t e r i a l s was a l s o noted i n t h i s study. The e f f e c t of v a r y i n g the c r o s s l i n k i n g agent c o n c e n t r a t i o n i n the PAAm g e l was found not t o s i g n i f i c a n t l y a l t e r the Lee-White c l o t t i n g times f o r the system. I n t e r e s t i n g e f f e c t s of other co-monomers i n c o r p o r a t e d i n t o the PAAm g e l s on the c l o t t i n g times were noted. Some o f these e f f e c t s a r e d i s c u s s e d i n S e c t i o n I I F. I n g e n e r a l , the PAAm system prepared w i t h t r i p l y r e c r y s t a l l i z e d monomer gave the best r e s u l t s , although systems c o n t a i n i n g dimethylaminoethy1 methacrylate were shown t o demonstrate e x c e l l e n t Lee-White c l o t t i n g times. The thrombogenicity o f these p a r t i c u l a r g e l s was found t o be extremely s e n s i t i v e t o c r o s s l i n k e r c o n c e n t r a t i o n and total gel solids. Further i n f o r m a t i o n about PAAm g e l s i s contained i n a r e c e n t l y p u b l i s h e d review c o v e r i n (4). I n p a r t i c u l a r th discussed i n r e l a t i o n t o v a r i o u s measures o f i t s b i o c o m p a t i b i l i t y . C. P o l y ( N - V i n y l - 2 - P y r r o l i d o n e ) . P o l y (N-vinyl-2-pyrrolidone) (P-NVP) i s a somewhat unique polymer i n t h a t , i n i t s uncrossl i n k e d form, i t i s extremely s o l u b l e i n water and i s a l s o s o l u b l e i n many other p o l a r and non-polar s o l v e n t s . Because o f i t s s t r o n g i n t e r a c t i o n w i t h water i t can be used f o r preparing g e l s which w i l l e x h i b i t h i g h water contents. P-NVP g e l s a r e a l s o o f i n t e r e s t f o r biomedical a p p l i c a t i o n s as the s o l u b l e polymer has had a long h i s t o r y o f use i n the medic a l and pharmaceutical f i e l d s . One of the most important uses f o r P-NVP s o l u t i o n s has been as a plasma expander (77). When i n f u s e d i n t r a v e n o u s l y P-NVP i s non-toxic and non-thrombogenic and can be used t o maintain c i r c u l a t o r y f l u i d volume i n cases o f s e vere i n j u r y o r trauma. Some r e t e n t i o n o f P-NVP i n the l i v e r , s p l e e n , lungs and kidneys has been noted (78). I n a review o f the world l i t e r a t u r e on P-NVP i n 1962 i t was concluded t h a t P-NVP could be used o r a l l y o r i n t r a v e n o u s l y w i t h complete s a f e t y (79). However, a t the present time P-NVP i s no longer used as a plasma expander i n humans because i t i s not metabolized and i s not r e t a i n e d i n c i r c u l a t i o n as w e l l as other plasma expanders (e.g., Dextrans). The r e s i s t a n c e o f P-NVP t o d i g e s t i o n by l y s o somal enzymes has been e x p l o i t e d i n a recent study i n which the k i n e t i c s o f uptake by p i n o c y t o s i s o f I l a b e l l e d P-NVP i n t o r a t y o l k sac c u l t u r e d i n v i t r o was measured(80). P-NVP has a l s o been used i n blood volume determinations (81) and i n the p r e s e r v a t i o n of blood and blood components (82). I n the pharmaceutical f i e l d i t has been used as a t a b l e t b i n d e r , a t a b l e t c o a t i n g and f o r the s o l u b i l i z a t i o n and s t a b i l i z a t i o n o f drugs. An extensive b i b l i o graphy l i s t i n g P-NVP medical and pharmaceutical a p p l i c a t i o n s i s a v a i l a b l e (83). Hydrogels c o n s i s t i n g only o f P-NVP have not o f t e n been described i n the b i o m e d i c a l l i t e r a t u r e p o s s i b l e because h i g h concentrations o f c r o s s l i n k e r (5-20%) are needed t o produce a 1

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m a t e r i a l w i t h u s e f u l mechanical p r o p e r t i e s . P-NVP g e l s c r o s s l i n k e d w i t h methylene b i s ( 4 - p h e n y l isocyanate) were evaluated f o r use as hemodialysis membranes (84). F a s t e r metabolic waste t r a n s f e r was obtained w i t h these membranes than w i t h conventional c e l l u l o s e f i l m s . P-NVP g e l s c r o s s l i n k e d w i t h 20%(w/w) methylenebisacrylamide have been considered f o r use as an implantable drug d e l i v e r y system (85). In v i t r o e v a l u a t i o n of the thrombogenicity of P-NVP g e l s and P-NVP-PAAm copolymer g e l s by the Lee-White t e s t showed some extension of c l o t t i n g times although problems w i t h r e s i d u a l NVP monomer i n the g e l s was noted (52). F i b r o b l a s t adhesiveness to P-NVP g e l s has a l s o been measured i n v i t r o (86). I t was determined t h a t i n c r e a s i n g the c o n c e n t r a t i o n of the g e l from 40% to 96% renders i t more adhesive to the c e l l s . The d i f f i c u l t i e s i n v o l v e d i n preparing homogeneous P-NVP m a t e r i a l s makes NVP an g r a f t i n g systems. A numbe cussed i n S e c t i o n I I I . D. P o l y e l e c t r o l y t e Complexes. P o l y e l e c t r o l y t e complexes are p o l y s a l t s formed by the c o r e a c t i o n of a c a t i o n i c polymer such as p o l y ( v i n y l benzyltrimethyl-ammonium c h l o r i d e ) and an a n i o n i c p o l y mer such as sodium p o l y ( s t y r e n e s u l f o n a t e ) . The complex formed from these two p a r t i c u l a r p o l y e l e c t r o l y t e s was developed by the Amicon Corporation and i s r e f e r r e d to as l o p l e x 101. I t has r e c e i v e d b i o m e d i c a l e v a l u a t i o n i n a number of s i t u a t i o n s ( 8 7 , 8 8 ) . Due to mechanical s t r e n g t h l i m i t a t i o n s p o l y e l e c t r o l y t e complexes have been g e n e r a l l y used as coatings on f a b r i c s and other supports. In order to be used f o r coatings the g e l must be s o l u b i l i z e d i n a complex, multicomponent s o l v e n t system u s u a l l y cont a i n i n g water, a p o l a r , water s o l u b l e o r g a n i c s o l v e n t , and a strong e l e c t r o l y t e . I o p l e x - s o l v e n t s o l u t i o n s have g e n e r a l l y been s t r o n g l y a c i d i c and have been shown to degrade c e r t a i n p l a s t i c s such as nylon-6,6 thus making the requirements f o r a s u i t a b l e subs t r a t e f o r the p o l y e l e c t r o l y t e complex more s t r i n g e n t (89). D i f f i c u l t i e s have a l s o been found i n the s t e r i l i z a t i o n of Ioplex m a t e r i a l s . Autoclave s t e r i l i z a t i o n of these complexes can r e s u l t i n d i s i n t e g r a t i o n of the g e l s t r u c t u r e (89). Gas s t e r i l i z a t i o n can leave entrapped ethylene oxide i n the m a t r i x . The problems encountered i n s t e r i l i z i n g these hydrogels have been reviewed(4,90). An advantage of these m a t e r i a l s i s the ease w i t h which a net charge ( a n i o n i c or c a t i o n i c ) can be i n c o r p o r a t e d i n the system. This i s done by adding s t o i c h i o m e t r i c a l l y g r e a t e r or l e s s e r amounts of one of the two polymeric components d u r i n g f o r m u l a t i o n . I t was determined u s i n g the i n v i v o vena cava r i n g t e s t that Ioplex 101 c o n t a i n i n g 0.5 meq. excess a n i o n i c component showed the g r e a t e s t thromboresistance (89). However, the performance of the n e u t r a l Ioplex g e l i n t h i s t e s t i n d i c a t e d t h a t i t i s perhaps only s l i g h t l y l e s s thromboresistant than the a n i o n i c g e l (4). C a t i o n i c Ioplexes and g e l s c o n t a i n i n g an increased number of a n i o n i c s i t e s

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were s i g n i f i c a n t l y more thrombogenic. E. P o l y ( v i n y l a l c o h o l ) . P o l y ( v i n y l a l c o h o l ) (PVA) i s a water s o l u b l e polymer formed by the h y d r o l y s i s of p o l y ( v i n y l acet a t e ) . C r o s s l i n k e d g e l s of PVA have found a number of uses i n the b i o m e d i c a l f i e l d . A c r o s s l i n k e d , h i g h l y porous sponge of PVA can be formed by r e a c t i n g formaldehyde w i t h s o l u b l e PVA and blowing a i r through the s o l u t i o n before the p o l y m e r i z a t i o n - c r o s s l i n k i n g process i s completed. This m a t e r i a l was commercially a v a i l a b l e under the name I v a l o n and had been e x t e n s i v e l y used i n h e r n i a treatment, duct replacement, c a r d i a c - v a s c u l a r surgery, p l a s t i c surgery, and r e c o n s t r u c t i v e surgery. H e a l i n g problems w i t h I v a l o n sponges were encountered and i t was concluded t h a t I v a l o n d i d not meet up to the e a r l y enthusias t h e t i c s would be more s a t i s f a c t o r A hydrogel c o n s i s t i n g of PVA and the a n t i c o a g u l a n t h e p a r i n c r o s s l i n k e d together w i t h glutaraldehyde/formaldehyde mixtures has been s y n t h e s i z e d and demonstrates low thrombogenicity i n i n v i t r o t e s t s (92). Experiments w i t h S l a b e l l e d heparin indicate that heparin does not l e a c h out o f the c r o s s l i n k e d g e l . A potent i a l problem w i t h t h i s m a t e r i a l i s t h a t , p o s s i b l y due t o the presence of h e p a r i n , i t shows a tendency t o adsorb blood p l a t e l e t s . PVA-heparin hydrogels have a l s o been evaluated f o r use as hemodialysis membranes (93). They show promise f o r t h i s a p p l i c a t i o n s i n c e the p e r m e a b i l i t y to "middle-molecular weight" molecules such as i n u l i n i s much h i g h e r f o r PVA-heparin hydrogels than f o r Cuprophan cellophane hemodialysis membranes. R a d i a t i o n c r o s s l i n k e d g e l s of PVA have been proposed f o r use as s y n t h e t i c c a r t i l a g e i n s y n o v i a l j o i n t s (94). The m a t e r i a l prepared f o r t h i s a p p l i c a t i o n i s annealed t o i n c r e a s e the c r y s t a l U n i t y and t h e r e f o r e the p h y s i c a l s t r e n g t h o f the hydrogel. This a p p l i c a t i o n f o r PVA g e l s i s discussed f u r t h e r i n S e c t i o n I I F. A PVA s u r f a c e w i t h a number o f immobilized biomolecules on i t was designed i n an e f f o r t t o simulate the n a t u r a l blood v e s s e l i n t i m a (95). This m a t e r i a l i s described i n S e c t i o n V. 3 5

F. A n i o n i c and C a t i o n i c Hydrogels. A n i o n i c and c a t i o n i c hydrogels are u s u a l l y formed by copolymerizing s m a l l amounts o f a n i o n i c o r c a t i o n i c monomers w i t h n e u t r a l hydrogel monomers (see Table I I ) . However, they can a l s o be prepared by modifying preformed hydrogels such as by the p a r t i a l h y d r o l y s i s of p o l y ( h y d r o x y a l k y l methacrylates) o r , i n the case o f p o l y e l e c t r o l y t e complexes, by adding an excess of the p o l y a n i o n o r p o l y c a t i o n component. The i n t e r e s t i n such hydrogel systems stems from observations on the s u r f a c e charges on blood c e l l s , blood v e s s e l w a l l s , and other t i s s u e types. Under normal c o n d i t i o n s the blood v e s s e l w a l l s and blood c e l l s have a negative charge. Sawyer has presented evidence which i n d i c a t e s that when t h i s charge i s a l t e r e d

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by pH changes, o r by drugs the tendency o f the system t o throm bose i s a l s o a l t e r e d (96). I t i s g e n e r a l l y thought t h a t negative l y charged s u r f a c e s should be l e s s thrombogenic than p o s i t i v e l y charged ones, (97) and experiments have been performed which support t h i s c o n t e n t i o n . However, r e s u l t s i n d i c a t i n g decreased thrombogenicity f o r p o s i t i v e l y charged s u r f a c e s have a l s o been presented (52*98)· The r o l e o f charge d e n s i t y (as opposed t o t o t a l charge) has been suggested as an important f a c t o r w i t h respect t o the thrombogenicity o f s u r f a c e s (97). A t the present time the importance o f s u r f a c e charge i n blood-hydrogel i n t e r a c t i o n s o r i n i n v i v o h e a l i n g i s not a t a l l c l e a r . Cerny, e t a l . , i n v e s t i g a t e d the h e a l i n g o f P-HEMA specimens, some o f which contained charged co-monomers, subcutaneously im planted i n s e v e r a l s p e c i e s o f l a b o r a t o r y animals (35). He found that i n four groups o f methacrylic acid, c a l c i f i c a t i o P-HEMA specimens) was i n h i b i t e d . B a r v i c , e t a l . , found t h a t P-HEMA g e l s c o n t a i n i n g 5 weight % o r g r e a t e r o f the co-monomer d i e t h y l a m i n o e t h y l methacrylate gave r i s e t o inflammatory r e a c t i o n s a f t e r three weeks o f i m p l a n t a t i o n i n r a t s (37). S p r i n c l , et a l . , i n a recent study, i n v e s t i g a t e d the h e a l i n g o f P-HEMA specimens c o n t a i n i n g s m a l l amounts o f copolymerized a n i o n i c (methacrylic a c i d ) and c a t i o n i c (Ν, N, dimethylaminoethyl meth a c r y l a t e ) monomers implanted subcutaneously f o r l o n g p e r i o d s (up t o 360 days) i n r a t s (39). They found t h a t the chemical composition o f the g e l s , wTFhin the c o n c e n t r a t i o n ranges s t u d i e d , showed no apparent e f f e c t on long term h e a l i n g . Thus, the o b s e r v a t i o n by B a r v i c , e t a l , , might o n l y be t r u e f o r short term i m p l a n t a t i o n . S p r i n c l , e t a l . , a l s o showed t h a t f o r P-HEMA g e l s , c a l c i f i c a t i o n apparently o n l y depended on the p h y s i c a l form o f the g e l ( i . e . , macroporous g e l s might c a l c i f y where microporous g e l s wouldn't) r a t h e r than on the chemical composition o f the g e l . Therefore, the i n h i b i t i o n o f c a l c i f i c a t i o n noted by Cerny, e t a l . , might be due t o d i f f e r e n c e s i n the p h y s i c a l s t r u c t u r e o f the gels he used r a t h e r than t o the presence o f 4% m e t h a c r y l i c a c i d . The i n v i t r o thrombogenicity of PAAm g e l s copolymerized w i t h dimethylaminoethyl methacrylate, t - b u t y l a m i n o e t h y l methacry l a t e , 2 - s u l f o e t h y l m e t h a c r y l a t e sodium s a l t , 2-hydroxy-3-methacryloloxypropyltrimethylammonium c h l o r i d e , a c r y l i c a c i d , meth a c r y l i c a c i d , 2 - v i n y l p y r i d i n e , 4 - v i n y l p y r i d i n e and 2-methyl5 - v i n y l p y r i d i n e was s t u d i e d by Halpern, et_ a l . , u s i n g the LeeWhite technique. Only the dimethylaminoethyl methacrylateacrylamide hydrogel showed a s i g n i f i c a n t e x t e n s i o n i n c l o t t i n g times. As was mentioned i n S e c t i o n I I D, p o l y e l e c t r o l y t e com plexes c o n t a i n i n g 0.5 meq. excess a n i o n i c component were found t o have the lowest thrombogenicity i n i n v i v o s t u d i e s (99). These two r e s u l t s are somewhat c o n t r a d i c t o r y as the dimethylaminoethyl methacrylate-acrylamide hydrogel i s p o s i t i v e l y charged a t p h y s i o l o g i c a l pH w h i l e the a n i o n i c p o l y e l e c t r o l y t e complex has a negative charge. Other s t u d i e s on the thrombogenicity o f

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charged hydrogels w i l l be d i s c u s s e d i n S e c t i o n I I I . A unique a p p l i c a t i o n f o r a c a t i o n i c hydrogel has been proposed by Bray and M e r r i l l (94,99). They constructed a s y n t h e t i c a r t i c u l a r c a r t i l a g e m a t e r i a l f o r use i n a s y n o v i a l j o i n t by simu l t a n e o u s l y r a d i a t i o n c r o s s l i n k i n g p o l y ( v i n y l a l c o h o l ) and r a d i a t i o n g r a f t i n g to the PVA chains a c a t i o n i c monomer. Such a mater i a l should s t r o n g l y adsorb n e g a t i v e l y charged h y a l u r o n i c a c i d and produce an "osmotically-enhanced, v i s c o u s g e l l a y e r of boundary l u b r i c a n t " (94). C a t i o n i c monomers which have been used f o r t h i s purpose are allyltrimethyl-ammonium bromide and 2hydroxy-3-methacryloxypropyltrimethylammonium c h l o r i d e . III.

Surface Coated Hydrogels There are a number of techniques which can be used to coat substrates with h y d r o p h i l i Aside from the c o n v e n t i o n a of the polymer, a l l other methods i n v o l v e covalent bonding ( g r a f t i n g ) of the h y d r o p h i l i c polymer to the s u b s t r a t e polymer chains. Table V Techniques f o r D e p o s i t i n g Hydrogel 1. 2. 3.

4.

Coatings

Dip-coat i n pre-polymer + s o l v e n t . Dip i n monomer(s) (+ s o l v e n t , polymer) then polymerize u s i n g c a t a l y s t + heat. P r e - a c t i v a t e s u r f a c e ("active vapor," i o n i z i n g r a d i a t i o n i n a i r ) then contact w i t h monomer(s) + heat to polymerize. Irradiate with ionizing radiation while i n contact w i t h vapor or l i q u i d s o l u t i o n of monomer ( s ) .

By c o v a l e n t l y bonding a hydrogel to the s u r f a c e of another polymer a new composite m a t e r i a l i s formed whose mechanical prope r t i e s more c l o s e l y resembles those of the base polymer than the t h i n g r a f t e d hydrogel l a y e r . The most e f f i c a c i o u s technique f o r p r e p a r i n g such m a t e r i a l s i n v o l v e s generating f r e e r a d i c a l s on a p l a s t i c s u r f a c e and then p o l y m e r i z i n g a monomer d i r e c t l y on that s u r f a c e . A number of techniques have been used f o r generating such r a d i c a l s on s u r f a c e s . Some of the more commonly used ones are l i s t e d i n Table VI. There are at l e a s t f i v e advantages to u s i n g g r a f t i n g t e c h niques f o r p r e p a r i n g b i o m e d i c a l hydrogels. The f i r s t and most obvious advantage i s the i n c r e a s e i n mechanical s t r e n g t h over the ungrafted hydrogel which can be obtained. A second advantage i s the permanence and d u r a b i l i t y which should be e x h i b i t e d by the c o v a l e n t l y bound hydrogel c o a t i n g as compared to coatings on devices prepared by d i p p i n g techniques. T h i r d , g r a f t p o l y m e r i z a -

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

RATNER AND H O F F M A N

Synthetic Hydrogels

17

T A B L E VI Techniques

ond Reoctions for Generoting Rodicols on Surfaces.

IONIZING RADIATION CH C H 3

CH

3

CH \ΛΛΛΛ

—

Co^

V/////

Electron Irrodiotion

CER1C+1Y ION OH OH OH 1 I I CH CH CH ////////// 2

2

Ο I CH

Ce

2

PEROXIDE FORMATION I

CH CH

Ç3 Ç 3 Ç 3 ///λ/λ/ / / / / / / / / ) / 0 /ionizing rodiotion H

H

3

CH3

2

CH

/)//////////

3

^

3

H

A,Fe

+ I

+

OH OH I I CH CH

2

2

0 C1

CH

3

"

_

2

CH-a

H

3

- +1 ) / / / / / / / / + OH +Fe

2

ACTIVE VAPOR ACTIVATION CH

3

^^3

^^3

)///)//)/

CH CH CH J , , ,I , >I ; +H 2

microwave generated Η·

3

3

2

U.V. GRAFTING C H

3

C

H

3

C

H

CH

3 photosensitizer

2

C H 3 CH3 γ γ + Η: photosensitizer

ΝΟΤΕ·· The precise noture of the radical intermediates formed has not been elucidated in most cases. Representations in this table show schematically radical species which might be formed.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

18

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

t i o n techniques make i t p o s s i b l e to prepare complex surfaces formed by s u c c e s s i v e g r a f t i n g s u s i n g d i f f e r e n t monomers. Fourth, the p r e p a r a t i o n of hydrogels g r a f t e d only on the s u r f a c e , p a r t i a l l y i n t o the s u b s t r a t e , or u n i f o r m l y throughout a hydrophobic m a t r i x can be e f f e c t e d by v a r y i n g the p o l y m e r i z a t i o n s o l v e n t and other g r a f t i n g parameters. The l a t t e r type of g r a f t e d hydrogel comprises an i n t e r e s t i n g c l a s s of m a t e r i a l s which should have mechanical and s u r f a c e p r o p e r t i e s r e f l e c t i n g the c h a r a c t e r i s t i c s of both the s u b s t r a t e and the hydrogel. F i n a l l y , u s i n g r a d i a t i o n to prepare a g r a f t e d h y d r o g e l , the a d d i t i o n of an i n i t i a t o r i s not necessary, thereby e l i m i n a t i n g one p o t e n t i a l source of contamination i n the f i n a l product. There are c e r t a i n u n d e s i r a b l e s i d e r e a c t i o n s which can occur w i t h g r a f t p o l y m e r i z a t i o n s , p a r t i c u l a r l y those i n i t i a t e d by i o n i z i n g r a d i a t i o n . Thes i n c l u d polyme degradation crosslinkin and the formation of unwante a c i d s ) . However, degradatio and c r o s s l i n k i n g ca be minimized by u s i n g low doses and the formation of most unwanted f u n c t i o n a l groups can be e l i m i n a t e d by e x c l u d i n g oxygen and r e a c t i v e solvents from the g r a f t i n g system. As has been noted above, g r a f t i n g c o p o l y m e r i z a t i o n techniques provide a convenient means f o r c o n t r o l l i n g composition, penetrat i o n and morphology of the g r a f t e d polymer. Such c o n t r o l should be u s e f u l f o r " t a i l o r i n g " a g r a f t e d polymer to a given biomedical a p p l i c a t i o n . D e t a i l e d d e s c r i p t i o n s of methods which have been used to vary the s u r f a c e c h a r a c t e r of g r a f t e d hydrogels f o r b i o medical uses have been d e s c r i b e d i n a few papers (100-102). Examples of some of the g r a f t i n g parameters which can be used to i n f l u e n c e the p r o p e r t i e s of r a d i a t i o n g r a f t e d hydrogels i n c l u d e r a d i a t i o n dose, dose r a t e , monomer c o n c e n t r a t i o n , g r a f t i n g s o l vent, temperature, and the presence of v a r i o u s metal ions i n the g r a f t i n g system. One of the e a r l i e s t a p p l i c a t i o n s of g r a f t p o l y m e r i z a t i o n techniques to the p r e p a r a t i o n of m a t e r i a l s f o r biomedical a p p l i c a t i o n s was reported i n 1964 by Yasuda and Refojo (103). They g r a f t e d N - v i n y l - 2 - p y r r o l i d o n e to s i l i c o n e rubber i n an e f f o r t to i n c r e a s e the h y d r o p h i l i c i t y of the rubber s u r f a c e . In 1966, L e i n i n g e r and co-workers discussed the p o s s i b i l i t y of g r a f t i n g v i n y l p y r i d i n e to a base p l a s t i c f o r use i n i m m o b i l i z i n g heparin to the s u r f a c e (104). L a i z i e r and Wajs i n 1969 described a transparent polymer prepared by g r a f t i n g NVP to a s i l i c o n e r e s i n which might be s u i t a b l e f o r contact l e n s a p p l i c a t i o n s (105). W i t h i n the l a s t f i v e yeais a number of groups have p u b l i s h e d papers or r e p o r t s d e s c r i b i n g g r a f t e d hydrogels designed f o r b i o medical a p p l i c a t i o n s . Much of the work i n t h i s f i e l d i s summari z e d i n Table V I I . There has been l i t t l e published on the i n v i v o e v a l u a t i o n of t i s s u e response to g r a f t e d hydrogels, i n a p r e l i m i n a r y , and as yet unpublished study, r a d i a t i o n g r a f t e d P-HEMA and PAAm hydrog e l s on s i l i c o n e rubber were found t o be w e l l t o l e r a t e d when

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Group

grafting

Electron irradiation, treatments

Acrylic Acid, Methacrylic Acid, Acrylamide, Ethylene S u l f o n i c A c i d , V a r i o u s E s t e r s and Imides

E l e c t r o n i r r a d i a t i o n o f preformed poly-electrolyte coating, C o mutual i r r a d i a t i o n C e r i c IV i o n g r a f t i n g , r a d i a t i o n g r a f t i n g (Mutual i r r a d i a t i o n tech.)

V i n y l A c e t a t e - c o - 2 % Crof.onic A c i d , NVP-co-2% A c r y l i c A c i d

A c r y l a m i d e , HEMA

HEMA

Polysciences, Inc.

U.S. Army M e d i c a l B i o e n g i n e e r i n g Research & Development L a b o r a t o r y

Univ. o f W i s c o n s i n

Hydromed S c i e n c e s

Union C a r b i d e

Corp.

(Mutual i r r a d i a t i o n

(Mutual i r r a d i a t i o n (Mutual i r r a d i a t i o n

6 0

6 0

6 0

Co

Co Co

HEMA

HEMA

6 0

technique)

technique)

technique)

grafting

technique)

A t o m i z e d gas p l a s m a technique

technique)

HEMA, A c r y l a m i d e

Institute

irradiation

Franklin

(Mutual i r r a d i a t i o n (Mutual

Co

6 0

Co

HEMA

Acrylamide

Kearney, e t a l .

6 0

technique)

Andrade, e t a l .

(Mutual i r r a d i a t i o n

6 0

Co

HEMA, NVP, A c r y l a m i d e , Meth acrylamide, Methacrylic a c i d , Ethyl methacrylate

chemical

technique)

Used

Hoffman, e t a l .

eta l .

Miller,

(pre-irradiation

Co

6 0

Radiation

Vinyl

Pyridine

irradiation

Electron

NVP

NVP

Columbus

G r a f t i n g Technique(s)

Monomer(s) Used

L a i z i e r and Wais

Battelle,

Yasuda and R e f o j o

Research

Grafted Hydrogels Prepared For Biomedical A p p l i c a t i o n s

Table V I I

applications

(113-115)

Artificial

(119)

(117) catheters

membrane

Dialysis IUD,

(118)

Burn d r e s s i n g

Blood oxygenator (hollow f i b e r ) , i n t r a (9, 115, aortic assist balloons, assist bladders, 116) a o r t i c implant tubes

h e a r t and o r g a n s

(9, 47, 112,113)

(111)

(102)

(100,101, 107-110)

(106)

(105)

(104)

(103)

Blood & t i s s u e compatible m a t e r i a l s , h e a r t a s s i s t d e v i c e s , I.U.D.

interface

h e a r t components

Blood compatible

Artificial

A r t i f i c i a l h e a r t components, c a t h e t e r s , k n i t t e d a r t e r y prostheses, blood & t i s s u e compatible i n t e r f a c e s

Non-thrombogenic s u r f a c e s

Contact lenses

Non-thrombogenic p l a s t i c s u r f a c e

Medical

Applications

sr

Ci

Ο

133

it

C/D

>

ο

ι

20

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

implanted both i n t r a p e r i t o n e a l l y and subcutaneously i n r a t s . The f i l m s were surrounded by a t h i n non-adhering f i b r o u s capsule a f t e r i m p l a n t a t i o n periods ranging from 5-10 days (120). I n an i n v i t r o study on adhesiveness of c h i c k embryo myoblasts to r a d i a t i o n g r a f t e d P-HEMA and N-VP hydrogels on s i l i c o n e rubber i t was found t h a t the c e l l s adhere very p o o r l y to g r a f t e d hydrogels and that the hydrogel g r a f t e d polymers always adhered fewer c e l l s than the ungrafted polymers (110). Whether such r e s u l t s are meaningful i n terms of i n v i v o c e l l - g r a f t e d hydrogel i n t e r a c t i o n s i s not yet clear. The blood c o m p a t i b i l i t y of g r a f t e d hydrogels has been examined i n a number of cases by the vena cava r i n g t e s t and r e n a l embolus r i n g t e s t . C e r t a i n g e n e r a l i z a t i o n s can be made from these results. 1. P-HEMA or P-NVP hydrogels g r a f t e d to s i l i c o n e rubber w i l l g r e a t l y reduce the thrombogenicit judged by the vena cav g 2. When evaluated by the vena cava r i n g t e s t s e v e r a l d i f f e r ent types of g r a f t e d hydrogels have been found to perform w e l l , but some thrombus i s u s u a l l y noted adhering to the r i n g , p a r t i c u l a r l y a f t e r the two week t e s t p e r i o d . An e x c e p t i o n to t h i s i s g r a f t e d polyacrylamide m a t e r i a l s which have, i n some cases, shown no thrombus a f t e r 14 days (4,121). Recent r e s u l t s , however, u s i n g a m o d i f i c a t i o n of the vena cava r i n g t e s t do show thrombus adhering to g r a f t e d acrylamide r i n g s i n four out of s i x cases ( 8 ) . A 60% sodium ionomer of p o l y ( v i n y l acetate-co-2% c r o t o n i c a c i d ) has, i n c e r t a i n t e s t s i t u a t i o n s , a l s o showed n e g l i g i b l e thrombus accumu l a t i o n a f t e r 14 days (114). 3. In the r e n a l embolus t e s t , although l i t t l e thrombus i s found w i t h i n most hydrogel t r e a t e d t e s t r i n g s , the kidneys almost always show moderate to e x t e n s i v e embolus damage ( 9 ) . Thus, the g e n e r a l l y low thrombogenicity r a t i n g of s y n t h e t i c hydrogels based on the vena cava r i n g t e s t , may, i n f a c t , be due to a low thromboadherance, i . e . a f l a k i n g o f f of thrombus from the hydrogel s u r f a c e . This aspect of hydrogel-blood i n t e r a c t i o n i s much i n need of f u r t h e r i n v e s t i g a t i o n . Recently p l a t e l e t adhesiveness to r a d i a t i o n g r a f t e d P-HEMA hydrogels on c e l l u l o s e a c e t a t e was measured i n an i n v i t r o t e s t c e l l . The per cent p l a t e l e t s adhering was found to decrease w i t h i n c r e a s i n g g r a f t l e v e l ( f i g u r e 1) (117). This trend was very s i m i l a r to t h a t noted f o r f i b r i n o g e n a d s o r p t i o n to P-HEMA g r a f t e d s i l i c o n e rubber s u r f a c e s ( f i g u r e 2) (109). Decreased adhesiveness of c e l l s to hydrogel g r a f t e d s u r f a c e s was a l s o noted i n the c e l l adhesion study d i s c u s s e d above (110). Again, these observat i o n s tend to support the i d e a t h a t hydrogels have a low thromboadherence.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RATNER AND H O F F M A N

Synthetic Hydrogeh

Ο.ΟΙ

2 GRAFT

3

6

4

(mg/cm2) ACS Advances in Chemistry Series

Figure 2. Fibrinogen

adsorption to radiation grafted HEMA silicone rubber ( 109 )

hydrogels on

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

IV.

C h a r a c t e r i z a t i o n o f the Imbibed Water C l e a r l y , the presence o f l a r g e amounts o f water w i t h i n a polymeric network i s not the s o l e f a c t o r c o n t r o l l i n g the biocomp a t i b i l i t y and thrombogenicity o f such m a t e r i a l s . Thus, g e l a t i n or some p o l y s a c c h a r i d e s which o f t e n have water contents o f 90% or h i g h e r are considered t o be r e l a t i v e l y thrombogenic m a t e r i a l s . A l s o , extremely "open" P-HEMA g e l s which e x h i b i t h i g h water cont e n t s are l e s s t i s s u e compatible than t i g h t e r P-HEMA g e l s w i t h lower water contents (39). S t i l l , the presence o f a s i g n i f i c a n t f r a c t i o n o f water i s considered important f o r the b i o c o m p a t i b i l i t y and low thrombogenicity o f those hydrogels which are biocompatible and "reasonably" non-thrombogenic (or non-thromboadherent) ( 4 ) . The nature o r o r g a n i z a t i o n o f the water w i t h i n the hydrated polymeric network may be one o f the f a c t o r s which w i l l i n f l u e n c e the i n t e r a c t i o n s that occu hydrogel. Problems concerning the o r g a n i z a t i o n of water a t the molecul a r l e v e l (water s t r u c t u r e ) are o f t e n extremely complex and t h e l i t e r a t u r e on the subject i s e x t e n s i v e . There are e x c e l l e n t r e views a v a i l a b l e on water s t r u c t u r i n g (122,123). A l s o , a number of review a r t i c l e s have been w r i t t e n on the b i o l o g i c a l i m p l i c a t i o n s o f s t r u c t u r e d water (124-126) and the p o t e n t i a l importance of the molecular o r g a n i z a t i o n of water t o the performance o f b i o m a t e r i a l s (127,128). The gross t o t a l water contents o f swollen hydrogels are most e a s i l y measured and most o f t e n reported. I n f o r m a t i o n about the molecular nature o f water w i t h i n the network i s not as easy t o o b t a i n ; such water may be (a) p o l a r i z e d around charged i o n i c groups, (b) o r i e n t e d around hydrogen bonding groups o r other d i p o l e s , (c) s t r u c t u r e d i n " i c e - l i k e " c o n f i g u r a t i o n s around hydrophobic groups, and/or (d) imbibed i n l a r g e pores as "normal" b u l k water. Attempts have been made t o separate the t o t a l g e l water content i n t o some o f these c a t e g o r i e s u s i n g NMR techniques (129). Based upon the NMR data g e l water contents would be d i v i d e d i n t o "bulk water" (category d ) , "bound water" ( c a t e g o r i e s a, b) and the remaining water, c a l l e d " i n t e r f a c e water" (category c ) . R e s u l t s f o r P-HEMA g e l s shown i n Table V I I I suggest that the f r a c t i o n s o f b u l k , bound and i n t e r f a c i a l water v a r y w i t h the t o t a l water content o f the g e l . I n c r e a s i n g f r a c t i o n s o f b u l k water and d e c r e a s i n g f r a c t i o n s of bound water are found as the water content o f the g e l i n c r e a s e s . The f r a c t i o n o f i n t e r f a c i a l water undergoes o n l y s m a l l changes w i t h changing g e l water cont e n t . S i m i l a r c o n c l u s i o n s were drawn concerning the s t a t e o f water i n P-HEMA g e l s which were i n v e s t i g a t e d u s i n g the techniques of d i l a t o m e t r y , s p e c i f i c c o n d u c t i v i t y and d i f f e r e n t i a l scanning c a l o r i m e t r y (130). Water s o r p t i o n s t u d i e s which p r o v i d e some i n s i g h t i n t o the k i n e t i c s and thermodynamics o f water i n t e r a c t i o n w i t h hydrogels have a l s o been performed (131 ,132).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RATNER AND H O F F M A N

1.

23

Synthetic Hydrogeh

Table V I I I The F r a c t i o n of Water i n P-HEMA Gels of D i f f e r e n t T o t a l Water Content (Wt %) [Data from Lee, Andrade and Jhon, (114)] Wt % of T o t a l Water i n the Gel f w

f,

20

25 30

35

40

45

50

55

F r a c t i o n of 0 0 0.09 0.21 0.30 0.37 0.42 Bulk Water 0 F r a c t i o n of 0 0.2 0.33 0.34 0.29 0.26 0.33 0.22 I n t e r f a c i a l Water F r a c t i o n of 1.0 0.8 0.67 0.57 0.50 0.44 0.40 0.36 Bound Water

60 0.47 0.20 0.33

Studies to date o g e l s are only p r e l i m i n a r of water o r g a n i z a t i o n on b i o l o g i c a l i n t e r a c t i o n s remain to be e l u c i d a t e d . I t should be noted t h a t the o r g a n i z a t i o n and content of g e l water w i l l vary s i g n i f i c a n t l y w i t h hydrogel composition, probably most o f t e n i n expected d i r e c t i o n s ( i . e . , g e l s w i t h higher water contents w i l l have lower f r a c t i o n s of bound and i n t e r f a c i a l water). V.

I m m o b i l i z a t i o n and Entrapment of B i o l o g i c a l l y A c t i v e Molecules on and W i t h i n Hydrogels f o r B i o m a t e r i a l A p p l i c a t i o n s . Hydrogels a r e , i n many r e s p e c t s , eminently s u i t e d f o r use as a base m a t e r i a l f o r " b i o l o g i c a l l y a c t i v e " b i o m a t e r i a l s . Examples of c l a s s e s of b i o l o g i c a l l y a c t i v e molecules which can be used i n c o n j u n c t i o n w i t h hydrogels are l i s t e d i n Table IX. Examples of b i o m e d i c a l a p p l i c a t i o n s f o r immobilized enzymes are presented i n Table X. There are a number of d i s t i n c t advantages f o r hydrogels i n these types of systems. Small molecules (drugs, enzyme subs t r a t e s ) can d i f f u s e through hydrogels and the r a t e of permeation can be c o n t r o l l e d by c o - p o l y m e r i z i n g the hydrogel i n v a r y i n g r a t i o s w i t h other monomers. Hydrogels may i n t e r a c t l e s s s t r o n g l y than more hydrophobic m a t e r i a l s w i t h the molecules which are immobilized to or w i t h i n them thus l e a v i n g a l a r g e r f r a c t i o n of the molecules a c t i v e (3)· Hydrogels can be l e f t i n contact w i t h blood or t i s s u e f o r extended p e r i o d s of time without causing r e a c t i o n making them u s e f u l f o r devices to be used i n long-term treatment of v a r i o u s c o n d i t i o n s . F i n a l l y , hydrogels u s u a l l y have a l a r g e number of p o l a r r e a c t i v e s i t e s on which molecules can be immobilized by r e l a t i v e l y simple c h e m i s t r i e s .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

24

HYDROGELS FOR MEDICAL AND RELATED

APPLICATIONS

Table IX B i o l o g i c a l l y A c t i v e Molecules which may be Entrapped or Immobilized i n Hydrogels Antibiotics Anticoagulants Anti-Cancer Drugs Antibodies Drug A n t a g o n i s t s Enzymes Contraceptives Estrous-Inducers A n t i - b a c t e r i a Agents

Biomedical A p p l i c a t i o n Immobilized Enzyme(s)

Application

Brinolase Urokinase Streptokinase Asparaginase,Glutaminase Carbonic Anhydrase, Catalase Urease Glucose Oxidase

Non-Thrombogenie Surface Non-Thrombogenic Surface Non-Thrombogenie Surface Leukemia Treatment

(133) (134) (135) (136)

Membrane Oxygenator A r t i f i c i a l Kidney Glucose S e n s o r - A r t i f i c i a l Pancreas A r t i f i c i a l Liver Blood A l c o h o l E l e c t r o d e Removal of A i r b o r n Infections

(137) (138)

Microsomal Enzymes A l c o h o l Oxidase DNase, RNase

Reference

(139) (140) (141) (142)

B i o l o g i c a l l y a c t i v e molecules can be immobilized w i t h i n hydrogels permanently, or t e m p o r a r i l y . I f the hydrogel system i s designed to r e l e a s e the entrapped b i o l o g i c a l l y a c t i v e molec u l e s at a p r e s e t r a t e , these m a t e r i a l s are w e l l s u i t e d f o r use as c o n t r o l l e d drug d e l i v e r y devices. I n the s i m p l e s t example of such a system, hydrogels can be s a t u r a t e d w i t h s o l u t i o n s of v a r ious a n t i b i o t i c s and other drugs which w i l l l e a c h out to the surrounding t i s s u e upon i m p l a n t a t i o n . A number of papers d e s c r i b i n g such hydrogel drug d e l i v e r y systems have already been mentioned (41-46,95). The r a t e of drug d e l i v e r y g e n e r a l l y decreases r a p i d l y w i t h simple homogeneous hydrogels saturated w i t h a drug s o l u t i o n . By u s i n g a b i o c o m p a t i b l e hydrogel membrane device f i l l e d w i t h a drug i n the form of a pure l i q u i d or s o l i d , constant drug d e l i v e r y r a t e s and extended treatment times can be obtained (143,144). A s t i l l more s o p h i s t i c a t e d approach i n v o l v e s the d e s i g n of a h y d r o p h i l i c polymer backbone c h a i n onto which a l t e r n a t e " c a t a l y s t groups and l a b i l e "drug" groups are 11

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.

RATNER AND H O F F M A N

Synthetic

Hydrogeh

25

HYDROPHILIC BACKBONE POLYMER

C=0 CATALYTIC—• Η Ν'· GROUP R Figure S.

C=0

ΗΝ·· I

R

I

+

0"

DRUG

Drug release from a polymeric chain controlled by intramolecular catalysis

bound (e.g. F i g u r e 3). The k i n e t i c s o f v a r i o u s i n t r a m o l e c u l a r l y c a t a l y z e d polymeric r e a c t i o n s have been described (145). Hydrogels which are prepared by the s o l u t i o n p o l y m e r i z a t i o n of a monomer i n the presence of a c r o s s l i n k i n g agent are w e l l s u i t e d f o r entrapping an a c t i v e biomolecule w i t h i n the network s t r u c t u r e . For the i m m o b i l i z a t i o n o f an enzyme by the entrapment technique, leakage o f the enzyme would be u n d e s i r a b l e . There f o r e , the "pore s i z e o r average i n t e r c h a i n d i s t a n c e o f the g e l should be s m a l l e r than the s i z e o f the a c t i v e enzyme. A "pore" s i z e o f 35 A o r s m a l l e r should be s u i t a b l e f o r r e t a i n i n g most entrapped enzymes (146). For comparison, homogeneous P-HEMA g e l s have estimated "pore" s i z e s o f approximately 4-5 A (5g,147)> w h i l e acrylamide g e l s might have "pore" s i z e s from 7 A - 17 A depending upon the method o f p r e p a r a t i o n (148). However, these pore s i z e s are probably low w i t h an e r r o r estimated a t greater than 25% (4). Recent r e p o r t s on enzyme entrapment i n c l u d e sys tems i n v o l v i n g glucose oxidase entrapped i n P-HEMA and P-NVP(149) glucoamylase, i n v e r t a s e , and 3-galactosidase entrapped i n p o l y (2-hydroxyethyl a c r y l a t e ) and poly(dimethylacrylamide) g e l s (150) and asparaginase and microsomal enzymes entrapped i n P-NVP g e l s (151). The entrapment o f heparin i n a PVA g e l was described i n S e c t i o n I I Ε (92). Techniques f o r the covalent i m m o b i l i z a t i o n o f a c t i v e mole c u l e s t o s u r f a c e s have been the subject o f a number o f recent i n depth reviews (140,152,153). A l a r g e number o f chemical t e c h niques which are p a r t i c u l a r l y a p p l i c a b l e f o r i m m o b i l i z a t i o n t o hydrogels have been developed. Figure 4 shows c h e m i s t r i e s u s e f u l f o r c o u p l i n g biomolecules t o g e l s c o n t a i n i n g c a r b o x y l groups. F i g u r e 5 i l l u s t r a t e s the probable r e a c t i o n s o c c u r r i n g during the i m m o b i l i z a t i o n o f a p r o t e i n t o a polymer which con t a i n s h y d r o x y l groups (154). The Ugi r e a c t i o n i s a four compo nent condensation r e a c t i o n which occurs between an amine, an aldehyde, a c a r b o x y l i c a c i d and an i s o c y a n i d e (155). I t i s o f i n t e r e s t f o r i m m o b i l i z a t i o n t o hydrogels because o f the many 11

Q

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6).

5)·

*

+

Figure 4.

-CC^H

-CO

•CO

i-CO?H +

4).

C H

i-COgH-h S 0 C (

3).

2) .

C

C

Η

/ " x

0

-

^

2

2

.CO^i^CH-CO-NHR

2

-C0 H

V

2

*2

e.g., PROTEIN - C 0 H

PROTEIN

PROTEIN-NH2

PROTEIN-NH

2

2

2

J o ON Η PROTEIN

^-C0 -CH CHOH-R-CHOH-CH -0 C-PROTEIN -CONH - PROTEIN

?

CO -OU-CHOH-R-CH-CH

ί

i - C O N H -

k02-N^J

CONH - PROTEIN

SURFACES 1

Chemistries useful for coupling biomolecules to gels containing carboxyl groups

Η

2

Νφ B F

PROTEIN-NH

V

2

OH

PROTEIN-NH'

ON POLYCARBOXYLIC

e.g., P R O T E I N - N H 2

m

PROTEINS

4-coce

A

— C H - R - C H - C H

V

2

2

1) . ij-copH 3-CO?H + R - C = N=C-R

IMMOBILIZING

2

ο

> ha r* Ο

w

> > ϋ

ο

a

M

S3

3

ο w

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to

1.

RATNER AND H O F F M A N

Synthetic Hydrogels

27

p o t e n t i a l r e a c t i o n s which might be used depending upon which f u n c t i o n a l groups are on the hydrogel and which are on the b i o molecule to be immobilized. The Ugi r e a c t i o n used t o couple a p r o t e i n - NH group t o a c a r b o x y l i c hydrogel i s shown i n F i g u r e 6. Glutaraldehyde i s o f t e n used f o r i m m o b i l i z a t i o n o f biomolecules to polyacrylamide hydrogels although the p r e c i s e mechanism o f c o u p l i n g i s not yet known (156). There have been s u r p r i s i n g l y few r e p o r t s on the development of m a t e r i a l s intended t o be both biocompatible and b i o l o g i c a l l y a c t i v e . Many a c t i v e biomolecules have been bound t o supports such as Sephadex and Sepharose (modified p o l y s a c c h a r i d e s ) which allow l a r g e amounts o f a c t i v e biomolecule to be immobilized but which would not be expected to show s i g n i f i c a n t b i o c o m p a t i b i l i t y . Devices s p e c i f i c a l l y constructed f o r the i m m o b i l i z a t i o n o f enzymes t o be used i n contac of such m a t e r i a l s as poly(methy c h l o r i d e o r polycarbonate (134), a l l o f which are considered to be r a t h e r thrombogenic s u r f a c e s (although c o l l o i d a l g r a p h i t e was used on some o f the s u r f a c e s , presumably to reduce thrombog e n i c i t y ) . One o f the e a r l i e s t papers d e s c r i b i n g an " a c t i v e " b i o m a t e r i a l prepared by combining r a d i a t i o n g r a f t polymerization plus biochemical and medical concepts i s by Hoffman, ert a l . , (135). In t h i s study s t r e p t o k i n a s e , albumin and heparin were immobilized on r a d i a t i o n g r a f t e d hydrogels based upon P-HEMA and P-NVP. Streptokinase immobilized v i a an "arm" demonstrated s i g n i f i c a n t fibrinolytic activity. Immobilized heparin, on the other hand, d i d not seem to r e t a i n b i o l o g i c a l a c t i v i t y when immobilized to these surfaces u s i n g e i t h e r BrCN o r carbodiimide c h e m i s t r i e s . Nguyen and Wilkes have d e s c r i b e d a f i b r i n o l y t i c s u r f a c e made by immobilizing b r i n o l a s e to E n z a c r y l , a p a r t i c u l a t e , crosslinked, modified polyacrylamide (133). S i g n i f i c a n t b r i n o l a s e a c t i v i t y was maintained. The authors suggest a g r a f t e d polymer u t i l i z i n g a c r y l i c a c i d and N - a c r y l o y l para-phenylene diamine as a more p r a c t i c a l m a t e r i a l f o r both immobilizing b r i n o l a s e and c o n s t r u c t ing u s e f u l d e v i c e s . Another approach to preparing a blood compatible s u r f a c e based upon the i m m o b i l i z a t i o n o f biomolecules to hydrogels has been taken by Lee, e t a l . (95). They prepared a three l a y e r support m a t e r i a l w i t h PVA a t the s u r f a c e . Onto the PVA they e s t e r i f i e d f i r s t , h a l f c h o l e s t e r o l e s t e r s of d i c a r b o x y l i c a c i d s and next, the h a l f s i a l i c a c i d e s t e r o f a longer chain dicarboxy l i c a c i d . The s u r f a c e was f i n a l l y t r e a t e d w i t h t i s s u e c u l t u r e medium t o c o n d i t i o n i t w i t h s a l t s and p r o t e i n s found i n the blood. The r a t i o n a l e behind the m a t e r i a l was to simulate the n a t u r a l blood v e s s e l i n t i m a . Vena cava r i n g t e s t s i n d i c a t e d g e n e r a l l y poor thromboresistance f o r these complex s u r f a c e s (113, 115). Renal embolus r i n g s were r e l a t i v e l y f r e e o f thrombus, but the kidneys o f t e s t animals were o f t e n massively i n f a r c t e d (9). I t has been proposed that an " a r t i f i c i a l pancreas" might be constructed by combining a blood glucose sensor w i t h feedback to 2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

28

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

0C0NH

2

Carbamate (inert) ^-0-C=N

OH BrCNL

Activation step

^OH

/ /

OH Imidocarbonate (reactive)

C = NH Intermediate cyanate structure

4- O - C - N H - Protein II NH -OH

Coupling step

H N-Protein

C = NH

Isourea derivative

N-substituted imidocarbonate

C = N - Protein

2

Imidocarbonate

;-0H Transactions—American Society for Artificial Internal Organs

Figure 5. Immobilization

of a protein to a polymer containing hydroxyl groups (135)

\

H® C=0

+

H N-PROTEIN 2

C = Ν-PROTEIN + H 0 2

1! ® C - N H - PROTEIN

^NH-PROTEIN

R' \ NH N

R

c

\

o

v

o

0'

PROTEIN

C

, -N R V

-°

Enzyme Engineering

Figure 6. The Ugi reaction as might be used for the of a protein to a surface ( 136 )

immobilization

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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RATNER AND H O F F M A N

Synthetic Hydrogels

29

an i n s u l i n d e l i v e r y system. P o t e n t i a l l y , other h e a l t h problems could a l s o be t r e a t e d u s i n g a combination of a blood sensor and a drug or hormone d e l i v e r y system. Enzymatic e l e c t r o d e s which can be designed to show h i g h s p e c i f i c i t y f o r a given type of molecule might be p a r t i c u l a r l y w e l l s u i t e d f o r use as blood sensors (157). Techniques have been r e c e n t l y described whereby an enzyme i s simultaneously entrapped w i t h i n a c r o s s l i n k e d hydrogel and coated onto a g l a s s e l e c t r o d e . Polyacrylamide hydrog e l s have been u t i l i z e d f o r t h i s a p p l i c a t i o n (158). Such e l e c t r o d e s might be expected to show both h i g h s p e c i f i c i t y and non-thrombogenicity making them p a r t i c u l a r l y w e l l s u i t e d f o r blood sensor a p p l i c a t i o n s . VI.

Conclusions Hydrogels as a c l a s t i l i t y and e x c e l l e n t performanc c a t i o n s . However, i n the 15 years s i n c e s y n t h e t i c hydrated polymeric networks were f i r s t proposed f o r b i o m a t e r i a l a p p l i c a t i o n s the " s u r f a c e has b a r e l y been s c r a t c h e d " w i t h respect to new types of hydrogels which might by s y n t h e s i z e d , fundamental knowledge concerning how and why hydrogels "work", and new b i o medical a p p l i c a t i o n s f o r these u s e f u l polymers. S p e c i f i c areas which are c l e a r l y i n need of f u r t h e r study before the f u l l p o t e n t i a l of b i o m e d i c a l hydrogels can be r e a l i z e d a r e : (1) Thrombogenicity of hydrogel s u r f a c e s , p a r t i c u l a r l y w i t h respect to emboli formation. (2) C e l l adhesion and i n t e r a c t i o n w i t h hydrated polymer networks, e s p e c i a l l y w i t h c e l l types such as p l a t e l e t s , leukocytes and f i b r o b l a s t s . (3) The s t r u c t u r e of water w i t h i n hydrogels and i t s p o t e n t i a l r e l a t i o n s h i p to b i o l o g i c a l i n t e r a c t i o n s . (4) The behavior of b i o l o g i c a l molecules immobilized to hydrogels. (5) The i n t e r a c t i o n of a n i o n i c and c a t i o n i c hydrogels w i t h blood and other b i o l o g i c a l systems.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22. 23. 24. 25. 26.

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AND HOFFMAN

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118. Meaburn, G.M., Cole, C.M. Hosszu, J.L., Wade, C.W., and Eaton, J., Abstracts - Fifth International Congress of Radiation Research, (1974), Seattle, Washington, July 14-20, 200. 119. Abrahams, R.A. and Ronel, S.H., Society Plast. Eng. - Technical Papers, (1975), 21, 570. 120. Lagunoff, D. and Horbett, T.A., unpublished observations. 121. Ratner, B.D., Hoffman, A.S. and Whiffen, J.D., unpublished observations. 122. Eisenberg, D. and Kauzmann, W., "The Structure and Properties of Water", Oxford University Press, N.Y.,(1969). 123. "Water - A Comprehensive Treatise", Vol. 1, ed. by F. Franks, Plenum Press, New York, (1972). 124. Drost-Hansen, W. in "Chemistry of the Cell Interface" Part B, ed. by p. 1. 125. Ling, G.N., Int. J. Neuroscience, (1970), 1, 129. 126. Klotz, I.M., in "Membranes and Ion Transport", Vol. 1, ed. by E.E. Bittar, John Wiley, Inc., N.Y.,(1970), p. 93. 127. Andrade, J.D., Lee, H.B., Jhon, M.S., Kim, S.W., and Hibbs, Jr., J.B., Trans. Am. Soc. Artif. Int. Organs, (1973), 19, 1. 128. Jhon, M.S. and Andrade, J.D., J. Biomed. Mater. Res., (1973), 7, 509. 129. Lee, H.B., Andrade, J.D. and Jhon, M.S., A.C.S. Polymer Preprints, (1974), 15(1), 706. 130. Lee, H.B., Jhon, M.S. and Andrade, J.D., J. Colloid Interface Sci., (1975), 51, 225. 131. Khaw, B., M.S. Thesis, (1973), University of Washington. 132. MacKenzie, A.P. and Rasmussen, D.H., in "Water Structure at the Water-Polymer Interface" (1972), ed. by H.H.G. Jellinek, Plenum Publishing Corp., N.Y., 146. 133. Nguyen, A. and Wilkes, G.L., J. Biomed. Mater. Res., (1974), 8, 261. 134. Kusserow, B.K., Larrow, R.W. and Nichols, J.E., Trans. Am. Soc. Artif. Int. Organs, (1973), 19, 8. 135. Hoffman, A.S., Schmer, G., Harris, C., Kraft, W.G., Trans. Am. Soc. Artif. Int. Organs, (1972), 18, 10. 136. Hersh, L.S., in "Enzyme Engineering, Vol. 2", ed. by E.K. Pye and L.B. Wingard, Jr., Plenum Press, N.Y., (1974), p. 425. 137. Broun, G., Tran-Minh, C., Thomas, D., Domurado, D. and Selegny, E., Trans. Am. Soc. Artif. Int. Organs, (1971), 17, 341. 138. Chang, T., "Artificial Cells", Charles C. Thomas, Springfield, Ill., (1972), p. 150. 139. Guilbault, G.G. and Lubrano, G., Anal. Chim. Acta., (1973), 64, 439. 140. Eiseman, B. and Soyer, T., Transplant. Proc., (1971), 3, 1519.

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In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2 Vapor Pressure and Swelling Pressure of Hydrogels MIGUEL F. REFOJO Eye Research Institute of Retina Foundation, Boston, Mass. 02114

H y d r o g e l s c o n s i s t o f two c o m p o n e n t s : the polymer network, which i s constant i n q u a n t i t y is variable. At e q u i l i b r i u t h e w a t e r i n t h e g e l and t h e w a t e r s u r r o u n d i n g t h e g e l a r e e q u a l . The a d d i t i o n t o t h e s o l u t i o n s u r r o u n d i n g t h e g e l o f m a c r o m o l e c u l e s t h a t are too l a r g e to penetrate the gel lowers the chemical p o t e n t i a l of the water i n the s o l u t i o n . Water t h u s moves o u t o f t h e g e l and t h e n e t w o r k c o n t r a c t s , d e c r e a s i n g t h e c h e m i c a l p o t e n t i a l o f the water i n the network to the v a l u e o f the water i n the solution. Therefore, at e q u i l i b r i u m the osmotic pressure of the macromolecular s o l u t i o n equals the s w e l l i n g pressure of the hydrogel. The d e g r e e o f h y d r a t i o n t h a t can be a c h i e v e d by e q u i l i b r a t i o n w i t h a m a c r o m o l e c u l a r s o l u t i o n c a n a l s o be o b t a i n e d by compressing the g e l under a mechanical p r e s s u r e e q u i v a l e n t i n magnitude t o the osmotic p r e s s u r e o f the macromolecular s o l u t i o n . The m e c h a n i c a l p r e s s u r e r a i s e s t h e c h e m i c a l p o t e n t i a l o f t h e w a t e r i n t h e g e l , so w a t e r exudes f r o m t h e g e l u n t i l e q u i l i b r i u m i s reached. Thus t h e e q u i l i b r i u m w a t e r c o n t e n t depends on t h e mechanical pressure a p p l i e d to the g e l . The s w e l l i n g phenomena o f g e l s have been t h e o b j e c t o f t h e r m o d y n a m i c a n a l y s i s (1_,2). The o s m o t i c p r e s s u r e a t t r i b u t e d t o the polymer network ( π ) i s the d r i v i n g f o r c e o f s w e l l i n g . The s w e l l i n g p r o c e s s d i s t e n d s t h e n e t w o r k , and i s c o u n t e r a c t e d by t h e e l a s t i c c o n t r a c t i l i t y o f the s t r e t c h e d polymer network ( p ) . Hence t h e s w e l l i n g p r e s s u r e o f n o n i o n i c h y d r o g e l s ( Ρ ) i s t h e r e s u l t o f t h e i m b i b i t i o n o f s o l v e n t d r i v e n by an o s m o t i c p r e s s u r e , c o u n t e r a c t e d by t h e c o n t r a c t i l i t y o f t h e n e t w o r k w h i c h tends to expel the s o l v e n t : Ρ = π - p. A t e q u i l i b r i u m , π = ρ, and t h e s w e l l i n g p r e s s u r e i s z e r o (P = 0 ) . S w e l l i n g p r e s s u r e can be d e f i n e d as t h e p r e s s u r e e x e r t e d by a g e l when s w e l l i n g i s c o n s t r a i n e d b u t s w e l l i n g s o l v e n t i s a v a i l able. In g e n e r a l , t h e f o l l o w i n g e m p i r i c a l r e l a t i o n s h i p (I), d e v e l o p e d by P o s n j a k i n 1912 (3)> a p p l i e s t o a s w e l l i n g s u b stance:

37

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Ρ = k X C

(I)

where Ρ i s t h e s w e l l i n g p r e s s u r e , k and η a r e c o n s t a n t s whose v a l u e s a r e u s u a l l y between 2 and 3 , and c i s t h e p o l y m e r n e t w o r k concentration. Since n>l, expression (I) i n d i c a t e s the w e l l known f a c t t h a t t h e s w e l l i n g p r e s s u r e , P, i n c r e a s e s r a p i d l y w i t h the c o n c e n t r a t i o n of the polymer i n the g e l . In t h i s p a p e r , t h e s w e l l i n g p r e s s u r e o f two c l a s s e s o f hydrogels of i n t e r e s t to ophthalmology i s i n v e s t i g a t e d . The f i r s t c l a s s i s a g r o u p o f h i g h l y h y d r a t e d h y d r o g e l s (above a b o u t 80% w a t e r a t s w e l l i n g e q u i l i b r i u m i n w a t e r ) w h i c h i s i n t e n d e d f o r s u r g i c a l use. The s e c o n d c l a s consist f hydrogel d manufacture contact l e n s e s 40 t o 75% w a t e r a t s w e l l i n g e q u i l i b r i u E f f e c t o f t h e E x t e r n a l S o l u t i o n upon t h e H y d r a t i o n o f Hydrogels. The m a g n i t u d e o f e q u i l i b r i u m s w e l l i n g o f a h y d r o g e l i n an aqueous medium i s d e t e r m i n e d by t h e c h e m i c a l p o t e n t i a l o f water i n the outside s o l u t i o n . The c h e m i c a l p o t e n t i a l o f w a t e r i n t h e o u t s i d e s o l u t i o n i s d e t e r m i n e d by t h e n a t u r e and c o n c e n t r a t i o n of the solutes i n the s o l u t i o n . The s o l u t e s , d e p e n d i n g on t h e i r m o l e c u l a r s i z e , may o r may n o t p e n e t r a t e t h e p o l y m e r network. Some s o l u t e s w h i c h c a n p e n e t r a t e t h e n e t w o r k may i n t e r a c t w i t h t h e p o l y m e r s e g m e n t s , m o d i f y i n g t h e i r s t r e t c h i n g and contracting properties. Nevertheless, a l l solutes i n the gel w a t e r a f f e c t t h e g e l by l o w e r i n g t h e c h e m i c a l p o t e n t i a l o f i t s water. Solution t o n i c i t y i s a b i o l o g i c a l concept. It is related to osmotic p r e s s u r e , but i t l a c k s e x a c t p h y s i c o c h e m i c a l meaning. T h u s , v a r i o u s i s o t o n i c s o l u t i o n s may s w e l l o r d e s w e l l h y d r o g e l s d e p e n d i n g on t h e p e n e t r a t i o n and i n t e r a c t i o n o f t h e s o l u t e s w i t h t h e n e t w o r k segments ( £ ) ( F i g . 1 ) . S w e l l i n g Pressure o f High Water-Content G l y c e r y l Methac r y l a t e H y d r o g e l s and t h e i r O p h t h a l m i c A p p l i c a t i o n s . The s w e l 1 ing pressure-volume r e l a t i o n s h i p of p o l y ( g l y c e r y l methacrylate) h y d r o g e l s (PGMA) i s o f p r a c t i c a l i n t e r e s t f o r t h e d e v e l o p m e n t o f s w e l l i n g or expanding s u r g i c a l i m p l a n t s . A swelling implant i s a d e v i c e t h a t can be p l a c e d i n s i d e an o r g a n o r t i s s u e t h r o u g h a r e l a t i v e l y small i n c i s i o n . By i m b i b i n g a v a i l a b l e body f l u i d i t w i l l s w e l l t o f i l l a c a v i t y or to a l t e r the form of the s u r rounding t i s s u e s (5). To be u s e f u l , a s w e l l i n g i m p l a n t must s w e l l t o s e v e r a l tTmes i t s d r y volume u n d e r t h e c o n d i t i o n s o f implantation. In some a p p l i c a t i o n s , t h e i m p l a n t must e x e r t s u f f i c i e n t s w e l l i n g pressure to counteract the c o n s t r a i n i n g pressure of the surrounding t i s s u e s . Of i n t e r e s t i s a c o h e r e n t v i t r e o u s s u b s t i t u t e w h i c h c o u l d be used t o f i l l t h e v i t r e o u s c a v i t y o f t h e eye ( £ ) , and i n some c a s e s , t h e e n t i r e e y e b a l l (_7).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10

2CH

30Η

.9%

NaCI 2

CaCI 13% 2

UREA 1.63°/o

GLUCOSE 5.51%

SERUM

SERUM VITREOUS ULTRAFILTRATE

Figure 1. PGMA hydrogel, 98% H 0 at equilibrium swelling in distilled water. The bars repre sent the equilibrium swelling of the same hydrogel in diverse isotonic solutions and physiological fluids.

2

m

1 50Η

Τ- 6θΗ

Ζ ζ

80

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

40

A n o t h e r t y p e o f s w e l l i n g i m p l a n t i s used i n ophthalmology i n t h e s c l e r a l b u c k l i n g procedure i n r e t i n a l detachment s u r g e r y (8). G l y c e r y l m e t h a c r y l a t e monomer (GMA) p r e p a r e d by h y d r o T y s i s o f g l y c i d y l m e t h a c r y l a t e (9·) y i e l d s GMA w i t h s m a l l amounts o f some s t i l l u n i d e n t i f i e d c r o s s l i n k i n g a g e n t . However, h y d r o g e l s w i t h r e p r o d u c i b l e p h y s i c a l and c h e m i c a l p r o p e r t i e s c a n be o b t a i n e d f r o m GMA p r e p a r e d by t h i s p r o c e d u r e . Polymerization is c a r r i e d o u t i n g l a s s molds f i l l e d w i t h aqueous s o l u t i o n s o f GMA w i t h a redox i n i t i a t o r . The monomer d i l u t i o n i n t h e p r e p o l y m e r m i x t u r e determines the e q u i l i b r i u m degree of s w e l l i n g of the r e s u l t i n g hydrogel. The s w e l l i n g p r e s s u r e o f PGMA h y d r o g e l s was o b t a i n e d by e q u i l i b r a t i o n i n s o l u t i o n s o f d e x t r a n ( P h a r m a c i a , mol wt 2 3 6 , 0 0 0 ) , The PGMA s p e c i m e n s were p l a c e d i n d e x t r a n s o l u t i o n s and a l l o w e d to e q u i l i b r a t e in t i g h t l d jar t temperature E q u i l i b r i u m s w e l l i n g wa i n g on t h e c o n c e n t r a t i o , specimen. When e q u i l i b r i u m s w e l l i n g was r e a c h e d , t h e d e x t r a n c o n c e n t r a t i o n was d e t e r m i n e d f r o m a l i q u o t s o f t h e s o l u t i o n . The o s m o t i c p r e s s u r e o f t h e d e x t r a n a t d i f f e r e n t c o n c e n t r a t i o n s was determined osmometrically (4). The d e x t r a n m o l e c u l e s Tn w a t e r s o l u t i o n have an e l l i p s o i d a l shape. The d i a m e t e r o f t h e d e x t r a n m o l e c u l e s u s e d i n t h e s e e x p e r i m e n t s i s a b o u t 270 Â ( 1 0 ) . The d e x t r a n m o l e c u l e s a r e n o t l i k e l y t o p e n e t r a t e i n t o PGMTThydrogels b o t h b e c a u s e a ) t h e average pore s i z e o f the hydrogels i s s m a l l e r than the s i z e of t h e d e x t r a n m o l e c u l e s ( f o r i n s t a n c e , PGMA h v d r o g e l s o f 94% H 0 have an a v e r a g e p o r e d i a m e t e r o f a b o u t 124 A)(1_1_), and b) when à h i g h l y hydrated gel i s placed i n a dextran s o l u t i o n , the osmotic d e h y d r a t i o n o f the gel i s f a s t e r than the d i f f u s i o n o f the dextran molecules i n t o the g e l . As a g e l d e h y d r a t e s , p o r e s i z e i s r e d u c e d , f u r t h e r l i m i t i n g p e n e t r a t i o n by l a r g e d e x t r a n m o l e c u l e s . F i g u r e 2 g i v e s t h e s w e l l i n g p r e s s u r e o f s e v e r a l PGMA h y d r o gels versus the " s w e l l i n g r a t i o " (q). The s w e l l i n g r a t i o i s d e f i n e d as t h e volume o f t h e s w o l l e n g e l o v e r t h e volume o f t h e same g e l i n t h e d r y s t a t e . The " s w e l l i n g r a t i o " was c a l c u l a t e d f r o m t h e " d e g r e e o f s w e l l i n g " ( γ ) , t h e d e n s i t y o f t h e s w o l l e n g e l ( d ) , and t h e d e n s i t y of the dry gel ( d ) , according to (II): 2

0

γ i s the r a t i o of the weights of the swollen gel to the dry gel (£). The d e n s i t i e s were o b t a i n e d f r o m F i g u r e 3 , w h i c h g i v e s t h e d e n s i t y o f a PGMA h y d r o g e l v e r s u s t h e w e i g h t f r a c t i o n o f w a t e r i n t h e s w o l l e n g e l (Cw = % H 0 / 1 0 0 ) . D e n s i t i e s w e r e d e t e r m i n e d by t h e h y d r o s t a t i c w e i g h i n g method. G e l s were weighed both i n a i r , 2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

Vapor Pressure and Swelling

REFOJO

5

10 15 20

30

40

41

Pressure

50

60

70

125.2

q = VOL. SWOLLEN GEL + VOL. DRY G E L

Figure 2. Swelling pressure vs. "swelling ratio" of several PGMA hydrogels. The equilibrium swelling of the PGMA hydrogels in distilled water is given for each swelling pressure curve in the graph.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

42

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

and immersed i n η - h e p t a n e a t room t e m p e r a t u r e , Cw b y :

γ

=

γ i s r e l a t e d to

TO

The d e n s i t y o f t h e d i f f e r e n t PGMA h y d r o g e l s was o b t a i n e d from F i g u r e 3 assuming t h a t the d e n s i t i e s o f the d r y g e l s ( x e r o g e l s ) and h y d r o g e l s were n o t a f f e c t e d a p p r e c i a b l y by t h e amount of crosslinkage. The r e s u l t s g i v e n i n F i g u r e 2 show t h a t a s u b s t a n t i a l amount o f w a t e r i s removed f r o m h i g h l y h y d r a t e d h y d r o g e l s ( j e l l i e s ) when t h e y a r e s u b j e c t e d t o a s l i g h t c o m p r e s s i o n . Thus, the volume o f PGMA h y d r o g e 1 2 5 . 6 ) was more t h a n h a l v e Hg o s m o t i c p r e s s u r e . T h e s e j e l l i e s exude l i q u i d w a t e r when t h e y a r e a l l o w e d t o s t a n d u n d e r t h e i r own w e i g h t i n t h e a i r . As t h e water content i n the hydrogel decreases, the pressure r e q u i r e d to compress w a t e r o u t o f t h e g e l i n c r e a s e s . H e n c e , PGMA w i t h 95% H 0 by w e i g h t (q = 30) a t e q u i l i b r i u m i n w a t e r l o s t a b o u t one t h i r d o f i t s volume o f w a t e r (q = 20) u n d e r o n l y a b o u t 4 mm Hg, b u t PGMA h y d r o g e l w i t h 82% H 0 by w e i g h t (q = 6.9) l o s t p r a c t i c a l l y no w a t e r u n d e r t e n t i m e s as much o s m o t i c p r e s s u r e . The r e l a t i o n s h i p o f h y d r a t i o n by w e i g h t , Cw, and s w e l l i n g r a t i o by volume ( q ) i s g i v e n b y : 2

2

q

"

(

T^CW~

(IV)

H e n c e , when t h e v a l u e o f Cw i s n e a r o n e , s u c h as i n j e l l i e s , s m a l l d i f f e r e n c e s i n h y d r a t i o n r e p r e s e n t s u b s t a n t i a l volume changes. The s w e l l i n g p r e s s u r e p r o p e r t i e s o f j e l l i e s and h y d r o g e l s a r e i m p o r t a n t f r o m t h e p o i n t o f v i e w o f t h e two k i n d s o f s w e l l i n g i m p l a n t s m e n t i o n e d a b o v e , a v i t r e o u s s u b s t i t u t e and a s c l e r a l b u c k l i n g d e v i c e . A v i t r e o u s s u b s t i t u t e must m i m i c t h e n a t u r a l v i t r e o u s body, which i s a h i g h l y h y d r a t e d j e l l y . The g e l i s implanted i n i t s dry s t a t e i n t o the e y e b a l l through the s m a l l e s t p o s s i b l e i n c i s i o n . Then i t a b s o r b s a v a i l a b l e i n t r a o c u l a r f l u i d , s w e l l i n g f r e e l y as l o n g as i t does n o t a d j o i n t h e w a l l s o f t h e eye, u n t i l i t f i l l s the v i t r e o u s c a v i t y . F u l l y swollen, the i m p l a n t must o c c u p y t h e v i t r e o u s c a v i t y w h i l e e x e r t i n g a minimum of pressure against the extremely s e n s i t i v e r e t i n a . The s e c o n d t y p e o f s w e l l i n g i m p l a n t i s p l a c e d on t h e o u t s i d e o f t h e e y e b a l l , i n t h e s c l e r a . T h i s i m p l a n t , upon s w e l l i n g , e x e r t s p r e s s u r e t o b u c k l e t h e w a l l o f t h e eye i n w a r d , t h e r e b y approximating the choroid that c a r r i e s the blood supply to a

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

REFOJO

43

Vapor Pressure and Swelling Pressure

detached r e t i n a . Such an i m p l a n t must be s o f t t o a v o i d p r e s s u r e n e c r o s i s i n t h e s c l e r a , b u t n o t so f r a g i l e t h a t i t w i l l c r u m b l e under p r e s s u r e . About a f i v e - f o l d s w e l l i n g a g a i n s t t h e c o n s t r a i n ing t i s s u e i s s u f f i c i e n t . For t h i s a p p l i c a t i o n , a hydrogel w i t h 80 t o 85% w a t e r c o n t e n t a t e q u i l i b r i u m s w e l l i n g i n w a t e r a p p e a r s t o be most u s e f u l . V a p o r P r e s s u r e and S w e l l i n g P r e s s u r e o f H y d r o g e l C o n t a c t Lens M a t e r i a l s . S i n c e W i c h t e r l e and L i m ( 1 2 ) f i r s t p r o p o s e d t h e use o f h y d r o g e l s f o r c o n t a c t l e n s e s and otTiêr m e d i c a l d e v i c e s , many new h y d r o g e l s have been d e v e l o p e d ( 1 3 ) . The f i r s t commerc i a l h y d r o g e l c o n t a c t l e n s e s w e r e made o f s l i g h t l y c r o s s l i n k e d p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) (PHEMA), w h i c h i s s t i l l t h e m a t e r i a l most o f t e n used i n t h e s o f t l e n s i n d u s t r y . A second compound used t o make h y d r o g e (VP), i n t h e form o f a L e n s e s w h i c h c o n t a i n V P , b u t no HEMA a r e a l s o made, s u c h as a c o p o l y m e r o f m e t h y l m e t h a c r y l a t e and V P , P(MMA/VP) ( 1 5 ) . As d i f f e r e n t h y d r o g e l c o n t a c t l e n s e s become a v a T T a b l e , i t i s o f i n t e r e s t t o i n v e s t i g a t e and t o compare t h e i r r e l a t i v e w a t e r retention. Differences i n hydration at swelling equilibrium are i m p o r t a n t i n t h e e v a l u a t i o n o f t h e o p t i c a l and p h y s i o l o g i c a l performance o f t h e l e n s e s . This study determined the e q u i l i b r i u m s w e l l i n g o f s e v e r a l hydrogel c o n t a c t l e n s m a t e r i a l s under o s m o t i c and m e c h a n i c a l p r e s s u r e , as w e l l as t h e w a t e r a c t i v i t y o f t h e h y d r o g e l s under v a r i o u s s w e l l i n g c o n d i t i o n s . 1. D e t e r m i n a t i o n o f S w e l l i n g P r e s s u r e o f a PHEMA H y d r o g e l by E q u i l i b r i u m S w e l l i n g i n D e x t r a n S o l u t i o n . PHEMA I was o b t a i n e d by s o l u t i o n p o l y m e r i z a t i o n ( 1 6 ) . S t e q u i l i b r i u m s w e l l i n g i n d i s t i l l e d w a t e r , i t c o n t a i n s 40% H 0 by w e i g h t on a w e t b a s i s . PHEMA I s p e c i m e n s were p l a c e d i n d e x t r a n - 4 0 s o l u t i o n s and a l l o w e d t o e q u i l i b r a t e i n t i g h t l y capped j a r s a t room t e m p e r a t u r e [ D i f f e r e n t v a l u e s have been r e p o r t e d f o r a v e r a g e p o r e d i a m e t e r o f PHEMA I h y d r o g e l s ; t h e maximum i s 35 S ( 1 3 ) . D e x t r a n - 4 0 i n w a t e r has a m o l e c u l a r d i a m e t e r o f a b o u t 105 Â ( 1 0 ) ] . Equilibrium s w e l l i n g was r e a c h e d a f t e r two months. After e q u i l i b r a t i o n , the d e x t r a n c o n c e n t r a t i o n i n t h e j a r was d e t e r m i n e d g r a v i m e t r i c a l l y . The o s m o t i c p r e s s u r e ( π , i n a t m . ) o f d e x t r a n (mol wt 2 6 , 0 0 0 ) was calculated according to equation (V): 2

π = A-jC + A c

2

2

+ A c

(V)

3

3

where c i s t h e c o n c e n t r a t i o n ( i n g » c m " ) and A = 0 . 8 5 2 a t m » c m . g " , A = 1 3 . 5 2 a t m - c m g ~ , and A = 6 6 . 8 a t m - c m - g " a r e t h e v i r i a l c o e f f i c i e n t s a c c o r d i n g t o V i n k ( V 7 ) . The r e s u l t s o f t h e s w e l l i n g p r e s s u r e o f PHEMA I o b t a i n e d by t h i s p r o c e d u r e a r e g i v e n in Figs. 4,5. 3

3

x

1

6

2

2

9

3

3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS 1.4 -η

~t ι » ι » » » 11111 » 1111111M 111 f 1111111111 f 11111 111 > π 11111 i i ι » 111111 π 11 n 111 » 11 M » η ι n n I n ι n m 1 I M 11| 10

20

30

40

50

I

60

70

80

90

100

H 0 2

Figure 3. Density of PGMA hydrogel, 96% H 0 at equilibrium swelling, vs. hydration of the gel. The curve was printed by a computer using the least squares method applied to the data points. 2

Φ

I I I I I I

ι

I!

5°'

a of

SI

1/

/

Q

·/# 'ο

^ / /

4 I

Figure 4. Dehydration of two PHEMA hydrogeh under osmotic and mechanical pressure

- ι — ι — ι — r -1 -2 - 3 -4 WEIGHT

*. H

2

0 LOST

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

REFOJO

Vapor Pressure and Swelling Pressure

45

2. D e t e r m i n a t i o n o f S w e l l i n g P r e s s u r e o f a PHEMA H y d r o g e l by E q u i l i b r i u m S w e l l i n g Under M e c h a n i c a l C o m p r e s s i o n . PHEMA I I was o b t a i n e d by b u l k p o l y m e r i z a t i o n ( 1 6 ) . I t c o n t a i n s 3 8 . 5 % H 0 a t e q u i l i b r i u m swelling i n d i s t i l l e d water. C i r c u l a r pieces o f PHEMA I I h y d r o g e l 20 mm i n d i a m e t e r (1 t o 2 mm t h i c k ) were p l a c e d between two s i n t e r e d g l a s s d i s k s i n a w a t e r b a t h u n d e r i r o n w e i g h t s ( f i v e t o t e n k i l o g r a m s ) , w h i c h were s e p a r a t e d f r o m t h e u p p e r s i n t e r e d g l a s s d i s c by a p l a s t i c c y l i n d e r p r o t r u d i n g above t h e s u r f a c e o f t h e w a t e r b a t h . The w a t e r b a t h c o n t a i n i n g t h e h y d r o g e l d i s c and t h e w e i g h t s on t o p o f i t were k e p t i n a c l o s e d chamber t o p r e v e n t e v a p o r a t i o n . Equilibrium swelling of t h e h y d r o g e l was o b t a i n e d a f t e r s e v e r a l weeks o f c o m p r e s s i o n . The r e l a t i o n s h i p between h y d r a t i o n and s w e l l i n g p r e s s u r e o f PHEMA I I o b t a i n e d by t h i s p r o c e d u r e i s shown i n F i g s . 4 , 5 . While a s l i g h t pressur water-content hydrogel w a t e r by o s m o t i c o r m e c h a n i c a l means f r o m h y d r o g e l s h a v i n g l o w w a t e r - c o n t e n t , s u c h as t h e commonly u s e d PHEMA c o n t a c t l e n s h y d r o g e l s , w h i c h c o n t a i n a b o u t 40% w a t e r a t e q u i l i b r i u m s w e l l i n g ( F i g s . 4 , 5 ) . S i m i l a r r e s u l t s a r e expected from hydrogels c o n t a i n i n g l e s s t h a n 80% w a t e r a t e q u i l i b r i u m ( F i g . 2 ) . Most h y d r o g e l c o n t a c t l e n s m a t e r i a l s c o n t a i n a b o u t 35 t o 75% w a t e r a t equilibrium in physiological saline solution. I t requires subs t a n t i a l p r e s s u r e t o remove w a t e r f r o m h y d r o g e l l e n s e s , w h i c h i s a d v a n t a g e o u s b e c a u s e i f l i d p r e s s u r e were t o s q u e e z e w a t e r f r o m the l e n s e s i n t h e eye t h e i r performance would s u f f e r . Of c o u r s e , t h e o p t i c a l p r o p e r t i e s , t h e s h a p e , and t h e s i z e o f a h y d r o g e l l e n s a r e a l l d e p e n d e n t on i t s w a t e r - c o n t e n t . 2

3. D e t e r m i n a t i o n o f Water A c t i v i t y i n H y d r o g e l s . One way to f a c i l i t a t e water l o s s from hydrogels i s t o decrease t h e r e l a t i v e humidity; t h i s decreases the chemical p o t e n t i a l o f the water vapor i n t h e s u r r o u n d i n g atmosphere t o a low v a l u e . It i s well known t h a t h y d r o g e l s c a n l o s e w a t e r r a p i d l y by e v a p o r a t i o n . When t h i s happens, t h e network, which i s under e l a s t i c t e n s i o n , w i l l contract. As t h e h y d r o g e l d e h y d r a t e s , t h e c h e m i c a l p o t e n t i a l o f t h e w a t e r r e m a i n i n g i n t h e g e l d e c r e a s e s and i s m a n i f e s t e d a s an i m b i b i t i o n p r e s s u r e , which i s equal i n magnitude t o t h e osmotic o r m e c h a n i c a l p r e s s u r e needed t o compress t h e g e l t o t h e same p a r t i a l l y dehydrated s t a t e . The w a t e r r e t e n t i o n , o r w a t e r e s c a p i n g t e n d e n c y ( f u g a c i t y ) o f h y d r o g e l s was d e t e r m i n e d ( F i g s . 6 , 7 ) by m e a s u r i n g t h e e q u i l i b r i u m r e l a t i v e h u m i d i t y (% ERH) o f t h e h y d r o g e l s a t 3 2 ° C , w h i c h i s approximately the surface temperature of the eye. F o u r d i f f e r e n t h y d r o g e l s were u s e d i n t h e s e e x p e r i m e n t s : ( a ) a PHEMA h y d r o g e l w i t h 4 2 . 5 % H 0 a t e q u i l i b r i u m s w e l l i n g ; ( b ) a c o p o l y m e r o f m e t h y l m e t h a c r y l a t e and v i n y l p y r r o l i d o n e , P(MMA/ V P ) , used i n t h e manufacture o f hydrogel c o n t a c t l e n s e s under t h e t r a d e name S a u f I o n ( c o n t a i n i n g a b o u t 70% H 0 a t e q u i l i b r i u m s w e l l i n g i n d i s t i l l e d w a t e r ) ; ( c ) a c o p o l y m e r o f HEMA and VP 2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

46

-/•A-r 35

36

37

3 8 39

τ — ι 38 39

37

1 — - r 4 0 41

W E I G H T 7o H 2 O

Figure 5. Swelling pressure of two PHEMA hydrogels equilibrated under mechanical pres sure (PHEMA II, 38.5% H 0) and osmotic pressure (PHEMA I, 40% H 0), respectively 2

2

WATER ACTIVITY ia ) w

Figure 6.

Water sorption isotherms of hydrogel contact lens materials

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

REFOJO

47

Vapor Pressure and Swelling Pressure

[P(HEMA/VP)], known as PHP and a l s o used i n c o n t a c t l e n s e s . It c o n t a i n s a b o u t 45% w a t e r a t e q u i l i b r i u m s w e l l i n g i n w a t e r ; and (d) a c o p o l y m e r o f MMA and GMA [P(GMA/MMA)] w h i c h c o n t a i n s 4 1 % water a t e q u i l i b r i u m s w e l l i n g . The e q u i l i b r i u m r e l a t i v e h u m i d i t y ( w a t e r a c t i v i t y ) was determined w i t h t h e hydrogel i n a c l o s e d c o n t a i n e r having a c a l i b r a t e d h u m i d i t y and t e m p e r a t u r e s e n s o r c o n n e c t e d by c a b l e t o a r e c e i v e r (Hygrodynamics U n i v e r s a l Hygrometer I n d i c a t o r , A m e r i can I n s t r u m e n t C o . , S i l v e r S p r i n g s , M a r y l a n d ) . The chamber c o n t a i n i n g t h e h y d r o g e l and t h e s e n s o r was m a i n t a i n e d i n a c o n s t a n t t e m p e r a t u r e d r y i n c u b a t o r a t 32°C. When r e l a t i v e h u m i d i t y r e a c h e d e q u i l i b r i u m , t h e w e i g h t o f t h e g e l was r e c o r d e d . The o p e r a t i o n was r e p e a t e d f o r d i f f e r e n t s t a t e s o f h y d r a t i o n t o o b t a i n t h e isotherms i n which t h e weight o f water sorbed per u n i t o f d r y p o l y m e r w e i g h t was p l o t t e d w i t h r e f e r e n c e t o w a t e r a c t i v i t y ( F i g . 6 ) . The w a t e c o n d i t i o n s , e x c e p t f o r P(HEMA/VP), resorp tion conditions. The i s o t h e r m s o b t a i n e d have t h e s t a n d a r d s i g m o i d shape o f w a t e r s o r p t i o n i n p o l y m e r s . F i g u r e 7 shows t h e same r e s u l t s o f w a t e r a c t i v i t y v e r s u s w a t e r c o n t e n t i n t h e h y d r o g e l s , e x p r e s s e d i n p e r c e n t h y d r a t i o n on a w e t b a s i s , w h i c h i s c o n v e n t i o n a l l y used i n hydrogel l i t e r a t u r e . ERH ( r e l a t i v e h u m i d i t y o f t h e s p a c e a r o u n d t h e h y d r o g e l , when m o i s t u r e w i l l n e i t h e r l e a v e n o r e n t e r t h e h y d r o g e l ) i s a r a t i o o f e x i s t i n g p a r t i a l vapor pressure o f water i n t h e hydrogel (P ) t o t h e s a t u r a t i o n w a t e r v a p o r p r e s s u r e i n t h e a i r (P ). It i s given by:

% ERH =

15S. χ

100

(VI)

P /Ps i s , o f course, the "water a c t i v i t y " ( a ) i n t h e hydrogel a t the given temperature. Thus, t h e water a c t i v i t y i n hydrogels a t d i f f e r e n t l e v e l s o f h y d r a t i o n c a n be d e t e r m i n e d d i r e c t l y and c o u l d be u s e d t o c a l c u l a t e t h e s w e l l i n g p r e s s u r e ( P ) o f t h e hydrogels a t d i f f e r e n t hydrations according t o : e

w

P = P l n i w w

(VII)

where R i s t h e u n i v e r s a l gas c o n s t a n t , Τ t h e a b s o l u t e t e m p e r a t u r e , and V ^ t h e p a r t i a l m o l a r volume o f w a t e r . Under t h e e x p e r i mental c o n d i t i o n s ( F i g . 7 ) , small d i f f e r e n c e s i n water a c t i v i t y i n t h e h y d r o g e l s w e r e n o t d e t e c t a b l e a t h y d r a t i o n s above a b o u t 30% w a t e r . Not o n l y t h e hydrogels a t e q u i l i b r i u m s w e l l i n g i n w a t e r , b u t l i q u i d w a t e r a s w e l l , gave an ERH j u s t b e l o w t h e t r u e v a l u e o f 100%. T h i s i s a l i m i t a t i o n o f t h e h y g r o s e n s o r u s e d .

Amcnccn Chemical Socisîy Library 1155 16th St. N. 11

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Washington. 0. C. Society: 20036Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

B e c a u s e o f t h e l a r g e v a l u e o f RT/V ( a b o u t 1390 a t m . a t 32°C) a v e r y s m a l l v a p o r - p r e s s u r e decrease can r e s u l t i n a s u b s t a n t i a l increase in swelling pressure. Thus, the hygrometer t e c h n i q u e c a n n o t be used t o d e t e r m i n e t h e s w e l l i n g p r e s s u r e o f h y d r o g e l s near e q u i l i b r i u m h y d r a t i o n . The s o r p t i o n i s o t h e r m s o f f o u r h y d r o g e l s a r e g i v e n i n F i g u r e 6. The s i g m o i d shape o f t h e c u r v e s may be an i n d i c a t i o n o f t h e t h r e e c l a s s e s o f w a t e r w h i c h , a c c o r d i n g t o L e e , J h o n , and A n d r a d e ( 1 8 ) , may be p r e s e n t i n t h e s e h y d r o gels. Because o f t h e d i f f i c u l t y o f o b t a i n i n g t h e e q u i l i b r i u m r e l a t i v e humidity of a x e r o g e l , the s e c t i o n of the curves at l o w e r h y d r a t i o n a r e n o t as s h a r p l y d e f i n e d as t h e o t h e r two sections. The ERH i s a measure o f " f r e e w a t e r - v a p o r p r e s s u r e " w h i c h i s , i n e s s e n c e , a measurement o f t h e " f r e e d o m o f w a t e r " o r i t s "escaping tendency" (19) ments do g i v e a good i n d i c a t i o o f h y d r a t i o n i n h y d r o g e l s , t h a t i s , w a t e r i n t h e aqueous phase o f t h e h y d r o g e l w i t h t h e same v a p o r p r e s s u r e as l i q u i d w a t e r a t t h e same t e m p e r a t u r e . Water a c t i v i t y v e r s u s h y d r o g e l h y d r a t i o n ( F i g . 7) shows t h a t t h e w a t e r o f h y d r a t i o n i n h y d r o g e l s above a b o u t 25 t o 30% has a p p r o x i m a t e l y t h e same v a p o r p r e s s u r e as l i q u i d w a t e r . F i g u r e 8 , t h u s , r e p r e s e n t s t h e amounts o f " f r e e " w a t e r ( a - l ) and somewhat " b o u n d " w a t e r ( a < l ) i n h y d r o g e l s as r e p l o t t e d f r o m F i g u r e 6. The amount o f " b o u n d " w a t e r , a b o u t 30% o f w a t e r i n t h e h y d r o g e l s , i s s i m i l a r t o t h e amounts t h a t L e e , J h o n , and A n d r a d e (18) a s s i g n e d as " b o u n d " p l u s " i n t e r f a c i a l " w a t e r . The r e s t o f the water of hydration i n the hydrogels i s " f r e e " or " b u l k " water. T h u s , t h e c o n t a c t l e n s m a t e r i a l s examined a l l seem s u b j e c t t o l o s i n g s u b s t a n t i a l amounts o f w a t e r by e v a p o r a t i o n . The f a c t t h a t a b o u t 30% o f t h e w a t e r i s r e t a i n e d more t e n a c i o u s l y i n t h e l e n s m a t e r i a l s does n o t seem t o have any p r a c t i c a l i m p o r t a n c e f r o m t h e p o i n t o f v i e w o f w a t e r r e t e n t i o n o f h y d r o g e l l e n s e s and o p t i c a l performance. Of c o u r s e , f r e q u e n t b l i n k i n g and good t e a r s u p p l y a r e e s s e n t i a l f o r good r e s u l t s w i t h a l l h y d r o g e l c o n t a c t lenses. W

w

w

O s m o t i c E f f e c t s Due t o t h e Aqueous Phase o f a H y d r o g e l Contact Lens. In a d d i t i o n t o t h e s w e l l i n g p r e s s u r e ( o r i m b i b i t i o n pressure) of h y d r o g e l s , which i s a p r o p e r t y of the polymer phase o f g e l s , t h e r e a r e o t h e r o s m o t i c e f f e c t s a t t r i b u t a b l e t o t h e aqueous p h a s e t h a t a r e o f p a r t i c u l a r i n t e r e s t i n t h e c o n t a c t lens f i e l d . Most o f t h e aqueous p h a s e o f a h y d r o g e l c a n be f r e e l y e x c h a n g e d w i t h t h e s u r r o u n d i n g aqueous media by d i f f u s i o n . The movement o f w a t e r , i o n s and o t h e r d i s s o l v e d s u b s t a n c e s i s r e s t r i c t e d t o some d e g r e e by f r i c t i o n w i t h t h e p o l y m e r n e t w o r k . H o w e v e r , due t o t h e l a r g e s u r f a c e a r e a o f c o n t a c t l e n s e s r e l a t i v e t o t h e i r t h i c k n e s s , most o f t h e aqueous phase w i l l i n t e r c h a n g e w i t h t h e t e a r s i n a few m i n u t e s .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

REFOJO

Vapor

Pressure and Swelling

49

Pressure

70

60

H

_i LU

Ο

s

5 0

RHEMA/VP) 1—1 PHEMA RGMA/MMA)

CL ^

40

WATER ACTIVITY ·

I » = 30

1

Η WATER ACTIVITY < 1

Figure 8. Amounts of "free" water (a ~ I) and "bound" water (a < 1) in hydrogel contact lens materials w

w

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

The aqueous phase i n a c o n t a c t l e n s can be i s o t o n i c , h y p o tonic or hypertonic with respect to tears. When a h y d r o g e l l e n s i s e q u i l i b r a t e d i n i s o t o n i c s a l i n e s o l u t i o n (0.9% N a C l ) , the aqueous phase o f t h e h y d r o g e l i s i s o t o n i c t o t h e normal t e a r s . When s u c h a l e n s i s p l a c e d i n t h e e y e , t h e r e w i l l be an i n t e r change o f i t s aqueous phase and t h e t e a r f i l m , b u t t h e t e a r t o n i c i t y w i l l n o t change i n t h e p r o c e s s o f e q u i l i b r a t i o n o f t h e lens. The p o s s i b l e change i n s i z e and o p t i c s o f a l e n s f r o m e q u i l i b r i u m s w e l l i n g i n i s o t o n i c s o l u t i o n and i n t e a r s i s n e g l i gible. I f the hydrogel lens i s wetted w i t h tap or d i s t i l l e d water p r i o r t o p l a c i n g i t i n t h e e y e , t h e aqueous phase o f t h e l e n s w i l l be h y p o t o n i c t o t e a r s . Water w i l l move f r o m t h e l e n s t o t h e tears. The l e n s w i l l t h e n c o n t r a c t , o f t e n a d h e r i n g t e n a c i o u s l y t o t h e c o r n e a and c a u s i n t h e aqueous phase o f t h i s o t o n i c i t y i s r e a c h e d and t h e n t h e l e n s w i l l r e l e a s e f r o m i t s adhesion to the cornea. I f t h e l e n s i s p l a c e d i n a s o d i u m c h l o r i d e s o l u t i o n o f more t h a n 0 . 9 % , t h e aqueous phase o f t h e h y d r o g e l w i l l be h y p e r t o n i c to t e a r f i l m . As t h e l e n s i s p l a c e d i n t h e e y e , w a t e r w i l l be drawn o s m o t i c a l l y f r o m t h e t e a r f i l m i n t o t h e l e n s , b u t s a l t w i l l a l s o d i f f u s e from the lens i n t o the t e a r . T h i s w i l l change t h e t o n i c i t y of the tears to a hypertonic s t a t e . The l e n s e f f e c t can be q u i t e l a r g e as t h e t o t a l volume o f t h e t e a r f i l m and t h e t e a r meniscus i s roughly comparable to the water c o n t e n t of a hydrogel lens. The h y p e r t o n i c t e a r s w i l l d e h y d r a t e t h e c o r n e a l e p i t h e l i u m r e s u l t i n g i n o c u l a r d i s c o m f o r t ( i t c h i n g ) to the p a t i e n t . As t h e i s o t o n i c i t y of the tears i s r e - e s t a b l i s h e d through d i l u t i o n , the sensation of comfort i s again r e s t o r e d . The e x i s t e n c e o f a t h i n aqueous f i l m between a h y d r o g e l l e n s and t h e c o r n e a l e p i t h e l i u m i s a m a t t e r o f c o n t r o v e r s y . If the lens i s i n d i r e c t contact w i t h the cornea, the osmotic e f f e c t due t o t h e aqueous phase o f t h e h y d r o g e l l e n s w i l l be a c t i n g d i r e c t l y upon t h e c o r n e a l e p i t h e l i u m w i t h t h e same r e s u l t s d i s cussed above.

Abstract The swelling pressure of two types of hydrogels, which were classified according to application and hydration, were investigated. 1. Poly(glyceryl methacrylate) (PGMA) hydrogels which are intended for surgical uses in the eye, are divided into two subgroups, a) hydrogels of about 80 to 85%H Oat equilibrium swelling, for scleral buckling procedures in retinal detachment surgery, and b) hydrogels of above 98%H Oat equilibrium swelling, for vitreous implantation. The swelling pressure-volume relationship of these hydrogels was determined with dextran solutions. 2. Hydrogel contact lens materials (40-75%H Oat equilibrium). The swelling pressure of PHEMA hydrogels was determined by equilibration under osmotic and mechanical pressure. 2

2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Although slight pressure will remove water from highly hydrated hydrogels, it is very difficult to expel water from medium hydra tion (40-80% HO) hydrogels. The equilibrium relative humidity at different states of hydration of PHEMA, P(HEMA/VP), P(MMA/VP), and P(GMA/MMA) was investigated. The results show that up to 30% HO, the water activity in the hydrogels is below the activity for pure water. However, all water above 30% hydration up to equilibrium swelling has the same vapor pressure as pure water. 2

2

Acknowledgements The author is indebted to Dr. F. Holly for his helpful discussion. Ms. F.L. Leong provided technical assistance. A. Seidman provided editorial assistance. This study was supported b PHS Grant EY-00327 f th National Eye Institute Literature Cited 1. 2. 3.

Katchalsky, Α.: Prog. Bioph. (1954) 4:1 Rijke, A.M., and Prins, W.: J. Polym. Sci. (1962) 59:171 Posnjak, E.: Kolloidchem. Beih. (1912) 3:417, in M.R. Kruyt, Ed. Colloid Science II. Elsevier Publishing Co., New York, 1949,p557 4. Refojo, M.F.: Anales Qui. (Madrid) (1972) 68:697 5. Refojo, M.F.: J. Biomed. Mater. Res. Symposium (1971) 1:179 6. Daniele, S., Refojo, M.F., Schepens, C.L., and Freeman, H.M.: Arch. Ophthal. (1968) 80:120 7. Dohlman, C.H., Refojo, M.F., Webster, R.G., Pfister, R.R., and Doane, M.G.: Arch. Ophthal. (Paris) (1969) 29:849 8. Calabria, G.A., Pruett, R.C., and Refojo, M.F.: Arch. Ophthal. (1971) 86:77 9. Refojo, M.F.: J. Appl. Polym. Sci. (1965) 9:3161 10. Senti, F.R., Hellman, N.N., Ludwig, N.M., Babcock, G.E., Tobin, R., Glass, C.A., and Lamberts, B.L.: J. Polym. Sci. (1955) 17:527 11. Refojo, M.F.: J. Appl. Polym. Sci. (1965) 9:3417 12. Wichterle, O. and Lim, D.: Nature (1960) 185:117 13. Refojo, M.F.: Encycl. Polymer. Sci. Technol. Supplement (in press) 14. Seiderman, M.: U.S. Patent 3,721,657 (1973) 15. Frankland, J.D., and Highgate, D.J.: Ger. Offen. (1973) No. 2,312,470 [in Chem. Abstr. 80:27872z] 16. Refojo, M.F., and Yasuda, H.: J. Appl. Polymer Sci. (1965) 9:2425 17. Vink, H.: European Polym. J. (1971) 7:1411 18. Lee, M.B., Jhon, M.S., and Andrade, J.D.: J. Colloid and Interface Sci. (1975) 51:225 19. Quinn, F.C.: Reprint No. 472, Am. Instrument Co., Div. Travenol Labs, Silver Springs, Md. In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

3 The Role of Proteoglycans and Collagen in the Swelling of Connective Tissue F. A. MEYER, R. A. GELMAN, and A. SILBERBERG Weizmann Institute of Science, Rehovot, Israel

Connective tissue provide th chemical environment fo most body cells and th tion. It varies in composition according to this function and characteristically involves a f i b r i l l a r skeletal network composed mainly of collagen f i b r i l s and the so-called ground substance , a highly disperse system of proteoglycans, macromolecules involving both protein and carbohydrate chains. Bone, cartilage, vessel walls, skin, tendon, umbilical cord and synovial fluid are typical examples of connective tissues. A l l of them are characterized by a low cell content. The major biological roles fulfilled by connective tissues in the body are physical in nature. They involve mechanical function such as motion and transport and (chemical) communication between cells. In its mechanical role connective tissue transmits energy from the muscles permitting the performance of work and the absorbance of stresses from the environment thereby protecting delicate structures. Clearly, the physico-chemical properties and the structure of connective tissue are the important factors fixing these features. The s t r u c t u r e and chemistry o f the i n d i v i d u a l connective t i s s u e components v i z . c o l l a g e n , e l a s t i n and proteoglycans can be summarized as f o l l o w s : Collagen (1). Collagen i s a f i b r o u s p r o t e i n which i s formed by the assembly o f monomer u n i t s o f t r o p o c o l l a g e n . Tropocollagen i s a rod shaped molecule V300 nm long and 1.4 nm i n diameter w i t h a molecular weight o f 300,000. I t c o n s i s t s o f three chains of equal l e n g t h . F i v e t r o p o c o l l a g e n molecules can pack t o give a m i c r o f i b r i l , the u n i t s are l o n g i t u d i n a l l y staggared around t h e m i c r o f i b r i l a x i s . M i c r o f i b r i l s pack i n a t e t r a g o n a l l a t t i c e t o give f i b r i l s . There i s a gap between consecutive t r o p o c o l l a g e n s i n the f i b r i l which together w i t h the s t a g g e r i n g gives c o l l a g e n a c h a r a c t e r i s t i c 65 nm repeat d i s t a n c e . S t a b i l i t y o f the s t r u c t u r e i s due i n p a r t t o the q u a r t e r s t a g g e r i n g , which optimizes secondary bond i n t e r a c t i o n s and covalent c r o s s l i n k s as w e l l 52

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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ET AL.

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occur between and w i t h i n t r o p o c o l l a g e n u n i t s . The h i g h l y ordered compact arrangement w i t h i n the f i b e r gives i t a r i g i d character. Large v a r i a t i o n s are seen i n the degree o f c r o s s l i n k i n g , f i b r i l diameter, f i b r i l o r g a n i z a t i o n and f i b r i l c o n c e n t r a t i o n i n v a r i o u s t i s s u e s . Moreover, four g e n e t i c a l l y d i s t i n c t types of c o l l a g e n e x h i b i t i n g some t i s s u e s p e c i f i c i t y have been char a c t e r i z e d which i n p a r t may e x p l a i n the v a r i a t i o n s o f c o l l a g e n morphology seen. Ε las t i n (2) . E l a s t i n i s g e n e r a l l y i n v o l v e d w i t h t h e f i b r i l l a r network. I t contains an unusually h i g h p r o p o r t i o n o f non-polar amino a c i d s and the amino a c i d s , desmosine and i o d o desmosine, which may be unique t o t h i s p r o t e i n . A corpuscular s t r u c t u r e has been propose the peptide chain i s f o l d e l i n k i n g occurs a t the s u r f a c e between such u n i t s . The b a s i s o f •the e l a s t i c nature o f t h i s p r o t e i n a r i s e s from the work r e q u i r e d t o u n f o l d the hydrophobic r e g i o n s . Ground Substance Components (3_) . The major ground substance components are proteoglycans which c o n s i s t o f g l y c o s aminoglycans attached t o p r o t e i n . The most abundant g l y c o s aminoglycans are h y a l u r o n i c a c i d , c h o n d r o i t i n s u l f a t e , keratan s u l f a t e , and dermatan s u l f a t e . The glycosaminoglycans are l i n e a r chains c o n s i s t i n g o f r e p e a t i n g n e g a t i v e l y charged d i saccharide u n i t s . The p r o t e o g l y c a n , h y a l u r o n i c a c i d , i s o f h i g h molecular weight and i n s o l u t i o n adopts a very voluminous random c o i l c o n f i g u r a t i o n . I t i s a s s o c i a t e d w i t h a s m a l l amount of p r o t e i n (<0.5%) which i s n o t important f o r t h e i n t e g r i t y o f the molecule. This i s the dominant proteoglycan found i n loose connective t i s s u e , e.g. s k i n , v e s s e l w a l l s , heart v a l v e s and u m b i l i c a l cord and i s present a t concentrations o f l e s s than 1% based on t i s s u e volume. I n loose connective t i s s u e t h e system i s bathed i n a medium which contains about 3% serum p r o t e i n . The t i s s u e i s maintained i n a steady s t a t e i n contact w i t h the m i c r o c i r c u l a t i o n o f the blood and t h e lymph system. Exchange w i t h blood i n v o l v e s the n o n - s e l e c t i v e f i l t r a t i o n o f water and low molecu l a r weight c o n s t i t u e n t s ( s a l t , m e t a b o l i t e s , etc.) through the e n d o t h e l i a l w a l l o f the c a p i l l a r y system, t h e i r r e a b s o r p t i o n on t h e venous s i d e and a one way t r a n s f e r o f serum albumin and other macromolecular plasma c o n s t i t u e n t s t o the t i s s u e . These macromolecules are recovered predominantly through the lymph system, which i s formed i n open channels i n d i r e c t and open contact w i t h t i s s u e and i s returned t o t h e blood through an a c t i v e pumping system. The b l o o d , on the other hand, communi cates w i t h the t i s s u e through a p a r t i a l l y s e l e c t i v e membrane represented by the v e s s e l w a l l . I n response t o p a t h o l o g i c a l

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changes, t i s s u e w i l l s w e l l and d e s w e l l , passing from one steady s t a t e t o another. We have i n v e s t i g a t e d the s t r u c t u r a l o r g a n i z a t i o n o f loose connective t i s s u e and have e s t a b l i s h e d the r o l e s of v a r i o u s conn e c t i v e t i s s u e components i n e f f e c t i n g t i s s u e volume changes. These r e s u l t s could be of i n t e r e s t i n the development of hydrog e l s f o r medical a p p l i c a t i o n and w i l l here be reviewed. Experimental U m b i l i c a l cord (Wharton's J e l l y ) was used i n these s t u d i e s . The methods have been d e s c r i b e d (4_,5_,6) and w i l l here only be summarized. Tissue s l i c e s (some 300-600 mg i n weight) were incubated i n s m a l l volumes o f s w e l l i n g medium ( s a l i n e ) cont a i n i n g added macromolecules or other c o n s t i t u e n t s Weights both of the t i s s u e and was the c o n c e n t r a t i o n o way both weight (volume) changes could be c a l c u l a t e d and the d i s t r i b u t i o n o f the macromolecular species determined. Using v a r i o u s enzymes, s p e c i f i c m o d i f i c a t i o n s i n t o t i s s u e s t r u c t u r e could be i n t r o d u c e d and evaluated. Results The most important r e s u l t s can be reviewed as f o l l o w s : 1. Tissue S l i c e s Incubated i n Serum Albumin S o l u t i o n s (up t o about 12%) i n Ringer's P h y s i o l o g i c a l S a l i n e . The f i n a l e q u i l i b r i u m volume of t i s s u e i s l a r g e r than the i n i t i a l volume a t a l l c o n c e n t r a t i o n s (Figure 1 ) . The dependence o f the e q u i l i b rium s w e l l i n g r a t i o on serum albumin c o n c e n t r a t i o n i s given i n F i g u r e 2. I f e x t r a p o l a t i o n of the data i s p e r m i t t e d , no s w e l l i n g would occur a t about 15% serum albumin. Only a s m a l l f r a c t i o n o f the h y a l u r o n i c a c i d (as measured by u r o n i c a c i d release) i s l o s t during incubation. 2. Treatment o f the Samples w i t h T e s t i c u l a r Hyaluronidase. This produces a t i s s u e of a b o l i s h e d s w e l l i n g tendency and p r a c t i c a l l y no volume changes occur a g a i n s t any of the serum albumin c o n c e n t r a t i o n used (Figure 2 ) . I f p r e v i o u s l y s w o l l e n samples are e n z y m a t i c a l l y t r e a t e d , they d e s w e l l t o the o r i g i n a l volume (Figure 3 ) . 3. Treatment of Tissue w i t h P r o t e o l y t i c Enzymes, For Example, T r y p s i n . This produces a f i n a l e q u i l i b r i u m volume l a r g e r than the c o n t r o l where no t r y p s i n treatment has been used (Figure 4 ) . A t r y p s i n treatment f o l l o w e d by a hyaluronidase treatment, or v i c e v e r s a , r e s u l t s i n the same f i n a l e q u i l i b r i u m volume which i s , however, l a r g e r than the o r i g i n a l volume o f the t i s s u e sample. The same f i n a l volume r e s u l t s i r r e s p e c t i v e of when the enzymes were added d u r i n g the s w e l l i n g process (Figure 4 ) .

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Figure 1. The degree of swelling (β) with respect to the initial tissue volume as a func tion of time. Incubated at 4° at neutral pH in solutions of bovine serum albumin whose ini tial concentration (a = o) is given in percent (w/v). In the case of the more concentrated solutions there is an initial volume decrease. Eventually all samples are swelling, and the final equilibrium volume is larger than the initial. t

2

a* (gm /

100 ml)

Figure 2. The equilibrium degree of swelling (β*) plotted against external serum albumin concentration (a*) at equilibrium for the intact (Q) and hyaluronidase-treated (Φ) tissue. Note that in contrast to the intact tissue, treatment with hyaluronidase produces a tissue of practically no swelling tendency. The dependence of l/β* on a* is linear. So plotted, the data show no swelling ten dency of intact tissue at about a* = 15%.

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1

H Y A L U R O N I D A S E (H) A D D I T I O N (RINGER'S

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2.0 4°C)

03.

Τ

J

1

1.5

\

_

1.0

Figure 3. The swelling (β) as a function of time for intact tissue (Q>) and for tissue treated with hyaluronidase (Φ). Treatment with the enzyme occurs at the times indi cated by the arrows. Such treatment the tissue to its original volume of the degree of swelling of the tissue.

Intact

e

——·

t Η

ι—Γ AND I HHYALURONIDASE(H) Y TRYPSIN(T) ADDITION

° Τ (Control)

30

Ά

/ H\

\ . HtT

20

Λ

H(Control) TIME (min)

Figure 4. The swelling of tissue (β) as a func tion of time for tissue treated with either trypsin or hyaluronidase (Ç)) or a combination of the two (Φ) at the times indicated by the arrows. Note that the equilibrium value of β after tryp sin treatment is 3.2 as compared with a value of about 2.1 for intact tissue (see Figure 3). Tryp sin treatment followed by hyaluronidase treat ment or vice versa gives the same equilibrium value of β independent of the time and order of treatment.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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T r y p s i n , chymotrypsin and pronase each r e s u l t s i n a s p e c i f i c f i n a l e q u i l i b r i u m volume. 4. Treatment o f the Tissue w i t h Glutaraldehyde. T h i s abolishes the tendency o f t i s s u e t o s w e l l . The t i s s u e volume i s f i x e d a t the volume a t which the treatment was a p p l i e d . 5. I n t e r a c t i o n o f the Tissue w i t h High Molecular Weight Poly(L-Lysine). A t p h y s i o l o g i c a l s a l t concentrations t h i s causes one-to-one b i n d i n g o f the polybase by the a c i d groups 6f the proteoglycans. I n t h i s c o n d i t i o n the proteoglycans are e x t e n s i v e l y p r o t e c t e d from subsequent hyaluronidase a c t i o n (5). The i n t e r a c t i o n a l s o serves t o freeze i n the c o n f i g u r a t i o n i n which the t i s s u e f i n d s i t s e l f a t the time o f i n t e r a c t i o n . Thus when i n c u b a t i n g the t i s s u e i n a p o l y (jj-lysine) s o l u t i o n , normal s w e l l i n (du t s a l i n p e n e t r a t i o n ) d t i s s u e f i x a t i o n (due t h i g h e r the p o l y ( L - l y s i n e ) , p e n e t r a t i o n and the c l o s e r i s the u l t i m a t e volume, t o which the t i s s u e s w e l l s , t o the o r i g i n a l volume o f the t i s s u e sample (Figure 5). As the i o n i c s t r e n g t h o f the suspending medium i s increased t o about 0.5 M, p o l y ( L - l y s i n e ) i s excluded t o t a l l y from the t i s s u e and no e f f e c t whatsoever on t i s s u e volume i s experienced (Figure 6 ) . An i n v e s t i g a t i o n o f the t i s s u e by e l e c t r o n microscopy (4_) shows t h a t the c o l l a g e n f i b e r s are randomly d i s t r i b u t e d i n space and are composed o f bundles o f p a r a l l e l f i b r i l s some 54 nm i n diameter and some 54 nm a p a r t . Discussion These r e s u l t s are c o n s i s t e n t w i t h the f o l l o w i n g f u n c t i o n a l model (4): I n the u s u a l k i n d o f g e l , the network acts both as the s w e l l i n g agent and the source o f the e l a s t i c r e s t r a i n i n g f o r c e ; i n connective t i s s u e these two f u n c t i o n s are separated. The c o l l a g e n based f i b r i l l a r network i s the mechanical s p r i n g and i s thermodynamically not a c t i v e , whereas the p r o t e o g l y c a n component a c t s as the a c t i v e s w e l l i n g agent but i s not d i r e c t l y forming the network. I f the p o t e n t i a l o f the s o l v e n t medium i s too low, f l u i d w i l l enter the t i s s u e and d i l u t e the p r o t e o g l y cans. Since the p r o t e o g l y c a n s , however, are mechanically enmeshed w i t h the f i b r i l l a r network, the d r i v i n g f o r c e i s t r a n s f e r r e d t o the f i b e r network and thus produces i t s expansion. When the proteoglycans are digested away the c o l l a g e n f i b e r system returns the t i s s u e t o i t s o r i g i n a l volume and water content. Hence the unstressed s t a t e o f the f i b e r network, i . e . the mechanical r e f e r e n c e s t a t e , i s the n a t i v e s t a t e o f the tissue. Treatment w i t h p r o t e o l y t i c enzymes a l t e r s the f i b r i l l a r system and s h i f t s i t s f o r c e l e s s s t a t e t o one o f higher volume. Reaction w i t h gluteraldehyde "freezes" i n the p a r t i c u l a r deformation s t a t e o f the system and no volume change a t a l l

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Figure 5. Dependence the degree of swelling (β) on poly(L-lysine) con centration in 0.15M NaCl at neutral pH. Note that poly (L-lysine) binds strongly to proteo glycans at this sodium chloride concentration and freezes in the volume of the tissue at which it interacts with the polysaccharide. Hence, only at high poly(L-lysine) concentrations (fast polybase penetration into the tissue) is the poly(L-lysine) able to abolish swelling.

2.0

Figure 6. The ultimate degree of swelling (β) as a function of salt concentration (A) and salt concentration in the presence of 1 mg/ml of poly(h-lysine) (B) at neutral pH. Note that there is interaction with poly(Llysine) when the sodium chloride concen tration is less than 0.5M. At a concentration of 1 mg/ml poly(L-lysine) the freezing in occurs only after some swelling has already taken place (see Figure 5).

0.5 NaCl Concentration

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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occurs. I n t e r a c t i n g the t i s s u e w i t h p o l y ( L - l y s i n e ) produces a one-to-one complex w i t h the proteoglycans and s w e l l i n g i s r e s t r i c t e d . This i s probably because the proteoglycans being complexed are no longer o s m o t i c a l l y a c t i v e . Simultaneously the i n t e r a c t i o n s t i f f e n s the proteoglycans and c r o s s l i n k s them i n t o a network so t h a t no f u r t h e r volume change can occur. I t i s o f i n t e r e s t , u s i n g n o n - i n t e r a c t i n g macromolecular probes (6_,7) t h a t most o f the t i s s u e space, o u t s i d e t h a t ex cluded by the c o l l a g e n f i b r i l s , i s a c c e s s i b l e . Tissue i s thus a c o l l a g e n f i b r i l " g e l " which i s o s m o t i c a l l y a c t i v e because o f the presence of the proteoglycans. These may form an independent network, but are more l i k e l y l i n k e d through t h e i r p h y s i c a l involvement w i t h the c o l l a g e n system. The f i n d i n g t h a t t i s s u e i s not i n e q u i l i b r i u m w i t h a p r o t e i n s o l u t i o n o f compositio i n t e r s t i t i a l f l u i d , confirm t a i n t i s s u e , i n v i v o , i n a "dehydrated" s t a t e . I t may be assumed t h a t t h i s "dehydrated" s t a t e o f t i s s u e i s maintained by the blood flow through exchange processes across the endo t h e l i a l b a r r i e r which a c t s as a semipermeable membrane w i t h respect t o macromolecules. The s w e l l i n g tendency o f t i s s u e due t o the proteoglycans i s thus o f f s e t p e r m i t t i n g the c o l l a g e n f i b e r network, as i t grows and assembles, t o be i n a f o r c e - f r e e state. The r e s u l t s obtained i n the present study d e s c r i b e t h e behavior o f u m b i l i c a l cord. There are i n d i c a t i o n s t h a t other types o f connective t i s s u e tend t o e x h i b i t s i m i l a r behavior {8) but not a l l forms o f connective t i s s u e a r e n e c e s s a r i l y expected t o f a l l i n t o t h i s p a t t e r n . The d i v i s i o n i n t o an o s m o t i c a l l y a c t i v e and i n a c t i v e network system i s b e l i e v e d , however, t o be g e n e r a l l y c o r r e c t . f

Literature Cited 1. Traub, W. and Piez, K.A. Adv.Ρrot.Chem. (1971) 25, 243. 2. Partridge, S.M., in: "The Comparative Molecular Biology of Extracellular Matrices", H.C. Slavkin ed. (1972), AcademicPress, New York and London. 3. Laurent, T.C. Pflűgers Arch. (1972), 336 (Suppl.) S21. 4. Meyer, F.A. and Silberberg, A. Microvascular Res. (1974) 8, 263. 5. Gelman, R.A. and Silberberg, A. Connective Tissue Res., accepted for publication. 6. Meyer, F.A., Koblenz, M. and Silberberg, Α., to be published. 7. Meyer, F.A. and Silberberg, A. Pflűgers Arch. (1972) 336 (Suppl.), S37. 8. Hedbys, B.O. Exp.Eye Res. (1961) 1, 81.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

4 The Role of Water in the Osmotic and Viscoelastic Behavior of Gel Networks MU SHIK JHON,* SHAO MU MA, SACHIKO HATTORI, DONALD E. GREGONIS, and JOSEPH D. ANDRADE Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 The existence of an ordered structure at water/solid inter faces has been generall certain preferred orientation independen neighboring molecules. In the case of water the word "structured" should not be misinterpreted; we do not mean that water possess long range orderness. By "structure" we mean the orderness rela tive to that in bulk water. In a strict sense, even the molecules in bulk water are structured because of hydrogen bonding and other near neighbor interactions. Drost-Hansen (1_) has discussed a three-layer model for the structure of water near certain water/solid interfaces. According to this model, water molecules near the solid surface are struc tured; those sufficiently far away from the surface have bulk water structure and those in between have decreasing orderness as a function of distance from the interface. Others (2,3) have indicated the existence of three states of water in natural macro molecular gels (2) or in membranes of cellulose acetate (3). Jhon and Andrade (4) proposed a three-state model of water in hydrogel systems. They suggested that three classes of water exist in hy drogels, namely X water (bulk water), Ζ water (bound water), and Y water (intermediate forms or interfacial water). Following this, Lee, Jhon and Andrade (5,6) tested the model by thermal expansion, specific conductivity, differential scanning calorimetry, and proton spin-lattice nuclear magnetic relaxation studies for poly(hydroxyethyl methacrylate) (PHEMA gels). Very recently, Choi, Jhon and Andrade {7) again extended the model by means of thermal expansion, specific conductivity, and dielectric relaxation studies for (2,3-dihydroxypropyl methacrylate) gels (DHPMA gels). In this paper theories for the osmotic and viscoelastic behavior of hydrogels are developed in terms of water structure. Some experimental results on the viscoelastic behavior of hydrogels are presented. *0n leave from Korea Advanced Institute of Science, Seoul, Korea; to whom correspondence should be addressed. 60

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S w e l l i n g and O s m o t i c

Pressure

A c c o r d i n g t o F l o r y (8), t h e n e t w o r k s t r u c t u r e mav have s e v e r a l roles. I n a s o l v e n t t h e n e t w o r k d i s s o l v e s and t a k e s t h e r o l e o f a solute. In a s o l u t i o n i t p e r m i t s t h e p a s s a g e of solvent mole c u l e s and keeps o u t o t h e r d i s s o l v e d m a t e r i a l s a n d , h e n c e , a c t s as a membrane. As t h e n e t w o r k s w e l l s t h e p o l y m e r c h a i n s a r e e l o n g a t e d and e x e r t a f o r c e i n o p p o s i t i o n t o t h e s w e l l i n g . In t h i s c a s e , t h e n e t w o r k a c t s as a p r e s s u r e g e n e r a t i n g d e v i c e . His t h e o r y o f s w e l l i n g i s based on t h e b a l a n c i n g o f t h e o s m o t i c p r e s s u r e by t h e m e c h a n i c a l c o n t r a c t i o n . To o b t a i n h i s f o r m a l e x p r e s s i o n , F l o r y u t i l i z e s the Flory-Huggins polymer s o l u t i o n t h e o r y a s s u m i n g random m i x i n g between s o l u t e and s o l v e n t and a rigid lattice. The e x p r e s s i o n i n c l u d e s two t e r m s an e n t r o p y t e r m ( c o m b i n a t i o n a l ) an (non-combinational). Later c o r r e s p o n d i n g s t a t e t h e o r y f o r p o l y m e r s o l u t i o n s w i t h a more rigorous expression f o r t h e non-combinational c o n t r i b u t i o n than the Flory-Huggins approach. However, t h e c o m b i n a t i o n a l t e r m i s s t i l l used i n i t s o r i g i n a l f o r m . In t h e s w e l l i n g o f h y d r o g e l s , t h e random m i x i n g a s s u m p t i o n between h i g h p o l y m e r and w a t e r i s n o t g e n e r a l l y v a l i d , b e c a u s e o f t h e h i g h d e g r e e o f s t r u c t u r i n g o f w a t e r i n some g e l n e t w o r k s . The P r i g o g i n e t h e o r y , w h i c h i s l i m i t e d t o n o n - p o l a r and m o d e r a t e l y p o l a r s y s t e m s w i t h no h y d r o g e n b o n d i n g , i s a l s o n o t g e n e r a l l y applicable. In t h i s p a p e r , a new s e m i - e m p i r i c a l i n t e r a c t i o n p a r a m e t e r , Δ , i s i n t r o d u c e d and used i n an e q u a t i o n o f s w e l l i n g b a s e d on a " S o l u b i l i t y M o d e l " (TOJ t o a v o i d t h e a b o v e - m e n t i o n e d d i f f i c u l t i e s . The e q u i l i b r i u m c o n d i t i o n f o r i s o t r o p i c s w e l l i n g (9J c l a s s i c a l l y requires that 2

\ 3Ni/T,P

\ 3Ni / T , P

L

1

J

where A F and àF ^ a r e t h e f r e e e n e r g y o f m i x i n g and e l a s t i c b e h a v i o r , r e s p e c t i v e l y , and N i i s t h e mole f r a c t i o n o f s o l v e n t . The " S o l u b i l i t y M o d e l " (10) s t i l l c a r r i e s t h e same e l a s t i c t e r m , b u t t h e f i r s t t e r m i s m o d i f i e d . The p r o c e s s o f t r a n s f e r r i n g one mole o f w a t e r from b u l k t o g e l network i n v o l v e s t h r e e s t e p s ( F i g u r e 1 ) : m

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(energy t o d i g holes i n solvent)

1st

Step ΔΗ (heat o f vaporization)

3rd

Step Δ (polymer-solvent interaction) 2

Water i n bulk

Water i n |Gel Network

Figure 1. A schematic of the process of transferring one mole of water from bulk to gel network

F i r s t Step. To b r i n g a mole o f w a t e r f r o m t h e b u l k phase t o t h e vapor phase: T h i s r e q u i r e s an e n e r g y w h i c h e q u a l s t h e h e a t o f vaporization o f water, Δ Η : ν

ΔΗ

γ

= ΔΗ °

+ £

ν

T

Cp d T ,

[2]

where Δ Η ° i s t h e h e a t o f v a p o r i z a t i o n a t some r e f e r e n c e t e m p e r a t u r e , T , and Cp i s t h e s p e c i f i c h e a t . Second s t e p . To b r i n g w a t e r m o l e c u l e s from t h e vapor phase t o t h e e x i s t i n g g e l water: A t o r n e a r e q u i l b r i u m t h e g e l n e t w o r k i s expanded w i t h w a t e r , w h i c h c o n s i s t s o f X , Y and Ζ t y p e s , f i l l i n g t h e empty spaces. The e n e r g y , Δ ι , r e q u i r e d i n t h i s s t e p i s f o r c r e a t i n g a mole o f c a v i t y o f t h e s i z e o f t h e s o l u t e m o l e c u l e ( w a t e r i n v a p o r ) ag a i n s t t h e s o l v e n t ( g e l w a t e r ) s u r f a c e t e n s i o n : γ

Δ! = 4 i r r 2

2

(γ Ζ + γ Y + γ Χ ) / ( 1 ζ y χ

+ Κ),

where 4lïr

[3]

i s the surface area of the water molecule; γ , γ , χ y γ , a r e t h e s u r f a c e t e n s i o n s and Χ , Υ, Ζ a r e t h e w e i g h t f r a c t i o n s o f t h e three types o f water, Χ , Υ, Ζ r e s p e c t i v e l y . The t e r m 1/(1 + K) needs a l i t t l e more e x p l a n a t i o n . Jhon, G r o s h , Ree and E y r i n g ( 1 1 ) p r o p o s e d a model i n w h i c h ( b u l k )

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

4.

JHON ET AL.

Water in the Behavior of Gel Networks

63

w a t e r i s v i s u a l i z e d as c o n t a i n i n g a t l e a s t t w o s o l i d - l i k e s t r u c t u r e s , t h e i c e - I - l i k e open s t r u c t u r e a n d t h e i c e - I I I - l i k e c l o s e d s t r u c t u r e , i n e q u i l i b r i u m w i t h each o t h e r and w i t h t h e g a s - l i k e m o l e c u l e s (A g a s - l i k e m o l e c u l e r e f e r s t o a m o l e c u l e w h i c h i s s u r r o u n d e d by h o l e s ) . A t e r m 1/(1 + K) i s i n t r o d u c e d i n e q u a t i o n [ 3 ] on t h e a s s u m p t i o n t h a t t h e e n e r g y o f c a v i t y formation i s neglible i n the i c e - I - l i k e part of the l i q u i d s t r u c t u r e , and Κ i s t h e e q u i l i b r i u m c o n s t a n t between i c e - I - l i k e and i c e - I I I - l i k e d o m a i n s . Third step. To b r i n g w a t e r m o l e c u l e s in g e l water t o gel network: T h i s s t e p r e q u i r e s an e n e r g y o f i n t e r a c t i o n between t h e p o l y m e r m o l e c u l e s a n d t h e s u r r o u n d i n g water molecules, Δ ( 1 2 ) : 2

A =fn 2

/~

U(R) < S

>

dR.

[4]

where A and Β d e n o t e w a t e t h e number d e n s i t y o f s o l v e n t m o l e c u l e s ; f i s t h e q u a n t i t y w h i c h takes account o f t h e f a c t t h a t t h e d i s t r i b u t i o n o f s o l v e n t m o l e c u l e s o f t h e p o t e n t i a l minimum i s d e n s e r t h a n t h e a v e r a g e d e n s i t y ; U(R) i s t h e i n t e r m o l e c u l a r p o t e n t i a l o f A and Β m o l e c u l e s a t a s e p a r a t i o n d i s t a n c e , R; a n d ^ / \ + R ^ the <

>

1

+r

S

v

a v e r a g e v a l u e o f t h e s u r f a c e a r e a . To e v a l u a t e U(R) a n d /\ ^ 3 /\ » the reader should consult the c i t e d references (10, < s

>

+

+

v

12), A l t h o u g h t h e new i n t e r a c t i o n p a r a m e t e r Δ i s d i f f i c u l t t o e v a l u a t e d i r e c t l y , i t c a n be o b t a i n e d f o r a v a i l a b l e s y s t e m s i n the i n p u t d a t a i n E q u a t i o n [ 2 ] , E q u a t i o n [ 3 ] and second terms i n Equation [ 1 ] a r e provided. Experiments t o support t h i s s e m i - e m p i r i c a l s o l u b i l i t y t h e o r y w o u l d be f i r s t , t o d e t e r m i n e t h e i n t e r a c t i o n p a r a m e t e r , Δ , f o r v a r i o u s g e l - w a t e r s y s t e m s . T h e s e c a n be o b t a i n e d b y m e a s u r i n g s w e l l i n g d e g r e e s as t h e o n l y i n p u t d a t a , t h e r e s t being a v a i l a b l e i n the l i t e r a t u r e . S w e l l i n g e x p e r i m e n t s on PHEMA n e t w o r k s made o f c o n t r o l l e d p u r i t y a r e underway. The o b t a i n e d Δ w i l l be compared t o t h e F l o r y i n t e r a c t i o n p a r a m e t e r χ ( 8 ) . O t h e r s o l v e n t s y s t e m s s u c h as a l c o h o l s s h o u l d a l s o be studied. S e c o n d , a phase d i a g r a m may be c o n s t r u c t e d f o r t h e water-polymer system. P e r f e c t symmetry a t t h e c o n s o l u t e t e m p e r a t u r e w i l l s u p p o r t t h e h y p o t h e s i s o f random m i x i n g o f w a t e r and p o l y m e r m o l e c u l e s . D e v i a t i o n s from i t would i n d i c a t e support of the s o l u b i l i t y theory. The t h r e e - s t a t e model o f w a t e r s t r u c t u r e c a n a l s o be a p p l i e d t o osmotic pressure o f polymer s o l u t i o n s . The u s u a l e x p r e s s i o n f o r o s m o t i c p r e s s u r e c a n be u s e d e x c e p t t h a t t h e volume s h o u l d be r e p l a c e d by V ( X f x ) , where f x i s t h e r a t i o between t h e s o l u b i l i t y o f s o l u t e i n X o r b u l k w a t e r t o t h a t i n g e l w a t e r . We b e l i e v e t h a t o n l y t h e p o r t i o n o f w a t e r a v a i l a b l e f o r o s m o t i c p r e s s u r e s h o u l d be i n c l u d e d i n t h e o s m o t i c p r e s s u r e e q u a t i o n . T y i n g both s w e l l i n g and o s m o t i c p r e s s u r e e x p e r i m e n t s t o g e t h e r w i t h t h e t h r e e - s t a t e t h e o r y , one may 2

2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

64

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

perform a s w e l l i n g and/or osmotic p r e s s u r e experiment o f pure w a t e r as a f u n c t i o n o f t e m p e r a t u r e . As w a t e r i s h e a t e d f r o m - 30°C t o 1 0 0 ° C , any change i n s w e l l i n g a n d / o r o s m o t i c p r e s s u r e w o u l d be p a r t l y due t o m e l t i n g o f i c e - I - l i k e c l u s t e r s ( 1 1 ) . Viscoelastic

Properties

The e f f e c t o f s o l v e n t on t h e v i s c o e l a s t i c b e h a v i o r o f h y d r o g e l s has been w i d e l y r e p o r t e d i n t h e l i t e r a t u r e ( 1 3 , 1 4 , 1 5 ) . In c r e e p and s t r e s s r e l a x a t i o n m e a s u r e m e n t s , t h e r e t a r d a t i o n t i m e and r e l a x a t i o n t i m e a r e f u n c t i o n s o f s o l v e n t . In dynamic s t u d i e s t h e s o l v e n t r e d u c e s t h e γ r e l a x a t i o n p r o c e s s and s h i f t s the β process to lower temperatures. Furthermore, the c o n c e n t r a t i o n and t h e n a t u r e o f low m o l e c u l a r w e i g h t compounds a f f e c t t h e s i z e and shap w e l l as t h e a p p a r e n t a c t i v a t i o s t u d i e s , C i n t h e M o o n e y - R i v l i n e n u a t i o n i s a f f e c t e d by t h e solvent. H y d r o g e l s , i n t h e r u b b e r y r e g i o n , behave much l i k e r u b b e r . T h e r e f o r e , i n t h i s study, a theory f o r the s t r e s s - s t r a i n r e l a t i o n i n h y d r o g e l s was d e v e l o p e d by m o d i f y i n g t h e t h e o r y of rubber e l a s t i c i t y . Consider a f r e e l y o r i e n t i n g chain which c o n t a i n s η segments. The f o r o e F needed t o m a i n t a i n t h e c h a i n a t an a v e r a g e e l o n g a t i o n L i s g i v e n by t h e expression: 2

F

L

= i7 * Φ

or

τ-Μ,ιφ.

[5]

The s t r e s s σ needed t o m a i n t a i n a r u b b e r n e t w o r k a t h i g h e l o n g a t i o n i s g i v e n by (16) σ/vkT = 1 n\*{a/n ) h

-α"

2

,

[6]

where v , a , l , k and Τ a r e , r e s p e c t i v e l y , t h e number o f c h a i n s p e r u n i t v o l u m e , t h e e x t e n s i o n r a t i o , t h e seament l e n g t h , B o l t z m a n n ' s c o n s t a n t , and t h e a b s o l u t e t e m p e r a t u r e . L and L* a r e t h e L a n g e v i n f u n c t i o n and i n v e r s e L a n g e v i n f u n c t i o n , r e s p e c t i v e l y w h i c h a r e d e f i n e d by χ = L ( y ) = c o t h y - 1/y and y = L * ( x ) , r e s p e c t i v e l y . F o r a h y d r o g e l w i t h m a i n l y X w a t e r , i t i s r e a s o n a b l e t o assume t h a t t h e p o l y m e r c h a i n s can r o t a t e f r e e l y and E q u a t i o n s [ 5 ] and [ 6 ] a p p l y . However, f o r h y d r o g e l s w i t h m o s t l y Ζ w a t e r , due t o t h e c o n s t r a i n e d s t a t e , o n l y two l i m i t e d c o n f o r m a t i o n s can o c c u r , i . e . , i n t e r n a l i s o m e r i z a t i o n between two c o n f o r m e r s k e e p i n g t h e p o s i t i o n o f e a c h c h a i n end f i x e d . In m a g n e t i c t h e o r y , i t i s known t h a t t h e m a g n e t i c f i e l d s h i f t s t h e r e l a t i v e amount o f two o r i e n t a t i o n s . F o r M mag n e t i c d i p o l e s e a c h o f w h i c h can e x i s t e i t h e r i n t h e d i r e c t i o n o f t h e m a g n e t i c f i e l d , H, o r a g a i n s t t h e f i e l d , t h e r e l a t i o n between t h e m a g n e t i z a t i o n , I, and t h e m a g n e t i c f i e l d t a k e s Q

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

4.

Water

JHON ET AL.

in the Behavior

of Gel

65

Networks

t h e f o r m [ 1 7 ] I/m M = t a n h (|γ ), where m i s t h e m a g n e t i c moment. In p o l y m e r e l a s t i c i t y t h e f o r c e p l a y s a c o r r e s p o n d i n g role. The e q u i v a l e n t e x p r e s s i o n f o r p o l y m e r e l a s t i c i t y c a n be written as: 7(

.

hL\

£ e

°

F

/

k

0

e

We s h a l l

-£ F/kT

T

" *oF/kT ;

k T

refer to Ζ ( ^ γ )

e

JUF/kT

-

t

a

n

h

^/kT).

ill

as t h e Ζ f u n c t i o n .

F o r h y d r o g e l s w i t h m o s t l y Y w a t e r s t r u c t u r e , we e x p e c t t h a t b o t h t h e L f u n c t i o n and t h e Ζ f u n c t i o n s h o u l d f a i l t o a p p l y and a Y f u n c t i o n s h o u l

π YUoF/kT) = /

s i n e cose tanh ( H F cose/kT)

de .

0

[8]

0 In t h i s c a s e , n o t o n l y two o r i e n t a t i o n s ( c o n f o r m e r ) a r e p e r m i t t e d , b u t t h e p o s i t i o n o f each c h a i n end i s n o t f i x e d . With t h e same argument t h e n u m e r i c a l v a l u e o f a Y f u n c t i o n l i e s between t h e c o r r e s p o n d i n g X f u n c t i o n and Ζ f u n c t i o n . H e n c e , e q u a t i o n [ 6 ] c a n be m o d i f i e d as f o l l o w s : α/vkT = i r\ lXL*(a/n ) h

+ W*{a/n )

h

h

+ ZZ*(a/n^) - a " \

[9]

where Y * , Z* a r e t h e i n v e r s e Y f u n c t i o n and i n v e r s e Ζ f u n c t i o n , respectively. To t e s t o u r h y p o t h e s i s , t h e s t r e s s - e l o n g a t i o n c u r v e s f o r three poly(hydroxyethy1 methacrylate) gels with d i f f e r e n t w a t e r c o n t e n t s were o b t a i n e d f r o m s t r e s s - s t r a i n measurements a t room t e m p e r a t u r e ( 2 3 ° C ) ( F i g u r e 2 ) . The o b s e r v e d d a t a a r e g i v e n by t h e s o l i d l i n e s i n t h e f i g u r e . The v a l u e s o f X , Y and Ζ a r e t a k e n f r o m Lee ( 5 , 6 ) . C h o o s i n g η = 100 (we f o u n d t h a t r e s u l t s o b t a i n e d f o r η = 100 and η = 1000 do n o t d i f f e r s i g n i f i c a n t l y ) , t h e v a l u e s o f σ c a n be c a l c u l a t e d as a f u n c t i o n o f a . The c o n s t a n t s , v k T , needed f o r f i t t i n g t h e e x p e r i m e n t a l c u r v e s a r e 1.09 χ 1 0 d y n e s / c m ( G e l I, 45% w a t e r ) ; 6 . 0 3 χ 1 0 d y n e s / c m ( G e l I I , 31% w a t e r ) ; and 2 . 5 9 χ 1 0 d y n e s / c m ( G e l I I I , 2 9 . 9 % w a t e r ) . The c a l c u l a t e d p o i n t s a r e i n d i c a t e d i n t h e figure. G e l s I and I I were p r e p a r e d w i t h t h e i n d i c a t e d amount o f w a t e r i n t h e p o l y m e r i z a t i o n m i x w h i l e G e l I I I was p r e p a r e d w i t h 100% h y d r o x y e t h y l m e t h a c r y l a t e monomer and t h e n s w e l l e d t o 29.9% water c o n t e n t . Although t h e w a t e r c o n t e n t i n G e l II and G e l I I I a r e a b o u t t h e same, t h e f o r m e r i s c o n s i d e r a b l y tougher than t h e l a t t e r . I t i s p o s s i b l e t h a t , a t 23°C, t h e behavior o f Gel II i s n o t w i t h i n i t s r u b b e r y r e g i o n , s i n c e t h e a g r e e m e n t between t h e c a l c u l a t e d and measured a v a l u e s f o r t h i s g e l i s l e s s 7

2

2

7

7

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

66

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

4.

JHON ET AL.

Water in the Behavior of Gel Networks

s a t i s f a c t o r y t h a n t h o s e f o r t h e o t h e r two

67

gels.

Summary A theory i s developed to i n t e r p r e t the osmotic, s w e l l i n g and v i s c o e l a s t i c b e h a v i o r o f h y d r o g e l n e t w o r k s i n t e r m s o f a t h r e e - s t a t e model o f w a t e r s t r u c t u r e . For i s o t r o p i c s w e l l i n g under e q u i l i b r i u m c o n d i t i o n s , F l o r y assumed random m i x i n g between t h e s o l v e n t m o l e c u l e s and t h e p o l y m e r m o l e c u l e s . Since water molecules i n hydrogels possess h i g h e r degrees o f orderness than those i n the b u l k , i t i s b e l i e v e d t h a t the s o l u b i l i t y t h e o r y (10) s h o u l d be used i n s t e a d o f t h e c l a s s i c a l Flory theory. T h i s i s because s o l u b i l i t y t h e o r y c o n s i d e r s the f r e e energy of mixing of water w i t h the gel network, which i n c l u d e s the hea required to c r e a t e hole i n t e r a c t i o n between w a t e r and p o l y m e r . In o u r t h e o r y o n l y t h e p o r t i o n o f w a t e r a v a i l a b l e f o r o s m o t i c p r e s s u r e was i n c l u d e d i n the osmotic pressure e q u a t i o n . For the v i s c o e l a s t i c b e h a v i o r o f h y d r o g e l s , t h e t h e o r y o f r u b b e r e l a s t i c i t y was m o d i f i e d t o accommodate t h e e f f e c t o f t h r e e t y p e s o f w a t e r on t h e i r s t r e s s strain relationship. P o l y m e r c h a i n s w i t h m a i n l y X w a t e r can r o t a t e f r e e l y , t h o s e w i t h m a i n l y Ζ w a t e r can o n l y have two r e s t r i c t e d c o n f o r m a t i o n s and t h o s e w i t h m a i n l y Y w a t e r can have intermediate behavior. In a c t u a l c a s e s , t h e c o n t r i b u t i o n s f r o m a l l t h r e e t y p e s o f w a t e r s h o u l d be c o n s i d e r e d s i n c e t h e y a r e c o e x i s t i n g i n any p o l y m e r - w a t e r s y s t e m . A few e x p e r i m e n t s a r e proposed. A c c o r d i n g t o some e x p e r i m e n t a l r e s u l t s , t h e t h e o r y p r o v i d e s good a g r e e m e n t .

Acknowledgement We g r a t e f u l l y a c k n o w l e d g e s u p p o r t o f t h i s work by NIH HL1692K)! and NSF G r a n t GH33996X.

Grant

Abstract A three-state model of water structure in hydrogels has been extended to describe the osmotic, swelling and viscoelastic be havior of gel networks. The solubility theory modification of the classical Flory theory is proposed to explain the osmotic and swelling behavior of gel networks. In describing the viscoe lastic behavior of hydrogels, three functions, governed by the three types of water, are used to explain the stress-strain relations in the rubbery region.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

68

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Literature Cited 1. Drost-Hansen, W., Indust. Eng. Chem. (1969) 61, 10. 2. Aizawa, Μ., and Suzuki, S., Bull. Chem. Soc. Japan (1971) 44, 2907. 3. Krishnamurthy, S., McIntyre, D., and Santee, E. R., Jr., J. Polym. Sci. (1973) 11, 427. 4. Jhon, M. S., and Andrade, J. D., J. Biomed. Mater. Res. (1973) 7, 509. 5. Lee, H. B., Andrade, J. D., and Jhon, M. S., Polymer Preprints (1974) 15, 706. 6. Lee, H. B., Jhon, M. S., and Andrade, J. D., J. Colloid and Interface Sci. (1974) 51, 225. 7. Choi, S. H., Jhon M S. and Andrade J D. Submitted for publication. 8. Flory, P. J. "Principle Polyme Chemistry, University Press, Ithaca, New York (1953). 9. Prigogine, I., Belleman, Α., and Englert-Chowles, Α., J. Chem. Phys. (1956) 24, 518. 10. Jhon, M. S., Eyring, H., and Sung, Y. K., Chem. Phys. Letters (1972) 13, 36. 11. Jhon, M. S., Grosh, J., Ree, T., and Eyring, H., J. Chem. Phys (1966) 44, 1465. 12. Kihara, T., and Jhon, M. S., Chem. Phys. Letters (1970) 7, 559. 13. Janacek, S., and Kolarik, J., Coll. Czech. Chem. Comm. (1965) 30, 1597. 14. Janacek, J., and Ferry., J. D., J. Poly. Sci. (1969) Part A-2, 7, 1681. 15. Ilavsky, M., and Prins, W., Macromolecules (1970) 3, 415. 16. Bueche, F., J. Appl. Poly. Sci. (1960) 4, 107. 17. Hill, T. L., "An Introduction to Statistical Thermody namics," Addison-Wesley Publishing Company, Inc., Reading and London (1960).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5 Permeability as a Means to Study the Structure of Gels N. WEISS and A. SILBERBERG Weizmann Institute of Science, Rehovot, Israel

Permeation flow of the suspending medium past a non-moving gel matrix can also be interpreted th translatio f th l substance through the stationar permeation coefficient bears some relationship to the sedimentation coefficient of an isolated macromolecule of the same chemical build-up as the gel substance. There should, in fact, be a one-to-one relationship between the two if the macromolecule and the gel network are both freely draining, i.e. if the hydrodynamic effects are linearly additive and the resistance to flow per gram of gel substance is the same as the translational friction coefficient per gram of the free macromolecule. Non-linear (in segment concentration) hydrodynamic interactions between the parts of the macromolecular chains will, however, affect the flow resistance and the permeation rates will depend upon the structural arrangements of the network strands. Hence measurements of permeability can be used for an analysis of gel structure. The main difficulty in the way of using this approach has been the lack of suitable experimental equipment which would avoid large pressures and large pressure gradients in the gel. A device will be described and results obtained with it discussed. Experimental The principle of the apparatus is illustrated in Figure 1. Two compartments are involved. Flow is from 1 to 2. Volume displacement is achieved by bending of a thin metal diaphragm by means of a piston driven by a linear actuator. Volume displacement is measured by means of a LVDT (linear variable differential transformer) connected to the piston and calibrated in terms of volume displacements in a separate set up involving a fine glass capillary. Pressure is measured in each compartment by means of sensitive, very small volume change pressure transducers. The cells are filled to completion by rotatory valves on each compartment. 69

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

70

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Volume displacements o f the order o f 10~^ ml are aimed a t and such s m a l l volume changes correspond approximately t o the volume changes t h a t might be expected from the response o f t h e pressure t r a n s d u c e r , the f i n i t e compliance o f the w a l l s and the c o m p r e s s i b i l i t y of the f l u i d . To overcome t h i s d i f f i c u l t y the pressure i n compartment 1 i s maintained equal t o ambient by means of a feedback c i r c u i t which i s d r i v e n by pressure transducer 1 and actuates the volume displacement device 1. Hence there i s no compression of the f l u i d i n compartment 1 and the w a l l s o f c e l l 1 are not under s t r e s s . A s i m i l a r feedback c i r c u i t keeps the pressure i n compartment 2 a t a s e l e c t e d l e v e l ΔΡ = P 2 ~ l ambient by s u i t a b l y a d j u s t i n g the volume displacement device 2. While volume changes d e r i v i n g from the causes l i s t e d above do a r i s e i n compartment 2; they are not important f o r the volume measurement s i n c e o n l be known. An o u t l i n e drawing o f the instrument i s given i n F i g u r e 2 and the feedback c o n t r o l c i r c u i t s are d e s c r i b e d i n Figure 3. The g e l s s t u d i e d i n the present i n v e s t i g a t i o n (1) are based on acrylamide co-polymerized i n water w i t h N-N' methylene b i s acrylamide as c r o s s l i n k i n g agent, ammonium persulphate as i n i t i a t o r and Ν,Ν,Ν',N -tetramethy1 ethylenediamine as regu l a t o r . Since t h i s r e a c t i o n i s s t r o n g l y i n f l u e n c e d by oxygen the r e a c t i o n was c a r r i e d out under n i t r o g e n and i n s o l u t i o n s through which n i t r o g e n had been bubbled t o s a t u r a t i o n . N e v e r t h e l e s s , even i f these p r e c a u t i o n s were taken, the upper l a y e r s o f any g e l produced tended t o be o f d i f f e r e n t s t r u c t u r e . To a v o i d such e f f e c t s i n t e r f e r i n g w i t h t h e g e l samples a c t u a l l y t e s t e d and i n order t o produce w e l l d e f i n e d and w e l l anchored g e l b l o c k s the f o l l o w i n g procedure was adopted. The s p e c i a l mould i l l u s t r a t e d i n F i g u r e 4 was used. The r e a c t i o n mixture was f i l l e d i n t o t h e mould under n i t r o g e n , t h e t h i c k n e s s o f the f i n a l g e l l a y e r being c o n t r o l l e d by spacer as shown. The upper and lower p a r t s was then removed as e x p l a i n e d i n the t e x t t o the F i g u r e and the cup w i t h the g e l l a y e r i n p l a c e was clamped between the two c e l l s o f the instrument. The g e l mixtures prepared are c h a r a c t e r i z e d by the concent r a t i o n o f monomer and the percentage o f c r o s s l i n k i n g reagent i n the monomer mixture. Measurements a t one pressure were completed w i t h i n one minute i n general and c o u l d be reproduced w i t h great accuracy. Sedimentation r e s u l t s were obtained on f r a c t i o n s o f l i n e a r polyacrylamide i n a Model E U l t r a c e n t r i f u g e a t t h e concentrations shown and were c o r r e c t e d f o r pressure by e x t r a p o l a t i o n u s i n g the method of E l i a s (2). The polymers were s y n t h e s i z e d u s i n g the same r e a c t i o n procedure as f o r the g e l s but l e a v i n g out the m u l t i f u n c t i o n a l monomer. p

o

1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

v

e

r

WEISS A N D SILBERBERG

Permeability VALVE 2

VOLUME DISPLACEMENT 2

Figure 1.

in Gel Study VALVE 1

VOLUME DISPLACEMENT 1

Schematic outline of permeability apparatus

Figure 2. Outline drawing of permeability apparatus. 1, half-cell; 2, rotatory valve; 3, connecting ring; 4, connection for pressure transducer; 5, volume displacement device; 6, LVDT; 7, linear actuator; 8,9, base.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR M E D I C A L A N D R E L A T E D

APPLICATIONS

RECORDER

Figure 3. Feedback P23D pressure transducer amplifier/indicator 311-A; OA, Kepco bipolar operational power supply BOP 36-1.5 (M); LA, Derritron VP.2 MM vibrator; LVDT, Sanborn Linearsyn differential transformer 585 DT-050; C, permeability half-cell. (l

6

7

Figure 4. Special mould for preparation of gel blocks. 1, Clamping screws; 2, upper clamp; 3, holes for screws; 4, escape hole for excess gel mixture; 5, upper defining surface for gel block; 6, spacer ring defining block thickness; 7, cell with perforated bottom for gel block; 8, perforation; 9, seal; 10, lower clamp.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

73

Permeability in Gel Study

WEISS A N D SILBERBERG

T h e o r e t i c a l R e l a t i o n s h i p between Permeation C o e f f i c i e n t Kç. and the Sedimentation Constant ( j ) The f l u x J p e r u n i t c r o s s - s e c t i o n J

= ( K / n ) (-Vp) g

o f the g e l i s given by

,

(1)

where Vp i s the a p p l i e d pressure g r a d i e n t , K i s the permeation c o e f f i c i e n t and η i s the v i s c o s i t y o f the suspending medium o f the g e l . I t should be noted t h a t s

Κ /η = L , (2) s ρ where Lp i s the h y d r a u l i c volume flow permeation c o e f f i c i e n t used i n membrane a n a l y s i s . I d e f i n e d as the r e c i p r o c a u n i t volume o f suspending medium, i . e . Κ /η = L = 1/f . s p w

(3)

The f r i c t i o n c o e f f i c i e n t f i s d e f i n e d as the f o r c e exerted on the immobile g e l m a t r i x , p e r u n i t volume o f the suspending medium, a t u n i t r e l a t i v e v e l o c i t y . I f the suspending medium i s a t r e s t ( i n the mean) and the g e l substance ( i n t h e same c o n f i g u r a t i o n as before) i s moving r e l a t i v e t o i t , we can d e f i n e a f r i c t i o n f a c t o r f g which i s the force which has t o a c t on u n i t volume o f g e l substance f o r i t t o move w i t h u n i t v e l o c i t y r e l a t i v e t o the suspending medium. Since the f o r c e which would be r e q u i r e d t o move the g e l substance through the suspending medium a t a c e r t a i n v e l o c i t y must equal the f o r c e t o move the suspending medium through the g e l substance at the same (mean) v e l o c i t y , we can w r i t e ( 4 ) : w

(1/f ) = (1/f )((1-φ)/φ) w g

.

(5)

I f , i n the case o f movement o f the g e l substance through the suspending medium, the f o r c e a c t i n g upon the g e l substance de r i v e s from a c e n t r i f u g a l f i e l d ( u l t r a c e n t r i f u g e ) i t may be questionable whether t h e g e l membrane w i l l not change c o n f i g u r a tion (the c e n t r i f u g a l but not the hydrodynamic f o r c e s are u n i formly d i s t r i b u t e d ) . I f , n e v e r t h e l e s s , we make t h i s i d e n t i f i c a tion λ

2 ν/ω r = s = ( l - p v ) / f ν , (6) g where ν i s the v e l o c i t y o f movement, ω i s the angular v e l o c i t y , r i s the l o c a t i o n , from the c e n t e r , o f the phase boundary i n the u l t r a c e n t r i f u g e , ρ i s the d e n s i t y o f the s o l u t i o n and ν i s the s o l u t e p a r t i a l s p e c i f i c volume. Whereas s c a l c u l a t e d from

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

74

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

eqn. (6) may not t h e r e f o r e match s measured i n an a c t u a l e x p e r i ment, i t nevertheless p r o v i d e s a r e n d i t i o n of t h e p e r m e a b i l i t y data i n a form which can be compared w i t h sedimentation v a l u e s . Hence combining equations ( 3 ) , (5) and (6), K /n s

= L =

p

= l / f =

(1/f

w

(sv/U-pv))

)(1-φ)/φ

((1-φ)/φ)

.

(7)

Equation (7) can be used t o c a l c u l a t e an e f f e c t i v e K , L o r f value from a measurement of s , o r an e f f e c t i v e s (or f ) value from a measurement of K or Lp. Since the volume f r a c t i o n φ can be w r i t t e n s

p

w

g

s

φ

= cv

where c i s the c o n c e n t r a t i o n o f t h e g e l substance i n weight p e r u n i t volume, we can r e w r i t e equation (7) as f o l l o w s cK

s

= η (s) ( l - c v ) / ( l - p v )

.

(9)

An equation i d e n t i c a l t o t h i s has been d e r i v e d by M i j n l i e f f and Jaspers ( 5 ) . I t d i f f e r s from equation (9) o n l y i n t h a t t h e f a c t o r (1-cv)/(1-pv) i s w r i t t e n i n the e q u i v a l e n t form ( l - v / v ) where v i s the p a r t i a l s p e c i f i c volume o f the s o l v e n t . M i j n l i e f f and Jaspers attached p h y s i c a l s i g n i f i c a n c e t o t h e i r r e s u l t b e l i e v i n g t h a t permeation and sedimentation r e s u l t s could be compared i n t h i s way. I t i s important t o note, however, t h a t equation (9) can h o l d o n l y i n the u n l i k e l y event t h a t e x a c t l y the same system i s used both i n determining K and s. In p a r t i c u l a r , data obtained from sedimentation s t u d i e s , o f u n c r o s s l i n k e d m a t e r i a l , should n o t , when used i n (9) be expected t o match K , measured on a c r o s s l i n k e d system even of the same o v e r a l l c o n c e n t r a t i o n and even i f as i n our case the e x p e r i mental s-values were c o r r e c t e d f o r t h e h i g h pressure i n t h e ultracentrifuge. On the other hand, as already p o i n t e d o u t , equation (9) represents a u s e f u l means o f t r a n s l a t i n g permeation i n t o e f f e c t i v e sedimentation r e s u l t s and v i c e v e r s a . I n p a r t i c u l a r , one may look upon sedimentation data of independent macromolecules as corresponding t o zero c r o s s l i n k d e n s i t y . w

w

s

s

Results The r e s u l t s c a l c u l a t e d as c K e i t h e r from equation ( 1 ) , i n the case o f permeation d a t a , o r from equation (9) i n the case of sedimentation are represented i n F i g u r e 5. Sedimentation r e s u l t s depended on molecular weight. The data shown i s f o r a sample o f 1.57x10^ M.W. A t concentrations s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

WEISS A N D

Permeability

SILBERBERG

75

in Gel Study

above the minimum i n the curve, however, the molecular weight dependence of the sedimentation data was n e g l i g i b l e . Discussion M o l e c u l a r , S t r u c t u r a l I n t e r p r e t a t i o n of K

s

Since permeation flow occurs r e l a t i v e t o the g e l m a t r i x , the system i s hydrodynamically two phase: the f l o w i n g suspending medium and the immobile g e l substance. I f a l l the suspending medium i s f l o w i n g e q u a l l y f r e e l y around a l l p a r t s o f the g e l substance we can r e p l a c e the l a t t e r by a system o f e q u a l l y s i z e d spheres, evenly strung out along the strands o f the network. I f there are ν such spheres p e r u n i t volume o f g e l substance and each has a f r i c t i o n c o e f f i c i e n f

= vf ο

g

(10)

and combining equations ( 7 ) , cK

s

( 8 ) and

(10)

= η (1/vf )(l-cv)/v ο

(11)

.

Since i n the extreme f r e e d r a i n i n g case envisaged, f by d e f i n i t i o n does not depend upon the d e n s i t y of c r o s s l i n k s c K / should be independent o f c r o s s l i n k d e n s i t y as w e l l . On the o t h e r hand, i f we assume some c l u s t e r i n g o f the seg ments around p o i n t s , f o r example, i n r e g i o n s where the c r o s s l i n k d e n s i t y l o c a l l y i s above average, these regions w i l l a c t t o h i n d e r flow through them (become non-draining) and a more appro p r i a t e p i c t u r e t o use i s t h a t o f a number o f o v e r s i z e d spheres, equal t o the number of c l u s t e r e d r e g i o n s , a c t i n g as f r i c t i o n centers (Figure 6 ) . I f t h e r e are again ν such spheres p e r u n i t volume of g e l substance each c o n t a i n i n g a l e n g t h I o f g e l network chain and each of e f f e c t i v e Stokes r a d i u s a, Q

s

f

(12)

= 67rna

ο

and ν =

1/AA

,

(13)

where A i s the molecular c r o s s - s e c t i o n o f the g e l network chains. Combining equations ( 1 1 ) , ( 1 2 ) and ( 1 3 ) cK

g

(14)

= [ (A/6TT) ( l - c v ) / v ] ( V a ) .

I n equation ( 1 4 ) the term i n square b r a c k e t s , but not (&/a), i s independent of the degree o f c r o s s l i n k i n g . Hence K as a func t i o n o f the degree of c r o s s l i n k i n g i s determined s o l e l y by the r a t i o (l/a.) . s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

0

0.05

0.10

0.15

C(gm/cm ) 3

Figure 5. Specific permeability K c = s-rj(l — (v)/(l — pv) as a function of concentration c for aqueous polyacrylamide gels. Parameter: percent crosslinking monomer per ordinary monomer added. 0% corresponds to non-crosslinked material, and the data represent sedimentation results corrected for pressure. s

A

S

Figure 6. Freely and partially draining gel. V = (Al/cv) (1 — cv); V, effective volume of gel containing one hydrodynamic resistance center of radius a and a length of polymer chain I. (i) Freely draining case; (ii) partially draining case.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

77

Permeability in Gel Study

WEISS A N D SILBERBERG

T h i s r a t i o i s a measure o f t h e segment d i s t r i b u t i o n a r o u n d each p o i n t o f r e s i s t a n c e . I f one c a n p a c k a l a r g e amount o f c h a i n ( l a r g e i) i n t o a s m a l l volume ( s m a l l a) p e r m e a t i o n r a t e s w i l l be h i g h . I f , on t h e o t h e r hand, t h e l e n g t h I approaches the dimensions o f t h e c h a i n d i a m e t e r , a , as w e l l , w i l l t e n d towards t h i s v a l u e . For a given concentration, c, therefore, t h e l o w e s t p e r m e a t i o n r a t e s w i l l o c c u r when i/a - 1 and t h e c h a i n s a r e f r e e l y d r a i n i n g . Where t h e r e a r e s t r u c t u r a l i n h o m o g e n e i t i e s , however, f o r example a l o c a l c l u s t e r i n g o f c r o s s l i n k s , (£/a) » 1 (many segments g r o u p e d i n n o n - d r a i n i n g s p a c e s ) p e r meation r a t e s w i l l be h i g h . T h i s can be seen even b e t t e r i f e q u a t i o n (14) i s r e a r r a n g e d t o g i v e

Κ

s

= V/6i\a

(15)

Here V = ( 1 - c v ) /(cv/Al)

(16)

i s t h e volume o f s u s p e n d i n g medium a s s o c i a t e d w i t h e a c h e f f e c t i v e s p h e r e o f h y d r o d y n a m i c r a d i u s a ( F i g u r e 6 ) . C l e a r l y Kg w i l l b e t h e l a r g e r , t h e l a r g e r V and t h e s m a l l e r a , o r i n o t h e r w o r d s , K w i l l be l a r g e i f t h e r e i s l o c a l c r o w d i n g o f t h e g e l network a r o u n d an e f f e c t i v e c r o s s l i n k l e a v i n g l a r g e v o l u m e s o f s u s p e n d i n g medium f r e e o f s e g m e n t s , o r , t r i v i a l l y , i f we go f r o m a m i c r o - p o r o u s network t o a macro-porous network k e e p i n g t h e amount o f g e l s u b s t a n c e f i x e d . The r e s u l t s o f F i g u r e 5 i n t e r p r e t e d as %/a f r o m e q u a t i o n (14) a r e u s e d i n F i g u r e 7. The f o l l o w i n g r e l a t i o n s h i p was u s e d t o e v a l u a t e A/v : s

A / v = Μ /Ν b ο A

(17)

where M i s t h e m o l e c u l a r w e i g h t , b t h e l e n g t h o f t h e monomer u n i t a n d N i s A v o g a d r o ' s number. W i t h MQ = 71 gm a n d b = 2 . 5 x l 0 ~ cm, A / v i s f o u n d t o b e 4 . 7 1 x l 0 ~ g m / c m . Since ν = 0 . 7 0 1 ml/gm t h e c r o s s - s e c t i o n A = 3 . 3 x l 0 ~ ^ c m . Q

A

8

1 5

1

4TT

2

3

I f we p u t V = y - R and assume t h a t t h e volume f r a c t i o n o f polymer i n the non-draining region o f r a d i u s a i s φ we f i n d that α 1

I r -

3

φ _

7-

r

where fL - 1 - 4 (R-a) i s t h e a c t u a l l e n g t h o f c h a i n i n v o l v e d with the non-draining region. I t follows that Q

(V a

[1-4

a

- l)/£ 1 a

= φÎsi.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

(19)

78

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 7. Permeability data of Figure 5 recalculated according to Equation (14) in relation to the model of Equation (19)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

5.

WEISS A N D SILBERBERG

Permeability

79

in Gel Study

We w i l l assume t h a t i n the r e g i o n where the r e s u l t s i n Figure 5 come together a t h i g h e r c o n c e n t r a t i o n s , the o v e r a l l c o n c e n t r a t i o n φ j u s t matches the c o n c e n t r a t i o n φ ^ i n the c r o s s l i n k r e g i o n s . This suggests t h a t φ ^ - 0.2 and we make t h e f u r t h e r assumption t h a t t h i s w i l l now a l s o be the c o n c e n t r a t i o n i n the c r o s s l i n k regions a t a l l lower o v e r a l l concentrations as w e l l . With t h i s we can c a l c u l a t e the dependence of l/a on φ f o r given R/a u s i n g equation (19). The r e s u l t s a r e i n d i c a t e d by the dotted l i n e s i n F i g u r e 7. The f u l l l i n e s i n t h i s f i g u r e are the r e s u l t s from F i g u r e 5 redrawn using equation (14) f o r il/a and equation (8) f o r φ. I t w i l l be seen, i n t e r e s t i n g l y , t h a t £/a - 1 f o r the noncross l i n k e d polymers but t h a t i t l i e s between 10 and 100 f o r t h e c r o s s l i n k e d systems. Fro th r o s t e f line t f i x e d R/ values we f i n d t h a t p o r o s i t i n c r e a s e d . A t the sam l a r g e region f r e e l y d r a i n i n g ( i n c r e a s i n g R/a) more and more polymer segments w i l l be found o u t s i d e the e f f e c t i v e c r o s s l i n k r e g i o n . This w i l l e v e n t u a l l y tend t o decrease p e r m e a b i l i t y and accounts f o r t h e maximum i n the curves. Since ΙΑ = φ(4ττ/3^^* we f i n d u s i n g equation (19) , α

α

a

= A[£/a - 4 (- - 1 ) ] / ψ- φ _ . a ο ci. 15

(20)

2

We have c a l c u l a t e d t h a t A = 3 . 3 x l 0 ~ cm and assumed t h a t Φσΐ °· · Hence equation (20) can be used together w i t h t h e data i n Figure 7.to c a l c u l a t e a f o r each experimental p o i n t . I t turns o u t t h a t a, the r a d i u s o f the e f f e c t i v e c r o s s l i n k r e g i o n / ranges from about 1.5 nm f o r the g e l s of low degree o f c r o s s l i n k i n g t o 7 nm f o r the g e l s a t the high degree of c r o s s l i n k i n g which we have measured. Hence p e r m e a b i l i t y measurements can be used t o c h a r a c t e r i z e the i n t e r n a l arrangement o f g e l s . =

2

Acknowledgment The a i d i n p a r t from a grant by the I s r a e l N a t i o n a l Academy o f Sciences and Humanities i n support o f t h i s research i s g r a t e f u l l y acknowledged.

Literature Cited 1. Weiss Ν., Ph.D. Thesis, Feinberg Graduate School, Weizmann Institute of Science, Rehovot 1975. 2. Elias H.G., Makromol.Chem. (1959) 29 30. 3. Weiss Ν. and Silberberg A., Second International Congress of Biorheology, Rehovot, 1975. Biorheology, (1975) 12 107. 4. Spiegler K.S., Trans.Farad.Soc. (1958) 54 1408. 5. Mijnlieff P.F. and Jaspers W.J.R., Trans.Farad.Soc. (1971) 67 1837.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6 Diffusion through Hydrogel Membranes I.

Permeation of Water Through Poly(2-hydroxyethyl methacrylate) and Related Polymers

S. J. WISNIEWSKI, D. E. GREGONIS, S. W. KIM, and J. D. ANDRADE Departments of Applied Pharmaceutical Sciences and Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112

H y d r o g e l s a r e known f o r t h e i r u n i q u e p h y s i c a l p r o p e r t i e s and potential biomedical a p p l i c a t i o n s some p o l y ( 2 - h y d r o x y e t h y been p r e v i o u s l y i n v e s t i g a t e d (1-4). R a t n e r and M i l l e r {5) have shown t h a t p o l y HEMA membranes have a h i g h p e r m e a b i l i t y t o u r e a due t o i n t e r a c t i o n between t h e s o l u t e and membrane, a n d , i n a d d i t i o n , c o n s i d e r t h e e x i s t e n c e o f p o r e s i n t h e p o l y HEMA g e l s t r u c t u r e , w i t h t h e w a t e r r e g i o n s o f t h e g e l a c t i n g as " p o r e s " for solute transport. R e c e n t l y Chen f o u n d (6_) t h a t t h e a b s o r p t i o n o f w a t e r by d e h y d r a t e d p o l y HEMA was a f u n c t i o n o f c r o s s ! i n k e r c o n t e n t . In h i s s t u d y d i f f u s i o n c o e f f i c i e n t s were c a l c u l a t e d f r o m a b s o r p t i o n k i n e t i c data using F i c k ' s law. Several f a c t o r s i n f l u e n c e the s t r u c t u r e o f h y d r o g e l s and s u b s e q u e n t l y t h e i r d i f f u s i o n c h a r a c teristics. These f a c t o r s i n c l u d e i n i t i a t o r , c r o s s l i n k i n g a g e n t s , c r o s s l i n k e r c o n t e n t , e q u i l i b r i u m w a t e r c o n t e n t and i m p u r i t i e s . Yasuda e t a l . ( 1 , 1 1 ) have u t i l i z e d a t h e o r y , b a s e d on a f r e e - v o l u m e c o n c e p t o f d i f f u s i v e t r a n s p o r t , f o r h y d r a t e d homogeneous membranes. They c o n c l u d e d t h a t t h i s c o n c e p t e x c e l l e n t l y e x p l a i n s t h e d i f f u s i v e t r a n s p o r t p a r a m e t e r s as a f u n c t i o n o f water c o n t e n t over a wide h y d r a t i o n range. In t h i s s t u d y , we have been c o n c e r n e d w i t h v a r y i n g c r o s s l i n k e r c o n c e n t r a t i o n s and w a t e r c o n t e n t . I t i s hoped t h a t t h i s d a t a w i l l p r o v i d e a d d i t i o n a l i n f o r m a t i o n on t h e b a s i c t r a n s p o r t mechanisms f o r w a t e r i n d i f f e r e n t g e l s y s t e m s and on t h e r o l e o f water i n the transport of other s o l u t e s . Experimental H y d r o x y e t h y l m e t h a c r y l a t e was o b t a i n e d as a g i f t f r o m Hydron L a b o r a t o r i e s (New B r u n s w i c k , New J e r s e y ) and was used w i t h o u t a d d i t i o n a l p u r i f i c a t i o n . M e t h o x y e t h y l m e t h a c r y l a t e and m e t h o x y e t h o x y e t h y l m e t h a c r y l a t e were p r e p a r e d i n o u r l a b o r a t o r i e s by base c a t a l y z e d t r a n s e s t e r i f i c a t i o n o f m e t h y l m e t h a c r y l a t e w i t h

80

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6. wisNiEwsKi E T A L .

81

Diffusion through Hydrogel Membranes

t h e c o r r e s p o n d i n g a l c o h o l . E t h y l e n e g l y c o l d i m e t h a c r y l a t e (Monomer P o l y m e r L a b o r a t o r i e s , P h i l a d e l p h i a , P e n n s y l v a n i a ) and tetraethylene glycol dimethacrylate (Polysciences, Warrington, P e n n s y l v a n i a ) were p u r i f i e d by base e x t r a c t i o n t o remove i n h i b i t o r f o l l o w e d by d i s t i l l a t i o n . A z o b i s m e t h y l i s o b u t y r a t e , p r e p a r e d by t h e method o f M o r t i m e r Ç 7 ) , was used t o i n i t i a t e p o l y m e r i z a t i o n a t a c o n c e n t r a t i o n o f 7.84 m m o l e s / l i t e r monomer. Crosslinker c o n c e n t r a t i o n was c a l c u l a t e d on a m o l e - t o - m o l e b a s i s w i t h HEMA. The d e s i r e d monomer s o l u t i o n was m i x e d w i t h 45% v/v d i s t i l l e d w a t e r and p o l y m e r i z e d between g l a s s p l a t e s a t 60°C f o r 24 h o u r s . A g l a s s d i f f u s i o n c e l l , w h i c h c o n t a i n s two compartments o f e q u a l volume (175 m l ) , was d e s i g n e d . Each chamber was s t i r r e d a t a c o n s t a n t r a t e t o reduce boundary l a y e r e f f e c t s . A t t h e b e g i n n i n g o f each e x p e r i m e n t , one compartment was f i l l e d w i t h t r i t i a t e d w a t e r , t h e o t h e r was f i l l e t r i t i a t e d w a t e r was m o n i t o r e times. These a l i q u o t s were p l a c e d i n 10 ml o f l i q u i d s c i n t i l l a t i o n " c o c k t a i l " ( A q u a s o l , New E n g l a n d N u c l e a r Company) and counted i n a Packard S c i n t i l l a t i o n c o u n t e r . The t h i c k n e s s o f t h e w e t membrane was measured u s i n g a l i g h t m i c r o m e t e r (Van Kauren Company). Membrane d e n s i t i e s were measured by w e i g h i n g s e c t i o n s o f w e t membrane o f known v o l u m e . Water c o n t e n t was o b t a i n e d f r o m t h e d i f f e r e n c e i n w e i g h t o f w e t and d r y membrane ( d r i e d t o c o n s t a n t w e i g h t u n d e r vacuum a t a b o u t 6 0 ° C ) . A l l e x p e r i m e n t s were r u n a t 23°C ± 1 ° C . Results

(8)

and D i s c u s s i o n

The e q u a t i o n used t o t r e a t o u r d a t a was d e r i v e d e l s e w h e r e and i s g i v e n as f o l l o w s : In

(1 - 2 C / C ) = T

Q

(J

+ \)

AUt,

where C t = H 0 c o u n t a t t i m e t ; C = H 0 c o u n t a t t i m e 0 ; V i = V = compartment volume = 175 m l ; A = membrane c o n t a c t a r e a = 14.9 c m ; U = p e r m e a b i l i t y ( c m / s e c ) ; and t = t i m e ( s e c o n d s ) . A p l o t o f In (1 - 2 C t / C ) v s . t i m e w i l l y i e l d a s t r a i g h t l i n e w i t h slope = -(1/V + 1/V )AU. S u b s t i t u t i n g our values of A , V and V g i v e s U = - 5 . 8 7 χ s l o p e (cm/sec). The d i f f u s i o n c o e f f i c i e n t s a r e g i v e n by D = Ud/Kp, where d = w e t membrane t h i c k n e s s and Kp i s t h e p a r t i t i o n c o e f f i c i e n t . By d e f i n i t i o n Kp = w a t e r c o n c e n t r a t i o n i n membrane/water c o n c e n t r a t i o n i n b u l k , w h i c h f o r o u r c a s e r e d u c e s t o Kp = ( j v | / y ) f · * 3

3

2

0

2

2

2

0

X

x

2

2

p

p

W

a n c

P^ a r e t h e w e t membrane d e n s i t y and t h e d e n s i t y o f w a t e r a t 23°C, r e s p e c t i v e l y . wet membrane and

W i s the weight f r a c t i o n of water i n the f

i s e q u a l t o W /W , where W i s t h e w e i g h t o f W

M

w

w a t e r and W i s t h e w e i g h t o f t h e w e t membrane. M

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

82

HYDROGELS

FOR MEDICAL

AND RELATED APPLICATIONS

Table 1 l i s t s the o b t a i n e d parameters f o r the v a r i o u s g e l s used. The d a t a f o r t h e ^ 0.2 m o l e - % d i e s t e r c r o s s ! i n k e r p o l y HEMA g e l s and u n r e p o r t e d d a t a on g e l s a p p r o x i m a t e l y 3-4 t i m e s t h i c k e r i n d i c a t e t h a t the p e r m e a b i l i t y U i s roughly p r o p o r t i o n a l t o 1/d, w h i l e D i s e s s e n t i a l l y i n d e p e n d e n t o f t h i c k n e s s . Variat i o n s i n D f o r t h e same g e l s a p p e a r t o be m a i n l y due t o e r r o r s i n t h e t h i c k n e s s measurement and a s m a l l d e g r e e o f n o n - u n i f o r m i t y in thickness. W.p and Kp d e c r e a s e as c r o s s l i n k i n g c o n t e n t i n c r e a s e s , as expected.

However,

Kp seems t o i n c r e a s e a g a i n a t

approximately

6 mole-% c r o s s ! i n k e r . The i n c r e a s i n g Kp o f t h e h i g h e r c r o s s l i n k e d g e l s may be t h e r e s u l t o f g r e a t e r i n t e r a c t i o n between w a t e r and a h i g h l y c r o s s l i n k e d p o l y m e r n e t w o r k . This water, a r e l a t i v e l y small f r a c t i o f o r transport or i s involve (13,14), A p l o t o f d i f f u s i o n c o e f f i c i e n t s v s . p e r c e n t EGDMA and TEGDMA c r o s s ! i n k e r s i s shown i n F i g u r e 1. The d i f f u s i o n c o e f f i c i e n t s d e c r e a s e as t h e c r o s s ! i n k e r c o n t e n t i n c r e a s e s , a p p r o a c h i n g a l i m i t i n g v a l u e a t about 6 mole-% c r o s s l i n k e r . This behavior may i n d i c a t e a " p a r t i t i o n " t y p e membrane. Diffusion coefficients r a p i d l y i n c r e a s e w i t h d e c r e a s i n g c r o s s l i n k e r c o n t e n t below about 2.5 m o l e - % c r o s s l i n k e r , s u g g e s t i n g t h e d e v e l o p m e n t o f " l o o s e pores." Two b a s i c mechanisms have been c o n s i d e r e d i n e x p l a i n i n g s o l u t e t r a n s p o r t t h r o u g h a p o l y m e r membrane: 1) a m i c r o p o r o u s t y p e , w h i c h can a c t as a s i e v e w i t h t h e s o l u t e m o l e c u l e s b e i n g t r a n s p o r t e d t h o u r g h t h e m i n u t e p o r e s o f t h e membrane, and 2) a " p a r t i t i o n " t y p e membrane, w h i c h f u r t h e r a c t s t o s l o w t h e d i f f u s i o n p r o c e s s due t o t h e i n t e r a c t i o n between d i f f u s i n g s o l u t e and membrane m a t r i x o r membrane w a t e r . C r a i g ' s work (9) showed t h a t , i n g e n e r a l , a l i n e a r r e l a t i o n s h i p e x i s t s between m o l e c u l a r w e i g h t and h a l f t i m e r a t e s o f t r a n s f e r f o r p o r o u s membranes. However, t h e e f f e c t o f m o l e c u l a r w e i g h t on t h e h a l f t i m e r a t e s t h r o u g h c e r t a i n " p a r t i t i o n " membranes i n d i c a t e d no such t r e n d (10). R e c e n t l y , Chen ( 6 j has d e s c r i b e d t h r e e d i f f e r e n t d i f f u s i o n mechanisms f r o m h i s w a t e r a b s o r p t i o n s t u d i e s : 1) a d i s s o l u t i o n mechanism f o r h i g h e r c r o s s l i n k i n g c o n t e n t , 2) a p o r e f l o w mechanism f o r l o w c r o s s l i n k i n g c o n t e n t , and 3) an i n t e r m e d i a t e mechanism a t i n t e r m e d i a t e c r o s s l i n k e r c o n c e n t r a t i o n s . We have found s i m i l a r r e s u l t s from our s t u d y . Water d i f f u s e s t h r o u g h t h e c r o s s l i n k e d p o l y HEMA membranes v i a a p r e d o m i n a n t l y p o r e mechanism f r o m 0% c r o s s l i n k e r t o a p p r o x i m a t e l y 2.5 m o l e - % c r o s s l i n k e r . Above 4 m o l e - % c r o s s l i n k e r , w a t e r t r a n s p o r t i s m a i n l y c o n t r o l l e d by t h e i n t e r a c t i o n o f w a t e r w i t h t h e g e l m a t r i x . The i n t e r m e d i a t e r e g i o n l i e s between 2.5 t o 4 m o l e - % . A d e s c r i p t i o n o f t h e homogeneous membrane model and t h e t h e o r e t i c a l assumptions i n v o l v e d i n the d e r i v a t i o n of the r e l a t i o n between d i f f u s i o n c o e f f i c i e n t s and w a t e r c o n t e n t i s f o u n d

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6.

WISNIEWSKI ET AL.

TABLE Diffusion

I.

C o e f f i c i e n t s , P e r m e a b i l i t i e s and H y d r o g e l

45% H2O-HEMA EGDMA-mole-% 7.5 5.3 3.8 2.3 0.75 0.38 =0.02

83

Diffusion through Hydrogel Membranes

„ _D

u

d(cm)

P(g/cc)

_f

Composition

D χ 10 (cm /sec)

U χ 10* (cm/sec)

6

2

0.0777 0.0713 0.0717 0.0696 0.0739 0.0679 0.071 0.058 0.0638 0.0682

1.26 1.22 1.20 1.20 1.25 1.27

0.381 0.348 0.355 0.376 0.403 0.417

0.481 0.426 0.427 0.452 0.505 0.531

2.3 2.3 2.4 2.6 3.0 3.3

1.4 1.4 1.4 1.7 2.1 2.6

1.30 1.22

0.420 0.418

0.547 0.511

3.4 3.3

2.9 2.5

0.07-76 0.0736 0.0702 0.0697 0.0793

1.20 1.22 1.23 1.23 1.33

0.405 0.368 0.377 0.402 0.418

0.488 0.450 0.465 0.496 0.557

2.5 2.6 2.7 3.1 3.4

1.6 1.6 1.8 2.2 2.4

0.0660 0.0694 0.0686 0.0901 0.0733 0.0747

1.22 1.25 1.29 1.11 1.20 1.20

0.0348 0.173 0.308 0.631 0.504 0.444

0.0426 0.217 0.397 0.702 0.605 0.534

0.23 0.58 1.6 7.6 5.1 3.9

0.015 0.18 0.94 5.9 4.2 2.8

45% H2O-HEMA TEGDMA-mole-% 7.5 4.6 2.5 1.4 0.46

Copolymers: Volume-% MEMA 33% HEMA-67% MEMA 67% HEMA-33% MEMA MEEMA 33% HEMA-67% MEEMA 67% HEMA-33% MEEMA

MEMA

- methoxyethyl

methacrylate

MEEMA - m e t h o x y e t h o x y e t h y l HEMA

- hydroxyethyl

methacrylate

methacrylate

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

84

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

elsewhere

(11 ), and t h a t r e l a t i o n i s g i v e n as f o l l o w s :

In D/D = β χ ( 1 - α ) / ( 1 + x a ) , Q

where χ = ( 1 - K p ) / K p ,

a = V / V , β = V*/V , D = experimental p

w

w

d i f f u s i o n c o e f f i c i e n t w i t h p a r t i t i o n c o e f f i c i e n t Kp, D s i o n c o e f f i c i e n t o f water i n pure w a t e r , V

w

Q

= diffu

= f r e e volume i n

u n i t volume o f p u r e w a t e r , V = f r e e volume i n u n i t volume o f Ρ p o l y m e r p h a s e , and V * = a c h a r a c t e r i s t i c volume p a r a m e t e r d e s c r i b i n g t h e d i f f u s i o n o f a permeant m o l e c u l e i n t h e medium. It i s possible to rearrange t h i s equation i n order t o obtain a l i n e a r plot. That i s

A p l o t o f (In D/Do)" v s . x " s h o u l d y i e l d a s t r a i g h t l i n e w i t h s l o p e = - 1 / β ( 1 - α ) and i n t e r c e p t = - α / β ( 1 - α ) . T a b l e I I g i v e s v a l u e s o f _ ( l n D/Do) and x " f o r t h e g e l s s t u d i e d , u s i n g D = 2 . 4 χ 1 0 " c m / s e c f r o m (12). The v a l u e s f o r p o l y HEMA, u n c r o s s ! i n k e d (^ 0 . 0 2 m o l e - % ) , a r e an a v e r a g e o f f o u r d i f f e r e n t g e l s . Figure 2 i s a p l o t o f (In D/Do)" v s . x " f o r a l l b u t t h e c r o s s l i n k e d gels. L e a s t s q u a r e s f i t y i e l d s v a l u e s o f α = 0 . 6 9 and β = 11 with a c o r r e l a t i o n c o e f f i c i e n t of -0.998. The i n t e r c e p t y i e l d s a v a l u e o f 1.6 χ 1 0 " c m / s e c f o r t h e h y p o t h e t i c a l z e r o w a t e r content polymer. I n c l u s i o n o f c r o s s l i n k e d p o l y HEMA g e l s y i e l d s v a l u e s o f α = 0 . 7 2 and β = 13 w i t h a c o r r e l a t i o n c o e f f i c i e n t o f -0.988. Even t h o u g h a f a i r c o r r e l a t i o n i s o b t a i n e d w i t h t h e i n c l u s i o n o f t h e c r o s s l i n k e d g e l s , t h e s e g e l s seem t o e x h i b i t a t r e n d o f t h e i r own. These r e s u l t s and p r e v i o u s r e s u l t s ( 1 1 ) i n d i c a t e t h a t s t r u c t u r a l d i f f e r e n c e s o f t h e monomers used may p l a y some r o l e i n t h e d i f f u s i o n p r o c e s s o t h e r t h a n j u s t d e t e r mining water content. Changing o f t h e w a t e r c o n t e n t o f s w o l l e n g e l s f o r p u r e s y s t e m s by v a r y i n g p o l y m e r i z a t i o n w a t e r c o n c e n t r a t i o n may shed some l i g h t on s t r u c t u r a l e f f e c t s i n t h e d i f f u s i o n process. This study i s being continued. 1

1

1

1

0

5

2

1

7

1

2

Acknowledgements T h i s work was s u p p o r t e d by NHLI G r a n t No. HL 1 6 9 2 1 - 0 1 . The donations o f generous q u a n t i t i e s o f h y d r o x y e t h y l m e t h a c r y l a t e f r o m Hydro Med S c i e n c e s , I n c . , i s g r a t e f u l l y a c k n o w l e d g e d .

Abstract Water transport through fully swollen poly (2-hydroxyethyl methacrylate) (poly HEMA) hydrogels, containing varying concentra tions of ethylene glycol dimethacrylate (EGDMA) and tetraethylene

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

6.

Diffusion through Hydrogel Membranes

WISNIEWSKI ET AL.

TABLE Values o f (In D/D )"

1

Q

(Using D

Q

Hydrogel (Copolymers i n volume-%) MEMA 33% HEMA-67% MEMA 67% HEMA-33% MEMA MEEMA 33% HEMA-67% MEEMA 67% HEMA-33% MEEMA HEMA

and x "

II. 1

f o r Hydrogels

= 2.4 χ 1 ( f

5

cm /sec) 2

IniD/D^"

1

Studied 1 2

χ

1

= K, D

-0.22 -0.27 -0.37 -0.87

0.0445 0.277 0.658 2.36

-0.51

1.09

-0.43 -0.43 -0.43 -0.45 -0.48 -0.50

0.927 0.742 0.745 0.825 1.02 1.13

-0.44 -0.45 -0.46 -0.49 -0.51

0.953 0.818 0.869 0.984 1.26

M o l e - % EDGMA 7.5 5.3 3.8 2.3 0.75 0.38

M o l e - % TEGDMA 7.5 4.6 2.5 1.4 0.46

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

86

10.00

•

EGDMR

+

TEGDMR

00

θ

ο

+

ω ο

ε mm

ε

CROSSL INKER

ο

,

Η.

mm

2

mm

Β

2)0

a

ο

+

° 2.

m

2 .3 0

2 .3 0

2 .Β 0

D

Χ

10

0

α

ο

and TEGDMA

in

PHEMA

MEMR

•

-

3 . 5Γ0

(cm / s e c )

Figure 1. Plot of diffusion coefficients vs. mole % EGDMA hydrogel

0

3 . 2 0

.2

MEMR-HEMR

Χ κεΜΠ

. Η

Η

MEEMR—HEMR

Μ

MEEMR

c

.ε

Β

l ^0 0.

0

I . S

1 . 0

y

Figure 2. Plot of (In D/Do)'

1

2 . 0

2 . E

-1

vs. X' for hydrogels swollen to different water concentrations 1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

equilibrium

6.

wisNiEwsKi E T A L .

Diffusion through Hydrogel Membranes

87

glycol dimethacrylate (TEGDMA) crosslinkers, was investigated using tritiated water and a designed diffusion cell. Diffusion coefficients obtained in these experiments decrease as the concentration of crosslinker increases. This decrease is very sharp at crosslinker concentrations of 0-2.5 mole-%. At higher crosslinker concentrations (2.5-7.5 mole-%), the diffusion coefficients appear to reach a limiting value. This study indicates that a crosslinked poly HEMA membrane may provide both "partition" and "pore" mechanisms of solute transport based on crosslinker concentration. Hydrogels of varying water content were prepared to investi gate the relationship between water content and diffusion coefficient. Poly(methoxyethyl methacrylate) (poly MEMA), poly(methoxyethoxyethyl methacrylate) (poly MEEMA) and copolymers of MEMA and MEEMA wit with water content rangin results of the diffusion experiments were examined in light of a free-volume model of diffusive transport. Excellent theoreticalexperimental correlation was obtained for the hydrogels used in this study. Literature Cited 1. Yasuda, H., Lamaze, C., and Ikenberry, L., Makromol. Chem. (1968) 118, 19. 2. Spacek, P., and Kubin, M., J. Poly. Sci., Part C, (1967) 16, 705. 3. Ikenberry, L., Yasuda, H., and Clark, H., Chem. Eng. Prog. Sym. Ser. (1968) 64 (84) 69. 4. Refojo, M. F., J. Appl. Poly. Sci. (1964) 9, 3417. 5. Ratner, B. D., and Miller, I. F., J. Biomed. Matl. Res. (1973) 7, 353. 6. Chen, R. Y. S., Polymer Preprints (1974) 15, No. 2, 387. 7. Mortimer, G. Α., J. Org. Chem. (1964) 30, 1632. 8. Mah, M. Y., Master's Thesis, University of Utah, 1972. 9. Craig, L. C., and Konigsberg, W., J. Phys. Chem. (1961) 65 116. 10. Lyman, D. J., Trans. Am. Soc. Art. Int. Org. (1964) 10, 17. 11. Yasuda, H., Lamaze, C. E., and Peterlin, Α., J. Poly, Sci., A-2 (1971) 9, 1117. 12. Wang, J. H., Robinson, C. V., and Edelman, I. S., J. Amer. Chem. Soc. (1953) 75, 466. 13. Chang, Y. J., Chen, C. T., and Tobolsky, J. Poly. Sci., Poly. Phys. Ed., (1974), 12, 1. 14. Hoffman, A. S., Modell, M., and Pan, P., J. Appl. Poly. Sci., 14, 285 (1970).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7 The Chemistry of Some Selected Methacrylate Hydrogels DONALD E. GREGONIS, CHWEN M. CHEN, and JOSEPH D. ANDRADE Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112

H y d r o g e l s have been d e s c r i b e d as b i o c o m p a t i b l e m a t e r i a l s Q J Polymers o f hydroxyethy s y n t h e t i c hydrogels ( 2 J a r e w i d e l y used i n c o r r e c t i v e c o n t a c t l e n s e s ( 3 ) ; i t has been used as a c o a t i n g f o r c a t h e t e r s and o t h e r m e d i c a l d e v i c e s ( 4 - 6 J . In o u r i n v e s t i g a t i o n s , we wanted t o s t u d y a v a r i e t y o f h y d r c i p i r i l i c m e t h a c r y l a t e polymers t o e v a l u a t e t h e i r b i o l o g i c a l behavior i n r e l a t i o n t o t h e c o n c e n t r a t i o n and t y p e o f g r o u p s i n c o r p o r a t e d i n to the polymer. In o r d e r t o p u r s u e t h i s g o a l , a t h o r o u g h u n d e r standing o f the bulk properties of the hydrogels i s r e q u i r e d . In t h i s work t h e e q u i l i b r i u m w a t e r c o n t e n t o f t h e g e l s i s r e g u l a t e d by v a r y i n g c o p o l y m e r r a t i o s . Charged g r o u p s a r e i n c o r p o r a t e d i n t o t h e p o l y m e r by c o p o l y m e r i z a t i o n w i t h a c i d i c o r b a s i c m e t h a c r y l a t e s . The monomers t h a t a r e b e i n g i n v e s t i g a t e d a r e shown i n T a b l e I. The amount o f w a t e r i n t h e e q u i l i b r a t e d p o l y m e r s c o v e r e d t h e r a n g e f r o m 3 . 5 % t o g r e a t e r t h a n 90% ( T a b l e I I ) . For t h i s s t u d y , h y d r o x y e t h y l m e t h a c r y l a t e was s e l e c t e d as t h e f u n d a m e n t a l monomer b e c a u s e o f p a s t work and a v a i l a b i l i t y . TABLE

I.

H y d r o p h i l i c M e t h a c r y l a t e Monomers

1. 2. 3. 4. 5. 6. 7.

R i = OH, R = H; h y d r o x y e t h y l m e t h a c r y l a t e (HEMA). R i = 0CH CH 0H, R = H; h y d r o x y e t h o x y e t h y l m e t h a c r y l a t e ( H E E M A l R i = 0CH CH20CH2CH 0H,R = H; h y d r o x y d i e t h o x y e t h y l m e t h a c r y l a t e (HDEEMA). R i = -OCH3, R = H; m e t h o x y e t h y l m e t h a c r y l a t e (MEMA). R i = 0CH CH 0CH , R = H; m e t h o x y e t h o x y e t h y l m e t h a c r y l a t e (MEEMA). R i = 0CH CH 0CH CH 0CH , R = H; m e t h o x y d i e t h o x y e t h y l m e t h a c r y l a t e (MDEEMA). R i = CH2OH, R = OH; 2 , 3 - d i h y d r o x y p r o p y l methacrylate(DHPMA). 2

2

2

2

2

2

2

2

2

2

3

2

2

2

2

2

3

2

2

88

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

GREGONis E T AL.

89

Methacrylate Hydro gels TABLE I I . Water S w e l l i n g o f P u r e Homopolymers

Equilibrium 1.

pHEMA

-

0.40*

5.

pMEEMA

2.

pHEEMA

-

0.80

6.

pMDEEMA

3.

pHDEEMA

-

>0.90

7.

pDHPMA

4.

pMEMA

-

0.62 -

>0.90 0.70

t o >0.90

H)

0.035

*Water f r a c t i o n (W ) = weight o f water i n polymer/weight hydrated polymer f

Monomers (See T a b l e

of

I)

The l a b o r a t o r y s y n t h e s i s o f t h e s e monomers i s a c c o m p l i s h e d by t h e f o l l o w i n g c l a s s i c a l c h e m i c a l r e a c t i o n s : 1. Reaction of methacryl c h l o r i d e with the corresponding alcohol :

*

" - τ * * — U n i *

(Monomers 1-7) 2. T r a n s e s t e r i f i c a t i o n o f methyl m e t h a c r y l a t e w i t h t h e corresponding a l c o h o l : 0 ^ — ! L ' 3.

0 + R0H 0CH

τ > Η o r base

3

0

H

+ »

4. Acid catalyzed hydrolysis methacrylate (8-9).

of

0 C ^ ( / N/

0

0 —

> '

+ CH-0H OR

(Monomers 1-7) of g l y c i d y l methacrylate (7):

Acid catalyzed hydrolysis 0

t 7

(Monomer 7) isopropylidineglyceryl

0 0 J

LL^V-C

0

^

,+CH~C-CH~ TKOH OH (Monomer 7) 0

H

0

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

90

HYDROGELS

FOR MEDICAL

A N D RELATED APPLICATIONS

A t h o r o u g h s u r v e y o f h y d r o p h i l i c m e t h a c r y l a t e monomers c o v e r i n g b o t h l a b o r a t o r y and i n d u s t r i a l s y n t h e s i s i s a v a i l a b l e (10). M o d e r a t e amounts o f monomer (0.1 t o 1 £ . ) s a t i s f i e s most o f our r e q u i r e m e n t s . A d e s c r i p t i o n o f o u r p u r i f i c a t i o n scheme i s p r e s e n t e d b e l o w . Normal p r e p a r a t i v e c h r o m a t o g r a p h i c t e c h n i q u e s a r e i m p r a c t i c a l f o r t h e p u r i f i c a t i o n o f t h i s q u a n t i t y o f monomer. The p r e p a r a t i o n o f monomers 1, 2 , and 3 i s c o m p l i c a t e d by the formation of dimethacrylate e s t e r s . These d i e s t e r s a r e u n d e s i r e d i n t h e p r o d u c t b e c a u s e t h e y a c t as c r o s s l i n k i n g a g e n t s . They may be removed f r o m t h e p r o d u c t by e x t r a c t i o n t e c h n i q u e s . Since the d i e s t e r s are l e s s s o l u b l e i n water than the c o r r e s p o n d i n g m o n e s t e r s , t h e i m p u r e monomer i s d i s s o l v e d i n w a t e r ( M : l r a t i o , v/v) and e x t r a c t e d w i t h a n o n - p o l a r s o l v e n t , s u c h as c a r b o n t e t r a c h l o r i d e , benzene o r p e t r o l e u m e t h e r (1J_). The l e s s water s o l u b l e d i e s t e r s ar d e s i r e d monomer i s i s o l a t e w i t h s o d i u m c h l o r i d e and e x t r a c t i o n w i t h a more p o l a r s o l v e n t , u s u a l l y methylene c h l o r i d e . E t h e r s o l v e n t s a r e n o t used f o r any e x t r a c t i o n u n l e s s t h e y have been r i g o r o u s l y p u r i f i e d . A n h y d r o u s e t h e r s r e a d i l y a i r o x i d i z e t o f o r m p e r o x i d e s , w h i c h a r e good p o l y m e r i z a t i o n i n i t i a t o r s . The monomer i s c a r e f u l l y e x t r a c t e d t o remove a c i d i c and b a s i c i m p u r i t i e s and t h e n e l u t e d t h r o u g h Grade II n e u t r a l a l u m i n a t o remove o t h e r p o l a r i m p u r i t i e s . The monomers s h o u l d f i n a l l y be p u r i f i e d by vacuum d i s t i l l a tion. The vacuum s y s t e m must be a b l e t o c a r r y o u t d i s t i l l a t i o n s as l o w as 0.1 mm Hg. p r e s s u r e . Inhibitor is required to deter p o l y m e r i z a t i o n when t h e p r o d u c t i s d i s t i l l e d . Cuprous c h l o r i d e o r c o p p e r powder i s r o u t i n e l y added t o t h e d i s t i l l a t i o n f l a s k and o f f e r s t h e a d v a n t a g e o f n o t c o - d i s t i l l i n g w i t h t h e monomer. H y d r o q u i n o n e o r p - m e t h o x y p h e n o l a r e more e f f e c t i v e i n h i b i t o r s and a r e r e q u i r e d i n d i s t i l l a t i o n s above 1 0 0 ° C , b u t t h e y c o - d i s t i l l with the product. A n i t r o g e n bleed i n t o the d i s t i l l a t i o n f l a s k l a r g e l y e l i m i n a t e s the d e l e t e r i o u s e f f e c t s o f oxygen. Table III g i v e s t h e b o i l i n g p o i n t s o f t h e monomers,

1. 2. 3. 4. 5. 6. 7.

TABLE III. B o i l i n g P o i n t s o f H y d r o p h i l i c M e t h a c r y l a t e Monomers Monomer B o i l i n g P o i n t , °C Hydroxyethyl methacrylate(HEMA) 6 9 ° @ 0.1 mm Hg H y d r o x y e t h o x y e t h y l m e t h a c r y l a t e 9 7 - 9 9 ° @ 1 mm Hg ( 2 ) (HEEMA) H y d r o x y d i e t h o x y e t h y l m e t h a c r y l - 1 2 0 - 1 2 2 ° @ 0 . 4 mm Hg a t e (HDEEMA) Methoxyethyl methacrylate(MEMA) 5 7 ° @ 8 mm Hg M e t h o x y e t h o x y e t h y l m e t h a c r y l a t e 6 5 - 7 0 ° @ 0.1 mm Hg (MEEMA) M e t h o x y d i e t h o x y e t h y l m e t h a c r y l - 9 5 - 1 0 1 ° @ 0.1 mm Hg a t e (MDEEMA) 2 , 3 - d i h y d r o x y p r o p y l m e t h a c r y l a t e 1 4 0 ° @ 0.6 mm Hg (DHPMA)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

(£)

7.

GREGONis E T AL.

Methacrylate Hydrogeh

91

Gas c h r o m a t o g r a p h y ( G C ) , h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC) and t h i n - l a y e r c h r o m a t o g r a p h y (TLC) (1_2) a r e used f o r analysis of purity. C e r t a i n i m p u r i t i e s t h a t c a n n o t be d e t e c t e d by one t e c h n i q u e a r e e a s i l y d e t e c t e d by a n o t h e r t e c h n i q u e . Both UV a b s o r b a n c e and r e f r a c t i v e i n d e x a r e m o n i t o r e d w i t h t h e h i g h pressure l i q u i d chromatograph. The m e t h a c r y l a t e s y s t e m o f f e r s a w i d e v a r i e t y o f monomers, w h i c h we c a l l g e l m o d i f i e r s , t o c o p o l y m e r i z e w i t h t h e h y d r o p h i l i c monomers t o change t h e c h a r a c t e r i s t i c s o f t h e g e l . For example, copolymerization with methacrylic acid introduces negatively charged groups i n t o the g e l . D i m e t h y l a m i n o e t h y l m e t h a c r y l a t e may be used t o i n t r o d u c e p o s i t i v e c h a r g e s i n t o t h e g e l . Some o f t h e c o m m e r c i a l l y a v a i l a b l e monomers used f o r g e l m o d i f i c a t i o n a r e l i s t e d i n F i g u r e 1. M o d i f i e r s 11-14 i n F i g u r e 1 a r e n o t d i s c u s sed f u r t h e r i n t h i s p a p e r Methacrylate diester d i e s t e r s f u n c t i o n as c r o s s ! i n k i n g a g e n t s . The c r o s s ! i n k e r s t h a t we have u s e d a r e t h e d i m e t h a c r y l a t e s o f e t h y l e n e g l y c o l (EGDMA) and t e t r a e t h y l e n e g l y c o l (TEGDMA)(2^133 · The l a t t e r i s more s o l u b l e i n w a t e s Initiators M e t h a c r y l a t e s may be p o l y m e r i z e d by e i t h e r r a d i c a l o r anionic i n i t i a t o r s (14). r a d i c a l i n i t i a t i o n we s e l e c t e d a c l a s s o f a z o i n i t i a t o r s . These a z o i n i t i a t o r s a r e r e a d i l y a v a i l a b l e and decompose a t a u n i f o r m r a t e u n a f f e c t e d by s o l v e n t o r i n d u c e d d e c o m p o s i t i o n ( 1 5 ) . The a z o i n i t i a t o r s may be m o d i f i e d so t h a t t h e end g r o u p s i n t r o d u c e d i n t o t h e p o l y m e r c h a i n a r e v e r y s i m i l a r t o t h e mer u n i t . They t h e r m a l l y decompose a t a r e a s o n a b l e t e m p e r a t u r e (16) and c a n be decomposed a t l o w t e m p e r a t u r e s by UV l i g h t . In t h i s s t u d y , e s t e r s f r o m a z o b i s i s o b u t y r o n i t r i l e (AIBN) were p r e p a r e d by t h e f o l l o w i n g method ( 1 7 ) . Mild a c i d a l c o h o l y s i s o f AIBN c o n v e r t s t h e n i t r i l e g r o u p t o i m i n o e s t e r s a l t s , which p r e c i p i t a t e from the r e a c t i o n s o l u t i o n . The p r o d u c t i s i s o l a t e d by f i l t r a t i o n and t h e i m i n o - e s t e r g r o u p i s hydrolyzed to form the e s t e r (Figure 2 ) . A z o b i s ( m e t h y l i s o b u t y r a t e ) (Compound 16) has been p r e v i o u s l y prepared (17-19). More w a t e r s o l u b l e a z o i n i t i a t o r s were p r e p a r e d f r o m t h e m o n o m e t h o x y g l y c o l s (Compounds 17 and 1 8 ) . I n i t i a t o r a z o b i s ( m e t h o x y d i e t h o x y e t h y l i s o b u t y r a t e ) (Compound 18) i s completely water s o l u b l e . In o t h e r work we have shown t h a t a l l t h r e e AIBN d e r i v a t i v e s i n i t i a t e p o l y m e r i z a t i o n o f HEMA a t an e q u a l r a t e , w h i c h i s s l i g h t l y f a s t e r t h a n AIBN ( 2 0 ) . For bulk p o l y m e r i z a t i o n , 7.84 m i c r o m o l e s o f i n i t i a t o r a r e used p e r m i l l i l i t e r o f monomer. T h i s r a t i o i s k e p t c o n s t a n t and does n o t v a r y with water content. For p o l y m e r i z a t i o n o f high w a t e r c o n t e n t g e l s , t h e more w a t e r s o l u b l e d e r i v a t i v e s o f AIBN a r e u s e d . P r o v i d i n g no a c t i v e h y d r o g e n s a r e p r e s e n t i n t h e monomer o r s o l v e n t , a n i o n i c i n i t i a t o r s may be u s e d . These c o n d i t i o n s p r o h i b i t p o l y m e r i z a t i o n o f t h e h y d r o x y l i c monomers 1, 2 , 3 , 7 o r F

o

r

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

92

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

t h e use o f w a t e r o r a l c o h o l s as s o l v e n t s . The a n i o n i c i n i t i a t o r s a r e o r g a n o m e t a l l i c compounds, s u c h as η - b u t y l l i t h i u m , o r s t r o n g b a s e s , s u c h as l i t h i u m t - b u t o x i d e . Although there are disadvan t a g e s t o t h e s e i n i t i a t o r s , t h e y a r e used t o p o l y m e r i z e m e t h a c r y l a t e s i n a wide range o f t a c t i c i t i e s from c h a i n s having high s y n d i o t a c t i c t r i a d s to chains having high i s o t a c t i c t r i a d s . Our group i s i n v e s t i g a t i n g such polymers t o determine t h e e f f e c t o f t a c t i c i t y on t h e p r o p e r t i e s o f t h e h y d r o g e l s (21). F o r c e r t a i n s t u d i e s s o l u b l e p o l y m e r s were r e q u i r e d ( 2 2 ) . R a d i c a l p o l y m e r i z a t i o n of the m e t h a c r y l a t e system w i t h o u t s o l v e n t g i v e s a p o l y m e r t h a t s w e l l s t o a h i g h d e g r e e i n good s o l v e n t s , b u t does n o t d i s s o l v e . I t i s f e l t t h a t some c r o s s ! i n k i n g o f t h e p o l y m e r o c c u r s by r a d i c a l c h a i n t r a n s f e r mechanisms ( 2 3 ) . S o l u b l e m e t h a c r y l a t e p o l y m e r s may be o b t a i n e d w i t h a r a d i c a l i n i t i a t o r at high d i l u t i o n s o f th monomers i n e t h a n o l ( l : 1 0 , v / v ) f o r s o l v e n t c a s t i n g (24) and g e l c o a t i n g s ( 2 2 ) . P r e p a r a t i o n o f Gel

Membranes

Gel membranes w e r e r e q u i r e d f o r o u r d i f f u s i o n s t u d i e s (13) and were used f o r o u r work on w a t e r s w e l l i n g o f g e l s . Polymeri z a t i o n o f t h e monomer was i n i t i a t e d by a z o b i s ( m e t h y l i s o b u t y r a t e ) u s i n g t h e same c o n c e n t r a t i o n used f o r t h e p o l y m e r i z a t i o n o f b u l k g e l s , 7.84 m i c r o m o l e s p e r m i l l i l i t e r o f monomer. This r a t i o i s i n d e p e n d e n t o f w a t e r c o n c e n t r a t i o n . A l l g e l m o d i f i e r s and c r o s s l i n k i n g a g e n t s a r e e x p r e s s e d as m o l a r q u a n t i t i e s i n t e r m s o f t h e monomer v o l u m e . The membranes were p r e p a r e d by p o l y m e r i z a t i o n o f t h e monomer s o l u t i o n between f l a t p l a t e s u s i n g a s i l i c o n e rubber spacer to r e g u l a t e t h i c k n e s s . The p o l y m e r i z a t i o n c o n d i t i o n s w e r e s t a n d a r i z e d a t 60°C f o r 24 h r . A t l o w w a t e r c o n c e n t r a t i o n s (Wf < 0 . 2 0 ) , p o l y e t h y l e n e o r p o l y p r o p y l e n e mold p l a t e s a r e used; a t higher water c o n c e n t r a t i o n s , g l a s s p l a t e s are used. T h i s i s b e c a u s e g e l s p o l y m e r i z e d a t low w a t e r c o n c e n t r a t i o n a d h e r e strongly to the glass surface. P o l y m e r i z a t i o n o f membranes on a g l a s s s u r f a c e i s p r e f e r e d s i n c e i t has a more u n i f o r m s u r f a c e . The t h i c k n e s s o f t h e g e l membranes i s 0 . 7 5 mm u n l e s s o t h e r w i s e stated. S y n t h e s i s and P o l y m e r i z a t i o n o f C ^ - l a b e l e d HEMA To d e t e r m i n e t h e r e l a t i v e s t a b i l i t y o f t h e m e t h a c r y l a t e g e l s i n v a r i o u s m e d i a , C * - l a b e l e d HEMA was s y n t h e s i z e d by r e a c t i n g m e t h a c r y l c h l o r i d e and 1 , 2 - C * - e t h y l e n e g l y c o l , u s i n g e x c e s s c a r r i e r ethylene g l y c o l to prevent the formation of excessive amounts o f C - l a b e l e d d i e s t e r ( e t h y l e n e g l y c o l d i m e t h a c r y l a t e ). The C *-HEMA was p u r i f i e d u t i l i z i n g t h e " s a l t i n g o u t " t e c h n i q u e described e a r l i e r . The p r o d u c t was d i l u t e d w i t h c a r r i e r HEMA and was t h e n d i s t i l l e d by m i c r o - v a c u u m t e c h n i q u e s . The d i s t i l l a t e was d e t e r m i n e d t o have a s p e c i f i c a c t i v i t y o f 9.1 χ 1 0 dpm/ml. l l

l l

1 1 +

ll

5

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

GREGONis E T AL.

93

Methacrylate Hydrogels

To c h e c k t h e r a d i o i s o t o p i c p u r i t y , a 1 ml a l i q u o t was c h r o m a t o g r a p h e d on 100g Grade I I a l u m i n a . The column was e l u t e d w i t h a l i n e a r g r a d i e n t f r o m 100% p e t r o l e u m e t h e r (250ml) t o 5% m e t h a n o l in d i e t h y l e t h e r (250ml). The r a d i o a c t i v i t y e l u t e d i n one p e a k , t h e f r a c t i o n s were combined and e v a p o r a t e d . The p r o d u c t e x h i b i t e d an i d e n t i c a l i n f r a - r e d s p e c t r u m t o c o n t r o l HEMA. To i n i t i a t e t h e p o l y m e r i z a t i o n o f C - H E M A , a z o b i s ( m e t h y l i s o b u t y r a t e ) was added ( 7 . 8 4 m i c r o m o l e / m l ) . The monomer was p o l y m e r i z e d i n a p o l y p r o p y l e n e m o l d . The mold d i m e n s i o n s w e r e 50mm χ 50mm χ 1mm. The s t a n d a r d p o l y m e r i z a t i o n c o n d i t i o n s w e r e 60°C f o r 24 h o u r s u n l e s s o t h e r w i s e n o t e d . A f t e r t h i s t i m e t h e mold was c o o l e d t o 0 ° C , and t h e C *-pHEMAwas removed and w e i g h e d . The C p H E M A w a s t h e n p l a c e d i n v a r i o u s s o l v e n t s a t 3 7 ° C . A l i q u o t s were removed a t v a r i o u s t i m e i n t e r v a l s and c o u n t e d u s i n g l i q u i d s c i n t i l l a t i o n techniques t h e s o l v e n t was c a l c u l a t e a c t i v i t y i n the polymer. F i g u r e 3 shows t h e p e r c e n t r a d i o a c t i v i t y e x t r a c t e d w i t h t i m e f o r some t y p i c a l g e l s . G e l s 1 and 2 a r e d u p licates. G e l 6 was p o l y m e r i z e d u n d e r i d e n t i c a l c o n d i t i o n s b u t was e x t r a c t e d w i t h 95% e t h a n o l . G e l 9 was p o l y m e r i z e d w i t h 45% w a t e r and e x t r a c t e d w i t h w a t e r . The r a d i o a c t i v i t y o f a l l g e l s e x t r a c t e d w i t h w a t e r l e v e l e d o u t a f t e r one d a y , b u t t h e r a d i o a c t i v i t y e x t r a c t e d w i t h e t h a n o l ( G e l 6) a p p e a r e d t o i n c r e a s e a f t e r t h i s t i m e a t a b o u t 0.5%/week. T a b l e IV p r o v i d e s a c o m p l e t e l i s t o f t h e C -HEMA p o l y m e r i z a t i o n c o n d i t i o n s , e x t r a c t i o n s o l v e n t s , and t h e p e r c e n t r a d i o a c t i v i t y e x t r a c t e d . The p e r c e n t r a d i o a c t i v i t y e x t r a c t e d was c a l c u l a t e d as t h e a v e r a g e o f t h e i s o t o p e found i n s o l u t i o n a t p o i n t s i n time from 4 days t o 5 weeks. llf

ll

l l L

1If

TABLE I V . J4 E l u t i o n o f R a d i o a c t i v i t y from C -pHEMA Gels E q u i l i b r a t e d i n Various Solvents % R a d i o a c t i v i t y in Solvent S o l v e n t a f t e r 5 wks. Gel# P o l y m e r i z a t i o n C o n d i t i o n s D i s t i l l e d H 0 4.7% + 0.13 1 24 h o u r s θ 60°C 24 h o u r s @ 60°C 2 D i s t i l l e d H 0 4 . 8 % + 0.08 24 h o u r s @ 60°C 3.6% + 0.27 Human R e f e r 3 e n c e Serum D i s t i l l e d H 0 4.8% + 0.14 3 1/2 h o u r s & 60°C D i s t i l l e d H 0 2.6% + 0.41 66 h o u r s @ 60°C 24 h o u r s @ 60°C 9.1% ± 0.90 95% E t h a n o l 24 h o u r s @ 60°C D i s t i l l e d H 0 2.9% + 0 . 3 0 ( G e l c o n t a i n e d 1% u n l a b e l e d EGDMA) D i s t i l l e d H 0 2 . 3 % ± 0.11 24 h o u r s @ 60°C ( G e l c o n t a i n e d 5% u n l a b e l e d EGDMA) D i s t i l l e d H 0 1.7% ± 0 . 0 9 24 h o u r s @ 60°C ( G e l c o n t a i n e d 45% H 0 v / v ) ,tf

2

2

2

2

2

2

2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

94

HYDROGELS FOR MEDICAL AND RELATED 0

0

Η

0

NH

OH

9

io

(MA A)

U

2

12

11

n

APPLICATIONS

(MA)

(DMAEMA)

0

0 n

\

0CH 13

14

15

(GMA)

(MAN)

(MMA)

3

MONOMERS - FOR GEL MODIFICATION Figure 1.

H*

- Ν=ΝC=N

Commercially available gel modifiers

ROH

C=N ι OR

C=N

C=N ι \, OR Η

+

+

H

AIBN

IMINOESTER SALT

16.

R=CH

17.

R = CH CH 0CH

18.

R= C H C H 0 C H C H 0 C H C H 0 C H

2

2

OR

AZOBISISOBUTYROESTER

2

2

AZOBISMETHOXYETHYL ISOBUTYRATE

3

2

Figure 2.

Figure 3.

C=0

OR

AZOBISMETHYLISOBUTYRATE

3

2

C=0

2

2

3

AZOBISMETHOXYDIETHOXYETHYLISOBUTYRATE

Preparation of azobisisobutyrate esters

Radioactivity extracted at given time for C-HEMA gel 14

TIME (WEEKS)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

GREGONis E T AL.

Methacrylate Hydrogeh

95

The amount o f u n p o l y m e r i z e d C *-HEMA o r C *-pHEMA t h a t d i s s o l v e d i n t h e s o l u t i o n s i s g i v e n i n T a b l e IV. The b e t t e r s o l v e n t , e t h a n o l (#6) c o n t a i n e d more r a d i o a c t i v i t y t h a n t h e poorer s o l v e n t , water (#1,2). At shorter polymerization times, more r a d i o a c t i v i t y was f o u n d i n t h e s o l v e n t t h a n a f t e r l o n g p o l y m e r i z a t i o n t i m e s (#4 v s . #5). T h e r e d i d n o t a p p e a r t o be any s i g n i f i c a n t d i f f e r e n c e , h o w e v e r , between G e l s 1, 2 and 4 . With h i g h e r p e r c e n t c r o s s l i n k e r , t h e amount o f r a d i o a c t i v i t y t h a t i s f o u n d i n t h e s o l v e n t d e c r e a s e d (#1, 7 and 8 ) . I t was somewhat s u r p r i s i n g t o f i n d t h a t t h e l e a s t r a d i o a c t i v i t y was f o u n d i n t h e s o l v e n t when C *-HEMA was p o l y m e r i z e d w i t h 45% w a t e r (#9). These p r e l i m i n a r y studies are c o n t i n u i n g . I s o l a t i o n and i d e n t i f i c a t i o n o f t h e e x t r a c t e d r a d i o a c t i v i t y w i l l be n e c e s s a r y t o d e t e r m i n e i f i t i s due t o u n r e a c t e d monomer o r t o p o l y m e r s . ll

ll

ll

Water S w e l l i n g o f pHEM

(£s)

I s o t r o p i c s w e l l i n g i s assumed i n t h e g e l s . Linear and w a t e r f r a c t i o n (Wf) a r e d e f i n e d as f o l l o w s : £

= length of equilibrium swollen length of dry gel

S

y

= weight of water i n weight of hydrated

f %*s

%W

= W

f

χ

gel

gel gel

= wet l e n g t h - d r y l e n g t h wet l e n g t h f

swelling

m

100

E x p e r i m e n t s t o d e t e r m i n e t h e k i n e t i c s o f l i n e a r s w e l l i n g and w a t e r f r a c t i o n o f t h e g e l were p e r f o r m e d . The pHEMA g e l s p o l y merized w i t h d i f f e r e n t water c o n c e n t r a t i o n s reached e q u i l i b r i u m w a t e r c o n t e n t a f t e r 24 h o u r s . At e q u i l i b r i u m the water f r a c t i o n (Wf) i n t h e pHEMA g e l was 0 . 4 0 ± 0 . 0 5 ( F i g u r e 4 ) , w h e t h e r o r n o t t h e HEMA was p o l y m e r i z e d w i t h w a t e r . The same e x p e r i m e n t s were n o t done w i t h MEEMA s i n c e t h i s monomer i s n o t v e r y s o l u b l e i n w a t e r , h o w e v e r , a n h y d r o u s pMEEMA r e a c h e d an e q u i l i b r i u m w a t e r f r a c t i o n o f 0.62 a f t e r 48 h o u r s . The k i n e t i c s o f l i n e a r s w e l l i n g ( £ s ) do n o t f o l l o w t h e same p a t t e r n as t h e k i n e t i c s o f w a t e r u p t a k e as measured by t h e w a t e r fraction (W ). T h i s i s f o u n d i n b o t h pHEMA ( F i g u r e 5) and pMEEMA gels (Figure 6). F o r a pHEMA g e l p o l y m e r i z e d w i t h o u t w a t e r and t h e n a l l o w e d t o e q u i l i b r a t e i n w a t e r , t h e w a t e r f r a c t i o n (Wf) increased to 0.20, h a l f the f i n a l e q u i l i b r i u m water f r a c t i o n , but t h e l i n e a r s w e l l i n g ( i l s ) was measured t o be 1 . 0 1 5 , o n l y 5% o f t h e f i n a l equilibrium l i n e a r swelling (Figure 5). An a n a l o g o u s r e s u l t i s o b t a i n e d f o r pMEEMA g e l s ( F i g u r e 6 ) . In e f f e c t , t h e r e i s a much g r e a t e r i n i t i a l amount o f w a t e r t a k e n i n t o t h e g e l t h a n w o u l d be a n t i c i p a t e d f r o m j u s t t h e l i n e a r s w e l l i n g measurement. f

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

~05~

"

ΙΌ

15

20" 2A

TIME (HOURS) Figure 4.

Water uptake of pHEMA polymerized at different water contents

TIME (HOURS) Figure 5. Percent equilibrium hydration (0.40 w = 100% ) and percent equilibrium linear swelling (1.165 l = 100%) plotted vs. time for pHEMA gel f

8

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

7.

GREGONis E T AL.

Methacrylate

Hydrogeh

97

S i m i l a r r e s u l t s a r e o b s e r v e d by R e f o j o f o r pHEMA and o t h e r g e l s (25), A b s e n c e o f l i n e a r s w e l l i n g a t low w a t e r f r a c t i o n s i s o b s e r v e d by us i n a n a l t e r n a t i v e e x p e r i m e n t w i t h pHEMA g e l s . HEMA was p o l y m e r i z e d a t v a r i o u s w a t e r c o n c e n t r a t i o n s a n d t h e n allowed t o e q u i l i b r a t e i n d i s t i l l e d water (Figure 7 ) . I t i s f o u n d t h a t when HEMA i s p o l y m e r i z e d w i t h a w a t e r f r a c t i o n (Wf) o f 0.1 o r l e s s (Wf o f 0.1 i s 25% o f t h e e q u i l i b r i u m W f ) , t h e g e l s t i l l s w e l l s t o t h e same d e g r e e as an a n h y d r o u s pHEMA g e l . T h e s e r e s u l t s i n d i c a t e t h a t t h e r e i s f r e e volume o r " v o i d s " i n t h e h y d r o g e l s , and t h i s volume i s f i l l e d w i t h w a t e r m o l e c u l e s before the g e l i s able t o e x h i b i t any l i n e a r expansion. This c o n c e p t has been used t o e x p l a i n t h e s w e l l i n g and m e c h a n i c a l b e h a v i o r o f 3 - k e r a t i n ( 2 6 ) and o t h e r b i o p o l y m e r s (27_). S i m i l a r s t u d i e s on o t h e r h y d r o p h i l i c g e l s a r e i n p r o g r e s s . Copolymers In o r d e r t o o b t a i n g e l s t h a t w o u l d s w e l l i n w a t e r a s p e c i f i e d amount, c o p o l y m e r s o f t h e h y d r o p h i l i c monomers w e r e i n v e s t i gated. A t t h e same t i m e t h e w a t e r s o l u b i l i t y o f t h e comonomers was a l s o s t u d i e d . I t was hoped t h a t monomer s o l u b i l i t y b e h a v i o r w o u l d b e u s e f u l t o e x p l a i n some a s p e c t s o f s w e l l i n g o f t h e polymer. F i g u r e 8 shows HEMA-MEMA comonomers and c o p o l y m e r s and t h e i r r e l a t i o n s h i p t o s o l u b i l i t y and s w e l l i n g i n w a t e r . Water has a maximum s o l u b i l i t y o f 3.5% ( v / v ) i n MEMA monomers. HEMA monomer i s i n f i n i t e l y s o l u b l e i n w a t e r . The comonomer s o l u t i o n s e x h i b i t a w a t e r s o l u b i l i t y t h a t i n c r e a s e s s l i g h t l y a s t h e amount o f HEMA i s i n c r e a s e d up t o 40% MEMA-60% HEMA. A t t h a t p o i n t t h e w a t e r s o l u b i l i t y i n c r e a s e s g r e a t l y u n t i l 20% MEMA-80% HEMA where t h e comonomers become i n f i n i t e l y s o l u b l e . The c o p o l y m e r s o f MEMA-HEMA a p p e a r t o have a 1 i n e a r w a t e r f r a c t i o n (Wf) r e l a t i o n s h i p f r o m 0 . 0 3 5 w a t e r f o r p u r e pMEMA t o 0.40 f o r p u r e pHEMA. F i g u r e 9 shows t h e r e l a t i o n s h i p between MEEMA-HEMA and w a t e r s o l u b i l i t y and between t h e i r c o p o l y m e r s and t h e d e g r e e o f s w e l l i n g in water. I n t h i s c a s e , MEEMA monomer d i s s o l v e s o n l y a b o u t 8% w a t e r and a s HEMA monomer i s a d d e d , t h e amount o f w a t e r t h a t i t dissolves increases sharply. Near a 6 0 : 4 0 HEMA:MEEMA r a t i o , w a t e r becomes i n f i n i t e l y s o l u b l e i n t h e comonomers. The w a t e r s w e l l i n g o f t h e c o p o l y m e r s show t h e o p p o s i t e r e l a t i o n s h i p . pMEEMA s w e l l s t o 63% H 0 , a n d a s i n c r e a s i n g amounts o f HEMA a r e i n c o r p o r a t e d i n the copolymer, the degree o f s w e l l i n g decreases u n t i l a 40% H 0 u p t a k e i s r e a c h e d f o r pHEMA. These above r e s u l t s i n d i c a t e t h a t t h e h y d r o p h i l i c i t y o f t h e monomer and t h e d e g r e e o f c r o s s l i n k i n g o f t h e p o l y m e r ( 2 8 ) a r e not the only f a c t o r s t h a t determine the degree o f water s w e l l i n g of t h e polymer. In t h e m e t h a c r y l a t e s y s t e m , we have r a t i o n a l i z e d t h a t the length o f the e s t e r chain i s a t h i r d f a c t o r t h a t should be t a k e n i n t o a c c o u n t when d i s c u s s i n g s w e l l i n g . This i s probably due t o t h e f a c t t h a t t h e l o n g e s t e r f u n c t i o n a l i t y d e c r e a s e s t h e packing energy o f the m e t h a c r y l a t e polymer. 2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TIME (HOURS) Figure 6. Percent equilibrium hydration (0.62 w = 100% ) and percent equilibrium linear swelling (1.37 l = 100%?) plotted vs. time for pMEEMA gel f

8

Z

0

20

40

60

80

^rOC

PERCENT SWELLING

Figure 7. Plot of percent linear swelling vs. the initial amount of water in HEMA gel. Gel polymerized without water swells to 100%. 100

-

ο Ο »C0M0N0MER

/

•

/

= COPOLYMER

PERCENT HEMA

Figure 8. Water solubility of MEMA-HEMA comonomers (v/v) and the equilibrium water weight fraction, w , of MEMA-HEMA co polymers f

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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GREGONis E T AL.

Methacrylate

HEMA-Ionic M e t h a c r y l a t e

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I t h a s p r e v i o u s l y been shown t h a t marked s w e l l i n g i n w a t e r of crosslinked poly(methacrylic acid) gels i s a function o f the degree o f n e u t r a l i z a t i o n (29, 3 0 , 31). We needed t o know t h e e f f e c t o f s w e l l i n g i n h y d r o g e l s as a f u n c t i o n o f c h a r g e i n c o r porated i n t o t h eg e l . Forthis investigation, methacrylic acid (MAA) i s u s e d t o i n t r o d u c e a n i o n i c g r o u p s and d i m e t h y l aminoethy1 m e t h a c r y l a t e (DMAEMA) i s used t o i n t r o d u c e c a t i o n i c g r o u p s i n pHEMA. The g e l s w e r e p r e p a r e d by a d d i n g m o l a r amounts o f MAA a n d / o r DMAEMA t o t h e HEMA monomer a l o n g w i t h 40% v / v w a t e r a n d were p o l y m e r i z e d a s membranes u s i n g s t a n d a r d c o n d i t i o n s . The g e l s w e r e e q u i l i b r a t e d f o r two d a y s i n d i s t i l l e d w a t e r b e f o r e t h e e q u i l i b r i u m w a t e r f r a c t i o n (Wf) was d e t e r m i n e d . As shown i n F i g u r e 10 d 1 1 pHEMA g e l polymerized with up t o 5 m o l a r p e r c e n t MA e q u i l i b r i u m water conten pur pHEM g e p o l y m e r i z e a t i d e n t i c a l c o n d i t i o n s (Wf = 0 . 4 0 ) . This i s not s u r p r i s i n g s i n c e c a r b o x y l i c a c i d s a n d t e r t a r y amines a r e weak a c i d s a n d b a s e s and do n o t i o n i z e t o an a p p r e c i a b l e e x t e n t a t n e u t r a l p H . To f o r m c h a r g e d g r o u p s , t h e MAA-HEMA c o p o l y m e r s w e r e e q u i l i b r a t e d i n pH 10 NaOH s o l u t i o n f o r t w e l v e h o u r s and t h e DMAEMA-HEMA c o p o l y m e r s w e r e e q u i l i b r a t e d i n pH 3 HC1 s o l u t i o n f o r t w e l v e hours. A f t e r t h i s time they are e q u i l i b r a t e d i n d i s t i l l e d water f o r two d a y s , c h a n g i n g t h e w a t e r s e v e r a l t i m e s i n t h i s p e r i o d t o remove e x c e s s i o n s f r o m t h e g e l s . The c o n v e r s i o n o f MAA t o i t s c a r b o x y l a t e s a l t i n t h e g e l s d r a m a t i c a l l y increases the e q u i l i b r i u m water content o f t h e g e l (Figure 10). F o r e x a m p l e , t h e HEMA g e l c o n t a i n i n g o n l y 2 m o l a r p e r c e n t MAA e q u i l i b r a t e d w i t h a w a t e r f r a c t i o n g r e a t e r t h a n 0 . 9 0 . C o n v e r t i n g t h e HEMA-DMAEMA g e l t o i t s h y d r o c h l o r i d e s a l t d i d n o t show t h e same d r a m a t i c s w e l l i n g e f f e c t , even t h o u g h t h e w a t e r f r a c t i o n i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f DMAEMA. The 5 m o l a r p e r c e n t DMAEMA g e l w a t e r f r a c t i o n measured 0 . 4 6 ( F i g u r e 11). I t i s known t h a t t e r t a r y amines a c t a s c h a i n t r a n s f e r agents w i t h the m e t h a c r y l a t e system ( 3 2 , 3 3 ) . In t h e s e e x p e r i m e n t s , t h e t e r t a r y amine w o u l d a l s o become p a r t o f t h e p o l y m e r chain. The e f f e c t o f t h i s w o u l d g i v e t h e DMAEMA-HEMA g e l s a higher c r o s s l i n k d e n s i t y , p o s s i b l y accounting f o r i t s lower degree o f s w e l l i n g i n w a t e r . F i g u r e 12 shows t h e e q u i l i b r i u m w a t e r c o n t e n t o f HEMA g e l s c o n t a i n i n g e q u a l m o l a r q u a n t i t i e s o f MAA and DMAEMA. When e q u i l i b r a t e d i n d i s t i l l e d water the water f r a c t i o n o f the g e l i n c r e a s e d s l i g h t l y w i t h i n c r e a s i n g m o l a r p e r c e n t MAA-DMAEMA t o a b o u t 0 . 5 w a t e r f r a c t i o n f o r t h e 5 m o l a r p e r c e n t g e l . The c a r b o x y l a t e g r o u p s were t h e n c o n v e r t e d t o t h e i r s o d i u m s a l t a n d t h e amino g r o u p s were c o n v e r t e d t o f r e e amines by e q u i l i b r a t i n g t h e g e l i n pH 10 NaOH s o l u t i o n f o r one d a y . The g e l s w e r e r e e q u i l i b r a t e d i n d i s t i l l e d w a t e r f o r two d a y s and a much g r e a t e r s w e l l i n g d e g r e e was o b s e r v e d . The 5 p e r c e n t MAA-DMAEMA g e l had

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•• COMONOMER = COPOLYMER

Figure 9. Water solubility of MEEMAHEMA comonomers (v/v) and the equi librium water weight fraction, w , of MEEMA-HEMA copolymers

20

f

111 0 4 -

- Ο

- Ο

40

60

PERCENT HEMA

- Ο

-Ο

Ο

Ο

MOLAR PERCENT MAA IN HEMA GEL

Figure 10. Equilibrium water weight fraction, w , of HEMA-MAA copolymers f

CH •

· IONIZED

FORM

3

- N H CI " CH

3

ÇH Ο = UN-IONIZED

3

FORM - N

,^ • · « · · ^

~~

1

2

o

-ο

ο

3

4

5

MOLAR PERCENT DMAEMA IN HEMA GEL Figure

11. Equilibrium water weight fraction, w , of HEMA-DMAEMA copolymers f

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

GREGONis E T A L .

Methacrylate

1

2

Hydrogeh

3

4

5

EQUAL MOLAR PERCENT MAA AND DMAEMA IN HEMA GEL

Figure 12. Equilibrium water weight fraction, w , of HEMA-MAA-DMAEMA terpolymers. MAA and DMAEMA are polymerized at equal molar concentrations. f

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a water fraction measuring 0.81 (Figure 12). This same effect was not observed when the amine was converted to its hydrochloride salt by equilibrating the gel in pH 3 hydrochloric acid followed by two days equilibration in distilled water. The 5 percent MAA-DMAEMA had a water fraction of only 0.53 (Figure 12). It is not known at present why the carboxylate salt gel swells to a greater degree than the amine hydrochloride gel. Polymerization of the amine salt of DMAEMA with HEMA would possibly reduce the crosslink density due to chain transfer mechanisms found in free tertary amine groups. Careful experiments have to be designed in this regard since it is known that changing the pH changes the reactivity ratios of ionic methacrylates (34). Future experiments will be directed towards a correlation of equilibrium water fraction charge and groups and gels. Acknowledgements We gratefully acknowledge NIH Grant HL 16921-01 for financial support of this research, and Hydro Med Sciences, Inc., for generous donations of pure hydroxyethyl methacrylate. Abstract A summary of hydrophilic methacrylate monomer synthesis is discussed. Radical polymerization of the methacrylate system was accomplished by azobisisobutyronitrile (AIBN) derivatives. Some new water soluble AIBN derivatives were prepared and used. Copolymers of various hydrophilic methacrylate monomers afford a method to control water uptake. Some water swelling parameters of poly(hydroxyethyl methacrylate) and poly(methoxyethoxyethy1 methacrylate) are described. The water uptake as measured by the water fraction is shown not to be a linear function of the degree of swelling. The water swelling behavior of poly(hydroxyethyl methacrylate) containing ionic groups is described using methacrylic acid for anionic groups and dimethylaminoethyl methacrylate for cationic groups. The synthesis of C -hydroxyethyl methacrylate is reported. After the radiolabeled monomer was polymerized, the amount of radioactivity in various solvents was measured. 14

Literature Cited 1. 2. 3. 4. 5.

Wichterle, O., and Lim, D., Nature (1960) 185, 117. Wichterle, O., Encyclopedia of Polymer Sci., (1971) 15, 273. Wichterle, O., U. S. Pat. 3,361,858 (1968). Majkus, V., Horakova, Z., Vymula, F., and Stol, M., J. Biomed. Mat. Res. (1969) 3, 443. Refojo, M. F., J. Biomed, Mat. Res. (1969) 3, 333. In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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103

Taylor, G. R., Warren, T. C., Murray, D. G., and Prins, W., J. Surgical Res. (1971) 11, 401. Refojo, M. F., J. Appl. Polymer Sci. (1965) 9, 3161. Fegley, V. W., and Roland, S. P., U. S. Patent 2,680,735. Rohm & Haas Co., British Patent 852,384 (1965). Yocum, R. H. and Nyquist, Ε. B., "Functional Monomers," Marcel Dekker, New York, Vol. 1, 1973, pp 299-487 and Vol. 2 (1974). Horsley, L. H., and Sheetz, D. P., U. S. Patent 3,162,677. Brinkman, U. A. Th., Van Schaik, T. A. M., deVries, G., and deVisser, A. C., this symposium. Wisniewski, S. J., Gregonis, D. E., Kim, S. W., and Andrade, J. D., this symposium. Lenz, R. W., "Organic Chemistry of Synthetic High Polymers," John Wiley & Sons Ne York 1967 244 Pryor, W. Α., "Fre pp. 128-133. Walling, C., "Free Radicals in Solution," John Wiley and Sons, New York, 1957, p. 511. Mortimer, G. Α., J. Org. Chem. (1965) 30, 1632. Thiele, J., and Hauser, K., Ann. (1896) 290, 1. Lim, D., Coll. Czech. Chem. Comm. (1968) 33, 1122. Ma, S. Μ., Gregonis, D. E., Chen, C. Μ., and Andrade, J. D., this symposium. Russell, G. Α., Dalling, D. K., Gregonis, D. E., deVisser, A. C., and Andrade, J. D., this symposium. Ma, S. Μ., Gregonis, D. E., Van Wagenen, R., and Andrade, J. D., this symposium. Flory, P. J.,"Principles of Polymer Chemistry," Cornell University Press, Ithaca, New York, 1953, p. 384. Sorenson, W. R., and Campbell, T. W., "Preparative Methods of Polymer Chemistry," Interscience Publications, New York, 1961. Refojo, M. F., Contact and Intraocular Lens Med. J., (1975) 1, 153. Bradbury, J. H., and Leeder, J. D., J. Appl. Polymer Sci., (1963) 7, 533. Hiltner, Α., Case Western Reserve University, Cleveland, Ohio, personal communication. Flory, P. J., "Principles of Polymer Chemistry," Cornell University Press, Ithaca, New York, 1953, p. 581. Schaefgen, J. R. and Trivisonno, C. F., J. Am. Chem. Soc., (1951) 73, 4580. Schaefgen, J. R. and Trivisonno, C. F., J. Am. Chem. Soc., (1952) 74, 2715. Katchalsky, A. and Eisenberg, H., J. Polymer Sci., (1951) 6, 145. Lenz, R. W., "Organic Chemistry of Synthetic High Polymers," John Wiley and Sons, New York, 1967, p. 355.

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Shalati, M. D. and Scott, R. Μ., Macromolecules, (1975) 8, 127. Alfrey, M. D., Overberger, C. G., and Penner, S. H., J. Am. Chem. Soc., (1953) 75, 4221.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8 Analysis and Purification of 2-Hydroxyethyl Methacrylate by Means of Thin-Layer Chromatography U. A. TH. BRINKMAN, T. A. M. VAN SCHAIK, and G. DEVRIES Department of Analytical Chemistry, Free Reformed University, Amsterdam, The Netherlands A. C. DEVISSER

Department of Materials Science, Free Reformed University, Amsterdam, The Netherlands T h r e e - d i m e n s i o n a l networks o f h y d r o p h i l i c methac r y l a t e polymers, hav f u l materials f o r Implants and o t h e r prothèses a r e b e i n g p r e p a r e d from such h y d r o g e l s o f t e n r e i n f o r c e d w i t h a f a b r i c s t r u c t u r e . The r e s e a r c h program o f o u r departments aims a t the development o f a h y d r o g e l t h a t l j combined w i t h a f i l l e r can s e r v e t o s u b s t i t u t e h a r d t i s s u e (bone), and 2 ) promotes c a l c i f i c a t i o n and/or bone growth i n i t s m a t r i x , so t h a t t h e i n i t i a l l y s o f t m a t e r i a l i s f i l l e d up by t h e body i t s e l f w i t h h a r d t i s s u e . As h y d r o g e l , p o l y ( h y d r o x y e t h y l m e t h a c r y l a t e ) (PHEMA), has been s e l e c t e d which can be p r e p a r e d from t h e commerc i a l l y a v a i l a b l e monomer, 2 - h y d r o x y e t h y l m e t h a c r y l a t e (HEMA) by v a r i o u s methods ( 3 - 5 ) . PHEMA, which has been w i d e l y s t u d i e d and d i s c u s sed f o r m e d i c a l a p p l i c a t i o n s , has l a r g e l y been accept s (1*6) b i o c o m p a t i b l e , s a f e and s t a b l e m a t e r i a l f o r m e d i c a l u s e . I n o r d e r t o p r e p a r e a g e l t h a t meets such r e q u i r e m e n t s , t h e i n i t i a l monomer s h o u l d be o f h i g h p u r i t y . The c o m m e r c i a l l y a v a i l a b l e HEMA c o n t a i n s r e l a t i v e l y l a r g e p r o p o r t i o n s o f m e t h a c r y l i c a c i d (MAA) and t h e d i e s t e r e t h y l e n e d i m e t h a c r y l a t e (EDMA); t h e l a t t e r can be formed from HEMA by t r a n s e s t e r i f i c a t i o n . Other contaminants may a l s o be p r e s e n t , as i s d i s c u s s e d below. The removal o f EDMA from HEMA, as w e l l as MAA and i t s o t h e r e s t e r s , i s p a r t i c u l a r l y d e s i r a b l e , because t h e p r o p o r t i o n o f d i e s t e r i n t h e p o l y m e r i z i n g mixture a f f e c t s the nature o f the network formed (1)· S i n c e t h e r a t e o f both t h e p o l y m e r i z a t i o n and t h e t r a n s e s t e r i f i c a t i o n r e a c t i o n s t r o n g l y i n c r e a s e s w i t h i n c r e a s i n g temperature, t e c h n i q u e s such as d i s t i l l a t i o n and gas chromatography do n o t appear t o be e n t i r e l y f a v o r a b l e f o r t h e u l t i m a t e p u r i f i c a t i o n and a n a l y s i s o f HEMA samples. f

a

s

a

105

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T h e r e f o r e , i n t h e p r e s e n t s t u d y , t h i n - l a y e r chromato graphy ( t i c ) has been s e l e c t e d f o r both q u a l i t a t i v e a n a l y s i s and s m a l l - s c a l e p r e p a r a t i v e work. M a t e r i a l s and Methods 2 - H y d r o x y e t h y l m e t h a c r y l a t e was p u r c h a s e d from BDH ( P o o l e , England) and Merck (Darmstadt, W. Germany); t h e s e p r o d u c t s have a y e l l o w c o l o r . C o l o r l e s s h i g h l y pure (> 99%) HEMA was o b t a i n e d as a g i f t from Hydro Med S c i e n c e s (New Brunswick, N.J., U.S.A.). E t h y l e n e d i m e t h a c r y l a t e was o b t a i n e d from BDH ( P o o l e , England) and Koch and L i g h t (Colnbrook, Eng land). Methacrylic acid ethylene g l y c o l 2-hydroxy propyl methacrylate and £-methoxyphenol D i e t h y l e n e g l y c o l m e t h a c r y l a t e was s y n t h e s i s e d (8) by m i x i n g a p p r o p r i a t e amounts o f d i e t h y l e n e g l y c o l (855 g) and methyl m e t h a c r y l a t e (500 g) a t a temperature o f 60°C, a d d i n g a 4 Ν s o l u t i o n o f sodium methanolate i n methanol (10 g) and h e a t i n g t h e r e a c t i o n m i x t u r e f o r a p e r i o d o f time o f 30 min. S u b s e q u e n t l y , t h e m i x t u r e was poured i n t o water (400 g ) , washed w i t h n-hexane and e x t r a c t e d t w i c e w i t h d i e t h y l e t h e r . A f t e r r e p e a t e d washings w i t h w a t e r , t h e e t h e r e x t r a c t was d r i e d and d i e t h y l e n e g l y c o l m e t h a c r y l a t e was d i s t i l l e d a t r e d u ced p r e s s u r e . A l l f u r t h e r c h e m i c a l s used i n t h i s study were r e a g e n t - g r a d e p r o d u c t s , and were used w i t h o u t f u r t h e r purification. T h i n - l a y e r chromatography was c a r r i e d o u t i n Hellendahl s t a i n i n g j a r s , using precoated s i l i c a g e l p l a t e s ( K i e s e l g e l 60 F 9 5 4 ' o ^^ * a p p r o p r i a t e s i z e (4 χ 8 cm ) . Spots were a p p l i e d w i t h a p o i n t e d paper wick and chromatography i s c a r r i e d o u t i n a n o n - s a t u r a t e d atmosphere. A f t e r development o v e r a l e n g t h o f r u n o f a p p r o x i m a t e l y 7 cm, d e t e c t i o n i s done u s i n g t h e me thods r e p o r t e d below. R e p r o d u c i b i l i t y o f t h e Rp v a l u e s i n t h e s o l v e n t systems used i n t h e p r e s e n t study was satisfactory. P r e p a r a t i v e - s c a l e t h i n - l a y e r chromatography was c a r r i e d o u t on 20 χ 20 cm2 2-mm t h i c k p r e c o a t e d s i l i c a g e l p l a t e s ( K i e s e l g e l 60 F 2 5 4 , Merck). 100-150 mg HEMA, as a 20% (v/v) s o l u t i o n i n e t h a n o l , were a p p l i e d w i t h a Camag chromatocharger equipped w i t h a d i s p o s a b l e p l a s t i c s y r i n g e . Development o v e r a d i s t a n c e o f 15-17 cm, w i t h o u t p r i o r a c c l i m a t i s a t i o n , i s done i n a normal r e c t a n g u l a r tank. M e r c

c

u

t

n

t

o

t

n

e

y

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BRiNKMAN E T A L .

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107

R e f r a c t i v e i n d i c e s were measured on a t h e r m o s t a t t e d (20 + 0.1°C) Abbe r e f r a c t o m e t e r . I R - s p e c t r a were run on a Shimadzu IR 400 s p e c t r o m e t e r , u s i n g c e l l s w i t h NaCl windows. R e s u l t s and D i s c u s s i o n Reference Substances. As has been s t a t e d i n t h i s i n t r o d u c t i o n , HEMA g e n e r a l l y c o n t a i n s s e v e r a l i m p u r i t i e s * * 9 · EDMA, m e t h a c r y l i c a c i d and e t h y l e n e g l y c o l . S i n c e m e t h a c r y l i c a c i d and i t s d e r i v a t i v e s tend t o p o l y m e r i z e even a t room temperature, an i n h i b i t o r such as h y d r o q u i n o n e , £-methoxyphenol, p h e n o t h i a z i n e o r o c t y l p y r o c a t e c h o l i u s u a l l added t thes d u c t s (9). A c c o r d i n samples o b t a i n e d fro 200 pp p-methoxyphenol, and 100 ppm hydroquinone p l u s 100 ppm £-methoxyphenol, r e s p e c t i v e l y ; HEMA from Hydro Med S c i e n c e s , I n c . , c o n t a i n s 36 ppm £-methoxyphenol. Hydroquinone i s a l s o p r e s e n t i n EDMA and m e t h a c r y l i c a c i d samples. M e t h y l m e t h a c r y l a t e may a l s o be p r e s e n t as an i m p u r i t y i n c o m m e r c i a l l y a v a i l a b l e HEMA, s i n c e i t i s one o f t h e s t a r t i n g m a t e r i a l s i n i t s s y n t h e s i s . However, d u r i n g t i c , i t e v a p o r a t e s o f f from t h e c h r o matoplate (b.p., 100°C) and escapes d e t e c t i o n i n q u a l i t a t i v e a n a l y s i s . In p r e p a r a t i v e - s c a l e work, such e v a p o r a t i o n d u r i n g development ensures i t s removal. By d e v e l o p i n g a sample o f pure methyl m e t h a c r y l a t e and s p r a y i n g w i t h water (JL0) immediately a f t e r t h e r u n , we have shown i t s Rp v a l u e t o be c o n s i d e r a b l y h i g h e r than t h a t o f HEMA i n b o t h s o l v e n t systems recommended below. As r e g a r d s t h e presence o f water, one i s r e f e r r e d t o F i g . 3 and t h e accompanying t e x t . As a consequence o f t h e above, i n a d d i t i o n t o HEMA, 5 compounds were i n c l u d e d i n o u r s t u d y , v i z . EDMA, m e t h a c r y l i c a c i d , e t h y l e n e g l y c o l , hydroquinone and £-methoxyphenol ( c f . T a b l e I ) . The p u r i t y o f t h e samples o b t a i n e d as r e f e r e n c e s u b s t a n c e s was t e s t e d , u s i n g two o r more o f t h e s o l v e n t systems d i s c u s s e d below. The samples were found t o be c h r o m a t o g r a p h i c a l l y pure^ a p a r t from t h e presence o f an i n h i b i t o r , w i t h t h e e x c e p t i o n o f EDMA. Here, t i c w i t h n-hexane d i e t h y l e t h e r (1:1) as mobile phase r e v e a l e d t h e p r e s e n c e o f some e i g h t a d d i t i o n a l s p o t s ( F i g s , l a and l b ) . These contaminants were removed by d i s t i l l a t i o n a t reduced p r e s s u r e (b.p. o f EDMA a t 4 mm Hg, 90°C) o r , p r e f e r a b l y , by p r e p a r a t i v e - s c a l e t i c ( F i g s , l c and d ) . e

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

±

4

2

0.2

0.6

0.6

0.6

KMnO

0.60.6

0.60.6

15

60

15

100

I

with

0.2

0.2

Echtblausalz B

(yg/μΐ)

0.2

0.2

D i a z o t i s e d sulphan i l i c acid

20 F o r HEMA, d has a v a l u e o f 1.070-1.074 U 4 ) . T h e r e f o r e , l y g contaminant/μΐ o f HEMA r o u g h l y c o r r e s p o n d s w i t h 1000 ppm o r 0.1 wt.%.

15

-

Ethylene g l y c o l

£-Methoxyphenol

30

Methacrylic acid

15

30

Ethylene dimethacrylate (EDMA)

Hydroquinone

30

UV

Detection l i m i t

2-Hydroxyethyl methacrylate (HEMA)

Name

T a b l e I . D e t e c t i o n l i m i t s o f HEMA and some o f i t s contaminants,

8.

2-Hydroxyethyl

BRiNKMAN E T AL.

Methacrylate

109

With t h e former t e c h n i q u e , a d d i t i o n o f hydroquinone o r o f a m i x t u r e o f KC1, B 1 C I 3 and B 1 I 3 (1_1) i s n e c e s sary i n order t o prevent polymerization. Detection. From among a l a r g e number o f non s p e c i f i c methods o f i d e n t i f i c a t i o n , t h r e e d e t e c t i o n methods were s e l e c t e d f o r f u r t h e r r e s e a r c h . Under U.V.l i g h t (254 nm) a l l compounds b u t e t h y l e n e g l y c o l a r e v i s i b l e as dark s p o t s on a f l u o r e s c e n t background. Treatment w i t h i o d i n e vapor (red-brown s p o t s on a y e l l o w - w h i t e background) i s a s u i t a b l e means t o d e t e c t the g l y c o l t o o . U n f o r t u n a t e l y , t h e s e n s i t i v i t y o f t h e combined U . V . / I p r o c e d u r e i s r a t h e r low (cf:. T a b l e I ) . T h e r e f o r e as an a l t e r n a t i v e , d e t e c t i o n has been done by s p r a y i n g w i t h a White s p o t s a r e forme a l l compounds e x c e p t £-methoxyphenol, which shows up as a p a l e orange s p o t . S i n c e KKn04 r e a c t s w i t h i m p u r i t i e s i n t h e acetone t o form Μηθ2, which mars t h e detec t i o n , t h e r e a g e n t s o l u t i o n s h o u l d be f r e s h l y p r e p a r e d . Keeping i n mind t h a t c h e c k i n g t h e p u r i t y o f HEMA i s done by t i c o f u n d i l u t e d samples, one may c o n c l u d e from t h e d a t a i n T a b l e I t h a t s p r a y i n g w i t h KMn04 a l l o w s t h e d e t e c t i o n o f i m p u r i t i e s down t o a t l e a s t 0.1%. Since the i n h i b i t o r s are present i n the v a r i o u s samples o f HEMA, EDMA and m e t h a c r y l i c a c i d i n concen t r a t i o n s o f up t o 0.0 2% o n l y , they w i l l escape d e t e c t i o n w i t h t h e above p r o c e d u r e s . T h e r e f o r e , two a l t e r n a t i v e methods o f i d e n t i f i c a t i o n (12,13) have been t e s t e d : 1) s p r a y i n g w i t h a f r e s h l y p r e p a r e d 0.5% s o l u t i o n o f t h e d i a z o r e a g e n t E c h t b l a u s a l z Β i n water, and e i t h e r s p r a y i n g w i t h 0.1 Ν NaOH o r , p r e f e r a b l y , exposure t o vapor o f NH3 ; 2) s p r a y i n g w i t h a m i x t u r e o f 0.09 g s u l p h a n i l i c a c i d d i s s o l v e d i n 10 ml 1.1 Ν HC1 and 10 ml o f an aqueous 4.5% NaN02 s o l u t i o n , k e p t a t 0°C and d i l u t e d w i t h an e q u a l volume o f a 10% Na2C03 s o l u t i o n immediately b e f o r e use. With both r e a g e n t s , brownish s p o t s show up on a n e a r l y white back ground. As can be seen from t h e d a t a i n T a b l e I , t h e use o f even these r e a c t i o n s b a r e l y s u f f i c e s f o r t h e i d e n t i f i c a t i o n o f the i n h i b i t o r s i n methacrylic a c i d and i t s d e r i v a t i v e s . F o r t u n a t e l y , improved r e s u l t s a r e o b t a i n e d i f s u l p h a n i l i c a c i d i s used t o d e t e c t t h e p h e n o l i c compounds by a drop t e s t p r o c e d u r e , i n which 1 drop o f t h e d i a z o t i s e d r e a g e n t i s mixed w i t h 1 drop o f sample ( d i s s o l v e d i n a minimum amount o f e t h a n o l , i f n e c e s s a r y ) and 1 drop o f a 10% Na2C03 s o l u t i o n . The d e t e c t i o n l i m i t now i s a p p r o x i m a t e l y 0.01% f o r h y d r o quinone ( g r e e n - t o - y e l l o w s p o t s ) , and 0.001% f o r η methoxyphenol ( r e d - t o - g r e e n s p o t s ) . 2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

S o l v e n t Systems. A f t e r s e v e r a l t r i a l runs o f the f o u r main components w i t h s i n g l e - s o l v e n t systems r a n g i n g from n o n - p o l a r (n-hexane, cyclohexane) t o p o l a r s o l v e n t s ( d i e t h y l e t h e r , m e t h y l i s o b u t y l ketone (MIBK), a c e t o n e ) , m i x t u r e s o f n-hexane w i t h d i e t h y l e t h e r o r ( a n ) o t h e r c o m p o n e n t ( s T were s e l e c t e d f o r f u r t h e r r e s e a r c h . S a t i s f a c t o r y r e s u l t s were o b t a i n e d w i t h n-hexane - d i e t h y l e t h e r (1:1, v/v) and n-hexaneMIBK - n - o c t a n o l (9:2:1, v/v) ( F i g . 2a and d ) . As a drawback, however, i t was o b s e r v e d t h a t the s p o t due t o m e t h a c r y l i c a c i d tends t o t a i l , e s p e c i a l l y i n the l a t t e r s o l v e n t system. Such t a i l i n g , which i s p a r t i c u l a r l y harmful i n p r e p a r a t i v e - s c a l e t h i n - l a y e r or column chromatography, can e f f e c t i v e l y be reduced by a d d i n g an a c i d i c componen n a t i v e s ' have been i n v e s t i g a t e d i s s a t u r a t e d w i t h an e q u a l volume o f 25% (v/v) H N O 3 . When a h i g h e r p e r c e n t a g e o f a c i d i s used, decomposit i o n o f one o r more o f the compounds under i n v e s t i g a t i o n o c c u r s d u r i n g development, as e v i d e n c e d by the o c c u r r e n c e o f a brown s t r e a k i n g zone on the chromatogram. 2) Development i s done on a s i l i c a g e l p l a t e soaked f o r 15 min w i t h 1% H 2 S O 4 , and s u b s e q u e n t l y d r i e d o v e r n i g h t a t 6 0 ° C . As i s m a n i f e s t from the d a t a i n F i g . 2, w i t h each o f these t e c h n i q u e s , the r e s u l t s i n d e e d improved. F o r q u a l i t a t i v e a n a l y s i s , development w i t h a HNOj s a t u r a t e d mobile phase s h o u l d be p r e f e r r e d t o t i c on an a c i d - i m p r e g n a t e d s u p p o r t , which i s a more time consuming p r o c e d u r e . From F i g . 2 i t i s e v i d e n t t h a t n-hexane - MIBK - n - o c t a n o l (9:2:1, s a t d . w i t h 25% H N O 3 ) i s a v e r y p o w e r f u l s o l v e n t system f o r the r e s o l u t i o n o f the f o u r main components. A d d i t i o n o f n i t r i c a c i d t o n-hexane - d i e t h y l e t h e r (1:1) a l s o improves the s e p a r a t i o n o f HEMA from m e t h a c r y l i c a c i d ; however, the s p o t s due t o m e t h a c r y l i c a c i d and EDMA now show an a p p r e c i a b l e o v e r l a p . T h e r e f o r e , as a t e n t a t i v e conc l u s i o n , we recommend the use o f both n-hexane d i e t h y l e t h e r (1:1) and n-hexane - MIBK - n - o c t a n o l (9:2:1, s a t d . w i t h 25% H N O 3 ) f o r q u a l i t a t i v e a n a l y s i s . F o r p r e p a r a t i v e - s c a l e t i c , chromatography on H 2 S O 4 -impregnated s i l i c a g e l has been p r e f e r r e d t o development w i t h a HNC^-saturated mobile phase, because s u l p h u r i c a c i d , but not n i t r i c a c i d , has a n e g l i g i b l y s m a l l s o l u b i l i t y i n the s o l v e n t used t o e l u t e HEMA from the s u p p o r t m a t e r i a l ( c f . b e l o w ) . n-hexane d i e t h y l e t h e r (1:1) has been s e l e c t e d as m o b i l e phase r a t h e r than the n-hexane - MIBK - n - o c t a n o l m i x t u r e , because o f the r e l a t i v e l y l a r g e time o f development o f the l a t t e r s o l v e n t system ( a p p r o x i m a t e l y 4 v s . 2.5 h

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8.

BRINKMAN ET AL.

111

2-Hydroxyethyl Methacrylate

EDMA

Figure 1. Thin-layer chromatograms of EDMA samples, (a) EDMA from BDH; (b) EDMA from Koch and Light; (c) redis silica gel/n-hexane-diethyl

ether (1:1).

3

α

b

c

d

β

Figure 2. Tic of HEMA, EDMA, methacrylic acid (MA), and ethylene glycol (EG) in the systems: (a) silica gel/n-hexane-diethyl ether (1:1); (b) silica gel/n-hexane-diethyl ether (1:1, satd. with 25% HN0 ); (c) H S0^-impregnated silica gel/n-hex ane-diethyl ether (1:1); (d) silica gel/n-hexaneMIBK-n-octanol (9:2:1); (e) silica gel/n-hexaneMIBK-n-octanol (9:2:1, satd. with 25%> HN0 ). , acid (1), n-octanol (2) and MIBK (3) front. 3

2

3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

112

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

f o r a 16-cm r u n ) . Due t o t h e low v o l a t i l i t y o f n - o c t a n o l , i t s complete removal from t h e t h i n l a y e r i s n o t e a s i l y e f f e c t e d , and p a r t o f i t w i l l be e l u t e d t o g e t h e r w i t h HEMA. Next, a t t e n t i o n was p a i d t o t h e s e l e c t i o n o f a s o l v e n t s u i t e d t o e l u t e t h e s e p a r a t e d component(s) from t h e s i l i c a g e l . A t f i r s t s i g h t , t h i s c h o i c e does not appear t o be v e r y c r i t i c a l , because HEMA d i s s o l v e s f r e e l y i n s o l v e n t s such as water, acetone, d i e t h y l e t h e r , t e t r a c h l o r o m e t h a n e and, l e s s s o , n-hexane. However, when u s i n g any o f t h e f i r s t t h r e e h i g h l y p o l a r s o l v e n t s , t h e a c i d used t o impregnate t h e s i l i c a g e l i s e x t r a c t e d a l o n g w i t h HEMA. Moreover, when d e v e l o p ment i s c a r r i e d o u t on s i l i c a g e l plates, zinc o r i g i n a t i n g from t h p r e s e n t as f l u o r e s c i n s a l t , i s a l s o e x t r a c t e d . The presence o f both t h e a c i d and i t s z i n c s a l t i n p u r i f i e d HEMA have been demons t r a t e d by t i c on p l a i n s i l i c a g e l , w i t h n-hexane d i e t h y l e t h e r (1:1) as d e v e l o p i n g s o l v e n t . Both compounds remain a t t h e o r i g i n and a r e i d e n t i f i e d by s p r a y i n g w i t h a s u i t a b l e a c i d - b a s e i n d i c a t o r , e.g. thymol b l u e (pH range, 1.2-2.8), and 4 - ( 2 - p y r i d y l a z o ) r e s o r c i n o l , r e s p e c t i v e l y . As a consequence, t h e r e l a t i v e l y n o n - p o l a r t e t r a c h l o r o m e t h a n e i s p r e f e r r e d as eluent. When e x a m i n i n g t h e t i c d a t a i n o r d e r t o e v a l u a t e systems s u i t a b l e f o r column chromatography, one s h o u l d c o n s i d e r t h a t development w i t h a HNO^-saturated mobile phase causes t h e f o r m a t i o n o f an a c i d f r o n t a t Rp v a l u e s o n l y s l i g h t l y h i g h e r than those o f HEMA ( F i g . 2b and e ) . C o n s e q u e n t l y , i n chromatography on columns e q u i l i b r a t e d w i t h HNC^-saturated s o l v e n t systems, t h e s e p a r a t i o n o f HEMA from contaminants h a v i n g h i g h e r Rp v a l u e s , presumably w i l l be r a t h e r m a r g i n a l . T h e r e f o r e , p r e f e r e n c e s h o u l d be g i v e n t o chromatography on H 2 S O 4 impregnated s i l i c a g e l ; f o r t h e same reasons as mentioned above, n-hexane - d i e t h y l e t h e r (1:1) i s a g a i n recommended as mobile phase. Application. T h i n - l a y e r chromatograms o f a l l r e f e r e n c e substances and HEMA samples a r e p i c t u r e d i n F i g . 3. F u r t h e r i n f o r m a t i o n i s r e c o r d e d i n T a b l e I I . The chromatograms demonstrate t h a t - a p a r t from the i n h i b i t o r s - e t h y l e n e g l y c o l and/or o t h e r p o l a r m a t e r i a l , EDMA and m e t h a c r y l i c a c i d (BDH sample o n l y ) are p r e s e n t i n t h e HEMA samples from Merck and BDH. D e t e c t a b l e amounts o f m e t h a c r y l i c a c i d and EDMA a r e absent from HEMA o b t a i n e d from Hydro Med S c i e n c e s .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

60 40

pM

MA

£-Methoxyphenol

Methacrylic

DGMA

EG/P

HEMA

Diethyleneglycol methacrylate

Ethylene g l y c o l / polar material

Cannot be d e t e c t e d : c f . t e x t .

Drop t e s t

Hy

HEMA

Hydroquinone

acid

70

EDMA

EDMA

00

10

25

25

Pure compounds

Abréviations (cf. F i g . 3)

Compound

hRp o f / i n :

gel/n-hexane - d i e t h y l e t h e r ( 1 : 1 )

00

+

+

+

10

00

10

25

-

25

60

—

HEMA (Hydro)

60

70

HEMA (Merck)

00

10

25

*

40

60

70

HEMA (BDH)

T a b l e I I . Rp d a t a f o r HEMA and i t s p r i n c i p a l c o n t a m i n a n t s i n t h e system

+

-

—

-

25

40

-

Methacrylic acid

silica

114

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

S t i l l , even t h i s h i g h l y pure p r o d u c t c o n t a i n s a t l e a s t two i m p u r i t i e s . The s p o t w i t h Rp 0.0 a g a i n may be a t t r i b u t e d t o e t h y l e n e g l y c o l and/or o t h e r p o l a r m a t e r i a l . As f o r t h e second s p o t , a compound d i s p l a y i n g Rp a p p r o x i m a t e l y 0.10 i n both chromatographic s o l v e n t systems, has a l s o been o b s e r v e d i n t h e HEMA samples o b t a i n e d from Merck and BDH. M i l l i g r a m amounts have been c o l l e c t e d by p r e p a r a t i v e - s c a l e t i c and c o lumn chromatography. On t h e b a s i s o f t h e mass spec trum (m/e v a l u e o f 144; p r e s e n c e o f 3 oxygen atoms) the unknown compound was i n i t i a l l y thought t o be h y d r o x y p r o p y l m e t h a c r y l a t e . However, comparison o f t h e t i c b e h a v i o r o f t h e unknown compound and a sample o f pure 2 - h y d r o x y p r o p y l m e t h a c r y l a t e demonstrated o u r h y p o t h e s i s t o be i n c o r r e c t o b t a i n e d from Hydr dent t h a t HEMA samples may c o n t a i n s e v e r a l t e n t h s o f a p e r c e n t o f d i e t h y l e n e g l y c o l m e t h a c r y l a t e . The l a t t e r p r o d u c t has been s y n t h e s i s e d i n o u r l a b o r a t o r y ( c f . above) and has been shown t o be i d e n t i c a l w i t h the unknown compound by both t i c and mass s p e c t r o metry . I n a d d i t i o n , we note t h a t a sharp s e p a r a t i o n o f HEMA and d i e t h y l e n e g l y c o l m e t h a c r y l a t e i s o b t a i n e d by c a r r y i n g o u t development w i t h pure d i e t h y l e t h e r i n s t e a d o f n-hexane - d i e t h y l e t h e r (1:1). The Rp v a l u e s now a r e 0.80 and 0.55 r e s p e c t i v e l y . As r e g a r d s t h e i n h i b i t o r s , w i t h n-hexane d i e t h y l e t h e r (1:1), hydroquinone d i s p l a y s an Rp v a l u e a p p r o x i m a t e l y e q u a l t o t h a t o f HEMA and cannot be detected with e i t h e r Echtblausalz Β o r d i a z o t i s e d s u l p h a n i l i c a c i d . When u s i n g n-hexane - MIBK - η o c t a n o l (9:2:1, s a t d . w i t h 25% H N O 3 ) as mobile phase, hydroquinone has a s l i g h t l y f a s t e r m i g r a t i o n r a t e than has HEMA, i t s s p o t o v e r l a p p i n g t h a t o f m e t h a c r y l i c a c i d . However, d e t e c t i o n o f s m a l l amounts o f h y d r o quinone even now i s n o t s u c c e s s f u l . I n s e p a r a t e expe r i m e n t s , we have demonstrated t h a t t h i s i s due t o s e v e r e s t r e a k i n g o f t h e hydroquinone s p o t , e f f e c t e d by t h e p r e s e n c e o f t h e l a r g e e x c e s s o f HEMA. As a consequence, hydroquinone i s d e t e c t e d i n i s o l a t e d i n s t a n c e s ( m e t h a c r y l i c a c i d ! ) o n l y . No such d i f f i c u l t i e s a r e e n c o u n t e r e d w i t h p-methoxyphenol; w i t h t h i s i n h i b i t o r , i d e n t i f i c a t i o n by t i c i s s u c c e s s ful. *As r e g a r d s t h e d i s c r e p a n c y between t h e mass o f d i e t h y l e n e g l y c o l m e t h a c r y l a t e (174) and t h e v a l u e quo t e d above (144), i t i s a well-known f a c t t h a t i n mass spectrometry p o l y g l y c o l s e a s i l y lose a p a r t o f t h e i r m o l e c u l e h a v i n g a mass o f 30; i . e . , t h e m/e v a l u e o f 144 s h o u l d be a t t r i b u t e d t o Im - C H o ] . 2

+

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8.

BRiNKMAN E T A L .

2-Hydroxyethyl Methacrylate

115

S t i l l , w i t h both compounds, i n view o f t h e r a t h e r u n f a v o r a b l e d e t e c t i o n l i m i t s quoted i n T a b l e I , i t i s recommended t o a p p l y s e v e r a l m i c r o l i t e r s o f sample s o l u t i o n t o t h e c h r o m a t o p i a t e , u s i n g development o v e r a d i s t a n c e o f 10-15 cm. A l t e r n a t i v e l y , p r e c o n c e n t r a t i o n o f t h e i n h i b i t o r s by means o f t r e a t m e n t o f t h e sample s o l u t i o n w i t h Amberlyst A-27 r e s i n ( c f . below) may be used. I n c o n c l u s i o n , as i s e v i d e n t from t h e d a t a i n T a b l e I I , t h e drop t e s t p r o c e d u r e w i t h s u l p h a n i l i c acid i s e x c e l l e n t l y s u i t e d t o detect the presenc e , though n o t t h e t y p e , o f an i n h i b i t o r . Comparison o f t h e o v e r a l l p i c t u r e o f t h e s e r i e s o f chromatograms o b t a i n e d w i t h n-hexane - d i e t h y l e t h e r (1:1), and t h e s e r i e s o b t a i n e d w i t h n-hexane MIBK - n - o c t a n o l (9:2:1 strates that a smalle when d e v e l o p i n g w i t h t h e l a t t e r s o l v e n t system. T h i s i s m a i n l y due t o t h e f a c t t h a t n - o c t a n o l has low v o l a t i l i t y and cannot e a s i l y be removed from t h e c h r o matogram p r i o r t o i d e n t i f i c a t i o n . As a consequence, upon s p r a y i n g w i t h a KMn04 s o l u t i o n , t h e c o l o r o f t h e background t u r n s brown r a t h e r r a p i d l y , thus o b s c u r i n g s e v e r a l o f t h e s m a l l e r s p o t s . I n summary, n-hexane d i e t h y l e t h e r (1:1) s h o u l d be p r e f e r r e d f o r q u a l i t a t i v e a n a l y s i s , both on account o f i t s f a s t e r m i g r a t i o n r a t e , and t h e b e t t e r d e t e c t i o n a c h i e v e d f o r a c t u a l l y a l l components p r e s e n t i n t h e HEMA and EDMA samples. P r e p a r a t i v e - s c a l e t i c on ^ S C ^ - i m p r e g n a t e d s i l i c a g e l has s u c c e s s f u l l y been c a r r i e d o u t w i t h HEMA samp l e s from Hydro Med S c i e n c e s and BDH. ( F o r a l l compounds s t u d i e d , t h e Rp v a l u e s i n t h i s system a r e e q u a l t o , o r s l i g h t l y h i g h e r than t h o s e i n t h e system s i l i c a gel/n-hexane - d i e t h y l e t h e r , s a t d . w i t h 25% HNCK; c f . F i g . 2 ) . A f t e r development w i t h n-hexane - d i e t h y l e t h e r (1:1) and e l u t i o n w i t h t e t r a c h l o r o m e t h a n e , t h e samples t u r n o u t t o be c h r o m a t o g r a p h i c a l l y pure. I f hydroquinone has been added t o t h e HEMA sample (Merck\ t h i s i n h i b i t o r w i l l s t i l l be p r e s e n t a f t e r chromatog r a p h i c p u r i f i c a t i o n (£f. above). G e n e r a l l y , t h e p r e s e n c e o f an i n h i b i t o r i s n o t h a r m f u l s i n c e i t can be removed q u a n t i t a t i v e l y from t h e PHEMA g e l by p r o l o n g e d washing w i t h water. However, s h o u l d q u a n t i t a t i v e removal o f hydroquinone a t an e a r l y stage be i m p e r a t i v e , then two c o n s e c u t i v e t r e a t m e n t s o f t h e sample t o be p u r i f i e d w i t h Amberlyst A-27 r e s i n (Rohm and Haas, P h i l a d e l p h i a , Pa. 19105, U.S.A.) s u f f i c e t o remove up t o 0.2% o f hydroquinone, and a l s o o t h e r p o l a r compounds; t h e s e can s u b s e q u e n t l y be e l u t e d w i t h methanol. The c o n c e n t r a t e d e x t r a c t so o b t a i n e d i s e x c e l l e n t l y s u i t e d t o d e t e c t t h e presence o f i n h i b i -

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Table I I I . η

v a l u e s f o r HEMA and some o f i t s

contaminants Compound

η

HEMA

1.4525

I'

Diethyleneglycol methacrylate

1.4568

8

n

ri.4549 U.4549-1.4553

EDMA Ethylene

I,

2, 4 1

glycol

Methacrylic Methyl

Ref.

acid

methacrylate

1.4314

1> 1

1.4142

5

t o r ( s ) , as was r e f e r r e d t o above. A f i n a l word about two f u r t h e r c r i t e r i a which a r e used t o a s s e s s t h e p u r i t y o f HEMA and EDMA, v i z . t h e r e f r a c t i v e i n d e x and t h e i n f r a r e d spectrum. A c c o r d i n g t o o u r e x p e r i e n c e s , n $ v a l u e s , which a r e r a t h e r gene r a l l y quoted i n t h e l i t e r a t u r e , a r e n o t v e r y r e l i a b l e c r i t e r i o n , since the i m p u r i t i e s normally present i n HEMA e x h i b i t n2Q v a l u e s which a r e both h i g h e r (EDMA and d i e t h y l e n e g l y c o l m e t h a c r y l a t e ) and lower (metha c r y l i c a c i d and e t h y l e n e g l y c o l ) than a r e those o f HEMA i t s e l f . As an i l l u s t r a t i o n , a s e r i e s o f d a t a i s recorded i n Table I I I . Besides, the r e f r a c t i v e index o f HEMA s t r o n g l y d e c r e a s e s w i t h i n c r e a s i n g water con t e n t , as i s demonstrated i n F i g . 4. I n f r a r e d s p e c t r o scopy has been recommended t o check t h e p u r i t y o f EDMA. Our r e s u l t s i n d i c a t e t h a t compounds such as HEMA and e t h y l e n e g l y c o l can be d e t e c t e d down t o app r o x i m a t e l y 1 wt.% due t o t h e s t r o n g a b s o r p t i o n o f ^ t h e i r h y d r o x y l groups (broad band a t 2800-3600 cm ) . However, one s h o u l d keep i n mind t h a t t h e i n f r a r e d spectrum does n o t e a s i l y a l l o w a c o n c l u s i o n r e g a r d i n g the n a t u r e o f t h e c o n t a m i n a n t ( s ) - e.g. HEMA, e t h y l e n e g l y c o l o r water. B e s i d e s , a compound such as metha c r y l i c a c i d goes u n d e t e c t e d a t t h e 1% l e v e l . 2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

8.

BRiNKMAN E T AL.

117

2-Hydroxyethyl Methacrylate

HEMA

DGMA EG/P

Figure 3. Tic of three HEMA samples on silica gel, using n-hexane-diethyl ether (1:1) (a-c), and n-hexane-M IBK-n-octanol (9:2:1, satd. with 25% HNO ) (d-f) as mobile phases. HEMA samples obtained from BDH (a,d), Merck (b,e), and Hydro Med Sciences (c,f). For abbreviations, see Table II. , acid (1), n-octanol (2), and MIBK (3) front. s

1.45

1.40

1.35

100

80

60

40

20

0 % HEMA

0

20

40

60

80

1 0 0 % water

Figure 4. Dependence of n of HEMA

D

20

on water content

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

118

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Abstract Poly(hydroxyethyl methacrylate) is generally accepted as a biocompatible, safe and stable hydrogel for medical use. The present paper describes the use of tlc for the analysis and small-scale preparation of the initial monomer, 2-hydroxyethyl methacrylate. Tlc on silica gel, with n-hexane - diethyl ether (1:1, v/v) and/or n-hexane - MIBK - n-octanol (9:2:1, v/v, satd. with 25% HNO) as mobile phase is recommen ded for qualitative analysis. Preparative-scale work is preferably carried out on HSO-impregnated silica gel, development being done with n-hexane - diethyl ether (1:1). Inhibitors are detected by means of tlc and of a drop test procedur with diazotised sulpha nilic acid. The natur several commercially available HEMA samples is dis cussed, n D values are reported for the system HEMA- water. 3

2

4

20

Literature Cited 1. Wichterle, O., and Lim, D., Nature (1960) 185, 117. 2. Wichterle, O., and Lim, D., U.S. Patent, (1961) 2, 976, 576. 3. Kopecek, J., Jokl, J., and Lim, D., J. Polymer Sci., [C] (1968) 16, 3877. 4. Refojo, M.F., J. Appl. Polymer Sci., (1965) 9, 3161. 5. Goedberg, A.I., Fertig, J., and Stanley, Η., U.S. Patent, (1962) 3, 059, 024. 6. Refojo, M.F., and Yasuda, H., J. Appl. Polymer Sci., (1965) 9, 2425. 7. Hasa, J., and Janácek, J., J. Polymer Sci., [C] (1967) 16, 317. 8. Kopecek, J., Thesis, Inst. Macromol. Chem., Prague, (1965) p. 35. 9. Mark, M.F., Gaylord, N.G., Bikales, N.M., (eds), "Encyclopedia of Polymer Science and Technology", Vol. 15, 1971, 273. 10. Zweig, G., and Sherma, J. (eds), "Handbook of Chromatography", CRC Press, Interscience, New York, 1971. 11. Crawford, J.W.C., U.S. Patent, (1939) 2, 143, 941. 12. Jatzkewitz, Η., and Lenz, U., Hoppe-Seylers Z. Physiol. Chem., (1956) 305, 53. 13. Jatzkewitz, Η., Hoppe-Seylers Z. Physiol. Chem., (1953) 292, 99. 14. Merck, Ε., Chemicalien, Reagenzien (1974), Darmstadt.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9 Rotational Viscometry Studies of the Polymerization of Hydrophilic Methacrylate Monomers SHAO M. MA, DONALD E. GREGONIS, CHWEN M. CHEN, and JOSEPH D. ANDRADE Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 T h e r e i s c o n s i d e r a b l e l i t e r a t u r e on t h e m e c h a n i c a l b e h a v i o r of poly (hydroxyethyl methacrylate k n o w l e d g e , no s t u d y ha polymer during the course of p o l y m e r i z a t i o n . I n t h i s s t u d y we a t t e m p t e d t o use t h i s e a s i l y o b t a i n e d p r o p e r t y t o o b t a i n knowledge c o n c e r n i n g (1) t h e r e l a t i v e r a t e o f p o l y m e r i z a t i o n w i t h v a r i o u s i n i t i a t o r s ; ( 2 ) t h e e f f e c t o f p o l y m e r i z a t i o n t i m e on t h e f l o w p r o p e r t i e s o f PHEMA; ( 3 ) t h e r e l a t i o n s between t h e f l o w c u r v e , m o l e c u l a r w e i g h t and t h e m o l e c u l a r w e i g h t d i s t r i b u t i o n . At a given temperature the v i s c o s i t y (η) v s . shear r a t e ( § ) c u r v e f o r c o n c e n t r a t e d p o l y m e r s o l u t i o n s and p u r e p o l y m e r s depends on t h e m o l e c u l a r w e i g h t and m o l e c u l a r w e i g h t d i s t r i b u t i o n o f t h e system. However, an i n c r e a s e i n v i s c o s i t y o f t h e p o l y m e r i z a t i o n mixture indicates only that conversion i s i n c r e a s i n g . How v i s c o s i t y changes d u r i n g t h e c o u r s e o f p o l y m e r i z a t i o n f r o m N e w t o n i a n t o n o n - N e w t o n i a n as a f u n c t i o n o f m o l e c u l a r w e i g h t o r m o l e c u l a r weight d i s t r i b u t i o n i s not t o t a l l y understood. Q u a l i t a t i v e l y , t h e p o l y m e r i z a t i o n m i x t u r e behaves as a N e w t o n i a n f l u i d d u r i n g t h e e a r l y s t a g e o f p o l y m e r i z a t i o n . As t h e degree o f p o l y m e r i z a t i o n o r t h e c o n c e n t r a t i o n o f t h e p o l y m e r i c f r a c t i o n i n c r e a s e s , t h e v i s c o s i t y a l s o i n c r e a s e s and t h e s y s t e m becomes n o n - N e w t o n i a n . Polymers w i t h broad m o l e c u l a r weight d i s t r i b u t i o n s show a h i g h e r v i s c o s i t y dependency on s h e a r r a t e ( 2 , 3 ) ; non-Newtonian b e h a v i o r s t a r t s t o o c c u r a t l o w e r shear r a t e s than s i m i l a r polymers w i t h narrow m o l e c u l a r weight d i s tributions. T h e r e f o r e , the e f f e c t of p o l y m e r i z a t i o n time a t t h i s s t a g e w o u l d depend on t h e p o l y d i s p e r s i t y o f t h e p o l y m e r p r o d u c e d . F u r t h e r p o l y m e r i z a t i o n e n c o u n t e r s b r a n c h i n g and g e l f o r m a t i o n . G r a e s s l e y (4) r e p o r t e d t h a t l o n g - c h a i n b r a n c h i n g a f f e c t s t h e v i s c o s i t y - s h e a r r a t e c u r v e i n a s i m i l a r way as b r o a d e n i n g o f t h e molecular weight d i s t r i b u t i o n . However, t h e e f f e c t s o f b r a n c h i n g c a n n o t be s e p a r a t e d f r o m t h e e f f e c t o f p o l y d i s p e r s i t y i n v i s c o m e t r y measurements. The v i s c o s i t y o f a b r a n c h e d p o l y m e r may be h i g h e r o r lower than a l i n e a r polymer. T h i s depends on w h e t h e r the molecular weight i s higher or lower than the m o l e c u l a r weight

119

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120

HYDROGELS

FOR MEDICAL

AND RELATED APPLICATIONS

of t a n g l i n g segments. Because o f t h e c o m p l i c a t e d n a t u r e o f t h e p r o b l e m , no s i n g l e t h e o r y can a d e q u a t e l y d e s c r i b e t h e m e c h a n i c a l behavior of the p o l y m e r i c system during the course of p o l y m e r i z a tion. However, , s e v e r a l t h e o r i e s ( 5 - 7 ) have w o r k e d w e l l f o r l i n e a r p o l y m e r s , w h i c h m i g h t be t h e c a s e f o r PHEMA p r o d u c e d w i t h very l i t t l e c r o s s l i n k i n g agent i n a r e l a t i v e l y poor s o l v e n t s u c h as w a t e r . The p r e s e n t i n v e s t i g a t i o n c o n s i s t s o f : (1) f l o w c u r v e s o b t a i n e d f o r h y d r o x y e t h y l m e t h a c r y l a t e (HEMA)-water m i x t u r e s , p o l y m e r i z e d a t 60°C as f u n c t i o n s o f p o l y m e r i z a t i o n t i m e and i n i t i a t o r ; (2) t h r e e t h e o r i e s o f n o n - N e w t o n i a n v i s c o s i t y , t h e B u e c h e - H a r d i n g method ( 5 j , R e e - E y r i n g a c t i v a t e d - s t a t e t h e o r y (6) and B a r t e n e v ' s e m p i r i c a l method ( 7 ) , a r e b r i e f l y d e s c r i b e d and t h e f l o w p a r a m e t e r s o f o u r s y s t e m s a r e a n a l y z e d w i t h t h e s e t h e o r i e s ; (3) t h e r e l a t i o n r a t e and between f l o w c u r v e d i s t r i b u t i o n and p o l y m e r i z a t i o n t i m e a r e d i s c u s s e d . Material

and

Methods

The m o n o m e r - s o l v e n t m i x t u r e used i n t h i s s t u d y c o n s i s t e d o f s i x p a r t s o f HEMA and t h r e e p a r t s o f w a t e r , by v o l u m e . The i n i t i a t o r s used i n c l u d e ammonium p e r s u l f a t e , a z o b i s i s o b u t y r o n i t r i l e (AIBN), azobis(methyl i s o b u t y r a t e ) , azobis(methoxyethyl i s o b u t y r a t e ) , and a z o b i s ( m e t h o x y d i e t h o x y e t h y l i s o b u t y r a t e ) . A c o n c e n t r a t i o n o f 5.71 m m o l / l i t e r was used f o r ammonium p e r s u l f a t e and 5.21 m m o l / l i t e r f o r t h e o t h e r s . The s y n t h e s i s , p u r i f i c a t i o n and c h e m i c a l c h a r a c t e r i z a t i o n o f t h e PHEMA a r e g i v e n e l s e w h e r e (8). I t s h o u l d be n o t e d t h a t a l t h o u g h HEMA monomer i s c o m p l e t e l y m i s c i b l e w i t h w a t e r , PHEMA i s n o t w a t e r s o l u b l e . A Haake R o t o v i s c o r o t a t i o n a l v i s c o m e t e r was u s e d f o r v i s c o s i t y measurement. P o l y m e r i z a t i o n was c a r r i e d o u t i n a c o a x i a l c y l i n d e r s e n s o r s y s t e m , w i t h a MV cup and a MVI b o b . Temperature was k e p t a t 60°C w i t h a Lauda K-2/R c i r c u l a t i n g b a t h . F l o w c u r v e s were d e t e r m i n e d as f u n c t i o n s o f t i m e a t s h e a r r a t e s f r o m 0 t o 685 s e c " . To a v o i d permanent m e c h a n i c a l b r e a k d o w n , l o w e r r a t e s were used as t h e p o l y m e r i z a t i o n i n c r e a s e d . Non-Newtonian

shear

Viscosity

In n o n - N e w t o n i a n f l o w t h e c h a n c e i n a p p a r e n t v i s c o s i t y as a f u n c t i o n o f s h e a r r a t e ( § ) g e n e r a l l y t a k e s t h e f o r m η η

0

= f(xS),

(η)

Cl]

where η i s t h e v i s c o s i t y a t z e r o s h e a r r a t e , § > and τ i s t h e c h a r a c t e r i s t i c r e l a x a t i o n t i m e , which i s molecular weight-depend ent. 0

0

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

Polymerization of Hydrophilic Methacrylate Monomers

M A E T AL.

121

Many t h e o r i e s have been p r o p o s e d t o e x p l a i n n o n - N e w t o n i a n b e h a v i o r i n condensed systems. M o l e c u l a r t h e o r i e s b a s e d on t h e e q u i v a l e n c e h y p o t h e s i s ( 9 j , on t h e e n t a n g l e m e n t c o n c e p t ( 1 0 ) , and on t h e a c t i v a t e d - s t a t e model (6) have g a i n e d c o n s i d e r a b l e acceptance. Bueche-Harding Standard Curve. Bueche i n t r o d u c e d a s h e a r r a t e dependence t o Rouse t h e o r y ( 1 1 ) . A c c o r d i n g t o h i s h y p o t h e s i s , macromolecules i n s o l u t i o n under dynamic d e f o r m a t i o n a r e assumed t o behave s i m i l a r l y t o t h o s e u n d e r s t e a d y s h e a r i n g . The change i n v i s c o s i t y w i t h s h e a r r a t e s i s c o n s i d e r e d as due t o t h e r e s u l t s o f d e f o r m i n g and r o t a t i n g o f t h e c o i l i n g p o l y m e r m o l e c u l e s under a s h e a r i n g f o r c e . Below a c e r t a i n c h a r a c t e r i s t i c t i m e , t ^ , (which equals a p p r o x i m a t e l y t h e r e c i p r o c a l o f z e r o shear r a t e , s ) the v i s c o s i t s h e a r r a t e , w h i l e abov y approache value. T h i s r e l a x a t i o n t i m e c a n be c a l c u l a t e d f r o m t h e p r o p e r t i e s of the system at low shear r a t e s . Bueche has i g n o r e d t h e e f f e c t o f c h a i n e n t a n g l e m e n t s on n o n - N e w t o n i a n b e h a v i o r and assumed t h a t t h e l o c a l p r o p e r t i e s o f t h e s y s t e m , s u c h as t h e r e l a x a t i o n t i m e d i s t r i b u t i o n , a r e i n d e pendent o f i t s s t a t e o f m o t i o n . H i s t h e o r y , t h e r e f o r e , does n o t c o r r e l a t e well with experimental data (4, 12-14). G r a e s s l e y ( 1 0 ) p r o p o s e d a t h e o r y by a s s u m i n g t h a t t h e v i s c o s i t y o f a p o l y m e r i c s y s t e m i s c o n t r o l l e d by i n t e r m o l e c u l a r c h a i n e n t a n g l e m e n t s and t h a t an i n c r e a s e i n s h e a r i n d u c e s changes i n t h e n e t w o r k o f e n t a n g l e m e n t s and hence c a u s e s t h e v i s c o s i t y to decrease. T h i s e n t a n g l e m e n t a p p r o a c h has a sound theoretical basis. However, i n f o r m a t i o n c o n c e r n i n g t h e p o l y d i s p e r s i t y and t h e e n t a n g l e m e n t d e n s i t y a r e needed t o c a r r y o u t actual computation. For a l i n e a r c o i l i n g polymer, B u e c h e - H a r d i n g , l a t e r , s u g g e s t e d a method f o r d e t e r m i n i n g i t s a b s o l u t e m o l e c u l a r w e i g h t f r o m i t s f l o w c u r v e ( 5 j . In t h i s method t h e y u s e d a s t a n d a r d curve which f o l l o w s the e m p i r i c a l equation 0

n / n = 1.00 + 0 . 6 0 ( T S ) o

3 / 4

,

[2]

t o match t h e i r e x p e r i m e n t a l d a t a . The v a l u e o f η and s a r e d e t e r m i n e d by s u p e r i m p o s i n g t h e s t a n d a r d c u r v e i n t h e f o r m o f l o g ( η / η ο ) v s . l o g ( S T ) w i t h an e x p e r i m e n t a l c u r v e i n t h e f o r m o f l o g ( η ) v s . l o g ( s ) w h i l e b o t h c u r v e s were p l o t t e d on t h e same scale. The m o l e c u l a r w e i g h t o f t h e p o l y m e r i s t h e n c a l c u l a t e d from t h e e x p r e s s i o n 0

M = 7T NckT/12n

0

s , [3] ο o where N, c , k, and Τ a r e A v o g a d r o ' s number, t h e c o n c e n t r a t i o n o f p o l y m e r i n g / c c , B o l t z m a n n ' s c o n s t a n t , and t h e a b s o l u t e t e m p e r a ture, respectively. 2

o

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

122

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

B u e c h e s r e l a x a t i o n t i m e {tw = l / s ) , as p o i n t e d o u t by G r a e s s l e y ( 1 5 ) , g o v e r n s t h e m a g n i t u d e o f t h e s h e a r r a t e when t h e v i s c o s i t y begins to decrease. I t s h o u l d be an i m p o r t a n t p a r a meter f o r those p r o p e r t i e s i n which the l o n g e r r e l a x a t i o n times a r e d e t e r m i n i n g f a c t o r s . The B u e c h e - H a r d i n g s t a n d a r d c u r v e , w h i c h a g r e e d m o d e r a t e l y w e l l w i t h d a t a on u n f r a c t i o n a t e d p o l y s t y r e n e i n benzene and p o l y ( m e t h y l m e t h a c r y l a t e ) (PMMA) i n c h l o r o f o r m , s h o u l d p r e d i c t t h e n o n - N e w t o n i a n b e h a v i o r o f any l i n e a r p o l y m e r i c system w i t h not too broad a m o l e c u l a r weight d i s t r i b u tion (5). PHEMA and PMMA have t h e same backbone s t r u c t u r e . S i n c e t h e s h e a r r a t e used i n t h i s s t u d y i s r e l a t i v e l y l o w ; and t h e s y s t e m i s e x p e c t e d t o have low e n t a n g l e m e n t d e n s i t y ( w a t e r i s a p o o r s o l v e n t ) , low b r a n c h i n g and c r o s s l i n k i n g ( t h e d i e s t e r c o n c e n t r a t i o n i s l o w ) , t h e B u e c h e - H a r d i n g method s h o u l d work w e l l f o r our system. 1

0

R e e - E y r i n g s ' s A c t i v a t e d - S t a t e M o d e l . T h i s model (6) assumes t h a t t h e r e e x i s t s i g r o u p s o f f l o w u n i t s w h i c h d i f f e r i n r e l a x a t i o n t i m e and i n g e o m e t r i c a l d i m e n s i o n s . Some o f t h e s e flow u n i t s are Newtonian, others are non-Newtonian. A Newtonian f l o w u n i t i s a m o l e c u l e o r a group o f m o l e c u l e s i s o l a t e d f r o m o t h e r u n i t s , w h i l e a non-Newtonian u n i t i s a Newtonian u n i t bonded ( o r e n t a n g l e d ) w i t h a n o t h e r N e w t o n i a n u n i t o r u n i t s . T h u s , f o r f l o w o f n o n - N e w t o n i a n u n i t s , t h i s bond ( o r e n t a n g l e ment) must be b r o k e n ( o r d i s e n t a n g l e d ) . Based on t h i s c o n c e p t , t h e g e n e r a l i z e d v i s c o s i t y e q u a t i o n i s χ . β. Σ , -^-1 i

η =

1

sinh"

β.s

1

,

Γ4]

β^

a

where χ · i s t h e f r a c t i o n a l a r e a o c c u p i e d by t h e i t h f l o w u n i t on t h e s h e a r s u r f a c e , and η

cu = (λ X X 3 ) . / 2 k T

;

[5]

β. = [(A/X )2k ]T

;

[6]

2

l

Ί

1

1

a., and β. a r e t h e c h a r a c t e r i s t i c s h e a r volume d i v i d e d by kT w h i c h i s r e l a t e d t o t h e i n v e r s e o f t h e s h e a r modulus and t h e r e l a x a t i o n t i m e , r e s p e c t i v e l y , β./α· i s t h e N e w t o n i a n v i s c o s i t y . λ is

the jumping d i s t a n c e ,

λ ι , λ , and λ 2

3

are the m o l e c u l a r

d i m e n s i o n s o f a f l o w u n i t , k' i s t h e j u m p i n g f r e q u e n c y o f t h e f l o w u n i t when t h e r e i s no s t r e s s . According to the theory of rate processes (16): k

'

=

ΊΪ

IT

e

x

p

<- o e

/ k T )

»

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

[

7

]

9.

M A ET AL.

Polymerization of Hydrophilic Methacrylate Monomers

123

where h i s P l a n c k ' s c o n s t a n t ; Gt and G a r e t h e p a r t i t i o n f u n c t i o n s o f t h e a c t i v a t e d and t h e i n i t i a l s t a t e , r e s p e c t i v e l y ; and ε is the a c t i v a t i o n energy. C o n s i d e r i n g t h e HEMA-HoO m i x t u r e , one can assume t h e p r e s e n c e o f two t y p e s o f f l o w u n i t s , t h e N e w t o n i a n t y p e ( u n s t r u c t u r e d w a t e r , HEMA monomer, and t h e l o w e r members o f HEMA p o l y m e r s \ and t h e n o n - N e w t o n i a n t y p e ( h i g h e r members o f PHEMA w i t h bonded water). For these c o n s i d e r a t i o n s , Equation [4] takes the form "

η n

γ η V s

+ +

ΐΕΐ ~^Γ Χ

+ 2&2 ~i

Here, n i s the s o l v e n t v i s c o s i t y . v e r y s m a l l , one has s

o

n

Χ

SJnh" g S Bis 1

2

·

When t h e r a t e o f s h e a r

[8] is

V

T h i s t h e o r y makes no a s s u m p t i o n s as t o t h e s p e c i f i c m o l e c u l a r conformations or c o n f i g u r a t i o n s to the flow u n i t s . Also, i t r e c o g n i z e s t h e d i s c r e t e boundary between f l o w u n i t s and medium and between s e p a r a t e f l o w u n i t s . To o b t a i n a r e l a t i o n s h i p between t h e r e l a x a t i o n t i m e and m o l e c u l a r w e i g h t ( 1 7 ) , e q u a t i o n [ 6 ] can be r e w r i t t e n as

£ „ | . η. β-Αψκτ k

,

[10]

where A F . i s t h e s t a n d a r d f r e e e n e r g y o f a c t i v a t i o n p e r mole o f ith flow unit. F o r a c o n d e n s e d phase t h i s f r e e e n e r g y a p p r o x i mately equals the Helmholtz f r e e energy which i s r e l a t e d t o the p a r t i t i o n f u n c t i o n , f . , by A. = - k T £n f j . Introducing

equation [11] l «

T

i n t o [ 1 0 ] one

[11] has

i '

[12]

i A c c o r d i n g t o Ma, Jhon and E r y i n g ( 1 8 ) , t h e p a r t i t i o n f u n c t i o n f o r l i n e a r p o l y m e r s can be s e p a r a t e d i n t o two p a r t s , a p a r t d e p e n d e n t on c h a i n e n t a n g l e m e n t and a p a r t i n d e p e n d e n t o f c h a i n entanglement. The f i r s t p a r t can be w r i t t e n as ,Mx 4/3 m\

K

l

,Mx2 m£ ·

Here M, mi and m a r e t h e m o l e c u l a r w e i g h t s o f t h e p o l y m e r , t h e k i n e t i c segment and t h e t a n g l i n g segment, r e s p e c t i v e l y . In c o n c e n t r a t e d s o l u t i o n s and i n p u r e p o l y m e r i c s y s t e m s , 2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

124

HYDROGELS

FOR MEDICAL

A N D RELATED APPLICATIONS

k i n e t i c segments can move i n t o n e i g h b o r i n g v a c a n c i e s i n d e p e n d e n t l y except f o r the r e s t r i c t i o n t h a t they remain t i e d t o g e t h e r w h i l e a l l t a n g l i n g segments e x c e p t one a r e o b l i g e d t o f o l l o w around the randomly l o c a t e d t a n g l i n g p o i n t s . We e x p e c t t h a t f l o w u n i t s , w h i c h c o n t a i n m o l e c u l e s w i t h H > m , are mostly non-Newtonian i n b e h a v i o r . F o r two s y s t e m s w i t h t h e same m o l e c u l a r w e i g h t i t f o l l o w s t h a t 2

(62)1

(f ) 2

(m )î

2

(Γ )?

2

=

2

=

(62)2

=

.

Ml

(f )i 2

[13]

(Â )i 2

The s u b s c r i p t 2 i n s i d e t h e p a r e n t h e s e s r e f e r s t o t h e n o n N e w t o n i a n f l o w u n i t s and t h subcript 1 d 2 outsid th parentheses r e f e r to system [13] i n d i c a t e s t h a t the average l e n g t h or the m o l e c u l a r w e i g h t o f t h e t a n g l i n g segments i s p r o p o r t i o n a l t o t h e s q u a r e r o o t o f t h e r e l a x a t i o n t i m e o f t h e f l o w u n i t s w h i c h c o n t a i n s u c h segments. T h i s w o u l d a p p r o x i m a t e l y be t h e c a s e when t h e same d e a r e e o f p o l y m e r i z a t i o n was r e a c h e d f r o m d i f f e r e n t i n i t i a l HEMA-water mixtures. On t h e o t h e r hand we can w r i t e (19) Bp = 6

S

(M/m ) (M/rn^ 2

1

/

3

and

^s a

p

(MM,)

[14] (M/m ) 2

where t h e s u b s c r i p t s ρ and s r e f e r t o p r o p e r t i e s f o r t h e p o l y m e r and u n a t t a c h e d k i n e t i c s e g m e n t s , r e s p e c t i v e l y . I f the degree of p o l y m e r i z a t i o n i s s u f f i c i e n t l y l a r g e , i . e . , i f M > m , the v a l u e o f nu , m s h o u l d n o t be a f f e c t e d by f u r t h e r p o l y m e r i z a tion. It follows that 2

2

(0p)ti

where t i and t

2

(M

V t ,

" '

V t .

.

•

)

t

(M t

' ) V

t

2

r e f e r t o two d i f f e r e n t p o l y m e r i z a t i o n

times.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

M A ET AL.

9.

Results

Polymerization

of Hydrophilic

Methacrylate

Monomers

125

and D i s c u s s i o n

The v i s c o s i t y o f HEMA-water m i x t u r e s a t 60°C i s p l o t t e d against t h e r a t e o f shear a t various p o l y m e r i z a t i o n times f o r f i v e i n i t i a t o r s (Figure 1 ) . A c c o r d i n g t o Nakajima ( 2 0 ) , t h e s t e a d y - s t a t e f l o w c u r v e s f o r e i g h t p o l y e t h y l e n e s a m p l e s have a s i m i l a r shape t o t h e i r c o r r e s p o n d i n g c u m u l a t i v e f r a c t i o n a t i o n curves. He assumed t h a t EWi be t h e c u m u l a t i v e w e i g h t f r a c t i o n and B. be t h e r e l a t i v e c h a i n l e n g t h a t EWi = H ( a f r a c t i o n a l number) and c o n c l u d e d t h a t n/n

o

= H = EWi

s

= KB.

[16]

and H

[17]

W i t h t h r e e a d j u s t a b l e p a r a m e t e r s , n o , K, and a , he showed t h a t w i t h h i s d a t a t h e f l o w c u r v e and f r a c t i o n a t i o n c u r v e a r e e q u i v a l e n t f o r most o f t h e r a n g e . The amount o f c r o s s l i n k e r ( d i e s t e r ) p r e s e n t i n o u r system i s l e s s than 0.01%. Equations [ 1 6 ] and [ 1 7 ] , which apply s a t i s f a c t o r i l y t o l i n e a r polymers, a r e probably a l s o a p p l i c a b l e t o t h e c u r r e n t system. To p r o v e t h i s , a f r a c t i o n a t i o n c u r v e f o r PHEMA has t o be o b t a i n e d i n d e p e n d e n t l y through o t h e r measurements. Note t h a t i n t h e p o l y m e r i z a t i o n s y s t e m employed h e r e i n t h a t a d i e s t e r c o n c e n t r a t i o n i n e x c e s s o f 0 . 0 3 5 % w i l l r e s u l t i n an i n s o l u b l e , c r o s s l i n k e d p o l y m e r . F l o w p a r a m e t e r s were c a l c u l a t e d f r o m t h e B u e c h e - H a r d i n g s t a n d a r d c u r v e , from E q u a t i o n [ 8 ] and from B a r t e n e v ' s e m p i r i c a l e q u a t i o n , t h e l a t t e r t a k i n g t h e form log n/n

0

= -K

1

T

w

,

[18]

where τ

i s t h e shear s t r e s s , w The B u e c h e - H a r d i n g s t a n d a r d c u r v e r e p r e s e n t s c o m p o s i t e v i s c o s i t y data f o r p o l y d i s p e r s e polymer systems. Our data can be s u p e r i m p o s e d o n t o t h i s s t a n d a r d c u r v e a n d f o r m a s i n g l e master curve. F i g u r e 2 i s an e x a m p l e . The v a l u e s o f τ ^ , s a n d η o b t a i n e d h e r e a f t e r a r e g i v e n i n T a b l e I. The p o l y m e r c o n c e n t r a t i o n i n m o l e / l i t e r i s c a l c u l a t e d from Equation [ 3 ] . Because o f t h e l a c k o f d a t a c o n c e r n i n g t h e v a l u e o f c a s a function of polymerization time, theabsolute values o f molecular w e i g h t a r e n o t computed. However, two p o i n t s c a n be c o n c l u d e d from o u r d a t a : 1) o u r e x p e r i m e n t a l c u r v e s c o r r e l a t e w e l l w i t h t h e Bueche-Harding standard c u r v e , i n d i c a t i n g polymers p r o d u c e d d u r i n g t h e c o u r s e o f measurement a r e p r o b a b l y a l l l i n e a r p o l y m e r s ; a n d 2 ) a s s u m i n g h a l f o f t h e i n i t i a l HEMA monomer ( 0 . 7 g / c c ) was c o n v e r t e d i n t o p o l y m e r s a t t h e end o f o u r m e a s u r e ment, t h e v i s c o s i t y a v e r a g e m o l e c u l a r w e i g h t r e a c h e d a v a l u e o f e

0

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

ΙΟ

1

ΙΟ

ιο

2

3

s, SHEAR RATE, seer

1

Figure la. Flow curves of HEMA-water mixtures at 60°C. Initiator: ammonium persulfate.

Figure lb.

Flow curves of HEMA-water 60°C. Initiator: AIBN.

mixtures at

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

M A E T AL.

Polymerization

of Hydrophilic

Methacrylate

Monomers

Figure Id. Flow curves of HEMA-water mixtures at 60°C. Initiator: azobis (methoxyethyl isobutyrate).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

127

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

1

Γ ι

ι 11 1 1

j

1 — Γ Τ "

600 sec. 900 sec. ο 1030 sec. ο 1100 sec. α Δ

—

—

10

I 1 1 1 10"

I

I

1 111 1 1 10'

Log ( x s )

Figure 2. A superimposed plot of experimental data and Bueche-Harding standard curve. Initiator: ammonium per sulfate.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

Polymerization of Hydrophilic Methacrylate Monomers

MA ET AL.

TABLE

I.

The E f f e c t o f P o l y m e r i z a t i o n T i m e , t , on t h e F l o w P a r a m e t e r s ( B u e c h e - H a r d i n g Method) f o r HEMA-water M i x t u r e s Solvent:

Water (η

Concentration:

t (sec) (1)

600 1030 1100 (2)

1100 1515 1900 2300 (3)

(4)

Initiator: 930 1300 1600

(5)

1100 1500 1900

60°C)

water

(poise)

1000 C/M (mole/liter)

persulfat

27.5 4.8 3.1

10 -5 10 -5 10

1.57 4.46 4.9

13.03 213.0 357.3

53.7 3.5 1.7 0.9

34.7 2.1 0.96 0.72

3.0 44.67 134.9 281.8

26.3 2.2 0.91

4.92 91.2 496.6 954.0

27.5 3.0 0.53

X X X X

10 10 10' 10'

10.6 151.4 626.0

7.2 97.7 758.5

7.46 8.26 2.08 3.03

X

6 10 -6 10 -5 10 5 10

χ χ χ

10"! lO ? ΙΟ

X X X

isobutyrate)

Azobis(methoxydiethoxyethyl 0.0364 0.333 1.887

7.07 6.8 1.03 1.1

isobutyrate)

Azobis(methoxyethyl 0.038 0.455 1.099

Initiator:

•1. )

Azobis(methyl 0.0288 0.476 1.042 1.389

900 1300 1700 1900

poise at

Azobisisobutyronitrile

.0186 .286 .588 1.111

Initiator:

(sec

Ammoniu .0364 .208 .323

Initiator:

3

6 p a r t s HEMA/3 p a r t s

(sec)

Initiator:

= 4.665 χ 1 0 "

1.22 1.46 2.51

-

- 3

isobutyrate) 8.77 1.29 1.75

χ χ χ

10~î 10"? 1θ"°

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

130

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TABLE

II.

The E f f e c t o f P o l y m e r i z a t i o n T i m e , t , on t h e F l o w P a r a m e t e r s ( E y r i n g ' s T h e o r y ) f o r HEMA-Water M i x t u r e s Solvent:

W a t e r (η

Concentration: t

(sec) (1) 600 900 1030 1100 (2) 900 1100 1300 1515 1700 1900 2100 2300 (3) 800 900 1100 1300 1500 1700 1900

ν

= 4.665 χ 1 0 "

3

p o i s e a t 60°C)

6 p a r t s HEMA/3 p a r t s

+

n

Χ

ι

β

o s

ι

αι

n

Initiator:

(sec)

1.2 4.66 6.21 7.37

0.077 0.124 0.213 0.294

2

Correlation

2

Coefficient

η

ο

(poise)

x χ χ χ

10p 10, 10, 10

0.981 0.999 1.000 1.000

10.56 60.91 141.08 230.24

0.96 0.996 0.999 1.000 0.998 1.000 1.000 1.000

0.46 2.24 8.03 22.69 47.89 77.4 159.95 212.15

0.912 0.999 0.984 1.000 1.000 1.000 1.000

1.30 3.21 11.04 41.93 113.96 308.38 871.8

Azobisisobutyronitrile

0.09 0.28 0.55 0.64 0.86 0.999 0.42 1.28 Initiator:

Bz

(dyne/cm )

Ammoniu

1.31 3.14 8.92 13.34 Initiator:

X2/012

water

5.0 6.8 9.53 1.46 1.68 2.19 2.12 2.05

χ χ χ χ χ χ χ χ

Azobis(methyl

.13 .28 1.33 1.06 1.70 3.54 15.13

ίο] 1θ{ 10, 10, 10, 10, 10, 10^

0.0074 0.0288 0.0785 0.151 0.280 0.340 0.754 1.03

isobutyrate)

1.58 χ 10^ 1.02 χ 1 0 , 1.84x10, 1.91 χ 1 0 , 2.94 χ 1 0 , 4.14 χ 10, 4 . 4 0 χ 10^

0.0074 0.0287 0.0528 0.214 0.381 0.736 1.755

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

M A ET AL.

Table

t (sec) (4) 600 930 1100 1300 1400 1515 1600 (5) 900 1100 1300 1500 1700 1900

II.

Polymerization of Hydrophilic Methacrylate Monomers

131

(cont.)

+

W Initiator:

XiEi ai

2

62 (sec)

Azobiz(methoxyethyl

0.32 1.48 0.55 1.13 1.54 4.65 9.50 Initiator:

X2/122 (dyne/cm )

2.28 χ ίο] 5.57 χ 1 0 , 2.43x10, 3.09 χ 1 0 , 3.8 4.23x10 4 . 3 3 χ 10

1.05 χ 10? 1.25 χ 1 0 , 1.49x10, 2.71 χ 1 0 , 3.09 χ 1 0 , 3.42 χ 10^

2

0 (poise)

isobutyrate) 0.0675 0.216 0.0878 0.258

0.94 0.990 1.000 1.000

1.86 13.51 21.90 80.72

1.002

1.000

442.99

Azobis(methoxydiethoxyethyl

0.043 0.28 1.02 1.04 1.71 4.72

Correlation Coefficient

0.01 0.0346 0.140 0.179 0.526 1.44

isobutyrate) 1.000 0.997 1.000 0.997 1.000 1.000

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1.096 4.62 21.87 49.62 164.15 497.32

132

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TABLE

III.

The E f f e c t o f P o l y m e r i z a t i o n T i m e , t , on t h e Flow P a r a m e t e r s ( B a r t e n e v ' s M e t h o d ) f o r HEMA-Water M i x t u r e s Solvent:

Water (η

Concentration: t (sec) (1)

Initiator: 600 900 1030 1100

(2)

Initiator: 900 1100 1300 1515 1700 1900 2100 2300

(3)

Initiator: 800 900 1100 1300 1500 1700 1900

(4)

Initiator: 600 930 1100 1300 1400 1515 1600

= 4.665 χ Ι Ο

- 3

p o i s e a t 60°C)

6 p a r t s HEMA/3 p a r t s w a t e r , K'

χ 10

o (poise) n

Correlation Coefficient

Ammonium p e r s u l f a t e 0.52 0.32 0.214 0.216

138.38 299.77

0.957 0.999

Azobisisobutyronitrile 1.64 1.47 1.22 1.17 1.14 1.06 1.40 1.30

0.518 2.47 8.51 27.98 62.68 126.34 377.28 419.89

0.945 0.978 0.968 0.986 0.993 1.000 0.997 0.994

Azobis(methy1 i s o b u t y r a t e ) 1.53 1.31 0.581 1.03 0.816 0.596 0.394

1.73 3.94 13.48 59.74 200.34 556.69 1046.6

Azobis(methoxyethyl 1.07 0.917 0.778 0.755 0.629 0.510 0.473

1.000 0.998 0.913 0.999 0.996 0.999 0.989

isobutyrate)

0.99 9.37 29.25 133.75 222.29 389.16 633.97

0.771 0.938 0.986 0.998 0.999 0.995 0.994

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

MA ET AL.

e III.

Polymerization

of Hydrophilic

Methacrylate

Monomers

(cont.) t (sec)

Initiator: 900 1100 1300 1500 1700 1900

3 K' χ 10

0

(poise)

Azobis(methoxydiethoxyethyl 1.64 1.18 0.877 0.812 0.804 0.72

1.47 5.67 22.67 78.49 297.38 876.56

Correlation Coefficient isobutyrate) 0.998 0.977 0.979 0.489 0.999 0.999

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

134

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

the order of TO . W i t h s u c h a h i g h m o l e c u l a r w e i g h t one w o u l d e x p e c t t h e v i s c o s i t y o f t h e s y s t e m t o be much h i g h e r . However, s i n c e w a t e r i s a p o o r s o l v e n t f o r PHEMA, a l a r g e p o r t i o n o f t h e p o l y m e r m o l e c u l e m i g h t r e m a i n u n e n t a n g l e d a n d , as a c o n s e quence, r e s u l t i n a lower v i s c o s i t y . The p a r a m e t e r s x n + χ ι $ ι / α ι » x / a and β a l l i n c r e a s e w i t h an i n c r e a s e i n p o l y m e r i z a t i o n t i m e ( T a b l e I I ) a t a g i v e n temperature (60°C). The q u a n t i t y x n + x i B i / α ι i n c r e a s e s w i t h i n c r e a s i n g p o l y m e r i z a t i o n time because X i i s c o n c e n t r a t i o n dep endent. The q u a n t i t y x s h o u l d i n c r e a s e w i t h b o t h an i n c r e a s e i n p o l y m e r c o n c e n t r a t i o n and i n c r e a s e d m o l e c u l a r w e i g h t . Thus, t h e i n c r e a s e o f x / a w i t h p o l y m e r i z a t i o n t i m e seems t o be natural since a i s a c h a r a c t e r i s t i c p r o p e r t y f o r f l o w u n i t 2. The r e l a x a t i o n t i m e , β , i s a q u a n t i t y w h i c h i s i n d e p e n d e n t o f c o n c e n t r a t i o n a t low c o n c e n t r a t i o n t r a t i o n at high c o n c e n t r a t i o n s c r e a s e s w i t h p o l y m e r i z a t i o n t i m e . The e a r l y i n c r e a s e i s p r o b a b l y due t o an i n c r e a s e i n m o l e c u l a r w e i g h t a l o n e , w h i l e t h e l a t e r i n c r e a s e i s due t o changes i n c o n c e n t r a t i o n and i n molecular weight. O r i g i n a l l y , we a p p l i e d a method d e v e l o p e d by G a b r y s h e t a l . (21) t o d e t e r m i n e t h e s e p a r a m e t e r s . However, we were u n a b l e t o d e t e r m i n e x n + X i 3 i / c t i m e a n i n g f u l by s u c h a method. The p a r a m e t e r s g i v e n i n T a b l e 11 were d e t e r m i n e d by using a computer program to o b t a i n a best s t r a i g h t l i n e f i t f o r a η vs. s i n h " B s / B s p l o t . The s q u a r e o f t h e c o r r e l a t i o n c o e f f i c i e n t f o r s u c h a f i t i s g r e a t e r t h a n 0 . 9 8 w i t h a few e x c e p t i o n s , p r e s u m a b l y due t o g r e a t e r e x p e r i m e n t a l e r r o r s when s o l u t i o n s are d i l u t e . 7

0

s

2

0

2

2

s

2

2

2

2

0

s

β

1

2

2

In B a r t e n e v ' s e x p r e s s i o n K i n c r e a s e s w i t h i n c r e a s i n g b r e a d t h o f m o l e c u l a r w e i g h t d i s t r i b u t i o n and η i n c r e a s e s w i t h increasing molecular weight. T a b l e I I I shows t h a t t h e c o r r e l a t i o n between o u r d a t a and B a r t e n e v ' s e x p r e s s i o n i s n o t v e r y s a t i s f a c t o r y ( t e n c u r v e s have t h e s q u a r e o f c o r r e l a t i o n c o e f f i c i e n t l e s s than 0 . 9 8 ) . However, t h e changes i n K and η w i t h p o l y m e r i z a t i o n time f o l l o w s a reasonable t r e n d . Bueche-Harding's η i s g r e a t e r than E y r i n g ' s . The r a t i o between them i s r a n g i n g f o r m 1.23 t o 2.18 ( w i t h one e x c e p t i o n ) . Bartenev's η s t a r t e d with a value close to that of E y r i n g ' s , i n c r e a s e d s t e a d i l y and f i n a l l y p a s s e d b o t h Bueche-Harding's and E y r i n g ' s v a l u e . B u e c h e ' s r e l a x a t i o n t i m e , x , and E y r i n g ' s r e l a x a t i o n t i m e , β , a r e o f t h e same o r d e r o f m a g n i t u d e , p r e sumably b o t h and β measure t h e m o l e c u l a r t i m e c o n s t a n t c o n t r o l l i n g non-Newtonian b e h a v i o r . From E q u a t i o n [ 3 ] , one o b t a i n s 1

0

1

0

0

0

D

2

2

T

b

~

Ό

M 00

[19]

CT

In a c o n c e n t r a t e d p o l y m e r s o l u t i o n , we can assume β

2

= β , and

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

MA ET AL.

no = 3 / α . $

$

Polymerization of Hydrophilic Methacrylate Monomers

Then, from e q u a t i o n

135

[14],

no Comparing

Equation g

2

[19] w i t h Equation » cx M b

1 / 3

[20],

i t follows

that

.

[21]

E x p e r i m e n t a l l y t h e m o l e c u l a r w e i g h t e x p o n e n t i s l e s s t h a n one and t h e c o n c e n t r a t i o n e x p o n e n t i s g r e a t e r t h a n one [ 2 2 ] . Graess l e y , e t a l . [T3] suggested t h a t the c h a r a c t e r i s t i c r e l a x a t i o n t i m e be p r o p o r t i o n a l t o M/cT a t low e n t a n g l e m e n t d e n s i t y and proportional to l / c T a t h a t the entanglement d e n s i t Hence, b o t h τ. and 32 s h o u l d g o v e r n . Figure 3 gives the p l o t of E y r i n g ' s zero shear v i s c o s i t y a g a i n s t t h e p o l y m e r i z a t i o n t i m e . The r e l a t i o n s h i p between I n no and In t i s l i n e a r f o r s a m p l e s 2 , 3 and 5 and can be f i t t e d w i t h two s t r a i g h t l i n e s f o r sample 4. Because t h e r e a r e n o t enough d a t a p o i n t s , no s u c h f i t t i n g was a t t e m p t e d f o r sample 1. A t t h e same l e n g t h o f p o l y m e r i z a t i o n t i m e , we can c o n c l u d e that: (a) t h e p o l y m e r i z a t i o n i n i t i a t e d by ammonium p e r s u l f a t e i s t h e f a s t e s t ; and (b) t h e p o l y m e r i z a t i o n s i n i t i a t e d by t h e t h r e e AIBN d e r i v a t i v e s have c o m p a r a b l e r a t e s w h i c h a r e s l i g h t l y f a s t e r t h a n t h e r a t e i n d u c e d by AIBN i t s e l f . Similar conclusions can be drawn by u s i n g B u e c h e - H a r d i n g s o r B a r t e n e v ' s z e r o s h e a r viscosity. 2

1

Conclusions From v i s c o m e t r y d a t a we can c o n c l u d e t h e f o l l o w i n g : PHEMA p r o d u c e d f r o m HEMA-water m i x t u r e s w i t h l e s s than 0.01% c r o s s l i n k e r i s mostly l i n e a r polymer. (2) The r e l a t i v e r a t e o f p o l y m e r i z a t i o n i n i t i a t e d w i t h t h e various i n i t i a t o r s are of the f o l l o w i n g order: ammonium s u l f a t e » AIBN d e r i v a t i v e s > A I B N ; (3) Both R e e - E y r i n g ' s t h e o r y o f n o n - N e w t o n i a n f l o w and B u e c h e H a r d i n g ' s method can d e s c r i b e t h e b e h a v i o r o f HEMA-water mixtures d u r i n g the course of p o l y m e r i z a t i o n . Bartenev's e m p i r i c a l e x p r e s s i o n works l e s s w e l l presumably because t h e r a t i o n/no f o r o u r s y s t e m i s s t i l l some f u n c t i o n o f m o l e c u l a r w e i g h t and c a n n o t be assumed t o depend on s h e a r s t r e s s alone; (4) The m o l e c u l a r w e i g h t o f l i n e a r PHEMA can be o b t a i n e d by u s i n g t h e B u e c h e - H a r d i n g method w i t h i n d e p e n d e n t i n f o r mation concerning c o n c e n t r a t i o n of the polymer. However, we have n o t c a r r i e d o u t c o n c e n t r a t i o n measurements i n t h i s

(1)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

136

HYDROGELS

FOR

MEDICAL

AND RELATED

APPLICATIONS

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9. M A E T AL.

Polymerization

of Hydrophilic

Methacrylate

Monomers

137

s t u d y and o n l y t h e r a t i o VM i s e s t i m a t e d . The r e l a t i v e m o l e c u l a r w e i g h t a t d i f f e r e n t p o l y m e r i z a t i o n t i m e s c a n be obtained from E y r i n g ' s r e l a x a t i o n t i m e ; (5) Nakagima's theory (20) c o r r e l a t e s t h e f l o w curve w i t h t h e cumulative molecular weight d i s t r i b u t i o n curve o f l i n e a r polymers. One c a n e x p l o r e t h i s t h e o r y f u r t h e r and s e e whether i t i s a p p l i c a b l e t o o u r systems. The work p r e s e n t e d h e r e i s o n l y a p r e l i m i n a r y s t u d y . The d a t a o b t a i n e d w i t h a Haake R o t o v i s c o v i s c o m e t e r a r e n o t a s u f ficient test. To f u r t h e r t e s t t h e a p p l i c a b i l i t y o f t h e t h e o r i e s requires that: ( 1 ) e x p e r i m e n t a l d a t a be o b t a i n e d o v e r a w i d e range o f r a t e s o f s h e a r , a t s e v e r a l d i f f e r e n t t e m p e r a t u r e s and u s i n g v a r i o u s s o l v e n t s ; ( 2 ) c o n c e n t r a t i o n and p o l y d i s p e r s i t y be d e t e r m i n e d s i d e by s i d e w i t h v i s c o m e t r i c measurement s o t h a t r e s u l t s f r o m t h e s e measurement

Abstract Flow curves for hydroxyethyl methacrylate-water mixtures were determined as functions of polymerization time and initia tor. The non-Newtonian behavior of these systems was analyzed by a Bueche-Harding standard curve, by the Ree-Eyring generalized viscosity equation and by Bartenev's empirical equation. The relative rate of polymerization initiated with AIBN and various AIBN esters and the relationships between flow curves, molecular weight, molecular weight distribution and polymerization time are discussed. Literature Cited 1. Janacek, J., J. Macromol. Sci.-Revs. Macromol. Chem. (1973) C9 (1) 1-47. 2. Rudd, J. F., J. Poly. Sci. (1960) 44, 459. 3. Sabia, R., J. Appl. Poly. Sci. (1964) 8, 1053. 4. Graessley, W. W., and Prentice, J. S., J. Poly. Sci., A-2 (1968) 6, 1887. 5. Bueche, F., and Harding, S. W., J. Poly. Sci. (1958) 32, 177. 6. Ree, T., Eyring, H., J. Appl. Phys. (1955) 26, 793, 800. 7. Bartenev, G. Μ., Vysokomolekyluarnye Soedineniya (1964) 6, 2155. 8. Gregonis, D., Chen, C. M., and Andrade, J. D., this symposium. 9. Bueche, F., F. J. Chem. Phys. (1954) 22, 1570. 10. Graessley, W. W., J. Chem. Phys. (1965) 43, 2696. 11. Rouse, P. E., J. Chem. Phys. (1953) 21, 1272. 12. Ballman, R. L., and Simon, R. M., J. Poly. Sci. A-2 (1964) 2, 3557. 13. Graessley, W. W., Hazleton, R. L., and Lindeman, L. R., Trans. Soc. Rheology, (1967) 11, 267.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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14. Shih, C. K., Tran. Soc. Rheology (1970) 14, 83. 15. Graessley, W. W., J. Chem. Phys. (1967) 47, 1942. 16. Glasstone, S., Laidler, K., and Eyring, Η., "The Theory of Rate Processes," p. 483, McGraw Hill Book Company, New York, 1941. 17. Jhon, M. S., and Eyring, H., private communication. 18. Ma, S. M., Eyring, H., and Jhon, M. S., Proc. Natl. Acad. Sci. (USA) (1974) 71, 3096. 19. Eyring, H., Ree, T., and Hirai, N., Proc. Natl. Acad. Sci. (USA) (1958) 44, 1213. 20. Nakajima, Ν., Proc. 5th Intl. Cong. on Rheology, Ed. by Shigeharu Onogi, (1968) 4, 295. 21. Gabrysh, A. F., Eyring, H., Shimizu, M., and Asay, J., J. Appl. Phys. (1963) 34 261 22. DeWitt, T. W., Markovitz J., J. Colloid Sci

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10 The Effect of Tacticity on the Thermal Behavior of Various Poly(methacrylate esters) G. A. RUSSELL, D. E. GREGONIS, A. A. DEVISSER, and J. D. ANDRADE Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 D. K. DALLING Department of Chemistry, University of Utah, Salt Lake City, Utah 84112 T a c t i c i t y , t h e s t e r e o c h e m i c a l placement o f pendant s i d e chains along t h e polyme important f a c t o r i n determinin Such p r o p e r t i e s as c r y s t a l l i z a b i l i t y , s o l u b i l i t y , m e l t i n g p o i n t , g l a s s t r a n s i t i o n t e m p e r a t u r e , e t c . have been c o r r e l a t e d w i t h t h e t a c t i c i t y o f a g i v e n polymer ( 1 , 2 , 3 , 4 ) . Introduction of ZieglerN a t t a c a t a l y s t s and o t h e r c a t a l y s t s y s t e m s c a p a b l e o f c o n t r o l l i n g t a c t i c i t y d u r i n g s y n t h e s i s has opened up new a r e a s f o r c o m m e n ç a i d e v e l o p m e n t based on s y s t e m a t i c v a r i a t i o n o f m o l e c u l a r c o n f i g u r a tion. L i t t l e work has been r e p o r t e d , h o w e v e r , c o r r e l a t i n g c h a i n t a c t i c i t y t o t h e s u r f a c e and i n t e r f a c i a l p r o p e r t i e s o f p o l y m e r s . R e c e n t work ( 5 j s u g g e s t s t h a t r e o r i e n t a t i o n o f t h e c h a i n s i n t h e s u r f a c e zone o f t h e p o l y m e r c a n a f f e c t w e t t a b i l i t y o f t h e s u r f a c e If t h a t i s indeed t h e case, t h e t a c t i c i t y o f t h e polymer chains may w e l l have an i n f l u e n c e on w e t t a b i l i t y , s i n c e t h e b a r r i e r s t o chain r o t a t i o n are a function of t a c t i c i t y . Also, the a v a i l a b i l i t y o f h y d r o p h i l i c and h y d r o p h o b i c s i t e s f o r i n t e r a c t i o n a t t h e i n t e r f a c e i s i n f l u e n c e d by t h e c h a i n c o n f i g u r a t i o n and c o n formation. C o n s e q u e n t l y , i t was f e l t t h a t a s t u d y o f t h e i n f l u e n c e o f t a c t i c i t y on b u l k p h y s i c a l and i n t e r f a c i a l p r o o e r t i e s i n t h e p o l y ( h y d r o x y e t h y l m e t h a c r y l a t e ) (pHEMA)/water s y s t e m m i g h t i n d i c a t e some means o f a l t e r i n g t h e i n t e r f a c i a l p r o p e r t i e s o f t h e p o l y m e r by c o n t r o l o f i t s t a c t i c i t y d u r i n g s y n t h e s i s . In t h i s p a p e r we r e p o r t p r e l i m i n a r y r e s u l t s o f some e x p e r i m e n t s i n w h i c h v a r i o u s methods o f a l t e r i n g t a c t i c i t y d u r i n g s y n t h e s i s have been examined and t h e i r e f f e c t c o r r e l a t e d w i t h changes i n t h e thermal p r o p e r t i e s of t h e r e s u l t i n g polymers. S y n t h e s i s o f m e t h y l and o t h e r a l k y ! m e t h a c r y l a t e s o f h i g h s t e r e o r e g u l a r i t y , e i t h e r o f high i s o t a c t i c content o r high s y n d i o t a c t i c c o n t e n t , i s w e l l - k n o w n (6_). U n f o r t u n a t e l y , t h e organomet a l l i c i n i t i a t o r s used f o r p r o d u c t i o n o f s t e r e o r e g u l a r m e t h a c r y l ates are highly r e a c t i v e t o the hydroxyl f u n c t i o n a l i t y i n the s i d e c h a i n o f HEMA. C o n s e q u e n t l y , some means o f b l o c k i n q t h e h y d r o x y l g r o u p d u r i n g p o l y m e r i z a t i o n was r e q u i r e d . Methoxyethyl m e t h a c r y l a t e (MEMA) was c h o s e n as a model f o r a H E M A - l i k e monomer

139

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w i t h i t s h y d r o x y l g r o u p p r o t e c t e d , even t h o u g h t h e m e t h y l e t h e r l i n k a g e i s t o o s t a b l e t o p e r m i t h y d r o l y s i s back t o pHEMA. A s e c o n d p r o t e c t e d HEMA d e r i v a t i v e used was t r i m e t h y l s i l y l e t h y l m e t h a c r y l a t e (TMSEMA), whose t r i m e t h y l s i l y l p r o t e c t i o n g r o u p can be c l e a v e d r e a d i l y , f o r m i n g HEMA. T h i s a l l o w s p o l y m e r i z a t i o n o f TMSEMA u s i n g an a n i o n i c c a t a l y s t t o p r o d u c e a s t e r e o r e g u l a r pTMSEMA, f o l l o w e d by h y d r o l y s i s t o s t e r e o r e g u l a r pHEMA. P o l y m e r s o f HEMA, MEMA, and TMSEMA were s y n t h e s i z e d by a v a r i e t y o f t e c h n i q u e s , t h e i r t a c t i c i t y was d e t e r m i n e d by C - N M R s p e c t r o m e t r y , and t h e v a r i a t i o n i n t a c t i c i t y was c o r r e l a t e d w i t h changes i n t h e DSC thermograms o f e a c h p o l y m e r . 13

Experimental Monomer P r e p a r a t i o n A l l d i thi stud exceDt TMSEMA were o b t a i n e d c o m m e r c i a l l y methoxyethyl methacrylat (MEMA) supplie y c a l C o . ; 2 - h y d r o x y e t h y l m e t h a c r y l a t e (HEMA) was s u p p l i e d by Hydro-Med S c i e n c e s . TMSEMA was s y n t h e s i z e d i n o u r l a b o r a t o r y by a p r o c e d u r e t o be d e s c r i b e d e l s e w h e r e (7). The h y d r o a u i n o n e i n h i b i t o r was removed f r o m t h e c o m m e r c i a l monomers by e x t r a c t i o n w i t h aqueous NaOH. The monomer was t h e n d r i e d o v e r MgSOi* and d i s t i l l e d from L i A l a n d CuCl. The d i s t i l l e d p r o d u c t was t h e n s t o r e d a t 4°C o v e r 5A m o l e c u l a r s i e v e u n t i l u s e d . The s t r u c t u r e s o f t h e monomers and a b b r e v i a t i o n s used a r e shown i n F i g u r e 1. The p o l y m e r i z a t i o n c o n d i t i o n s used a r e summarized i n T a b l e I. Anionic Polymerization. MMA, MEMA and TMSEMA were each p o l y m e r i z e d a n i o n i c a l l y i n dry t o l u e n e a t -78 C u s i n g η - b u t y l l i t h i u m as i n i t i a t o r . These c o n d i t i o n s had been shown p r e v i o u s l y t o p r o d u c e pMMA and o t h e r a l k y ! m e t h a c r y l a t e s c o n t a i n ing a high percentage of i s o t a c t i c t r i a d s (8). In a l l c a s e s t h e r e a c t i o n was t e r m i n a t e d by a d d i t i o n o f m e t h a n o l , and t h e p o l y m e r p r e c i p i t a t e d by a n o n - s o l v e n t , p e t r o l e u m e t h e r f o r pMMA and pMEMA, w a t e r f o r pTMSEMA. The c r u d e p r o d u c t s were t h e n r e d i s s o l v e d i n benzene (pW1A and pMEMA) o r some s u i t a b l e s o l v e n t , and c e n t r i f u g e d t o remove c r o s s - l i n k e d p o l y m e r and p r e c i p i t a t e d L i OH. The r e s u l t i n g p r o d u c t was t h e n d r i e d o v e r n i g h t i n vacuo a t a b o u t 80°C. Free R a d i c a l P o l y m e r i z a t i o n . Free r a d i c a l p o l y m e r i z a t i o n of HEMA and MEMA were a c c o m p l i s h e d by a d d i n g 1.^ ηα/ml o f a z o b i s m e t h y l i s o b u t y r a t e (AMIB) t o t h e d e g a s s e d monomer. The monomer/ i n i t i a t o r s o l u t i o n was i n j e c t e d i n t o a s p l i t p o l y p r o p y l e n e mold and p l a c e d i n an oven a t 80°C f o r 20 h o u r s . The r e s u l t i n g p o l y mer s h e e t ( 3 - 4 mm t h i c k ) was t h e n removed f o r f u r t h e r c h a r a c t erization. S i n c e t h e pHEMA p r o d u c e d by b u l k p o l y m e r i z a t i o n was t o o h i g h l y c r o s s - l i n k e d t o be r e d i s s o l v e d f o r d e t e r m i n a t i o n o f i t s t a c t i c i t y , t h e monomer, s o l v e n t ( g e n e r a l l y p y r i d i n e ) and i n i t i a t o r were added d i r e c t l y t o a 10 mm NMR t u b e a l o n g w i t h 0 . 1 - 0 . 2 ml o f p - d i o x a n e as an i n t e r n a l s t a n d a r d and t h e r e s u l t i n g m i x t u r e was p o l y m e r i z e d i n s i t u by p l a c i n g t h e t u b e i n t o t h e oven a t 80°C

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10.

RUSSELL ET AL.

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141

f o r 20 h o u r s . TMSEMA was p o l y m e r i z e d i n d r y t o l u e n e u s i n g AMIB (TMSEMA/AMIΒ 1:100) as i n i t i a t o r . The p o l y m e r f o r m e d was p r e c i p a t e d i n p e t r o l e u m e t h e r and d r i e d o v e r n i g h t a t 80°C i n v a c u o . TABLE

I.

MONOMERS USED AND POLYMERIZATION CONDITIONS* Monomer Methylmethacrylate

(MMA)

Approx. Yield

Initiator

Solvent

Τ (°C)

n-BuLi n-BuLi

Toluene THF

-78° -78°

80% 20%

60°

>95%

None

2-Hydroxyethylmethacrylate (HEMA)

AMIB

trimethylsilylethylmetha c r y l a t e (TMSEMA)

AMIB n-BuLi

Toluene Toluene

60°C >90% -78° 20%

methoxyethylmethacrylate

n-BuLi AMIB

Toluene None

-78° 60°

*n-BuLi AMIB UV

: : :

30% >95%

η-butyl l i t h i u m azobismethylisobutyrate ultraviolet radiation

One low t e m p e r a t u r e p o l y m e r i z a t i o n o f HEMA was c a r r i e d o u t i n an e f f o r t t o p r o d u c e a h i g h l y s y n d i o t a c t i c pHEMA. I t was p e r formed by d i s s o l v i n g HEMA and AMIB i n methanol and e x p o s i n g t h e s o l u t i o n t o a 254 nm UV s o u r c e f o r s e v e n h o u r s a t -60°C. The r e s u l t i n g p o l y m e r was t h e n p r e c i p i t a t e d i n t o l u e n e and d r i e d o v e r n i g h t a t 60°C i n vacuo p r i o r t o s u b s e q u e n t c h a r a c t e r i z a t i o n . T a c t i c i t y Determination. Samples f o r t a c t i c i t y d e t e r m i n a t i o n were p r e p a r e d by p l a c i n g 0 . 8 - 1 . 0 g o f t h e d r i e d p o l y m e r i n t o a 10 mm NMR t u b e and a d d i n g 1.5-2 ml o f an a p p r o p r i a t e s o l v e n t a l o n g w i t h 0 . 1 - 0 . 2 ml o f p - d i o x a n e as an i n t e r n a l r e f e r e n c e . The t u b e was c a p p e d , and t h e s o l v e n t was a l l o w e d t o s w e l l o r d i s s o l v e the polymer i n the tube. CDC1 was used f o r pMMA and pMEMA, and p y r i d i n e f o r pHEMA and pTMSEMA. The p r o t o n d e c o u p l e d 25,2 MHz C s p e c t r u m was t h e n o b t a i n e d u s i n g a V a r i a n X L F T - Ι Ο Π NMR s p e c t r o m e t e r i n t h e F o u r i e r T r a n s f o r m mode. Spectra obtained at a m b i e n t t e m p e r a t u r e c o n t a i n e d peaks w h i c h were t o o b r o a d t o resolve. C o n s e q u e n t l y , t h e samples were h e a t e d as h i g h as p r a c t i c a l w i t h o u t causing r e f l u x i n g of s o l v e n t . Probe t e m p e r a t u r e s ranged f r o m 50°C f o r MMA and pMEMA i n CDC1 t o 70°C f o r ρHEMA and pTMSEMA i n p y r i d i n e . A t e l e v a t e d t e m p e r a t u r e s s h a r p s n e c t r a were o b t a i n e d i n w h i c h e a c h c a r b o n a b s o r p t i o n was r e s o l v e d . 3

1 3

3

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A s s i g n m e n t o f t h e peaks i n t h e pMMA s p e c t r a were b a s e d on p u b l i s h e d spectra (10). A s s i g n m e n t o f peaks e x h i b i t i n g s h i f t s due t o t a c t i c i t y f o r pMEMA, pHEMA, e t c . were made by a n a l o g y t o pMMA. Thermal A n a l y s i s . D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y (DSC) c u r v e s f o r e a c h p o l y m e r were o b t a i n e d o v e r t h e r a n g e - 5 0 to +250 C u s i n g a DuPont Model 990 Thermal A n a l y s i s S y s t e m . Samples were w e i g h e d and p l a c e d i n c o v e r e d aluminum sample p a n s , t h e n p l a c e d i n t o t h e DSC. Each sample was a n n e a l e d above i t s g l a s s t r a n s i t i o n temperature f o r approximately 5 minutes, then c o o l e d t o t h e s t a r t i n g t e m p e r a t u r e f o r the thermogram. The s a m p l e was t h e n h e a t e d a t 10 C/minute u n d e r a n i t r o g e n a t m o s p h e r e . An empty aluminum sample pan was used as a r e f e r e n c e mass f o r e a c h r u n . Results

and

Discussion

pMMA. F i g u r e 2 c o n t r a s t s t h e C - N M R s p e c t r a o f two d i f f e r e n t s a m p l e s o f pMMA, one p r o d u c e d by f r e e r a d i c a l i n i t i a t i o n and t h e o t h e r by low t e m p e r a t u r e a n i o n i c p o l y m e r i z a t i o n . Each peak i n the spectrum i s assigned to the v a r i o u s carbons i n the polymer, and t h e c h e m i c a l s h i f t o f each r e l a t i v e t o t h e p - d i o x a n e s i n g l e t a t 0 . 0 ppm i s shown. Of p a r t i c u l a r i n t e r e s t a r e t h e t h r e e peaks w h i c h c o r r e s p o n d t o t h e a - C H c a r b o n atoms w h i c h a r e i n t h e c e n t e r o f i s o t a c t i c ( i ) , h e t e r o t a c t i c (h) and s y n d i o t a c t i c ( s ) triads, respectively. S i m i l a r s p l i t t i n g i s observed f o r the c a r b o n y l and q u a t e r n a r y c a r b o n a t o m s , a l t h o u g h t h e peaks a r e l e s s w e l l - r e s o l v e d than the a - C H peaks. The a r e a u n d e r e a c h o f t h e peaks i s p r o p o r t i o n a l t o t h e number o f c a r b o n atoms i n e a c h t y p e of t r i a d i n the sample. H e n c e , a r a t i o o f t h e peak a r e a s y i e l d s t h e r e l a t i v e amounts o f each t y p e o f t r i a d . Since the a-CH peaks a r e s p l i t r e l a t i v e l y f a r a p a r t , t h e y have been used t o c a l c u l a t e t a c t i c i t i e s in t h i s study. The f r e e r a d i c a l pMMA s p e c i m e n c o n t a i n s 52%s / 41% h/ 7% i t r i a d s , b a s e d on t h e r e l a t i v e peak a r e a s . The a n i o n i c pMMA c o n t a i n s 10% s/ 19% h/ 71% i triads. The peak a r e a s and c h e m i c a l s h i f t s o f b o t h pMMA s a m p l e s , as w e l l as t h o s e f o r t h e o t h e r p o l y m e r s s t u d i e d , a r e l i s t e d i n T a b l e I I . F i g u r e 3 c o n t r a s t s t h e DSC thermograms o f t h e same two polymers. C l e a r l y , the predominantly i s o t a c t i c a n i o n i c polymer e x h i b i t s d i f f e r e n t thermal behavior from t h a t o f the predominantly s y n d i o t a c t i c f r e e r a d i c a l polymer. The i s o t a c t i c p o l y m e r shows a g l a s s t r a n s i t i o n a t 76 C, whereas t h e s y n d i o t a c t i c p o l y m e r has a Tg o f 110 C. The t h e r m a l b e h a v i o r o f t h e s p e c i m e n s can t h u s be c o r r e l a t e d w i t h t h e t a c t i c i t y o b s e r v e d by NMR. 3

3

3

pMEMA, The b e h a v i o r o f pMMA d e s c r i b e d above c o r r e s p o n d s w e l l w i t h t h a t r e p o r t e d p r e v i o u s l y (1_). C o n s e q u e n t l y , i t was e x p e c t e d t h a t p o l y m e r s o f MEMA p r o d u c e d by f r e e r a d i c a l and a n i o n i c methods w o u l d show a c o r r e s p o n d i n g d i f f e r e n c e i n t a c t i c i t y . The a - C H p o r t i o n s o f t h e C - N M R s p e c t r a o f two d i f f e r e n t pMEMA s a m p l e s a r e shown i n F i g u r e 4. The f i r s t s p e c i m e n was p r o d u c e d u s i n g AMIB as a f r e e r a d i c a l i n i t i a t o r , w h i l e t h e s e c o n d was 13

3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Thermal Behavior of Poly(methacrylate esters)

RUSSELL E T AL. H

/CH c=c

x

/CH

3

0-CH -CH -OH

O-CH3

2

METHYLMETHACRYLATE (MMA)

H

CH C=C

N

7

H

2

2

2

2-HYDROXYETHYLMETHACRYLATE (HEMA)

3

0-CH -CH

3

\

/ 3 C=C C H

0-CH -CH -0-Si-CH I CH

-OCH3

2

2

3

3

TRIMETHYLSILYLETHYLMETHACRYLATE (TMSEMA)

2-METHOXYETHYLMETHACRYLATE (MEMA)

Figure 1.

Monomer structure and nomenclature

-0-CH3

Free Radical pMMA

110.8 ppm

0.0 ppm

50.5 ppm

Anionic pMMA

Ai 109.4 ppm

Figure 2.

0.0 ppm

-45.0 ppm

C NMR spectra of poly(methyl methacrylate)

13

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

-47.7

—

from

-48.5

— -50.3

-50.2

-49.3

-49.4

-49.4

-49.5

-50.5

-45.7

45.4 71%

<1%

<1%

21.1 9%

137 30%

6 4%

<1%

14 7%

57.1 71%

8.1 10% 109.9 110.1

— —

11.9 18%

77.5 45%

28.8 33%

7.2 11%

95.0 54%

55.8 66%

--

—

109.5

109.4

110.2

110.3

110.8

—

3 1 . 2 189.6 13% 78%

--

110.8

110.2

57.3 38% 164.4 1 6 5 . 0 35% 35%

110.8

110.7

110.8

110.4

110.2

110.0

109.7

109.4

89.1 58%

5 4 . 0 107.9 33% 66%

88.5 109.3 42% 51%

15.6 19%

3

TABLE I I . f o r V a r i o u s a - C H and C a r b o n y l T r i a d s * A r e a : a-CH3 6c=o(ppm]1 s_ h i_ h

* i = isotactic; h = heterotactic; s = syndiotactic * * C h e m i c a l s h i f t s e x p r e s s e d as ppm f r o m d i o x a n e s i n g l e t a t 0 ppm

pMEMA (anionic pMMA)

pMEMA (free radical)

pMEMA (anionic)

-47.6

-48.5

-45.9

pHEMA (UV-low

-47.5

—

-44.5

pHEMA (anionic-from pTMSEMA)

temp)

-44.6

pHEMA (free r a d i c a l f r o m pTMSEMA)

-47.6

-48.2

-45.8

pMMA (commercial)

pHEMA (free radical)

-45.6

-45.0

3

pMMA (anionic)

Polymer

Chemical S h i f t s 6a-CH (ppm)** h

3

3

CDC1

3

CDC13

CDC1

Pyridine

Pyridine

Pyridine

Pyridine

CDC1

CDC13

50°

50°

50°

70°

70°

70°

70°

Ambient

Ambient

Sol vent Temperature(°C)

RUSSELL ET AL.

Thermal Behavior of Poly (methacrylate

_l

-40

0

40

80

esters)

L_

120

160

200

TEMPERATURE (°C)

Figure 3.

DSC thermograms of poly(methyl methacrylate)

Free Radical pMEMA

Anionic pMEMA

Transeste rified Anionic pMMA X MEMA

Figure 4. a-CH portions of C-NMR spectra of poly(methoxyethyl methacrylate) 3

13

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

146

produced a n i o n i c a l l y i n t o l u e n e at -78°C. Both p o l y m e r s c o n t a i n a p r e p o n d e r a n c e o f s y n d i o t a c t i c and h e t e r o t a c t i c t r i a d s , w i t h few i s o t a c t i c t r i a d s , as l i s t e d i n T a b l e I I . In an e f f o r t t o show t h a t no i r r e g u l a r i t i e s i n c h e m i c a l s h i f t had c a u s e d t h e a n i o n i c p o l y m e r t o a p p e a r s y n d i o t a c t i c when i t was a c t u a l l y i s o t a c t i c , a sample o f t h e i s o t a c t i c pMMA d e s c r i b e d above was t r a n s e s t e r i f i e d t o pMEMA. The a - C H p o r t i o n o f i t s C - N M R s p e c t r u m i s shown i n Figure 4c. S i n c e i t shows t h e same c h e m i c a l s h i f t s and r e l a t i v e peak a r e a s as t h e p a r e n t pMMA, i t must be c o n c l u d e d t h a t t h e h i g h s y n d i o t a c t i c c o n t e n t o f t h e a n i o n i c pMEMA i s n o t an a r t i f a c t . R a t h e r , i t must be due t o some i n t e r a c t i o n between t h e oxygen o f t h e methoxy s i d e c h a i n and t h e a n i o n i c g r o w t h c e n t e r w h i c h changes t h e l o c a l d i e l e c t r i c c o n s t a n t and a l t e r s t h e g e g e n i o n s e p a r a t i o n , t h u s f a v o r i n g s y n d i o t a c t i c p l a c e m e n t o f i n c o m i n g monomer u n i t s . Similar effect MEMA i n i t i a t e d by t - b u t y l l i t h i u s t u d y , f e w e r t h a n 10% i s o t a c t i c t r i a d s were f o u n d a t e i t h e r t e m p e r a t u r e . The f a c t t h a t b o t h t h e a n i o n i c and f r e e r a d i c a l pMEMA specimens are p r e d o m i n a n t l y s y n d i o t a c t i c i s r e f l e c t e d i n t h e i r DSC t r a c e s , shown i n F i g u r e 5. Both show an i n f l e c t i o n a t 25°C w i t h no m e l t i n g a p p a r e n t , and b e g i n t o decompose above 2 5 0 ° C . 13

3

pHEMA. F i g u r e 6 shows t h e a - C H p o r t i o n o f t h e C - N M R s p e c t r a o f pHEMA p r o d u c e d by f o u r d i f f e r e n t m e t h o d s . The r e l a t i v e t a c t i c i t i e s c a l c u l a t e d f r o m t h e peak a r e a s a r e g i v e n i n Table II. The d a t a show t h a t pHEMA p r o d u c e d a t e l e v a t e d t e m p e r a t u r e s w i t h AMIB ( F i g u r e 6 ( a ) ) c o n t a i n s p r e d o m i n a n t l y s y n d i o t a c t i c (66%) and h e t e r o t a c t i c t r i a d s ( 3 3 % ) , w i t h few i s o t a c t i c t r i a d s (< 1%). F i g u r e 6 ( b ) shows s i m i l a r r e s u l t s f o r a p o l y m e r f o r m e d f r o m TMSEMA u s i n g AMIB as i n i t i a t o r , t h e n s u b s e q u e n t l y h y d r o l y z e d t o pHEMA. S y n d i o t a c t i c (58%) and h e t e r o t a c t i c (38%) t r i a d s p r e d o m i n a t e o v e r i s o t a c t i c (4%) i n t h i s c a s e as w e l l . The t h i r d p o l y m e r , shown i n F i g u r e 6 ( c ) , was p o l y m e r i z e d a n i o n i c a l l y a t -78°C i n t o l u e n e f r o m TMSEMA, t h e n h y d r o l y z e d t o pHEMA. Here we see a s i g n i f i c a n t p r o p o r t i o n o f i s o t a c t i c t r i a d s (30%) f o r t h e f i r s t t i m e , w i t h t h e r e m a i n d e r e v e n l y d i v i d e d between s y n d i o t a c t i c (35%) and h e t e r o t a c t i c t r i a d s ( 3 5 % ) . The f o u r t h pHEMA, shown i n F i g u r e 6 ( d ) , was p r o d u c e d by UV i n i t i a t i o n u s i n g AMIB a t - 7 8 C. As t h e f i g u r e s h o w s , a h i g h l y s y n d i o t a c t i c p o l y m e r has been p r o d u c e d , h a v i n g 78% s y n d i o t a c t i c t r i a d s , w i t h 9% i s o t a c t i c t r i a d s and 13% h e t e r o t a c t i c t r i a d s . The DSC thermogram o f each p o l y m e r i s shown i n F i g u r e 7. The g l a s s t r a n s i t i o n o f t h e pTMSEMA p r o duced a n i o n i c a l l y i s 13° l o w e r t h a n t h a t o f f r e e r a d i c a l pTMSEMA, i n d i c a t i n g t h a t an i n c r e a s e i n i s o t a c t i c t r i a d s l e a d s t o a d e c r e a s e i n T g , j u s t as i t d i d w i t h pMMA. F u r t h e r work w i l l be r e q u i r e d t o i n c r e a s e t h e i s o t a c t i c c o n t e n t o f pHEMA p o l y m e r s i n o r d e r t o f i l l o u t t h e f u l l range f r o m h i g h l y s y n d i o t a c t i c pHEMA t o p r e d o m i n a n t l y i s o t a c t i c pHEMA. However, i t now a p p e a r s l i k e l y t h a t s y s t e m a t i c v a r i a t i o n o f t a c t i c i t y w i l l be a c h i e v a b l e i n t h e f u t u r e and may p r o v i d e a means o f v a r y i n g m e c h a n i c a l , 13

3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

RUSSELL E T AL.

Thermal

Behavior

Anionic

-50

0

of Poly (methacrylate

esters)

pMEMA

50

100

150

200

250

T E M P E R A T U R E (°C)

Figure 5. DSC thermograms of poly(methoxyethyl methacrylate )

Free Radical pHEMA

Free Radical pTMSEMA Hydrolyzed to pHEMA

Anionic pTMSEMA Hydrolyzed to pHEMA

Low Temperature UV-initiated pHEMA

- " ... Ά L :

Figure 6. a-CH portions of C~ NMR spectra of poly(hydroxyethyl methacrylate) 3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

ure 7. DSC thermograms of poly(hydroxyethyl methacrylate) and derivatives

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

10.

RUSSELL ET AL.

Thermal Behavior of Poly(methacrylate esters)

t h e r m a l and s u r f a c e p r o p e r t i e s i n t h e pHEMA/water s y s t e m future studies.

149

for

Conclusions Based on t h e d a t a p r e s e n t e d a b o v e , we c o n c l u d e : 1.

2.

3.

4.

C - N M R p r o v i d e s a method f o r d i r e c t l y d e t e r m i n i n g t h e t a c t i c i t y of methacrylate polymers. This e l i m i n a t e s the need f o r l e n g t h y h y d r o l y s i s t o p o l y ( m e t h a c r y l i c a c i d ) and r e - e s t e r i f i c a t i o n t o pMMA. M e t h a c r y l a t e - b a s e d p o l y m e r s d i s p l a y s i g n i f i c a n t changes i n t h e i r DSC thermograms as a r e s u l t o f changes i n tacticity. Methacrylate ester interact with anioni content of the r e s u l t i n g polymers. Whether t h e e f f e c t o f t h e h e t e r o a t o m s can be r e d u c e d a t t e m p e r a t u r e s b e l o w -78°C o r by s t e r i c a l l y h i n d e r i n g t h e h e t e r o a t o m d u r i n g p o l y m e r i z a t i o n r e m a i n s t o be s e e n . T e c h n i q u e s a r e now a v a i l a b l e f o r p r o d u c i n g s o l u b l e pHEMA polymers having a v a r i e t y of t a c t i c i t i e s . The e f f e c t o f t h a t t a c t i c i t y v a r i a t i o n on i n t e r f a c i a l and b u l k p r o p e r t i e s w i l l be t h e s u b j e c t o f f u t u r e w o r k .

13

Acknowledgements: T h i s work was s u p p o r t e d by NIH G r a n t HL 1 6 9 2 1 - 0 1 . We t h a n k Hydro Med S c i e n c e s , I n c . , New B r u n s w i c k , N. J . f o r g e n e r o u s d o n a t i o n s o f p u r e HEMA monomer.

Abstract In this study a series of hydroxyethyl methacrylate (HEMA) and methoxyethyl methacrylate (MEMA) polymers of varying tacticities was produced. Both free radical and anionic initiators were used and their effect on tacticity determined. Tacticity of each polymer was measured by ratioing the C-NMR peak areas for the a-CH carbon atoms in isotactic, syndiotactic, and heterotactic triads. Both the a-CH and carbonyl carbon peaks exhibit splitting due to tacticity, but the a-CH peak was better resolved. Chemical shifts for both atoms appear to be independent of the ester side chain for the polymers studied. The thermal behavior of each polymer was observed over the range -50° to +250°C by differential scanning calorimetry (DSC). Differences in melting point and glass transition of the polymers were relatable to the tacticity measured by C-NMR. This indicates potential for some control of the properties of pHEMA and other related hydrogels by control of tacticity during synthesis. 13

3

3

3

13

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Literature Cited 1. Bovey, F. A. and Tiers, G. V. D., J. Poly. Sci., 44, (1960), 173. 2. Matsuzaki, K., Ishida, Α., and Tateno, N., J. Poly. Sci., Part C, (1967), 16, 2111 3. Goode, Ν. E., Owens, F. H., Fellman, R. P., Snyder, W. H. and Moore, J. E., J. Poly. Sci., (1960), 46, 317 4. McCrum, N. G., Read, Β. E. and Williams, G., "Anelastic and Dielectric Effects in Polymeric Solids", pp. 249-55, John Wiley & Sons, New York, 1967. 5. Holly, F. J. and Refojo, M. F., J. Biomed. Materials Res., (1975) 9, 315. 6. Lenz, R. W., "Organic Chemistry of Synthetic High Polymers", pp. 439-41, Interscience 7. DeVisser, A. C., Unpublishe 8. Chlanda, F. P., and Donamura, L. G., J. Appl. Poly. Sci., (1971 ), 15, 1195. 9. Lando, J. B., Litt, M., Kumar, N. G., and Shimko, T. M., J. Poly. Sci., (1974), Symposium Series No. 44, 203. 10. Bovey, F. Α., High Resolution NMR of Macromolecules, pp. 80-82, Academic Press, New York, 1972. 11. Trekoval, J., Vlcek, P., and Lim, D., Coll. Czech. Chem. Comm., (1971), 36, 3032.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11 Calcification and Bone Induction Studies in Heterogeneous Phosphorylated Hydrogels J. G. N. SWART Department of Oral Surgery, Free University, Amsterdam, The Netherlands A.A.DRIESSENandA.C.DEVISSER Department of Materials Science, Free University, Amsterdam, The Netherlands Development o f p o l y ( h y d r o x y e t h y l methacrylate) poly(HEMA) , h y d r o g e l f i e l d has up t o dat replacement o f s o f t - t i s s u e s t r u c t u r e s and o r g a n s , e.g. the s o f t c o n t a c t l e n s , Because o f i t s r e l a t i v e l y poor m e c h a n i c a l p r o p e r t i e s t h e h y d r o g e l as such can n o t be a p p l i e d as subs t i t u t e f o r h a r d t i s s u e . However, based on t h e f i n d i n g s t h a t c a l c i f i c a t i o n has o c c u r r e d i n heterogeneous poly(HEMA) h y d r o g e l s ( 1 - 6 ) , Calnan e t a l . (3) s u g g e s t e d t h a t t h e h y d r o g e l p o s s i b l y c o u l d promote t h e d e p o s i t i o n of c a l c i u m s a l t s i n i t s matrix, thus being a " c h a l l e n ger" f o r c a l c i f i c a t i o n . Some a u t h o r s ( 2 , 4 ) r e p o r t e d bone f o r m a t i o n f o l l o w ing the occurrence o f c a l c i f i c a t i o n . C a l c i f i c a t i o n and bone i n d u c t i o n appeared t o be a c c e l e r a t e d by t h e p r e s e n c e o f m e t h a c r y l i c a c i d (MAA) groups i n t h e g e l (£). S p r i n c l e t a l . (6), however, r e p o r t e d t h a t modif i c a t i o n o f a poly(HEMA) g e l by i n c o r p o r a t i o n o f MAA (up t o a mole r a t i o o f MAA/HEMA 1 : 5 ) o r d i m e t h y l a m i n o e t h y l m e t h a c r y l a t e (DMAEMA) d i d n o t a f f e c t t h e c a l c i f i c a t i o n p r o c e s s , whereas C e r n i j e t a l . (2) found t h a t i n c o r p o r a t i o n o f 4 % MAA i n h i b i t e d c a l c i f i c a t i o n . From t h e above r e s u l t s i t i s e v i d e n t t h a t t h e e f f e c t o f MAA, i n c o r p o r a t e d i n t h e h y d r o g e l , on c a l c i f i c a t i o n o r bone f o r m a t i o n has n o t been unambiguously e s t a b l i s h ed y e t . P o s s i b l y , o t h e r f a c t o r s than c h e m i c a l m o d i f i c a t i o n such as pore s i z e o f t h e g e l , a n i m a l s p e c i e s and i m p l a n t a t i o n s i t e , p l a y a more dominant r o l e i n the o c c u r r e n c e o f t h e s e phenomena. A h y d r o g e l t h a t , i n a d d i t i o n t o c a l c i f i c a t i o n , c o u l d i n d u c e bone f o r m a t i o n i n i t s m a t r i x would have g r e a t p o t e n t i a l f o r r e s t o r a t i o n o f l a r g e d e f e c t s i n bone t i s s u e . Such a m a t e r i a l c o u l d f o r example be a p p l i e d t o f a c i l i t a t e t h e h e a l i n g p r o c e s s i n t h e p o s t e x t r a c t i o n a l v e o l u s o r bone i n 3

151

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growth i n l a r g e c y s t s . On t h e o t h e r hand, c a l c i f i c a t i o n i s o f t e n an u n d e s i r a b l e phenomenon i n a h y d r o g e l s e r v i n g as s o f t t i s s u e s u b s t i t u t e s i n c e i t a f f e c t s t h e f u n c t i o n a l and e s t h e t i c p r o p e r t i e s o f t h e m a t e r i a l adversely. In t h i s case, i n h i b i t i o n o f c a l c i f i c a t i o n by c h e m i c a l m o d i f i c a t i o n would be b e n e f i c i a l . In view o f t h e above c o n s i d e r a t i o n s o u r o b j e c t i v e i s t o study c a l c i f i c a t i o n and bone f o r m a t i o n i n h e t e r o geneous h y d r o g e l s as f u n c t i o n o f t h e i r c h e m i c a l and p h y s i c a l p r o p e r t i e s and t o f i n d means t o c o n t r o l these p r o c e s s e s by s u i t a b l e m o d i f i c a t i o n o f t h e h y d r o g e l . The study d e s c r i b e d h e r e a f t e r d e a l s w i t h an experiment d e s i g n e d t o determine t h e e f f e c t o f i n c o r p o r a t i o n o f the phosphate group i heterogeneou poly(HEMA) l on i t s c a l c i f i c a t i o n - i n d u c i n The h y d r o g e l s h o u l d be heterogeneous because i t was found t h a t a s u f f i c i e n t l y l a r g e pore s i z e (> 40ym) (A) i s a p r e r e q u i s i t e f o r ingrowth o f t h e s u r r o u n d i n g t i s s u e which i n t u r n can l e a d t o c a l c i f i c a t i o n and f o r m a t i o n o f bone. The phosphate group was s e l e c t e d t o be b u i l t i n t h e g e l because t h i s group i s one o f t h e b u i l d i n g b l o c k s o f c a l c i u m h y d r o x y a p a t i t e t h e main i n o r g a n i c c o n s t i t u e n t o f bone and, a l t h o u g h b e i n g p r e s e n t as HEMA-phosphate, s h o u l d have a h i g h a f f i n i t y towards calcium ions. M a t e r i a l and Methods HEMA-phosphate was p r e p a r e d by r e a c t i n g HEMA (Hydro Med S c i e n c e s , I n c . , U.S.A., p u r i t y min. 99.2%) w i t h phosphorus p e n t o x i d e (Merck,Darmstadt, Germany, p u r i t y min. 98%) i n a 1:1 mole r a t i o i n d i c h l o r o m e t h a ne a t 0 - 5°C. The r e a c t i o n proceeds almost q u a n t i t a t i v e l y . A f t e r removal o f s o l i d s by c e n t r i f u g a t i o n and e v a p o r a t i o n o f t h e s o l v e n t i n vacuo a t 20 C and 1 mm Hg, a m i x t u r e o f mono- and d i - e s t e r was o b t a i n e d i n a mole r a t i o o f 2 t o 1 assuming t h a t t h e amount o f t r i e s t e r formed i s n e g l i g i b l e . Ethyleneglycol d i m e t h a e r y l a t e (EGDMA) (Merck) was p u r i f i e d by d i s t i l l a t i o n a t 83 C and 1.5 mm Hg. HEMA was used as o b t a i n e d from Hydro Med S c i e n c e s . P r e p a r a t i o n o f t h e h y d r o g e l s was performed i n s e a l e d ampoules o f 9 mm i n n e r d i a m e t e r . The v a r i o u s monomers were d i s s o l v e d i n a Tyrode s o l u t i o n c o n t a i n i n g 1% (w/w) o f ammonium p e r s u l f a t e as i n i t i a t o r . Polymer i z a t i o n was c a r r i e d o u t a t 50 C d u r i n g 24 hours. o

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.

SWART E T A L .

Composition follows :

Heterogeneous

Phosphorylated

Hydrogels

o f the p o l y m e r i z a t i o n mixtures

Hydrogel

HEMA

H 1 H 2

19.8 17.8

EGDMA HEMA( c r o s s l i n k e r ) phosphate 0.2 0.2

— 2.0

153

i s as

Tyrode s o l .

80% w/w 80% w/w

The g e l s were k e p t i n d i s t i l l e d water f o r s e v e r a l days and then b o i l e d t w i c e i n d i s t i l l e d water t o r e move low m o l e c u l a r weight s u b s t a n c e s . Then t h e g e l s were soaked i n s t e r i l e s a l t s o l u t i o n and c u t i n t o d i s c s about 2 mm t h i c k and 9 mm i n d i a m e t e r These d i s c s wer the back muscle o f d i s c s o f t h e same h y d r o g e l and one p o c k e t was f i l l e d w i t h g e l a t i n foam ( S p o n g o s t a n , F e r r o s a n , Will-Pharma N.V., H o l l a n d ) as a c o n t r o l . E x c i s i o n f o l l o w e d a f t e r p e r i o d s o f 3 days up t o 24 weeks. The s k i n was c u t away and t h e i m p l a n t s removed w i t h t h e s u r r o u n d i n g muscle t i s s u e . The removed t i s s u e b l o c k s were p l a c e d f o r 10 minutes between gauze s t r i p s s a t u r a t e d w i t h i s o t o n i c s a l i n e t o allow the c o n t r a c t i l e p r o p e r t i e s t o become q u i e s c e n t . T h e r e a f t e r t h e e x c i s e d t i s s u e was f i x e d i n 10% b u f f e r e d f o r m a l i n . A f t e r f i x a t i o n and d e h y d r a t i o n i n a b s o l u t e a l c o h o l t h e b i o p s i e s were embedded i n p a r a f f i n and c u t i n t o s e c t i o n s o f 6 ym. These s e c t i o n s were s t a i n e d w i t h t h e Haematoxylin and e o s i n s t a i n . C a l c i u m s a l t s were v i s u a l i z e d a c c o r d i n g t o Von Kossa. Pore s i z e o f t h e m a t e r i a l s was determined by scanning e l e c t r o n microscopy. R

R e s u l t s and D i s c u s s i o n M a c r o s c o p i c a l l y a l l i m p l a n t s were a c c e p t e d and w e l l t o l e r a t e d ; no s i g n s o f s e v e r e i n f l a m m a t i o n o r i m p l a n t r e j e c t i o n c o u l d be o b s e r v e d , ( f i g u r e 1 ) . Microscopic i n v e s t i g a t i o n revealed the following: 3 days a f t e r i m p l a n t a t i o n a s l i g h t edema and i n v a s i o n o f g r a n u l o c y t e s and mononuclear c e l l s i n t o a l l i m p l a n t s was o b s e r v e d . The p h o s p h o r y l a t e d hydrog e l s showed l e s s c e l l u l a r i n f i l t r a t i o n and s t a i n e d b a s o p h i l i c p r o b a b l y because o f t h e a c i d phosphate group. Some o f t h e c o n t r o l g e l a t i n foams (Spongostan ) showed i n v a s i o n o f f i b r o b l a s t s and mononuclear c e l l s as w e l l as hyperaemia around t h e i m p l a n t s as a s i g n o f acute inflammatory response. R

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

154

In the one week b i o p s i e s t h e r e was an i n c r e a s e i n c e l l u l a r ingrowth o f about 0.2 mm f o r the p o l y (HEMA) i m p l a n t s . The p h o s p h o r y l a t e d i m p l a n t s h a r d l y showed any ingrowth o r s i g n s o f i n f l a m m a t i o n . A f t e r two weeks we saw a f u r t h e r i n c r e a s e o f ingrowth up t o a maximum o f 1 mm i n t o the poly(HEMA) specimen. The i m p l a n t s were s l i g h t l y t h i c k e r (3 mm) and f o r the f i r s t time b a s o p h i l i c c l u s t e r s c o u l d be seen as the e a r l i e s t s i g n o f c a l c i f i c a t i o n ( f i g u r e 2 ) . The p h o s p h o r y l a t e d poly(HEMA) i m p l a n t s behaved q u i t e i n d i f f e r e n t l y towards the t i s s u e . There was p r a c t i c a l l y no f i b r o u s l i n i n g around the i m p l a n t , i t even seemed t o l a y d i r e c t l y upon the muscle f i b r e s . In the 4 week b i o p s i e s the "Spongostan " g e l a t i n foam i m p l a n t s showe tissue proliferatio g i a n t c e l l s and c a p i l l a r i e s dominated the p i c t u r e . The g e l a t i n foam g r a d u a l l y d i s a p p e a r s as a r e s u l t o f p h a g o c y t o s i s by macrophages. In the poly(HEMA) h y d r o g e l s b a s o p h i l i c g r a n u l e s are s c a t t e r e d a l l through the i m p l a n t . The p h o s p h o r y l a t e d i m p l a n t s were w e l l t o l e r a t e d , and s t i l l showed p r a c t i c a l l y no ingrowth o r encapsulation. 8 weeks the "Spongostan " g e l a t i n foam has completely disappeared, with only small strands of f i b r o u s s c a r t i s s u e l e f t . The poly(HEMA) i m p l a n t s showed c o n s i d e r a b l y more f i b r o b l a s t s than 4 weeks bef o r e . The b a s o p h i l i c g r a n u l e s w i t h i n t h e s e i m p l a n t s formed l a r g e r c o l o n i e s . F r e q u e n t l y , a t h i n f i b r o u s c a p s u l e surrounded the p h o s p h o r y l a t e d i m p l a n t s , which s t i l l h a r d l y showed any ingrowth. A t 16 and 20 weeks whole g r a n u l a r f i e l d s were o b s e r v e d i n the poly(HEMA) i m p l a n t s . These f i e l d s p a r t i c u l a r l y appeared i n the edges o f the i m p l a n t . Where t h e s e g r a n u l a r f i e l d s were p r e s e n t , t i s s u e i n growth d i d not take p l a c e o r seemed t o be p r e v e n t e d . Some o f the poly(HEMA) i m p l a n t s showed l e s s b a s o p h i l i c g r a n u l e s , but c o n s i d e r a b l y more t i s s u e i n g r o w t h . C a l cium d e t e r m i n a t i o n by the Von Kossa s t a i n r e v e a l e d t h a t the s o c a l l e d b a s o p h i l i c g r a n u l e s were c a l c i f i e d ( f i g u r e 3). ^ 24 week b i o p s i e s , the l a s t p e r i o d e v a l u a t e d , showed some i n t e r e s t i n g f e a t u r e s . A rontgenogram (60 kv, 3 mA, 0.10 sec) o f b i o p s i e s from the 5 r a t s out o f t h i s group showed a r a d i o paque o u t l i n e i n the poly(HEMA) i m p l a n t s , p h o s p h o r y l a ted poly(HEMA) i m p l a n t s d i d not show t h i s phenomenon. R

A

T

f

t

e

r

e

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

SWART E T AL.

Heterogeneous

Phosphorylated

Hydrogels

Figure 1. Phosphorylated poly(HEMA) implants in the back of a rat 24 weeks after the implantation showing the excellent biocompatibility of this material

Figure 2. Photomicrograph of a poly(HEMA) implant section, 14 days after implantation. For the first time calcified granules (arrows) appear within the poly(HEMA) implants. (Haematoxylin and eosin stain, magnif. X39)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 3a. Photomicrograph of a poly(HEMA) implant 20 weeks after insertion showing areas of dense calcification (1) and areas of massive tissue ingrowth (2) with only sparse calcification. The center (3) of the implant shows only a slight ingrowth of cells into the pores of the implant material. (Haematoxylin and eosin stain, magnification X20)

Figure 3b. Same section stained according to Von Kossa for calcium determination. With this method calcified tissues stain black and uncalcified tissues red, which shows plain grey on this photomicrograph.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.

SWART E T A L .

Heterogeneous

Phosphorylated

Hydrogels

157

The Von Kossa s t a i n proved the p r e s e n c e o f c a l c i u m w i t h i n the poly(HEMA) i m p l a n t s which showed r a d i o paque l i n e s i n the roentgenogram and the absence o f c a l c i u m s a l t d e p o s i t s i n the non-radiopaque specimen. The p h o s p h o r y l a t e d i m p l a n t s showed o n l y a t h i n f i b r o u s l i n i n g and i n g r o w t h , i f any, p r e d o m i n a n t l y i n the o u t e r margin ( f i g u r e 4 ) . In one r a t we o b s e r v e d a remarkable macroscopic d i f f e r e n c e between two poly(HEMA) i m p l a n t s t h a t were c h e m i c a l l y i d e n t i c a l ; one i m p l a n t had a w h i t e a s p e c t and a normal s i z e ; the o t h e r i m p l a n t showed an i n c r e a s e i n s i z e and the same c o l o u r as the s u r r o u n d i n g t i s s u e . The m i c r o s c o p i c p i c t u r e r e v e a l e d t h a t the white i m p l a n t was mor i m p l a n t and showed l e s ence might be the r e s u l t o f a d i f f e r e n t b l o o d s u p p l y (figure 5). In o r d e r t o r e l a t e the r e s u l t s w i t h the p h y s i c a l s t r u c t u r e o f the g e l s , the pore s i z e o f the v a r i o u s i m p l a n t s was determined by s c a n n i n g e l e c t r o n m i c r o s copy. The poly(HEMA) i m p l a n t s had pores from 30 - 70 ym, w h i l e the p h o s p h o r y l a t e d h y d r o g e l s p o s s e s s e d pores from 70 t o o v e r 100 ym ( f i g u r e 6 ) . The i n h i b i t i o n o f t i s s u e i n f i l t r a t i o n i n the phosphate c o n t a i n i n g g e l s i s s u r p r i s i n g because one would e x p e c t t h a t s u b s t a n t i a l i n g r o w t h would o c c u r i n an i m p l a n t h a v i n g such a l a r g e pore s i z e . Wether t h i s phenomenon i s due t o the h i g h e r a c i d i t y i n t h e s e g e l s , as compared t o the poly(HEMA) g e l s , o r the n a t u r e o f the phosphate group i t s e l f i s not known and needs f u r t h e r study. Conclusions Summarizing the r e s u l t s we can c o n c l u d e t h a t : 1. I n c o r p o r a t i o n o f the phosphate group i n h e t e r o g e neous poly(HEMA) h y d r o g e l s when i m p l a n t e d i n t r a m u s c u l a r l y i n r a t s , does not promote c a l c i f i c a t i o n o r bone f o r m a t i o n , on the c o n t r a r y i t seems t o p r e v e n t c a l c i f i c a t i o n and t i s s u e i n g r o w t h . T h i s may make the p h o s p h o r y l a t e d poly(HEMA) h y d r o g e l s a better material for soft tissue substitution than r e g u l a r poly(HEMA), a l t h o u g h i t i s t o e a r l y y e t t o make d e f i n i t e c o n c l u s i o n s and recommendations. 2. None o f the i m p l a n t s showed bone o r c a r t i l a g e f o r mation. The c a l c i f i c a t i o n o c c u r i n g w i t h i n the p o l y (HEMA) i m p l a n t s i s most l i k e l y due t o c a l c i f i c a t i o n o f degenerate t i s s u e ( d y s t r o p h i c c a l c i f i c a t i o n ) as the r e s u l t o f i n s u f f i c i e n t b l o o d s u p p l y .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 4a. Photomicrograph of a phosphorylated poly(HEMA) imphnt section 24 weeks after insertion showing the thin fibrous lining of two or three cell layers and rather superficial tissue ingrowth.

Figure 4b. Photomicrograph of a section of the same material which has been excised after 14 days shows almost the same picture. (Haematoxylin and eosin stain, magnification X 85)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

SWART ET AL.

Heterogeneous Phosphorylated Hydrogels

Figure Sa. Poly(HEMA) implants out of the same rat 24 weeks after implantation. In one implant (1) there is a diffuse ingrowth of tissue practically without calcification. The other implant (2) shows colonies of calcification and only tissue ingrowth in the outer margin of the implant. (Haematoxylin and eosin stain, magnification X 21)

Figure 5b. Comparable section, stained according to Von Kossa, proving the presence of calcium salts which stain black (arrows)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 6a. Scanning electron micrograph of poly(HEMA) having pore sizes from 80-70 μ

Figure 6b. Scanning electron micrograph of phosphorylated poly(HEMA) having pores from 70 to over 100 μ

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.

SWART E T A L .

Heterogeneous Phosphorylated Hydrogels

161

3. No lymphocytes were seen in the implants which indicates that poly(HEMA) and phosphorylated poly (HEMA) are immunologically inert materials. 4. A large variation in calcification within the regular poly(HEMA) implants has been observed. Variation in blood supply with implant site possi bly plays an important role in this phenomenon. Abstract The effect of chemical modification of a hetero geneous poly(hydroxyethyl methacrylate) hydrogel on calcification in its matrix has been studied by intra muscular implantation in Wistar rats. Ten percent phosphorylated hydroxyethy total amount of monomer containing 80% (w/w) water. These gels which exhibit poor mechanical properties can not be used as direct replacement for hard tissue, but may have potential as calcification "challenger" for living tissue. Scanning electron micrographs show that the gels have pores with an average diameter 70 - 100 μm. Histological examination of the implants after 3 days up to 24 weeks revealed that the poly(HEMA) and the phosphorylated poly(HEMA) gels were very well tolerated by the organism. Tissue ingrowth and calcification occurred in the poly(HEMA) gels, but bone formation was not found. The phosphorylated gels were encapsulated by a thin fibrous lining whereas ingrowth only sporadically and to a slight extent was observed; calcification did not take place. Literature Cited 1. Kliment, Κ., Stol, Μ., Fahoun, K., Stockar, Β., J . Biomed. Mater. Res. (1968), 2, 237. 2. Winter, G.D., and Simpson, B . J . , Nature (1969), 223, 88. 3. Calnan, J . S . , Pflug, J.J., Chhabra, A.S., Raghypatli, N., Brit. J . Plastic Surgery,(1971) ,24, 113. 4. Smahel, J.,Proserova, J.,Behounkova, Ε . , Acta Chirurgiae Plasticae (1971), 13, 193. 5. Sprincl, L.,Vacik, J.,Kopecek, J., J. Biomed. Mater. Res. (1973), 7, 123. 6. Sprincl, L.,Kopecek, J.,Lim, D., Calc.Tiss.Res. (1973), 13, 63. 7. Cernij, Ε . , Chromeĉek, R., Opleta, Α., Papousek, F. Otoupalová, J., Scripta Medica (1970).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

12 Continuous Monitoring of Blood Glucose Using Gel Entrapped Glucose Oxidase K. F. O'DRISCOLL and A. KAPOULAS Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada A. M. ALBISSER and R. GANDER Medical Engineering Department, Hospital for Sick Children, Toronto, Ontario, Canada

The use of enzymes as efficient and highly specific cat alysts in chemical analysi during the past twent cedures for the immobilization of enzymes by physical occlusion within a hydrophilic polymeric matrix offers new opportunities for the practical use of enzymes in chemical analysis (1,2). Glucose oxidase is an intracellular enzyme exhibiting a high specificity for β-D-glucose. As the only sugar normally present in the bloodstream is D-glucose, immobilized glucose oxidase would seem ideally suited for determining blood sugar on a con tinuous basis during surgery or for diabetics. This report describes a practical system for the analysis of glucose concentration in both simple and complex biological fluids, using glucose oxidase enzyme immobilized by occlusion within a polymeric, hydrophilic matrix. In designing a system for such a purpose, there were two basic criteria to be con sidered. First, results must be rapidly obtained and be repro ducible. Second, flow through the system must be kept to a minimum since the ultimate use involves blood flow from a human body. In the system we have devised the oxygen concentration of a sample solution is continuously measured, after passing through immobilized glucose oxidase, by means of a commercially available polarographic electrode. The amount of oxygen consumed by oxid ation of glucose is related to the glucose concentration of the sample by means of a calibration graph. The linearity of the graph in the range of 10-150 ppm glucose, permits a single point calibration graph. The design of the electrode holder insures high linear velocity of liquid past the electrode membrane and results in a minimum pressure drop. Thus oxygen concentrations recorded are 98% or better of the true oxygen concentration of liquids. Both the procedure and associated hardware are simple in design and operation and have demonstrated exceptional operation al stability. Blood, continuously withdrawn from a dog and 162

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

12.

Ο DRISCOLL ET AL.

Monitoring of Blood Glucose

163

and d i l u t e d 3 0 - f o l d has been u s e d , and t h e s y s t e m has been s a t i s factorily operated f o r f i v e hours c o n t i n u o u s l y . With synthetic glucose solutions t h e s y s t e m has been o p e r a t e d f o r up t o 60 h o u r s c o n t i n u o u s l y w i t h o u t need f o r r e c a l i b r a t i o n . One r e a c t o r has been used w i t h t h e same g e l i n i t f o r more t h a n 200 h o u r s o v e r a t h r e e month p e r i o d w i t h o u t a n y d e t e c t a b l e l o s s o f e n z y m a t i c a c t i v i t y . The o n l y i n s t a b i l i t i e s o f t h e s y s t e m we have o b s e r v e d have been a s s o c i a t e d w i t h t h e e l e c t r o n i c e q u i p m e n t used and t e m p e r a t u r e v a r i a t i o n s r a t h e r t h a n w i t h t h e i m m o b i l i z e d enzyme c o l u m n . Work in progress i s aimed a t e l i m i n a t i n g s u c h p r o b l e m s s o t h a t t h i s system may become the glucose sensing p o r t i o n o f an a r t i f i c i a l pancreas. The S y s t e m The p r o t o t y p e system t h r e e p r i n c i p a l components i n s e q u e n c e : 1. An a e r a t i o n c o i l , c o n s i s t i n g o f 0 . 2 3 cm I.D. g l a s s t u b e , 45 cm l o n g , f o l l o w e d by a d e b u b b l e r . 2. A 0 . 5 cm I.D. f i x e d bed r e a c t o r , containing 1.2 gm ( d r y ) o f hydrated g e l i n which i s the immobilized glucose o x i d a s e . 3. The d e t e c t o r a s s e m b l y , i n c o r p o r a t i n g (in a specially designed electrode holder) a membrane covered polarographic e l e c t r o d e , (Beckman I n s t r u m e n t Company 39533 0 S e n s o r ) , f o r t h e m e a s u r e ment o f d i s s o l v e d o x y g e n i n t h e e f f l u e n t f r o m t h e r e a c t o r . Solution transport through t h e system i s achieved by a Watson-Marlow (Buckinghamshire, England), Multichannel, Variable Speed P e r i s t a l t i c Pump. 2

Procedure The g l u c o s e oxidase been d e s c r i b e d (3). Calibration

immobilization

i n a poly(HEMA) g e l has

Graph.

Sodium a c e t a t e b u f f e r solution (0.1 M, pH 5.6) containing 0.5% NaCl and 1.3 χ 1 0 % KCN i s pumped t h r o u g h t h e s y s t e m f o r a t l e a s t f i v e m i n u t e s and t h e oxygen c o n c e n t r a t i o n of the o u t l e t stream i s recorded. Synthetic g l u c o s e samples o f d i f f e r e n t c o n centrations a r e prepared, allowed t o e q u i l i b r a t e f o r twenty four h o u r s and t h e n pumped through t h e system a t a f i x e d f l o w r a t e o f c a . 1 ml/miη. The e f f l u e n t oxygen c o n c e n t r a t i o n o f t h e s e s o l u t i o n s i s c o n t i n u o u s l y recorded, t a k i n g approximately 3 - 5 minutes to reach a steady s t a t e . A graph of 0 concentration a t steady s t a t e , v£. glucose concentration, is linear and e s t a b l i s h e s t h e r e l a t i o n between total glucose concentration and p e r c e n t c o n v e r s i o n . Flow rate s h o u l d be c h e c k e d and m a i n t a i n e d c o n s t a n t f o r obvious reasons. F i g u r e 2 shows c a l i b r a t i o n c u r v e s f o r two r e a c t o r s containing _ l f

2

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slightly oxidase.

different

Serum

activity

levels

of

gel

entrapped

glucose

Samples.

Individual serum samples d i l u t e d t h i r t y - f o l d , thus having glucose c o n c e n t r a t i o n s between 0.01 - 0.15 mg/ml may be a n a l y z e d by t h e same method used t o o b t a i n t h e c a l i b r a t i o n g r a p h . The steady s t a t e oxygen c o n c e n t r a t i o n i s r e l a t e d to i n i t i a l total g l u c o s e c o n c e n t r a t i o n by means o f a c a l i b r a t i o n g r a p h . For the c a l i b r a t i o n graph, i t i s i m p o r t a n t t o use g l u c o s e solutions of the same i o n i c s t r e n g t h as t h e s o l u t i o n used for d i l u t i o n o f serum samples because d i s s o l v e d oxygen c o n c e n t r a t i o n i n l i q u i d s i s a f u n c t i o n o f i o n i c s t r e n g t h and because i o n s such as h a l i d e s d e p r e s s t h e a c t i v i t Continuous

Glucose A n a l y s i s

of

Blood.

Oxygen c o n c e n t r a t i o n o f a b l o o d s t r e a m i s much l o w e r t h a n t h e oxygen c o n c e n t r a t i o n o f a i r saturated liquids. Moreover, red c e l l s continue to m e t a b o l i z e thus consuming oxygen and g l u c o s e . Several chemicals e x i s t which depress or stop the r a t e of m e t a b o l ism o f red c e l l s . Some o f t h e s e chemicals also d e a c t i v a t e the immobilized enzyme, g l u c o s e o x i d a s e . Thus a s o l u t i o n t o be used for the d i l u t i o n of b l o o d must n o t c o a g u l a t e o r hemolyze t h e b l o o d , must m a i n t a i n a c o n s t a n t pH, and must n o t s e r i o u s l y i n h i b i t t h e enzyme a c t i v i t y and t h e r e b y a p p e a r to reduce the glucose c o n v e r s i o n o r oxygen c o n c e n t r a t i o n . R e c o g n i z i n g t h e s e c o n s t r a i n t s , t h e b l o o d s t r e a m , 0.05 m l / m i n . i s m i x e d w i t h 0.05 m l / m i n . o f h e p a r i n s o l u t i o n i n a c a t h e t e r and drawn o f f c o n t i n u o u s l y . T h i s s t r e a m , 0.1 m l / m i n . , i s m i x e d a t t h e beginning of the a e r a t i o n c o i l w i t h 1.15 m l / m i n . o f d i l u e n t s o l u tion containing the f o l l o w i n g chemicals: sodium a c e t a t e buffer 0.1 M, pH = 5 . 6 , 0.5% sodium c h l o r i d e , 0.2% s o d i u m f l u o r i d e , 0.13 mg/100 ml p o t a s s i u m cyanide. This solution satisfies t h e above criteria and has f u n c t i o n e d s a t i s f a c t o r i l y as a d i l u e n t s o l u t i o n f o r continuous blood glucose a n a l y s i s . P o t a s s i u m c y a n i d e i s added to depress the a c t i v i t y of c a t a l a s e , e x i s t i n g i n the blood stream, which c a t a l y z e s t h e d e c o m p o s i t i o n o f hydrogen p e r o x i d e t o oxygen and w a t e r , t h u s introducing a serious e r r o r i n measurement o f s t e a d y s t a t e oxygen c o n c e n t r a t i o n of the r e a c t a n t stream. Sodium f l u o r i d e suppresses the red c e l l a c t i v i t y toward oxygen. D i l u e n t s o l u t i o n and t h e b l o o d s t r e a m a r e m i x e a w i t h 2.5 m l / m i n . a i r and t r a n s p o r t e d t o t h e r e a c t o r t h r o u g h t h e a e r a t i o n c o i l and a s s o c i a t e d t u b i n g . The r e a c t o r c o n t a i n e d 1.3 gms ( d r y ) g e l w i t h an a c t i v i t y o f 12 u n i t s / g m . D e b u b b l i n g and s t r e a m splitting takes p l a c e a t the i n l e t o f the i m m o b i l i z e d glucose oxidase r e actor. The r e a c t a n t s t r e a m goes t h r o u g h t h e packed bed r e a c t o r and t h e n t o t h e o x y g e n e l e c t r o d e f l o w c e l l . I t s oxygen c o n c e n t r a t i o n i s r e c o r d e d c o n t i n u o u s l y as a f u n c t i o n of time. Glucose

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ODRiscoLL E T A L .

Monitoring of Blood Glucose Air

1 1

AIR DILUENT SAMPLE

Gel Entrapped Enzyme

Waste

-Multi-Channel Peristaltic Pump Amplifier

Figure 1. Schematic of glucose analyzer with gel entrapped glucose oxidase

OOI

004

002

0 06

GLUCOSE SOLUTION,

Figure 2. Calibration

E v

0 08

010

mg/mf

plots for (a) reactor No. 1 and (b) reactor No. 2

F

^J 10 mg %

A

Β

Figure 3.

Monitoring of dog blood glucose

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

c o n c e n t r a t i o n o f the r e a c t i o n stream i s o b t a i n e d from the steady s t a t e oxygen c o n c e n t r a t i o n by means o f t h e c a l i b r a t i o n graph. There i s no a p p a r e n t e f f e c t o f t h e b l o o d on e i t h e r t h e oxygen e l e c t r o d e o r t h e i m m o b i l i z e d enzyme a c t i v i t y . Flexible t u b i n g f r o m t h e r e a c t o r t o t h e oxygen electrode f l o w c e l l must be k e p t as s h o r t as p o s s i b l e i n o r d e r t o reduce t h e oxygen diffusion from a i r to the r e a c t a n t stream, thus r e s u l t i n g i n a l o w e r s e n s i t i v i t y o f g l u c o s e measurements. Uncom p e n s a t e d e r r o r s may be i n t r o d u c e d i f t h e oxygen c o n c e n t r a t i o n o f the r e a c t a n t stream i s l e s s than a i r s a t u r a t i o n l e v e l s . T h i s can be c h e c k e d by on l i n e measurements o f oxygen concentration of this s t r e a m a f t e r d e b u b b l i n g , u s i n g a n o t h e r oxygen e l e c t r o d e or by b y p a s s i n g the stream from the g l u c o s e oxidase reactor a f t e r d e b u b b l i n g and r e c o r d i n g i t s oxygen c o n c e n t r a t i o n . Results

and

Discussion

F i g u r e 3 shows t h e r e s u l t s o f m o n i t o r i n g the blood glucose of a dog. P o i n t A represents the s t a b l e l e v e l a f t e r approximat e l y 3 hours o f m o n i t o r i n g . A t t h a t t i m e , the blood d i l u e n t being used was changed t o i n c l u d e 1% g l u c o s e , a r e s p o n s e being noted a f t e r a few m i n u t e s b e g i n n i n g a t p o i n t Β and s t a b i l i z i n g i n 10 minutes. The d i l u e n t was changed back to the glucose free solution a t C and s t e a d y s t a t e r e a c h e d a g a i n a t D. Over a t i m e p e r i o d o f 5 m i n u t e s (E - F) 5 gms o f g l u c o s e were i n j e c t e d i n t o t h e dog and t h e heightened blood s u g a r was c o n t r o l l e d by t h e d o g ' s p a n c r e a s ( n o t e t h e maximum a t p o i n t G ) . The e x p e r i m e n t was t e r m i n a t e d when t h e l e v e l had r e t u r n e d t o normal a t p o i n t H. T h i s e x p e r i m e n t , and many o t h e r s w i t h b l o o d s e r a , show t h e c o m p a t i b i l i t y o f t h e s y s t e m w i t h w h o l e b l o o d . The s o l u t i o n s used for diluent successfully compensate for the hemoglobin and c a t a l a s e c o n t e n t o f t h e b l o o d w i t h o u t a f f e c t i n g t h e enzyme a c t i v ity. No a d v e r s e " a d s o r p t i o n e f f e c t s " as d e s c r i b e d by G u i l b a u l t (4) have been o b s e r v e d w i t h t h e s e g e l s . Acknowledgement S u p p o r t o f t h i s r e s e a r c h by t h e N a t i o n a l R e s e a r c h C o u n c i l Canada i s a p p r e c i a t e d .

of

Literature Cited (1) Hicks, G.P., and Updike, S.J., Anal. Chem. (1966), 38, 726. (2) Weetall, H.H., Anal. Chem. (1974), 46, 602A. (3) Hinberg, I., Kapoulas, Α., Korus, R., and O'Driscoll, K.F., Biotech. & Bioeng. (1974), 16, 159. (4) Guilbault, G.G., Chem. and Eng. News (October 2, 1972), 45.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13 The Slow Release of Hydrocortisone Sodium Succinate from Poly(2-hydroxyethyl methacrylate) Membranes J.M.ANDERSON, T. KOINIS,T.NELSON,M.HORST, andD.S.LOVE Department of Macromolecular Science and Department of Anatomy, Case Western Reserve University, Cleveland Ohio 44106

Love and c o - w o r k e r s have s u g g e s t e d t h a t t h e e n d o c r i n e system p l a y s a key r o l and s k e l e t a l m u s c l e d i f f e r e n t i a t i o To d e t e r m i n e t h e v a l i d i t y o f t h i s h y p o t h e s i s , a t t e m p t s t o i n j e c t i n t r a v e n o u s l y s e r i a l d o s e s o f s p e c i f i c hormonal a g e n t s i n t o h y p o p h y s e c t o m i z e d c h i c k embryos were made. T h i s method o f d r u g d e l i v e r y was u n s a t i s f a c t o r y as dosage l e v e l s were v a r i a b l e , s e r i a l i n j e c t i o n i n t o t h e c h o r i o a l l a n t o i c membrane was d i f f i c u l t and c o n s t a n t m a n i p u l a t i o n o f t h e c h i c k embryo r e s u l t e d i n a h i g h r a t e o f m o r t a l i t y . These d i f f i c u l t i e s were overcome by u s i n g h y d r o g e l membranes w h i c h gave a s l o w , s u s t a i n e d r e l e a s e o f d r u g t o t h e c h i c k embryo. This paper, t h e f i r s t o f a s e r i e s , w i l l d e a l w i t h t h e d e v e l o p m e n t and i n v i t r o a n d i n v i v o r e l e a s e b e h a v i o r o f h y d r o c o r t i s o n e sodium s u c c i n a t e from c r o s s l i n k e d p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) membranes. In r e c e n t y e a r s , t h e c o n c e p t o f a s u s t a i n e d r e l e a s e s y s t e m , w h i c h , upon i m p l a n t a t i o n i n t h e b o d y , w o u l d r e l e a s e b i o l o g i c a l l y a c t i v e l e v e l s o f d r u g f o r p r o l o n g e d t i m e p e r i o d s has r e c e i v e d i n c r e a s e d a t t e n t i o n . S u s t a i n e d r e l e a s e s y s t e m s have been d e v e l o p e d and examined f o r t h e i r p o t e n t i a l a p p l i c a t i o n i n t h e t r e a t m e n t o f glaucoma ( 3 ) , t r i c h o m a ( 4 ) , n a r c o t i c a d d i c t i o n ( 5 J , m a l a r i a ( 6 J , d i a b e t e s ( 7 J , c a n c e r ( 8 ) and c o n t r a c e p t i o n (9_» 1 0 , 11). Most o f t h e s e s y s t e m s have used s i l i c o n e r u b b e r a s t h e m a t r i x m a t e r i a l i n w h i c h t h e d r u g was e i t h e r embedded o r contained. I n a d d i t i o n , s i l i c o n e r u b b e r has been e x t e n s i v e l y s t u d i e d i n r e g a r d s t o i t s t r a n s p o r t p r o p e r t i e s and t h o s e f a c t o r s which c o n t r o l o r a l t e r t h e t r a n s p o r t o f drugs through t h e matrix (12, 1 3 , H ) . P o l y T 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) and o t h e r h y d r o g e l t y p e p o l y m e r s have a l s o been e x a m i n e d f o r p o t e n t i a l u s e i n s u s t a i n e d d e l i v e r y s y s t e m s (]_5). As t h e d i f f u s i v i t y o f d r u g s f r o m t h e p o l y m e r membranes i s p r o b a b l y t h e most i m p o r t a n t c r i t e r i a f o r the polymer, a s i d e from i t s b i o l o g i c a l c o m p a t i b i l i t y , t h e h y d r o g e l m a t e r i a l s have e x c e l l e n t p o t e n t i a l a s t h e i r p h y s i c a l

167

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

c h a r a c t e r i s t i c s (degree o f h y d r a t i o n , c r o s s l i n k d e n s i t y , p o r o s i t y , e t c . ) can be e a s i l y a l t e r e d and c o n t r o l l e d t o v a r y the r a t e o f drug d i f f u s i o n . Through c o n t r o l l e d a l t e r a t i o n o f t h e amount o f c r o s s ! i n k i n g a g e n t , monomer t o w a t e r r a t i o , and the c o n d i t i o n s of p o l y m e r i z a t i o n , hydrogel s t r u c t u r e s ranging f r o m compact g e l s t o c e l l u l a r sponges w i t h v a r y i n g p h y s i c a l p r o p e r t i e s c a n be o b t a i n e d (16», 1 7 ) . L e v o w i t z and c o - w o r k e r s have shown t h a t s u t u r e s and c a t h e t e r s c o a t e d w i t h an e t h y l e n e g l y c o m e t h a c r y l a t e g e l i n w h i c h c e p h a l o t h i n was i n c o r p o r a t e d d e c r e a s e d t h e r a t e o f i n d u c e d i n f e c t i o n when used i n a n i m a l s t u d i e s (18.). In v i t r o r e l e a s e r a t e s were shown f o r n o r e t h a n d r o l o n e and 5 - f l u o r o u r a c i l and r e f e r e n c e was made t o t h e s u s t a i n e d r e l e a s e o f a n t i b i o t i c s , l o c a l a n e s t h e t i c s and C o r t i s o l . Human s t u d i e s u s i n g Hydron ® cephalothin urethral catheter o f b a c t e r i u r i a t h a t wa controls. In a d d i t i o n t o s e r v i n g as a v e h i c l e f o r t h e d e l i v e r y o f a w i d e v a r i e t y o f a n t i b i o t i c s s u c h as n e o m y c i n , a m p i c i l l i n , t e t r a c y c l i n e , e t c . , Hydron © c o a t e d c a t h e t e r s i n w h i c h c e p h a l o t h i n was a b s o r b e d were e f f i c a c i o u s a g a i n s t s u s c e p t i b l e b a c t e r i a a f t e r b e i n g s t o r e d s t e r i l l y f o r up t o s i x months ( 1 9 ) . The use o f s o f t c o n t a c t l e n s e s f o r p r o l o n g i n g t h e a c t i o n of drugs a d m i n i s t e r e d d r o p - w i s e o r f o r s u s t a i n e d r e l e a s e o f d r u g s has been i n v e s t i g a t e d . Kaufman and c o - w o r k e r s have shown t h a t s o f t c o n t a c t l e n s e s can p r o l o n g t h e t h e r a p e u t i c a c t i o n o f p o l y m i x i n B, α - p h e n y l e p h r i n e , and p i l o c a r p i n e when t h e s e d r u g s a r e a d m i n i s t e r e d d r o p - w i s e t o human and a n i m a l e y e s . In a d d i t i o n , these s t u d i e s p o i n t out the f a c t t h a t n e a r - t o x i c drug l e v e l s do n o t have t o be a d m i n i s t e r e d t o p r o v i d e t h e r a p e u t i c e f f i c a c y when s o f t c o n t a c t l e n s e s a r e used t o m e d i a t e and p r o l o n g t h e d e l i v e r y o f d r u g s ( 2 0 ) . The above a u t h o r s were a l s o a b l e t o show t h a t c o r n e a l u l c e r s i n r a b b i t e y e s , p r e v i o u s l y i n f e c t e d w i t h McKrae h e r p e s v i r u s , h e a l e d f a s t e r w i t h 5 - i o d o - 2 - d e o x y u r i d i n e d r o p s m e d i a t e d by a s o f t c o n t a c t l e n s t h a n w i t h 5 - i o d o - 2 d e o x y u r i d i n e drops a l o n e . Podos e t a l . have i n v e s t i g a t e d t h e a d m i n i s t r a t i o n o f p i l o c a r p i n e , t o d e c r e a s e o c u l a r h y p e r t e n s i o n and r e l i e v e m i o s i s and o t h e r glaucoma symptoms, by t h e use o f s o f t c o n t a c t l e n s e s p r e - s o a k e d i n p i l o c a r p i n e (2j_). T h e i r r e s u l t s a r e e n c o u r a g i n g and p o i n t t o t h e need f o r f u r t h e r work i n r e t a r d i n g t h e r a t e o f drug r e l e a s e from presoaked l e n s e s . Pre-soaking the l e n s e s , w h i l e p r o v i d i n g f o r easy p r e p a r a t i o n , u s u a l l y r e s u l t s i n too rapid release of the drug. Abrahams and Ronel (22) u s i n g p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) h y d r o g e l s , have e x a m i n e d t h e e f f e c t t h a t c o p o l y m e r c o m p o s i t i o n and i o n o g e n i c g r o u p s on t h e h y d r o g e l c h a i n have on the i n v i t r o release behavior of c y c l a z o c i n e , a n a r c o t i c a n t a g o n i s t , f r o m s u c h v a r i e d f o r m s as c a p s u l e s , b a r r i e r - f i l m c o a t e d t a b l e t s and b u l k p o l y m e r i z e d r o d s . Using capsular devices t h e r e l e a s e o f c y c l a z o c i n e c o u l d be i n c r e a s e d m a r k e d l y by

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13.

ANDERSON ET AL.

Hydrocortisone

Sodium

Succinate

169

a d j u s t i n g t h e i o n o g e n i c group c o n t e n t ( m e t h a c r y l i c a c i d ) o f t h e h y d r o g e l f r o m 0 . 3 % t o 2.4%. Homogeneous b u l k p o l y m e r i z e d r o d s t o w h i c h a c o a t i n g o f a c o p o l y m e r o f 80% e t h o x y e t h y l m e t h a c r y l a t e and 20% h y d r o x y e t h y l m e t h a c r y l a t e has been a p p l i e d , p r o v i d e d z e r o - o r d e r r e l e a s e i n e x c e s s o f one month whereas u n c o a t e d r o d s e x h i b i t e d a f i r s t - o r d e r r e l e a s e p a t t e r n w i t h t h e t o t a l amount o f c y c l a z o c i n e b e i n g r e l e a s e d i n l e s s t h a n 10 d a y s . O t h e r h y d r o g e l s y s t e m s s u c h a s p o l y a c r y l a m i d e and p o l y v i n y l p y r r o l i d o n e have a l s o been examined f o r t h e i r p o t e n t i a l i n p r o v i d i n g s u s t a i n e d r e l e a s e (23^, 2 4 , 2 5 ) . These h y d r o g e l s have been shown t o r e l e a s e s u c h b i o l o g i c a l l y a c t i v e s u b s t a n c e s as p r o s t a g l a n d i n s , i m m u n o g l o b u l i n s , l u t e i n i z i n g hormone, a l b u m i n , i n s u l i n , and e t h i n y l o e s t r a d i o l . In summary, h y d r o p h i l i c p o l y m e r s s u c h a s p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) have p o t e n t i a d e l i v e r y systems. The systems f o r s u s t a i n e d o r slow r e l e a s e o f b i o l o g i c a l l y a c t i v e a g e n t s a r e t h e e a s e by w h i c h t h e h y d r o g e l s c a n be c h e m i c a l l y modified t o control t h e i r physical properties, i . e . , t h e i r r e l e a s e behavior, the minimal formation o f f i b r o u s capsule which may impede d r u g r e l e a s e i n v i v o , and t h e a b i l i t y t o r e l e a s e p o l a r and l a r g e m o l e c u l a r w e i g h t compounds s u c h a s p e p t i d e hormones. However, f u r t h e r c h a r a c t e r i z a t i o n o f t h e r e s p e c t i v e h y d r o g e l s i s n e c e s s a r y and much r e m a i n s t o be done i n i n v e s t i g a t i n g those f a c t o r s such as h y d r a t i o n , c r o s s l i n k d e n s i t y , p o r o s i t y , drug-polymer i n t e r a c t i o n s , e t c . which w i l l c o n t r o l t h e r e l e a s e o f any d r u g . M a t e r i a l s and Methods F i l m s c o n t a i n i n g h y d r o c o r t i s o n e s u c c i n a t e were p r e p a r e d by f i l m c a s t i n g polymer s o l u t i o n s c o n t a i n i n g h y d r o c o r t i s o n e sodium s u c c i n a t e , ^ - h y d r o c o r t i s o n e s o d i u m s u c c i n a t e , Hydron © p o l y m e r Type N, a n d , when d e s i r e d , known amounts o f p h o t o s e n s i t i v e p o l y m e r c r o s s ! i n k i n g a g e n t , ammonium d i c h r o m a t e . F o r each f i l m , nanograms o f h y d r o c o r t i s o n e sodium s u c c i n a t e p e r m i l l i g r a m d r y f i l m and m i l l i g r a m s o f p o l y m e r c r o s s l i n k e r p e r m i l l i g r a m o f d r y f i l m were c a l c u l a t e d . The f i l m s were c a s t on g l a s s p l a t e s and when f i l m s c o n t a i n i n g t h e p h o t o s e n s i t i v e c r o s s l i n k i n g a g e n t were p r e p a r e d , f i l m c a s t i n g was c a r r i e d o u t i n a d a r k r o o m u s i n g a W r a t t e n OA f i l t e r . C r o s s l i n k e d p o l y m e r f i l m s were p r e p a r e d by i r r a d i a t i n g w i t h u l t r a v i o l e t l i g h t t h o s e f i l m s w h i c h c o n t a i n e d t h e p h o t o s e n s i t i v e c r o s s l i n k i n g a g e n t f o r 18 h o u r s a t 26°C. Hydron © p o l y m e r - Type Ν and t h e p h o t o s e n s i t i v e c r o s s l i n k i n g a g e n t , ammonium d i c h r o m a t e , were g r a c i o u s l y p r o v i d e d by D r . Sam Rone! o f t h e Hydron L a b o r a t o r i e s , I n c . , New B r u n s w i c k , New J e r s e y . H y d r o c o r t i s o n e s o d i u m s u c c i n a t e was o b t a i n e d a s S o l u - C o r t e f f r o m t h e U p j o h n Company, K a l a m a z o o , M i c h i g a n , a n d 3 H - h y d r o c o r t i s o n e sodium s u c c i n a t e was p u r c h a s e d f r o m New England N u c l e a r , Boston, Massachusetts.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

170

D i s c s f o r t h e d i f f u s i o n s t u d i e s were p r e p a r e d by r e s w e l l i n g t h e p o l y m e r f i l m s s l i g h t l y w i t h e t h a n o l ( t o make t h e f i l m s p l i a b l e ) and t h e d i s c s were t h e n c u t f r o m t h e f i l m . The d i s c d i a m e t e r was 0.61 cm (240 m i l s ) and t h e d r y t h i c k n e s s v a r i e d f r o m 0 . 0 2 3 t o 0.028 cm (9-11 m i l s ) . Wet t h i c k n e s s e s were measured i m m e d i a t e l y f o l l o w i n g t h e c o m p l e t i o n o f e a c h d i f f u s i o n s t u d y and v a r i e d f r o m 0 . 0 3 3 cm t o 0.041 cm ( 1 3 - 1 6 m i l s ) . T a b l e 1 summarizes t h e d a t a on t h e i n v i t r o h y d r o c o r t i s o n e sodium s u c c i n a t e d i s c s and i n c l u d e s nanograms o f h y d r o c o r t i s o n e sodium s u c c i n a t e per m i l l i g r a m dry d i s c , weight percent c r o s s l i n k e r i n t h e r e s p e c t i v e d i s c s , and t o t a l nanograms o f h y d r o c o r t i s o n e sodium s u c c i n a t e per d i s c . To most c l o s e l y a p p r o x i m a t e t h e i n v i v o d i f f u s i o n s y s t e m i n which a d i s c o f the polymer f i l m c o n t a i n i n g the d e s i r e d drug i s p l a c e d on a s m a l l c u embryo, t h e f o l l o w i n g i t w e n t y - f i v e ml E r l e n m e y e r f l a s k w i t h a g r o u n d f l a t t o p and s i d e arm f o r s a m p l e r e m o v a l was u s e d . A c e l l u l o s e mi H i p o r e f i l t e r ( 0 . 6 5 ym p o r e d i a m e t e r ) was s e a l e d w i t h a c e t o n e t o t h e t o p o f t h e E r l e n m e y e r t o f u n c t i o n as a p o r o u s s u p p o r t f o r t h e p o l y m e r d i s c and t h e s i d e arm was capped w i t h a r u b b e r s t o p p e r . The E r l e n m e y e r was f i l l e d w i t h R i n g e r ' s s o l u t i o n and t h e p o l y m e r d i s c was p l a c e d on t h e m i l l i p o r e f i l t e r . A glass cap, to prov i d e a c o n s t a n t h u m i d i t y and t e m p e r a t u r e e n v i r o n m e n t was p l a c e d o v e r t h e m i l l i p o r e f i l t e r and p o l y m e r d i s c . The i n v i t r o d i f f u s i o n a p p a r a t i were t h e n p l a c e d i n a 38°C i n c u b a t o r and removed o n l y f o r s a m p l i n g p u r p o s e s . One ml a l i q u o t s were removed f r o m t h e E r l e n m e y e r s a t e a c h t i m e p o i n t and t h e E r l e n m e y e r s r e f i l l e d w i t h R i n g e r ' s s o l u t i o n t o b r i n g t h e s o l u t i o n l e v e l s up to the m i l l i p o r e f i l t e r . A l l i n v i t r o d i f f u s i o n experiments were r u n i n t r i p l i c a t e . D i f f u s i o n o f t h e t r i t i a t e d h y d r o c o r t i s o n e s u c c i n a t e was d e t e r m i n e d as f o l l o w s : 1 ml a l i q u o t s o f t h e d e s o r b i n g Howard R i n g e r s s o l u t i o n were w i t h d r a w n f r o m t h e i n v i t r o s y s t e m s a t a p p r o p r i a t e t i m e i n t e r v a l s and m i x e d w i t h 5 ml o f T r i t o n X-100 toluene s c i n t i l l a t i o n f l u o r in a glass s c i n t i l l a t i o n v i a l . C o u n t i n g was c a r r i e d o u t i n a S e a r l e I s o c a p 300 preprogrammed f o r t r i t i u m u s i n g samples c h a n n e l s r a t i o . Results

and

Discussion

The d i f f u s i o n o f h y d r o c o r t i s o n e s o d i u m s u c c i n a t e f r o m h y d r o g e l d i s c s v a r y i n t h e i r c r o s s l i n k d e n s i t y i s shown i n F i g u r e 1. In g e n e r a l , t h e d i f f u s i o n c u r v e s show a r a p i d r e l e a s e o f h y d r o c o r t i s o n e sodium s u c c i n a t e i n t h e e a r l y p a r t o f t h e e x p e r i m e n t , 0 t o 24 h o u r s . This rapid release is d e c r e a s e d by i n c r e a s i n g t h e c r o s s l i n k d e n s i t y and a t 24 h o u r s , t h e 0% c r o s s l i n k e d d i s c s have r e l e a s e d c a . 75% o f t h e s t e r o i d , t h e 5.1% c r o s s l i n k e d d i s c s have r e l e a s e d c a . 30% o f t h e s t e r o i d , and t h e 9.9% c r o s s l i n k e d d i s c s have r e l e a s e d c a . 16% o f t h e steroid. I t i s a p p a r e n t t h a t t h e 0% c r o s s l i n k e d d i s c s have a f i r s t order r e l e a s e p a t t e r n i n which the r a t e of s t e r o i d

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13.

ANDERSON ET AL.

Hydrocortisone

Sodium

Succinate

171

d i f f u s i o n i s d e p e n d e n t upon t h e amount o f s t e r o i d s t i l l c o n tained i n the disc. I n a d d i t i o n , t h e 0% c r o s s l i n k e d d i s c s have r e l e a s e d c a . 90% o f t h e s t e r o i d w i t h i n 4 8 h o u r s . F i g u r e 2 shows e i g h t d a y r e l e a s e p a t t e r n s f o r t h e c r o s s l i n k e d d i s c s w i t h a t w e l v e d a y r e l e a s e p a t t e r n shown f o r t h e 9.9% c r o s s l i n k e d d i s c s . T h e s e d a t a a r e an e x t e n s i o n o f t h o s e shown i n F i g u r e 1 a n d s u p p o r t t h e s u g g e s t i o n t h a t t h e 9 . 9 % crosslinked discs e x h i b i t a nearly constant rate o f steroid release. The c u r v e f o r t h e 9.9% c r o s s l i n k e d d i s c s i s n o t s t r i c t l y l i n e a r b u t does shown l i n e a r i t y f r o m d a y 3 t o d a y 12 of t h e experiment. Since t h e i n vivo experiments r e q u i r e s t e r o i d r e l e a s e f o r a p e r i o d o f e i g h t d a y s , d a y 10 t o d a y 18 o f t h e c h i c k embryo g e s t a t i o n p e r i o d , i t a p p e a r s t h a t t h e 9 . 9 % c r o s s l i n k e d d i s c s c a n s u c c e s s f u l l y be used t o d e l i v e r a r e l a t i v e l y c o n s t a n t amount f steroid da f o t h i tim period. To d e t e r m i n e t h e e f f e c t o f t h e i n i t i a l c o n c e n t r a t i o n o f s t e r o i d i n t h e d i s c on t h e s t e r o i d r e l e a s e p a t t e r n , 5% c r o s s l i n k e d d i s c s c o n t a i n i n g 5 0 , 5 0 0 nanograms o f s t e r o i d p e r m i l l i g r a m o f d i s c , 2,140 nanograms o f s t e r o i d p e r m i l l i g r a m o f d i s c and 1,090 nanograms o f s t e r o i d p e r m i l l i g r a m o f d i s c were prepared and t h e i r r e s p e c t i v e r e l e a s e p a t t e r n s examined o v e r a f i v e day p e r i o d . The i n i t i a l c o n c e n t r a t i o n o f s t e r o i d a p p e a r e d t o have l i t t l e e f f e c t on t h e p e r c e n t r e l e a s e d p e r t i m e interval. In an e f f o r t t o p l a c e t h e e x p e r i m e n t a l r e s u l t s on a q u a n t i f i a b l e b a s i s f o r t h e purpose o f d e t e r m i n i n g d i f f u s i o n c o e f f i c i e n t s , a q u a s i - s t e a d y s t a t e approacn was t a k e n . This method was i n i t i a l l y p l a c e d on a f i r m s c i e n t i f i c b a s i s by B a r n e s ( 2 6 ) a n d r e c e n t l y G a r r e t t and Chemburkor {27) have a p p l i e d t h i s technique t o drug d i f f u s i o n across polymer membranes. A p p l i e d t o o u r i n v i t r o system, t h i s approach r e q u i r e s t h a t (1) t h e c o n c e n t r a t i o n s o f the d i f f u s i n g d i s c and the desorbing s o l u t i o n s a r e n o t held c o n s t a n t ; (2) t h e c o n c e n t r a t i o n o f the d i f f u s i n g d i s c , i n i t i a l l y Co, decreases as t h e c o n c e n t r a t i o n , Cj-, o f t h e d e s o r b i n g s o l u t i o n i n c r e a s e s ; a n d (3) t h e c o n c e n t r a t i o n v a l u e s o f b o t h t h e d i s c a n d t h e d e s o r b i n g s o l u t i o n a p p r o a c h t h e same e q u i l i b r i u m v a l u e w i t h t i m e . In t h e a p p l i c a t i o n o f t h i s method, Barnes s t r e s s e d t h a t t h e r a t i o o f t h e membrane volume t o d e s o r b i n g s o l u t i o n volume s h o u l d be l e s s than 0.1. S i n c e o u r membrane v o l u m e / d e s o r b i n g s o l u t i o n volume r a t i o i s 0 . 0 0 3 , t h e method s h o u l d be a p p l i c a b l e and i n t h e a b s e n c e o f anomalous p a r t i t i o n i n g e f f e c t s a l l t h e d r u g i n t h e membrane w i l l e v e n t u a l l y d i f f u s e i n t o t h e d e s o r b i n g s o l u t i o n . The f o l l o w i n g e q u a t i o n f o r t h e q u a s i - s t e a d y - s t a t e d i f f u s i o n was used where V] i s t h e volume o f t h e d e s o r b i n g s o l u t i o n , c a . 30 m i s , V2 i s t h e volume o f t h e d i f f u s i o n d i s c , c a . 0.01 m l , C i s t h e i n i t i a l c o n c e n t r a t i o n o f s t e r o i d in the diffusing disc, Ct i s theconcentration of steroid i n t h e desorbing s o l u t i o n a t time t , X i s t h e thickness o f t h e 0

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Figure 2. The effect of crosslinking on hydrocortisone succinate in vitro release. Percent by weight crosslinker: O, 0%;Ç), 1.0%; • ,

5.1%; and V,9.9%.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13.

173

Hydrocortisone Sodium Succinate

ANDERSON E T AL.

s w o l l e n d i s c , S i s t h e s u r f a c e a r e a o f t h e d i s c and D i s t h e apparent d i f f u s i o n constant: D S In o

X V.

I t

F i g u r e 3 shows t h e p l o t s o f l o g C V 2 / C V 2 - V - | C ç v e r s u s t i m e i n d a y s and t h e l i n e a r i t y f o r e a c h g i v e n c r o s s - l i n k d e n s i t y i s c o n s i s t e n t w i t h t h e e x p e c t a t i o n s o f t h e above e q u a t i o n f o r quasi-steady state d i f f u s i o n were c a l c u l a t e d f r o m t h T a b l e 1. 0

TABLE 1: Nanograms per Milligram Disc

1,090 2,140 5,000 5,200 5,350 4,750 4,990 4,640 50,500

0

IN VITRO HYDROCORTISONE-SUCCINATE DISCS

Total Nanograms per Disc

13 DxlO L/cm s e c . +

Weight% Crosslinker

7,700 9,800 24,000 28,100 25,300 30,200 28,300 20,900 293,000

5.0 5.0 0 0.1 0.5 1.0 5.0 9.9 4.9

51.0 42.0 213.0 90.0 44.0 8.3 43.0

Y a s u d a , Lamaze, a n d I k e n b e r r y ( 2 8 ) have shown t h a t d i f f u s i o n t h r o u g h h y d r o p h i l i c p o l y m e r s , s u c h as p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) , c a n be d e s c r i b e d by t h e f r e e volume theory of d i f f u s i o n . In t h e f r e e volume t h e o r y , t h e d i f f u s i o n c o e f f i c i e n t c a n be e x p r e s s e d by D <* e x p - ( V * / V f ) where V f i s t h e f r e e volume i n t h e s a m p l e and V * i s a c h a r a c t e r i s t i c volume r e q u i r e d t o accomodate t h e d i f f u s i n g s t e r o i d m o l e c u l e s . A s s u m i n g a l i n e a r v a r i a t i o n o f t h e f r e e volume w i t h t h e h y d r a t i o n , H, and an i n v e r s e l i n e a r v a r i a t i o n between t h e h y d r a t i o n and t h e c r o s s l i n k d e n s i t y , o r i n o u r c a s e t h e w e i g h t p e r c e n t c r o s s l i n k e r , W, t h e f o l l o w i n g e q u a t i o n r e s u l t s : log D = log D

ô

- Κ · W + κ'

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

174

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where Κ i s a c o n s t a n t . The v a l i d i t y o f t h e above e q u a t i o n w i t h i t s a c c o m p a n y i n g a s s u m p t i o n s i s s u p p o r t e d by t h e l i n e a r i t y o f the p l o t log D versus W using the apparent d i f f u s i o n constants f o u n d i n T a b l e 1 f o r t h e 5,000 ng p e r mg d r y f i l m membranes (Figure 4). As Love and c o w o r k e r s (]_, 2) have p r e v i o u s l y shown, t h e f e t a l e n d o c r i n e system p l a y s an i m p o r t a n t r o l e i n t h e d e v e l o p ment o f s k e l e t a l m u s c l e i n c h i c k embryos by r e g u l a t i n g t h e r a t e o f myoblast p r o l i f e r a t i o n , the f u s i o n o f myoblasts t o form m y o t u b e s , and t h e m a t u r a t i o n o f myotubes t o f o r m m a t u r e m u s c l e fibers. We have c h o s e n t o examine t h i s c o n v e r s i o n f r o m m y o b l a s t t o myotube t o m u s c l e by m e a s u r i n g t h e e f f e c t t h a t t h e s l o w r e l e a s e d h y d r o c o r t i s o n e s u c c i n a t e has on t h e m u s c l e enzymes: p h o s p h o r y l a s e , p h o s p h o g l u c o m u t a s e , and g l u c o s e - 6 - p h o s p h a t e dehydrogenase. In g e n e r a l t h a c t i v i t f glucose-6-phosphat dehydrogenase i n the d e v e l o p i n o f t h e r a t e o f DNA s y n t h e s i s and t h u s r o u g h l y measures t h e m y o b l a s t r e p l i c a t i o n o r p r o l i f e r a t i o n . On t h e o t h e r h a n d , p h o s p h o r y l a s e and p h o s p h o g l u c o m u t a s e a c t i v i t i e s i n d i c a t e t h e l e v e l of anaerobic metabolism i n the developed muscle. Thus, d e v e l o p m e n t and m a t u r a t i o n o f t h e e m b r y o n i c c h i c k m u s c l e c a n be shown by i n c r e a s i n g a c t i v i t y o f p h o s p h o r y l a s e and p h o s p h o g l u c o m u t a s e and d e c r e a s i n g a c t i v i t y o f g l u c o s e - 6 - p h o s p h a t e dehydrogenase. In o r d e r t o t e s t t h e a b i l i t y o f t h e h y d r o c o r t i s o n e s u c c i n a t e - H E M A d i s c s t o f u n c t i o n b i o l o g i c a l l y i n an i n v i v o s y s t e m , r e p r e s e n t a t i v e d i s c s were i m p l a n t e d on h y p o p h y s e c t o m i z e d c h i c k embryo c h o r i o a l l a n t o i c membranes a t day 10 o f t h e g e s t a t i o n p e r i o d . Comparison w i t h t h r e e d i f f e r e n t t y p e s o f embryos were made: normal e m b r y o s , embryos i n w h i c h t h e p i t u i t a r y g l a n d s were removed ( h y p o p h y s e c t o m i z e d ) a t day 2 o f t h e g e s t a t i o n p e r i o d , and embryos w h i c h were h y p o p h y s e c t o m i z e d a t day 2 and a t day 14 a p i t u i t a r y g l a n d was t r a n s p l a n t e d t o t h e embryo. A l l embryos were s a c r i f i c e d a t day 18 o f t h e g e s t a t i o n p e r i o d , and t h e e n z y m a t i c a c t i v i t i e s o f p h o s p h o r y l a s e , p h o s p h o g l u c o m u t a s e , and g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e were d e t e r m i n e d . Data on t h e e f f e c t o f h y d r o c o r t i s o n e - s u c c i n a t e p o l y ( 2 h y d r o x y e t h y l m e t h a c r y l a t e ) i m p l a n t s on e n z y m a t i c a c t i v i t y i n d e v e l o p i n g muscle a r e found i n T a b l e 2. As t h e c o n c e n t r a t i o n of hydrocortisone s u c c i n a t e i n the implanted d i s c i s i n c r e a s e d , t h e a c t i v i t i e s o f p h o s p h o r y l a s e and p h o s p h o g l u c o m u t a s e a l s o i n c r e a s e s u g g e s t i n g t h a t t h e e f f e c t o f hypophysectomy i s r e v e r s e d by t h e s e s l o w - r e l e a s e i m p l a n t s . A t t h e same t i m e , the a c t i v i t y o f g l u c o s e - 6 - p h o s p h a t e dehydrogenase i s d e c r e a s e d . T h i s t r e n d , as d i s c u s s e d e a r l i e r , i s i n d i c a t i v e o f a d e c r e a s e d r a t e o f DNA s y n t h e s i s o r o f m y o b l a s t p r o l i f e r a t i o n . T h u s , t h e h y d r o c o r t i s o n e s u c c i n a t e r e v e r s e s t h e e f f e c t o f hypophysectomy.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ANDERSON ET AL.

j I

Hydrocortisone

Sodium

Succinate

ι

ι

5

10

WEIGHT PERCENT CROSS-LINKER

Figure 4. Free volume diffusion—variation of the apparent diffusion con stant, D, with the weight percent crosslinker

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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176

TABLE 2:

EFFECT OF HYDROCORTISONE SUCCINATE HEMA IMPLANTS ON ENZYMATIC ACTIVITY IN DEVELOPING MUSCLE Phosphoryll a s e b*

Phosphoglucoroutase*

Normal

273

2200

12

Hydrocortisone Succinate 41000 20000 2000

390 170 101

1970 1330 610

20 31 60

82

420

63

152

1000

30

Hypophysectomized Pituitary Transplant * **

Glucose-6-P0^ Dehydrogenase

Nanomoles p e r m i n u t e p e r m i l l i g r a m DNA a t day 1 8 . T o t a l nanograms i n p o l y m e r i m p l a n t , 0% c r o s s l i n k i n g .

F i n a l l y , t o t e s t t h e v a l i d i t y o f o u r i n v i t r o s y s t e m and i t s a b i l i t y t o i m i t a t e t h e i n v i v o s y s t e m , d i f f u s i o n e x p e r i m e n t s were c a r r i e d o u t u s i n g 2 , 0 0 0 ng h y d r o c o r t i s o n e s u c c i n a t e p e r mg d r y f i l m d i s c s d e s c r i b e d i n T a b l e 1. The i n v i t r o d i s c s c o n t a i n e d 5% c r o s s l i n k e r and t h e i n v i v o d i s c s c o n t a i n e d 0% c r o s s l i n k e r . I t i s e a s i l y seen i n F i g u r e 5 t h a t t h e i n v i v o d i s c s r e l e a s e t h e d r u g a t a s l o w e r r a t e t h e n do t h e i n v i t r o d i s c s even t h o u g h the i n vivo d i s c s c o n t a i n no c r o s s l i n k e r and w o u l d be e x p e c t e d to r e l e a s e the h y d r o c o r t i s o n e s u c c i n a t e a t a f a s t e r r a t e than t h e i n v i t r o d i s c s w h i c h c o n t a i n 5% c r o s s l i n k e r . Microscopic e x a m i n a t i o n o f t h e i m p l a n t e d d i s c and t h e u n d e r l y i n g c h o r i o a l l a n t o i c membrane d u r i n g t h e e x p e r i m e n t and up t o t e n d a y s i m p l a n t a t i o n r e v e a l s no f i b r o u s c a p s u l e f o r m a t i o n w h i c h m i g h t decrease the r a t e of r e l e a s e . In a d d i t i o n , t h e v a s c u l a r bed i n t h e c h o r i o a l l a n t o i c membrane u n d e r l y i n g t h e i m p l a n t e d d i s c a p p e a r s t o be w e l l d e v e l o p e d and does n o t a p p e a r t o undergo any change d u r i n g t h e p e r i o d o f i m p l a n t a t i o n . F u r t h e r c o n f i r m a t i o n o f t h i s i s c u r r e n t l y b e i n g o b t a i n e d by h i s t o l o g i c a l e x a m i n a t i o n o f t h e c h o r i o a l l a n t o i c membrane. In summary, p o l y ( 2 - h y d r o x y e t h y l m e t h a c r y l a t e ) membranes c o n t a i n i n g h y d r o c o r t i s o n e s u c c i n a t e have been shown t o s l o w l y r e l e a s e s t e r o i d i n v i t r o and i n v i v o . This r e l e a s e i s depende n t upon t h e w e i g h t p e r c e n t o f c r o s s l i n k e r o f t h e i m p l a n t e d d i s c . Further experiments are being conducted to e l u c i d a t e the r o l e of membrane h y d r a t i o n , c o n c e n t r a t i o n o f d r u g , t y p e and q u a n t i t y o f c r o s s l i n k e r , and p a r t i t i o n i n g e f f e c t s o f t h e d r u g between t h e p o l y m e r , t h e h y d r o g e l w a t e r phase and t h e d e s o r b i n g s o l u t i o n .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

ANDERSON ET AL.

100

Hydrocortisone

Sodium

Succinate

γ

1

2

3

4

5

Time, Days

Figure 5. Comparison of in vitro and in vivo release of hydrocortisone succinate (2,000 ng per mg dry film)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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178

ABSTRACT Poly(2-hydroxyethyl methacrylate)membranes containing hydrocortisone succinate have been shown to exhibit slow relase behavior in vitro and in vivo. Apparent diffusion constants, obtained from a quasi-steady-state approach, correlate well with weight percent crosslinker suggesting a free volume type of diffusion. In vivo experiments designed to show the effect of replacement therapy in hypophysectomized chick embryos were carried out. Using muscle enzymes, it has been shown that implanted discs of poly(2-hydroxyethyl methacrylate) containing hydrocortisone succinate can reverse the effects of hypophysectomy (pituitary gland removal).

Literature Cited 1. Love, D. S., Stoddard, F. J., and Grasso, J. Α., Developmental Biology, (1969), 20, pp. 563-582. 2. Love, D. S. and Eisenberg, J., to be published. 3. Leaders, F. E., Hecht, G., Van Hoose, Μ., and Kellogg, Μ., Annals of Opthalmology, (1973), 5(5), pp. 513-522. 4. Dohlman, C. H., Langston-Pvan, and Rose, J., Annals of Opthalmology, (1972), pp. 823-832. 5. Yolles, S. in "Controlled Release of Biologically Active Agents", A. C. Tanquary and R. E. Lacey, Ed., Plenum Press, New York, New York, 1974, pp. 177-193. 6. Fu, J. C., Kale, Α. Κ., and Moyer, D. L., Journal of Biomedical Materials Research, (1973), 7, pp. 71-78. 7. Fu, J. C., Kale, A. K. and Moyer, D. L., Biomaterials Medical Devices and Artificial Organs, (1973), 1(4). 8. Schmidt, V., Zapol, W., Prensky, W., Wonders, T., Wodinsky, J., and Kitz, R., Transactions of the American Society for Artificial Internal Organs, (1972), 18, pp. 45-52. 9. Tatum, H. J., Contraception, (1970), 1(4), pp. 253-263. 10. Kincl, F. Α., and Rudel, H. W., Acta Endocrinologica, (1971),66,Accompanies Supplementum 151, pp. 5-30. 11. Dzuik, P. J., Cook, B., Niswender, G. D., Kaltenback, C. C., and Doane, Β. B., American Journal of Veterinary Research, (1968), 29(12), pp. 2415-2417. 12. Most, C. F. Jr., Journal of Biomedical Materials Research, (1972), 6, pp. 3-14. 13. Most, C. F., Journal of Applied Polymer Science, (1970), 14, pp. 1019-1024. 14. Roseman, T. J., Journal of Pharmaceutical Sciences, (1972), 61, No. 1., pp. 46-50. 15. Abrahams, R. Α., Ronel, S. Η., and Gould, F. E., Abstract, Eighth Annual Scientific Session of the Association for the Advancement of Medical Instrumentation, Medical Instrumentation, (1973), 7(1), p. 41.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

13.

ANDERSON ET AL.

Hydrocortisone

Sodium

Succinate

179

16. Refojo, M. F., Journal of Applied Polymer Science, (1965), 9, p. 3161. 17. Sprincl, L., Kopecek, J., and Lim, D., Calcified Tissue Research (1973), 13, pp. 63-72. 18. Levowitz, B. S., LaGuerre, J. N., Calem, W. S., Gould, F. E., Francis, E., Scherrer, J., and Schoenfeld, H., Transactions of American Society for Artificial Internal Organs, (1968), 14, p. 82. 19. Lazarus, S. M., LaGuerre, J. N., Kay, H., Weinberg, S., and Levowtiz, B. S., Journal of Biomedical Materials Research, (1971), 5, p. 129. 20. Kaufman, H. E., Votila, M. H., Cassett, A. R., Wood, T. O., and Varnell, E. D., "Medical Uses of Soft Contact Lenses", Chap. 22, Ed. by A. R. Gassett and Η. Ε. Kaufman, Soft Contact Lens, C. V Mosley Co. St Louis Mo. (1972) 21. Podos, S. M., Becker American Journal of Opthalmology, (1972), 73(3), p. 336. 22. Abrahams, R. A. and Ronel, S. H., Journal of Biomedical Materials Research, (1975), 9, pp. 355-366. 23. Davis, Β. K., Noske, I. and Chang, M. C., Acta Endocrinologica, (1972), 70, pp. 385-395. 24. Davis, Β. Κ., Proceedings of the National Academy of Sciences U. S. Α., (1974), 71(8), pp. 3120-3123. 25. Balin, H., Halpern, B. D., Davis, R. H., Akkapeddi, M. K., and Kyriazis, G. Α., Journal of Reproductive Medicine, (1974), 13(6), pp. 208-212. 26. Barnes, C., Physics, (1934), 5(1), pp. 4-8. 27. Garrett, E. R., and Chemburkar, P. B., Journal of Pharmaceutical Sciences, (1968), Vol. 57, No. 6, p. 949. 28. Yasuda, H., Lamaze, C. E. and Ikenberry, L. D., Die Macromolekulare Chemie, (1968), 118, pp. 19-35; (1969), 125, pp. 108-118.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

14 Controlled Release of Fluoride from Hydrogels for Dental Applications D. R. COWSAR, O. R. TARWATER, and A. C. TANQUARY Southern Research Institute, Birmingham, Ala. 35205

A c e n t u r y ago E r h a r d t ( 1 ) recommended o r a l a d m i n i s t r a t i o n of potassiu and t o c h i l d r e n d u r i n d e n t a l c a r i e s by h a r d e n i n g t o o t h s t r u c t u r e . Numerous s t u d i e s s i n c e then have demonstrated c o n c l u s i v e l y the e f f e c t i v e n e s s o f f l u o r i d e p r o p h y l a x i s f o r r e d u c i n g the incidence of carious l e s i o n s i n teeth. Methods f o r a d m i n i s t e r i n g f l u o r i d e now i n c l u d e the f l u o r i d a t i o n o f d r i n k i n g water, the i n g e s t i o n o f f l u o r i d e t a b l e t s , the i n c o r p o r a t i o n o f f l u o r i d e i n t o mouthwashes and d e n t i f r i c e s , and the t o p i c a l a p p l i c a t i o n o f f l u o r i d e s o l u t i o n s and g e l s . (2^,3) When f l u o r i d e i s p r e s e n t i n d r i n k i n g water a t the optimum l e v e l , which i s about 1 ppm o f f l u o r i d e added as sodium f l u o r i d e o r sodium f l u o r o s i l i c a t e , the i n c i d e n c e o f c a r i o u s l e s i o n s i s reduced by 50 t o 60%. However, a l a r g e p o r t i o n o f the p o p u l a t i o n does not have a c c e s s t o p u b l i c water supp l i e s , and s i n c e many communities have e l e c t e d not t o employ c o n t r o l l e d f l u o r i d a t i o n o f water, a major segment o f the p o p u l a t i o n must r e l y on a l t e r n a t i v e means, which are l e s s e f f i c i e n t , t o o b t a i n the a n t i c a r i e s b e n e f i t s of f l u o r i d e . D u r i n g the p a s t s e v e r a l y e a r s c o n s i d e r a b l e p r o g r e s s has been made i n o p t i m i z i n g the t h e r a p e u t i c e f f e c t i v e ness (potency) o f p h a r m a c e u t i c a l s and o t h e r b i o l o g i c a l l y - a c t i v e agents by s i m p l y i m p r o v i n g t h e i r d e l i v e r y t o the t a r g e t organs o r o r g a n i s m s . Controlled-release f o r m u l a t i o n s t h a t d e l i v e r s m a l l amounts o f drugs a t c o n s t a n t r a t e s f o r l o n g t i m e s are u s u a l l y much more e f f e c t i v e than c o n v e n t i o n a l l y - a d m i n i s t e r e d medicaments. While not a l l drugs are amenable t o c o n t r o l l e d r e l e a s e , the c o n t r o l l e d d e l i v e r y o f f l u o r i d e appears p r a c t i c a l as w e l l as h i g h l y d e s i r a b l e . An i n t r a o r a l , c o n t r o l l e d release device could provide continuous t o p i c a l a p p l i c a t i o n o f f l u o r i d e t o t o o t h s u r f a c e s f o r s i x months t o 180

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

14.

COWSAR E T A L .

Fluoride

Release from

Hydrogels

181

a y e a r o r more depending upon d e s i g n . Moreover, a d e v i c e t h a t can be a p p l i e d o r a t t a c h e d i n the mouth by s i m p l e p r o c e d u r e s would p e r m i t an e c o n o m i c a l a n t i c a r i e s t r e a t m e n t a p p l i c a b l e t o v i r t u a l l y a l l segments o f the p o p u l a t i o n . T h i s paper d e s c r i b e s r e s e a r c h t o d e v e l o p a cont r o l l e d - r e l e a s e d e l i v e r y system t h a t w i l l r e l e a s e i n o r g a n i c f l u o r i d e i n t o the o r a l c a v i t y a t p r e determined r a t e s o f 1.0, 0.5, 0.2, and 0.02 mg/day f o r s i x months w i t h o u t maintenance o r adjustment. Once the system i s d e v e l o p e d , the optimum f l u o r i d e r e l e a s e r a t e and the e f f i c a c y o f t h i s method o f t r e a t ment can be d e t e r m i n e d c l i n i c a l l y . Our approach has been t o f a b r i c a t e r e s e r v o i r cont r o l l e d - r e l e a s e devices(£ which c o n t a i n s a s u p p l and a c o a t i n g , which s e r v e s as a membrane t o c o n t r o l d i f f u s i o n ( r e l e a s e ) o f f l u o r i d e . The r a t e o f r e l e a s e o f f l u o r i d e i s determined by the geometry (area and t h i c k n e s s ) o f the membranes and by the p e r m e a b i l i t y parameters o f the s p e c i f i c a c r y l i c copolymers. The f o l l o w i n g equation i s a simple expression o f F i c k ' s law t h a t d e s c r i b e s the s t e a d y - s t a t e r a t e o f r e l e a s e o f f l u o r i d e from such r e s e r v o i r d e v i c e s . dM dt where:

=

dM _ dt A = hcoat = Dcoat

T S f J f

( o - K s

f

C

f

)

rate of release o f fluoride

a r e a o f the d e v i c e t h i c k n e s s o f the c o a t i n g diffusion coefficient of fluoride i n the p o l y m e r i c c o a t i n g = saturation s o l u b i l i t y of fluoride i n the c o a t i n g = c o n c e n t r a t i o n o f f l u o r i d e i n the receiving f l u i d (saliva) = partition coefficient of fluoride between the p o l y m e r i c c o a t i n g and the r e c e i v i n g f l u i d

=

C

S

C

f

K

f

In o r d e r t o d e s i g n d e v i c e s t h a t r e l e a s e f l u o r i d e a t the s p e c i f i e d r a t e s o f 1.0, 0.5, 0.2, and 0.02 mg/24 h r , we needed t o use, as r a t e - c o n t r o l l i n g c o a t i n g s , polymers t h a t had a p p r o p r i a t e v a l u e s o f D, C , and Kf f o r sodium f l u o r i d e . S e v e r a l y e a r s ago, Yasuda and co-workers (5^) d e t e r m i n e d the d i f f u s i o n a l parameters f o r sodium c h l o r i d e i n a s e r i e s o f h y d r a t e d a c r y l i c copolymers and h y d r o g e l s . V a r i o u s copolymers o f m e t h y l s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

182

m e t h a c r y l a t e (MMA), g l y c e r y l m e t h a c r y l a t e (GMA), h y d r o x y e t h y l m e t h a c r y l a t e (HEMA), h y d r o x y p r o p y l metha c r y l a t e (HPMA), and g l y c i d y l m e t h a c r y l a t e (GdMA) p r o v i d e d a s e r i e s o f h y d r a t e d membrane m a t e r i a l s h a v i n g e q u i l i b r i u m water c o n t e n t s r a n g i n g from 1 0 t o 7 0 % . Yasuda found t h a t t h e d i f f u s i o n c o e f f i c i e n t s o f NaCl i n t h e s e membranes v a r i e d o v e r f i v e o r d e r s o f magnitude w i t h changes i n t h e e q u i l i b r i u m l e v e l s o f h y d r a t i o n , and t h a t t h e v a r i a n c e i n d i f f u s i v i t y was v i r t u a l l y independent o f t h e s p e c i f i c c h e m i c a l i d e n t i t i e s ( s t r u c t u r e s ) o f t h e copolymers. On t h e b a s i s o f Y a s u d a s f i n d i n g s , we a n t i c i p a t e d s i m i l a r v a r i a n c e i n t h e d i f f u s i v i t y o f sodium f l u o r i d e i n h y d r a t e d a c r y l i c copolymers. S p e c i f i c a l l y , we a n t i c i p a t e d t h a t copolymer and MMA i n r a t i o s r a n g i n (HEMA/MMA) would p r o v i d e a s e r i e s o f c o r e and c o a t i n g m a t e r i a l s w i t h v a l u e s o f D, Cs, and Kf s u i t a b l e f o r f l u o r i d e - r e l e a s i n g systems. And s i n c e MMA and HEMA homopolymers a r e known t o be t i s s u e c o m p a t i b l e and t o x i c o l o g i c a l l y s a f e i n t h e o r a l environment, we a n t i c i p a t e d no problems i n d e m o n s t r a t i n g t h e b i o c o m p a t i b i l i t y and s a f e t y o f d e v i c e s based on HEMA/MMA copolymers. 1

M a t e r i a l s and Methods A c r y l i c Copolymers. Random copolymers o f HEMA and MMA were p r e p a r e d i n aqueous e t h a n o l s o l u t i o n s by mixi n g HEMA and MMA monomers i n v a r i o u s molar r a t i o s and i n i t i a t i n g t h e i r p o l y m e r i z a t i o n w i t h a redox c a t a l y s t . A l l c o p o l y m e r i z a t i o n s were c a r r i e d o u t e s s e n t i a l l y as follows: To a 1 - l i t e r , g l a s s - s t o p p e r e d b o t t l e were added 9 5 0 ml o f a 3 - t o - 2 m i x t u r e o f e t h a n o l and water and 5 0 g o f a m i x t u r e o f p u r i f i e d h y d r o x y e t h y l metha c r y l a t e (Hydron L a b o r a t o r i e s , New Brunswick, New J e r s e y ) and methyl m e t h a c r y l a t e ( P o l y s c i e n c e s , I n c . , W a r r i n g t o n , P a . ) . N i t r o g e n was then b u b b l e d t h r o u g h the m i x t u r e f o r 5 0 min t o purge i t f r e e o f oxygen. P o l y m e r i z a t i o n was i n i t i a t e d by a d d i n g 0 . 1 2 5 g o f K S 0 and 0 . 2 5 g o f N a S 0 t o t h e m i x t u r e , and t h e b o t t l e was s e a l e d . A f t e r t e n days a t room temperature, the copolymer was p r e c i p i t a t e d by p o u r i n g t h e v i s c o u s p o l y m e r i z a t e i n t o an e x c e s s ( 3 0 0 0 ml) o f water. The w h i t e r e s i n was washed t h o r o u g h l y w i t h water, i s o l a t e d by f i l t r a t i o n , and d r i e d a t 5 0 ° C i n vacuo. T h i s p r o c e d u r e was used t o p r e p a r e HEMA/MMA c o p o l y mers h a v i n g molar r a t i o s o f 2 0 / 8 0 , 3 0 / 7 0 , 4 0 / 6 0 , 5 0 / 5 0 , 6 0 / 4 0 , and 7 5 / 2 5 . Polymer y i e l d s ranged from 4 1 t o 100% o f theory. I n h e r e n t v i s c o s i t i e s were determined 2

2

5

2

2

5

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

COWSAR ET AL.

14.

Fluoride Release from Hydrogeh

183

for each copolymer. These values ranged from 0.39 t o 2.2 d l / g when m e a s u r e d a t a c o n c e n t r a t i o n o f 0 . 5 % i n a 60:40 (v/v) m i x t u r e o f acetone and £-dioxane a t 30°C. Most o f o u r e v a l u a t i o n s o f t h e copolymers were c a r r i e d o u t o n f i l m s p e c i m e n s t h a t we p r e p a r e d b y s p i n casting.(6) An example o f t h e general procedure f o r f a b r i c a t i n g t h i n f i l m s o f t h e a c r y l i c copolymers i s as follows: E x a c t l y 3 g o f c o p o l y m e r was d i s s o l v e d i n 25 ml o f a 60:40 (v/v) m i x t u r e o f acetone and £ - d i x o a n e . T h i s s o l u t i o n was poured i n t o a 3 - i n . - d i a m e t e r by l-in.-deep, T e f l o n - l i n e d , spin-casting cup which r o t a t e d a t 3500 r p m u n t i l t h e s o l v e n t h a d e v a p o r a t e d . The c y l i n d r i c a l f i l m was t h e n l i f t e d f r o m t h e c u p a n d cut transversely to give a highly uniform, rectangular film 15-mil thick, 1-in some c a s e s , t h e t h i c k n e s a d d i n g more o r l e s s s o l u t i o n t o t h e s p i n - c a s t i n g c u p . Permeability Parameters. The d i f f u s i o n c o e f f i c i e n t s f o r sodium f l u o r i d e i n t h e hydrated a c r y l i c copolymers were determined by a m o d i f i c a t i o n o f t h e d e s o r p t i o n m e t h o d o f Y a s u d a e t a l . (5_) In p r i n c i p l e , the d e s o r p t i o n method i n v o l v e s f i r s t e q u i l i b r a t i n g a t h i n membrane o f t h e t e s t m a t e r i a l i n a s o l u t i o n o f the t e s t solute (fluoride i n t h i s case), and then i m m e r s i n g t h e s o l u t e - l o a d e d membrane i n a f r e s h a l i q u o t of r e c e i v i n g f l u i d and measuring the rate o f increase i n concentration o f the solute i n the f l u i d (or the r a t e o f d e s o r p t i o n o f t h e s o l u t e from t h e membrane). A plot of the concentration of the solute i n the r e c e i v i n g f l u i d vs t h e square root o f time i s prepared f r o m t h e d a t a , a n d t h e d i f f u s i o n c o e f f i c i e n t , D, i s c a l c u l a t e d from t h e l i n e a r p o r t i o n o f t h e curve by the following formula.

, w

h

„ e

r

e

and

:

d(Mt/Moo)

= ditVh) Mt Moo h t

= mass o f s o l u t e r e l e a s e d a t t i m e = mass o f s o l u t e r e l e a s e d a t t i m e = t h i c k n e s s o f t h e membrane = time

t 0 0

F i l m specimens o f t h e 30/70, 40/60, a n d 50/50 HEMA/MMA c o p o l y m e r s w e r e e q u i l i b r a t e d a t 3 7 ° C i n a s a t u r a t e d aqueous s o l u t i o n o f sodium f l u o r i d e . The e q u i l i b r a t i n g s o l u t i o n was m a i n t a i n e d a t s a t u r a t i o n

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

184

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

by d e s o r p t i o n o f sodium f l u o r i d e from p e l l e t s o f HEMA polymer t o which s o l i d sodium f l u o r i d e had been added b e f o r e i t was p o l y m e r i z e d i n b u l k . This procedure i s a s i m p l e way o f m a i n t a i n i n g s a t u r a t i o n o f t h e s o l u t i o n w i t h o u t c o n t a m i n a t i n g t h e s u r f a c e s o f f i l m specimens w i t h p a r t i c l e s o f sodium fluoride.(J) After equilibra t i o n had been a c h i e v e d (1 o r 2 d a y s ) , t h e f i l m samples were removed and b l o t t e d t o remove s u r f a c e s o l u t i o n . The f i l m s were t h e n p l a c e d i n a p o l y e t h y l e n e b o t t l e c o n t a i n i n g 50 ml o f water which had been e q u i l i b r a t e d a t 37°C. The f l u o r i d e s p e c i f i c - i o n e l e c t r o d e (Orion Model 96-09) was i n s e r t e d i n t h e s o l u t i o n , and t h e b o t t l e was shaken v i g o r o u s l y . The m i l l i v o l t o u t p u t o f the s p e c i f i c - i o n e l e c t r o d e was r e c o r d e d c o n t i n u o u s l y on a s t r i p - c h a r t r e c o r d e r Concentratio yj \H p l o t were made from t h e samples were t h e n e q u i l i b r a t e d severa additiona 50-ml volumes o f water t o a l l o w e x t r a c t i o n o f a l l o f the f l u o r i d e . The d e s o r b i n g s o l u t i o n s and t h e e x t r a c t i n g s o l u t i o n s were a n a l y z e d t o g i v e t h e t o t a l mass, Moo, of f l u o r i d e released. The f i l m s were then weighed wet and d r i e d i n vacuo. The d i f f u s i o n c o e f f i c i e n t s , D, were c a l c u l a t e d by the d e s o r p t i o n e q u a t i o n . The s a t u r a t i o n c o n c e n t r a t i o n , C / o f sodium f l u o r i d e i n t h e h y d r a t e d copolymers was c a l c u l a t e d by d i v i d i n g t h e Moo v a l u e by t h e volume, A χ h, o f t h e e q u i l i b r a t e d sample. The p a r t i t i o n c o e f f i c i e n t , K f , o f f l u o r i d e between t h e polymer and t h e r e c e i v i n g f l u i d was c a l c u l a t e d from Cs and Cf, t h e s a t u r a t i o n c o n c e n t r a t i o n o f sodium f l u o r i d e i n t h e f l u i d (determined by a n a l y z i n g t h e s a t u r a t e d e q u i l i brating solution). S

The e q u i l i b r i u m water c o n t e n t (degree o f h y d r a t i o n ) was c a l c u l a t e d from t h e wet and d r y w e i g h t s o f t h e f i l m s . Water C o n t e n t = Wet Weight - Dry Weight Wet Weight Other Methods. Methods f o r f a b r i c a t i n g d e v i c e s and f o r d e t e r m i n i n g t h e i r f l u o r i d e - r e l e a s e r a t e s i n v i t r o and i n v i v o a r e d e s c r i b e d i n o t h e r s e c t i o n s . The s y n t h e t i c s a l i v a f o r i n v i t r o e v a l u a t i o n s was a d i l u t e solution of electrolytes: 0.6 mM C a C l ; 1.8 mM NaH POi*; 12 mM NaHC0 ; and 1.0 mM HC1. Human s a l i v a was c o l l e c t e d from v o l u n t e e r s . 2

2

3

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

14.

COWSAR ET AL.

Fluoride

Release from

Hydrogels

185

Device Design To r e l e a s e f l u o r i d e a t a c o n s t a n t r a t e f o r a p r o l o n g e d p e r i o d , a d e v i c e must c o n t a i n a r e s e r v o i r o f f l u o r i d e s a l t s e p a r a t e d from the r e c e i v i n g f l u i d ( s a l i v a ) by a r a t e - c o n t r o l l i n g membrane t h a t i s permeable t o f l u o r i d e ions. To be permeable t o f l u o r i d e i o n s , a membrane must become h y d r a t e d when immersed i n aqueous media; and t o c o n t r o l f l u o r i d e r e l e a s e , i t s degree o f h y d r a t i o n must be e a s i l y r e p r o d u c e d . The a c r y l i c h y d r o g e l s r e p r e s e n t a c l a s s o f polymers t h a t can be f o r m u l a t e d t o p r o v i d e membrane m a t e r i a l s h a v i n g a wide range o f degrees o f h y d r a t i o n . For our f l u o r i d e - r e l e a s i n g d e v i c e s , we chose t o use copolymers of hydroxyethyl methacrylat a c r y l a t e (MMA). In F i g u r e 1 we show t h a t the e q u i l i b r i u m water c o n t e n t o f HEMA/MMA copolymer membranes v a r i e s d i r e c t l y w i t h the mole % o f the h y d r o p h i l i c monomer (HEMA) but t o d i f f e r e n t d e g r e e s above and below t h e e q u i m o l a r ratios. S e v e r a l v a l u e s f o r HEMA/MMA copolymers t h a t were r e p o r t e d by Yasuda, e t a_l. (5) are a l s o shown i n F i g u r e 1. Copolymers w i t h 50 mole % o r l e s s o f HEMA are p r e f e r r e d f o r b o t h the c o r e m a t r i x and the r a t e c o n t r o l l i n g membranes o f o u r d e v i c e s . Two d i f f e r e n t copolymers were r e q u i r e d f o r t h e devices. The one f o r t h e c o r e had t o be h i g h l y p e r meable t o sodium f l u o r i d e , and the one f o r the r a t e c o n t r o l l i n g c o a t i n g had t o be l e s s permeable. Values o f D, C r and Kf were d e t e r m i n e d f o r t h r e e copolymer compositions: 30/70, 40/60, and 50/50 HEMA/MMA. T a b l e I shows the d i f f u s i o n c o e f f i c i e n t , D, t h e s a t u r a t i o n s o l u b i l i t y , C , the p a r t i t i o n c o e f f i c i e n t , Kf, and the c a l c u l a t e d p e r m e a b i l i t y , P, f o r sodium f l u o r i d e when water was the e q u i l i b r a t i n g ( r e c e i v i n g ) fluid. The v a l u e s were e s s e n t i a l l y the same when s y n t h e t i c s a l i v a and human s a l i v a were used as r e c e i v ing f l u i d s . On the b a s i s o f t h e s e d a t a we chose t h e 50/50 HEMA/MMA copolymer f o r the c o r e m a t r i x m a t e r i a l and the 30/70 HEMA/MMA copolymer f o r the c o a t i n g materials . S i n c e the f l u o r i d e - r e l e a s i n g d e v i c e s a r e e x p e c t e d t o be worn by c h i l d r e n as w e l l as a d u l t s , we d e s i g n e d the f i n a l d e v i c e s so t h a t they would be as s m a l l as possible. The main f a c t o r r e s t r i c t i n g the o v e r a l l s i z e was t h e amount o f f l u o r i d e s a l t t h a t was r e q u i r e d t o s u s t a i n the d e s i r e d d a i l y r a t e s o f r e l e a s e f o r s i x months. From our work w i t h d e s i g n p r o t o t y p e s , we d e t e r m i n e d t h a t the r a t e o f r e l e a s e o f f l u o r i d e from t r i l a m i n a t e d e v i c e s remains c o n s t a n t u n t i l a p p r o x i S

s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

11.1

13.8

20.3

40/60

50/50

Degree o f hydration, % water

30/70

Copolymer, r a t i o of HEMA/MMA

Table I.

1

63 χ Ι Ο "

26 χ Ι Ο "

8 χ 10"

2

D, cm sec~

3.3 χ 10"**

8

1

3.1 χ 10" *

2.5 χ 10""

3

8

8

s

C , gF/cm Κ

0.017

0.015

2

107 χ 10"

39 χ 10"

9.6 χ 10"

Ρ = DK cm sec"

Values

0.012

Experimentally-Determined

14.

COWSAR E T A L .

Fluoride

Release

from

187

Hydrogels

m a t e l y 80% o f t h e f l u o r i d e i n t h e c o r e i s e x h a u s t e d . With t h i s i n mind, we d e s i g n e d t h e f i n a l d e v i c e s t o c o n t a i n a 20% e x c e s s o f f l u o r i d e s a l t . The c o r e o f t h e d e v i c e was d e s i g n e d t o c o n s i s t o f powdered sodium f l u o r i d e d i s p e r s e d i n a m a t r i x o f HEMA/MMA copolymer. The copolymer m a t r i x s e r v e s two functions. F i r s t , i t p r o v i d e s a medium o f f i x e d geometry and water c o n t e n t i n which t h e f l u o r i d e s a l t d i s s o l v e s p r i o r t o d i f f u s i n g through the r a t e - c o n t r o l l i n g membranes. T h i s d e s i g n e n s u r e s t h a t t h e concent r a t i o n of dissolved fluoride a t the i n t e r i o r surfaces o f t h e r a t e - c o n t r o l l i n g membrane w i l l remain c o n s t a n t throughout the l i f e t i m e o f the d e v i c e . And second, the copolymer m a t r i x p r o v i d e s a degree o f s a f e t y t o prevent c a t a s t r o p h i r e l e a s f th f th de vice i f the r a t e - c o n t r o l l i n l y p u n c t u r e d , t o r n , o r worn t h r o u g h . Since the core i s a c t u a l l y a monolithic c o n t r o l l e d - r e l e a s e device,(4) s t r e s s f a i l u r e o f t h e r a t e - c o n t r o l l i n g membrane w i l l r e s u l t i n a high rate o f f l u o r i d e release f o r a short p e r i o d u n t i l t h e f l u o r i d e s a l t i n t h e immediate v i c i n i t y of the f a u l t i s depleted. Then, t h e c o r e m a t r i x m a t e r i a l w i l l become r a t e c o n t r o l l i n g , and t h e r e l e a s e o f f l u o r i d e from t h e d e v i c e w i l l o c c u r a t a low, declining rate. Cores f o r t r i l a m i n a t e d e v i c e s t h a t are prepared from t h e 50/50 HEMA/MMA copolymer t o c o n t a i n 80% (w/w) o f sodium f l u o r i d e have a l o a d i n g d e n s i t y o f 1.9 g o f sodium f l u o r i d e p e r cm o f c o r e . Cores p r e p a r e d t o c o n t a i n 62% (w/w) o f sodium f l u o r i d e i n 50/50 HEMA/MMA copolymer have a l o a d i n g d e n s i t y o f 1.53 g o f sodium f l u o r i d e p e r cm o f c o r e . These d e n s i t y f a c t o r s were used t o c a l c u l a t e t h e minimum c o r e volumes r e q u i r e d for the four devices. From t h e minimum c o r e volumes we c a l c u l a t e d " a r b i t r a r y " l i n e a r dimensions f o r t h e c o r e s , and from t h e s e we c a l c u l a t e d t h e membrane s u r face areas. Then, we used t h e v a l u e s o f D, C r and Kf t h a t were determined f o r t h e 30/70 HEMA/MMA copolymer and t h e F i c k ' s Law e q u a t i o n t o c a l c u l a t e t h e t h i c k n e s s o f t h e r a t e - c o n t r o l l i n g membranes r e q u i r e d t o g i v e t h e desired rates o frelease of fluoride. Two a l t e r n a t i v e d e s i g n s p e c i f i c a t i o n s f o r each o f t h e f o u r f i n a l dev i c e s a r e g i v e n i n T a b l e I I . S i m i l a r v a l u e s c a n be calculated for cylindrical, spherical, or irregular devices. S i n c e t h e r a t e - c o n t r o l l i n g membranes a r e very t h i n , the device dimensions a r e very n e a r l y the core dimensions. 3

3

S

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 62 80

62 80

95

95

9.5

9.5

50/50

50/50

50/50

50/50

0.2 mg/day

0.2 mg/day

0.02 mg/day

0.02 mg/day

0.005

0.006

0.049

0.063

0.12

80

240

50/50

0.5 mg/day

0.16

62

240

50/50

0.5 mg/day

0.25

80

50/50

1 mg/day

475

50/50

1 mg/day

0.31

3

Volume, cm .

0.3

0.4

2.0

2.5

1.9

2.5

3.9

4.8

Length, cm

Specifications

Loading, % w/w

Core

62

Core Copolymer

Wt NaF Contained, mg

0.3

0.4

0.5

0.5

0.8

0.8

0.8

0.8

Width, cm

0.05

0.037

0.05

0.05

0.08

0 .08

0.08

0.08

Thickness, cm

Membrane

30/70

30/70

30/70

30/70

30/70

30/70

30/70

30/70

Membrane Copolymer

DESIGN SPECIFICATIONS FOR FLUORIDE-RELEASING DEVICES

475

Fluoride Release Rate

Table I I .

0.18

0.32

2.0

2.5

3.0

4.0

6.2

7.7

2

Area, cm

0.016

0.028

0.017

0.022

0.011

0.014

0.011

0.013

Thickness, cm

Specifications

ο 2

$

t—t

•t> r n

α >

H M

>

w r

ο

>

π >

2

w

ο sa

ο M r

X

§Ο

00 00

14.

COWSAR E T AL.

Fluoride

Release from

Hydrogels

189

F a b r i c a t i o n o f Devices The key t o f a b r i c a t i n g t r i l a m i n a t e d e v i c e s h a v i n g p r e c i s e , predetermined r a t e s o f r e l e a s e i s i n accur a t e l y p r e p a r i n g the f l u o r i d e - c o n t a i n i n g c o r e s t o s p e c i f i e d dimensions. To do t h i s , we had s t a i n l e s s s t e e l r e p l i c a s o f the cores s p e c i f i e d i n Table I I p r e p a r e d i n our machine shop. U s i n g t h e s e r e p l i c a s , we p r e p a r e d m u l t i - c a v i t y s i l i c o n e r u b b e r molds f o r t h e cores. M o l d i n g m i x t u r e s were p r e p a r e d by t h o r o u g h l y m i x i n g powdered sodium f l u o r i d e i n a p p r o p r i a t e amounts i n t o v i s c o u s s o l u t i o n s o f 50/50 HEMA/MMA copolymer i n 60:40 a c e t o n e - d i o x a n e . Cores were molded by a p p l y i n g t h e f l u o r i d e m i x t u r e s by s p a t u l a i n t o t h the s o l v e n t t o e v a p o r a t the c o r e s from the mold, they s t i l l c o n t a i n e d a s m a l l amount o f r e s i d u a l dioxane and were p l i a b l e . Two s t a i n l e s s - s t e e l w i r e s were a t t a c h e d t o each c o r e t o p r o v i d e a means f o r l a t e r a t t a c h i n g the d e v i c e t o an a p p l i a n c e i n a d o g s mouth. The l a r g e r i b b o n - s h a p e d c o r e s were a l s o c u r v e d s l i g h t l y w h i l e they were s t i l l p l i a b l e , and t h e n t h e y were a l l o w e d t o d r y . A s k e t c h of a c u r v e d c o r e w i t h the w i r e s a t t a c h e d i s shown a s F i g u r e 2. The c o r e s were then c o a t e d w i t h r a t e - c o n t r o l l i n g membranes by d i p p i n g them r e p e a t e d l y i n t o a 12% (by weight) s o l u t i o n o f 30/70 HEMA/MMA copolymer i n 60:40 acetone-dioxane. A f t e r each d i p , t h e c o a t i n g was a l l o w e d t o d r y i n a i r f o r a p p r o x i m a t e l y 20 min. Each d i p p i n g i n c r e a s e d the c o a t i n g t h i c k n e s s by a p p r o x i mately 0.001 cm. Thus, a p p r o x i m a t e l y 14 d i p s were r e q u i r e d t o produce a c o a t i n g t h i c k n e s s o f 0.014 cm. The a c t u a l t h i c k n e s s was measured w i t h a micrometer. A f t e r t h e l a s t d i p p i n g , t h e d e v i c e s were d r i e d i n a i r f o r 1 h r and t h e n p l a c e d i n a vacuum oven a t 60°C overnight. 1

In

V i t r o E v a l u a t i o n o f Devices

A c o n s t a n t - t e m p e r a t u r e f l o w system was used f o r e v a l u a t i n g the r e l e a s e r a t e s o f f l u o r i d e from the devices. The d e v i c e s were suspended by a s t a i n l e s s s t e e l wire i n a 30-ml-capacity, polyethylene flow c e l l h a v i n g i n l e t and o u t l e t p o r t s f o r the r e c e i v i n g f l u i d . I n i t i a l l y , h i g h - p u r i t y d e i o n i z e d water was pumped a t 0.85 ml/min (1220 ml/day) from a t h e r m o s t a t e d (37°C) r e s e r v o i r , t h r o u g h the f l o w c e l l ( a l s o h e l d a t 3 7 ° C ) , and i n t o a c o l l e c t i o n v e s s e l . L a t e r , d e v i c e s were

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MOLE PERCENT OF HEMA IN COPOLYMERS

Figure 1.

Equilibrium water content for HEMA/MMA

copolymers

STAINLESS STEEL

Figure 2.

Core for fluoride-releasing device

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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e v a l u a t e d w i t h s y n t h e t i c and n a t u r a l s a l i v a s as t h e receiving fluids. A t regular i n t e r v a l s the receiving f l u i d i n t h e c o l l e c t i o n v e s s e l was a s s a y e d f o r f l u o r i d e v i a a c a l i b r a t e d s p e c i f i c - i o n e l e c t r o d e (Orion Model 96-09), and t h e volume o f f l u i d c o l l e c t e d d u r i n g t h e time i n t e r v a l was r e c o r d e d . Figure 3 d e p i c t s the flow system. A l t h o u g h o n l y one f l o w c e l l i s shown i n F i g u r e 3, we u s u a l l y o p e r a t e d s i x c e l l s simultaneously. The f l u o r i d e - r e l e a s i n g d e v i c e s a l l have s i m i l a r p o l y m e r i c c o r e s l o a d e d w i t h powdered sodium f l u o r i d e . The copolymers f o r t h e c o r e m a t r i x were d e s i g n e d t o be h i g h l y permeable t o f l u o r i d e . The r a t e o f r e l e a s e o f f l u o r i d e from t h e d e v i c e s i s c o n t r o l l e d by t h e mem branes which s u r r o u n d t h e c o r e s . To demonstrate t h e v a l i d i t y o f t h i s desig concept placed disk shaped (1.18-cm-diamete 50/50 HEMA/MMA copolymer and c o n t a i n i n g 40 mg o f f l u o r i d e i n t h e f l o w c e l l and d e t e r m i n e d t h e r a t e o f r e l e a s e o f f l u o r i d e i n t o d e i o n i z e d water a t 37°C. Most o f t h e f l u o r i d e was r e l e a s e d d u r i n g t h e f i r s t day, and v i r t u a l l y a l l o f t h e f l u o r i d e was r e l e a s e d d u r i n g f o u r days. When 0.023-cm-thick, r a t e - c o n t r o l l i n g mem branes o f 30/70 HEMA/MMA copolymer were p r e s s u r e l a m i n a t e d on b o t h s i d e s o f a s i m i l a r c o r e , and t h e edges o f t h e l a m i n a t e were s e a l e d w i t h a c o a t i n g o f 30/70 copolymer, a d e v i c e t h a t r e l e a s e d f l u o r i d e a t a n e a r l y - c o n s t a n t r a t e o f a p p r o x i m a t e l y 0.8 mg/day was obtained. F i g u r e 4 shows t h e c u m u l a t i v e r e l e a s e o f f l u o r i d e from t h e c o r e a l o n e and from t h e complete trilaminate device. Long-term, i n v i t r o e v a l u a t i o n s o f two o f t h e f i n a l d e v i c e s were i n i t i a t e d soon a f t e r they were f a b r i c a t e d . The d e v i c e s were suspended i n t h e f l o w c e l l s and e l u t e d c o n t i n u o u s l y a t 37°C w i t h s y n t h e t i c s a l i v a f l o w i n g a t a p p r o x i m a t e l y 0.85 ml/min. The r e l e a s e - r a t e d a t a t h a t have been o b t a i n e d so f a r a r e shown i n F i g u r e s 5 and 6. D e v i c e 7935-29-C ( F i g u r e 5) was d e s i g n e d t o r e l e a s e f l u o r i d e a t a r a t e o f 0.2 mg/day. The c o r e was a 2.5 χ 0.5 χ 0.05-cm r i b b o n l o a d e d w i t h 62% o f sodium fluoride. The c o a t i n g t h i c k n e s s i s 0.022 cm. Over a t e s t p e r i o d o f 59 days t h e average r a t e o f r e l e a s e o f f l u o r i d e has been 0.19 mg/day. D e v i c e 7935-29-G ( F i g u r e 6) was d e s i g n e d t o r e l e a s e f l u o r i d e a t a r a t e o f 0.5 mg/day. I t has a 1.9 χ 0.8 χ 0.08-cm c o r e l o a d e d w i t h sodium f l u o r i d e a t a l e v e l o f 80%. The c o a t i n g t h i c k n e s s i f 0.011 cm. Over a t e s t p e r i o d o f 63 days t h e d e v i c e has r e l e a s e d f l u o r i d e a t an average r a t e o f 0.49 mg/day. The r a t e o f r e l e a s e o f f l u o r i d e from t h e s e d e v i c e s has been shown t o be e s s e n t i a l l y independent o f t h e

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Figure S. Diagram of constant-temperature flow system

0

5

10

15 TIME , days

20

25

30

Figure 4. Cumulative release of fluoride from a core alone and from a pressure-laminated device

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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c o m p o s i t i o n and pH o f t h e r e c e i v i n g f l u i d . In t e s t s w i t h s y n t h e t i c s a l i v a , human s a l i v a , and water h a v i n g v a l u e s o f pH r a n g i n g from 4 . 8 t o 7 . 8 , t h e f l u o r i d e r e l e a s e r a t e v a r i e d by l e s s than ± 0 . 0 3 mg/day f o r a dev i c e w i t h a d a i l y mean r e l e a s e r a t e o f 0 . 4 7 mg/day. In V i v o E v a l u a t i o n o f D e v i c e s Two f l u o r i d e - r e l e a s i n g d e v i c e s i d e n t i c a l t o t h o s e e v a l u a t e d i n v i t r o were a l s o e v a l u a t e d i n v i v o i n b e a g l e dogs. The d e v i c e s were a t t a c h e d v i a a s p e c i a l l y - d e s i g n e d i n t r a o r a l a p p l i a n c e t h a t was a t t a c h e d t o the upper c a n i n e t e e t h . F a b r i c a t i o n o f a f i x e d i n t r a o r a l a p p l i a n c e which would a l l o w f o r attachmen t r o l l e d - r e l e a s e devic i m p r e s s i o n s u s i n g an a l g i n a t e i m p r e s s i o n m a t e r i a l . Models made o f p l a s t e r o f p a r i s were poured and o r t h o d o n t i c bands f o r t h e upper c a n i n e s were c o n s t r u c t e d from 0 . 2 5 - i n . - w i d e , 0 . 0 0 2 - i n . - t h i c k s t a i n l e s s - s t e e l ribbon. The bands were hand f i t t e d and e l e c t r i c a l l y s p o t welded. A s e c t i o n o f s u r g i c a l a r c h b a r was adapted t o t h e c a s t s and s o l d e r e d t o t h e bands. The l a b i a l b a r was f i t t e d w i t h space l e f t f o r t h e o c c l u d i n g lower c a n i n e s . The l u g s were removed i n t h e a n t e r i o r part of the arch bar t o allow seating o f the f l u o r i d e r e l e a s i n g d e v i c e which was a t t a c h e d t o t h e f i x e d a p p l i a n c e by means o f s t a i n l e s s - s t e e l w i r e s hooked to the p o s t e r i o r lugs o f the arch bar (Figure 7 ) . S e a t i n g and z i n c oxyphosphate cementing o f t h e completed a p p l i a n c e were a c c o m p l i s h e d w i t h t h e dogs l i g h t l y a n e s t h e t i z e d w i t h Nembutal. Figure 8 i s a s k e t c h showing t h e completed a p p l i a n c e a t t a c h e d i n the b e a g l e ' s mouth. P r i o r t o attachment o f t h e f l u o r i d e - r e l e a s i n g dev i c e s , s a l i v a was c o l l e c t e d from two b e a g l e dogs and analyzed f o r f l u o r i d e . The dogs were g i v e n 0 . 0 7 ml o f a s o l u t i o n o f p i l o c a r p i n e ( 7 5 mg/ml) s . c . i n t h e nape o f t h e neck t o s t i m u l a t e s a l i v a f l o w . Saliva collect i o n by a s p i r a t i o n was begun immediately and c o n t i n u e d u n t i l a 2-ml sample had been c o l l e c t e d . Average s a l i v a f l u o r i d e l e v e l s were found t o be 0 . 0 1 5 ppm. D a i l y s a l i v a samples were c o l l e c t e d from t h e dogs a f t e r t h e d e v i c e s were i n p l a c e . S a l i v a was c o l l e c t e d w i t h and w i t h o u t p i l o c a r p i n e s t i m u l a t i o n . The i n v i v o d a t a (Table I I I ) show t h a t t h e f l u o r i d e - r e l e a s i n g devices elevated the s a l i v a f l u o r i d e l e v e l s s i g n i f i c a n t ly. The h i g h e r l e v e l s measured w i t h o u t p i l o c a r p i n e stimulation indicated that pilocarpine stimulation i n -

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

COWSAR ET AL.

Fluoride

Release from

Hydrogels

FLUORIDE - RELEASING DEVICE ν

SURGICAL

ARCH

Figure 7.

Intraoral orthodontic appliance with device attached

Figure 8. Fluoride-releasing device in place in a beagle's mouth

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creases s a l i v a flow rate but not the r a t e o f release o f f l u o r i d e from t h e d e v i c e s . T a b l e I I I . S a l i v a F l u o r i d e L e v e l s o f B e a g l e Dogs F i t t e d with I n t r a o r a l FluorideReleasing Devices

Saliva collected Before device Without p i l o c a r p i n e stimulation With p i l o c a r p i n e stimulation

F l u o r i d e c o n c e n t r a t i o n , ppm, i n s a l i v a o f dog w i t h device r e l e a s i n g a t 0 . 2 mg F/day 0 . 5 mg F/day 0.018

0.013

0.2 0.15

0.25

Abstract The continuous topical administration of fluoride to teeth from an intraoral controlled-release device is an untried method for reducing the occurrence of dental caries. Pharmacokinetic dose-response data can be obtained only after a delivery system has been developed. In the present study we are developing biocompatible, fluoride-containing devices that release fluoride in the oral environment at constant predetermined linear rates of 0.02 to 1.0 mg/day for at least six months without maintenance or adjustment. The trilaminate devices comprise a core of inorganic fluoride salt, which is dispersed in a copolymer (hydrogel) of hydroxyethyl methacrylate and methyl methacrylate, and outer layers (or coatings) of semipermeable, rate-controlling membranes, which are made from similar hydrated acrylic copolymers. Fluoride from the core diffuses through the membranes into the oral environment at a rate precisely controlled by the thickness, area, and permeability of the materials. The permeability parameters, which include the solubility of the fluoride salt in the polymeric core and in the outer membranes, the diffusion coefficients of fluoride in both materials, and the partition coefficient for fluoride between the outer membrane and saliva, have been quantitated; and prototype devices have been evaluated in vitro and in vivo in dogs.

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Acknowledgments T h i s work i s sponsored by t h e N a t i o n a l I n s t i t u t e o f D e n t a l R e s e a r c h under C o n t r a c t NOl-DE-42446. The a u t h o r s e x p r e s s t h e i r thanks t o Mrs. Melody D. H a m i l t o n , Mrs. Brenda H. P e r k i n s , Mr. J o e M. F i n k e l , Dr. Danny H. Lewis, and Dr. Lewis Menaker who c o n t r i b u t e d s i g n i f i c a n t l y t o t h i s work.

Literature Cited 1. Erhardt, Monatsschr. ration. Aerzte (Heil bronn) (1874), 19, p. 359. 2. Horowitz, H. S., Community Dent. Oral Epidemiol. (1973), 1, p. 104 3. Carlos, J. P., Ed. Fogarty International Center Series on Preventive Medicine, Vol. 1, DHEW Publication No. (NIH) 74-707, Washington, D. C., 1974. 4. Baker, R. W., and Lonsdale, Η. Κ., in "Controlled Release of Biologically Active Agents," pp. 15-71, A. C. Tanquary and R. E. Lacey, Ed., Plenum Press, New York, 1974. 5. Yasuda, H., Lemage, C. Ε., and Ikenberry, L. D., Die Makromolecular Chemie (1968), 118, p. 19. 6. Kaelble, D. Η., J. Appl. Polym. Sci., (1965), 9, p. 1209 7. Lacey, R. E., and Cowsar, D. R., in "Controlled Release of Biologically Active Agents," pp. 117144, A. C. Tanquary and R. E. Lacey, Ed., Plenum Press, New York, 1974.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

15 The Hydrogel-Water Interface A. SILBERBERG The Weizmann Institute of Science, Rehovot, Israel

There are very few investigations which deal with the gel -swellingmedium interfac solute components according y in treatments of gel chromatography. Contact angle measurements have been undertaken routinely (1), but the structure and the statistical mechanics of this type of interphase has received only a preliminary treatment (2). In selecting the hydrogel-water interface for special consideration, it will have to be realized that it does not possess features which are not characteristic of the gel-solvent interface in general and that the measure of our ignorance is probably about the same. This is provided, of course, that we put aside - as we shall do here - such special potential aspects as charged ionic surface groups and long range electrostatic interactions. Still, even without charge interactions, water is a rather special solvent and the uncharged polymeric species which are soluble, or swellable, are so, generally, because they possess groups which hydrate well, i.e. will form a hydrogen bond with water. Such groups are almost always also capable of forming hydrogen bonds with each other and solubility, in general, implies that the hydrogen bond with water is not too weak as compared with the bond dimerizing two groups. While in water both the solution and dimerization bond energies tend to be quite large, they are also about equal and their difference remains small. Solubility, i.e. sweliability, of the material will be assured if the interaction between the polymer segments in the aqueous environment is only mildly attractive, i.e. does not overfavor dimerization over hydration (see Figure 1). Similar considerations will apply to gels in general. Nevertheless, some special aspects govern aqueous gels which could cause them to behave differently to other gels.

198

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199

Energy Level Scheme

[ pp w

(a)

w

oo ~ 2 op] ~ ~ ^ w

(b)

Figure 1. Energy effects which control stability. w , energy level for polymer segment bound to solvent; w , energy level for polymer segment-polymer segment interaction; w , energy level for solvent-solvent interaction. According to the Flory-Huggins theory of polymer solutions, χ = [2w — w — w ]/2kT < 0.5 for solubility. The parameter χ is generally found to be in the range between 0 and 0.5. This implies, as the potential diagram (b) shows, that the "pp"-bond is probably stronger than the "op"-bond. If, how ever, it is too strong, then χ > 0.5, and the system is potentially unstable. Hence, the hydrogen bond between gel substance groups can be somewhat stronger than the hydro gen bond of this group with water but not very much stronger. op

pp

00

op

00

pp

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Features which Distinguish Hydrogels Whereas i n most non-polar solvents the mixing energies are due to van der Waals interactions only (both the individual energy level changes and their differences are small), i n aqueous environments we are dealing with relatively large energy effects. Hence the same percentage change can produce much larger effects on solubility i n the aqueous medium. Conformational changes and cooperative interactions between polymer and polymer and polymer and solvent are thus much more dramatic and much more readily precipitated i n aqueous media than i n other environments. A hydrogel can be regarded as a potentially unstable system whose state i s easily influenced by slight variations i n temperature,or pressure, or by minor chemical modifications of the aqueous environment (e.g. additio neutral salts or alcohols) To the extent that gradients exist i n the gel, particularly in the surface region, we might thus be faced with the p o s s i b i l i t y of large changes i n conformation (or degree of interaction between the segments of the gel substance) i n going through the i n t e r f a c i a l region between bulk homogeneous gel and bulk water. The Gel-Solvent Interface Gels are characterized by the possession of crosslinks. Generally speaking these are chemical units to which a number of chains, three or more, are linked i n some fashion either by permanent bonds or by bonds of a sufficiently long lifetime so that changes i n structure do not occur, or occur only very slowly under stress. These crosslinks, however, need not be defineable chemical links between separate chains, but could, for example, be more extensively organized regions containing a larqe amount of gel substance between-, which a small number of long coiling polymeric chains establish the connections and hold the gel together, topologically. These organized regions thus act as effective crosslinks and the chain segments between them as the effective network segments. © are comparing the actual gel with the classical homogeneous ideal, three-dimensional network model and try to establish correlations. These correlations w i l l depend upon the experiment considered and i t i s not to be expected that the division into effective crosslinks and effective network segments need necessarily be unique. The kind of d i v i sion which w i l l account for the elastic modulus may not correspond to the division best suited to permeation studies. The main points to remember are that gel structure i s not necessarily homogeneous,that not each chemical crosslink i s an effective crosslink,but that a meaningful separation into structural elements i s nevertheless possible. If the presence of the crosslinks defines the presence of the gel, the zone where the crosslink density (number of w

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crosslinks per unit volume) goes from i t s value i n the gel interior to i t s value, zero, i n the water phase,represents the gel surface, i . e . the interface with the solvent bulk phase. Ideally we could picture this transition as geometrically abrupt, but there i s , i n fact, l i t t l e reality to an i n t e r f a c i a l zone more sharply located than the mean distance between crosslinks in the bulk. The macromolecular fuzz, i . e . the region involving the freely coiling chain segments between crosslinks w i l l also terminate at the interface, and i n part, smooth out the coarse grain of the crosslink density distribution. Even then i t i s most unlikely that the gel surface can be much smoother than the displacements characteristic of the intercrosslink distance. Swelling Equilibrium Since we are considerin swelling equilibrium has been established. This means essent i a l l y that the concentration of water i n the gel has been adjusted, by suitably contracting or expanding the distance between crosslinks, by, i n other words, coiling or uncoiling the macromolecular network segment chains, so that solvent chemical potential i s the same inside and out. If this experiment were done using a solution containing a f i n i t e number of unlinked macromolecules i n place of the gel, the system would go to i n f i n i t e dilution. I f , however, the volume of polymer solution were confined inside a closed membrane, which does not permit the polymer component to move out, water w i l l enter, or w i l l tend to enter, the solution space raising the pressure u n t i l the water chemical potential i s matched. Mechanically, this build-up of pressure i s possible because the hydrostatic pressure can be compensated by a stress in the membrane. I f the membrane i s r i g i d (i.e. possesses i n f i n i t e elastic modulus) compensation of forces can be achieved without volume change. In more practical situations the membrane has a f i n i t e elastic modulus, w i l l tend to distend slightly and some swelling w i l l take place. The point to note i s that a real pressure difference i s established between solution and swelling medium and that the water molecules i n the solution phase are "aware" of i t by being forced closer together, i.e. being somewhat more compressed than i n the pure water phase at ambient pressure. In the case of the equilibrium swelling of a gel, the gel material would act as i t s own membrane. The question arises, therefore, whether a hydrostatic pressure difference exists between the interior of the gel and the water phase and whether a pressure gradient accompanies the concentration transition through the interface. This i s a more d i f f i c u l t question to answer than i t should be. F i r s t of a l l the literature i s f u l l of terms which suggest

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t h a t such a pressure a r i s e s . Moreover, mechanical work can be done as a r e s u l t o f s w e l l i n g . Thus s w e l l i n g p r e s s u r e seems t o be an a p p r o p r i a t e term t o apply when one wishes t o c h a r a c t e r i z e the force which would have t o be exerted on the g e l i n order t o prevent i t from s w e l l i n g i n an experiment analogous t o the membrane-polymer s o l u t i o n experiment j u s t d i s c u s s e d . I n t h e l a t t e r case, t o o , one speaks o f a p r e s s u r e , the osmotic p r e s s u r e , which can be t r a n s l a t e d i n t o a r e a l p r e s s u r e e f f e c t when a membrane i s employed t o r e s i s t a change i n volume o f the s o l u t i o n phase, i . e . t o prevent d i l u t i o n . Osmotic pressure i s , however, not an i n h e r e n t mechanical f e a t u r e o f the s o l u t i o n , b u t o n l y a method o f e x p r e s s i n g i t s c o n c e n t r a t i o n . To achieve a r e a l p r e s sure e f f e c t we have t o perform an experiment which w i l l depend upon the presence of a mechanical d e v i c e , t h e membrane, and another phase, t h e pur s o l v e n t Onl the d i f f e r e n c e be generated Now e x a c t l y the same i s t r u e when a g e l i s s t u d i e d under s i m i l a r c o n d i t i o n s . A porous r i g i d support f o r the g e l i s r e q u i r e d . M e c h a n i c a l l y i t i s the e q u i v a l e n t o f t h e membrane i n the osmotic p r e s s u r e experiment. Through i t the g e l i s p u t i n t o contact w i t h the water phase, and by i t s d e v i c e t h e volume o f the g e l i s confined mechanically. P r e c i s e l y as i n the osmotic pressure measurement system, water w i l l tend t o e n t e r , compression of the water molecules w i l l occur, the h y d r o s t a t i c p r e s s u r e w i l l r i s e , and the chemical p o t e n t i a l o f water i n s i d e the compartment w i l l r i s e t o match t h a t o f t h e water o u t s i d e . As i n the case o f osmotic e q u i l i b r i u m , s o l v e n t chemical p o t e n t i a l i s balanced by a r e a l p r e s s u r e r i s e and the mechanical i n t e r a c t i o n between the g e l and i t s porous r i g i d support p r o v i d e s the means by which water compression i s achieved. There i s o n l y one d i f f e r e n c e . The pressure e f f e c t i s not c a l l e d osmotic p r e s s u r e b u t s w e l l i n g pressure. The s i t u a t i o n o f s w e l l i n g p r e s s u r e measurement i s , however, not the s i t u a t i o n o f s w e l l i n g e q u i l i b r i u m . I n t h a t case expans i o n i s u n r e s i s t e d and w i l l stop only when c o n f o r m a t i o n a l changes i n the network segments have so d i s t o r t e d the s t a t i s t i c a l mec h a n i c a l p a r t i t i o n f u n c t i o n o f the system and so d i l u t e d the system t h a t the e f f e c t o f the g e l substance s o l u t e on the s o l v e n t chemical p o t e n t i a l i s reduced t o zero. S o l v e n t chemical p o t e n t i a l i s balanced by changes i n s o l u t e chemical p o t e n t i a l alone and the need f o r a p r e s s u r e r i s e on the s o l u t i o n i s o b v i a t e d . Indeed, i t would be m e c h a n i c a l l y i m p o s s i b l e t o have such a p r e s s u r e a r i s e without a d e v i c e which could compensate t h i s e f f e c t m e c h a n i c a l l y . True enough t h e network i s d i s t e n d e d but there i s no f o r c e i n the network segments a t s w e l l i n g e q u i l i b r i u m . We can c u t such a g e l a t any p o i n t and no volume change w i l l a r i s e . Only then i n c o n t a c t w i t h pure s o l v e n t w i l l f o r c e s a r i s e when the network i s m e c h a n i c a l l y d i s t o r t e d out o f the c o n f i g u r a t i o n i n which i t i s i n thermodynamic e q u i l i b r i u m . Only then w i l l

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i t s segments c a r r y f o r c e . I n contact w i t h a pure s o l v e n t phase, t h e r e f o r e , the c o n f i g u r a t i o n of the network i n i t s s w e l l i n g e q u i l i b r i u m s i t u a t i o n i s the reference unstressed n o n d i s t o r t e d c o n f i g u r a t i o n . Out o f contact w i t h pure s o l v e n t , the g e l w i l l have been synthesized,or formed i n other ways, a t a c o n c e n t r a t i o n above s w e l l i n g e q u i l i b r i u m . For volume p r e s e r v i n g deformations from t h i s s t a t e the system w i l l tend t o r e t u r n t o i t as i t s then reference s t a t e , but only i f out of contact w i t h s o l v e n t . The reference s t a t e of the system i s thus the e q u i l i b r i u m s t a t e of the system under the environmental c o n s t r a i n t s imposed. I t has nothing t o do w i t h whether or not the degree of c o i l i n g of the network segments by chance corresponds t o the conformational d i s t r i b u t i o n o f t h a t polymer species i n f r e e s o l u t i o n . We can, moreover, change the reference s t a t e by changing the o u t s i d e medium. I f aqueous s o l u t i o n were use too b i g t o enter the g e l , a d i f f e r e n t s w e l l i n g e q u i l i b r i u m would be reached and the reference s t a t e would correspond t o t h a t s i t u a t i o n . Hence any s t a t e can be a reference s t a t e , but the o u t s i d e medium may have t o possess r a t h e r s p e c i a l p r o p e r t i e s i n each case. I t i s the approach used by F l o r y i n h i s treatment of the i s o l a t e d macromolecule (3). Here the macromolecule i s considered as a microscopic g e l p a r t i c l e and s w e l l i n g t o e q u i l i b r i u m i s allowed t o occur. The s w o l l e n s t a t e of the macromolecule and not i t s Gaussian d i s t r i b u t i o n s t a t e i s now the normal conforma t i o n a l d i s t r i b u t i o n o f the segments i n t h a t s o l v e n t medium. Segment D i s t r i b u t i o n i n the I n t e r f a c e The s w e l l i n g of the macroscopic g e l can thus be considered analogously t o the F l o r y approach. The p r i n c i p a l d i f f e r e n c e d e r i v e s from the f a c t t h a t the segment d i s t r i b u t i o n i s r a t h e r d i f f e r e n t t o s t a r t w i t h . S w e l l i n g w i l l occur i n most cases of good s o l v e n t s and the θ-conditions of i d e a l i n s o l u b i l i t y w i l l not be d i f f e r e n t s i n c e the θ-point corresponds, as b e f o r e , t o the v a n i s h i n g of the second v i r i a l c o e f f i c i e n t i . e . t o the t r a n s i t i o n t o c r i t i c a l l y a t t r a c t i v e polymer-polymer i n t e r a c t i o n s . While we can assume t h a t network segment d i s t r i b u t i o n i n s i d e the g e l i s i s o t r o p i c t h i s d i s t r i b u t i o n goes t o zero over some i n t e r f a c i a l r e g i o n of a s l a b t h i c k n e s s corresponding approximately t o the i n t e r c r o s s l i n k d i s t a n c e . A number of problems a r e , however, here encountered. The d i s t r i b u t i o n of conformations o f a network segment between f i x e d c r o s s l i n k s , a l l o w i n g f o r the e x c l u s i o n of conformations due t o the simultaneous presence of other such network segments, i s not adequately known. S i m i l a r l y , the d i s t r i b u t i o n o f dangling chain ends from the outermost l a y e r o f c r o s s l i n k p o i n t s ( t a k i n g i n t o account t h a t here too there are network segments going

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from these c r o s s l i n k s t o i n t e r i o r c r o s s l i n k p o i n t s and w i l l i n h i b i t conformational freedom) has not y e t been d e r i v e d . I t may be assumed t h a t t h i s d i s t r i b u t i o n has some s i m i l a r i t y t o t h e d i s t r i b u t i o n o f chains t e r m i n a l l y attached t o a r i g i d plane surface (4). Account w i l l , however, have t o be taken o f the f a c t t h a t the outermost l a y e r o f c r o s s l i n k s does not l i e i n one plane and t h a t t h e dangling c h a i n ends are not uniform i n length. We may expect, t h e r e f o r e , t h a t the outermost l a y e r o f a g e l surface i s much t h i c k e r than one i n t e r c r o s s l i n k d i s t a n c e and t h a t the s u r f a c e phase w i l l thus be much more d i f f u s e than the i n t e r i o r s i t u a t i o n o f the g e l . I t should be noted t h a t using θ-conditions f o r the s w e l l i n g w i l l give cases where c h a i s t a t i s t i c t closel h th i d e a l Gaussian random walk R e l a t i o n s h i p between Bulk and Surface S t r u c t u r e Laser s c a t t e r i n g r e s u l t s from g e l s ( 5 , 6 ) and some recent p e r m e a b i l i t y s t u d i e s ( 7 ) both confirm the i d e a t h a t the i n t e r n a l s t r u c t u r e o f g e l s does not n e c e s s a r i l y conform t o the simple con cept t h a t each c r o s s l i n k molecule i n c o r p o r a t e d i n t o the network s t r u c t u r e produces one independent c r o s s l i n k . Many such l i n k s apparently are wasted simply producing l a r g e r c h a i n s . Others occur t o o c l o s e t o each other and may only serve t o extend t h e r e g i o n which mechanically w i l l a c t as a s i n g l e c r o s s l i n k , a l l chemical c r o s s l i n k s i n t h a t r e g i o n a c t i n g as one. Hence many g e l s r e a c t as though they had f a r fewer e f f e c t i v e c r o s s l i n k s than apparently i n c o r p o r a t e d and as though they had much wider openings i n them than would be suspected from the amount o f gel substance present. E f f e c t s l i k e these, a coarsening o f the g r a i n o f the g e l , may a l s o be expected t o a f f e c t the surface zone commensurately. Hydrodynamic E f f e c t s Macromolecules, p h y s i c a l l y adsorbed o r c h e m i c a l l y attached (at one or two p o i n t s ) t o s o l i d support surfaces produce a d i f f u s e macromolecular zone. I n attempts t o measure the t h i c k ness of such l a y e r s by the change i n dimensions they produce, i t i s found t h a t the t h i c k n e s s e s so measured are e f f e c t i v e l y much l a r g e r than those determined by other methods (9/10). There i s thus a hydrodynamic e f f e c t which produces an e x t r a energy d i s s i p a t i o n i n the f l u i d l a y e r s a d j o i n i n g t h e macro molecular s u r f a c e . Confirmation t h a t flow p a s t a g e l , o r g e l - l i k e , s u r f a c e i s a s s o c i a t e d w i t h an e x t r a energy d i s s i p a t i o n was obtained from s t u d i e s o f the r e s i s t a n c e t o flow through c y l i n d r i c a l

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channels i n a gel {11) . Compared with channels of the same dimensions with r i g i d walls, the cylinder i n the gel allows only a reduced throughput. The effect could be correlated with the elastic modulus of the gel and the thickness of the gel layer. I t seems to be due to oscillations excited i n the interface ( 1 2 ) . It i s to be hoped that the interesting properties of the hydrogel surface which have led to an increasing number of applications i n a variety of fields w i l l also stimulate more systematic work on their physico-chemical and mechanical attributes.

Literature Cited: 1. Holly F.J. and Refoj 426. 2. Silberberg A. Polymer Preprints (1970) 11, 1289. 3. Flory P.J. "Principles of Polymer Chemistry" (1953) Cornell Univ.Press, Ithaca, p.519. 4. Hesselink F.Th. J.Phys.Chem. (1971) 75, 65. 5. Dusek K. and Prins W. Adv.Polymer Sci. (1969) 6, 1-102. 6. Pines E. and Prins W. Macromolecules (1973) 6, 888. 7. Weiss N. and Silberberg A. Biorheology (1975) 12, 107. 8. Weiss N. and Silberberg A. This book. 9. Priel Z. and Silberberg A. Polymer Preprints (1970) 11, 1405. 10. Silberberg A. "Colloques Internationaux du C.N.R.S. No. 233 (1975) p.81. 11. Lahav J., Eliezer N. and Silberberg A. Biorheology (1973) 10, 595. 12. Hansen R.J. and Hunston D.L. J.Sound and Vibration (1974) 34, 297.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

16 Probing the Hydrogel/Water Interface JOSEPH D. ANDRADE, ROBERT N. KING, and DONALD E. GREGONIS Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112

The i n t e r f a c i a l p r o p e r t i e s o f gel/water i n t e r f a c e s are important i n the biomedical i n the areas o f blood c o m p a t i b i l i t y c e l l adhesion. The gel/water i n t e r f a c e may a l s o be important i n the i n t e r f a c i a l chemistry o f c e l l membranes (1). Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e on gel/water i n t e r f a c e s . Many o f the c l a s s i c a l s u r f a c e chemical methods i n v o l v i n g s o l i d / l i q u i d i n t e r f a c e s are i n a p p l i c a b l e t o the g e l / l i q u i d i n t e r f a c e , because the " s o l i d " i s h i g h l y deformable and, i n g e n e r a l , t h e l i q u i d i s h i g h l y d i f f u s i b l e i n the s o l i d . G e l s , by d e f i n i t i o n , are not s o l s so c l a s s i c a l l i q u i d - l i q u i d i n t e r f a c e techniques are not g e n e r a l l y a p p l i c a b l e . G e l / l i q u i d i n t e r f a c e s are thus e x p e r i m e n t a l l y and t h e o r e t i c a l l y somewhat f r u s t r a t i n g . Nevertheless, they are o f such great p r a c t i c a l and s c i e n t i f i c importance t h a t they merit c a r e f u l study. S i l b e r b e r g has considered the g e l / s o l v e n t i n t e r f a c e , l a r g e l y i n a t h e o r e t i c a l sense (2,3). The o b j e c t i v e o f t h i s paper i s t o consider a v a r i e t y o f experimental methods which can provide i n f o r m a t i o n on gel/water i n t e r f a c e s . Much o f the d i s c u s s i o n w i l l be b r i e f and perhaps shallow - but that i s l a r g e l y the present s t a t e o f a f f a i r s w i t h regard t o g e l / l i q u i d i n t e r f a c e s . Topography - S t r u c t u r e The f i r s t "measurement" which should be made on a s u r f a c e i s gross examination, f o l l o w e d by microscopic examination. I s the surface rough? Does i t have an apparent s t r u c t u r e o r morphology? I s there any apparent o r i e n t a t i o n ? Such questions must be answered before any subsequent surface c h a r a c t e r i z a t i o n can be meaningful as v i r t u a l l y a l l surface c h a r a c t e r i z a t i o n techniques are surface roughness o r topography dependent. A l though a v a r i e t y o f techniques are a v a i l a b l e f o r measuring the topography o f hard s o l i d surfaces (£), they are l a r g e l y inapp l i c a b l e t o gel surfaces. 206

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O p t i c a l microscopy i s very u s e f u l (5) and can be used t o study i n s i t u surface topography u s i n g phase or i n t e r f e r e n c e c o n t r a s t o r w i t h water immersion o p t i c s . Often, however, one r e q u i r e s higher m a g n i f i c a t i o n and must r e s o r t t o the scanning e l e c t r o n microscope (SEM) or t o the t r a n s m i s s i o n e l e c t r o n micro scope (TEM). Simple a i r d r y i n g o f the g e l f o l l o w e d by metal c o a t i n g o f t e n shows s u b s t a n t i a l d i f f e r e n c e s i n g e l topography. Figure 1 shows a series of r a d i a t i o n - g r a f t e d poly(hydroxyethyl methacrylate) (PHEMA) coatings on polypropylene (6). The gross d i f f e r e n c e s i n topography are q u i t e evident and s t r o n g l y a f f e c t the surface o r interfacial characterizations. A i r d r y i n g i s seldom adequate f o r studying g e l s u r f a c e s , p a r t i c u l a r l y f o r l e s s r i g i d g e l s . Most g e l s e x h i b i t gross changes i n s t r u c t u r e an to the xerogel s t a t e . able f o r examining d e l i c a t e b i o l o g i c a l s t r u c t u r e s by SEM and TEM, most g e n e r a l l y i n v o l v e f i x a t i o n or c r o s s - l i n k i n g s t e p s , f o l l o w e d by dehydration, and c r i t i c a l p o i n t d r y i n g from Freon o r l i q u i d CO^ (7). We would p r e f e r t o avoid such procedures w i t h g e l s . The most accepted and common method o f observing h i g h l y deformable gels i s by f r e e z e - e t c h i n g . The sample i s r a p i d l y f r o z e n , o f t e n i n l i q u i d Freon, and f r a c t u r e d under l i q u i d n i t r o g e n . The f r a c t u r e surface can then be d i r e c t l y observed i n a c o l d stage or c o l d stub-equipped SEM o r i t can be r e p l i c a t e d c o l d and the r e p l i c a examined by SEM or TEM (7,8). Cluthe has examined r a d i a t i o n c r o s s - l i n k e d p o l y ( e t h y l e n e oxide) (PEO) g e l s c o n t a i n i n g 1 t o 10% polymer (9) by freeze etch r e p l i c a t i o n . He showed that the s t r u c t u r e s observed from embed ded and s e c t i o n e d g e l s were s i m i l a r t o those o f the f r e e z e - e t c h r e p l i c a t e d samples. He observed and discussed " c e l l u l a r " s t r u c tures f o r the PEO g e l s . He a l s o noted t h a t the " f i b r i l l a r " s t r u c t u r e s observed i n the TEM by others may r e s u l t from a i r drying a r t i f a c t s . Blank and Reimschuessel (10) r e c e n t l y reviewed v a r i o u s g e l s t r u c t u r e t h e o r i e s and presented photographs o f polyacrylamide, p o l y ( e t h y l e n e o x i d e ) , and p o l y ( v i n y l a l c o h o l ) g e l s . They con s i d e r e d the l i q u i d phase i n the g e l i n terms o f f r e e l i q u i d , c a p i l l a r y or pore r e t a i n e d l i q u i d , and polymer adsorbed l i q u i d , i . e . , a t h r e e - l a y e r model. This i s s i m i l a r t o the "Χ, Y, Z" water hypothesis o f g e l water (11). The s o l i d phase was c o n s i d ered i n terms o f a c e l l u l a r or m i c e l l a r theory and a f i b r i l l a r theory (also c a l l e d the "brush-heap" theory ( 9 ) ) . T h e i r photo graphs c l e a r l y document roughness on the 10 micron l e v e l i n most of the gels examined. High r e s o l u t i o n s t u d i e s on the s t r u c t u r e of g e l a t i n g e l s are a l s o a v a i l a b l e (12,13). Geymayer has r e viewed the problems i n v o l v e d i n high r e s o l u t i o n s t u d i e s o f freeze etched g e l s , i n c l u d i n g c o o l i n g r a t e s , segregation and aggregation o f d i f f u s i b l e s o l u t e s , and i n s t r u m e n t a t i o n (14).

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Figure 1. SEM photos of HEMA grafts on polypropylene (original magnification, 3150 X (A) Control; (B) 15% HEMA, 0.25 Mrad; (C) 15% HEMA, 1 Mrad; (D) 20% HEMA, 0.5 Mrad.

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We have c a r r i e d out some very p r e l i m i n a r y s t u d i e s of PHEMA gels u s i n g f r e e z e - e t c h SEM techniques. These s t u d i e s are too premature to make any conclusions as t o g e l surface or bulk s t r u c t u r e , but they do permit some conclusions about experimental methodology. One can freeze dry a sample and observe i t d i r e c t l y i n the SEM at ambient temperatures (Figure 2a). This i s g e n e r a l l y uns a t i s f a c t o r y due t o charging. One can f r e e z e - d r y , coat the sample at ambient temperature, and observe at ambient temperatures (Figure 2b). One can freeze the sample and observe i t at l i q u i d n i t r o g e n temperatures (charging i s not a s e r i o u s problem as i c e i s a f a i r conductor); the sample can be s l o w l y warmed and the i c e sublimes away r e v e a l i n g the s t r u c t u r e (Figure 2c). The sample of Figure 2c was mounted as i n d i c a t e d i n Figure 3. I t i s c l e a r , even ed PHEMA g e l , of r e l a t i v e l or freeze d r y i n g and ambient temperature examination may not r e v e a l the r e a l s t r u c t u r e . Freeze e t c h i n g c e r t a i n l y has a r t i f a c t s a l s o , but at t h i s stage i t appears t o be the method of choice f o r s y n t h e t i c aqueous g e l s (14). A more r e l i a b l e procedure than o u t l i n e d i n Figure 3 i s t o place the sample onto a temperature c o n t r o l l e d stage. Such stages are a v a i l a b l e f o r most SEM s. The samples can be observed f r o z e n , freeze-etched, micromanipulated, and, i f d e s i r e d , coated under c o n t r o l l e d temperature c o n d i t i o n s . Figure 4 i s a sequence of photographs of a 70% H2O - 30% PHEMA opaque g e l , mounted and f r a c t u r e d under l i q u i d n i t r o g e n and examined i n the ETEC Bio-SEM (ETEC, Inc., Hayward, C a l i f o r n i a ) . The topography observed may be r e l a t e d t o the r a t e o f freeze e t c h i n g . We have not examined transparent g e l s - s t u d i e s are i n progress. Matas, e t . a l . , however, reported some SEM s t u d i e s o f h y d r o p h i l i c contact lenses (15). Surface scratches and roughness could be r e a d i l y seen - no apparent bulk s t r u c t u r e or p o r o s i t y could be seen. The surface roughness of the g e l s can be determined by stereo p a i r SEM photographs and stereophotogrammetric a n a l y s i s (16). f

f

!

Contact Angle Methods Contact angle methods are widely used f o r measuring surface tensions or f r e e energies of l i q u i d s and, l e s s r i g o r o u s l y , o f s o l i d s . I n t e r f a c i a l tensions can o f t e n be obtained by contact angle methods. The t r a d i t i o n a l techniques f o r measurement of contact angles r e l y on determination of the s t a t i c l i q u i d p r o f i l e encountered at the three-phase l i n e (TPL) of the s o l i d , a l i q u i d , and a second f l u i d which may be l i q u i d or vapor. These i n c l u d e (17) (a) the t i l t i n g p l a t e method, (b) the s e s s i l e drop method,

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Figure 2. A PHEMA gel containing 7.5% ethylene glycol dimethacrylate crosslinker. The gel is opaque and contains 38.1% wt water. (A) Freeze-dried, stored ambient, and examined ambient; (B) freeze-dried, coated ambient, and examined ambient; (C) frozen; freeze etched; examined cold.

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Figure 3. Cold stub and method of mounting and fracturing gel samples for freeze etch SEM examination

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Figure 4. 30% PHEMA-70% water opaque gel freeze fractured in LN and observed in the ETEC BioSEM before and during freeze etching. (A) Initial fracture surface and mold surface (top) showing ice crystals due to improper sample handling during fracture and admission to the SEM. (B) A groove produced by the micromanipulator. A cylindrical or cellular morphology is evident in the newly fractured zone. A similar but more random or disturbed morphology is evident outside of the micromanipulated zone. (C) Higher magnification of the micromanipulated zone showing "cells" which appear to be rupturing due to freeze etching. 2

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4. (D) A view of original fracture surface after extensive freeze etching areas as (A)). Note similarity with Β and C. (E) A view prior to complete etching. Note similarity to those regions of (B) outside the micromanipulated All photos courtesy of ETEC, Inc., Hayward, Calif. All samples uncoated.

Figure 5. The same as in Figure 4. (A) Examined via the method of Figure 3. (B) Freeze-dried, coated ambient, and examined ambient.

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(c) the c a p t i v e bubble ( s e s s i l e bubble) method, (d) the drop d i mension method and (e) the Wilhelmy p l a t e method. These methods have been adequately described elsewhere (17-20) and have been e x t e n s i v e l y used to i n v e s t i g a t e a l a r g e number of three-phase systems. A l l of the above methods e s s e n t i a l l y r e l y on the c l a s s i c a l contact angle or Young-Dupree equation: Y

SV

= ^SL

where:

+

Y

LV

C O s G

e

'

(I)

= solid/vapor i n t e r f a c i a l tension Y

S L

= s o l i d / l i q u i d i n t e r f a c i a l tension

JJJJ

= liquid/vapor i n t e r f a c i a l tension;

θ

= equilibriu

Several b a s i c assumptions are inherent i n t h i s equation. The l i q u i d must be considered i n c o m p r e s s i b l e , f l u i d , and coherent (25). F l u i d i t y means t h a t the a p p l i c a t i o n of a shear s t r e s s to the l i q u i d must be accompanied by a shear s t r a i n , provided the s t r e s s i s maintained beyond the molecular r e l a x a t i o n time. Co herence i m p l i e s t h a t the l i q u i d r e s i s t s a t e n s i l e s t r e s s u n t i l the s t r e s s magnitude i s s u f f i c i e n t t o cause rupture. The sur face t e n s i o n of a l i q u i d i s macroscopic evidence o f coherence. The s o l i d surface i n contact w i t h the l i q u i d must be con s i d e r e d smooth, homogeneous, i s o t r o p i c and, most i m p o r t a n t l y , non-deformable, i . e . , r i g i d i n the sense that s t r e s s g r a d i e n t s at the TPL are i n s u f f i c i e n t t o deform i t s i g n i f i c a n t l y . Equation 1 assumes t h a t thermodynamic e q u i l i b r i a , at a l l three i n t e r f a c e s ( s o l i d / l i q u i d , l i q u i d / v a p o r , and s o l i d / v a p o r ) , have been a t t a i n e d (21-24). The k i n e t i c i n t e r p r e t a t i o n r e l i e s on the measurement of dynamic contact angles and o b s e r v a t i o n of the r e s u l t a n t contact angle h y s t e r e s i s . The k i n e t i c s of surface w e t t i n g (or nonwetting) make use of the f a c t t h a t the l i q u i d / v a p o r i n t e r f a c e w i l l change i t s shape and t o t a l area as needed under the i n f l u ence o f the u n d e r l y i n g s u r f a c e i n order t o maintain a constant curvature at e q u i l i b r i u m . This d i r e c t l y r e s u l t s from the Laplace equation o f c a p i l l a r i t y (17): Δ Ρ

where:

= \v

\

+

>

W

ΔΡ = pressure d i f f e r e n t i a l between the concave and con vex s i d e s o f the i n t e r f a c e ; γ^γ

= l i q u i d / v a p o r i n t e r f a c i a l t e n s i o n ; and

R,,

R~ = p r i n c i p l e

r a d i i of curvature.

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D i f f e r e n c e s i n ΔΡ from r e g i o n t o r e g i o n over the i n t e r f a c e correspond t o g r a d i e n t s i n h y d r o s t a t i c ' p r e s s u r e w i t h i n the under l y i n g bulk l i q u i d , and t h e r e f o r e tend t o cause motion i n d i r e c t i o n s which w i l l d i m i n i s h these g r a d i e n t s . Such motion o r y i e l d i n g o f the i n t e r f a c e t o meet the L a p l a c i a n ΔΡ requirement i s n e a r l y always accompanied by a displacement o f the TPL which causes an instantaneous change i n the shape and/or area o f the l i q u i d / v a p o r i n t e r f a c e . The dynamic contact angle i s an i n s t a n taneous measure o f the angle between the l i q u i d / v a p o r i n t e r f a c e and the s o l i d / l i q u i d i n t e r f a c e w h i l e the TPL i s moving. Dynamic contact angles may be measured by e i t h e r spontaneous spreading o r f o r c e d spreading o f a l i q u i d on a s u r f a c e . In the spontaneous spreading method, the contact angle i s observed as a f u n c t i o n o f time and o f d i s t a n c e t r a v e r s e d by the TPL as a drop o f l i q u i d spontaneousl brium contact angle. I extent t o which the γ_,., γ__, and y cos θ. ^ are imbalanced. SV SL 'LV mst. In f o r c e d spreading, the l i q u i d / v a p o r i n t e r f a c e i s moved r e l a t i v e t o the s o l i d surface at a s e r i e s o f constant v e l o c i t i e s by ap p l i c a t i o n o f an e x t e r n a l f o r c e . At each v e l o c i t y the contact angle reaches a steady s t a t e value and a r e l a t i o n s h i p between the TPL v e l o c i t y and contact angle i s thus e s t a b l i s h e d . In t h i s case the d r i v i n g f o r c e i n which the TPL undergoes displacement i s the r e s u l t o f a pressure-curvature imbalance (25). The importance o f dynamic contact angle measurements l i e s i n the phenomenon o f contact angle h y s t e r e s i s , i . e . , the a b i l i t y to change the observed contact angle o f a l i q u i d on a surface without subsequent displacement o f the TPL. This may be exhib i t e d on many s u r f a c e s by measuring the instantaneous contact angle w h i l e , f o r example, i n c r e a s i n g and decreasing the s i z e o f the s e s s i l e drop. Comparison o f the r e s u l t i n g curves o f advanc ing and receeding contact angles i n the case o f most surfaces w i l l show t h a t a h y s t e r e s i s loop i s formed. The most common types o f n o n - i d e a l i t y are e i t h e r a homogeneous but g e o m e t r i c a l l y rough s u r f a c e o r a g e o m e t r i c a l l y smooth but heterogeneous sur face (19). One may a l s o have both h e t e r o g e n e i t i e s and s u r f a c e roughness. P r a c t i c a l l y speaking then, both s u r f a c e roughness and/or s u r f a c e h e t e r o g e n e i t y , both o f which give r i s e t o contact angle v a r i a t i o n s , can be the major undetected cause o f contact angle h y s t e r e s i s . S w e l l i n g , a b s o r p t i o n , and molecular r e o r i e n t a t i o n a t the i n t e r f a c e can a l s o lead t o apparent h y s t e r e s i s (26). The g e l surface may be capable o f molecular r e o r i e n t a t i o n during dynamic contact angle measurements, as has been suggested (28). The g e l / l i q u i d i n t e r f a c e poses s t i l l f u r t h e r d e v i a t i o n s from i d e a l i t y which have yet t o be considered. In a d d i t i o n t o p o s s i b i l i t i e s o f surface roughness and surface h e t e r o g e n e i t y , the t y p i c a l g e l surface i s extremely deformable. Thus i n the case o f contact angle c h a r a c t e r i z a t i o n o f the g e l s u r f a c e , the v e r t i c a l components o f s u r f a c e t e n s i o n should cause a p p r e c i a b l e rXT

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d i s t o r t i o n i n the g e l surface at the TPL and could cause gross m i s r e p r e s e n t a t i o n s of the angular measurements. This e f f e c t would thus s i g n i f i c a n t l y i n f l u e n c e dynamic measurements and thus contact angle h y s t e r e s i s . The contact angles o f l i q u i d s at deformable s o l i d surfaces have been t h e o r e t i c a l l y t r e a t e d by s e v e r a l i n v e s t i g a t o r s (23,27), but have yet to be e x p e r i m e n t a l l y a p p l i e d to the surfaces o f g e l systems. Interface

Potentials

I d e a l l y we would l i k e to be able t o probe the e l e c t r i c a l double l a y e r at g e l / s o l u t i o n i n t e r f a c e s . Perhaps the most s t r a i g h t f o r w a r d way i s to use e l e c t r o k i n e t i c methods (29,30) g e n e r a l l y e l e c t r o p h o r e s i s (31), streaming p o t e n t i a l (32), or e l e c t r o o s m o s i s (29). Such measurement allo t calculat the p o t e n t i a l at the shea number of assumptions Perhaps the b i g g e s t problem i s the assumption i n v o l v i n g the nature o f the f l u i d dynamic boundary l a y e r i n such s t u d i e s and the p o s i t i o n of the shear plane. Even i f the gel surface i s p e r f e c t l y smooth, we s t i l l have the problem of d e f i n i n g an i n t e r f a c e p o s i t i o n f o r a g e l c o n s i s t i n g of h i g h l y mobile segments and chains at the i n t e r f a c e (Z, 3). The shear plane could be o u t s i d e the i n t e r f a c i a l zone, w i t h i n the zone and f r e e d r a i n i n g , or w i t h i n the i n t e r f a c i a l zone and non-free d r a i n i n g , as d i s c u s sed by Brooks (33), and others (34). These same problems are present i n v i s c o m e t r i c or rhéologie c h a r a c t e r i z a t i o n of g e l / liquid interfaces. One must be p a r t i c u l a r l y c a r e f u l with streaming p o t e n t i a l and e l e c t r o o s m o s i s measurements w i t h respect to other f l u i d dynamic assumptions, p a r t i c u l a r l y entrance e f f e c t s and the establishment of p a r a b o l i c flow p r o f i l e s (35). One can a l s o o b t a i n surface p o t e n t i a l i n f o r m a t i o n u s i n g g e l / a i r measurements, such as w i t h a v i b r a t i n g reed e l e c t r o s t a t i c m i l l i v o l t m e t e r (36). These methods are commonly used to characte r i z e monomolecular f i l m s at the l i q u i d / a i r i n t e r f a c e (37), i n c l u d i n g s y n t h e t i c polymers (38). Such methods cannot be e a s i l y a p p l i e d t o the g e l / s o l u t i o n i n t e r f a c e , however. The i n t e r p r e t a t i o n of g e l / s o l u t i o n e l e c t r o k i n e t i c data i s f a r from s t r a i g h t f o r w a r d . One must of course consider a c l a s s i c a l treatment i n terms of f i x e d charges on the polymer " s u r f a c e " and double l a y e r counterions. In a d d i t i o n , the g e l w i l l absorb and p a r t i t i o n ions from s o l u t i o n , even i f the gel i s l a r g e l y "uncharged" i n terms of f i x e d polymer charges, ( t h i s w i l l be discussed l a t e r ) . I f ions are p a r t i t i o n e d between the gel and the s o l u t i o n , i n t e r f a c e p o t e n t i a l s w i l l r e s u l t which w i l l , of course, i n f l u e n c e e l e c t r o k i n e t i c measurements (39).

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Adsorption o f Polymers One can study the a d s o r p t i o n o f polymers at gel/water i n t e r faces. Much o f the work i n t h i s area has i n v o l v e d plasma p r o t e i n adsorption (40-42). We have discussed the p r o t e i n adsorption behavior of g e l s p r e v i o u s l y , i n terms of i n t e r f a c i a l f r e e energies and water s t r u c t u r e c o n s i d e r a t i o n s (1). One can o b t a i n i n f o r m a t i o n on the nature o f gel/water i n t e r faces by adsorbing g e l molecules on other s u b s t r a t e s and then c h a r a c t e r i z i n g the adsorbed polymer/water i n t e r f a c e . A l a r g e l i t e r a t u r e on polymer adsorption i s a v a i l a b l e , i n c l u d i n g the study of water-soluble polymers (43). The t h e o r i e s of c o l l o i d a l p a r t i c l e s t a b i l i z a t i o n by adsorbed n o n - i o n i c polymers v i a e n t r o p i e and e n t h a l p i c r e p u l s i o n (44,45) may be u s e f u l i n understandin ions . E f f e c t s of s t e r e o r e g u l a r i t y on i n t e r f a c i a l behavior have been observed i n poly(methyl m e t h a c r y l a t e ) , p o l y ( i s o p r o p y l a c r y l a t e ) , p o l y ( 2 - v i n y l p y r i d i n e 1-oxide) and other polymers (46,67). The adsorbed l a y e r * can then be c h a r a c t e r i z e d by the i n s t r u mental techniques discussed i n t h i s paper. Partitioning Gels are very s u b t l e probes of t h e i r environment. They w i l l p a r t i t i o n ions and other s o l u t e s and s w e l l or deswell i n response to t h e i r s o l u t i o n environments. Of p a r t i c u l a r importance t o t h e i r surface p r o p e r t i e s i s i o n p a r t i t i o n i n g i n the g e l s , which may s i g n i f i c a n t l y i n f l u e n c e i n t e r f a c i a l p o t e n t i a l and i n t e r f a c i a l tension studies. Ion p a r t i t i o n i n g i n gels has been observed f o r c e l l u l o s e (47), c r o s s - l i n k e d dextrans (48,49), p o l y ( h y d r o x y e t h y l methacryl a t e ) g e l s (50), and others. Of p a r t i c u l a r i n t e r e s t t o us i s the i o n c o n c e n t r a t i o n p r o f i l e o f the g e l / s o l u t i o n i n t e r f a c e . I d e a l l y we would l i k e to know the concentrations i n the e l e c t r i c a l double l a y e r , i n the gel/water i n t e r f a c i a l r e g i o n , and i n the s u b - i n t e r f a c i a l zone, perhaps t o 1 or 2 microns below the surface. Such measurements are d i f f i c u l t t o make by conventional techniques. One approach i s t o r a p i d l y freeze the gel/water i n t e r f a c e , f r a c t u r e i t , and perform an e l e c t r o n microprobe a n a l y s i s or energy d i s p e r s i v e a n a l y s i s of x-rays (EDAX) i n the SEM u s i n g a c o l d stage t o maintain the sample below -130°C t o avoid i c e c r y s t a l l i z a t i o n and consequent i o n segregation (51). U n f o r t u n a t e l y the s p a t i a l r e s o l u t i o n i s l i m i t e d to 0.1 t o 1.0 micron or l a r g e r , making a high r e s o l u t i o n i n t e r f a c i a l r e g i o n p r o f i l e very d i f f i c u l t . Microautoradiography of f r o z e n samples i s a l s o p o s s i b l e , but i s plagued by t e c h n i c a l d i f f i c u l t i e s . More s p e c u l a t i v e methods of measuring c o n c e n t r a t i o n p r o f i l e s w i l l be discussed later.

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Probing the Outermost Zone There are very few methods w i t h which t o probe the outermost p a r t o f the g e l / s o l u t i o n i n t e r f a c e . We have a l r e a d y d i s c u s s e d the problems w i t h contact angle measurements. Most o p t i c a l spectroscopy methods, p a r t i c u l a r l y IR, probe a zone o f the order of microns i n depth (next s e c t i o n ) . Although e l l i p s o m e t r y can i d e a l l y measure a f i l m t h i c k n e s s of a few Angstroms (52), i t may be d i f f i c u l t t o apply t o g e l s because o f the lack o f s i g n i f i c a n t r e f r a c t i v e index d i f f e r e n c e s between the g e l and the surrounding solution. A v a r i e t y of techniques are a v a i l a b l e f o r d i r e c t l y examining the nature o f the s u r f a c e i t s e l f . Only a l i m i t e d number o f these can be d i s c u s s e d here - see Reference 53 f o r the o t h e r s . The two techniques most promisin are e l e c t r o n spectroscop copy f o r chemical a n a l y s i s ) and secondary i o n mass spectroscopy (SIMS). Both techniques i n v o l v e high vacuum environments. The sample must be f r o z e n i n l i q u i d n i t r o g e n , g e n e r a l l y at Τ < -130°C. Experience w i t h such methods on aqueous and b i o l o g i c a l samples i s very l i m i t e d at present. ESCA was developed l a r g e l y by the e f f o r t s of Siegbahn and h i s c o l l a b o r a t o r s i n Sweden (54). ESCA i s based on the p r e c i s e measurement of the k i n e t i c energy of e l e c t r o n s e j e c t e d from the sample by the a c t i o n o f i n c i d e n t r a d i a t i o n , u s u a l l y x-ray or UV. The b i n d i n g energy of the e l e c t r o n p r i o r t o e j e c t i o n (Eg) i s obtained from the measured k i n e t i c energy (E, ):

where E ^

i s the energy o f the monoenergetic e x c i t a t i o n r a d i a t i o n

(commonly Mg Κα, 1254 eV), and C i s an instrument constant, which i s r e a d i l y determined e x p e r i m e n t a l l y . The h i g h p r e c i s i o n o f the Ε]ς measurement allows one t o not only i d e n t i f y the elements present i n the s u r f a c e but a l s o t o i d e n t i f y t h e i r o x i d a t i o n s t a t e . The p h o t o e l e c t r o n s p e c t r a can be s h i f t e d by up t o 10 eV, depending on the o x i d a t i o n s t a t e of the element (54,55). Modern ESCA instruments sample an area o f the order o f sev e r a l mm . The volume sampled depends on the p h o t o e l e c t r o n escape depth f o r the sample. The escape depth i s o f the order o f 5 t o 15 A over the energy range o f i n t e r e s t f o r most metals (58). Data f o r polymers and low d e n s i t y s o l i d s are not r e a d i l y a v a i l a b l e , though escape depths o f the order of 50 t o 100 A are gen e r a l l y accepted (58). The sampling depth can be decreased by decreasing the angle the escaping e l e c t r o n s make w i t h the sample s u r f a c e . This procedure i s commonly c a l l e d the " g l a n c i n g angle" technique (59). A n o n d e s t r u c t i v e depth p r o f i l e over the 0-50 A range i n polymers can be obtained by i n t e n s i t y r a t i o s o f emitted e l e c t r o n s o f d i f f e r e n t k i n e t i c energies (58). 2

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The SIMS technique u t i l i z e s a focused i o n beam which i s r a s t e r e d or scanned across the s u r f a c e , s p u t t e r i n g o f f the outer 0 to 15 or so Angstrons of the surface and a n a l y z i n g the s p u t t e r ed ions by a very s e n s i t i v e mass spectrometer. A l l elements and t h e i r i n d i v i d u a l isotopes can be detected. An elemental o r i s o t o p i c image of the surface can be obtained w i t h b e t t e r than one micron r e s o l u t i o n . The i n t e r f a c e can be p r o g r e s s i v e l y i o n etched away and reanalyzed, p r o v i d i n g a compositional a n a l y s i s i n t o the sample w i t h about 100 A depth r e s o l u t i o n . SIMS i s i n r e a l i t y a d e s t r u c t i v e technique, as the i o n beam c o n t i n u a l l y s p u t t e r s away the outer surface. The SIMS method has been e x t e n s i v e l y a p p l i e d to i n o r g a n i c samples (56). Very l i m i t e d a p p l i c a t i o n to b i o l o g i c a l samples i s a l s o underway (57). The q u a l i t a t i v e i n t e r p r e t a t i o s t r a i g h t f o r w a r d . Dept however, i s s p e c u l a t i v e at t h i s time and r e q u i r e s a great deal of e m p i r i c a l c a l i b r a t i o n work before i t can be very u s e f u l . Probing the Subsurface Zone The most common method i s the use of m u l t i p l e i n t e r n a l r e f l e c t i o n i n f r a r e d spectroscopy (60). Some depth dependence i s a v a i l a b l e using d i f f e r e n t angles of i n c i d e n c e , though the d i s tance probed i s i n the micron range. C a r e f u l s t u d i e s have given monolayer s e n s i t i v i t i e s of known monolayers deposited d i r e c t l y on the i n t e r n a l r e f l e c t i o n elements ( I R E s ) . The recent development o f F o u r i e r transform i n f r a r e d spectroscopy (FT-IR) (61) overcomes many of the i n t e n s i t y and s e n s i t i v i t y l i m i t a t i o n s of conventional d i s p e r s i v e i n f r a r e d spectroscopy, p a r t i c u l a r l y f o r aqueous s o l u t i o n s (62,65). FT-IR can a l s o be used i n the m u l t i p l e i n t e r n a l r e f l e c t i o n mode (64). To our knowledge FT-IR has not yet been a p p l i e d t o g e l / s o l u t i o n i n t e r face s t u d i e s , however. Raman spectroscopy should be very u s e f u l i n c h a r a c t e r i z i n g g e l / s o l u t i o n i n t e r f a c e s d i r e c t l y , as water i s an i d e a l solvent f o r Raman s t u d i e s (65). Elemental a n a l y s i s of the subsurface zone can be accomplished by energy d i s p e r s i v e a n a l y s i s of x-rays i n the SEM, as prev i o u s l y mentioned, or by the more accurate wavelength d i s p e r s i v e a n a l y s i s of most e l e c t r o n microprobes (51). Subsurface penetrat i o n i s i n the micron r e g i o n and can be adjusted somewhat by v a r y i n g the i n c i d e n t e l e c t r o n beam energy. Again the sample must be r a p i d l y frozen to l i q u i d n i t r o g e n temperatures and maintained below -130°C f o r a n a l y s i s (51). f

Further

Discussion

Other techniques are a v a i l a b l e f o r study of the gel/water or g e l / s o l u t i o n i n t e r f a c e . Most s o l i d surface c h a r a c t e r i z a t i o n

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techniques (53) can, i n p r i n c i p l e , be a p p l i e d t o the frozen g e l surface. P r a c t i c a l l y a l l o f the microscopic and h i s t o l o g i c techniques used f o r the study o f c e l l surfaces can a l s o be app l i e d , i n c l u d i n g s e l e c t i v e f i x a t i v e s , s t a i n s , e t c . ( 7 ) . Some o f the methods used i n the study o f l i q u i d / l i q u i d i n t e r f a c e s (17,66) can be a p p l i e d t o g e l i n t e r f a c e s . We have a l s o ignored many techniques which are g e n e r a l l y a p p l i c a b l e only f o r high surface area systems. We have d i s c u s s e d p r i m a r i l y those techniques with which we are f a m i l i a r o r which we are c o n s i d e r i n g t o apply t o the study o f gel/solution interfaces. Conclusions A v a r i e t y o f method solution interfaces. D i r e c t i n s i t u methods i n c l u d e : 1. rhéologie o r v i s c o m e t r i c a n a l y s i s 2. ellipsometry 3. contact angles 4. e l e c t r o k i n e t i c s 5. i n f r a r e d spectroscopy 6. Raman spectroscopy 7. o p t i c a l microscopy Dry g e l / a i r i n t e r f a c e methods i n c l u d e : 1. i n f r a r e d spectroscopy 2. scanning, t r a n s m i s s i o n , and o p t i c a l microscopy 3. surface p o t e n t i a l 4. contact angles 5. ESCA 6. SIMS Frozen g e l surfaces can be studied by: 1. scanning, t r a n s m i s s i o n , or o p t i c a l microscopy 2. e l e c t r o n microprobe o r EDAX 3. ESCA 4. SIMS and many o f the other methods. These methods permit one t o probe the g e l / s o l u t i o n i n t e r f a c e w i t h respect t o : 1. i n t e r f a c e energetics 2. i n t e r f a c e p o t e n t i a l s 3. i n t e r f a c e chemical groups and o r i e n t a t i o n s 4. i n t e r f a c e s t r u c t u r e o r morphology 5. i n t e r f a c e elemental composition The subsurface zone can be analyzed f o r : 1. i n t e r f a c e chemical groups and o r i e n t a t i o n s 2. i n t e r f a c e elemental composition In a d d i t i o n , s o l u b l e g e l molecules can be studied as adsorbed f i l m s at s o l i d / l i q u i d i n t e r f a c e s or l i q u i d / a i r i n t e r f a c e s

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Acknowledgements S t i m u l a t i n g d i s c u s s i o n s w i t h S. W. Kim, M. S. Jhon, Y. K. Sung and D. L. Coleman have been most h e l p f u l . The personnel of ETEC, I n c . , Hayward, C a l i f o r n i a were most generous w i t h B i o SEM time w i t h which our f i r s t f r e e z e - e t c h s t u d i e s were done. The t e c h n i c a l a s s i s t a n c e o f R. Middaugh, G. Iwamoto, and D. Kramer i s appreciated. This work has been supported by NIH Grant HL 16921-01.

Abstract The experimental characterization of gel/water interfaces is briefly discussed. Interfacial characteristics discussed in clude topography/morphology interfacial tensio fre interface potential, adsorption nature of the gel surface, and the nature of the subsurface region. Techniques briefly discussed include microscopy, con tact angle methods, electrokinetic methods, surface potentials, infrared spectroscopy - including Fourier transform and Raman, x-ray photoelectron spectroscopy (ESCA), and secondary ion mass analysis (SIMS). Most of these techniques are discussed in suggestive and speculative terms as so few have been applied to the gel/water interface. A variety of techniques are available for studying the gel/water interface either in situ or in the frozen state. Literature Cited 1. Andrade, J. D., Lee, H. B., Jhon, M. S., Kim, S. W., and Hibbs, Jr., J. Β., Trans. Amer. Soc. Artif. Internal Organ., (1973) 19, 1. 2. Silberberg, Α., Polymer Preprints, (1970) 11, 1289. 3. Silberberg, Α., This Symposium. 4. Whitehouse, D. J., in P. F. Kane and G. B. Larrabee, eds., "Characterization of Solid Surfaces," Plenum, 1974. 5. McCrone, S. C., in P. F. Kane and G. B. Larrabee, eds., "Characterization of Solid Surfaces," Plenum, 1974. 6. Lee, Η. Β., Shim, H. S. and Andrade, J. D., Polymer Pre prints, (1972) 13, 729. 7. Koehler, J. D., ed., "Advanced Techniques in Biological Electron Microscopy," Springer-Verlag, 1973. 8. "Freeze Etch Replication," Ε. M. Ventions, Rockville, Mary land. 9. Cluthe, C. Ε., "Microscopical Study of the Structure of Radiation Cross-linked Aqueous Poly-(ethylene oxide) Gels and Frozen Cryoprotective Agents," Ph.D. Thesis, Cornell University, May 1972. 10. Blank, Z. and Reimschuessel, A. C., J. Materials Science, (1974) 9, 1815.

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11. Jhon, M. S. and Andrade, J. D., J. Biomed. Materials Res., (1973) 7, 509. 12. Belavtseva, Y. M., Titova, Y. F., Braudo, Y. Y. and Tolstoguzov, V. B., Biophysics, (1974) 19, #1, 15. (English translation of Biofizika: 19: No. 1 (1974) 19 13. Bohoneck, J., Coll. and Polymer Sci., (1974) 252, 417. 14. Geymayer, W. F., J. Polymer Sci. Symposium, (1974) 44, 25. 15. Matas, B. R., Spencer, W. H., and Hayes, T. L., Arch. Ophthal., (1972) 88, 287. 16. Johari, O. and Samudra, Α. V., in P. F. Kane and G. B. Larrabee, eds., "Characterization of Solid Surfaces," Plenum, 1974. 17. Adamson, A. W., "Physical Chemistry of Surfaces," 2nd ed., Wiley, 1967. 18. Bikerman, J. J., "Physical Surfaces," Academi Press Ne York, 1970. 19. Neumann, A. W., Adv. Coll. Interface Sci., (1974) 4, 105. 20. Wu, S., J. Macromol. Sci.-Revs. Macromol. Chem., (1974) C10, 1, 1. 21. Bikerman, J. J., Proc. 2nd Intern. Congress on Surface Activity III, (1957) 125. 22. Johnson, R. Ε., J. Phys. Chem., (1959) 63, 1655. 23. Lester, G. R., J. Coll. Sci., (1961) 16, 315. 24. Hansen, R. J. and Toong, Τ. Υ., J. Coll. Interf. Sci., (1971) 37, 196. 25. Schwartz, Α. Μ., Adv. in Coll. and Interf. Sci., (1975) 4, 349. 26. Timmons, C. O. and Zisman, W. Α., J. Coll. Interf. Sci., (1966) 22, 165. 27. Braudo, Υ. Υ., Michailow, Ε. N. and Tolstoguzov, V. Β., Phys. Chemie, Leipzig, (1973) 253, 369. 28. Holly, F. J. and Refojo, M. F., J. Biomed. Materials Res., (1975) 9, 315-326. 29. Davies, J. T. and Rideal, Ε. Κ., "Interfacial Phenomena," 2nd ed. Academic Press, 1963. 30. Shaw, D. J., "Electrophoresis" Academic Press, 1969. 31. Nordt, F. Knox, R. J., and Seaman, G. V. F., This Symposium. 32. Ma, S. M., Gregonis, D. Ε., Van Wagenen, R. and Andrade, J. D., This Symposium. 33. Brooks, D. E., J. Coll. Interf. Sci., (1973) 43 687. 34. Kavanagh, Α. Μ., Posner, Α. Μ., and Quirk, J. P., Faraday Disc., No. 59, (1975). 35. Van Wagenen, R., Andrade, J. D. and Hibbs, Jr., J. Β., submitted to the J. Electrochem. Soc. 36. Instruction Manual for Model 162 Electrostatic Millivolt Meter, Monroe Electronics, Inc., Middleport, New York. 37. Gaines, G. L., "Insoluble Monolayers at Liquid-Gas Inter faces," Interscience, 1966. 38. Beredick, Ν., In. B. Ke, ed., "Newer Methods of Polymer Characterization," Wiley, 1964.

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39. Johansson, G., Biochem. Biophys. Acta, (1970) 221, 387. 40. Holly, F. J. and Refojo, M. F., This Symposium. 41. Horbett, T. A. and Hoffman, A. S., Adv. in Chem. Series #145, "Applied Chem. at Protein Interfaces," R. E. Baier, ed., ACS 1975, 230. 42. Kim, S. W., unpublished work. 43. Lipatov, Yu, S. and Sergeeva, L. M., "Adsorption of Polymers," J. Wiley and Sons, 1974. (Translated from Russian by R. Kondor). 44. Bagchi, P., J. Coll. Interfac. Sci., (1974) 47, 86. 45. Hesselink, F. Th., Vrij, A. and Overbeek, J. Th. G., J. Phys. Chem., (1971) 75, 2094. 46. Botham, R. and Thies, C., J. Coll. Interf. Sci., (1969) 31, 1. 47. McGregor, R. and Ezuddin Κ Η. J Appl Polyme Sci. (1974) 18, 629. 48. Kalasz, J . , Nagy, J., and Knoll, J . , J. Anal. Chem., (1974) 272, 22. 49. Marsden, Ν. V. B., Naturwissenschaften, (1973) 60, 257. 50. Adamcova, Ζ., Coll. Czech. Chem. Comm., (1968) 33, 336. 51. Hall, T., Echlin, P., and Kaufmann, R., eds., "Microprobe Analysis as Applied to Cells and Tissues," Academic Press, 1974. 52. Muller, Η., Adv. Electrochem. and Electrochem. Engr., (1973) 9, 167. 53. Kane, P. F. and Larrabee, G. Β., eds., "Characterization of Solid Surfaces," Plenum, 1974. 54. Siegbahn, Κ., et.al., "ESCA--Atomic, Molecular and Solid State Structure Studied by Means of Electron Spectroscopy," Almquist and Wiksells, Publ., Uppsala, Sweden, 1967. 55. Dekeyser, W., Fiermans, L., Vanderkelen, G., and Vennik, J . , "Electron Emmission Spectroscopy," D. Reidel Publ. Co., 1973. 56. Werner, H. W., Surface Science, (1975) 47, 201. 57. Galle, P., in Reference 51. 58. Clark, D. T., Feast, W. J., Musgrove, W. K. R., Ritchie, I., J. Polymer Sci., Polymer Chem., (1975) 13, 857. 59. Fadley, C. S., Baird, R. J., Siekhaus, W., Novakov, T., and Bergstrom, S. A. L., J. Electron Spect. Related Phen., (1974) 4, 93. 60. Harrick, N. J., "Internal Reflection Spectroscopy," Interscience, 1967. 61. Koenig, J. L. and Tabb, D. L., Canad. Res. and Dev., (SeptOct, 1974) 25. 62. Low, M. J. D. and Yang, R. T., Spectrochem. Acta, (1973) 29A, 1761. 63. Low, M. J. D. and Yang, R. T., Spectroscopy Letters, (1973) 6, 299. 64. Yang, R. T., Low, M. J. D., Haller, G. L. and Fenn, J . , J. Coll. Interf. Sci., (1973) 44, 249.

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65. Buechler, E. and Turkevich, J., J. Phys. Chem. (1972) 76, 2325. 66. Albertsson, P. Α., "Partition of Cell Particles and Macromolecules," 2nd ed., Wiley, 1971. 67. Dobreva, Μ., Dancheva, N. and Holt, P. F., Brit. J. Ind. Med., (1975) 32, 224.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

17 Elimination of Electroosmotic Flow in Analytical Particle Electrophoresis F. J. NORDT, R. J. KNOX, and G. V. F. SEAMAN Department of Neurology, University of Oregon Health Sciences Center, Portland, Oreg. 97201

I n t e r e s t i n surface coatings which w i l l markedly reduce or e l i m i n a t e the z e t a p o t e n t i a p r a c t i c a l i s s u e of e l i m i n a t i n p h o r e s i s . I n a closed c y l i n d r i c a l g l a s s e l e c t r o p h o r e s i s chamber c o n t a i n i n g an e l e c t r o l y t e the negative charge a t the g l a s s w a l l r e s u l t s i n an i n c r e a s e i n c o n c e n t r a t i o n of c a t i o n s c l o s e t o t h i s s u r f a c e . A p p l i c a t i o n of an e x t e r n a l e l e c t r i c a l f i e l d r e s u l t s i n movement of f l u i d near the w a l l (electroosmosis) toward the cathode and a concurrent f o r c e d r e t u r n flow through the center of the tube. I t can be shown from hydrodynamics that there i s a c y l i n d r i c a l envelope ( s t a t i o n a r y l e v e l ) i n the chamber where no net f l o w of f l u i d occurs during e l e c t r o p h o r e s i s . F i g u r e 1 i l l u s t r a t e s the general f e a t u r e s of laminar e l e c t r o o s m o t i c f l u i d f l o w f o r a c l o s e d c y l i n d r i c a l tube i n c l u d i n g the p a r a b o l i c f l u i d f l o w p r o f i l e , regions of e l e c t r o o s m o t i c f l o w , r e t u r n f l u i d flow and l o c a t i o n of the s t a t i o n a r y l e v e l . In a n a l y t i c a l p a r t i c l e e l e c t r o p h o r e s i s true e l e c t r o p h o r e t i c v e l o c i t i e s of p a r t i c l e s may be measured a t the s t a t i o n a r y l e v e l w h i l e v e l o c i t i e s determined elsewhere i n the chamber w i l l be comprised of c o n t r i b u t i o n s from both e l e c t r o p h o r e s i s and e l e c t r o osmosis. I n p r e p a r a t i v e a p p l i c a t i o n s of e l e c t r o p h o r e s i s the boundary of a concentrated suspension (sample) becomes p a r a b o l o i d a l i n contour as a r e s u l t of electroosmosis of the suspending medium i n the chamber. The non-planar sample d i s t r i b u t i o n introduces d i f f i c u l t i e s i n separating or r e s o l v i n g p a r t i c l e populations which d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t y . I n analyt i c a l p a r t i c l e e l e c t r o p h o r e s i s the presence of e l e c t r o o s m o t i c f l o w r e q u i r e s that measurements be c a r r i e d out a t the s t a t i o n a r y l e v e l . Since t h i s l e v e l i s i n f i n i t e l y t h i n e l e c t r o o s m o t i c f l o w w i l l always c o n t r i b u t e t o experimental e r r o r . The w a l l charge i n e l e c t r o p h o r e s i s chambers a r i s e s from e i t h e r the i o n i z a t i o n of surface charge groups or as a consequence of r e d i s t r i b u t i o n of ions from the suspending medium (adsorption or d e s o r p t i o n ) . The w a l l charge may be reduced o r e l i m i n a t e d by: a) use of adherent or adhesive f i l m s ( 1 ) . 225

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Figure 1. Electroosmotic fluid flow in a closed cylindrical tube, (a) A longitudinal crossectional view of the fluid velocity profile and fluid streamlines for a tube with radius, R (fluid velocity is plotted in terms of V , the fluid flow at the tube wall), (b) A transverse crossection where maximum electroosmotic fluid flow is shown by dense shading, and the unshaded area shows the region of the stationary level where fluid flow tends to zero, (c) Fluid flow parabola which is a plot of fluid velocity vs. distance from the tube wall. s

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b) covalent bonding m a t e r i a l s ( 2 ) . c) p h y s i c a l adsorption of substances ( 3 ) . E l e c t r o p h o r e t i c t e s t i n g of the s t a b i l i t y and completeness of d i f f e r e n t surface treatments or coatings may be c a r r i e d out i n v a r i o u s ways: i ) A f t e r coating the i n s i d e of the e l e c t r o p h o r e s i s chamber the electroosmotic f l o w i s c a l c u l a t e d from experimental measurements of the e l e c t r o p h o r e t i c v e l o c i t i e s of stand ard p a r t i c l e s a t v a r i o u s d i s t a n c e s from the tube w a l l , i i ) The e l e c t r o p h o r e t i c m o b i l i t i e s of coated or modified p a r t i c l e s made from the same m a t e r i a l s as the e l e c t r o p h o r e s i s chamber are measured by standard a n a l y t i c a l electrophoresis. i i i ) The zeta p o t e n t i a l of coated tubes may be a l s o determined from e l e c t r o o s m o t i ments. The f i r s t approach i s the more d e s i r a b l e t e s t of any coating procedure but f o r screening purposes the second t e s t approach w i l l s i g n i f i c a n t l y reduce the time needed f o r the i n i t i a l survey or t e s t i n g of coatings or m o d i f i c a t i o n procedures. Theoretical The e l e c t r o p h o r e t i c m o b i l i t y , u, i s defined as the e l e c t r o p h o r e t i c v e l o c i t y , v, of a p a r t i c l e per u n i t f i e l d s t r e n g t h , χ: ν

/.\

The r e l a t i o n s h i p between e l e c t r o p h o r e t i c m o b i l i t y , u, and zeta p o t e n t i a l , ζ, f o r nonconducting p a r t i c l e s whose r a d i u s of curvature, a, i s l a r g e i n comparison to the e f f e c t i v e thickness of the e l e c t r i c a l double l a y e r , l/κ, i s u s u a l l y described a c c u r a t e l y f o r Ka > 300 by the Helmholtz-Smoluchowski equation (4):

where ε and η are the d i e l e c t r i c constant and v i s c o s i t y , respec t i v e l y , w i t h i n the e l e c t r i c a l double l a y e r which are assumed t o be the same as the bulk v a l u e s of the suspending medium. E x p e r i mental measurements of ν r e s u l t i n an observed e l e c t r o p h o r e t i c v e l o c i t y , v , which i s subject t o e r r o r as i s the experimentally derived e l e c t r o p h o r e t i c m o b i l i t y , u . A p p l i c a t i o n of an e l e c t r i c f i e l d t o a suspension of charged p a r t i c l e s contained i n a closed c y l i n d r i c a l chamber w i t h a charged w a l l r e s u l t s i n e l e c t r o p h o r e s i s of the p a r t i c l e s and electroosmotic flow of the suspending medium. The observed v e l o c i t y , v , of a p a r t i c l e i n the tube i s thus the sum of i t s e l e c t r o p h o r e t i c v e l o c i t y , v , and the v e l o c i t y of the suspending e

e

Q

e

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medium, v : w

v

=

o

v

e

+

v

w

<

ii:L

>

I t has been shown by Bangham et a l . (5) that the observed v e l o c i t y of the p a r t i c l e i s r e l a t e d to i t s d i s t a n c e , r , from the a x i s of the tube by the expression: v

o = v + v [|j£ " H

(iv)

s

e

where v i s the f l u i d v e l o c i t y adjacent t o the tube w a l l and R i s the r a d i u s of the tube. S o l u t i o n of the flow equation ( i v ) shows that at a d i s t a n c e r = 0.707R from the a x i s there i s no net f l o w of f l u i d , i . e . , a s t a t i o n a r In theory e l e c t r o p h o r e t i made at the s t a t i o n a r y l e v e l are not subject to e r r o r as a r e s u l t of f l u i d flow. However, i n p r a c t i c e e r r o r s a r i s e as a r e s u l t o f : a) the f i n i t e s i z e of the p a r t i c l e s which cannot be c o n t a i n ed i n an i n f i n i t e l y t h i n s t a t i o n a r y l e v e l ; b) f o c u s i n g e r r o r s a t the s t a t i o n a r y l e v e l (requirement f o r appropriate o p t i c a l c o r r e c t i o n s to r e c t i f y the e f f e c t s of r e f r a c t i o n and a b e r r a t i o n , depth of focus, and shape and s i z e of f o c a l f i e l d r e l a t i v e t o the r a d i u s of curvature of the s t a t i o n a r y l e v e l ) ; c) heterogeneous d i s t r i b u t i o n of charge on the tube w a l l r e s u l t i n g i n a s h i f t i n l o c a t i o n of the s t a t i o n a r y l e v e l ; d) Brownian motion and sedimentation of the p a r t i c l e ; and e) thermal convection a r i s i n g from J o u l e h e a t i n g . The magnitude of e r r o r s i n the e l e c t r o p h o r e t i c v e l o c i t y as a r e s u l t of f l u i d flow may be estimated from a d i f f e r e n t i a t e d form of equation ( i v ) : s

dv

0

= *g£

dr

(v)

D i v i s i o n of equation (v) by the f i e l d g r a d i e n t , χ, gives an expression f o r the change i n the experimentally observed e l e c t r o p h o r e t i c m o b i l i t y at s m a l l r a d i a l increments from the s t a t i o n a r y l e v e l i n terms of the e l e c t r o o s m o t i c m o b i l i t y , u : g

Au

4u r . = — f - Ar 2 q

ο n

I f the f r a c t i o n a l e r r o r i n u as 6, then: δ =

, .v (vi)

R

e

due to f l u i d flow i s defined

Au 4u r = Ar u^ R u~

—0

s

9

z

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

.. (vu)

17.

NORDT E T A L .

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Particle

Electrophoresis

229

From e x p r e s s i o n ( v i i ) one may c a l c u l a t e the maximum v a l u e of u which w i l l produce f r a c t i o n a l e r r o r s of l e s s than δ i n u at d i s t a n c e s up t o Ar from the s t a t i o n a r y l e v e l . s

e

M a t e r i a l s and Methods Corning #7740 b o r o s i l i c a t e p a r t i c l e s were used as a model system f o r screening the e f f e c t i v e n e s s of p o l y s a c c h a r i d e d e r i v a t i v e s as low z e t a p o t e n t i a l s u r f a c e coatings f o r g l a s s . A l l chemicals were reagent grade and the water was d i s t i l l e d t w i c e i n pyrex ware. The p a r t i c l e s were prepared by g r i n d i n g Corning #7740 g l a s s tubing w i t h water i n an aluminum oxide b a l l m i l l f o r 16 hours. P a r t i c l e s of s u i t a b l e s i z e f o r e l e c t r o p h o r e t i c measurements were obtained by repeated sedimentatio c l e s having a sedimentatio P a r t i c l e s thus obtained were <_ 5 ym i n diameter. These were t r a n s f e r r e d t o g l a s s c e n t r i f u g e b o t t l e s and cleaned w i t h hot aqua r e g i a f o l l o w e d by hot 6N HC1. P o s s i b l e organic contaminants were removed by s e q u e n t i a l treatment of the p a r t i c l e s w i t h t o l u ene and i s o p r o p a n o l . The p a r t i c l e s were then washed s i x times w i t h 0.015 M NaCl a t 60-70°C. Further c l e a n i n g treatments f a i l e d t o i n c r e a s e the e l e c t r o p h o r e t i c m o b i l i t y of the p a r t i c l e s which was monitored throughout the c l e a n i n g procedure. Coating M a t e r i a l s . D i e t h y l a m i n o e t h y l - m e t h y l c e l l u l o s e (DEAE-methylcellulose) was prepared from m e t h y l c e l l u l o s e (Dow Methocel MC, 8000 cps Premium, L o t MM-110786) by a s c a l e d down m o d i f i c a t i o n of the method of Peterson and Sober ( 6 ) . A i r o x i d a t i o n of carbonyl groups on the m e t h y l c e l l u l o s e was prevented by the a d d i t i o n of sodium borohydride (0.5 g per 100 g of methyl c e l l u l o s e ) . Acetone was used t o p r e c i p i t a t e the polymer a f t e r the a d d i t i o n of the 2 M NaCl. The product was then f i l t e r e d and e x h a u s t i v e l y d i a l y s e d against water t o pH 7 i n Union Carbide d i a l y s i s tubing. The r e s u l t i n g DEAE-methylcellulose was a c l e a r g e l which was l y o p h i l i z e d and d i s p e r s e d i n a Waring blender. The extent of m e t h y l c e l l u l o s e m o d i f i c a t i o n was c o n t r o l l e d by a d j u s t i n g the amount of d i e t h y l a m i n o e t h y l c h l o r i d e used i n the a l k y l a t i o n r e a c t i o n (6). Two batches of 60 grams each of methyl c e l l u l o s e were modified w i t h 7 grams and 35 grams, r e s p e c t i v e l y , of d i e t h y l a m i n o e t h y l c h l o r i d e . These preparations w i l l be r e f e r r e d t o as 'lower and 'higher* degree amino s u b s t i t u t e d methylcellulose derivatives. The n i t r o g e n contents of the DEAE d e r i v a t i v e s were d e t e r mined f o r the l y o p h i l i z e d m a t e r i a l s by the m i c r o - K j e l d a h l method (7). The e f f e c t i v e n e s s of m e t h y l c e l l u l o s e and i t s d e r i v a t i v e s as low z e t a p o t e n t i a l c o a t i n g agents was examined by suspending g l a s s p a r t i c l e s i n a 0.015 M NaCl s o l u t i o n c o n t a i n i n g a 0.1% (w/v) c o n c e n t r a t i o n of a given polymer and measuring the e l e c t r o 1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

230

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

p h o r e t i c m o b i l i t y of the g l a s s p a r t i c l e s at 25°C i n an a l l g l a s s c y l i n d r i c a l chamber apparatus equipped w i t h e i t h e r grey platinum e l e c t r o d e s (5) or w i t h r e v e r s i b l e Ag/AgCl e l e c t r o d e s as described by Seaman and Heard (8). As a t e s t of the t e n a c i t y of the c o a t i n g , the p a r t i c l e s were washed one to three times i n d i l u t e s a l i n e media. The e f f e c t of the washes was assessed by measuring the e l e c t r o p h o r e t i c m o b i l i t y of the p a r t i c l e s i n 0.015 M NaCl-NaHC0 , pH 7.2 ± 0.2 and noting any changes. I n attempts to improve the s t a b i l i t y of the c o a t i n g , e p i c h l o r o h y d r i n was employed as a p o t e n t i a l c r o s s l i n k i n g agent f o r DEAE-methylcellulose. A m o d i f i c a t i o n of F l o d i n ' s procedure (9) was used. A 0.2% (w/v) s o l u t i o n of the higher modified DEAEm e t h y l c e l l u l o s e was prepared to which g l a s s p a r t i c l e s were added to make a m i l k y white suspension l y c e n t r i f u g e d , the supernatan suspended i n 1.4 M sodium hydroxide to which sodium borohydride (0.5 g per 100 g DEAE-methylcellulose) had been added to prevent o x i d a t i o n of the polymer. This suspension was mixed w i t h r e d i s t i l l e d e p i c h l o r o h y d r i n , 0.2% w/v (Eastman Kodak), and incubated at 70°C i n a water bath f o r 4 hours. The p a r t i c l e s were subsequently c e n t r i f u g e d , washed three times i n 0.015 M NaCl-NaHCOg, pH 7.2 and subjected to e l e c t r o p h o r e s i s . The e f f e c t of f u r t h e r washes on the e l e c t r o p h o r e t i c m o b i l i t y of the p a r t i c l e s was monitored. F i n a l l y the i n t e r n a l surface of an a l l g l a s s c y l i n d r i c a l e l e c t r o p h o r e s i s chamber was coated w i t h DEAE-methylcellulose and then t r e a t e d w i t h e p i c h l o r o h y d r i n . The chamber was f i l l e d w i t h a 0.2% w/v s o l u t i o n of DEAE-methylcellulose i n 1.4 M sodium hydroxide c o n t a i n i n g 0.5 g sodium borohydride per 100 g DEAE-methylc e l l u l o s e and 10% (v/v) e p i c h l o r o h y d r i n . The chamber was incubated i n a 70°C water bath overnight. The chamber was then thoroughly r i n s e d out w i t h 1 M KCl and set up i n the u s u a l manner (10). The e f f e c t of the c o a t i n g on the e l e c t r o o s m o t i c f l o w was assessed by measuring the e l e c t r o p h o r e t i c m o b i l i t y of n a t i v e g l a s s p a r t i c l e s i n media of v a r y i n g i o n i c strengths a t a s e r i e s of d i s t a n c e s from the i n n e r chamber w a l l . 3

Results I n a n a l y t i c a l p a r t i c l e e l e c t r o p h o r e s i s m o b i l i t y measurements are i n p r i n c i p l e made at the s t a t i o n a r y l e v e l of the e l e c t r o phoresis chamber where no e l e c t r o o s m o t i c f l u i d f l o w i n f l u e n c e s the measurements. The p a t t e r n of e l e c t r o o s m o t i c f l u i d flow has been described f o r closed systems of v a r i o u s geometries (11) so t h a t i t i s p o s s i b l e to c a l c u l a t e the l o c a t i o n of the s t a t i o n a r y l e v e l f o r i d e a l c o n d i t i o n s . I n p r a c t i c e , m o b i l i t y measurements are only made i n the v i c i n i t y of the r e a l s t a t i o n a r y l e v e l f o r s e v e r a l reasons. T y p i c a l l y , the l o c a t i o n of the r e a l s t a t i o n a r y l e v e l i s not experimentally d e f i n e d , but r a t h e r i s c a l c u l a t e d f o r

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

17. NORDT ET AL.

Analytical

Particle

Electrophoresis

231

i d e a l c o n d i t i o n s from the electroosmotic flow equations and used i n the adjustment of the apparatus. A l a c k of appropriate o p t i c a l r e f r a c t i o n or a b e r r a t i o n c o r r e c t i o n s , small e r r o r s i n instrument s e t t i n g s , or s h i f t s of the l o c a t i o n of the r e a l s t a t i o n a r y l e v e l from the t h e o r e t i c a l l o c a t i o n due t o n o n i d e a l c o n d i t i o n s such as contamination of the chamber surface a l l lead to e r r o r s i n l o c a t i n g the r e a l s t a t i o n a r y l e v e l . Even i f the apparatus i s o p t i m a l l y focused, the p a r t i c l e s under examination w i l l not remain a t the s t a t i o n a r y l e v e l f o r the d u r a t i o n of the measurements f o r the reasons o u t l i n e d i n the t h e o r e t i c a l s e c t i o n , so that electroosmosis when present i s a s i g n i f i c a n t source of experimental e r r o r . The magnitudes of these e r r o r s i n the m o b i l i t y measurements when the p a r t i c l e s are not a t the s t a t i o n a r y l e v e l may be e s t i mated f o r small distance equation ( v i i ) . The r e s u l t Figure 2 which i s i n t e r p r e t e d as f o l l o w s . I f the e l e c t r o p h o r e t i c m o b i l i t y , u , i s measured f o r p a r t i c l e s a t 20 ym from the s t a t i o n a r y l e v e l i n a 2 mm diameter c a p i l l a r y , there w i l l be a f r a c t i o n a l e r r o r , 6, i n the m o b i l i t y v a l u e obtained, the magnitude of which w i l l depend on the r e l a t i v e magnitudes of the e l e c t r o osmotic m o b i l i t y , u , and u . Thus f o r a p a r t i c l e w i t h u = 2.0, and u = 2.1, the measured e l e c t r o p h o r e t i c m o b i l i t y a t 20 ym from the s t a t i o n a r y l e v e l would deviate from the true m o b i l i t y by a δ of 0.06 or 6%. I n a cleaned b o r o s i l i c a t e g l a s s c a p i l l a r y a t n e u t r a l pH, u ranges from 5-6 ym s e c " v o l t " cm a t an i o n i c s t r e n g t h of 0.015 and from 2 t o 3 a t 0.15 i o n i c s t r e n g t h. I t can be seen that electroosmotic m o b i l i t i e s of such magnitude give r i s e t o appreciable e r r o r s i n the u v a l u e s obtained f o r p a r t i c l e s a t distances from the s t a t i o n a r y l e v e l l i k e l y to be encoun tered experimentally. The e s t i m a t i o n i n Figure 2 i s p a r t i c u l a r l y u s e f u l i n e s t a b l i s h i n g what r e d u c t i o n of u w i l l reduce e r r o r s t o an acceptable degree f o r m o b i l i t y measurements made near the s t a t i o n a r y l e v e l . For m o b i l i t y measurements made a t the c a l c u l a t e d s t a t i o n a r y l e v e l i t i s not necessary t o reduce u to zero i n order f o r the e r r o r s to be small u n l e s s the e l e c t r o p h o r e t i c m o b i l i t i e s t o be measured are near zero. I n p r a c t i c e , any s u b s t a n t i a l r e d u c t i o n of u f a c i l i t a t e s data c o l l e c t i o n , reduces operator e r r o r s i n judging whether p a r t i c l e s a r e i n focus, and minimizes the i n f l u e n c e of changes i n the l o c a t i o n of the s t a t i o n a r y l e v e l due t o contamina t i o n of the w a l l surface by adsorbed substances o r t o heterogen eous d i s t r i b u t i o n of charge on the uncontaminated w a l l . I n p r e p a r a t i v e p a r t i c l e e l e c t r o p h o r e s i s or i n automated a n a l y t i c a l techniques where measurements a r e made a t l o c a t i o n s other than near the s t a t i o n a r y l e v e l , i t i s necessary t o reduce u t o zero. M e t h y l c e l l u l o s e and two preparations of DEAE-methylcellulose which each contained d i f f e r e n t l e v e l s of amino group s u b s t i t u t i o n were tested as coating agents f o r cleaned b o r o s i l i c a t e g l a s s . The n i t r o g e n content of the low and higher amino s u b s t i t u t e d e

s

e

e

s

1

1

s

e

s

s

g

g

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

232

HYDROGELS FOR

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m e t h y l c e l l u l o s e d e r i v a t i v e s were found to be 0.37 and 1.28 mg per gram, r e s p e c t i v e l y , by m i c r o - K j e l d a h l assay. The e l e c t r o p h o r e t i c m o b i l i t i e s of bare b o r o s i l i c a t e g l a s s p a r t i c l e s and p a r t i c l e s which have been subjected to v a r i o u s treatments are presented i n Table I . I t should be noted t h a t the r e l a t i v e l y high zeta p o t e n t i a l (y -70 mV) c a l c u l a t e d from equation ( i i ) f o r the bare g l a s s p a r t i c l e s w i l l r e s u l t i n an average pH at the surface of the g l a s s p a r t i c l e s of about 1.2 u n i t s lower than the bulk value (12). The washing experiments show that the m e t h y l c e l l u l o s e i s r e a d i l y washed o f f the p a r t i c l e s whereas the DEAE-methylcellulose i s adherent and survives washi n g . The weak a d s o r p t i o n of the m e t h y l c e l l u l o s e r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n zeta p o t e n t i a l of the b o r o s i l i c a t e p a r t i c l e s , a phenomenon which has been observed f o r other weak and r e v e r s i b l y adsorbe I n F i g u r e 3 the e l e c t r o p h o r e t i s h i p s are presented f o r n a t i v e b o r o s i l i c a t e g l a s s p a r t i c l e s and e p i c h l o r o h y d r i n c r o s s l i n k e d DEAE-methylcellulose coated p a r t i c l e s . Evidence f o r an a p p r e c i a b l e degree of c r o s s l i n k i n g i s provided by the formation of a w a t e r - i n s o l u b l e g e l - l i k e m a t e r i a l which develops a f t e r a d d i t i o n of e p i c h l o r o h y d r i n to the r e a c t i o n mixture. In c o n t r a s t the p r e c i p i t a t e of the polymer which i s produced by the a d d i t i o n of sodium hydroxide alone i s water s o l u b l e . The pH versus m o b i l i t y r e l a t i o n s h i p f o r bare g l a s s suggests the presence of two or more surface ionogenic groups. The c r o s s l i n k e d DEAE-methylcellulose coated p a r t i c l e s e x h i b i t a p o s i t i v e branch to the pH versus m o b i l i t y r e l a t i o n s h i p at low pH and an increase i n m o b i l i t y between pH 9 and 10 c o n s i s t e n t w i t h the presence of a p o s i t i v e group of pK ca. 9.5. Figure 4 shows the e l e c t r o p h o r e t i c v e l o c i t y data on bare g l a s s p a r t i c l e s as a f u n c t i o n of t h e i r r a d i a l p o s i t i o n w i t h i n the e l e c t r o p h o r e s i s tube coated w i t h e p i c h l o r o h y d r i n c r o s s l i n k e d DEAE-methylcellulose. Consistent e l e c t r o p h o r e t i c v e l o c i t i e s were obtained f o r up to 15 hours. C a l c u l a t i o n of the e l e c t r o osmotic flow at the w a l l of the chamber by means of equation ( i v ) y i e l d e d values which ranged between -0.09 and +0.12 ym sec" v o l t cm over the range of i o n i c strengths i n v e s t i g a t e d . The dashed l i n e i n Figure 4 represents the e l e c t r o p h o r e t i c v e l o c i t y of the bare g l a s s p a r t i c l e s i n an uncoated g l a s s chamber. Use of equation ( i v ) f o r the r e s u l t s obtained from a bare g l a s s chamber y i e l d an average value f o r the e l e c t r o o s m o t i c f l o w of +5.9 ym s e c " v o l t " cm corresponding to an e l e c t r o p h o r e t i c m o b i l i t y of -5.9 which agrees w e l l w i t h the observed value of -5.6 ym s e c " v o l t " cm f o r bare b o r o s i l i c a t e g l a s s p a r t i c l e s . A f t e r twenty-four hours the chamber coated w i t h c r o s s l i n k e d DEAEm e t h y l c e l l u l o s e showed an i n c r e a s e i n negative surface charge which r e s u l t e d i n an e l e c t r o o s m o t i c flow of +1.21 ym s e c " v o l t " cm f o r the aqueous 0.015 M NaCl medium as a r e s u l t of slow d e s o r p t i o n of the c o a t i n g . 1

- 1

1

1

1

1

1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

1

17.

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Electrophoresis

TABLE I ELECTROPHORETIC MOBILITIES OF BOROSILICATE GLASS PARTICLES FOLLOWING VARIOUS TREATMENTS 3

Observed u ± s (n)

Corrected u

Treatment

pH

None

7.2

-5.55

+

0.25 (80)

8.1 7.2 7.2

+0.60 +0.76 +0.70

+ +

+1.23

+

0.04 (20) 0.05 (20) 0.04 (20)

8.0 7.2 7.2

+1.21 +0.93 +0.96

+ + +

0.04 (20) 0.05 (20) 0.05 (20)

+2.65

13

Methylcellulose 1 χ wash c

DEAE M e t h y l c e l l u l o s e 1 χ wash 3 χ wash

4

DEAE M e t h y l c e l l u l o s e 1 χ wash 3 χ wash

e

c

c

c

c

a.

b.

c.

d. e.

1

1

E l e c t r o p h o r e t i c m o b i l i t y , û", i n ym s e c " v o l t " " cm ± sample standard d e v i a t i o n (n = no. of measurements) f o r p a r t i c l e s at 25°C i n 0.015 M NaCl only or w i t h added polymer. Corrected f o r the bulk medium v i s c o s i t y e f f e c t s of the polymer d e r i v a t i v e s which were present a t a c o n c e n t r a t i o n of 0.1% (w/v). Washed samples had been suspended i n > 100 volumes of 0.015 M NaCl-NaHC03 pH 7.2 ± 0.1 c o n t a i n i n g no coating m a t e r i a l , c e n t r i f u g e d and resuspended i n f r e s h s a l i n e the number of times i n d i c a t e d . Low l e v e l amino group s u b s t i t u t e d m e t h y l c e l l u l o s e . Higher l e v e l amino group s u b s t i t u t e d m e t h y l c e l l u l o s e .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

0

1.0

2.0 U

e

3.0

4.0

5.0

6.0

(ym- s e c v o l t - c m ) H

Figure 2. Estimated electroosmotic mobilities, u , which produce fractional errors, δ, in the absolute values of electrophoretic mobility, u at 20 μτη from the stationary level in a 2.0-mm bore capillary tube. s

e

-6.00 r

-Η—M-H

-5.00

Γ/2 = 0.015

Β -4.001-

I

-3.00 Γ ρ. -2.00

Γ/2 = 0.015 -\—\-

5 -1.00 0.00 ^ +1.00 2

3 4

5

6

7

8

9 10 II 12

PH Figure 3. The electrophoretic mobility of bare borosilicate glass par ticles ( ) and crosslinked DEAE-methylcellulose coated particles ( ) as a function of pH of the suspending medium. Error bars indicate the standard deviation. Migration toward the cathode is positive. The ionic strength of the suspending medium (T/2) is 0.015 g ions I.' . 1

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-11.00 r -10.00 h w Γ/2 = 0.015 -9.00 -8.00

\

-7.00 -6.00 -5.00 -4.00 -3.00

4 — $ — M - V i — f -

-2.00 -1.00 0.00 300 400

600

Γ/2=0.Ι0 Γ/2 = 0.Ι5

800 1000

Distance From Axis Of Electrophoresis Tube (pm) Figure 4. Electrophoretic velocities of bare glass particles ( ) in a crosslinked DEAE-methylcellulose coated cy lindrical glass electrophoresis chamber at various radial positions inside the tube. The dotted line represents the same system for an uncoated glass chamber. The ionic strengths (T/2) at which each series of measurements were carried out are indicated.

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Discussion The aim of the work reported i n t h i s paper i s the development of a s t a b l e coating w i t h low or zero z e t a p o t e n t i a l f o r the surface of e l e c t r o p h o r e s i s chambers i n order to minimize or e l i m i n a t e the p e r t u r b i n g i n f l u e n c e of electroosmosis. In surface m o d i f i c a t i o n or c o a t i n g work i t i s important that the m a t e r i a l to be coated be w e l l defined and capable of being cleaned by some standard procedure so that b a s e l i n e or c o n t r o l e l e c t r o k i n e t i c data are r e a d i l y o b t a i n a b l e . The use of p l a s t i c s such as p o l y methylmethacrylate ( p l e x i g l a s ) or polycarbonates i n coating s t u d i e s i s complicated by the v a r i o u s a n t i - o x i d a n t s and other a d d i t i v e s which r e s u l t i n wide v a r i a t i o n s i n the surface propert i e s f o r o s t e n s i b l y the same product obtained from v a r i o u s sources or l o t s . In compariso p l a s t i c s and t h e i r e v a l u a t i o c u l t to d e v i s e . Taking these f a c t o r s i n t o c o n s i d e r a t i o n as w e l l as the extensive use of g l a s s i n the f a b r i c a t i o n of e l e c t r o p h o r e s i s chambers, e f f o r t s were focused on c o a t i n g pyrex g l a s s tubes and p a r t i c l e s . The nature of b o r o s i l i c a t e g l a s s surfaces and the p h y s i c a l and chemical c r i t e r i a r e q u i r e d f o r an optimum c o a t i n g designed to e l i m i n a t e electroosmosis determine the s e l e c t i o n of p o t e n t i a l c o a t i n g agents. The negative charge of b o r o s i l i c a t e g l a s s surfaces i s reported to o r i g i n a t e from the i o n i z a t i o n of surface groups which are an i n t e g r a l p a r t of the g l a s s . Two types of s i t e s are present, one w i t h a pK of approximately 7 and the second w i t h a pK of 5.1 (14). The weaker a c i d i c s i t e i s due to the s i l a n o l group "^Si-OH, w h i l e the stronger a c i d i c s i t e i s "^B-OH. I t should be noted that the e l e c t r o p h o r e t i c m o b i l i t y of a p a r t i c l e and e l e c t r o o s m o t i c f l u i d flow i n a closed e l e c t r o p h o r e s i s apparatus are both m a n i f e s t a t i o n s of surface charge, i n the former case t h a t of the p a r t i c l e and i n the l a t t e r that of the chamber. The geometry of the chamber precludes the use of adhesive f i l m s to e l i m i n a t e the surface charge which leaves two a l t e r n a t i v e general approaches, namely: a) covalent bonding to surface groups to e l i m i n a t e the negative groups by chemical m o d i f i c a t i o n ( t o t a l charge zero)or to introduce p o s i t i v e groups such that the net charge i s zero, or a combination of these types of surface m o d i f i c a t i o n . b) to use substances, u s u a l l y macromolecules, which p h y s i c a l l y adsorb to the g l a s s s u r f a c e , thereby s h i f t i n g the e l e c t r o p h o r e t i c plane of shear out from the o r i g i n a l g l a s s surface to the new macromolecular s u r f a c e . By t h i s means the charge on the o r i g i n a l surface w i l l have a n e g l i g i b l e i n f l u e n c e on i o n d i s t r i b u t i o n at the new plane of s l i p during e l e c t r o p h o r e s i s (13). The v a s t number of surface r e a c t i o n s encompassed by the a

a

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above two approaches can be s e v e r e l y r e s t r i c t e d by an examination of the c r i t e r i a r e q u i r e d f o r a s a t i s f a c t o r y surface coating or m o d i f i c a t i o n . The f o l l o w i n g p r o p e r t i e s of the surface coating should be considered i n judging i t s general s u i t a b i l i t y and application limitations: i) Physical properties: a) transparent to l i g h t , a t l e a s t over the same range of wavelengths as the o r i g i n a l g l a s s . b) s m a l l coating t h i c k n e s s r e l a t i v e to the r a d i u s of the e l e c t r o p h o r e s i s tube. c) capable of being a p p l i e d u n i f o r m l y . d) an e l e c t r i c a l conductance not s i g n i f i c a n t l y greater than that of the p a r t i c l e suspending media. e) low f i x e d or acquired e l e c t r o s t a t i c charge i n b u f f e r s . f ) poor surfac g) nonadhesive examined i n the coated tube. i i ) Chemical and b i o l o g i c a l p r o p e r t i e s : a) h y d r o p h i l i c t o minimize adsorption of components from samples. b) compatible w i t h system to be examined, e.g., non-toxic towards l i v i n g c e l l s . c) not subject to a t t a c k or degradation by any b i o l o g i c a l specimens under t e s t . d) does not desorb or change the e l e c t r o k i n e t i c p r o p e r t i e s of the p a r t i c l e s under examination. i i i ) E l e c t r o k i n e t i c a l l y s t a b l e (15) f o r d u r a t i o n of s t u d i e s to: a) b u f f e r s up t o p h y s i o l o g i c a l i o n i c strengths (y 0.15 g ions l i t e r " " ) . b) pH range, 2 t o 11. c) temperature range ^ 0-50°C. d) shear r a t e s encountered when f i l l i n g or c l e a n i n g the e l e c t r o p h o r e s i s tube. Taking the above c r i t e r i a i n t o c o n s i d e r a t i o n p o s s i b l e coat ing m a t e r i a l s or surface m o d i f i e r s i n c l u d e : A. P o l y s a c c h a r i d e s [agarose; dextran; f i c o l l ; glycogen; m e t h y l c e l l u l o s e ; and s t a r c h ] . B. Methacrylates and acrylamides [2-hydroxyethylmethacrylate (HEMA) (16); 2,3-dihydroxypropylmethacrylate (DHPMA) (16); and p o l y a c r y l a m i d e ] . C. Polymeric a l c o h o l s [polyethylene g l y c o l (PEG); and p o l y v i n y l a l c o h o l (PVA)]. D. S i l a n e d e r i v a t i v e s as e i t h e r sub-layer agents or primary coatings [γ-glycidoxypropyltrimethoxysilane (Dow Corning, Z6040) ( 2 ) ; γ-aminopropyltriethoxysilane (Union Carbide, A-1100) ( 2 ) ; γ-methacryloxypropyltrimethoxysilane (Dow Corning, Z6030) ( 2 ) ; and v i n y l t r i m e t h o x y s i l a n e (Dow Corning ( 2 ) ] . To date, p o l y s a c c h a r i d e s have o f f e r e d many promising f e a t u r e s 1

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as c o a t i n g s . They are h y d r o p h i l i c , have s m a l l numbers o f charged groups and are g e n e r a l l y biocompatible. As a c l a s s they may be c r o s s l i n k e d and t h e i r r e s i d u a l charge groups e l i m i n a t e d or c h e m i c a l l y reduced by procedures such as those used by Porath et a l . (17) on agarose. I n a d d i t i o n polysaccharides may be d e r i v a t i z e d or o x i d i z e d and mixtures used to produce coatings w i t h e i t h e r a net negative, net p o s i t i v e or net zero charge. The work reported has been d i r e c t e d at developing a surface c o a t i n g m a t e r i a l which may be e a s i l y a p p l i e d d i r e c t l y to a cleaned b o r o s i l i c a t e g l a s s surface under m i l d c o n d i t i o n s to form a very t h i n s t a b l e l a y e r which n e u t r a l i z e s or masks the g l a s s surface charge. Unmodified dextran (molecular weight 2 χ 10 d a l t o n s ) and m e t h y l c e l l u l o s e (molecular weight 110,000) were e a s i l y washed from model g l a s s p a r t i c l e s and c o n d i t i o n s were not found f o r a p p l y i n g agaros t h i n l a y e r , even thoug of formaldehyde c r o s s l i n k e d m e t h y l c e l l u l o s e to e l i m i n a t e e l e c t r o osmotic flow i n h i s f r e e zone e l e c t r o p h o r e t i c equipment. These c o n s i d e r a t i o n s prompted the s y n t h e s i s of d i e t h y l a m i n o e t h y l d e r i v a t i v e s of these substances w i t h the r a t i o n a l e that the i n t r o d u c t i o n of c a t i o n i c groups i n t o the polymers should enhance t h e i r b i n d i n g to the g l a s s surface where subsequently the t h i n adsorbed l a y e r could be chemically c r o s s l i n k e d to provide f u r t h e r s t a b i l i z a t i o n i f necessary. Each DEAE-derivative was found to adsorb to b o r o s i l i c a t e g l a s s p a r t i c l e s and upon two to three washes i n 0.015 M NaCl, t h e i r m o b i l i t i e s were p o s i t i v e at n e u t r a l pH. Work on DEAE-dextran and on DEAE-agarose was not c a r r i e d beyond t h i s p o i n t , even though they appeared to be p o t e n t i a l l y useful. The e l e c t r o p h o r e t i c m o b i l i t y versus pH f o r the c l e a n boros i l i c a t e g l a s s p a r t i c l e s (Figure 3) i s s i m i l a r to that reported i n the l i t e r a t u r e . However extensive comparisons are d i f f i c u l t because of v a r i a t i o n s i n suspending media composition and i o n i c s t r e n g t h from worker to worker (19, 20, 21). The decrease i n m o b i l i t y which occurs below about pH 7 i s c o n s i s t e n t w i t h the presence of boranol and s H a n o i groups (14) and the constant m o b i l i t y w i t h i n c r e a s i n g pH above a value of 7, t y p i c a l behavior f o r an anionogenic h y d r o p h i l i c s u r f a c e . The f a i l u r e of the c r o s s l i n k e d DEAE-methylcellulose coating to reverse the net charge of the g l a s s p a r t i c l e s at n e u t r a l pH i s i n d i c a t i v e of incomplete coverage, i n s u f f i c i e n t amino group s u b s t i t u t i o n , and perhaps a l s o some c a r b o x y l group contamination. The pK of about 9.5 i s i n the expected range f o r DEAE-derivatives (22). The s m a l l i n c r e a s e i n m o b i l i t y as the i o n i z a t i o n of the p o s i t i v e group i s suppressed i n d i c a t e s a low degree of s u b s t i t u t i o n i n the methylc e l l u l o s e . Coverage of the g l a s s surface appears to have been s u f f i c i e n t l y extensive to e l i m i n a t e any obvious dependence of m o b i l i t y on pH over the range 4 to 7. The decrease i n z e t a p o t e n t i a l i f i t were to occur at a chamber w a l l i s s u f f i c i e n t to reduce e r r o r s i n the e l e c t r o p h o r e t i c m o b i l i t y of p a r t i c l e s w i t h 6

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m o b i l i t i e s of ^ 3 \im s e c " v o l t " cm from about 11% t o 2% i n 0.015 i o n i c s t r e n g t h media based on the c o n d i t i o n s given i n F i g u r e 2. The t r a n s f o r m a t i o n which occurs on c o a t i n g a chamber w i t h e p i c h l o r o h y d r i n t r e a t e d DEAE-methylcellulose i s depicted i n F i g u r e 4 where the dashed l i n e shows the extreme dependence of p a r t i c l e v e l o c i t y on p o s i t i o n i n the chamber f o r an uncoated g l a s s chamber. The f u l l l i n e s demonstrate f o r s e v e r a l d i f f e r e n t i o n i c strengths the l a c k of dependence of p a r t i c l e v e l o c i t y on p o s i t i o n i n the chamber f o r a DEAE-methylcellulose coated g l a s s chamber. A c o m p l i c a t i n g f a c t o r a t present i s the slow desorp t i o n of the c o a t i n g which leads t o the gradual reappearance of s i g n i f i c a n t e l e c t r o o s m o t i c flow and the p o s s i b i l i t y of contamina t i o n of any i n d i c a t o r p a r t i c l e s during the course of e l e c t r o p h o r e t i c measurements. coating w i t h s e v e r a l o to be worked out but these s t u d i e s p o i n t t o the p r o b a b i l i t y of developing s i n g l e stage coatings which are easy t o apply, do not r e q u i r e a sub-layer nor e l a b o r a t e pretreatment procedures and which w i l l remain e l e c t r o k i n e t i c a l l y s t a b l e f o r a p p r e c i a b l e periods of time. Acknowledgement: This i n v e s t i g a t i o n was supported by Contract No. NAS8-30887 from the N a t i o n a l Aeronautics and Space Administration.

Abstract Physical, chemical and practical criteria are discussed for glass surface treatments or coatings designed to minimize electroosmosis as a source of error in analytical particle electrophoresis. A rapid method for screening potential coating agents was developed in which the electrophoretic mobility was measured for cleaned borosilicate glass particles before and after coating treatments. DEAE-derivatives of agarose, dextran and methylcellulose adsorb directly to the particles and signifi cantly reduce their negative surface charges to small net posi tive charges at neutral pH. A coating procedure involving epichlorohydrin treatment of DEAE-methylcellulose is described which produces a coating of reasonable stability for glass electrophoresis chambers and significantly decreases errors in electrophoretic mobility measurements due to electroosmotic flow. Literature Cited (1) Strickler, Α., and Sacks, T., Ann. N.Y. Acad. Sci., (1973), 209, 497-514. (2) Lee, L.H., J. Coll. Inter. Sci., (1968), 27, 751-760. (3) Van Oss, C.J., Fike, R.M., Good, R.J., and Reinig, J.M., Anal. Biochem., (1974), 60, 242-251. (4) Smoluchowski, Μ., Bull. Acad. Sci. Cracovie, (1903), 182.

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(5) Bangham, A.D., Flemans, R., Heard, D.H., and Seaman, G.V.F., Nature, (1958), 182, 642-644. (6) Peterson, E.A., and Sober, H.A., J. Amer. Chem. Soc., (1956), 78, 751-755. (7) "Micro-Analyses in Medical Biochemistry 3rd Edition", p. 50, ed., E.J. King and I.D.P. Wootton, J. & A. Churchill Ltd., London, 1959. (8) Seaman, G.V.F., and Heard, D.H., Blood (1961), 18, 599-604. (9) Flodin, P., Dextran Gels and their Application in Gel Filtration, Ph.D. Dissertation, Uppsala, Sweden, 1962, pp. 14-24. (10) Seaman, G.V.F., in "The Red Blood Cell, Vol. II", ed. D. MacN. Surgenor, pp. 1135-1229, Academic Press, New York, 1975. (11) Abramson, H.A., Moyer, L.S., and Gorin, M.H., "Electrophoresis of Proteins an 57, Van Nostrand-Reinhold (12) Hartley, G.J., and Roe, J.W., Trans. Faraday Soc., (1940), 36, 101-109. (13) Brooks, D.E., and Seaman, G.V.F., J. Coll. Inter. Sci., (1973), 43, 670-686. (14) Hair, M.L., and Altug, I., J. Phys. Chem., (1967), 71, 42604263. (15) Heard, D.H., and Seaman, G.V.F., J. Gen. Physiol., (1960), 43, 635-654. (16) Andrade, J.D., Med. Instrm., (1973), 7, 110-120. (17) Porath, J., Janson, J.C., and Låås, T., J. Chromat., (1971), 60, 167-177. (18) Hjertén, S., "Free Zone Electrophoresis", p. 51, Almqvist and Wiksells Boktryckeri AB, Uppsala, 1967. (19) Abramson, H.A., J. Gen. Physiol., (1929), 13, 169-177. (20) Chattoraj, D.K., and Bull, H.B., J. Amer. Chem. Soc., (1959), 81, 5128-5133. (21) Mehrishi, J.N., and Seaman, G.V.F., Biochim. Biophys. Acta, (1966), 112, 154-159. (22) Peterson, E.A., "Cellulosic Ion Exchangers", p. 236, North Holland/American Elsevier, 1970.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

18 Streaming Potential Studies on Gel-coated Glass Capillaries SHAO M. MA, DONALD E. GREGONIS, RICHARD VAN WAGENEN, and JOSEPH D. ANDRADE Department of Materials Science and Engineering, University of Utah, Salt Lake City, Utah 84112 The s e p a r a t i o n o f l i v i n g c e l l s i n z e r o g r a v i t y e n v i r o n m e n t s appears t o o f f e r s i g n i f i c a n on e a r t h . The z e r o g r a v i t and d e n s i t y f l u c t u a t i o n p r o b l e m s . As d e m o n s t r a t e d i n A p o l l o 16 experiments a m a j o r p r o b l e m w i t h f r e e zone e l e c t r o p h o r e s i s i n a c l o s e d s y s t e m i s t h e d i f f e r e n t i a l f l u i d movement due t o t h e surface e l e c t r i c a l properties of the container (electroosmosis). As a c o n s e q u e n c e , t h e r e s o l u t i o n o f t h e s e p a r a t i o n and t h e a n a l y s i s i n v o l v e d a r e g r o s s l y compromised by t h e r e s u l t i n g " b u l l e t " shape f l o w p a t t e r n s . A s o l u t i o n t o t h i s problem i s t o produce s u r f a c e s w h i c h e x h i b i t e s s e n t i a l l y no e l e c t r o k i n e t i c p r o p e r t i e s when i n e q u i l i b r i u m w i t h t h e i r e n v i r o n m e n t . T h i s c a n be a c h i e v e d by n e u t r a l i z i n g t h e s u r f a c e c h a r g e o r by s h i f t i n g t h e h y d r o d y namic s h e a r p l a n e away f r o m t h e i n t e r f a c e by use o f h y d r o p h i l i c surface coatings. In t h i s s t u d y , g l a s s t u b e s were s u b j e c t e d t o v a r i o u s c o a t i n g s and s u r f a c e t r e a t m e n t s . In p a r t i c u l a r , i t was f e l t t h a t u n charged h y d r o p h i l i c m e t h a c r y l a t e polymer c o a t i n g s should decrease the e l e c t r o o s m o s i s . The o v e r a l l e f f e c t s o f t h e s e t r e a t m e n t s upon s t r e a m i n g p o t e n t i a l i s d i s c u s s e d . Apparatus The s t r e a m i n g p o t e n t i a l a p p a r a t u s has been d e s c r i b e d i n detail (2,3). I t i s an a l l - g l a s s s y s t e m u t i l i z i n g Ag/AgCl e l e c trodes. S t r e a m i n g f l u i d i s f o r c e d back and f o r t h f r o m one r e s e r v o i r t o the o t h e r through a g l a s s tube using p r e s s u r i z e d p u r e n i t r o g e n g a s . A g l a s s pH e l e c t r o d e and a t h e r m o m e t e r a r e p o s i t i o n e d i n one o f t h e r e s e r v o i r s . A K e i t h l e y model 616 d i g i t a l e l e c t r o m e t e r measures s t r e a m i n g p o t e n t i a l o r s t r e a m i n g current. A l l new and l a r g e g l a s s w a r e a r e c l e a n e d i n i t i a l l y i n c h r o m i c s u l f u r i c a c i d , thoroughly rinsed i n running, d i s t i l l e d water, soaked i n doubly d i s t i l l e d w a t e r , r e r i n s e d i n double d i s t i l l e d w a t e r and t h e n a i r d r i e d i n a d u s t - f r e e e n v i r o n m e n t . Small

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g l a s s w a r e i s c l e a n e d by r a d i o f r e q u e n c y glow d i s c h a r g e To c a r r y o u t t h e s t r e a m i n g p o t e n t i a l measurement e a c h r e s e r v o i r i s f i l l e d w i t h a b o u t 500 ml o f p h o s p h a t e b u f f e r a t pH 7.2 c o n t a i n i n g 0.01 M KC1. The e l e c t r o d e s a r e i n s e r t e d . The p o t e n t i a l i s t h e n measured as a f u n c t i o n o f d r i v i n g p r e s s u r e . The s t r e a m i n g p o t e n t i a l , E t , i s l i n e a r l y d e p e n d e n t on t h e p r e s s u r e d r o p , P, a c r o s s t h e t u b e , as g i v e n by ( 5 j s

r

_ 4πηΚ str " ~D ψ— where ζ i s t h e z e t a p o t e n t i a l , η and D a r e t h e v i s c o s i t y and d i e l e c t r i c constant, r e s p e c t i v e l y , i n the d i f f u s e p o r t i o n of the e l e c t r i c a l d o u b l e l a y e r , and Κ i s t h e b u l k s p e c i f i c c o n d u c t i v i t y of the e l e c t r o l y t e (assuming s u r f a c e conductance i s n e g l i g i b l e r e l a t i v e to bulk conductance) B a l l and F u e r s t e n a l i t e r a t u r e i n r e g a r d t o s t r e a m i n g p o t e n t i a l d a t a . They have c o n c l u d e d t h a t , due t o as y e t u n e x p l a i n e d f l o w and symmetry p o t e n t i a l s common t o a w i d e v a r i e t y o f e l e c t r o d e t y p e s , t h e slope of the l o c i of E ^ d a t a a t a number o f d r i v i n g p r e s s u r e s , P, i n o p p o s i t e f l o w d i r e c t i o n s , Δ Ε / Δ Ρ , s h o u l d be u t i l i z e d i n t h e above e q u a t i o n . T h i s has been t h e c a s e i n t h i s s t u d y . S t r e a m i n g p o t e n t i a l d a t a was o b t a i n e d by m e a s u r i n g s t r e a m i n g p o t e n t i a l s a t a d r i v i n g p r e s s u r e o f 2cm Hg, t h e n r e v e r s i n g t h e f l o w d i r e c t i o n and r e p e a t i n g t h e measurement. The d r i v i n g p r e s s u r e was i n c r e a s e d by 2cm Hg and s t r e a m i n g p o t e n t i a l s were a g a i n measured i n b o t h f l o w d i r e c t i o n s . T h i s p r o c e s s was r e p e a t e d u n t i l t h e d r i v i n g p r e s s u r e r e a c h e d 12cm Hg. The l o c i o f s t r e a m i n g p o t e n t i a l d a t a as a f u n c t i o n o f d r i v i n g p r e s s u r e were t h e n f i t t e d to a l i n e a r regression best f i t s t r a i g h t l i n e using a H e w l e t t P a c k a r d (Model 9820A) programmable c a l c u l a t o r . A b s o l u t e v a l u e s o f z e t a p o t e n t i a l c a l c u l a t e d f r o m t h e above e q u a t i o n may be e r r o n e o u s due t o a s s u m p t i o n s c o n c e r n i n g v a l u e s f o r d o u b l e l a y e r v i s c o s i t y , d i e l e c t r i c c o n s t a n t , and e n t r a n c e f l o w e f f e c t s r e s u l t i n g f r o m t h e l a r g e ID o f t h e t u b e s u t i l i z e d i n t h i s study (2,3). E

r

ζ

s

r

5 τ Γ

Materials T h i c k - w a l l e d " P y r e x " c a p i l l a r y t u b e s , a p p r o x i m a t e l y 0.2cm ID and 15cm l o n g , were u s e d . U n c o a t e d c a p i l l a r i e s had ΔΕ . /ΔΡ o f - 0 . 3 5 7 mV/cm Hg. D i f f e r e n t s i l a n e a d h e s i o n p r o m o t e r s (7) were used t o p r e t r e a t t h e g l a s s s u r f a c e s p r i o r t o g e l c o a t i n g . An i n v e s t i g a t i o n o f s e v e r a l s i l a n i z i n g r e a g e n t s u s i n g d i f f e r e n t p r o c e d u r e s showed a s l i g h t decrease in ΔΕ £ /ΔΡ f o r a l l the s i l a n e c o a t i n g s . The Δ Ε £ /ΔΡ v a l u e s were n o t s e n s i t i v e t o t h e o r g a n i c f u n c t i o n a l i t y o f trie s i l a n e . A v a r i e t y o f h y d r o p h i l i c p o l y m e r s were used as c o a t i n g m a t e r i a l s i n c l u d i n g m e t h y l c e l l u l o s e (Dow M e t h o c e l MC, p r e m i u m , s

5

Γ

$

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

z

r

18.

MA ET AL.

Streaming

Potential

243

Studies

4000 c p s ) ; h y d r o x y p r o p y l m e t h y l c e l l u l o s e (Dow M e t h o c e l , 9 0 H g , premium, 15000 c p s ) ; d e x t r i n ( M a t h e s o n , Coleman and B e l l ) and agarose (Biorad L a b s ) . H y d r o x y e t h y l m e t h a c r y l a t e was d o n a t e d by Hydro Med S c i e n c e s , I n c . M e t h o x y e t h y l m e t h a c r y l a t e and m e t h o x y e t h o x y e t h y l m e t h a c r y l a t e were p r e p a r e d i n o u r l a b o r a t o r i e s by base c a t a l y z e d t r a n s e s t e r i f i c a t i o n o f m e t h y l m e t h a c r y l a t e with the corresponding alcohol ( 8 ) . Methods

and R e s u l t s

The s i l a n e compounds were a p p l i e d u s i n g s t a n d a r d p r o c e d u r e s (6). A s i l a n e s o l u t i o n was r i n s e d t h r o u g h t h e t u b e s , f i r s t i n one d i r e c t i o n , t h e n i n t h e o t h e r , and t h e t u b e s were vacuum dried overnight. The s i l a n e compounds u s e d a n d t h e ΔΕ /ΔΡ values are given i n Tabl TABLE ΔΕ^/ΔΡ

Values

I.

f o r Silane

Coatings s t r (mV/cm Hg) A

SSStina

E

M

P

1,1,1,3,3,3-hexamethyldisilazine:triethylamine -0.242 Chlorotrimethylsilane:triethylamine (1:1) -0.221 D i c h l o r o d i m e t h y l s i l a n e : t r i e t h y l ami ne ( 1 : 1 ) -0.253 γ - m e t h a c r y l o x y p r o p y l t r i m e t h o x y s i l a n e : t r i e t h y l ami ne ( 1 : 1 ) - 0 . 2 3 4 Y-glycidoxypropyltrimethoxysilane:triethylamine (1:1) -0.229 γ-glycidoxypropyltrimethoxysilane:n-propylami ne ( 1 : 1 ) -0.230 Y-glycidoxypropyltrimethoxysilane:pH 4 aq. a c e t i c acid -0.241 The Y - g l y c i d o x y p r o p y l t r i m e t h o x y s i l a n e : t r i e t h y l a m i n e ( 1 : 1 ) s i l a n e c o a t i n g was s e l e c t e d f o r f u r t h e r t r e a t m e n t w i t h t h e neutral polysaccharides. I t was t h o u g h t t h a t c o a t i n g t h e p o l y s a c c h a r i d e s upon t h e e p o x y - s i l a n i z e d g l a s s was e i t h e r an a c i d o r base c a t a l y z e d p r o c e s s r e s u l t i n g i n c o v a l e n t a t t a c h m e n t o f t h e polysaccharide to the surface (Figure 1 ) . S t a n d a r d s o l u t i o n s o f v a r i o u s p o l y s a c c h a r i d e s were p r e p a r e d by d i s s o l v i n g i n e i t h e r 0 . 5 % HC1 o r 0 . 5 % Κ0Η. I f t h e p o l y s a c c h a r i d e was s o l u b l e , t h e v i s c o s i t y o f t h e s o l u t i o n was " r e g u l a t e d " by t h e a d d i t i o n o f p o l y s a c c h a r i d e u n t i l i t had t h e c o n s i s t e n c y o f a t h i c k syrup. O t h e r w i s e t h e p o l y s a c c h a r i d e was added t o c o m p l e t e saturation. The p o l y s a c c h a r i d e s o l u t i o n was p u l l e d t h r o u g h t h e t u b e s i n each d i r e c t i o n by a w a t e r - a s p i r a t o r p a r t i a l vacuum. The t u b e s were t h e n p l a c e d i n a vacuum oven a t 120°C f o r 12 h o u r s , u n l e s s o t h e r w i s e s p e c i f i e d . The t u b e s were e x h a u s t i v e l y r i n s e d w i t h d i s t i l l e d w a t e r and t h e Δ Ε ^ / Δ Ρ v a l u e s were o b t a i n e d . C o n s i s t e n t l y l o w e r A E s t r M P v a l u e s were o b t a i n e d when t h e p o l y s a c c h a r i d e s were a p p l i e d i n a c i d i c s o l u t i o n as compared t o

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

244

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

a.

Acid Catalyzed

-CH -0-CH

-CH-CH

?

Process l L

CH -0-CH -CH-CH 2

2

HO-Cellulose

2

w

ο

OH

^Cellulose

O-Cellulose

-CH -0-CH -CH-CH -OH

-CH -0-CH -CH-CH -OH 2

b.

2

Base C a t a l y z e d

HO"

+

2

2

+ "O-Cellulose - v

HOH

2

+

"O-Cellulose

-CHg-O-CHg-ÇH-CHg-O-Cellulose

V

"0

-CH -0-CH -CH-CH -0-Cellulose 2

2

Process

HO-Cellulose

-CH -0-CH -CH-CH 2

2

2

2

2

,

H

+

OH

Figure 1. Reaction schemes for the attachment of polysacchardies to epoxy-silane surfaces. A , acid catalyzed; B, base catalyzed.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

M A ET AL.

18.

Streaming

Potential

245

Studies

b a s i c s o l u t i o n ; h o w e v e r , when m e t h y l c e l l u l o s e i n d i s t i l l e d w a t e r was a p p l i e d t o t h e e p o x y - s i l a n i z e d g l a s s , a Δ Ε ς ^ / Δ Ρ v a l u e was o b t a i n e d comparable t o t h e r e s u l t s o b t a i n e d w i t h m e t h y l c e l l u l o s e i n a c i d s o l u t i o n . A t t h e moment t h e mechanism t h a t g o v e r n s attachment o f m e t h y l c e l l u l o s e t o t h e e p o x y - s i l a n i z e d g l a s s i s i n doubt. The t r e a t m e n t o f g l a s s s u r f a c e s w i t h j u s t m e t h y l c e l l u l o s e or γ-glycidoxypropyltrimethoxysilane does n o t g i v e t h e l o w ΔΕ ^ /ΔΡ values. T h i s phenomenon i s s t i l l b e i n g i n v e s t i g a t e d . F u r t h e r c o a t i n g o f m e t h y l c e l l u l o s e onto a m e t h y l c e l l u l o s e c o a t i n g showed no change i n Δ Ε * / Δ Ρ . The use o f 1 , 2 , 4 , 5 , 9 , 1 0 t r i e p o x y d e c a n e (TED) as a c r o s s l i n k e r i n t h e m e t h y l c e l l u l o s e s o l u t i o n s (5% TED i n 0 . 5 % H C 1 - m e t h y l c e l l u l o s e ) was i n v e s t i g a t e d . C u r i n g was a c c o m p l i s h e d b y h e a t i n g o v e r n i g h t a t 120°C i n vacuum. No s t a t i s t i c a l change was n o t e d f o r t h i s t r e a t m e n t o v e r normal m e t h y l c e l l u l o s e tubes (Tabl 5

Γ

$

TABLE

Γ

II.

E f f e c t s o f S i l a n e and P o l y s a c c h a r i d e C o a t i n g s

str (mV/cm Hg)

A

Coating (1) (2) (3) (4) (5) (6) (7) (8) (9)

(10) (11) (12)

Uncoated γ-glycidoxypropyltrimeth o x y s i l a n e : t r i e t h y l ami ne ( 1 : 1 ) P r o c e d u r e 2 , t h e n 0 . 5 % HC1 P r o c e d u r e 2 , t h e n 0 . 5 % KOH Methylcellulose in d i s t i l l e d Ο Procedure 2 , then m e t h y l c e l l u l o s e i n 0 . 5 % KOH Procedure 2, then m e t h y l c e l l u l o s e i n 0 . 5 % HC1 Procedure 2 , then m e t h y l c e l l u l o s e i n d i s t i l l e d H«0 M e t h y l c e l l u l o s e coated tubes ( P r o c e d u r e 7) r e c o a t e d w i t h m e t h y l c e l l u l o s e w i t h 5% 1 , 2 , 4 , 5 , 9 , 1 0 - t r i e p o x y d e c a n e on 0 . 5 % HC1 Procedure 2 , then h y d r o x y p r o p y l m e t h y l c e l l u l o s e i n 0 . 5 % HC1 Procedure 2 , then d e x t r i n i n 0 . 5 % HC1 Procedure 2 , then agarose i n 0 . 5 % HC1

Upon ΔΕ ^ /ΔΡ V a l u e s

E

/

A

P

Standard Deviation

-0.356

0.010

-0.310 -0.226 -0.261 -0.166

0.007

-0.130

0.012

-0.078

0.008

-0.058

0.002

-0.103

0.003

— —

0.090

-0.096 -0.245 -0.250

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

246

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

A simple water-polyfluorocarbon surface contact angle t e s t was used t o d e t e c t s u r f a c e - a c t i v e e x t r a c t a b l e s (9) f r o m t h e coated c a p i l l a r i e s . H i g h c o n t a c t a n g l e s were n o t e d w i t h a l l tubes. No s i g n i f i c a n t change i n t h e c o n t a c t a n g l e was e v i d e n t w i t h the methyl c e l l u l o s e - c o a t e d t u b e s , i n d i c a t i n g t h a t the m e t h y l c e l l u l o s e i s not r e a d i l y e x t r a c t e d from the s u r f a c e . To o b t a i n c a p i l l a r y t u b e s c o a t e d w i t h m e t h a c r y l a t e h y d r o g e l s , f i r s t s o l u b l e p o l y m e r s were p r e p a r e d . T h i s was a c c o m p l i s h e d by r a d i c a l i n i t i a t i o n o f t h e d e s i r e d monomer a t l o w d i l u t i o n (1 to 10, v/v) i n e t h a n o l . A s m a l l amount o f t h i s p o l y m e r s o l u t i o n i s a l l o w e d t o f l o w t h r o u g h t h e g l a s s c a p i l l a r y , and w i t h a l i t t l e c a r e and p a t i e n c e , a v e r y u n i f o r m c o a t o f t h e p o l y m e r c o u l d be d e p o s i t e d on t h e i n s i d e o f t h e c a p i l l a r y . T h e v i s c o s i t y o f t h e p o l y m e r s o l u t i o n was r e g u l a t e d by t h e a d d i t i o n o r e v a p o r a t i o n o f solvent. The t u b e s wer to e q u i l i b r a t e i n d i s t i l l e The monomers t h a t were i n v e s t i g a t e d were m e t h o x y e t h y l m e t h a c r y l a t e (MEMA), h y d r o x y e t h y l m e t h a c r y l a t e (HEMA) and m e t h o x y e t h o x y e t h y l m e t h a c r y l a t e (MEEMA). I t has been d e t e r m i n e d t h a t t h e s e p o l y m e r s s w e l l i n w a t e r t o i n c o r p o r a t e 3 . 5 % , 40% and 63% w a t e r , r e s p e c t i v e l y ( 9 ) . The c o r r e s p o n d i n g Δ Ε ^ / Δ Ρ v a l u e s are comparable to the best m e t h y l c e l l u l o s e values

TABLE

III).

III.

E f f e c t s of H y d r o p h i l i c Methacrylate Coatings

Coating

(Table

Upon Δ Ε _ / Δ Ρ

ΔΕ /ΔΡ /«χ//™ un\ (mV/cm Hg)

(1) U n c o a t e d t u b e s -0.290 (2) H y d r o x y e t h y l m e t h a c r y l a t e (HEMA) - 0 . 0 4 6 (3) M e t h o x y e t h y l m e t h a c r y l a t e (MEMA) - 0 . 0 5 8 (4) M e t h o x y e t h o x y e t h y l m e t h a c r y l a t e (MEEMA) -0.038 (5) HEMA w i t h 1% m e t h a c r y l i c a c i d (MAA) -0.080 (6) HEMA w i t h 3% MAA -0.100 (7) HEMA w i t h 10% MAA -0.113 (8) HEMA w i t h q u a t e r n i z e d 1% d i m e t h y l a m i n o e t h y l m e t h a c r y l a t e (DMAEMA) +0.070 (9) HEMA w i t h q u a t e r n i z e d 3% DMAEMA +0.074 (10) HEMA w i t h q u a t e r n i z e d 10% DMAEMA +0.087

$1

Values

Γ

Standard . j D

e

v

a

t

i

0.045 0.029 0.050 0.012 0.024 0.024 0.035 0.02* 0.004 0.014

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

o

n

18.

MA ET AL.

Streaming

Potential

247

Studies

To o b s e r v e t h e e f f e c t t h a t c h a r g e d g r o u p s i n t h e h y d r o p h i l i c p o l y m e r s had upon Δ Ε ^ / Δ Ρ v a l u e s , HEMA was p o l y m e r i z e d a t l o w d i l u t i o n w i t h v a r i o u s amounts o f m e t h a c r y l i c a c i d (MAA) and d i m e t h y l a m i n o e t h y l m e t h a c r y l a t e (DMAEMA). In t h e s t r e a m i n g s o l u t i o n b u f f e r e d t o pH 7 . 2 , m e t h a c r y l i c a c i d e x i s t s as t h e c h a r g e d s a l t , b u t d i m e t h y l a m i n o e t h y l m e t h a c r y l a t e e x i s t s as an u n c h a r g e d s p e c i e s . After polymerization of t h e HEMA-DMAEMA c o p o l y m e r , m e t h y l i o d i d e was added t o t h e p o l y m e r s o l u t i o n t o r e a c t w i t h t h e f r e e amino g r o u p s f o r m i n g q u a t e r n a r y ammonium i o d i d e s ( F i g u r e 2 ) . The c a p i l l a r y t u b e s were c o a t e d a s d e s c r i b e d f o r t h e o t h e r m e t h a c r y l a t e p o l y m e r s , c u r e d a t 60°C o v e r n i g h t and t h e n a l l o w e d t o s w e l l t o e q u i l i b r i u m i n e i t h e r d i s t i l l e d water o r streaming p o t e n t i a l b u f f e r . The MAA-HEMA c o p o l y m e r c o a t i n g s e x h i b i t e d a v e r a g e AE t /àP values which i n c r e a s e d methacrylic acid i n th f o r 3% MAA, and - 0 . 1 1 3 f o r 10% MAA. The q u a t e r n a r y DMAEMA-HEMA copolymer coatings produced a n e t p o s i t i v e e l e c t r o k i n e t i c s u r f a c e as i n d i c a t e d by i n c r e a s i n g l y p o s i t i v e Δ Ε ~ ^ / Δ Ρ v a l u e s w i t h i n c r e a s i n g p e r c e n t a g e o f DMAEMA; +0.070 t o r 1% DMAEMA, +0.074 f o r 3% DMAEMA, and +0.087 f o r 10% DMAEMA ( T a b l e I I I ) . 5

Γ

s

r

Γ

Discussion A c c o r d i n g t o B r o o k ' s model ( 1 0 ) t h e z e t a p o t e n t i a l i n t h e p r e s e n c e o f a d s o r b e d p o l y m e r ( h y d r o p h i l i c , u n c h a r g e d ) c o u l d be h i g h e r o r l o w e r than t h a t i n t h e absence o f polymer. The e f f e c t depends on t h e r e l a t i v e m a g n i t u d e o f t h e t h i c k n e s s o f t h e a d s o r b e d p o l y m e r l a y e r , d , and t h e t h i c k n e s s , d , w i t h i n w h i c h n o n z e r o f l u i d f l o w o c c u r s d u r i n g an e l e c t r o k i n e t i c e x p e r i m e n t . T h r e e a r e a s have been c i t e d : f

1.

2.

F o r a t o t a l l y f r e e d r a i n i n g adsorbed l a y e r , t h e l o c a t i o n o f t h e s h e a r p l a n e i s u n a f f e c t e d by t h e p r e s e n c e o f t h e adsorbed l a y e r , i . e . d = d . In t h i s c a s e , polymer adsorp t i o n c a u s e s an i n c r e a s e i n ' z e t a p o t e n t i a l . F o r a p a r t i a l l y f r e e d r a i n i n g adsorbed l a y e r , t h e shear plane i s s h i f t e d t o a p o s i t i o n w i t h i n t h e adsorbed polymer l a y e r (0 < d < d ) . I n t h i s c a s e p o l y m e r a d s o r p t i o n may c a u s e e i t h e r an i n c r e a s e o r a d e c r e a s e i n z e t a p o t e n t i a l . When f l o w i s t o t a l l y e x c l u d e d f r o m t h e a d s o r b e d l a y e r , i . e . , the shear plane i s s h i f t e d o u t s i d e t h e adsorbed l a y e r (df = 0 ) , polymer a d s o r p t i o n causes a decrease i n z e t a potential. f

3.

Our d a t a was o b t a i n e d f r o m a l a r g e v a r i e t y o f n e u t r a l p o l y mer c o a t i n g s ; a l l show a d e c r e a s e i n ΔΕ +.~/ΔΡ. Our n e u t r a l p o l y m e r c o a t i n g s were c a s t on g l a s s c a p i l l a r y s u r f a c e s and a r e t h i c k e r t h a n t h o s e o b t a i n e d by a d s o r p t i o n . A l t h o u g h one c a n n o t d i r e c t l y compare o u r c o a t i n g s w i t h t h e t h r e e c a s e s p r e s e n t e d b y §

C o - , ; . •·•·,

·; -

:

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Washington, DC, 1976. Washi^iei, D. C.Society: 2003 6

248

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

M A A - H E M A COPOLYMER

DMAEMA-HEMA COPOLYMER

°7

O-7 £~0H

/ - O H

Oy

Oy

/-N-CH

3

^ O H

QUATERNIZED HEMA

CH r 3

Figure 2.

Charged groups introduced into the HEMA

DMAEMA

COPOLYMER

polymer

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

18.

Streaming

MA ET AL.

Potential

249

Studies

B r o o k ' s ( 1 0 ) i t does a p p e a r t h a t i n o u r c a s e t h e s h e a r p l a n e has been s h i f t e d f a r away f r o m t h e i n t e r f a c e . C o n s i d e r t h e Δ Ε £ / Δ Ρ values given i n Table I I I f o r p o s i t i v e o r n e g a t i v e groups c o p o l y m e r i z e d w i t h HEMA. A t l o w s t r e a m i n g p o t e n t i a l v a l u e s , i n c r e a s e d e x p e r i m e n t a l e r r o r i s i n t r o d u c e d i n t o the measurements. A t a g i v e n P, E ^ changes w i t h t i m e , p a r t l y due t o a change i n t h e r e l a t i v e f l u i d l e v e l s i n t h e two r e s e r v o i r s w h i c h c a u s e s a change i n Ρ a n d p a r t l y due t o e l e c t r o d e d r i f t w i t h t i m e o f unknown c a u s e ( 2 - 3 ) . I t was o b s e r v e d t h a t t h e Δ Ε £ r e a d i n g s were more s t a b l e f o r s u r f a c e s w i t h h i g h s t r e a m i n g p o t e n t i a l s . As a c o n s e q u e n c e , t h e e r r o r i s l a r g e r when t h e a b s o l u t e Δ Ε ^ / Δ Ρ v a l u e i s s m a l l . A l t h o u g h d i f f e r e n t samples o f s i m i l a r c o a t i n g o r t h e same sample measured on d i f f e r e n t days do show l a r g e v a r i a t i o n s i n t h e i r Δ Ε ^ / Δ Ρ v a l u e s when t h e same sample was measured t h r e e t i m e s w i t h i quite consistent. Also ΔΕ 4- /ΔΡ v a l u e f o r s a m p l e s 5 - 1 0 f o l l o w e d t h e same t r e n d as g i v e n i n t a b l e I I I . T h e r e f o r e , we w o u l d l i k e t o a t t r i b u t e t h e l a r g e standard d e v i a t i o n values t o the v a r i a t i o n s i n experimental c o n d i t i o n s , s u r f a c e r o u g h n e s s , uneven c o a t i n g s , e t c . , and c o n s i d e r the e f f e c t o f i n c r e a s i n g a b s o l u t e streaming p o t e n t i a l values w i t h i n c r e a s i n g charge as r e a l . δ

s

r

$

$

Γ

Γ

Γ

Conclusions A decrease i n Δ Ε ^ / Δ Ρ

o c c u r s when t h e g l a s s s u r f a c e i s

s i l a n i z e d o r c o a t e d w i t h a n e u t r a l p o l y s a c c h a r i d e such as methylcellulose. S i l a n i z a t i o n f o l l o w e d by m e t h y l c e l l u l o s e t r e a t m e n t g i v e s an a l m o s t t e n - f o l d d e c r e a s e i n Δ Ε ^ / Δ Ρ o v e r uncoated " P y r e x " g l a s s . Both a c i d a n d n e u t r a l m e t h y l c e l l u l o s e s o l u t i o n s c o a t e d upon a γ - g l y c i d o x y D r o p y l t r i m e t h o x y s i l a n e base gives the lowest Δ Ε ^ / Δ Ρ values f o r the polysaccharides examined. B a s e - c o n t a i n i n g m e t h y l c e l l u l o s e d e p o s i t e d on a γ-glycidoxypropyltrimethoxysilane surface gives s l i g h t l y higher ΔΕ ^ /ΔΡ v a l u e s , p e r h a p s due t o t h e c o r r o s i v e a c t i o n o f t h e b a s e . No a d d i t i o n a l improvement i n Δ Ε ^ / Δ Ρ v a l u e s was o b t a i n e d when the m e t h y l c e l l u l o s e tubes a r e recoated w i t h m e t h y l c e l l u l o s e containing a crosslinker. H y d r o p h i l i c m e t h a c r y l a t e polymer c o a t i n g s a l s o lower Δ Ε ^ / Δ Ρ values about t e n - f o l d over u n t r e a t e d g l a s s c a p i l l a r i e s . $

Γ

By added q u a t e r n a r y ammonium g r o u p s t o t h e s e p o l y m e r c o a t i n g s , t h e s i g n o f ΔΕ /ΔΡ i s changed f r o m n e g a t i v e t o p o s i t i v e . It t

i s f e l t t h a t by a d j u s t i n g t h e amount o f c h a r g e d co-monomers, a z e r o Δ Ε 7 Δ Ρ v a l u e c o u l d be o b t a i n e d . 0 + ν

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

250

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Acknowledgements T h i s work has been s u p p o r t e d by t h e U. S. N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n , M a r s h a l l Space F l i g h t C e n t e r , H u n t s v i l l e , A l a b a m a , u n d e r C o n t r a c t No, 8 - 3 0 2 5 3 , D r . J . Patterson, Contract Monitor. The a s s i s t a n c e o f R. Middaugh i s g r a t e f u l l y acknowledged.

Abstract Streaming potential techniques were used to measure the interfacial electrical potential of glass capillaries. Different silanizing reagents were used to coat the glass capillaries and decrease the streaming potential values by a comparable amount i.e., about half that o ten-fold decrease in streaming potentia methyl cellulose is coated onto a γ-glycidoxypropyl silanized glass capillary. A comparable decrease is noted when hydroxyethyl methacrylate (HEMA), methoxyethyl methacrylate (MEMA) and methoxyethoxyethyl methacrylate (MEEMA) polymers are coated on the glass capillary. By adding anionic or cationic charged groups to the poly-HEMA coating, streaming potential values of opposite sign are obtained. Increasing the amount of methacrylic acid copolymerized with the HEMA monomer negatively increases the average ΔE /ΔP values. Increasing the amount of quaternized dimethyl aminoethyl methacrylate in the copolymer positively increases the average ΔE /ΔP values. str

str

Literature Cited 1. Snyder, R. S., Bier, M., Griffin, R. N., Johnson, A. J., Leidheiser, Jr., H., Micale, F. J., Vanderhoff, J. W., Ross, S., van Oss, C. J., Sep. Purific. Methods (1973) 2 (2), 259. 2. Van Wagenen, R., Ph.D. Dissertation, University of Utah, September 1975. 3. Van Wagenen, R., Andrade, J. D., and Hibbs, J. B., Jr., Submitted to J. Electrochem. Soc. 4. Hollahan, J. R., and Bell, A. T., Eds., "Techniques and Applications of Plasma Chemistry," Wiley, New York, 1974. 5. Davies, J. T., and Rideal, Ε. K., "Interfacial Phenomena," 2nd Ed., Academic Press, 1963. 6. Ball, B., and Fuerstenau, D. W., Miner. Sci. Eng., (1973) 5, 267. 7. Bascom, W. D., Macromolecules (1972) 5, 792. 8. Gregonis, D. E., Chen, C. Μ., and Andrade, J. D., "The Chemistry of Some Selected Methacrylate Hydrogels," this symposium.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

18.

MA ET AL.

Streaming

Potential

Studies

251

9. Baier, R. E., Gott, V. L., and Feruse, Α., Trans. Amer. Soc. Artificial Internal Organs (1970) 16, 50. 10. Brooks, D. Ε., J. Colloid and Interface Science, (1973) 43, 687.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19 Water Wettability of Hydrogels FRANK J. HOLLY and MIGUEL F. REFOJO Eye Research Institute of Retina Foundation, 20 Staniford Street, Boston, Mass. 02114

The matrix of hydrogels consists of hydrophilic macromolecules which are crosslinke Thus, a gel spontaneousl equilibrium hydration. This equilibrium water content of the gel depends on crosslink density and on the amount and nature of hydrophilic sites i n the macratolecular matrix. With the exception of poly (hydroxyethyl methacrylate) [PHEMA], which i s not soluble i n water, the matrix of hydrogels i s usually made up of water-soluble polymers. At equilibrium hydration a large fraction of hydrogels consist of water. Hydrogels containing over 95% water are not uncarmon. This i s probably why i t has been implicitly assumed that hydrogels have a hydrop h i l i c surface. In other words, water i s expected to spread spontaneously over the surface of hydrogels, at least when the surface i s f u l l y hydrated. Past Work on Hydrogel Wettability Acrylic hydrogels are f a i r l y new and their wettability has only been examined recently. However, there are several reports i n the literature i n which the wettability of gelatin gels are considered. The f i r s t systematic investigation of the wettability of hydrated and air-dried gelatin gels appeares to have been made by Pchelin and Korotkina (]L). They found a water contact angle of 98° on hydrated gelatin and a contact angle of 115° on the a i r dried gelatin film. These authors also observed that the wetta b i l i t y of the gelatin gels depended on the polarity of the mate r i a l adjacent to the gel surface while the gel was being formed. Thus gelatin formed against paraffin or a i r was hydrophobic, while gelatin formed against a clean glass surface was hydrophili c . Garrett (2) also found gelatin to be hydrophilic when formed against a glass surface. Braudo and coworkers (3) studied the wettability of gelatin gels as a function of the gelatin concentration. The water content of the gel varied from 14% for the air-dried gels up to 86% 252

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

HOLLY

AND REFOJO

Water

Wettability

of

Hydrogeh

253

for the most dilute gels studied. They found that the water cont act angle on these gels varied frcm the surprisingly high value of 123° for the most hydrated gel to 87° for the dehydrated gels. These authors offer an explanation for the anomalous wettability of gelatin gels based on the preferred orientation of the water molecules i n a surface free-water layer of the gel. I t i s hard to see, however, why an oriented water layer having the characteris t i c s of the surface layer of pure water would not be wetted by water. I t i s suspected that the high, obtuse water contact angles were obtained because of surface dehydration and these values were further increased due to the roughness of the surface. More recently the agarose gel was the subject of an investig ation as to water wettability (4). I f the gel was formed under water or had been exposed to water for long periods of time, the surface was hydrophilic th othe hand i f th surfac formed while exposed t When this hydrophobic layer was shaved off, the gel again had a hydrophilic surface. We have recently found (5) that gels made of the polymer PHEMA. appear to have a hydrophobic surface even when the gel i s f u l l y hydrated. The wettability of PHEMA. gels having equilibrium water contents between 31 and 42% has been found to be surpris ingly low: advancing contact angle values for water between 60 and 80° have been obtained by both the sessile-drop and the captive-bubble techniques. The receding contact angle values measured were found to be much lower but s t i l l larger than zero. Description of the Hydrogels Studied The wettability of several types of hydrogels, consisting of macrcmolecules more hydrophilic than PHEMA, was measured (Figure 1). Gels were prepared by simultaneous polymerization and crosslinking of purified monomers i n aqueous solutions i n molds con s i s t i n g of two glass plates separated by a silicone gasket (6). The copolymer of glyceryl methacrylate and methyl methacrylate [P(GMA/MMA) ] was obtained from Corneal Sciences, Inc. Boston, MA. P (GMA/MMA) was made by bulk polymerization. The gel surface was polished and then equilibrated with water. Poly (glyceryl methacrylate) [PGMA] forms gels that have twice the number of hydroxyl groups per monomer unit than does the PHEMA gel. Poly(hydroxyethyl acrylate) [PHEA] forms gels that are sim i l a r to PHEMA i n hydrophilic sites but this polymer does not have the hydrophobic, bulky methyl side group on the acrylic backbone. The preparation of the PHEMA gels was described previously (5). The crosslinking agents for PHEMA, PGMA, and PHEA are the diesters which are usually found accompanying the monoesters. Poly(acrylamide) [PAA] gels were prepared using Ν,Ν -methylene bisacrylamide as a crosslinking agent. In addition to these synthetic materials, a polysaccharide gel, agarose, consisting of D- and L-galactopyranose, was includ1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

254

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

ed i n the study along with another material not s t r i c t l y a hydro gel, poly (dimethyl siloxane) bulk-grafted with poly (vinyl pyrrolidone) [SID-g-PVP] (7), which was obtained from the firm E s s i l o r (Paris, France). The gels, with the exception of Ρ (GMA/MMA), were formed against glass, which was made hydrophobic for some of the PHEMA. gels by S i l i c l a d coating (5). The gels were thoroughly washed and stored i n d i s t i l l e d water (that was frequently changed) for at least one month prior to the wettability measurements. Under these conditions, the hydrophobic or hydrophilic nature of the glass surface i n contact with the gel during i t s formation did not seem to affect gel wettability. Method of Measurement of Wettability The contact angle er. The contact angle of the sessile droplets was determined i n a closed chamber (Ramè-Hart Environmental Chamber for Goniometer). Clean a i r was circulated through two consecutive gas-washing bottles f i l l e d with water and the chamber i n a closed system by a v a r i s t a l t i c pump i n order to keep i t saturated with respect to water vapor. The captive-bubble technique was also employed occasionally using a chamber designed for this technique i n order to guard against the slight p o s s i b i l i t y of surface dehydration. As with PHEMA (5), this l a t t e r technique yielded contact angle values that were consistently a few degrees higher ( ! ) than the data obtained with the sessile-drop technique. Both the advancing and the receding contact angles were determined. The sessile droplets (or captive bubbles) were s l i g h t l y increased or decreased i n size after the contact with the gel surface had been established, and u n t i l the angle remained constant upon additional change i n volume. Thus, the contact angles measured were s t a t i c rather than dynamic values as the terms seem to imply. Gel Wettability as Characterized by Water Contact Angles Figure 2 displays both the advancing and receding contact angle values obtained with water on the various gels as a function of the equilibrium water content of the gels. As a reference, the average advancing and receding contact angle values for poly(methyl methacrylate) [PMMA] are also shown i n the figure. I t i s clear from the graph that only the agarose gel of very high water content (over 96%) i s completely wetted by water. The next most wettable gel i s PAA as expected from i t s chemical composition. The wettability of PGMA and PHEA are similar indicating that the additional hydroxyl group of PGMA just about cancels the hydrophobic effect of the methyl group absent i n PHEA. The wettability of the PHEMA gels, having considerably lower equilibrium hydration, i s widely variable, but a l l PHEMA gels are

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

HOLLY

Water Wettability

A N D REFOJO

H I

R I

I "k H

1.

255

Hydrogels

1

I C

Ο

GEL

of

R

0

OR SOLI D PMMA

-CH

3

3

3

2.

PHEMA

-CH

3.

PGMA

-CH

4.

PHEA

-Η

-0CH CH 0H

5.

PAA

-Η

- NH

-OCHOHCH OH 0

9

0

9

6. P(GMA/MMA 7. SIL-g-PVP

PVP grafted

8. Agarose

polysaccharide (galactopyranose)

Figure

onto s i l i c o n e

1. Chemical composition of hydrogels investigated

T=25°C

Φ

ο ζ <

m

Φ

Φ

ϋ <

O Ο

20

A i

A ?ô

GEL

WATER

CON TENT

@§o<

( I)

Figure 2. Contact angle of water on various hydrogels as a function of hydration. V , Sil-g-PVP; 0, PHEMA; O , PGMA; P(GMA/MMA); • , PHEA; Δ , PAA; ®, agarose. Solid symbols indicate receding contact angles.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

256

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

less wettable than PGMA. or PHEA. Ρ (GMA/MMA) i s about as hydro phobic as PHEMA, while SIL-g-PVP having a rather low water con tent (6-12%) i s only as hydrophobic as the less wettable PHEMA gels. The average water wettability of the PHEMA gels as judged by the magnitude of the advancing contact angle i s comparable to that of PMMA (1.5% water). The receding contact angle values of water on the gels are a l l much lower than the advancing contact angles. Hence, quite large contact angle hysteresis was observed with the hydrogels. Four factors or processes have been recognized as being capable of causing contact angle hysteresis: surface roughness, physical interaction between s o l i d and l i q u i d (e.g. dissolution), chemical interaction between s o l i d and l i q u i d (e.g. hydrolysis), and stereochemical changes at the s o l i d - l i q u i d interface. A l l the hydrogels to appear glossy when th surface. Thus, the small degree of roughness present cannot account for the pronounced hysteresis. A l l the gels were washed repeatedly for prolonged periods of time prior to contact angle measurements, so water-soluble, surface-active contaminants as a possible cause of contact angle hysteresis can be eliminated. The hydrcgels were i n ttermc
In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

Water Wettability

H O L L Y A N D REFOJO

of

257

Hydrogels

enough to s h i f t so that no free-water lakes are expected to cross the periphery of the sessile water droplet. The data contained i n Figure 2 seem to indicate that for a given type of gel, the equilibrium water content of the gel has no direct effect on surface wettability. However, there i s some vague indication, especially with the gels of higher water content that hydrophobic!ty may increase with increasing water content. As already mentioned, a similar phenomenon was observed for gelatin by others ( 3 ) . The main factor determining water wettability appears to be the chemical structure of the polymeric network at the interface. The surface structure and orientation of the polymer matrix i s most l i k e l y affected by the polarity of the adjacent phase bound ary as well as by seme uncontrolled events relating to gel matrix formation. Water Wettability and Relative Contact Angle Hysteresis One way to circumvent the d i f f i c u l t i e s introduced by the unpredictable nature of the gel surface i s to characterize i t by the advancing contact angle of water rather than by the bulk para meter; the degree of equilibrium hydration. Furthermore, one may define the relative contact angle hysteresis (H ) as the d i f f e r ence between the advancing (Θ,) and the receding (Θ ) contact angles normalized with respect to the advancing contact angle. That i s , R

H

R

=

(©A -

O R ) /

Θ

Α

Then, the maximum value of hysteresis w i l l be equal to 1 whenever the receding angle i s zero, irrespective of the magni tude of the advancing contact angle. The value of the relative hysteresis of the contact angle i s shown i n Figure 4 as a function of the advancing contact angle of water. I t i s apparent from the graph that the value of H i n creases approximately i n a linear manner with the increasing contact angle, i . e . with decreasing wettability. This observation, camion for a l l the gels investigated, can be readily explained by our hypothesis. We have proposed that both the hydrophobic nature of the gel surface and the convert i b i l i t y to a more hydrophilic surface when i n contact with water are due to the segmental mobility and the amphipathic character of the polymer chains at the gel boundary. The more mobile the segments are for a given gel, the higher the advancing contact angle of water and the greater the difference between the advanc ing and receding angles w i l l be. Apparently this i s so, because at higher segmental mobility more hydrophobic groups can be ex posed and lined up at the surface when i t i s exposed to water R

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

258

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

WATER (OR

VAPOR /---LIQUID-

OCTANE)

WAT Ε RH

C 3 OH CH, CM, CH/OH OH - ; OH OH Q H ^ C

H

··········· ·· · · ·.!_·.· ·····«

HYDROGEL

50

60

ADVANCING

70

80

CONTACT

90

100

110

A N G L E (degree)

Figure 4. Relative contact angle hysteresis as a function of the advancing contact angle of water. ®, agarose; A , PAA; Sil-g-PVP; O, PHEA; PGMA; ·, PHEMA.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

19.

H O L L Y AND REFOJO

Water Wettability of Hydrogels

259

vapor (resulting i n a higher advancing contact angle of wetting), and more hydrophilic sites can be exposed to the adjacent l i q u i d water phase (resulting i n lower receding contact angles). In other words, increased segmental mobility of the surface polymer chains for a given gel would be manifested by both decreased wettability and enhanced contact angle hysteresis. I t i s possible to obtain a value for a "limiting advancing contact angle" for each gel by f i t t i n g a straight l i n e to the data points by the least square method and extrapolating i t to the maximum hysteresis value: H = 1, 0 = 0. While the empirical nature of the relationship and the considerable scatter i n the data make this limiting value of questionable importance and accuracy, i t i s possible to assign a physical meaning to i t . This limiting value of Θ, corresponds to the water wettability of a hypothetical gel or th composition which would hav mental mobility s u f f i c i e n t l tact angle. Since this limiting contact angle i s 114 for PHEMA, which value i s larger than can be obtained with water on the closely packed methyl groups i n the freshly cleaved surface of a hexatriacontane single crystal (111 ) (9), i t i s unlikely that a zero receding contact angle of water can ever be obtained on a PHEMA gel, no matter how much the segmental mobility may be i n creased by some suitable manipulation of the gel-forming tech nique. The extrapolated value of the advancing contact angle appears to be of a more r e a l i s t i c magnitude for the other gels. I t must be remembered, however, that these gels are considerably more hydrated than PHEMA, and this fact i s l i k e l y to decrease the surface density of the hydrophobic (and hydrophilic) groups and chain segments at the gel surface. I t i s of interest to note, however, that the maximum hysteresis observed with gels of d i f ferent types i s approximately the same, 0.8 - 0.9, and the high est was observed with the gel of the copolymer, Ρ (GMA/MMA). I t i s apparent from Figure 4 that the material SIL-g-PVP exhibits a behavior just the opposite to that of the hydrogels. The seemingly anomalous behavior of this material actually strengthens the foregoing argument based on the segmental mobili ty of the surface polymer chains i n the gels. In PVP grafted s i l icone the convertibility of the surface frcm hydrophobic to hydrophilic depends on the poly (vinyl pyrrolidone) content. The more PVP there i s , the more hysteresis i s expected. However, more PVP at the surface would result i n higher wettability, i . e . lower advancing contact angle, since silicone i s inherently hydrophobic. This i s why the straight l i n e , which f i t s the data exceptionally well, has a negative slope i n the graph. R

R

0

C r i t i c a l Surface Tension of the Hydrogels We were curious to find out the wettability of these hydro gels by pure organic liquids which are immiscible with water. The

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

260

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

eight liquids chosen and their surface tensions are shown i n Table I. They a l l have a negative spreading coefficient on water with the exception of t r i c r e s y l phosphate.

Table I Organic Liquids Used i n C r i t i c a l Surface Tension Deterniination

DIAGNOSTIC LIQUIDS

SURFACE TENSION dyne/cm

SPREADING COEFFICIENT ON WATER

Diiodomethane 1,1,2,2-Tetrabromoethane

48.3

negative

1-Bromonaphthalene

43.0

negative

Tricre sylphosphate

40.4

positive

1-Methylnaphthalene

38.7

negative

Nitromethane

36.1

negative

Dicyclohexylantine

33.8

negative

n-Hexadecane

27.1

negative

The contact angle data obtained on four different hydrogels were plotted as the cosine of the advancing angle versus the l i q u i d surface tension (Zisman Plot) and also as the adhesion tension (the product of the l i q u i d surface tension and the cosine of the contact angle), W , versus the l i q u i d surface tension (Wolfram Plot). This latter manner of contact angle presentation has been used by some investigators (10-13). I t has yielded a straight line of reasonably good f i t for nonpolar solids and either aqueous solutions of surfactants or for surfactants d i s solved i n a two-phase immiscible l i q u i d system. Wolfram (11,12) and later Lucassen-Reynders (13) have shown that the slope of the straight l i n e described by the data i n the adhesion tension versus surface tension plot i s equal to the ratio of the Gibbs surface excess concentration at the s o l i d l i q u i d interface (Γ ,) and at the liquid-vapor interface (Γ·. ) : T

1

slope = - Γ

3 ΐ

/Γ

1 ν

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Figure 5 shows the Zisman plot obtained for the PHEMA. gel of 40% equilibrium water content and Figure 6 shows the Wolfram plot for the same gel. These figures also include data obtained with poly (methyl methacrylate), which served as the control surface. As can be seen, i n both cases the scatter of the data i s considerable. The least square method has been used to obtain the straight lines shown i n the graph. These were used to obtain the c r i t i c a l surface tension value by extrapolating to cos 0 = 1 or W = γ ^ , for the Zisman plot and the Wolfram plot, respectively. These c r i t i c a l surface tension values are contained i n Table I I for several a c r y l i c hydrogels as well as for poly (methyl metha crylate) . Whenever possible, the gels representative of each type were chosen so that the equilibrium water contents were similar. T

ν

C r i t i c a l Surface Tension of Acrylic Hydrogels GEL OR SOLID

WATER CONTENT IN WT %

CRITICAL SURFACE TENSION * ZISMAN PLOT WOLFRAM PLOT

PMMA

1.5

38.5

38.9

PGMA

73.3

37.6

38.2

PHEMA

40.0

36.0

36.9

PHEA

71.9

34.1

35.6

PAA

77.7

32.9

34.8

i n dyne/cm

A l l the gels appear to have a c r i t i c a l surface tension value below that of PMMA. These c r i t i c a l surface tension values seem to decrease with increasing water wettability. The only exception to this rule i s PGMA, which has a somewhat higher c r i t i c a l surface tension than does PHEMA. This may be due to the presence of the two adjacent hydroxyl group on the glyceryl side chain. However, the difference i n the c r i t i c a l surface tension values are rather small and the scatter of data about the straight lines are rather large, so the reverse order of PHEMA and PGMA may not be r e a l . The tendency of the c r i t i c a l surface tension values of the gels to decrease with increasing water wettability, however, i s

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3 6.0 38.5 υ

θ = 0°

ό\Γ.---

\

T=25°C

50 LIQUID S U R F A C E

60 TENSION

70 dyne/cm

Figure 5. Critical surface tension of PHEMA obtained by the Zisman method using organic liquids. Solid symbols indicate values used to deter mine the straight lines.

30

40

50

LIQUID S U R F A C E

àO

70

TENSION

dyn«/cm

Figure 6. Critical surface tension of PHEMA obtained from the Wolfram plot using organic liquids. Solid symbols indicate values used to determine the straight lines.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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significant i n our opinion. I t probably indicates the existence of lakes of free water a t the surface of the gels i n increasing proportion. Such water would have a lew c r i t i c a l surface tension toward hydrophobic liquids, since the dispersion force component of water surface tension i s only about 22 dyne/cm (14). Criterion of Hydrophilicity Using Two-Liquid Systems The common definition of a hydrophilic s o l i d surface i s that water spontaneously spreads over i t , or that the contact angle o f water on the s o l i d i s zero. This implies that the energy of ad hesion of water to the s o l i d i s a t least as high as the energy o f cohesion of water. This definition i s based on systems, where only one type of l i q u i d i s assumed to be present, so the s o l i d cannot display a preferenc I t has been suggeste s o l i d or a gel surface should be made by water contact angle measurements i n the presence of another l i q u i d which i s nonpolar. The selection of η-octane as the nonpolar phase has an advantage. Hamilton demonstrated (16) that the change i n i n t e r f a c i a l tension due to polar interaction across the solid-^water interface can be calculated from the two-phase contact angle provided that the nonpolar l i q u i d i s η-octane. When there i s no polar interaction between the s o l i d and water, the water-octane contact angle i s independent of the s o l i d composition and i s given by cos Θ = (γ - Y ) / Y W

0

(

where y = 22.4 dyne/cm, y = 71.9 dyne/cm, and γ = 51.4 dyne/cm, as obtained i n our laboratory, are the surrace tension of water-saturated octane, that of octane-saturated water, and the inter f a c i a l tension a t the water-octane boundary, respect ively. The upper l i m i t for the water-octane contact angle obtain ed on an ideally nonpolar s o l i d i s thus equal to 164°. Water- in-octane contact angle values were obtained for the gels i n Table I I as well as for P1VMA and polyethylene. Table III contains the results together with the water-in-air contact angles obtained i n water-vapor-saturated a i r . A l l the contact angles i n Table III are the advancing type, i . e . the water drop l e t was increased i n size while resting on the surface and the angle i s taken through water, the denser phase as i s customary. The standard deviations are also included. I t i s reasonable to choose Θ = 90° for the water- in-oc tane contact angles as the arbitrary dividing line between hydrophilic and hydrophobic surfaces, since at this angle the s o l i d - l i q u i d i n t e r f a c i a l tensions are the same both with octane and water. As can be seen from the table, a l l the hydrogels investigated are hydrophilic to various degrees, since for a l l the gels this Q

w

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contact angle i s less than 90°. PHEMA. i s near the borderline dividing the hydrophilic and hydrophobic solids. Conversely, PMMA and polyethylene are hydrophobic.

Table III Water-in-Octane Contact Angle Values for Hydrogels and Solids

GEL OR SOLID

WATER CONTENT IN WT %

CONTACT ANGLE OF CONTACT ANGLE OF WATER IN AIR (°) WATER IN OCTANE (°)

Ρ Ε

0.0

PMMA

1.5

72.6±1.5

120.011.1

PHEMA

38.9*

59.5±2.3

88.011.3

PHEA

71.9

44.012.5

79.312.1

PGMA

73.3

41.311.3

63.311.6

PAA

77.7

10.110.9

11.511.5

* This PHEMA gel had a s l i g h t l y lower equilibrium water content than the one included i n Table I I .

In a basic sense, this c r i t e r i o n of hydrophilicity i s more meaningful than the water advancing (or receding) contact angle, since two l i q u i d - s o l i d i n t e r f a c i a l tensions are compared simul taneously. The water-in-air contact angles, on the other hand, provide information of practical importance. As a c r i t e r i o n of hydrophilicity, the c r i t i c a l surface tension obtained by organic liquids i s rather a useless quantity as shown by the fact that both polyethylene and poly (acrylamide) have similar c r i t i c a l sur face tension values. Conclusions In conclusion, i t was found that the surfaces of methacrylic and a c r y l i c hydrogels are a l l hydrophobic to a certain degree (even a t high equilibrium water content) as determined by the magnitude of the advancing contact angle of water on the f u l l y hydrated gel surface. The wettability i s primarily determined by

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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the nature of the polymer and also by the segmental mobility of the polymer chains at the gel boundary. A large relative contact angle hysteresis was found for a l l the gels, which increased with decreasing wettability for each series of gels. The gel water content a t equilibrium hydration was not found to have a pronounced effect on gel wettability, especially for gels of the lower hydration range. The c r i t i c a l surface tension obtained by hydrophobic organic liquids after the method of Zisman has not been found to be a useful parameter for the characterization of the water wettabil i t y of hydrogels, nevertheless i t shewed a consistent i f reversed relationship with water wettability as determined by the advancing contact angle of water. Although the water contact angle on gels i s of practical importance, the distinctio betwee hydrophili d hydro phobic surface perhaps by the s o l i d surface towar presenc , nonpolar l i q u i d . By such a criterion, a l l the gels investigated can be c l a s s i f i e d as hydrophilic. Acknowledgement We wish to thank Professor J.J. Bikerman for c a l l i n g our attention to the references on gelatin wettability. Ms. Fee-Lai Leong and Ms. Cheryl J . DeVine provided valuable technical assistance. This work was supported by PHS Research Grants No. EY-00208 and No. EY-00327, both from the National Eye Institute, National Institutes of Health.

ABSTRACT The water wettability of various methacrylic and acrylic hydrogels were determined by the sessile-drop and the captivebubble techniques. Surprisingly large advancing contact angles were obtained for all the gels, even though the surfaces were fully hydrated. The receding contact angles were much smaller than the advancing contact angles. The magnitude of the contact angle hysteresis appeared to increase with decreasing water wettability. It is proposed that large segmental mobility and amphipathic character of the polymer chains at the gel surface are responsible for the convertibility of the gel surfacefroma relatively hydrophilic to a hydrophobic one. The water contact angle measured in octane was less than 90° for all the gels studied. By this criterion all the hydrogels investigated have "hydrophilic" surfaces. Literature Cited 1. Pchelin, V.A. and Korotkina, I.I., Zh.Fiz.Khim., (1938) 12:50

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

2. Garrett, H.E.,"Aspects of Adhesion", Vol. 2, p. 18 (ed. Alner D.J.) University of London Press Ltd., London, 1966 3. Braudo, E.E., Tolstoguzov, V.B., and Nikitina, E.A., Colloid J. (1974) 36:191 4. Padday, J.F., personal communication 5. Holly, F.J. and Refojo, M.F., J. Biomed. Mater. Res. (1975) 9:315 6. Refojo, M.F., J. Appl. Polymer Sci. (1965) 9:3161 7. Laizier, J. and Wajs, G., U.S. Patent 3,700,573 (1972) 8. Ratner, B.D. and Miller, I.F., J. Polymer Sci. A-1(1972) 10:25 9. Shafrin, E.G. and Zisman, W.A.,"Contact Angle, Wetting, and Adhesion", p. 145 (ed. Fowkes, F.M.) Adv. Chem. Series #43, Am. Chem. Soc., Washington, D.C. 1964 10. Bargeman, D. and Van Voorst Vader, F., J. Coll. Interface Sci. (1973) 42:467 11. Wolfram, E., Plast 12. Wolfram, E., Kolloid Z. u. Z. f. Polymere (1966) 211:84 13. Lucassen-Reynders, E.H., J. Phys.Chem.,(1963) 67:969 14. Fowkes, F.M., Ind.Eng.Chem. (1964) 56:40 15. Glazman, Yu.M. and Fapon, I.D.,"Researches in Surface Forces" Proc. 2nd Surface Conf., Acad. Sci. USSR, Moscow, (1956) 2:232 16. Hamilton, W.C., J. Coll. Interface Sci. (1972) 40:219

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20 Water Wettability of Proteins Adsorbed at the Hydrogel-Water Interface FRANK J. HOLLY and MIGUEL F. REFOJO Eye Research Institute of Retina Foundation, 20 Staniford Street, Boston, Mass. 02114

The h y d r o g e l s i n g e n e r a l , and c r o s s l i n k e d p o l y (hydroxyethyl methacrylate p r e s e n t l y b e i n g use c o n s i d e r e d as b i o m a t e r i a l s o r c o a t i n g s f o r b i o m a t e r i a l s . The w a t e r - s o l u b l e s u r f a c e - a c t i v e s u b s t a n c e s i n the b l o o d plasma and t i s s u e f l u i d s i n c l u d i n g t e a r s a r e proteinaceous i n character. Therefore, the c h a r a c t e r i s t i c s o f t h e i n t e r f a c e between h y d r o g e l s and water and i t s i n t e r a c t i o n with d i s s o l v e d p r o t e i n s are o f importance. P a s t Work L i t t l e i s known o f t h e e n e r g e t i c s o f t h e h y d r o g e l water i n t e r f a c e . Water has an u n e x p e c t e d l y h i g h advanci n g c o n t a c t a n g l e (60-80°) on PHEMA g e l s u r f a c e s (1) and even g e l s o f more h y d r o p h i l i c polymers such as p o l y ( g l y c e r y l m e t h a c r y l a t e ) [PGMA], and p o l y ( h y d r o x y e t h y l a c r y l a t e ) [PHEA] w i t h t w i c e t h e e q u i l i b r i u m water c o n t e n t o f PHEMA e x h i b i t advancing c o n t a c t a n g l e s as h i g h as 40° (2^) . A s o l i d w i t h such a low water w e t t a b i l i t y i s e x p e c t e d t o have an i n t e r f a c i a l t e n s i o n o f cons i d e r a b l e magnitude a g a i n s t water. The low r e c e d i n g c o n t a c t a n g l e v a l u e on h y d r o g e l s , however, i n d i c a t e s t h a t t h i s i s n o t t h e c a s e . Due t o t h e c o n s i d e r a b l e segmental m o b i l i t y o f t h e s u r f a c e polymer c h a i n s o f t h e g e l m a t r i x , o r i e n t a t i o n a l changes a t t h e g e l - w a t e r i n t e r f a c e seems t o l e s s e n t h e i n t e r f a c i a l t e n s i o n s i g n i f i c a n t l y by i n c r e a s i n g t h e d e n s i t y o f t h e h y d r o p h i l i c s i t e s a t the s u r f a c e . Tension a t the gel-water i n t e r f a c e has been e s t i m a t e d t o be f a i r l y low by o t h e r s (_3) · I f such i s t h e c a s e , then t h e w a t e r - s o l u b l e p r o t e i n s are e x p e c t e d t o i n t e r a c t l i t t l e w i t h such an i n t e r f a c e and a d s o r p t i o n would p r o b a b l y e n t a i l l i t t l e i f any i r r e v e r s i b l e c o n f o r m a t i o n a l changes o r d e n a t u r a t i o n o f 267

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

the p r o t e i n m o l e c u l e s . P r o t e i n adsorbed a t h y d r o g e l - w a t e r i n t e r f a c e s has not been c h a r a c t e r i z e d i n d e t a i l . To o u r knowledge, no s y s t e m a t i c attempts have been made t o s t u d y t h e energ e t i c s i n c l u d i n g w e t t a b i l i t y o f p r o t e i n s adsorbed a t the h y d r o g e l - w a t e r i n t e r f a c e . M a t e r i a l s and Methods We have s t u d i e d t h e e f f e c t o f p r o t e i n a d s o r p t i o n on t h e water c o n t a c t a n g l e on PHEMA g e l s h a v i n g e i t h e r 38.9 o r 40.0% e q u i l i b r i u m water c o n t e n t . Some measurements have a l s o been made on a c r o s s l i n k e d PHEA g e l c o n t a i n i n g 89.9% water a t e q u i l i b r i u m h y d r a t i o n . These g e l s a r e d e s c r i b e d elsewher (2) i o n a l l y poly(methyl ene [PE] were a l s o used as a d s o r b e n t s f o r comparison. The p r o t e i n used i n t h i s s t u d y was b o v i n e serum albumin [BSA] (Cohn F r a c t i o n V, Sigma C h e m i c a l Co., S t . L o u i s , MO) w i t h a m o l e c u l a r weight o f 68,000 g/mole. A h i g h m o l e c u l a r weight, c o m m e r c i a l l y a v a i l a b l e g l y c o p r o t e i n , b o v i n e s u b m a x i l l a r y mucin [BSM] (type I , MW= 4.1x10 g/mole, Sigma C h e m i c a l Company, S t . L o u i s , MO) and egg lysozyme [LYZ] (as c h l o r i d e , MW=14,800 g/mole, M i l e s L a b o r a t o r i e s , Kankakee, IL) were a l s o used i n t h e s t u d y . F o r comparison purposes, s y n t h e t i c , w a t e r - s o l u b l e polymers were a l s o used i n t h e p r e l i m i n a r y measurements . C o n t a c t a n g l e s were measured by u s i n g a Ramè-Hart goniometer w i t h an e n v i r o n m e n t a l chamber so t h a t t h e h y d r o g e l sample c o u l d be k e p t i n an atmosphere s a t u r a t e d w i t h water vapor. S u r f a c e t e n s i o n o f t h e aqueous s o l u t i o n s was measured by t h e Wilhelmy method u s i n g a roughened p l a t i n u m b l a d e and a Cahn E l e c t r o b a l a n c e Mod e l RM-2. Both t h e s u r f ice t e n s i o n and t h e c o n t a c t a n g l e were m o n i t o r e d f o r t h i r t y m i n u t e s . Measurements made a t l o n g e r times i n d i c a t e d t h a t such a time i n t e r v a l was s u f f i c i e n t t o approximate e q u i l i b r i u m a t l e a s t f o r s o l u t i o n s w i t h c o n c e n t r a t i o n s 1 0 " wt% and above. 6

2

E f f e c t o f S y n t h e t i c and B i o p o l y m e r s on A d h e s i o n T e n s i o n The a d v a n c i n g (but s t a t i c ) c o n t a c t a n g l e f o r aqueous polymer s o l u t i o n s was determined on t h e PHEMA g e l , the PHEA g e l , PMMA, and p o l y e t h y l e n e (_2) . I n a d d i t i o n t o t h e s o l u t i o n s o f t h e t h r e e b i o p o l y m e r s a l r e a d y described, the following synthetic or modified natural polymers were employed: P o l y ( v i n y l a l c o h o l ) ( G e l v a t o l 20/90), p o l y ( e t h y l e n e oxide) (WSR-205), h y d r o x y e t h y l c e l l u l o s e , and p o l y ( v i n y l p y r r o l i d o n e ) . The polymers

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

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Water Wettability

REFOJO

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Proteins

269

were d i s s o l v e d i n d i s t i l l e d water t o a s o l u t i o n concent r a t i o n o f 0.1% by weight. Aqueous s o l u t i o n s o f sodium s u l f o s u c c i n a t e and methyl c e l l u l o s e (USP grade) a t 0.1% c o n c e n t r a t i o n were a l s o used on PMMA and PE. See T a b l e I f o r the s u r f a c e t e n s i o n o f t h e s e s o l u t i o n s .

Table I Surface

Tension

o f Aqueous S o l u t i o n s o f S y n t h e t i c and

B i o p o l y m e r s and S u r f a c t a n t *

SOLUTE

SURFACE TENSION

Poly(vinyl

alcohol)

52. ,6

Poly(vinyl

pyrrolidone)

67. ,5

Methyl c e l l u l o s e

55. ,4

Hydroxyethyl c e l l u l o s e

63. ,5

Poly(ethylene

60. ,7

oxide)

BSM

41.,2

B SA

52. ,8

LYZ

58. .0

Sodium s u l f o s u c c i n a t e

30, .8

(dyne/cm)

* a t 0.1% c o n c e n t r a t i o n

and a t30 minutes

F i g u r e 1 shows the r e s u l t s where the a d h e s i o n t e n s i o n o f the s o l u t i o n s t o the g e l o r s o l i d are p l o t t e d as a f u n c t i o n o f t h e s o l u t i o n s u r f a c e t e n s i o n . F o r each s u b s t r a t e , the polymer s o l u t i o n s y i e l d e d d a t a which appear t o e x h i b i t a l i n e a r dependence o f the a d h e s i o n t e n s i o n on the s o l u t i o n s u r f a c e t e n s i o n . The 45° broken l i n e on the graph r e p r e s e n t s zero c o n t a c t a n g l e (adhesion tension = s o l u t i o n surface tension). I t s i n t e r c e p t w i t h the s t r a i g h t l i n e , c a l c u l a t e d by the l e a s t square method f o r each s u b s t r a t e , r e p r e s e n t s a k i n d o f

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

" c r i t i c a l s u r f a c e t e n s i o n " o b t a i n e d w i t h t h e s e aqueous s o l u t i o n s f o r t h e p a r t i c u l a r s o l i d o r g e l . The s l o p e s o f t h e s t r a i g h t l i n e s f o r PE, PMMA, PHEMA, and PHEA a r e e q u a l t o -0.76, -0.18, +0.16, and +0.46, r e s p e c t i v e l y . I t has been r e p o r t e d (4-6,) t h a t aqueous s o l u t i o n s o f s u r f a c t a n t s , when p l a c e d on a n o n p o l a r s o l i d such as p a r a f f i n , form c o n t a c t a n g l e s t h a t e x h i b i t a g e n e r a l r e l a t i o n s h i p with respect t o the surface t e n s i o n o f the s o l u t i o n . The p r o d u c t o f t h e s u r f a c e t e n s i o n o f t h e s o l u t i o n and t h e c o s i n e o f t h e c o n t a c t a n g l e (adhesion tension, W ) v a r i e s l i n e a r l y with s o l u t i o n surface ten s i o n ( Υ χ ) such t h a t T

ν

W_ = γ Τ 'l F o r low m o l e c u l a r weight s u r f a c t a n t s , f o r which the Gibbs a d s o r p t i o n e q u a t i o n i s v a l i d , t h e s l o p e , A, has been shown (£,6) t o be e q u a l t o t h e n e g a t i v e r a t i o o f t h e s u r f a c e excess c o n c e n t r a t i o n o f t h e s u r f a c t a n t a t t h e s o l i d - w a t e r i n t e r f a c e (^ ±) and t h a t a t t h e s o l u t i o n - a i r i n t e r f a c e (Γ^ ) . Assuming t h a t t h e s o l u t e s u r f a c e excess c o n c e n t r a t i o n a t the s o l i d - v a p o r i n t e r f a c e i s z e r o , we may w r i t e : s

ν

A

r

r

= - sl/ lv

When A = -1, t h e e x t e n t o f a d s o r p t i o n a t t h e two i n t e r f a c e s i s t h e same and t h e a d h e s i o n energy o f t h e s o l u t i o n t o t h e s o l i d becomes independent o f t h e s o l u tion surface tension. Such a s i t u a t i o n has a c t u a l l y been o b s e r v e d (2). I f A = 0, t h e c o n c e n t r a t i o n o f t h e s o l u t e i s t h e same a t t h e s o l i d - s o l u t i o n i n t e r f a c e as i n t h e b u l k , i . e . t h e i n t e r f a c i a l excess c o n c e n t r a t i o n is zero, r ^ = 0. Such a s u r f a c e t e n s i o n dependence o f a d h e s i o n t e n s i o n was demonstrated by Lucassen-Reynd e r s (6) u s i n g t h e d a t a o f Smolders (8) o b t a i n e d i n a hydrogen gas - aqueous a n i o n i c s u r f a c t a n t s o l u t i o n mercury system a t t h e e l e c t r o c a p i l l a r y maximum (zero i n t e r f a c i a l charge d e n s i t y ) . P o s i t i v e s l o p e s would mean t h a t the s u r f a c e excess c o n c e n t r a t i o n o f the s o l u t e ( s ) i s n e g a t i v e , i . e . t h e s o l u t e c o n c e n t r a t i o n i s lower a t the i n t e r f a c e than i n t h e b u l k . F i g u r e 1 demonstrates t h a t t h e s u r f a c e - a c t i v e polymer s o l u t i o n s behave s i m i l a r l y t o s i m p l e s u r f a c t a n t s o l u t i o n s on n o n p o l a r s o l i d s . W h i l e t h e v a l i d i t y o f t h e Gibbs e q u a t i o n does n o t extend t o macromolecules, i t i s s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

HOLLY

Water Wettability of Proteins

A N D REFOJO

271

r e a s o n a b l e t o suppose t h a t t h e magnitude o f t h e s l o p e A r e f l e c t s i n some way t h e magnitude o f t h e i n t e r f a c i a l adsorption o f the polymeric solute o r a t l e a s t i t s e f f e c t on t h e i n t e r f a c i a l t e n s i o n . I f t h i s i s s o , then t h e r e s u l t s i n d i c a t e t h a t con s i d e r a b l e a d s o r p t i o n o f the polymeric s o l u t e takes p l a c e a t the p o l y e t h y l e n e - w a t e r i n t e r f a c e . T h i s i s ex p e c t e d i n view o f t h e l a r g e i n t e r f a c i a l t e n s i o n a t t h e p o l y e t h y l e n e - w a t e r boundary. T h i s e f f e c t i s c o n s i d e r a b l y l e s s f o r t h e more p o l a r PMMA s u r f a c e as shown by the s m a l l e r a b s o l u t e , b u t s t i l l n e g a t i v e , v a l u e o f A f o r PMMA. T h i s i s no doubt due t o t h e g r e a t e r i n t e r a c t i o n o f PMMA s u r f a c e w i t h water which competes w i t h the p o l y m e r i c s o l u t e s f o r t h e a v a i l a b l e a d s o r p t i o n sites. It i s quite interestin c h a r a c t e r i z e d by a s t r a i g h t l i n e w i t h a s l i g h t l y p o s i t i v e s l o p e , which may i n d i c a t e no d i f f e r e n c e between i n t e r f a c e and b u l k c o n c e n t r a t i o n o r even lower concen t r a t i o n a t t h e i n t e r f a c e than i n t h e b u l k . The s l o p e o f the l i n e f o r PHEA i s a l s o p o s i t i v e and even g r e a t e r than t h a t f o r PHEMA. The s c a t t e r o f t h e d a t a i s c o n s i d e r a b l e f o r each s o l i d and i t i s e s p e c i a l l y l a r g e f o r t h e g e l s . There a r e i n d i c a t i o n s (9) t h a t t h e type o f t h e s o l u t e s em ployed a l s o a f f e c t the p o s i t i o n o f the s t r a i g h t l i n e i n t h i s Wolfram p l o t (adhesion t e n s i o n v s . s u r f a c e t e n s i o n ) e s p e c i a l l y f o r multicomponent p o l y m e r i c s o l u t i o n s . Thus, c o n s i d e r i n g t h e wide spectrum o f t h e p o l y m e r i c s o l u t e s used i n t h i s s t u d y , t h e r e s u l t s f i t t h e s t r a i g h t l i n e s reasonably w e l l . F i l m Pressure

o f Biopolymers Adsorbed a t the I n t e r f a c e

Attempts have been made t o o b t a i n t h e f i l m p r e s sure o f t h e b i o p o l y m e r s a t the water-PHEMA g e l and water-polyethylene i n t e r f a c e s as a f u n c t i o n o f t h e s o l u t i o n c o n c e n t r a t i o n . The d e c r e a s e o f i n t e r f a c i a l t e n s i o n , due t o t h e a d s o r p t i o n o f t h e b i o p o l y m e r , i s the f i l m p r e s s u r e o f t h e a d s o r b a t e a t t h e i n t e r f a c e . I t i s g i v e n by t h e d i f f e r e n c e between t h e s o l u t i o n s o l i d and t h e s o l v e n t - s o l i d a d h e s i o n t e n s i o n s (10) :

ι

'sol'n

sol'n

f

w

w

The f i l m p r e s s u r e s o f albumin c a l c u l a t e d f o r t h e PHEMA g e l - water and t h e PE - water i n t e r f a c e s , t o gether with the f i l m pressures a t the w a t e r - a i r i n t e r -

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

272

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

i n t e r f a c e , a r e shown i n F i g u r e 2. The r e s u l t s show t h a t t h e f i l m p r e s s u r e o f albumin i s s i m i l a r a t t h e w a t e r - a i r and w a t e r - p o l y e t h y l e n e i n t e r f a c e s , e s p e c i a l l y near the p h y s i o l o g i c c o n c e n t r a t i o n range (0.1 - 6%). On t h e o t h e r hand, t h e f i l m p r e s s u r e o f albumin a t t h e PHEMA-water i n t e r f a c e i s n o t s i g n i f i c a n t l y d i f f e r e n t from z e r o w i t h i n t h e r a t h e r l a r g e d e v i a t i o n o f t h e d a t a , a l t h o u g h i t appears t o d e c r e a s e with decreasing s o l u t i o n concentration. The f i l m p r e s s u r e o f mucin and lysozyme show simi l a r v a r i a t i o n w i t h s o l u t i o n c o n c e n t r a t i o n a t t h e PEwater i n t e r f a c e . A t t h e PHEMA g e l - water i n t e r f a c e , however, t h e f i l m p r e s s u r e i s n e g a t i v e a t h i g h s o l u t i o n c o n c e n t r a t i o n s and approximates z e r o as t h e s o l u tion concentration i W e t t a b i l i t y o f B i o p o l y m e r s A d s o r b e d onto PHEMA and PE The c o n t a c t a n g l e o f t h e s e s s i l e d r o p l e t o f t h e b i o p o l y m e r s o l u t i o n s on PHEMA and PE s u b s t r a t e s t h a t had been exposed t o a b i o p o l y m e r s o l u t i o n o f t h e same c o m p o s i t i o n and c o n c e n t r a t i o n was d e t e r m i n e d . By u s i n g the same s o l u t i o n f o r t h e a d s o r b i n g medium and f o r t h e w e t t a b i l i t y measurement, we a v o i d e d g r o s s changes i n the i n t e r f a c i a l t e n s i o n a t t h e d r o p l e t - s u b s t r a t e bounda r y due t o d i s s o l u t i o n o r f u r t h e r d e p o s i t i o n o f t h e biopolymers. A f t e r t h e s u b s t r a t e had been i n t h e b i o p o l y m e r s o l u t i o n o f a g i v e n c o n c e n t r a t i o n f o r 1 hour, i t was g e n t l y b l o t t e d w i t h a g r e a s e - f r e e f i l t e r paper t o r e move t h e excess l i q u i d . Then t h e c o n t a c t a n g l e o f a s e s s i l e d r o p l e t was d e t e r m i n e d i n t h e e n v i r o n m e n t a l chamber 30 minutes a f t e r the d r o p l e t o f t h e same s o l u t i o n was d e p o s i t e d on t h e s u b s t r a t e . A f t e r w a r d s , t h e s u b s t r a t e s u r f a c e was r i n s e d w i t h d i s t i l l e d water, b l o t t e d , and t h e c o n t a c t a n g l e was determined u s i n g t h e same b i o p o l y m e r s o l u t i o n . T h i s measurement was r e p e a t e d a t f i v e d i f f e r e n t b i o p o l y m e r c o n c e n t r a t i o n s f o r both substrates. F i g u r e s 3 and 4 show t h e advancing c o n t a c t a n g l e s o b t a i n e d w i t h bovine serum albumin s o l u t i o n on albumin adsorbed on PHEMA g e l and p o l y e t h y l e n e as a f u n c t i o n o f s o l u t i o n c o n c e n t r a t i o n . These graphs a l s o c o n t a i n t h e d a t a o b t a i n e d w i t h albumin s o l u t i o n on t h e c l e a n , a l bumin-free s u r f a c e s . The r e s u l t s o b t a i n e d w i t h mucin and lysozyme a r e n o t shown here b u t a r e q u a l i t a t i v e l y similar (11). A l b u m i n adsorbed from s o l u t i o n s c o n t a i n i n g t h e p r o t e i n a t c o n c e n t r a t i o n s o f t h e same o r d e r o f magnitude as t h a t o f p h y s i o l o g i c ( t e a r and b l o o d plasma)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

HOLLY

Water Wettability

A N D REFOJO

of

Proteins

T=25°C ζ 4

ο °CO ζ

Hi 30·

Ο

20·

CO

I ο < 10

20

3

"70

Figure 1. Adhesion tension of aqueous polymer solutions to gels and solids as a function of the solution surface tension. (Large symbols indicate values obtained with pure water also showing standard deviation.)

T=25°C

Θ Θ Θ

Φ

Φ

4

Θ

10

Φ

10

10

SOLUTION CONCENTRATION ( %)

Figure 2. Film pressure of bovine serum albumin at three interfaces: O, water-air; • , water-polyethylene; Δ , water-PHEMA gel. (Vertical lines show standard deviation.)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

273

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

T-25°C

[3 <

φ

L

*

4

water

on clean su f a c e

ι

Φ

Φ

Φ

Φ

Φ

Φ

ίο"

ίο"

4

SOLUTION CONCENTRATION (%)

Figure 3. Wettability by albumin solutions of bovine serum albumin adsorbed onto PHEMA gels. Q, clean surface. Surface exposed to albu min solution for 1 hour and blotted only ([J) then rinsed in distilled water and again blotted (A).

-θ"

water on clean

I

surface

φ

Φ T=25°C

SOLUTION

CONCENTRATION

(%)

Figure 4. Wettability by albumin solutions of bovine serum albumin adsorbed onto polyethyl ene. 0> clean surface. Surface exposed to albu min solution for 1 hour then blotted only ( Q ) then rinsed in distilled water and again blotted (A)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

HOLLY

AND REFOJO

Water Wettability

of

Proteins

275

l e v e l s ; 0.1 and 1%, makes both t y p e s o f s u r f a c e s more w e t t a b l e . A t t h e s o l u t i o n c o n c e n t r a t i o n o f 1%, no d i f f e r e n c e i s d i s c e r n i b l e between t h e w e t t a b i l i t y o f a l bumin-coated PHEMA and p o l y e t h y l e n e . R i n s i n g d e c r e a s e s the w e t t a b i l i t y somewhat, b u t a t t h e s e c o n c e n t r a t i o n s , i t i s s t i l l g r e a t e r than t h a t o f the r e s p e c t i v e c l e a n surface. A t t h e s o l u t i o n c o n c e n t r a t i o n s o f 1 0 " and 1 0 " %, r i n s i n g does n o t d e c r e a s e w e t t a b i l i t y , i . e t h e adsorbed albumin l a y e r i s t h i n n e r b u t i t i s more t i g h t l y bound to b o t h PHEMA and PE. A t t h e c o n c e n t r a t i o n o f 1 0 " %, b o t h s u r f a c e s a r e s t i l l more w e t t a b l e than t h e r e s p e c t i v e clean surfaces. A t the concentration l e v e l o f 10" %, however, albumin a d s o r p t i o n does n o t seem t o change w e t t a b i l i t y . f o r th polyethylen d i a c t u a l l de creases w e t t a b i l i t A t t h e lowest c o n c e n t r a t i o n l e v e l s t u d i e d , 10" %, the w e t t a b i l i t y by albumin s o l u t i o n i s t h e same f o r t h e b l o t t e d , r i n s e d , o r c l e a n s u r f a c e s o f PHEMA and i t i s a p p r o x i m a t e l y t h e same as t h e water v a l u e on t h e c l e a n g e l s u r f a c e . F o r p o l y e t h y l e n e , o n l y t h e exposed and b l o t t e d s u r f a c e i s somewhat more w e t t a b l e than t h e c l e a n s u r f a c e , w h i l e t h e r i n s e d and t h e c l e a n s u r f a c e s a r e wetted by t h i s d i l u t e albumin s o l u t i o n t o about t h e same e x t e n t as c l e a n p o l y e t h y l e n e i s wetted by water. The r e c e d i n g c o n t a c t angle o f t h e r e s p e c t i v e s o l u t i o n s was Zound t o be e s s e n t i a l l y zero f o r mucin, a l bumin, and lysozyme adsorbed on PHEMA and p o l y e t h y l e n e from aqueous s o l u t i o n s h a v i n g a b i o p o l y m e r c o n c e n t r a t i o n 10" % or higher. 2

3

2

3

2

C r i t i c a l Surface

Tension

o f Adsorbed B i o p o l y m e r s

In t h e p a s t , we have determined t h e c r i t i c a l s u r f a c e t e n s i o n o f mucin (12) and t h e two o t h e r b i o p o l y mers (13^) u s i n g h y d r o p h o b i c , d i a g n o s t i c l i q u i d s a c c o r d ing t h e well-known method o f Zisman (L4). However, we b e l i e v e t h a t such a parameter has l i t t l e r e l e v a n c e t o w e t t a b i l i t y i n an aqueous system, e s p e c i a l l y i f t h e segmental m o b i l i t y o f t h e s u r f a c e polymer s t r u c t u r e i s l a r g e as i t i s w i t h h y d r o g e l s (2^) which a l s o appears t o be t h e case w i t h adsorbed b i o p o l y m e r s . T h e r e f o r e , we used t h e c o n t a c t a n g l e d a t a o b t a i n e d f o r c l e a n , b l o t t e d , and r i n s e d s u r f a c e s t h a t were d i s c u s s e d i n t h e p r e v i o u s s e c t i o n , and p l o t t e d them i n two ways: 1. c o s i n e o f t h e a d v a n c i n g a n g l e (Zisman p l o t ) , and 2. t h e a d h e s i o n t e n s i o n (Wolfram p l o t ) b o t h a g a i n s t t h e s o l u t i o n s u r f a c e t e n s i o n . Then - assuming a s t r a i g h t l i n e f i t - we e x t r a p o l a t e d t o zero c o n t a c t a n g l e u s i n g t h e l e a s t square method.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

276

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

The r e s u l t s o b t a i n e d w i t h albumin on PHEMA a r e shown i n F i g u r e s 5 and 6, i n a Zisman and Wolfram p l o t , r e s p e c t i v e l y . The albumin d a t a on p o l y e t h y l e n e a r e shown i n F i g u r e s 7 and 8. The pure water v a l u e w i t h t h e s t a n d a r d d e v i a t i o n shown a r e a l s o i n d i c a t e d on t h e s e graphs. F o r t h e albumin s o l u t i o n on a c l e a n s u r f a c e , t h e contact angle values obtained a t the f i v e d i f f e r e n t s o l u t i o n concentrations define a s t r a i g h t l i n e reason ably w e l l . F o r the albumin-coated s u r f a c e s , o n l y the c o n t a c t angle values obtained a t s o l u t i o n concentra t i o n s 1%, 0.1%, and 0.01% f a l l on t h e same s t r a i g h t line. S i m i l a r graphs (not shown here) can be o b t a i n e d f o r mucin and lysozyme t t h a t f o thes b i o p o l y mers, even on a c l e a w i t h t h e t h r e e most c o n c e n t r a t e d s o l u t i o n s gave a r e a s o n a b l y good f i t t o a s t r a i g h t l i n e ( 1 1 ) . Table I I contains the c r i t i c a l surface t e n s i o n v a l u e s obtained with the biopolymer s o l u t i o n s f o r the c l e a n s u b s t r a t e s , PHEMA and PE. F o r comparison, t h e t a b l e a l s o c o n t a i n s t h e c r i t i c a l s u r f a c e t e n s i o n ob t a i n e d w i t h h y d r o p h o b i c o r g a n i c l i q u i d s (2^) .

Table I I Critical Obtained

S u r f a c e T e n s i o n o f PHEMA and PE as by Aqueous Biopolymer

DIAGNOSTIC LIQUIDS

Organic

liquids

Solutions

CRITICAL SURFACE TENSION* POLYETHYLENE PHEMA 36.9

; 36. 0

34.7

; 32.5

BSM

sol η

(3 cone. )

21.8

; 21. 7

33.8

; 32.4

BSA

sol η

(5 cone. )

45.8

; 42. 5

36.4

; 23.3

LYZ

sol η

(3 cone. )

37.0

; 27. 8

29.8

;

6.5

* i n dyne/cm

(Wolfram ; Zisman)

Due t o t h e s m a l l number o f d a t a p o i n t s used t o c a l c u l a t e the s t r a i g h t l i n e s , the exact numerical value

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

Water

H O L L Y AND REFOJO

Wettability

of

Proteins

θ«0°

ο ο

T = 25°C

40

Figure 5. Zisman plot obtained by albumin solution on clean and albumin-coated PHEMA gel surfaces. O, clean surface. Sur face exposed to albumin solution then blotted only (•) then rinsed in distilled water and again blotted (A). Solid symbols indicate values used to obtain the straight lines. Water value is also shown.

T = 25°C

40

SO

60

SOLUTION SURFACE TENSION

70

| dyne/cm |

Figure 6. Wolfram plot of adhesion tension obtained by albumin solutions on clean and albumin-coated PHEMA gel surfaces. 0> clean surface. Surface exposed to albumin solution for 1 hour then blotted only (•) then rinsed in distilled water and again blotted (A)- Solid symbols indicate values used to obtain the straight lines. Water value is also shown.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

277

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 7. Zisman plot obtained by albumin solutions on clean and albumin-coated polyethylene. clean surface. Surface exposed to albumin solution for 1 water and again blotted (A)- Solid symbols indicate values used to obtain the straight lines. Water value is also shown.

SOLUTION

SURFACE

TENSION

dyne/cm

Figure 8. Wolfram plot of adhesion tension obtained by albumin solutions on clean and albumin-coated polyethylene. Q, clean surface. Surface exposed to albumin solution for 1 hour then blotted only (•) then rinsed in distilled water and again blotted (A)- Solid symbols indicate values used to obtain the straight lines. Water value is also shown.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

H O L L Y AND REFOJO

Water Wettability

of

279

Proteins

of t h e " c r i t i c a l s u r f a c e t e n s i o n " parameter i s o f l i m i t e d s i g n i f i c a n c e . However, a few i n t e r e s t i n g o b s e r v a t i o n s c a n be made: 1. The c r i t i c a l s u r f a c e t e n s i o n o f p o l y e t h y l e n e i s a p p r o x i m a t e l y t h e same i r r e s p e c t i v e o f whether t h e h y d r o p h o b i c l i q u i d s o r any o f t h e b i o p o l y mer s o l u t i o n s i s used p r o v i d e d t h a t i t was o b t a i n e d from t h e Wolfram p l o t . When t h e Zisman method i s used, o n l y t h e mucin s o l u t i o n s y i e l d a c r i t i c a l s u r f a c e t e n s i o n v a l u e s i m i l a r t o t h a t o b t a i n e d w i t h t h e hydrophob ic liquids. 2. F o r t h e PHEMA s u r f a c e , t h e h i g h e s t c r i t i c a l v a l u e (Wolfram) i s o b t a i n e d w i t h t h e albumin s o l u t i o n s . The lysozyme s o l u t i o n s y i e l d a v a l u e (Wolf ram) o f c r i t i c a l s u r f a c e t e n s i o n p r a c t i c a l l y i d e n t i c a l to t h e v a l u e o b t a i n e d w i t h hydrophobic l i q u i d s . The c r i t i c a l s u r f a c e t e n s i o n v a l u e f o r PHEMA o b t a i n e d by the mucin s o l u t i o n f i e l d component o f Table I I I contains the c r i t i c a l surface t e n s i o n v a l u e f o r t h e adsorbed b i o p o l y m e r s as determined by t h e aqueous b i o p o l y m e r s o l u t i o n s b o t h b e f o r e and a f t e r r i n s i n g . The c r i t i c a l s u r f a c e t e n s i o n v a l u e s o b t a i n e d by hydrophobic l i q u i d s a r e n o t shown. I n a p r e v i o u s study (12), t h e c r i t i c a l s u r f a c e t e n s i o n v a l u e f o r mucin a d sorbed on p o l y e t h y l e n e o r g l a s s was found t o be about 38 dyne/cm, w h i l e t h e c r i t i c a l v a l u e s f o r albumin and lysozyme adsorbed on p o l y e t h y l e n e were found t o be s l i g h t l y h i g h e r , between 39-41 dyne/cm (13).

Table I I I Critical

S u r f a c e T e n s i o n o f Biopolymers

Adsorbed

on PHEMA and P o l y e t h y l e n e

BIOPOLYMER

BSM

S o l u t i o n s (3 cone.) "

BSA

S o l u t i o n s (3 cone.) "

LYZ

after rinsing

after rinsing

S o l u t i o n s (3 cone.) "

after rinsing

i n dyne/cm

CRITICAL SURFACE TENSION OF ADSORBED BIOPOLYMER ON* POLYETHYLENE Ρ Η Ε M A 38.7

37.8

7 37.6

38. 4 r 38.3

33.7

1 34.7

52. 2 r 52.2

52.4

r 52.4

51. 7 r 51.6

51.1

7 50.9

45. 2 r 43.4

41.8

t 31.1

37. 4 r 30.0

35.0

r 21.6

38. 8

?

(Wolfram ; Zisman)

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

280

With t h e e x c e p t i o n o f lysozyme, t h e r e i s a good agreement between t h e c r i t i c a l v a l u e s o b t a i n e d from t h e Wolfram p l o t and t h o s e o b t a i n e d from t h e Zisman p l o t . S i n c e t h e c r i t i c a l s u r f a c e t e n s i o n v a l u e s o b t a i n e d from t h e Wolfram p l o t appear t o be more c o n s i s t e n t , i t ap p e a r s t h a t t h e former v a l u e s a r e more r e l e v a n t f o r aqueous s o l u t i o n s . I t may be mentioned h e r e t h a t t h e s e w e t t a b i l i t y d a t a were a l s o t r e a t e d a c c o r d i n g t o t h e e q u a t i o n o f Good and G i r i f a l c o ( 1 5 ) :

cosG = 2 Φ ( γ / γ -

- 1

By assuming t h a Φ, i s c o n s t a n t , t h e g straigh d a t a i n a cosO v s . ( Y T _ ) " ^ p l o t y i e l d e d c r i t i c a l s u r f a c e t e n s i o n v a l u e s by e x t r a p o l a t i n g t o cosO = 1. These c r i t i c a l s u r f a c e t e n s i o n s compared w e l l w i t h t h o s e ob t a i n e d from t h e Wolfram p l o t (see T a b l e s I and I I ) ex c e p t t h a t t h e l a t t e r v a l u e s appeared t o be somewhat more c o n s i s t e n t (11). It i s of considerable i n t e r e s t that the c r i t i c a l surface tension values f o r the unrinsed surfaces are the same f o r b o t h t h e PHEMA g e l and t h e p o l y e t h y l e n e s u b s t r a t e s . T h i s o b s e r v a t i o n i s i n l i n e w i t h t h e accep ted view c o n c e r n i n g t h e s h o r t range o f s u r f a c e f o r c e s . The a d s o r b e d albumin l a y e r has t h e h i g h e s t c r i t i c a l s u r f a c e t e n s i o n , 52 dyne/cm, w h i l e t h e adsorbed mucin l a y e r has a c r i t i c a l v a l u e o f about 38-39 dyne/cm. I t may be s i g n i f i c a n t t h a t t h e s e v a l u e s a r e a p p r o x i m a t e l y the same as t h e s u r f a c e t e n s i o n o f t h e more c o n c e n t r a t ed aqueous s o l u t i o n s o f t h e r e s p e c t i v e b i o p o l y m e r s . The c r i t i c a l s u r f a c e t e n s i o n o f t h e a d s o r b e d lysozyme seems t o f a l l between t h e s e two c r i t i c a l v a l u e s . v

Conclusions In c o n c l u s i o n , i t was found t h a t t h e two h y d r o g e l s , PHEm and PHEA w i t h e q u i l i b r i u m water c o n t e n t 40% and 90%, r e s p e c t i v e l y do n o t e x h i b i t a h i g h a d s o r p t i v i t y a t t h e water i n t e r f a c e f o r a number o f s y n t h e t i c and b i o p o l y m e r s . The w a t e r - g e l i n t e r f a c i a l t e n s i o n a t l e a s t does n o t seem t o change much i n t h e p r e s e n c e o f t h e s e s o l u t e s and may even i n c r e a s e i n c e r t a i n c a s e s . The f i l m p r e s s u r e o f albumin a t t h e PHEIVft. g e l water i n t e r f a c e i s s m a l l and approaches z e r o w i t h de c r e a s i n g s o l u t i o n c o n c e n t r a t i o n . On t h e o t h e r hand, t h e

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20.

HOLLY

AND

REFOJO

Water

Wettability

of

Proteins

281

f i l m p r e s s u r e o f mucin and lysozyme a t t h e PHEMk-water i n t e r f a c e appears t o be somewhat n e g a t i v e a t h i g h s o l u t i o n c o n c e n t r a t i o n , a p p r o x i m a t i n g zero w i t h d e c r e a s i n g s o l u t i o n c o n c e n t r a t i o n . I n o t h e r words, t h e d e c r e a s e o f water c o n t a c t a n g l e on t h e g e l r e s u l t i n g from t h e p r e s ence o f t h e b i o p o l y m e r i n t h e d r o p l e t may be a c c o u n t e d f o r s o l e l y by t h e change i n t h e s u r f a c e t e n s i o n o f wat e r due t o t h e a d s o r p t i o n o f t h e b i o p o l y m e r a t t h e s o l u tion-air interface. N e v e r t h e l e s s , b i o p o l y m e r s adsorbed on ΡHEM. g e l s from aqueous s o l u t i o n i n c r e a s e w e t t a b i l i t y . W h i l e t h e s o l u t i o n c o n t a c t a n g l e i n c r e a s e s somewhat a f t e r r i n s i n g at high s o l u t i o n c o n c e n t r a t i o n , the c r i t i c a l s u r f a c e t e n s i o n v a l u e o b t a i n e d by t h e s e s o l u t i o n s show l i t t l e change w i t h r i n s i n g f o r B SA and BSM adsorbed on PHEM^. The c r i t i c a l s u r f a c the aqueous b i o p o l y m e t e n s i o n v s . s o l u t i o n s u r f a c e t e n s i o n p l o t appear t o be the most c o n s i s t e n t among t h e d i f f e r e n t systems comp a r e d . N u m e r i c a l l y , f o r albumin, mucin, and lysozyme r e s p e c t i v e l y , i t i s e q u a l t o 52, 39, and 45 dyne/cm, when adsorbed on PHEMA. The c o r r e s p o n d i n g critical v a l u e s f o r t h e s e b i o p o l y m e r s adsorbed on p o l y e t h y l e n e are 52, 38, and 42 dyne/cm. A cknowledgement Ms. F e e - L a i Leong and Ms. C h e r y l J . DeVine p r o v i d ed v a u a b l e t e c h n i c a l a s s i s t a n c e . T h i s work was s u p p o r t ed by PHS R e s e a r c h G r a n t s No. EY-00208 and No. EY-00327, b o t h from t h e N a t i o n a l Eye I n s t i t u t e , N a t i o n a l I n s t i tutes of Health.

ABSTRACT The surface tension of synthetic and biopolymer solutions and the contact angle on two hydrogels, poly (hydroxyethyl methacrylate) and poly(hydroxyethyl acrylate), and two hydrophobic solids, polyethylene and poly(methyl methacrylate) were measured. The adhesion tension vs. the solution surface tension plot yielded an approximately straight line for each of the gels and solids. From the slope of the lines it was concluded that the effect of polymer adsorption on the gel-water interfacial tension is small as compared to that at the hydrophobic solid - water interface. Adsorbed biopolymers increased the wettability of PHEMA gel and polyethylene. Critical surface tension values for the adsorbed biopolymers were obtained from

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

282

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

the adhesion tension vs. surface tension plot using the aqueous contact angle data. They were found to be in dependent of the substrate, unaffected by rinsing (for PHEMA) and was similar in magnitude to the surface ten sion of the concentrated biopolymer solutions for albu min and mucin on PHEMA and polyethylene. Literature Cited: 1. Holly, F.J. and Refojo, M.F., J. Biomed. Mater. Res. (1975) 9:315 2. Holly, F.J. and Refojo, M.F., These Proceedings. 3. Andrade, J., Lee, J.B., Jhon, M.S., Kim, S.W., and Hibbs, J.R.,Jr., Trans. Amer. Soc. Int. Organs, (1973) 19:1 4. Wolfram, Ε., Plast 5. Bargeman, J., J. Coll. Interface Sci. (1973) 42:467 6. Lucassen-Reynders, E.H., J. Phys. Chem. (1963) 67: 969 7. Wolfram, E., Kolloid Z. u. Z. f. Polymere, (1966) 211:84 8. Smolders, C .A., Rec. trav. chim. (1961) 80:699 9. Holly, F.J., ARVO Abstracts, p. 109, Spring, 1975 10. Harkins, W.D.,"Rec. Adv. in Surf. Chem. and Chem. Phys.", p. 19 (ed. Moulton, F.R.), The Science Press, Lancaster, PA, 1939 11. Holly, F.J. and Refojo, M.F., manuscript in prep aration. 12. Holly, F.J. and Lemp, M.A., Exp. Eye Res. (1973) 11:239 13. Holly, F.J., unpublished results. 14. Fox, H.W. and Zisman, W.A., J. Coll. Sci. (1952) 7: 428 15. Good, R.J. and Girifalco, L.A., J. Phys. Chem. (1960)64:561

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21 Radiation-Induced Co-Graft Polymerization of 2-Hydroxyethyl Methacrylate and Ethyl Methacrylate onto Silicone Rubber Films TAKASHI SASAKI,* BUDDY D. RATNER, and ALLAN S. HOFFMAN Department of Chemical Engineering and Center for Bioengineering, University of Washington, Seattle, Wash. 98195

A number of hydroge compatibility and low thrombogenicity in in vivo and in vitro tests (1). The poor mechanical properties of hydrogels have encouraged the development of techniques for reinforcing hydro gels to make them suitable for use in biomedical devices. By radiation grafting monomers such as 2-hydroxyethyl methacrylate (HEMA), N-vinyl-2-pyrrolidone, and acrylamide onto strong, inert polymeric supports, materials have been produced which combine the desirable biocompatibility properties of the hydrogel systems with the good mechanical properties of the substrate polymer (2-8). A clear correlation between the surface hydrophilicity of radiation grafted hydrogels and their biocompatibility has not yet been firmly established. Many useful biomaterials demon strate a balance between hydrophilic and hydrophobic sites which might be important for biocompatibility. Some of these materials are summarized in Table I. In order to systematically investigate the interrelationship between the hydrophilic-hydrophobic composition of a polymeric material and biological interactions with that material, a series of well characterized radiation graft copolymers of poly(HEMA) and poly(ethylmethacrylate) (EMA) have been prepared. A prelim inary report on the preparation and characterization of these graft copolymers is presented here. Experimental Silicone rubber films, 1.9 cm. χ 3.8 cm. χ 10 mils thick (Silastic, type 500-3, Dow Corning Corp.) were cleaned with five minute sonications in 0.1% Ivory soap solution and then in distilled water (3 times). After washing they were equilibrated at *0n a leave of absence from the Takasaki Radiation Chemistry Research Establishment, J.A.E.R.I., Takasaki, Japan. 283

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Table I Thromboresistant Surfaces Which May Demonstrate a Balance Between H y d r o p h i l i c and Hydrophobic S i t e s .

Hydrophobic S i t e

Material blood v e s s e l w a l l

lipid

Hydrophilic Site

Reference

protein, polysaccharide

(9)

polyether blocks

(10)

copolyetherpolyurethanes

polyurethane blocks

perfluorobutyryl ethylcellulose

perfluorobutyry group

S t e l l i t e 21

tallow

steel

Poly(HEMA)

backbone methyl groups

h y d r o x y l groups

(?) (12)

carboxylic acid (8) v i n y l acetate poly(vinyl groups groups acetate-crotonic acid) 52% R.H. The i n i t i a l weight of the s i l i c o n e rubber (W ) was measured a t 52% R.H. H i g h l y p u r i f i e d HEMA was generously s u p p l i e d by Hydron L a b o r a t o r i e s , Inc. and was used as r e c e i v e d . EMA monomer was used a f t e r d i s t i l l i n g a t a reduced pressure. Reagent grade s o l v e n t s and chemicals were used as r e c e i v e d . The f i l m s were immersed i n monomer s o l u t i o n s and the system was deoxygenated by bubbling n i t r o g e n gas through the s o l u t i o n for 30 minutes. I r r a d i a t i o n was performed i n a ca. 20,000 C i Co-60 source. The i r r a d i a t i o n dose used was 0.25 Mrad unless otherwise s p e c i f i e d . A f t e r the i r r a d i a t i o n , the f i l m s were washed twice (30 min, and 2 hrs.) w i t h acetone^methanol 111 m i x t u r e , and then w i t h d i s t i l l e d water f o r 24 hours changing the water twice d u r i n g t h i s p e r i o d . The wet weight (W ) of the g r a f t e d f i l m s was measured by b l o t t i n g between two sheets of f i l t e r paper (Whatman #1) f o r t e n seconds under a constant p r e s s s u r e and then weighing. F i l m s were then d r i e d i n an evacuated d e s i c c a t o r c o n t a i n i n g magnesium p e r c h l o r a t e f o r more than 12 h r s . a t which p o i n t the dry weight (W^) was measured. The degree of g r a f t and the water content i n the g r a f t were defined as: S

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

SASAKI ET AL.

Radiation Induced Co-Graft Polymerization

285

wt. % g r a f t = [(W, - W )/W,] χ 100 α s α water content (%) = [(W - W,)/(W - W ) ] χ 100 w d w s Results A p r e l i m i n a r y experiment on the r a d i a t i o n induced bulk c o p o l y m e r i z a t i o n of HEMA and EMA showed that the p o l y m e r i z a t i o n r a t e decreases a b r u p t l y as the f r a c t i o n of EMA i n a HEMA-EMA monomer mixture i n c r e a s e s . S i m i l a r behavior was noted i n a cog r a f t p o l y m e r i z a t i o n of HEMA and EMA onto s i l i c o n e f i l m s i n acetone o r acetone-methanol (1:1 m i x t u r e ) , as i s shown i n F i g . 1. When the HEMA p o r t i o n i 90 v o l . %, p r e c i p i t a t i o

EMA

(vol 7·) IN MONOMER

MIXTURE

Figure 1. Co-graft polymerization of HEMA-EMA at various monomer compositions. Total monomer, 20 vol %.

I n surveying a p p r o p r i a t e s o l v e n t systems f o r the c o - g r a f t p o l y m e r i z a t i o n an a c c e l e r a t i v e e f f e c t by water on the g r a f t p o l y m e r i z a t i o n , e s p e c i a l l y i n a l c o h o l i c systems, was noted. F i g . 2 shows the e f f e c t of water on the g r a f t i n g r a t e of 1:1 (by volume) mixture of HEMA and EMA i n methanol (MeOH), ethanol (EtOH) and acetone. I t can be seen from the f i g u r e that the a d d i t i o n of water t o any o f these s o l v e n t s i n c r e a s e s the g r a f t i n g r a t e . Pure EtOH appears t o be somewhat s u p e r i o r t o pure MeOH f o r o b t a i n i n g higher l e v e l s of g r a f t , but the a c c e l e r a t i v e e f f e c t o f

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water i s more marked i n the methanolic system. Co-graft p o l y m e r i z a t i o n w i t h v a r i o u s monomer compositions was c a r r i e d out i n a MeOH-H 0 o r an EtOH-I^O system. F i g . 3 and 4 show the r e l a t i o n s h i p between the degree of g r a f t and monomer r a t i o i n these systems. In F i g . 3, i t should be noted that the wt.% g r a f t i s almost constant i n the range of 25 t o 75 v o l . % of EMA i n the monomer mixture a t two monomer c o n c e n t r a t i o n s . This tendency i s q u i t e d i f f e r e n t from that shown i n F i g . 1. I n the s o l u t i o n s c o n s i s t ing of only EMA, some p r e c i p i t a t i o n of homopolymer occurred though the v i s c o s i t y of the s o l u t i o n s themselves remained r a t h e r low. F i g . 4 shows another aspect of the c o p o l y m e r i z a t i o n behavior of t h i s system. The c o g r a f t i n g i n pure EtOH has a somewhat s i m i l a r tendency to tha the wt. % g r a f t decrease the monomer. The a d d i t i o n of water t o the monomer s o l u t i o n , however, changes the s i t u a t i o n e n t i r e l y . Thus, w i t h 7.5 v o l . % of water i n the s o l u t i o n , the wt. % g r a f t i s n e a r l y constant i n the range of 25 t o 100 v o l . % EMA i n the monomer mixture. A d d i t i o n of more water leads t o p r a c t i c a l l y the reverse trend from that seen i n pure EtOH. The e f f e c t of water on the g r a f t i n g r a t e o f EMA o r HEMA i n MeOH-H 0 and EtOH-H 0 i s shown i n F i g u r e 5 which i s a c r o s s p l o t of some of the data i n F i g u r e 4. The degree of g r a f t of pure EMA markedly i n c r e a s e s w i t h an i n c r e a s e of water i n the s o l u t i o n . P r e c i p i t a t i o n of pure EMA homopolymer occurred when the f r a c t i o n of water was more than 5% i n the MeOH-^O system o r 15% i n the EtOH-H 0 system. I n the e t h a n o l i c system, the a d d i t i o n of water to the HEMA s o l u t i o n shows a r e t a r d i n g e f f e c t . However, very l i t t l e e f f e c t on HEMA g r a f t l e v e l i s noted upon the a d d i t i o n o f water to the methanolic system i n the c o n c e n t r a t i o n range studied. F i g . 6 shows the g r a f t water content f o r those samples which were prepared w i t h v a r i o u s HEMA-EMA mixtures i n an EtOH-H 0 s o l vent system (7.5% of water i n the s o l u t i o n ) . The wt. % g r a f t f o r these samples are almost constant (~22%). I t can be seen from t h i s f i g u r e that the water content decreases w i t h i n c r e a s i n g EMA p o r t i o n s i n the monomer m i x t u r e . Upon dehydration and then r e h y d r a t i o n of g r a f t s contains l a r g e r p o r t i o n s of EMA, water contents are found t o drop even f u r t h e r . A f t e r t h i s treatment these f i l m s gave constant water c o n t e n t s , ca. 2-3% f o r 1/3 HEMAEMA g r a f t e d f i l m s and > 1% f o r pure EMA g r a f t e d f i l m s , r e g a r d l e s s of the g r a f t l e v e l . The water content of the g r a f t i s u s u a l l y independent of g r a f t l e v e l a t a g i v e n monomer r a t i o i f more than 50% HEMA i s present i n the monomer m i x t u r e . However, t h i s i s no longer observed f o r l a r g e r EMA p o r t i o n s . I n F i g . 7 the water contents of a number of HEMA-EMA g r a f t e d polymers a r e p l o t t e d a g a i n s t the wt. % g r a f t . I n cases where more than 50% o f the monomer 2

2

2

2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

SASAKI

E T AL.

Radiation

Induced

Co-Graft

Polymerization

WATER (vol.%)

Figure 2. Effect of water on the grafting of a 1:1 mixture of HEMA and EMA in various solvents. Dose, 0.25 mrad; total monomer, 25 vol %.

Ο

25 vol.% MONOMER

•

20 vol.% MONOMER

25

50

EMA IN

75

100

(vol.%)

MONOMER

MIXTURE

Figure 3. Co-graft polymerization of HEMA-EMA in MeOH-H Q (5 vol % H Q) 2

2

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287

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

SASAKI ET AL.

Radiation

Induced

25

Co-Graft

50

Polymerization

75

100

EMA (vol.%) IN MONOMER MIXTURE

Figure 6. Effect of monomer composition on the graft water content. Samples were prepared in EtOH—H Ù. Monomer, 25 vol %. H 0 = 7.5%; dose, 0.25 Mrad or 0.16 Mrad (HEMA). 2

2

SOLVENT MeOH - H 0

HEMA/EMA Vol. RATIO 4/0

3/1 2/2

1/3 0 / 4

φ

V

•

Δ

·

EtOH-H 0

Ο

V

•

Δ

Ο

Acetone-H 0

Φ

2

2

2

Π

Φ

—ι

1

1

1

ΙΟ

20

30

40

γ50

Wt. % GRAFT

Figure 7. Plot of water content vs. wt % graft. Total monomer, 25 vol %.

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290

mixture i s EMA the water content increases w i t h i n c r e a s i n g g r a f t level. Figure 8 shows the r e s u l t s of a 2 hour vena cava r i n g t e s t f o r a s e r i e s of HEMA-EMA c o - g r a f t e d r i n g s . The g r a f t l e v e l s of these samples are d i f f e r e n t and the water contents are l a r g e r than those f o r the f i l m s i n v e s t i g a t e d above, probably due t o d i f f e r e n c e s i n the f o r m u l a t i o n of s i l i c o n e rubbers. The r e s u l t s , however, c l e a r l y show that HEMA-EMA co-grafted r i n g s as w e l l as pure HEMA g r a f t e d r i n g s have much lower thrombogenicity than pure EMA g r a f t e d o r ungrafted s i l i c o n e rubber r i n g s . Discussion The pronounced e f f e c t of s m a l l amounts o f water on the g r a f t i n g of HEMA-EMA mixture a t t r i b u t e d to the Trommsdorf the non-solvent water to an a l c o h o l i c s o l u t i o n of EMA w i l l cause the growing chains to adopt a l e s s expanded conformation which hinders the chain t e r m i n a t i o n step to produce h i g h e r g r a f t i n g r a t e s . The s i t u a t i o n i s the r e v e r s e f o r pure HEMA systems as EtOH o r MeOH mixtures w i t h water are good s o l v e n t s f o r HEMA p o l y mer. T h i s e x p l a n a t i o n could account f o r the c o p o l y m e r i z a t i o n behavior shown i n F i g . 3 and 4. Changes i n the p a r t i t i o n i n g of the monomer between the s i l i c o n e rubber and the g r a f t i n g s o l u t i o n may a l s o i n f l u e n c e the g r a f t i n g behavior demonstrated by t h i s system upon the a d d i t i o n of water. The v a r i a t i o n i n water content w i t h degree of g r a f t f o r a given monomer r a t i o only a t f r a c t i o n s of EMA g r e a t e r than 50% i n the monomer mixture may be due t o a l t e r a t i o n s i n the p o r o s i t y of these g r a f t e d f i l m s . The water i n the solvent system may cause p r e c i p i t a t i o n o f EMA polymer as i t i s formed. This procès occurs simultaneously w i t h the s u r f a c e g r a f t i n g . Such p r e c i p i t a t e d polymers o f t e n have an open c e l l u l a r , porous s t r u c t u r e . Thus, the water content i n the g e l should be r e l a t e d t o the p o r o s i t y o f the g r a f t as w e l l as to the HEMA-EMA r a t i o and should t h e r e f o r e be h i g h l y dependent on the s o l v e n t c o n d i t i o n s a t the time of g r a f t i n g . Upon d r y i n g , such a porous s t r u c t u r e may c o l l a p s e and, due to hydrophobic i n t e r a c t i o n s between the poly(EMA) chains, be r e s i s t a n t t o r e h y d r a t i o n . T h i s s i t u a t i o n would account f o r the behavior seen a t h i g h EMA r a t i o s i n Figure 6 i n which the water contents of g r a f t e d f i l m s which have been d r i e d and then r e hydrated are found t o be lower than they were before d r y i n g . The i n v i v o e v a l u a t i o n of the thrombogenicity of s i l i c o n e rubber r i n g s by the vena cava r i n g t e s t ( f i g u r e 8) i s c o n s i s t e n t w i t h the idea that a balance o f hydrophobic and h y d r o p h i l i c s i t e s on s u r f a c e might be important f o r the blood c o m p a t i b i l i t y of m a t e r i a l s . Two other c o n c l u s i o n s can a l s o be drawn from t h i s experiment. F i r s t , low apparent thrombogenicity (as evaluated by the vena cava r i n g t e s t ) can be achieved u s i n g hydrogel systems which have low water contents ( i . e . dow to ~ 12% H 0 ) . 9

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

SASAKI ET AL.

Radiation

Induced

Co-Graft

291

Polymerization

EMA/HEMA COPOLYMER GRAFTS

MONOMER RATIO

GRAFT LEVEL (mg/cm2)

WATER IN GRAFT %

TWO HOUR IVC RING TESTS (Dr. J.D. Whiff en. U. Wise.)

(SILASTIC)

Ο

negl

EMA

2.2

-10

5.1

-12

0.8

^25

Ο •

Ο •

Ο •

Ο

0.8

- 4 6

ΟΠ3

Ο Π

Ο •

O

I5EMA/ 5EMA/

5 H E M A

| 5 H E M A

HEMA

d

O

I

D

I

I

I

I

ODODOÛQU

Figure 8. Results of 2-hr vena cava ring tests for silastic and various grafted copolymers on silastic

• Q

HEMA/EMA

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

292

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Second, the vena cava r i n g t e s t does not have the s e n s i t i v i t y to d i s t i n g u i s h among most of the g r a f t e d polymers used i n t h i s experiment. As the s e r i e s of g r a f t e d polymers d i f f e r d r a m a t i c a l l y i n water contents and s u r f a c e p r o p e r t i e s , some d i f f e r e n c e i n performanace between g r a f t e d HEMA and HEMA-EMA copolymers would be expected. The a d d i t i o n of 25% HEMA to the monomer mixture was found s u f f i c i e n t t o produce a s u r f a c e which, although possessing a low water content, i s s i g n i f i c a n t l y l e s s thrombogenic than the hydrophobic S i l a s t i c o r P o l y ( E M A ) - g - S i l a s t i c s u r f a c e s . The r e l a t i v e l y l i n e a r decrease i n water content w i t h i n c r e a s i n g EMA i n the monomer mixture (Figure 6) i s taken t o i n d i c a t e that both HEMA and EMA enter i n t o the g r a f t copolymer a t s i m i l a r r a t e s and that the g r a f t copolymer composition i s not d i f f e r e n t by a l a r g e degree from the monomer s o l u t i o n composition However t h i s experiment does r a i s e a i n t e r p r e t a t i o n of the r e s u l t s EMA are d i s t r i b u t e d between the s i l i c o n e rubber surface and the b u l k o f the s i l i c o n e rubber i s not p r e s e n t l y known. The p r e c i s e f r a c t i o n s o f HEMA and EMA i n the copolymer, and perhaps more important, the d i s t r i b u t i o n of HEMA and EMA groups a t the s u r f a c e of the g r a f t copolymer i n t e r f a c i n g w i t h the b l o o d , i s not known. F i n a l l y , the importance of the l e v e l of g r a f t on the apparent s u r f a c e thrombogenicity of the g r a f t polymer cannot be determined from the p r e l i m i n a r y vena cava r i n g i m p l a n t a t i o n experiment. These problems are c u r r e n t l y under study i n an e f f o r t to c l a r i f y the i n t e r p r e t a t i o n of these r e s u l t s and to determine the importance of a h y d r o p h i l i c - h y d r o p h o b i c balance on the thrombogenicity of m a t e r i a l s . I t should be emphasized that the vena cava r i n g t e s t does not d i s t i n g u i s h between non-thrombogenic m a t e r i a l s and those which are only non-thromboadherent. Thus, the t r u e thrombog e n i c i t y of the m a t e r i a l s remains t o be evaluated. Tests a r e underway which should a l l o w a more r e a l i s t i c and q u a n t i f i a b l e assessment o f the thrombogenicity of HEMA-EMA g r a f t e d m a t e r i a l s . An e v a l u a t i o n o f the b i o l o g i c a l response t o these g r a f t e d copolymers implanted i n s o f t t i s s u e i s a l s o underway. Conclusions A survey has been made o f v a r i o u s s o l v e n t systems f o r use i n the p r e p a r a t i o n of HEMA-EMA co-grafted s i l i c o n e rubber f i l m s by the r a d i a t i o n g r a f t i n g technique. The a d d i t i o n of water t o the s o l v e n t s i n v e s t i g a t e d has a marked a c c e l e r a t i v e e f f e c t on the g r a f t i n g r a t e of EMA. By the a d d i t i o n of a p p r o p r i a t e amounts of water, i t i s p o s s i b l e t o prepare co-grafted f i l m s w i t h the same l e v e l s of g r a f t throughout a wide range of HEMA/EMA r a t i o s . The e f f e c t of water on the g r a f t i n g process f o r t h i s system has been discussed and i t i s suggested that the changes i n g r a f t l e v e l s noted might be due t o the Trommsdorff e f e c t o r d i f f e r e n c e s i n the

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

21.

SASAKI ET AL.

Radiation

Induced

Co-Graft

Polymerization

293

i n the p a r t i t i o n i n g of the monomers between the s i l i c o n e rubber and the monomer s o l u t i o n . The water content of g r a f t e d f i l m s a t a given monomer composition i s independent of g r a f t l e v e l a t higher HEMA r a t i o s , but increases w i t h i n c r e a s e s i n the g r a f t l e v e l a t higher EMA r a t i o s . This was concluded t o be due t o p o r o s i t y a f f e c t s i n the graft. HEMA-EMA g r a f t e d copolymers on s i l i c o n e rubber and pure HEMA g r a f t s on S i l i c o n e rubber show low apparent thrombogenicity by the vena cava r i n g t e s t . Pure g r a f t e d EMA m a t e r i a l s were found t o be thrombogenic by t h i s t e s t . An a n a l y s i s of the exact copolymer composition of these f i l m s i s planned f o r the near f u t u r e . A l s o a wide range of a d d i t i o n a l s'tudies on the i n t e r a c t i o n of these m a t e r i a l s w i t h b i o l o g i c a l systems are Acknowledgement One of the authors (T.S.) would l i k e t o g r a t e f u l l y acknowledge the support of the Japanese Atomic Energy I n s t i t u t e , Takasaki, Japan. We would a l s o l i k e t o thank the U.S. Atomic Energy Commission, D i v i s i o n of B i o m e d i c a l and Environmental Research (Contract AT (45-1)-2225) f o r t h e i r generous support f o r these s t u d i e s , and Dr. James D. Whiffen of the U n i v e r s i t y of Wisconsin f o r the vena cava r i n g t e s t s .

Literature Cited 1. Bruck, S. D., J. Biomed. Mater. Res., (1973),7,387. 2. Hoffman, A. S. and Kraft, W. G., ACS Polymer Preprints, (1972), 13(2), 723. 3. Hoffman, A. S. and Harris, C., ACS Polymer Preprints, (1972), 13(2), 740. 4. Lee, H. B., Shim, H. S. and Andrade, J. D., ACS Polymer Preprints, (1972), 13(2), 729. 5. Ratner, B. D. and Hoffman, A. S., Preprints - ACS Division of Organic Coatings and Plastics Chemistry, (1973), 33(2), 386. 6. Kearney, J. J., Amara, I. and McDevitt, M. B., Preprints ACS Division of Organic Coatings and Plastics Chemistry, (1973), 33(2), 346. 7. Ratner, B. D. and Hoffman, A. S., J. Appl. Polymer Sci., (1974), 18, 3183. 8. Kwiatkowski, G. T., Byck, J. S., Camp, R. L., Creasy, W. S. and Stewart, D. D., "Blood Compatible Polyelectrolytes for Use in Medical Devices", Contract No. N01-HL3-2950T, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, July 1, 1972 June 30, 1973, PB 225-636.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

9. Baier, R. E., Dutton, R. C. and Gott, V. L., "Surface Chemical Features of Blood Vessel Walls and Synthetic Materials Exhibiting Thromboresistance" in "Surface Chemistry of Bio logical Systems", M. Black, ed., pp. 235-260, Plenum Press, N.Y., 1970. 10. Brash, J. L., Fritzinger, Β. K. and Bruck, S. D., J. Biomed. Mater. Res., (1973), 7, 313. 11. Peterson, R. J. and Rozelle, L. T., "Ultrathin Membranes for Blood Oxygenators", Contract No. N01-HB-1-2364, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, December 1974, PB 231 324/5WM. 12. Ratner, B. D. and Miller, I. F., J. Polymer Sci., (1972), A-1, 10, 2425.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22 The

Thermodynamics of Water Sorption in Radiation-

Grafted Hydrogels BERNARD KHAW,* BUDDY D. RATNER, and ALLAN S. HOFFMAN** Department of Chemical Engineering and Center for Bioengineering, University of Washington, Seattle, Wash. 98195 Hydrogels are lightly crosslinked, water swollen polymer networks and they have been suggested for or applied to a number of biomedical application (e.g. 1-3) Sinc bulk hydrogel dis play relatively poor mechanica it is of interest to coa stronge suppor Radiation graft copolymerization is a useful technique for preparing a covalently bonded coating of one polymer on or within the surface region of another polymer and i t has been used to graft hydrogels to stronger polymer supports (3,4,5). It has been hypothesized that both the content and "structural character" of the water which is sorbed in hydrogels can be important determinants of the biological response at the interface with a hydrogel (6,7,8). It has become important, therefore, to study the manner in which water molecules are taken up by these hydrogel-grafted films, in order to find a correlation between the structure and character of the water in these gels and their biocompatibility. From this knowledge i t may be possible to design improved biomaterials by adjusting the water sorption behavior to some predetermined ideal. One way of studying the mode in which water molecules are taken up by hydrogels is by means of the thermodynamic parameters for the sorption of water vapor into the hydrogels. This paper describes the thermodynamics of water sorption in three different hydrogels, which were radiation grafted to silicone rubber fi1ms. Materials and Methods Three monomers were used in these studies: Hydroxyethyl methacrylate (HEMA), generously supplied by Hydro-Med Sciences of New Brunswick, N.J. and used as received; N-vinyl pyrrolidone (N-VP) purchased from Matheson, Coleman and Bell and purified by double distillation; and ethylene glycol dimethacry1 ate (EGDMA) purchased from Monomer-Polymer Laboratories, Philadelphia, PA. and used as received. Films of Medical Grade Silastic were * Present Address: ITT Rayonier, Shelton, Wash. ** To whom reprint requests should be directed. 295

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

296

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

p u r c h a s e d f r o m Dow-Corning C o r p , T h e i r ( n o m i n a l ) t h i c k n e s s was lOmil. The method used f o r r a d i a t i o n g r a f t i n g has been d e s c r i b e d i n p r e v i o u s p u b l i c a t i o n s (3,*0 . The r a d i a t i o n dose used h e r e was 0.25 Mrad. A q u a r t z s p r i n g v a p o r s o r p t i o n a p p a r a t u s was used t o o b t a i n s o r p t i o n isotherms at d i f f e r e n t temperatures f o r the three different films. From t h e s e d a t a , t h e s t a n d a r d d i f f e r e n t i a l h e a t s , (ΔΗ°) e n t r o p i e s , (AS°) and f r e e e n e r g i e s (AG ) o f s o r p t i o n were c a l c u l a t e d b a s e d on t h e method o f Othmer, e t , a l , (9). 0

R e s u l t s and

Discussion

The t h r e e f i l m s c h o s e n f o r s t u d y a r e d e s c r i b e d i n T a b l e I. They w e r e c h o s e n b e c a u s f t h e i wid f wate content and a l s o b e c a u s e t h e y w e r measured w a t e r s o r p t i o T a b l e I. Hydrogel Grafted

Monomer S o l u t i o n Composition ( v o l . % ) HEMA

NVP

EGDMA

20 10 0

0 10 20

1 1 1

HEMA HEMA/NVP NVP

R e m a i n i n g 73%

a)

HYPROGEL/S1LASTIC FILMS STUDIED a

( v o l . ) i s a 1/1

Extent of G r a f t i n g (mg/cm ) 2

%Η 0 i n Hydroge1 2

4.40 3.18 2.64

b

26 50 61

(vol.) mixture of Methanol/H 0. 2

b)

As measured by b l o t t i n g and w e i g h i n g t h e g r a f t e d f i l m a f t e r e q u i l i b r a t i o n in H 0 ( s e e Ref. 4 ) . T y p i c a l i s o t h e r m s f o r S i l a s t i c and two o f t h e g r a f t e d f i l m s a r e shown i n F i g u r e 1 and 2. I t can be seen t h a t w a t e r s o r p t i o n in the S i l a s t i c i s n e g l i g i b l e i n comparison to t h a t i n the g r a f t e d hydrogels. I t i s a l s o c l e a r t h a t water s o r p t i o n i s always exo t h e r m i c , as e x p e c t e d . The d e r i v e d d a t a f o r s t a n d a r d d i f f e r e n t i a l h e a t s , e n t r o p i e s and f r e e e n e r g i e s o f w a t e r s o r p t i o n as a f u n c t i o n o f e x t e n t o f s o r p t i o n a r e shown i n F i g u r e s 3~5 f o r t h e t h r e e g r a f t e d f i l m s . I t s h o u l d be n o t e d t h a t a l l t h e v a l u e s f o r ΔΗ°, A S , and AG o b t a i n e d f o r t h e t h r e e h y d r o g e l f i l m s under s t u d y a r e more neg~ a t i v e than those f o r the condensation o f water vapor t o l i q u i d water; the values f o r the condensation of water vapor to l i q u i d w a t e r a t 26°C a r e : -AG° = 1.97 k c a l / m o l e , «AS° = 2 8 , 2 9 e.u, ( c a l deg" m o l e " ) and -ΔΗ° = 10.48 kcal/mole. This indicates that e n t h a l p y , e n t r o p y and f r e e e n e r g y changes more n e g a t i v e t h a n t h o s e f o r c o n d e n s a t i o n a r e a t work and t h a t , as one w o u l d e x p e c t , t h e s o r p t i o n p r o c e s s i s not m e r e l y w a t e r v a p o r c o n d e n s i n g on t h e f i l m s ; t h a t i s , h y d r a t i o n o r p o l a r i n t e r a c t i o n s between t h e h y d r o s p h i l i e g r o u p s i n t h e h y d r o g e l s and t h e w a t e r m o l e c u l e s a r e no doubt o c c u r r i n g . The same can be s a i d f o r t h e c u r v e s o b t a i n e d a t 2

0

1

1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

0

KHAW

ET

AL.

Water

Sorption in Radiation-Grafted

P°

Hydrogels

(mmHg)

Figure 1. Sorption isotherms for water vapor in ungrafted Silastic films

mgH20

SORBEΡ

Figure 2. Sorption isotherms for water vapor in two radia tion-grafted hydrogel/Silastic films

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS 210

,1.0

-I Ο

,

,

,

,

,

,-

2

mg g. DRY HYDROGEL 2

Figure 3. Standard differential heat of sorption of water vapor as a function of the extent of water sorption in three radiation-grafted hydro gel/Silastic films at 26° C

0

20

40

60

80

100

120

mg. H 0 SORBED g. DRY HYDROGEL 2

Figure 4. Standard differential entropy of sorption of water vapor as a function of the extent of water sorption in three radia tion-grafted hydrogel/Silastic films at26°C

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22.

KHAW

Water

ET AL.

Sorption

in Radiation-Grafted

Hydrogels

299

h i g h e r t e m p e r a t u r e s o f 31°, 37°, ^ 3 ° , 50° and 56°C; t h e c u r v e s d e r i v e d a t t h e s e t e m p e r a t u r e s showed e s s e n t i a l l y t h e same behav i o r a s a t 26°C. R e f o j o and Yasuda (1Q) a n d l a t e r Warren and P r i n s (JLL) showed t h a t when b u l k HEMA h y d r o g e l s were immersed i n w a t e r , t h e y d e s w e l l e d as t e m p e r a t u r e was r a i s e d up t o a b o u t 55 t o 60°C. These o b s e r v a t i o n s a l s o s u p p o r t an e x o t h e r m i c p r o c e s s o f w a t e r s o r p t i o n below 55 60°C. When t h e i n i t i a l w a t e r m o l e c u l e s a r e s o r b e d i n t h e g r a f t e d f i l m s they partake i n h i g h l y exothermic i n t e r a c t i o n s i n a l l t h r e e hydrogel f i l m s . P r o b a b l y t h e s e i n i t i a l i n t e r a c t i o n s i n HEMA o r HEMA/NVP f i l m s a r e l o c a l i z e d a t t h e most a c c e s s i b l e -OH g r o u p s ; the s t r o n g i n t e r a c t i o n and l o c a l i z a t i o n o f a w a t e r m o l e c u l e a t such a s i t e n o t o n l y e v o l v e s heat (high -ΔΗ ) but i t a l s o r e duces t h e e n t r o p y o f t h e s y s t e m s i g n i f i c a n t l y ( h i g h - A S ° ) a s the w a t e r m o l e c u l e l o s e the h i g h -ΔΗ° and - A S h y d r o p h o b i c bond f o r m a t i o n upon s o r p t i o n o f w a t e r m o l e c u l e s , which could " p l a s t i c i z e " l o c a l c h a i n motions. That i s , t h e i n t e r a c t i o n o f H 0 m o l e c u l e s w i t h a v a i l a b l e -OH s i t e s c o u l d p e r m i t a c e r t a i n amount o f i n c r e a s e d h y d r o p h o b i c g r o u p i n t e r a c t i o n s , w h i c h w o u l d o c c u r w i t h -ΔΗ° and - A S ° c h a n g e s . Then, i f any w a t e r m o l e c u l e s were i n v o l v e d , t h e s e c o u l d become more s t r u c t u r e d a r o u n d s u c h h y d r o p h o b i c d o m a i n s , c o n t r i b u t i n g t o -ΔΗ° a n d -Δ$° changes. I t i s most l i k e l y t h a t t h e p o l a r i n t e r a c t i o n s d o m i n a t e t h e i n i t i a l s o r p t i o n p r o c e s s i n t h e HEMA-contaiηing f i l m s and c a l c u l a t e d c l u s t e r i n g f u n c t i o n s r e f l e c t t h i s ( s e e below). In t h e NVP f i l m t h e r e i s p r o b a b l y a somewhat w e a k e r i n t e r a c t i o n and l o c a l i z a t i o n o f t h e H 0 m o l e c u l e a r o u n d t h e p y r r o l i d o n e c a r b o n y l o r n i t r o g e n g r o u p s , and t h e changes h e r e w i t h i n c r e a s i n g s o r p t i o n a r e l e s s m a r k e d , o v e r t h e range o f water contents s t u d i e d . As more w a t e r i s s o r b e d , t h e e v o l u t i o n o f h e a t and d e c r e a s e i n e n t r o p y b o t h d e c r e a s e i n m a g n i t u d e u n t i l most o f t h e r e a d i l y a c c e s s i b l e s o r p t i o n s i t e s a r e s a t u r a t e d , and t h e c u r v e s b e g i n t o l e v e l o f f . E v e n t u a l l y t h e w a t e r w i l l b e g i n t o p e n e t r a t e and s w e l l t h e b u l k o f t h e HEMA h y d r o g e l s , o p e n i n g up some new -OH s o r p t i o n s i t e s t o t h e H 0 m o l e c u l e s ; t h i s l e a d s t o an i n c r e a s e i n t h e ( d i f f e r e n t i a l ) exothermîcity and d e c r e a s e i n ( d i f f e r e n t i a l ) entropy o f sorption. T h i s c a n e x p l a i n t h e minima n o t e d i n b o t h t h e -ΔΗ° and - A S ° c u r v e s , f o r t h e HEMA c o n t a i n i n g h y d r o g e l s ; t h e s e minima o c c u r a t a p p r o x i m a t e l y 1 H 0 / 3 HEMA-OH f o r the HEMA s y s t e m and 1 H 0/1 HEMA-OH f o r t h e HEMA/NVP c o p o l y m e r system. A model has been p r o p o s e d f o r t h e s t r u c t u r e o f p o l y (HEMA) g e l s i n w h i c h many o f t h e OH-groups a r e t i e d up i n s t r o n g i n t r a m o l e c u l a r Η-bonded c r o s s l i n k s w h i c h f o r m o r g a n i z e d r e g i o n s i n t o w h i c h w a t e r c a n n o t e a s i l y p e n e t r a t e . (12) The r a t i o o f 1 H 0 m o l e c u l e p e r 3 HEMA-OH g r o u p s may i n d i c a t e t h a t t w o - t h i r d s o r more o f t h e h y d r o x y l g r o u p s i n poly(HEMA) a r e i n v o l v e d i n t h e s e r e g i o n s i n t h e d e h y d r a t e d g e l . The u p t u r n i n t h e -ΔΗ° a n d _

0

2

2

2

2

2

2

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- A S ° c u r v e s ( F i g u r e s 3 and k) f o r HEMA c o u l d i n d i c a t e t h a t a l t h o u g h t h e g e l s a r e s w e l l i n g and r e a r r a n g i n g , a l a r g e number o f f r e e OH-groups a r e not b e i n g opened up f o r e x o t h e r m i c H^bond~ ing w i t h t h e w a t e r . In t h e HEMA/NVP c o p o l y m e r f i l m s t h i s HEMA o r g a n i z e d s t r u c t u r e may be p a r t i a l l y b r o k e n up by t h e NVP rings and t h e r e f o r e not as many h y d r o x y l g r o u p s a r e o c c u p i e d i n t h e Hbond s t r u c t u r e . The NVP s y s t e m does not show any minimum (and may i n f a c t e x h i b i t a s m a l l maximum) i n t h e -ΔΗ° and -AS° c u r v e s . This may be due t o t h e l a c k o f a p o r o u s s t r u c t u r e i n t h i s g r a f t e d h y d r o gel system (discussed below), coupled w i t h s o r p t i o n of r e l a t i v e l y l a r g e amounts o f H 0. F i n a l l y a l l c u r v e s s h o u l d l e v e l o f f a t some e q u i l i b r i u m v a l u e o f -ΔΗ° and -AS° r e p r e s e n t i n g t h e combined p r o c e s s o f c o n d e n s a t i o n , and m i x i n g ( o r d i l u t i o n S i l a s t i c e l a s t i c network c r e a s e d f r e e e n e r g y due t o s o r p t i o n v s . i n c r e a s e d f r e e e n e r g y due t o n e t w o r k s w e l l i n g . The g r a d u a l d e c r e a s e i n -AG° w i t h i n c r e a s ing s o r p t i o n ( F i g u r e 5) i s a r e f l e c t i o n o f t h e a p p r o a c h o f t h e o v e r a l l s o r p t i o n process to s a t u r a t i o n e q u i l i b r i u m . The r e s u l t s i n t h i s s t u d y f o r t h e t h r e e h y d r o g e l f i l m s a r e c o m p a r a b l e t o t h o s e o b t a i n e d by Masuzawa and S t e r l i n g (13) for t h e s o r p t i o n o f w a t e r v a p o r by a g a r , carboxymethy1 c e l l u l o s e , g e l a t i n and m a i z e s t a r c h and a l s o t o t h o s e r e p o r t e d by Bettelheim and E h r l i c h (1k) f o r t h e s o r p t i o n o f w a t e r v a p o r by m u c o p o l y s a c charides. In a d d i t i o n , t h e p r e s e n c e o f maxima and minima and t h e c l o s e p a r a l l e l s between t h e shapes o f t h e e n t r o p y and e n t h a l py c u r v e s s e e n h e r e were a l s o o b s e r v e d by t h e s e a u t h o r s . Both g r o u p s a l s o r e p o r t a g r a d u a l d e c r e a s e i n -AG° w i t h i n c r e a s e i n mg. H 0 s o r b e d / g s o r b a n t , as s e e n h e r e i n o u r d a t a , and e x p e c t e d as s a t u r a t i o n w a t e r s o r p t i o n i s a p p r o a c h e d . The t e n d e n c y f o r w a t e r m o l e c u l e s t o c l u s t e r ( o r not t o c l u s t e r ) t o g e t h e r w i t h i n t h e g r a f t e d h y d r o g e l m a t r i c e s may be e s t i mated by c a l c u l a t i n g t h e " c l u s t e r i n g f u n c t i o n " f o r w a t e r , a f t e r t h e t e c h n i q u e o f Zimm and L u n d b e r g (15)» These c a l c u l a t i o n s have been made, a s s u m i n g no change i n volume on m i x i n g , and r e s u l t s a r e shown i n F i g . 6. I t can be seen t h a t a l l v a l u e s o f t h e c l u s t e r i n g f u n c t i o n are s i g n i f i c a n t l y negative, suggesting that t h e w a t e r m o l e c u l e s a r e s t r o n g l y s i t e bound a t w i d e l y s e p a r a t e d s i t e s w i t h i n the hydrogel m a t r i x . This is e s p e c i a l l y true f o r the HEMA-containing polymers. Data o f Zimm and L u n d b e r g (15) for c o l l a g e n a r e r e p r o d u c e d i n F i g . 6 and show a s i m i l a r b e h a v i o r t o t h e h y d r o g e l s s t u d i e d h e r e . To q u o t e t h e i r d i s c u s s i o n o f t h e s e data: "The i n i t i a l w a t e r i s t i g h t l y bound" and as more w a t e r i s a b s o r b e d " t h e s o r p t i o n p r o c e s s changes . . . f r o m one o f s o r p t i o n on a few h i g h l y s p e c i f i c s i t e s t o a d i f f u s e s w e l l i n g phenomenon". 05) The d i f f u s i o n c o e f f i c i e n t f o r w a t e r i n t h e h y d r o g e l g r a f t e d f i l m s may be c a l c u l a t e d f r o m t h e i n i t i a l s l o p e o f t h e s o r p t i o n o r d e s o r p t i o n c u r v e when i t i s p l o t t e d as ( e . g . , see Réf. 16): 2

2

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Water

KHAW ET AL.

Sorption

in Radiation-Grafted

Hydrogels

-Δο< (kcal ^mole)

\ *

NVP

HEMA/NVP

HEMA

g. DRY HYDROGEL

Figure 5. Standard differential free energy of sorption of water vapor as a function of the extent of water sorption in three radia tion-grafted hydrogel/Silastic films at 26°C

CLUSTERING FUNCTION

G

„ „ , — -
s

s

(Ideal Solution )COLLAGEN

HEMA

-10

-20

-3a

Ο

0.1 02 0.3 VOLUME FRACTION OF WATER (0|) Journal of Physical Chemistry

Figure 6. Clustering functions for water as a function of water content in the three grafted hydrogel films and in collagen (15). Calculated by the technique of Zimm and Lundberg ( 15 ).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

302

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

e Where

M M

t

e

= =

t £

amount o f H 0 s o r b e d a t t i m e t . amount o f H 0 s o r b e d a t e q u i l i b r i u m time fi1m thickness 2

(t =

2

T a b l e II p r e s e n t s t h e s e r e s u l t s , and i t can be seen t h a t t h e d i f f u s i v i t y o f H 0 i n t h e HEMA f i l m i s s i g n i f i c a n t l y h i g h e r t h a n i n e i t h e r o f t h e o t h e r two f i l m s , even f o r t h e d e s o r p t i o n p r o c ess . T a b l e II. PIFFUSIVITIE 2

2

Hydrogel Composition HEMA HEMA/NVP NVP

1 0

DIFFUSIVITIES, cm /sec χ 1 0 Sorption Desorption Average 24.1 5Λ 9.2

18.9 7Λ 6.9

21.5 6.4 8.1

The i n c r e a s e d r a t e o f d i f f u s i o n o f w a t e r v a p o r i n t o and o u t o f t h e HEMA g r a f t e d f i l m s may i n d i c a t e a more p o r o u s and open s t r u c t u r e f o r the g r a f t regions o f t h i s m a t e r i a l . The homop o l y m e r s u r r o u n d i n g t h e HEMA f i l m a f t e r i r r a d i a t i o n was a s o l i d w h i t e g e l . Such w h i t e HEMA g e l s formed i n p r e c i p i t a t i n g s o l v e n t s y s t e m s a r e o f t e n macroporous i n s t r u c t u r e ( 1 0 , 1 7 ) . T h u s , t h e g r a f t on t h e f i l m may a l s o be m a c r o p o r o u s . The homopolymers s u r r o u n d i n g t h e HEMA—N-VP c o p o l y m e r f i l m s and t h e p u r e N-VP f i l m s were c l e a r v i s c o u s l i q u i d s . In a n o n - p r e c i p i t a t i n g p o l y m e r i z a t i o n medium one w o u l d n o t e x p e c t t h e f o r m a t i o n o f an open macroporous s t r u c t u r e . Thus, the h i g h d i f f u s i v i t y o f w a t e r i n t o t h e HEMA g r a f t e d f i l m may be a r e f l e c t i o n o f t h e c o n d i t i o n s under w h i c h i t was p r e p a r e d . Conclus ions The thermodynamics o f t h e s o r p t i o n o f w a t e r v a p o r by t h r e e h y d r o g e l - g r a f t e d f i l m s have been s t u d i e d . The f i l m s were p r e p a r e d by t h e r a d i a t ion-înîtiated g r a f t i n g o f S i l a s t i c backbones i n monomer s o l u t i o n s c o n t a i n i n g 2 - h y d r o x y e t h y 1 m e t h a c r y l a t e (HEMA), N-vinyl pyrrolidone (NVP) and an e q u i v o l u m e t r i c m i x t u r e o f t h e s e two monomers. A f u s e d - q u a r t z s p r i n g v a p o r s o r p t i o n a p p a r a t u s was used i n t h i s s t u d y . The s t a n d a r d d i f f e r e n t i a l h e a t o f w a t e r v a p o r s o r p t i o n by e a c h f i l m was c a l c u l a t e d a t d i f f e r e n t t e m p e r a t u r e s b a s e d upon t h e measured w a t e r v a p o r s o r p t i o n d a t a . The s t a n d a r d d i f f e r e n t i a l e n t r o p i e s and s t a n d a r d f r e e e n e r g i e s o f w a t e r v a p o r s o r p t i o n were t h e n c a l c u l a t e d . Clustering functions

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

22.

KHAW

E T AL.

Water

Sorption

in Radiation-Grafted

Hydrogels

303

for

w a t e r were a l s o c a l c u l a t e d f r o m t h e s o r p t i o n i s o t h e r m s . D i f f u s i o n c o e f f i c i e n t s were c a l c u l a t e d f r o m t h e s o r p t i o n and desorption k i n e t i c data. A l l o f these data i n d i c a t e that t h e g r a f t e d HEMA s y s t e m s a r e more p o r o u s and more p e n e t r a b l e t o w a t e r m o l e c u l e s and a l s o s i t e - b i n d w a t e r m o l e c u l e s s t r o n g l y when com p a r e d t o N-VP s y s t e m s , and s u p p o r t a model f o r t h e HEMA p o l y m e r i n w h i c h s t r o n g i n t r a m o l e c u l a r Η-bonds a r e i m p o r t a n t t o i t s g e l structure. The w a t e r s o r p t i o n t h e r m o d y n a m i c and k i n e t i c s i n h y d r o g e l systems can y i e l d v a l u a b l e i n s i g h t i n t o t h e s t a t e o r " c h a r a c t e r " o f t h e i m b i b e d w a t e r and i t s i n t e r a c t i o n s w i t h t h e h y d r o g e l , a s w e l l as help t o understand b e t t e r the m i c r o s c o p i c s t r u c t u r e o f the h y d r o g e l . When t h e s e t e c h n i q u e s a r e c o u p l e d w i t h o b s e r v a t i o n s o f b i o l o g i c a l i n t e r a c t i o n s o f t h e s e s y s t e m s , o n e may be b e t t e r a b l e t o d e s i g n ne applications. Acknowledgment The s u p p o r t o f t h e U,S,E.R. D. A, D i v i s i o n o f B i o m e d i c a l a n d E n v i r o n m e n t a l R e s e a r c h ( A r t i f i c i a l H e a r t Program) C o n t r a c t A T ( 4 5 - 1 ) 2 2 2 5 and t h e NIGMS B i o e n g i n e e r i n g C e n t e r G r a n t No. GM16436-07 a r e g r a t e f u l l y a c k n o w l e d g e d .

Abstract The thermodynamics of water sorption in three different radiation-grafted hydrogels have been studied. Diffusion coeffi cients for water in these films were also calculated from the sorption and desorption kinetic data. The thermodynamic and kinetic data indicate that the grafted hydroxyethyl methacrylate (HEMA) films are more porous and penetrable to water molecules and their OH groups interact more strongly with water molecules when compared to grafted N-vinyl pyrrolidone (NVP) films. Strong site binding of water molecules is also reflected by the relative ly large negative values calculated for the water clustering function in all films, although the values are less negative for the poly NVP films than for the poly HEMA films. These data all support a model for the HEMA polymer in which strong intra molecular Η-bonds are important to its gel structure. Such data may be relevant to the biological interactions of hydrogels in the living system environment. Literature Cited (1) (a) Wichterle, O. and Lim, D., Nature, (1960), 165, 117. (b) Levowitz, B.S., et al., Trans.Amer.Soc.Artif.Int.Organs, (1968), 14, 82. (2) Andrade, J.D., et al., Ibid., (1971), 17, 222.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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(3) Hoffman, A.S., et al., Ibid., (1972), 18, 10. (4) Ratner, B.D. and Hoffman, A.S., ACS Org.Coatings and Plastics Chemistry Preprints, (1973), 33(2), 286; J.Appl.Polymer Sci., 18, 3183. (5) Hoffman, A.S., et al., in "Permeability of Plastic Films and Coatings," ed. H.B. Hopfenberg, Plenum Press, N.Y., (1974), 441. (6) Drost-Hansen, W., Ind.Eng.Chem., (1969), 61, 10. (7) Jhon, M.S. and Andrade, J.D., J.Biomed.Mtls.Res., (1973), 7, 509. (8) Hoffman, A.S., J.Biomed.Mtls.Res., Biomed.Mtls. Symposium No. 5, Part 1, (1974), 8, 77. (9) (a) Othmer, D.F., Ind.Eng.Chem., (1940), 32 841. (b) Othmer, D.F and Sawyer, F.G., Ibid., (1943), 35, 1269 (c) Othmer, D.F (d) Jocefowitz, S (10) Refojo, M.F. and Yasuda, H., J.Appl.Polymer Sci.,(1965),9, 2425. (11) Warren, T.C. and Prins, W., Macromolecules, (1972), 5, 506. (12) Ratner, B.D. and Miller, I.F., J.Polym.Sci., Part A-1, (1972), 10, 2425. (13) Masuzawa, M. and Sterling C., J.Appl.Polymer Sci., (1968), 12, 2023. (14) Bettelheim, F. and Ehrlich, S.H., J.Phys.Chem., (1963), 67, 1948. (15) Zimm, B.H. and Lundberg, J.L., J.Phys.Chem., (1956), 60, 425. (16) Crank, J. and Park, A.S., "Diffusion in Polymers," 16, Academic Press, N.Y., 1968. (17) Yasuda, H., Gochin, M. and Stone, W., Jr., J.Polymer Sci., (1966), 4, 2913. ,

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23 Biological and Physical Characteristics of Some Polyelectrolytes E. O. LUNDELL, G. T. KWIATKOWSKI, J. S. BYCK, F. D. OSTERHOLTZ, W. S. CREASY, and D. D. STEWART Union Carbide Corp., Chemicals and Plastics, Research and Development, Bound Brook, N. J. 08805

It has been recognized for some time that the surface charge of bloo their interactions wit largely on the results of Sawyer and coworkers (2), i t has also become apparent that surface charge, and electrochemical phenomena in general, can significantly influence blood/material interactions and that their control may be a necessary condition for development of a truly blood compatible material. Research stemming from ths concept, involving blood compatibility studies with metals and polymers, has led to the conclusion that a limiting level of negative charge density or negative potential may be required to achieve nonthrombogenic character, provided that other necessary conditions are also satisfied. This has prompted extensive research directed at development of anionic polyelectrolytes which might be expected to be blood compatible. As represented in Figure 1, the blood compatibility and, in particular, thromboresistance of carboxyl-containing copolymers would be expected to depend upon the interaction of several physical and chemical parameters. These include not only the total concentration of ionizable groups, the degree to which these are neutralized by various monovalent or divalent metal salts, but also the hydrophilicity of the total polymer system. This last property depends largely on the selection of the neutral comonomer. As was expected at the outset of this program, and has been confirmed by experimental results, none of these parameters can be considered to be a sufficient condition for blood compatibility. A l l must be controlled, however, as necessary conditions for compatibility.

305

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

306

CONCENTRATION OF I O N I Ζ A B L E GROUPS (WT. % ACID)

C0 M 2

20

CH CH 2

CHCH

15 10 5 30

60

DEGREE OF NEUTRALIZATION

ETHYLENE/ACRYLIC ACID, ETHYLENE/VINYL •VINYL ACETATE/CROTONIC

SULFONATE

ACID

•VINYL Ρ Y R R O L I D O N E / A C R Y L I C

ACID

Figure 1. Schematic of the surface charge density and hydrophilicity of some polyelec trolytes

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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METHODS BIOLOGICAL COMPATIBILITY TESTING B i o l o g i c a l t e s t data d e s c r i b e d r e p r e s e n t the r e s u l t s o f e v a l u a t i o n programs conducted by o t h e r i n v e s t i g a t o r s under c o n t r a c t t o the N a t i o n a l H e a r t and Lung I n s t i t u t e o f the N a t i o n a l I n s t i t u t e s o f H e a l t h . Meth ods employed by t h e s e t e s t e r s a r e summarized i n t h i s section. In

V i t r o T e s t Methods P a r t i a l Thromboplastin

Time.

Polymer sample tubes and s t i r r i n g p a d d l e s χ 100 mm) were s o l u t i o n c o a t e d on t h e e n t i r e i n t e r i o r s u r f a c e e x c e p t f o r a 0.5"-1.0" r i m a t t h e o r i f i c e . T h i s was a c c o m p l i s h e d by i n t r o d u c i n g 3 ml o f polymer s o l u t i o n i n t o the tube, f i t t i n g the tube t o a r o t a r y e v a p o r a t o r , t i l t i n g i t so t h a t the e n t i r e i n n e r s u r f a c e would be c o a t e d (except f o r the r i m a t the o r i f i c e ) , and a p p l y i n g heat t o the e x t e r i o r o f the e v a c u a t e d tube w h i l e b e i n g r o t a t e d . S t i r r i n g p a d d l e s were p r e p a r e d by s o l u t i o n c o a t i n g g l a s s r e c t a n g l e s (3 mm χ 18 mm χ 120 mm) o r by c u t t i n g e q u i v a l e n t s i z e s t r i p s from compression molded polymer s h e e t . To c a r r y o u t the p a r t i a l t h r o m b o p l a s t i n time t e s t , 25 ml o f n a t i v e whole b l o o d was drawn i n t o a s i l i c o n e c o a t e d (G. Ε. D r i - F i l m SC-87) g l a s s s y r i n g e and imme d i a t e l y t r a n s f e r r e d t o t h e polymer c o a t e d t u b e . The p a d d l e was employed t o s t i r the b l o o d sample f o r f o u r minutes a t 60 rpm. A t t h e end o f t h i s t i m e , the sam p l e was a n t i c o a g u l a t e d by a d d i t i o n o f sodium c i t r a t e and the c e l l u l a r elements were removed by c e n t r i f u g a t i o n (425 g) a t 4°C. P a r t i a l t h r o m b o p l a s t i n time o f the r e s u l t a n t p l a t e l e t - p o o r plasma was determined ... a c c o r d i n g t o the methods o f L a n g d e l l ( 3 . ) a n d Rodman. Stypven

Time.

a

Samples o f p l a t e l e t - p o o r plasma p r e p a r e d f o r de t e r m i n a t i o n o f p a r t i a l t h r o m b o p l a s t i n time were a l s o used t o measure Stypven time a c c o r d i n g t o the methods of M i a l e ( i ) and Hardisty.(£)

a

*

Dr. R. G. Mason, U n i v e r s i t y o f N o r t h C a r o l i n a , School of Medicine, Chapel H i l l , North C a r o l i n a

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

308

In V i v o T e s t Methods b Canine

Implantation Studies.

T e s t samples were i n the form o f vena cava ("Gott") r i n g s . These a r e 9 mm l o n g and have an o.d. o f 8 mm. Both i n f l o w and o u t f l o w o r i f i c e s were streamlined. E t h y l e n e / a c r y l i c a c i d copolymer and ionomer r i n g s had a w a l l t h i c k n e s s o f 1mm (6 mm i . d . ) , whereas a l l r e m a i n i n g samples had a w a l l t h i c k n e s s o f 0.5 mm (7 mm i . d . ) . The l a r g e r i . d . r i n g was the s t a n d a r d s i z e p r e s c r i b e d by G o t t . Vena cava r i n g s may be p r e p a r e d by compression m o l d i n g ( i . d . ) and machining ( o . d . ) , by i n j e c t i o n m o l d i n g o r by s o l u t i o n c o a t i n g o f t h e t e s t polymer ont polyethylene rings. t i a l groove midway between the ends o f each r i n g t o p e r m i t the r i n g t o be t i e d i n p l a c e a f t e r i m p l a n t a t i o n . T e s t s were conducted by i n s e r t i n g t h e r i n g , u s i n g a s p e c i a l i n s e r t i o n d e v i c e , i n t o the c a n i n e i n f e r i o r vena cava through a s m a l l i n c i s i o n i n the r i g h t a t r i u m . A wrapping was p l a c e d over t h e r i n g e x t e r n a l t o the b l o o d v e s s e l t o p r e v e n t c u l de sac f o r m a t i o n and the r i n g was t i e d i n p l a c e . I n i t i a l l y t h r e e samples o f polymer were each i m p l a n t e d f o r two h o u r s , a f t e r which time they were removed and examined f o r the p r e s e n c e of adherent thrombi. Polymers which were r a t e d h i g h l y t h r o m b o r e s i s t a n t on the b a s i s o f t h i s a c u t e t e s t were t h e n i m p l a n t e d i n t h e same manner f o r a p e r i o d o f two weeks. Canine

Implantation Studies.

Implant samples were i n the form o f 30 mm l o n g tubes w i t h an o.d. o f 9 mm and a w a l l t h i c k n e s s o f 1 (7 mm i . d . ) . Both i n f l o w and o u t f l o w o r i f i c e s were streamlined. Tubes may be f a b r i c a t e d by the s e v e r a l methods noted above f o r vena cava r i n g s . The tubes were c i r c u m f e r e n t i a l l y grooved near each end.

* Dr. V. L. G o t t , Johns Hopkins U n i v e r s i t y , Dept. o f Surgery, B a l t i m o r e , Maryland. c

* Dr. P. N. Sawyer, S t a t e U n i v e r s i t y o f New York, Downstate M e d i c a l C e n t e r , B r o o k l y n , New York.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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I m p l a n t a t i o n was i n the c a n i n e i n f e r i o r vena cava through an i n c i s i o n i n t h a t v e s s e l . The tube i s t i e d i n p l a c e a t each groove, w i t h one t i e p r o x i m a l and one t i e d i s t a l t o the i n c i s i o n . F o r each polymer, one tube was employed as an a c u t e (2 hour) t e s t and two tubes as c h r o n i c (14 days) i m p l a n t s . POLYMER SYNTHESIS E t h y l e n e / A c r y l i c A c i d Copolymers and Ionomers The f a m i l y o f e t h y l e n e / a c r y l i c a c i d copolymers seemed an e x c e l l e n t e x p e r i m e n t a l probe f o r a c c o m p l i s h i n g t h i s study f o r s e v e r a l r e a s o n s Copolymers cont a i n i n g 2-20% by weigh m a t e r i a l s and c o u l c o n v e r s i o n o f t h e s e polymers t o t h e i r p a r t i a l l y neut r a l i z e d a l k a l i m e t a l , a l k a l i n e e a r t h , and o r g a n i c amine s a l t s had been t h o r o u g h l y i n v e s t i g a t e d and c o u l d be c o n t r o l l e d over a range o f 0-70% n e u t r a l i z a t i o n ; and t h e s e polymers a r e tough, f l e x i b l e t h e r m o p l a s t i c s c a p a b l e o f b e i n g f a b r i c a t e d from s o l u t i o n o r i n the m e l t t o y i e l d h i g h l y s a t i s f a c t o r y t e s t samples. E t h y l e n e / V i n y l S u l f o n i c A c i d Copolymers and Ionomers E t h y l e n e / v i n y l s u l f o n i c a c i d copolymers were p r e p a r e d by a two-step p r o c e d u r e . I n the f i r s t s t e p , e t h y l e n e was c o p o l y m e r i z e d w i t h sodium v i n y l s u l f o n a t e i n a h i g h p r e s s u r e aqueous e m u l s i o n p o l y m e r i z a t i o n . The r e s u l t i n g f u l l y n e u t r a l i z e d sodium v i n y l s u l f o n a t e ionomers were c o n v e r t e d by t r e a t m e n t w i t h anhydrous HCL t o the d e s i r e d s u l f o n i c a c i d copolymers which c o u l d be t i t r a t e d w i t h base t o y i e l d p a r t i a l l y neut r a l i z e d ionomers. N - V i n y l P y r r o l i d o n e / A c r y l i c A c i d Copolymers and Ionomers (NVP/AA) In i n i t i a l s t u d i e s l e a d i n g t o p r e p a r a t i o n o f Nv i n y l p y r r o l i d o n e ( N V P ) / a c r y l i c a c i d (AA) copolymers, s y n t h e s i s was c a r r i e d o u t as a two-step p r o c e d u r e . I n the f i r s t s t e p , sodium a c r y l a t e was c o p o l y m e r i z e d w i t h NVP i n b u f f e r e d aqueous s o l u t i o n a t pH 7-8 a f t e r which the r e s u l t i n g f u l l y n e u t r a l i z e d ionomer was a c i d i f i e d t o y i e l d t h e d e s i r e d N-VP/AA copolymer. Potassium p e r s u l f a t e was employed as i n i t i a t o r i n the p o l y m e r i z a t i o n s t e p . A l t h o u g h the c o p o l y m e r i z a t i o n c o u l d be c a r r i e d o u t s u c c e s s f u l l y , attempts t o c a r r y o u t a c i d i f i c a t i o n with concentrated or half-concentrated

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h y d r o c h l o r i c a c i d , f o l l o w e d by d r y i n g a t e l e v a t e d temperatures, r e s u l t e d i n d e c o m p o s i t i o n and d i s c o l o r a t i o n of the product. A l t h o u g h t h e r e had i n i t i a l l y been c o n c e r n t h a t d i r e c t a c i d i f i c a t i o n might, t h e r e f o r e , not be p o s s i b l e , i t was s u b s e q u e n t l y found t h a t s a t i s f a c t o r y c o l o r l e s s polymers c o u l d be o b t a i n e d i f two p r e c a u t i o n s were t a k e n . F i r s t , a c i d i f i c a t i o n was c a r r i e d o u t a t temperatures below 10°C. Second, exh a u s t i v e p u r i f i c a t i o n o f the polymer, by d i s s o l v i n g i n water and r e p r e c i p i t a t i n g w i t h a c e t o n e , was re~q u i r e d t o remove a l l t r a c e s o f a c i d b e f o r e d r y i n g . I t was a l s o found t h a t t h i s p u r i f i c a t i o n p r o c e d u r e p r o duced a drop i n a c r y l i c a c i d c o n t e n t , as determined by t i t r a t i o n , presumably by removing i m p u r i t i e s o f a c r y l i c a c i d homopolymer p u r i t i e s , however, t h found t o have h i g h e r AA/NVP r a t i o s than i n the o r i g i n a l monomer f e e d . By r u n n i n g a p o l y m e r i z a t i o n t o p a r t i a l c o n v e r s i o n , w i t h c o n t i n u o u s f e e d o f monomers and c o n t i n u o u s removal o f r e a c t i o n m i x t u r e , i t was p o s s i b l e t o c o n v e n i e n t l y c o n t r o l u n r e a c t e d monomer r a t i o s t o p r e p a r e a u n i f o r m copolymer throughout the c o u r s e o f the r e a c t i o n . Vinyl Acetate/Crotonic Acid Ionomers

(VA/CA) Copolymers

and

V i n y l a c e t a t e and c r o t o n i c a c i d were p o l y m e r i z e d under aqueous e m u l s i o n c o n d i t i o n s a t 6 5 - 6 9 ° C ( s l i g h t v i n y l a c e t a t e r e f l u x ) , w i t h p o t a s s i u m p e r s u l f a t e emp l o y e d as i n i t i a t o r . S m a l l amounts o f sodium a c e t a t e were used as b u f f e r f o r the c r o t o n i c a c i d . Emulsions were p r e p a r e d w i t h f i n a l s o l i d s c o n t e n t o f a p p r o x i m a t e l y 50%. A t low c r o t o n i c a c i d (CA) c o n t e n t ( 8%), the r e a c t i o n was s l i g h t l y e x o t h e r m i c , whereas w i t h CA l e v e l s h i g h e r than 10% the r e a c t i o n was more s l u g g i s h . R e a c t i o n t i m e s ranged from 20-30 minutes f o r 5% CA copolymers t o 15-20 hours f o r copolymers c o n t a i n i n g ^15% c r o t o n i c a c i d . The polymer e m u l s i o n o b t a i n e d p r o v e d t o be v e r y s t a b l e and d i f f i c u l t t o c o a g u l a t e . I s o l a t i o n was b e s t a c c o m p l i s h e d by g r a d u a l a d d i t i o n o f methanol t o the t h i c k l a t e x a t 0-5°C, w i t h c o n t i n u o u s removal o f t h e s l i m y p r o d u c t . T h i s was d r i e d under vacuum t o y i e l d r e l a t i v e l y b r i t t l e polymer o f o n l y modest m o l e c u l a r w e i g h t . High m o l e c u l a r w e i g h t s a r e u n o b t a i n a b l e because o f the c h a i n t r a n s f e r a c t i v i t y of crotonic a c i d .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Coating o f P o l y e l e c t r o l y t e Hydrogels pylene

onto

311

Polypro-

The h y d r o p h o b i c PE s u r f a c e can be made w e t t a b l e w i t h water by t r e a t m e n t w i t h chromic a c i d " c l e a n i n g solution". The PE tubes were suspended i n a m i x t u r e of c o n c e n t r a t e d s u l f u r i c a c i d c o n t a i n i n g s o l i d c h r o m i um t r i o x i d e and t h e m i x t u r e s t i r r e d a t room temperat u r e f o r one day. The tubes were r i n s e d w e l l w i t h d i s t i l l e d water f o l l o w e d by a r i n s e w i t h methanol. After t h i s t r e a t m e n t the tubes were n o t a l l o w e d t o d r y and were touched o n l y w i t h methanol r i n s e d c l e a n r u b b e r gloves. They were s t o r e d u n t i l used i n a j a r c o n t a i n i n g methanol. The tubes were p l a c e d i n a F e r r i s wheel a p p a r a t u s and c o a t e Union C a r b i d e A-174 n o l e i t h e r by s p r a y i n g o r d i p p i n g . The tubes were d r i e d and heat t r e a t e d (80°/0.5 hr) i n an oven w h i l e t u r n i n g w i t h t h e i r c y l i n d r i c a l a x i s p a r a l l e l t o the axis of rotation. The tubes were then c o a t e d w i t h a s o l u t i o n o f polyelectrolyte. A f t e r the d e s i r e d amount o f p o l y e l e c t r o l y t e had been c o a t e d onto the PE r i n g the c o a t i n g was h e a t c u r e d (80°/l h r ) . VA/CA s o l u t i o n c o n t a i n i n g 10% by weight polymer i n methanol was used. Two complete immersions o f t h e tube i n the s o l u t i o n , f o l lowed by d r y i n g , produced a d e f e c t - f r e e c o a t i n g o f p r o per t h i c k n e s s . A f t e r each d i p , a d r o p l e t o f s o l u t i o n r o l l e d around i n s i d e the tube as i t i s r o t a t e d i n the 80°C oven u n t i l the s o l v e n t i s e v a p o r a t e d . I n the c a s e VP/AA, a s o l u t i o n c o n t a i n i n g 5% by w e i g h t polymer i n methanol was used. One immersion f o l l o w e d by s h a k i n g to remove any e x c e s s drops i s used t o i n s u r e t h a t the e n t i r e tube has some f i n i t e c o a t i n g o f t h e polymer. A f t e r e v a p o r a t i n g o f f the r e m a i n i n g s o l v e n t a 0.15 ml d r o p l e t o f the s o l u t i o n i s p l a c e d i n s i d e the tube f o l lowed by e v a p o r a t i o n o f s o l v e n t w i t h r o t a t i o n . For i r r a d i a t i o n c r o s s l i n k i n g the p r e s e n c e o f a c o n t r o l l e d amount o f water i n the c o a t i n g and the absence o f oxygen both enhanced the e f f i c i e n c y o f c r o s s l i n k i n g o f the c o a t i n g . Water c o u l d be added t o the c o a t i n g by e x p o s i n g t h e tubes t o a humid atmosphere. The a b s o r p t i o n o f 2% by weight water by a 20 ml w i t ness s t r i p was used a s a g u i d e . The dampened tubes were s e a l e d under n i t r o g e n o r a r g o n i n a PE bag and i r r a d i a t e d w i t h the Van de G r a a f f . The dosages r e q u i r e d were 35 megarads f o r VA/CA and 100 megarads f o r NVP/CA. However, v a r i a t i o n i n h y d r o g e l d e n s i t y were a t t a i n a b l e i n t h e NVP/AA case o v e r a range o f 80-200 megarads.

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D i r e c t Graft Polymerization of V i n y l Acetate/Crotonic A c i d from P o l y p r o p y l e n e The p o l y p r o p y l e n e o b j e c t s t o be c o a t e d w i t h hyd r o g e l were p l a c e d i n a f l a s k c o n t a i n i n g e i t h e r an aqueous e m u l s i o n o r an e t h a n o l s o l u t i o n o f v i n y l acet a t e , c r o t o n i c a c i d , sodium c r o t o n a t e and a s u r f a c t a n t ( T e r g i t o l 12-P-12). The c o n c e n t r a t i o n o f monomers was e i t h e r 25% o r 50% by weight, and the s u r f a c t a n t was 1% by weight. The s o l u t i o n (emulsion) was deoxygenated w i t h n i t r o g e n , p l a c e d i n a C o ^ s o u r c e and i r r a d i a t e d at a r a t e o f 50,000 r a d / h r w h i l e a g i t a t i o n was maint a i n e d by n i t r o g e n . A f t e r i r r a d i a t i o n the samples were removed from the f l a s k and t h o r o u g h l y washed w i t h e t h a n o l t o remove an were then e q u i l i b r a t e 6

RESULTS AND

DISCUSSION

I n i t i a l l y , an attempt was made t o e l u c i d a t e the r e l a t i o n s h i p o f the s u r f a c e charge d e n s i t y o f a mate r i a l to i t s hemocompatibility. Two systems, e t h y l e n e / a c r y l i c a c i d (EAA) and e t h y l e n e / v i n y l s u l f o n a t e (EVS), were chosen f o r s t u d y . No member o f the EVS s e r i e s o f copolymers and ionomers showed s i g n i f i c a n t thromboresistance (7) and o n l y two members o f the EAA s e r i e s (the 19% a c r y l i c a c i d copolymer and i t s 60% sodium ionomer) showed s i g n i f i c a n t t h r o m b o r e s i s t a n c e . (JzW To e l u c i d a t e the thrombogenic dependence on hyd r o p h i l i c i t y , two systems, N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d (NVP/AA) and v i n y l a c e t a t e / c r o t o n i c a c i d (VA/CA) copolymers and ionomers, were chosen f o r s t u d y . While no member o f the NVP/AA s e r i e s d i s p l a y e d any thrombor e s i s t a n c e (MLIJL?.) , members o f the VA/CA s e r i e s showed v a r i o u s degrees o f t h r o m b o r e s i s t a n c e . The 2% c r o t o n i c a c i d 60% sodium ionomer, i n p a r t i c u l a r , e x h i b i t e d a h i g h degree of t h r o m b o r e s i s t a n c e i n vena cava i m p l a n t studies. Ethylene/Acrylic Acids Polyelectrolytes A s e r i e s o f copolymers and ionomers was p r e p a r e d which r e p r e s e n t e d f o u r l e v e l s o f t o t a l c a r b o x y l conc e n t r a t i o n (4.0, 9.8, 14.7, and 19.2% a c r y l i c a c i d ) . A t each l e v e l , u n n e u t r a l i z e d copolymers were compared w i t h copolymers which had been c o n v e r t e d t o a p p r o x i m a t e l y 30% and 60% n e u t r a l i z e d ionomers. Counteranions i n v e s t i g a t e d i n c l u d e d l i t h i u m , sodium, potassium, magnesium, c a l c i u m , and d i m e t h y l e t h a n o l a m i n e . The

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Polyelectrolytes

313

polymers which comprise t h e s e r i e s o f twenty-one e t h y l e n e / a c r y l i c a c i d copolymers and ionomers were e x p e c t e d t o d i f f e r from one a n o t h e r i n a number o f ways. These d i f f e r e n c e s i n c l u d e d v a r i a t i o n i n measurable p h y s i c a l c h e m i c a l parameters by which such s u r f a c e p r o p e r t i e s as w e t t a b i l i t y and e l e c t r o c h e m i c a l b e h a v i o r can be c h a r a c t e r i z e d , as w e l l as v a r i a t i o n o f c h e m i c a l i n t e r a c t i o n s w i t h b l o o d on t h e m o l e c u l a r l e v e l . The c o n t a c t a n g l e s o f d i s t i l l e d water on t h e polymer s u r f a c e s ranged from a h i g h o f 8 7 ( s u r f a c e f r e e energy = 1 9 . 9 ergs/cm) f o r t h e u n n e u t r a l i z e d 9 . 8 % a c r y l i c a c i d copolymer t o a low o f 7 1 ( 3 1 . 6 ergs/cm) f o r h i g h l y n e u t r a l i z e d l i t h i u m and sodium ionomers o f t h e 1 9 . 2 % a c r y l i c a c i d copolymer. Zeta p o t e n t i a l s determined by Dr. Sawyer ana coworke r s (15/ ( T a b l e I) a large negative values c a l c o n d i t i o n s proposed f o r b l o o d c o m p a t i b i l i t y were satisfied. I n i t i a l e v a l u a t i o n s o f the thrombogenicity of these m a t e r i a l s was performed by Mason, e t a l . (S) , a t t h e U n i v e r s i t y o f N o r t h C a r o l i n a S c h o o l o f M e d i c i n e and Pennington (9^) a t B a t t e l l e Memorial I n s t i t u t e , u s i n g Stypven time and p a r t i a l t h r o m b o p l a s t i n time as a measure o f b l o o d c o m p a t i b i l i t y (Table I I ) . No member o f the s e r i e s o f twenty-one e t h y l e n e / a c r y l i c a c i d c o p o l y mers and ionomers appeared t o be h i g h l y t h r o m b o r e s i s t a n t on t h e b a s i s o f t h e d a t a . The v a r i a t i o n s i n p o l y mer s t r u c t u r e appear t o have no s i g n i f i c a n t e f f e c t on the Stypven time, s i n c e a l l polymers t e s t e d y i e l d e d values w i t h i n experimental error o f values obtained f o r the s i l i c o n i z e d g l a s s c o n t r o l s . I n t h e case o f t h e p a r t i a l t h r o m b o p l a s t i c t i m e s , however, t h e r e was f a r greater v a r i a b i l i t y . In v i r t u a l l y a l l cases, the p o l y mers appeared t o be more thrombogenic than t h e s t a n dards. I n o n l y two c a s e s — t h e 9 . 8 % a c r y l i c a c i d c o polymer and i t s 2 9 . 5 % n e u t r a l i z e d sodium ionomer — were v a l u e s seen w h i c h were s i g n i f i c a n t l y h i g h e r than t h e values f o r s i l i c o n i z e d glass. I n n e i t h e r c a s e , howe v e r , were h i g h v a l u e s r e g i s t e r e d by b o t h t e s t groups. In f a c t , t h e r e appeared t o be l i t t l e o b v i o u s c o r r e l a t i o n between t h e r e s u l t s o f t h e two p a r a l l e l s e t s o f tests. As an a d d i t i o n a l p o r t i o n o f t h e b a s i c i n v i t r o b l o o d c o m p a t i b i l i t y s t u d i e s performed a t B a t t e l l e Memorial I n s t i t u t e (9^) , a d h e s i o n o f e r y t h r o c y t e s and p l a t e l e t s t o t h e e x p e r i m e n t a l polymers was a l s o i n v e s tigated. The r e s u l t s o f t h e s e t e s t s i n d i c a t e d f a i r l y u n i f o r m b e h a v i o r throughout t h e s e r i e s o f polymers. In a l l c a s e s , adherence o f e r y t h r o c y t e s was v e r y l i g h t , w i t h some showing no adherence a t a l l .

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

314

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

P l a t e l e t adherence s l i g h t aggregation c a n t e x c e p t i o n was which showed heavy gregation.

ranged from l i g h t t o moderate, w i t h i n some c a s e s . The o n l y s i g n i f i the dimethylethanolamine s a l t , p l a t e l e t adherence w i t h heavy a g -

TABLE I ZETA POTENTIALS ACROSS SOME POLYELECTROLYTES SALINE/1000 SOLUTION INTERFACES AS OBTAINED FROM STREAMING POTENTIALS

dE/dP* % AA

% Ionome

9.8 19.2 19.2 19.2 19.2 19.2 19.2 19.2

0 0 27.0% 25.5% 61.0% 61.5% 61.5% 34.5%

Mg; Ca Na Mg Ca DMEA

325 350 355 315 390 340 365 325

-17.2 -18.5 -18.8 -16.8 -20.7 -18.0 -19.5 -17.5

* dE/dP i s t h e average s l o p e o f t h e s t r e a m i n g pot e n t i a l v e r s u s p r e s s u r e r e l a t i o n from which t h e zeta p o t e n t i a l i s c a l c u l a t e d . I n v i v o b l o o d c o m p a t i b i l i t y s t u d i e s were a l s o p e r formed on many o f t h e e t h y l e n e / a c r y l i c a c i d copolymers and ionomers by Sawyer and coworkers(14) a t S t a t e U n i v e r s i t y o f New York Downstate M e d i c a l C e n t e r and G o t t (jL?.) and coworkers a t Johns Hopkins U n i v e r s i t y (Table I I I ) . Sawyer's r e s u l t s i n d i c a t e t h a t two o f t h e polymers e x h i b i t e d a moderate l e v e l o f t h r o m b o r e s i s tance. These were t h e 19% a c r y l i c a c i d copolymer and i t s h i g h l y n e u t r a l i z e d sodium ionomer. In both cases, two hour and two week i m p l a n t s were e i t h e r thrombusf r e e a t t h e end o f t h e i m p l a n t p e r i o d o r c o n t a i n e d o n l y m i n i m a l j u n c t i o n a l t h r o m b i . Based on t h e o b s e r v a t i o n s i n t h e s e experiments(UD , t h e s e two polymers appeared t o be m o d e r a t e l y t h r o m b o r e s i s t a n t , whereas copolymers a t lower n e u t r a l i z a t i o n o r lower a c r y l i c a c i d c o n t e n t , as w e l l as c a l c i u m ionomers, were thrombogenic. The h i g h e s t l e v e l o f thrombogenic a c t i v i t y was found i n t h e magnesium and d i m e t h y l e t h a n o l a m i n e ionomers. In G o t t s t e s t s , a l l o f t h e polymers d i s p l a y e d s i g n i f i c a n t l e v e l s o f thrombogenic a c t i v i t y , w i t h o n l y l i m i t e d v a r i a t i o n 1

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL

E T AL.

Characteristics

of Polyelectrolytes

315

TABLE I I

IN VITRO BLOOD COMPATIBILITY OF ETHYLENE/ACRYLIC ACID POLYELECTROLYTES

A c r y l i c Acid Cor ent

% Ionome

Stypven Time*

P a r t i a l Thromboplastin Time* University of North C a r o l i n Battell

4.0%

0 24..0% Na 55..0% Na

— — —

— — —

109 99 101

9.8%

0 29.,5% Na 71..0% Na

98 98 97

76 115 95

137 83 89

14.7%

0 32., 0% Na 62.. 0% Na

97 98 98

106 85 74

101 99 86

19.2%

0 27.,0% L i 28.,0% Na 24.,0% Κ 27.,0% Mg 26.,5% Ca 34., 5% DMEA 65.,0% L i 61., 0% Na 40.,5% Κ 61., 5% Mg 61.. 5% Ca

100 98 103 101 99 103 104 98 96 100 107 97

74 78 62 59 79 68 84 78 89 94 65 74

92 70 94 83 91 78 103

··

* % of s i l i c o n i z e d glass control

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

—

— 109 99 104

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

% Neut.

0

29.0

71.0

0

62.0

0

% AA

9.8

9.8

9.8

14.7

14.7

19.2

—

Na

-

Ν,ι

Na

—

Cation

Q

φ φ φ φ

φ

H

HI HI

H

φ

Q

• i

H

I I

C C

φ

φ

t D H

φ

Q

I

• ϋ

L

φ

I

φ

• H Ρ

φ

•

•

φ

φ

t

I

φ

HI

Π

TABLE I I I

h

o

u

r

s

2

n o u r s

14 days

14 days

2 hours

18 hours

14 days

2 hours

14 days

14 days

2

14 days

14 days

2 hours

14 days

14 days

2 hours

14 days

14 days

Duration

I

k

Ο

Ο

L

I

j

Îi

Γ

L>

L

1

ι—

Ρ

,

-

—

0

%

1

I

I

1

1

90%

30%

50%

%

30%

junctional

clean

junctional

0

junctional

60%

10%

90%

1

1

9

%

5%

—

0

50%

20%

8

clean

1

J

1

1

1. ,ιτη 1

1

1

LT"

J B * ^ * _ J

I

Ο

©

Ο

Q

Ο

Ο

Ο

©

Ο

Ο

Ο

Ο

Ο

Ο

Q

Ο

% Occlusion

AND IONOMERS

Downstate Medical Center

OF ETHYLENE/ACRYLIC ACID COPOLYMERS

Johns Hopkins 2 hr". Implant

IMPLANTATION

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

26.5

34.5

61.0

60.5

61.5

61.5

19.2

19.2

19.2

19.2

19.2

27.0

19.2

19.2

Ca

Mg

Na

DMEA

Ca

Mg

•• •

EU ©

Ο • Ο •• ·

2 hours

Ο Ο

Ο 31 days 14 days

Ο

ο

2 hours

14 days

4 hours

2 hours

14 days

14 days

c

Ε»

c

Ο Ο C Ο C

Ο

2 hours

Ο

14 days

Ο cz

1

1

60%

Ο

50%

clean

junctional 50%

30%

90%

clean

clean

5% (jnl.)

2 0%

80%

60%

40%

10%

5%

60%

80%

Ο

©

14 days

2 hours

6 days

14 days

2 hours

14 days

14 days

TABLE I I I (Continued)

1

C<5

«δ*

CO

Ci

Ο

r

M

G d

318

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

w i t h i n the s e r i e s . S e v e r a l o f the polymers d i s p l a y e d some t h r o m b o r e s i s t a n c e , i n c l u d i n g the h i g h l y n e u t r a l i z e d sodium ionomer o f the 19% a c r y l i c a c i d copolymer. Ethylene/Vinyl

Sulfonate

PolyelectmLytes

The c h e m i s t r y o f the a n i o n i c m o i e t y o f a p o l y e l e c t r o l y t e must a l s o be c o n s i d e r e d as a f a c t o r r e g u l a t i n g blood c o m p a t i b i l i t y . Although carboxyl-cont a i n i n g polymers have been the p r i m a r y s u b j e c t s i n our i n v e s t i g a t i o n , comparison of t h e i r p h y s i o l o g i c a l a c t i v i t y w i t h the b l o o d c o m p a t i b i l i t y of analogous s u l f o n i c a c i d - c o n t a i n i n g copolymers and ionomers was also an e s s e n t i a l element o f the program. L i k e c a r b o x y l groups, s u l f o n i c a c i many n a t u r a l l y o c c u r r i n b l y , they a r e p r e s e n t i n h e p a r i n , i n h i g h c o n c e n t r a t i o n , as s u l f a t e h a l f - e s t e r s and are presumed t o p l a y a r o l e i n the b i o l o g i c a l a c t i v i t y o f t h a t compound. However, s u l f o n i c a c i d s are s t r o n g e r a c i d s t h a n t h e i r c a r b o x y l i c a c i d s a n a l o g u e s and would be e x p e c t e d t o d i f f e r i n t h e i r c h e m i c a l r e a c t i o n s w i t h plasma. In o r d e r t o a s s e s s the s i g n i f i c a n c e o f t h i s f a c t o r t o the development o f b l o o d c o m p a t i b l e p o l y e l e c t r o l y t e s , e t h y l e n e / v i n y l s u l f o n i c a c i d copolymers and ionomers were p r e p a r e d and b i o l o g i c a l l y e v a l u a t e d . I n i t i a l t e s t s o f two copolymers i n t h i s s e r i e s , and an ionomer of each, s u g g e s t e d t h a t no s i g n i f i c a n t d i f f e r e n c e s c o u l d be d e t e c t e d between the i n v i t r o b l o o d c o m p a t i b i l i t y o f t h e s e polymers ( p a r t i a l thromb o p l a s t i n time; Stypven t i m e , T a b l e I V ) , and analogous d a t a f o r s i m i l a r a c r y l i c a c i d copolymers and ionomers. The poor b l o o d c o m p a t i b i l i t y o f t h e s e p o l y e l e c t r o l y t e s was c o n f i r m e d by i n v i v o s t u d i e s o f two members o f the s e r i e s by G o t t and coworkers (Table V ) . ( 1 3 ) N-Vinyl Pyrrolidone/Acrylic

Acid

Polyelectrolytes

E t h y l e n e / a c r y l i c a c i d and e t h y l e n e / v i n y l s u l f o n a t e copolymers and ionomers r e p r e s e n t a s e r i e s o f p o l y e l e c t r o l y t e s which a r e h y d r o p h o b i c o r , a t most, o n l y s l i g h t l y h y d r o p h i l i c , depending on the c o n t e n t o f the h y d r o p h o b i c comonomer e t h y l e n e and the degree o f neutralization. I t i s known, however, t h a t n a t u r a l l y o c c u r r i n g p o l y e l e c t r o l y t e s , which i m p a r t n e g a t i v e s u r f a c e charge t o b l o o d v e s s e l w a l l s o r t o b l o o d c e l l s , are c o n s i d e r a b l y more h y d r o p h i l i c t h a n even the most h y d r o p h i l i c e t h y l e n e / a c r y l i c a c i d ionomers, due t o the p r e s e n c e o f l a r g e numbers o f p o l a r groups a l o n g the

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL

E T AL.

Characteristics

of

319

Polyelectrolytes

polymer c h a i n . (lâ'il.) I t was a l s o found t h a t c e r t a i n h y d r o g e l s (1£) and i o n i c i n t e r p o l y m e r s (12) e x h i b i t good p h y s i o l o g i c a l p r o p e r t i e s on exposure t o b l o o d . An appropriate continuation o f studies o f the r e l a t i o n s h i p of a n i o n i c groups and n e g a t i v e s u r f a c e c h a r g e t o t h e b l o o d c o m p a t i b i l i t y o f polymers, t h e r e f o r e , seemed t o be i n v e s t i g a t i o n o f p o l y e l e c t r o l y t e s more h y d r o p h i l i c than e t h y l e n e / a c r y l i c a c i d copolymers and ionomers. In o r d e r t o a c c o m p l i s h t h i s , v i n y l a c e t a t e and N - v i n y l p y r r o l i d o n e were employed i n s t e a d o f e t h y l e n e a s t h e n e u t r a l comonomer t o p r e p a r e c a r b o x y l - c o n t a i n i n g c o polymers. N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d copolymers were s e l e c t e d as a system t o be i n v e s t i g a t e d r e c o g n i z i n g t h a t t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f Nv i n y l p y r r o l i d o n e woul electrolytes. V i n y l p y r r o l i d o n e / a c r y l i c a c i d copolymers and ionomers were c r o s s l i n k e d by Van de G r a a f f i r r a d i a t i o n t o form water i n s o l u b l e h y d r o g e l s which s w e l l s i g n i f i c a n t l y i n aqueous media by i m b i b i n g l a r g e q u a n t i t i e s of water. The degree o f c r o s s l i n k i n g was i n c r e a s e d by i n c r e a s e d dosage. Among t h e f e a t u r e s c o n t r o l l e d by the d e n s i t y o f c r o s s l i n k i n g , t h e t i g h t n e s s o f t h e g e l d e t e r m i n e s t h e maximum s i z e o f s o l u t e m o l e c u l e s c a p a b l e o f p e n e t r a t i n g t h e p o l y e l e c t r o l y t e c o a t i n g as water i s imbibed. Samples o f N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d c o polymers and sodium ionomers were c o a t e d on h i g h dens i t y polyethylene. The 8% ionomers were s t u d i e d by Gott(17) and were shown t o be h i g h l y thrombogenic. The 2% ionomers were s t u d i e d a t t h e Utah B i o m e d i c a l T e s t L a b o r a t o r y and were a l s o h i g h l y thrombogenic (Table VI)(12). i n b o t h s e r i e s o f ionomers, t h e i m p l a n t r e s u l t s showed t o t a l o r near t o t a l o c c l u s i o n , o f t e n a c companied by a s c i t e s o r pulmonary embolism. TABLE IV IN VITRO BLOOD COMPATIBILITY OF ETHYLENE/VINYL ACID COPOLYMERS AND IONOMERS*

% Vinyl Sulfonic Acid

% Na Ionomer

Stypven Time**

4 0 91 4 40 99 8 0 94 8 40 100 * U n i v e r s i t y o f North C a r o l i n a ** % o f s i l i c o n i z e d g l a s s c o n t r o l

SULFONIC

Partial Thromboplastin Time** 67 87 100 90

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

320

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TABLE V IN VIVO BLOOD COMPATIBILITY OF ETHYLENE/VINYL SULFONIC ACID SODIUM IONOMERS*

Vinyl Sulfonic Acid

% Na Ionomer

4

2 hr. Test

WE m

40

E H

Q

40

a

* John Hopkins

o

University

TABLE VI THROMBORESISTANCE OF N-VINYL PYRROLIDONE/ACRYLIC ACID COPOLYMERS AND IONOMERS. VENA CAVA IMPLANTATION OF 8% ACRYLIC ACID-CONTAINING POLYMERS

% Neut.

Duration

0%

2 hours

30%

2 hours

Johns-Hopkins • •

φ

• 60%

2 hours

A

Ο

•Ι

·

α

Ο

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL ET AL.

Characteristics

Vinyl Acetate/Crotonic Acid

of

321

Polyelectrolytes

Polyelectrolytes

Since the N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d copolymers imbibed such l a r g e amounts o f water, t h e i n v e s t i g a t i o n o f p o l y e l e c t r o l y t e systems which had more modest water uptake was u n d e r t a k e n . V i n y l a c e t a t e , b e i n g l e s s h y d r o p h i l i c than N - v i n y l p y r r o l i d o n e , was chosen a s the comonomer i n an attempt t o p r e p a r e a h y d r o g e l w i t h a modest water uptake. V i n y l a c e t a t e and a c r y l i c a c i d c o u l d n o t be s a t i s f a c t o r i l y c o p o l y m e r i z e d because o f w i d e l y d i f f e r i n g r e a c t i v i t y r a t i o s , so v i n y l a c e t a t e / c r o t o n i c a c i d copolymers were, t h e r e f o r e , adopted as an acceptable substitute. As i t was found t h a t copolymers above 15% c r o t o n i c a c i d l e v e l were t o o b r i t t l e f o r conv e n i e n t h a n d l i n g , polymer p a t i b i l i t y studies a a c i d monomer r a t i o s . The c r o t o n i c a c i d c o n t e n t i n t h e s e m a t e r i a l s was determined by t i t r a t i o n o f a l i q u o t samples w i t h N/10 NaOH. I n a n a l o g y t o t h e E/AA p o l y e l e c t r o l y t e s , sodium ionomers were p r e p a r e d a t a p p r o x i mately 30% and 60% c o n v e r s i o n s . R a d i a t i o n t r e a t m e n t , u s i n g t h e Van de G r a a f f e l e c t r o n a c c e l e r a t o r , was i n v e s t i g a t e d as t h e method o f c h o i c e f o r c r o s s l i n k i n g t h e s e copolymers and ionomers t o c o n v e r t them t o w a t e r - s w e l l a b l e h y d r o g e l s . Experiments were c a r r i e d o u t i n which compression molded p l a q u e s , 0.02" t h i c k , were exposed t o v a r i o u s r a d i a t i o n doses r a n g i n g from 5 t o 75 megarads. Immersion o f the t r e a t e d samples i n water showed t h a t doses o f 30 t o 40 megarads were s u f f i c i e n t t o c o n v e r t 30% n e u t r a l i z e d

TABLE V I I IN VITRO BLOOD COMPATIBILITY OF VINYL ACETATE/CROTONATE COPOLYMERS AND IONOMERS

% Crotonic 5% 5% 5% 5% 5% 15% 15% 15%

Acid

% Na S a l t 0 0 30 60 60 0 30 60

(dry) (wet) (dry) (wet)

Stypven Time 106 105 82 105 110 105 94 90

P a r t i a l Thrombop l a s t i n Time 131 140 109 112 142 103 94 74

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

322

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TABLE V I I I THROMBORESISTANCE OF VINYL ACETATE/CROTONIC ACID COPOLYMERS AMD IONOMERS VENA CAVA IMPLANTATION OF 2% CROTONIC ACID-CONTAINING POLYMERS % Meut. L )

u

SUNY-Downstate

Johns fiopkins

r

• Ο • • οο mο• • • ο

4 days

5 days

10% occl. Junct. thrombus - Sacrif.

died of pulmonary embolism

7 days

ο

oc

14 days

• Ο

• ο

oc

90% occl. Junct. and laminar thrombus Junct. thrombus Sacrif.

Junct. thrombus Sacrif.

•

ο ο

•

4 days 6 days 14 days

2 hours

14 days

• • • • • • • α

ο

ο ο ο

•

80% occl. junct. and laminar thrombus

ΟΓ.

Junct. thrombus Sacrif.

Ο

Clean - sacrif.

ο

ο ο ο

• ο

OC

oc

Clean - sacrif. Clean - sacrif.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL ET AL.

Characteristics

of

Polyelectrolytes

TABLE V I I I IMPLANTATION

Crotonic

%

Ac[d

Neutralization

7.6

0

7.6

7.6

16.2

30

60

0

O F VINYL A C E T A T E / C ROTO NIC

Π3

30

60

a

2 hours

EZI

14 d a y s

EJ

:

2 hours

•

2 hours

EZI ED

G

16.2

%

Ζ

i

IONOMERS

D o w n s ta t e M e d i c a l C e n t e r

14 d a y s

i

AND SODIUM

Duration

£•

• • wm • • !• • Π3 • • • • •

• 16.2

Ο

(Continued)

ACID COPOLYMERS

Tohns H o p k i n s 2 h r . Implant

323

ο

•1 τ

r . .

ι

ο

β 0

3

14 d a y s

ο

Ί ]

Ο C

14 d a y s

2 hours 14 d a y s

ι

I

ο

Ι

Ί

Ο Ο Ο

3 J

c

14 d a y s

90%

40%

2 hours

I

10% clean

clean

ο

2 hours

90%

Ο

3 J

2 days

10%

·

γ

S hours

30% minimal

S 14 d a y s

7 days

Occlusion

I

I !

junctional

clean 30% 30%

ο

clean

ο

Junctional

Ο

clean

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

324

HYDROGELS

FOR MEDICAL

AND RELATED

APPLICATIONS

ionomers t o w a t e r - i n s o l u b l e m a t e r i a l s , b u t t h a t f o r t h e 60% n e u t r a l i z e d ionomers, i n s o l u b i l i z a t i o n was a c h i e v e d o n l y a f t e r exposure t o doses o f 75 megarads. In order t o e q u a l i z e r a d i a t i o n - i n d u c e d changes i n t h e polymers, 75 megarads was chosen as a s t a n d a r d dose f o r a l l v i n y l a c e t a t e / c r o t o n i c a c i d copolymers and ionomers. Irradia t i o n was c a r r i e d o u t s t e p w i s e i n 3-5 megarad doses t o m i n i m i z e s i d e e f f e c t s from o v e r h e a t i n g o f samples. M e c h a n i c a l p r o p e r t i e s have been measured on f i v e o f t h e s i x m a t e r i a l s a f t e r r a d i a t i o n and exposure t o water f o r a p p r o x i m a t e l y 100 h o u r s . A l l of the propert i e s i n d i c a t e d v e r y weak m a t e r i a l s ; t h u s , a s o l i d supp o r t m a t e r i a l was n e c e s s a r y t o p r o v i d e s u f f i c i e n t mec h a n i c a l s t r e n g t h f o r t e s t samples and end use a p p l i c a t i o n s (see Method The i n i t i a l i time and p a r t i a l t h r o m b o p l a s t i n time performed by Dr. R. G. Mason(8.) and Dr. C. J . P e n n i n g t o n . The r e s u l t s i n d i c a t e d t h a t s e v e r a l c o m p o s i t i o n s were more b l o o d c o m p a t i b l e under t h e s e c o n d i t i o n s than s i l i c o n i z e d g l a s s c o n t r o l s (Table V I I ) . Furthermore, i t was a l s o found t h a t b l o o d c o m p a t i b i l i t y c o u l d be f u r t h e r enhanced by p r e s w e l l i n g t h e p o l y e l e c t r o l y t e h y d r o g e l s by 30 minutes immersion i n 0.154 M N a C l . Nine members o f t h e v i n y l a c e t a t e / c r o t o n i c a c i d s e r i e s were e v a l u a t e d by Dr. P. N. Sawyer(14) as c o a t ings of p o l y o l e f i n r i n g s . These were copolymers cont a i n i n g 2, 8 and 16 p e r c e n t c r o t o n i c a c i d , as w e l l as the 30% and 60% sodium ionomers o f t h e s e copolymers. Dr. V. L. Gott^i^.) i m p l a n t e d s i x o f t h e n i n e members of the v i n y l a c e t a t e / c r o t o n i c a c i d p o l y e l e c t r o l y t e hydrogel s e r i e s . Copolymers c o n t a i n i n g a p p r o x i m a t e l y two p e r c e n t c r o t o n i c a c i d gave e x c e l l e n t two hour r e s u l t s a t a l l l e v e l s o f n e u t r a l i z a t i o n and p r o v i d e d f a i r l y good two week r e s u l t s (Table V I I I ) . I n t h e unn e u t r a l i z e d c a s e , two o f t h e a n i m a l s had c l e a n r i n g s a t f o u r and f i v e days, one d y i n g o f pneumonia and t h e o t h e r a p p e a r i n g t o d i e o f pulmonary embolism. The t h i r d r i n g i n t h i s s e r i e s thrombosed a t f o u r days. The two ionomers l o o k e d s u r p r i s i n g l y good a t two weeks, w i t h two o f t h e t h r e e r i n g s i n each case b e i n g f r e e o f thrombus. Comparison o f r e s u l t s from both groups sugg e s t e d t h a t t h e m a t e r i a l which c o n s i s t e n t l y demonstrat e d t h e most e n c o u r a g i n g t h r o m b o r e s i s t a n c e i s t h e 60% sodium ionomer o f v i n y l a c e t a t e / 2 % c r o t o n i c a c i d c o polymer. U s i n g Co r a d i a t i o n , i t has been p o s s i b l e t o g r a f t polymerize v i n y l a c e t a t e / c r o t o n i c acid/sodium c r o t o n a t e d i r e c t l y onto t h e p o l y p r o p y l e n e . T h i s has 6

0

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL ET AL.

Characteristics

of

Polyelectrolytes

325

p r o v i d e d an o p p o r t u n i t y t o c a r e f u l l y c o n t r o l t h e c o a t i n g t h i c k n e s s and water uptake o f t h e h y d r o g e l ( see T a b l e IX and X) i n o r d e r t o o p t i m i z e b i o l o g i c a l r e sults. I n v i v o s t u d i e s have been performed on samples p r e p a r e d w i t h v a r i o u s c o a t i n g t h i c k n e s s e s and water uptake v a l u e s . The G o t t vena cava i m p l a n t and Kusserow r e n a l embolus t e s t s h o w t h e p o l y ( v i n y l a c e t a t e - c o - 2 % - c r o t o n i c a c i d ) 60% sodium ionomer p r e p a r e d a t 0.1 Mrad (12 y c o a t i n g and 55% water uptake) t o be the most c o n s i s t e n t l y t h r o m b o r e s i s t a n t p o l y e l e c t r o l y t e hydrogel i n t h i s s e r i e s . s

TABLE IX COATING PARAMETER DOSE-SURFACTANT Dose (Megarads) 0.10 0.20 0.20 0.20 0.40 1.20 2.40 a.

Monomer Cone. (%)

Coating Thickness

50 50 25 25 25 25 25

12 30 7 13 30 32 65

(μ)

a

E q . Water Abs. (%) 50.0 33.4 53.8 28.0 29.6 18.7 9.7

a

a a

Approximate; c a l c u l a t e d from c o a t i n g weight TABLE X

COATING PARAMETERS AS A FUNCTION OF RADIATION DOSESURFACTANT PROMOTED IN WATER b

Dose (Megarads) 0.05 0.10 0.20 0.40 0.80 1.00 a. b.

Monomer Cone. (%) 25 25 25 25 25 25

Coating Thickness a

6 16 30 70-100 130-180 140-200 a

(μ)

Eq. Water Abs. (%) 111.3 55.6 16.4 3.3 3.2 3.3

Approximate; c a l c u l a t e d from c o a t i n g weight N i t r o g e n gas a g i t a t i o n d u r i n g i r r a d i a t i o n t o maintain emulsion

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

326

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

R e s u l t s o f vena cava i m p l a n t t e s t s on p o l y ( v i n y l c o - 2 % - c r o t o n i c a c i d ) 60% sodium ionomer samples p r e pared a t v a r y i n g g r a f t i n g doses a r e shown i n T a b l e X I . F o r those samples p r e p a r e d by e m u l s i o n g r a f t i n g p r o c e d u r e s , t h e r e appears t o be a minimum dose (0.1 Mrad) below which t h e samples become s i g n i f i c a n t l y l e s s nonthrombogenic. T h i s may be due e i t h e r t o a p h y s i c a l l y d i f f e r e n t h y d r o g e l produced a t t h e lowest dose o r t o a non-uniform c o a t i n g . A t p r e s e n t , we cannot d i s t i n g u i s h between t h e s e two p o s s i b i l i t i e s . I t i s noteworthy t h a t those samples p r e p a r e d a t 0.07 Mrad show a 43% i n c i d e n c e o f o c c l u s i o n a t two hours w h i l e t h o s e p r e p a r e d a t 0.1 and 0.4 Mrads show a 100% patency r a t e i n t h e same p e r i o d . The d a t a a l s o t e n d t o i n d i c a t e (with t h e e x c e p t i o Mrad) t h a t c o a t i n g more t h r o m b o r e s i s t a n t than t h o s e w i t h low water uptake v a l u e s . F o r samples p r e p a r e d a t 1.0 and 1.2 Mrad i n e t h a n o l (20% water u p t a k e ) , t h e r e was a 35% i n c i dence o f complete o c c l u s i o n a t two h o u r s . TABLE XI VENA CAVA IMPLANT TESTS G r a f t i n g Dose (Megarads) 0.07 0.1b 0.4 1.0 1.2 60. 0 b

C

C

d

a. b. c. d.

b

Water Uptake (%) 75 56 3 20 19 120

3

0

Results I X

2 5 3

-

2 2 2 3

2 3

2

10 5 1 2 4 1

X = Occluded, I = P a r t i a l O c c l u s i o n , 0 = Patent. Emulsion P o l y m e r i z a t i o n . Solution Polymerization i n Ethanol. Van de G r a a f f I r r a d i a t i o n o f Preformed Hydrogel. CONCLUSION

S y s t e m a t i c v a r i a t i o n o f t h e s u r f a c e charge dens i t y and h y d r o p h i l i c i t y o f p o l y e l e c t r o l y t e systems has y i e l d e d i m p o r t a n t i n f o r m a t i o n r e g a r d i n g nonthrombog e n i c s u r f a c e s . The r e s u l t s from experiments w i t h e t h y l e n e / a c r y l i c a c i d and e t h y l e n e / v i n y l s u l f o n a t e p o l y e l e c t r o l y t e s have shown a thrombogenic dependence on t h e c h e m i c a l n a t u r e o f t h e i o n i c moiety bound t o

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

23.

LUNDELL ET AL.

Characteristics

of

Polyelectrolytes

327

the polymer backbone and i t s c o u n t e r i o n . Evaluation of v i n y l a c e t a t e / c r o t o n i c a c i d and N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d p o l y e l e c t r o l y t e h y d r o g e l s has r e v e a l e d a thrombogenic dependence on b o t h t h e s u r f a c e charge den s i t y and t h e h y d r o p h i l i c i t y o f t h e p o l y e l e c t r o l y t e . In v i v o and i n v i t r o b l o o d c o m p a t i b i l i t y s t u d i e s e s t a b l i s h e d that the v i n y l acetate-co-2%-crotonic a c i d 60% sodium ionomer was t h e most t h r o m b o r e s i s t a n t mate r i a l i n i t s s e r i e s . Furthermore, the v i n y l a c e t a t e / c r o t o n i c a c i d h y d r o g e l system was s i g n i f i c a n t l y more t h r o m b o r e s i s t a n t than t h e N - v i n y l p y r r o l i d o n e / a c r y l i c a c i d h y d r o g e l system. A l s o , w i t h i n each s e r i e s i t has been shown t h a t t h e r e i s a thrombogenic dependence on the s u r f a c e charge d e n s i t y . These r e s u l t s imply t h a t the a n t i t h r o m b o g e n i increase i n d e f i n i t e l charge d e n s i t y o f t h e s u r f a c e i n c r e a s e s . R a t h e r , an optimum h y d r o p h i l i c i t y and charge d e n s i t y i s r e a c h e d , beyond which t h e s u r f a c e a g a i n becomes thrombogenic. Based on t h e r e s u l t s o f o u r s t u d i e s , we a r e con c e n t r a t i n g o u r e f f o r t s on o p t i m i z i n g t h e p r o m i s i n g v i n y l a c e t a t e - c o - 2 % - c r o t o n i c a c i d ionomer h y d r o g e l system. F u r t h e r r e s u l t s o f s t u d i e s on v i n y l a c e t a t e c o - 2 % - c r o t o n i c a c i d ionomer h y d r o g e l c o a t i n g s p r e p a r e d by t h e d i r e c t p o l y m e r i z a t i o n o f monomers from s u r f a c e s w i l l be r e p o r t e d s u b s e q u e n t l y .

LITERATURE CITED 1. Sawyer, P. N. and J. W. Pate, Surgery, (1953), 34, 191; Amer. J. Physiol., (1953), 103, 113, 175. 2. Sawyer, P. Ν . , ed., "Biophysical Mechanisms in Vas cular Hemostasis and Intravascular Thrombus", Appelton-Century-Crofts, New York, 1965. 3. Langdale, R. D . , J. Lab. C l i n . Med., (1953), 41, 637. 4. Rodman, N. F., An. J. Clin. Path., (1958), 29, 525. 5. Maile, J. P . , "Laboratory Medicine-Hematology", 2nd e d . , C. V. Mosby Co., St. Louis, 1962. 6. Hardisty, J., Brit. J. Haemat., (1966), 12, 764. 7. Gott, V. L . and R. E . Baier, "Materials Compatible with Blood", Contract PH43-68-84-2, April 1969. 8. Mason, R. G. and L . D. Ikenberry, "Antithrombogenic Surfaces: Platelet-Interface Reactions", Contract PH43-67-1416, September 1969. 9. Pennington, D. J., L . L . Peterson, J. H. Rotaru and J . B. Boatman, "Biological Evaluation of Prosthetic Materials III: In Vitro Evaluation of Cardiovascular Prosthetic Devices", Contract PH43-67-1404-F-3, April 1967.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

328

10. 11. 12. 13. 14. 15. 16.

17. 18. 19. 20.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Sawyer, P. N. and S. Srinivasan, "New Approaches in the Selection of Materials Compatible with Blood", Contract PH43-68-75, December 1969. Gott, V. L . and R. E . Baier, "Materials Compatible with Blood, V o l . I . " , Contract PH43-68-84, Septem ber 1973. Daniels, A. U. and J. D. Mortenson, "Vena Cava Ring Tests of Biomaterials i n Dogs", Contract NO1HB42979, November 1974. Lucas, T., S. Srinivasan and P. Ν. Sawyer, Trans. Amer. Soc., A r t i f . Int. Organs, (1970) 16, 1. Sawyer, P. Ν . , and S. Srinivasan, "New Approaches in the Selection of Materials Compatible with Blood". Contract PH43-68-75, December 1970. Gott, V. L . and with Blood", Contrac K i r k , J. E., "Mucopolysaccharides and Thromboplas tin in the Vessel Wall, In: Biophysical Mechanisms in Vascular Hemostasis and Intravascular Thrombo sis", Ed. P. N. Sawyer, Appelton-Century-Crofts, New York, 1965. Bruck, S. D . , "Blood Compatible Synthetic Polymers - An Introduction", Charles C. Thomas, S p r i n g f i e l d , Ill., 1974. Halpern, B. D . , R. Shibakawa, H. Cheng, and C. Cain, "Non-Clotting P l a s t i c Surfaces", Report PB178469, June 1967. Nelson, L . Μ., "Development of P o l y e l e c t r o l y t e Complexes as Anti-Thrombogenic Materials", Report PB-177694, January 1968. Kusserow, B. K . , R. W. Larrow and J. E . Nichols, "Analysis and Measurement of the Effects of Mate rials on Blood Leukocytes, Erythrocytes and P l a t e lets", Contract N01-HB-8-1427, March 1975.

ACKNOWLEDGEMENTS This work was supported by the Biomaterials Program of the Division of Blood Diseases and Resources, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland under Contract No. PH43-68-1388 and N01-HB-3-2950. Valuable and stimulating discussions were held with Dr. S. D. Bruck, Project Officer of the Biomaterials Program. Contributions of Drs. R. L. Camp, J. F. Gaasch and L. S. Gonsior are gratefully ac knowledged.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

24 Fibrous Capsule Formation and Fibroblast Interactions at Charged Hydrogel Interfaces J. J. ROSEN and D. F. GIBBONS Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106 L. A. CULP Department of Microbiology, Case Western Reserve University, Cleveland, Ohio 44106

Precise measuremen properties of an implanted material requires a well defined materials system and a method for accurately evaluating variations in the response. Ionogenic hydrogels have been used as a materials system in this study to evaluate the effect of charge on the early phases of fibrous capsule formation. Image analysis techniques were used to quantitatively measure the response. Specific aspects of fibroblast interactions with these materials were also investigated using cell culture techniques. The fibrous capsule is a layer of connective tissue formed as part of the biological response to an implanted foreign material. It consists primarily of collagen fibers, but may also contain a variety of inflammatory cell types and new capillaries, depending on the degree of the response(1). It has been previously demonstrated in vivo, that the addition of charged functional groups to a hydrogel network can significantly alter the early phases of the response (2). An understanding of the mechanisms responsible for these changes is an essential step in the development of materials which can fulfill the varied requirements encountered in soft-tissue implant applications. Although many attempts have been made to correlate cell attachment with properties of the substrate material in culture (.3 they have generally been limited by an ability to uniquely vary the specific material parameter of interest. The hydrophilic gel polymers based on hydroxyethyl methacrylate (HEMA) represent a class of well-characterized materials which are particularly well suited to isolate the effect of surface charge on cell attachment kinetics. The same hydrogel compositions were used in the implant study and for cell culture substrates. This allowed direct comparisons to be made between the effect of surface charge on 329

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

330

HYDROGELS FOR

MEDICAL AND

RELATED APPLICATIONS

t h e i n v i v o r e a c t i o n , and on t h e c e l l u l a r r e s p o n s e in vitro.

observed

Methods and M a t e r i a l s Monomers. 2 - H y d r o x y e t h y l m e t h a c r y l a t e as r e c e i v e d f r o m P o l y s c i e n c e s , I n c . , c o n t a i n e d between 0.25 and 0 . 4 0 p e r c e n t m e t h a c r y l i c a c i d as an i m p u r i t y , 2 0 0 - 4 0 0 ppm MEHQ as an i n h i b i t o r and had a pH o f 6.00 t o 6 . 2 0 . Adsorption with potassium carbonate (anhydrous) lowered the m e t h a c r y l i c a c i d c o n t e n t t o b e l o w 0.15 p e r c e n t and r e d u c e d t h e i n h i b i t o r s l i g h t l y t o 1 5 0 - 2 0 0 ppm. The monomer p u r i t y l e v e l s were d e t e r m i n e d u s i n g gas c h r o m a t o g r a p h y . The f i n a l pH was 6.85 t o 6 . 9 0 . Methacrylic a c i d ( P o l y s c i e n c e s , I n c . ) ( b . p . = 5 9 ° C , 10mm Hg.) and d i e t h y l ami no e t h y l m e t h a c r y l a t e (Rohm and Haas C o . ) ( b . p . = 9 0 ° C , 3 mm Hg.) were vacuum d i s t i l l e f i n a l p u r i t y was c h e c k e e t h y l e n e g l y c o l d i m e t h a c r y l a t e ( P o l y s c i e n c e s , I n c . ) was t h e c r o s s l i n k i n g a g e n t and was u s e d as r e c e i v e d . Polymerization. S h e e t s o f h y d r o p h i l i c g e l s were p r e p a r e d by s o l u t i o n p o l y m e r i z a t i o n between g l a s s p l a t e s w i t h 1.0 m i l l i m e t e r g l a s s s p a c e r s f o r t h e i m p l a n t m a t e r i a l s and 0.15 m i l l i m e t e r g l a s s spacers f o r the c e l l c u l t u r e s u b s t r a t e s . P o l y m e r i z a t i o n p r o c e e d e d f o r 24 h o u r s a t room t e m p e r a t u r e . The r e a c t i o n mix c o m p o s i t i o n s a r e g i v e n i n T a b l e I. The g e l s were removed f r o m t h e g l a s s by s o a k i n g i n d i s t i l l e d w a t e r . All h y d r o g e l s were washed i n d i s t i l l e d w a t e r w h i c h was changed d a i l y , f o r a minimum o f t h r e e w e e k s . E q u i l i b r i u m water content o f t h e g e l s was d e t e r m i n e d on f u l l y h y d r a t e d s a m p l e s by m e a s u r i n g t h e p e r c e n t w e i g h t l o s s a f t e r vacuum d r y i n g a t 120°C u n t i l no f u r t h e r w e i g h t l o s s was o b s e r v e d . In V i v o . I m p l a n t s p e c i m e n s , m e a s u r i n g 7 χ 15 mm., were c u t f r o m t h e washed g e l s . The s a m p l e s were r e e q u i l i b r a t e d i n p h o s p h a t e b u f f e r e d s a l i n e s o l u t i o n f o r t h r e e days and b o i l e d f o r 30 m i n u t e s p r i o r t o i m p l a n t a t i o n . S p r a g u e - D a w l e y male r a t s , w e i g h i n g between 300 and 350 grams were used f o r a l l e x p e r i m e n t s . S u b c u t a n e o u s i m p l a n t s were p l a c e d away f r o m t h e i n c i s i o n i n a p o c k e t opened p r e c i s e l y a t t h e f a c i a l a y e r o f t h e d o r s a l s u r f a c e . The i m p l a n t s were removed w i t h t h e s u r r o u n d i n g t i s s u e s a f t e r v a r i o u s t i m e s f r o m one t o t w e n t y - o n e d a y s , f i x e d i n f o r m a l i n and p r e p a r e d f o r h i s t o l o g y by s t a n d a r d t e c h n i q u e s . Sham e x p e r i ments i n w h i c h t h e e n t i r e s u r g i c a l p r o t o c o l was p e r f o r m e d , b u t w i t h no m a t e r i a l i m p l a n t e d , s e r v e d as c o n t r o l s . The t i s s u e r e s p o n s e was e v a l u a t e d q u a l i t a t i v e l y on t h e b a s i s o f c e l l t y p e s , d e g r e e o f g r a n u l a t i o n , m o r p h o l o g y o f c o l l a g e n o r g a n i z a t i o n and i n t e g r i t y of implant m a t e r i a l . A modified tri-chrome stain d i f f e r e n t i a t e d t h e c o l l a g e n f i b e r s f r o m s u r r o u n d i n g f e a t u r e s and provided the b a s i s f o r q u a n t i t a t i v e a n a l y s i s of the f i b r o u s c a p s u l e . C o m p u t o r - a s s i s t e d image a n a l y s i s u s i n g a M i l l i p o r e p a r t i c l e c o u n t i n g m i c r o s c o p e was u s e d t o measure t h e mean

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

24.

ROSEN ET AL.

Fibrous

Capsules

and

Fibroblasts

331

d i s t a n c e i n m i c r o n s f r o m t h e m a t e r i a l i n t e r f a c e t o t h e edge o f the f i b r o u s c a p s u l e , t h e percentage o f t h e capsule c r o s s s e c t i o n a l area a c t u a l l y f i l l e d w i t h c o l l a g e n f i b e r s , and t h e t o t a l c o l l a g e n area i n square microns p e r u n i t l e n g t h along t h e m a t e r i a l i n t e r f a c e . A m o d i f i e d n u c l e a r s t a i n was u s e d t o c o u n t t h e number o f c e l l s p r e s e n t p e r u n i t a r e a a d j a c e n t t o t h e m a t e r i a l i n t e r f a c e . A l l measurements were made a t s e v e n l o c a t i o n s a d j a c e n t t o t h e i n t e r i o r s u r f a c e o f t h e i m p l a n t , and were r e p e a t e d on two s e c t i o n s f o r e a c h m a t e r i a l a n d e a c h i m p l a n t time. The r e s u l t s a r e r e p o r t e d as t h e mean o f t h e s e m e a s u r e ments. In V i t r o . A l l c e l l c u l t u r e e x p e r i m e n t s were p e r f o r m e d u s i n g t h e S w i s s 3T3 c e l l l i n e , c o n t a c t i n h i b i t e d mouse f i b r o blasts. O n l y c e l l s between t h e i r 1 0 t h and 2 0 t h p a s s a g e were used f o r t h i s s t u d y . Cell m i n i m a l e s s e n t i a l mediu t i m e s t h e normal c o n c e n t r a t i o n o f amino a c i d s and v i t a m i n s (MEMX4), p e n i c i l l i n ( 2 5 0 u n i t s / m l ) , s t r e p t o m y c i n ( 0 . 2 5 m g / m l ) , and 10 p e r c e n t d o n o r c a l f s e r u m . The DNA o f t h e s o u r c e c e l l s was l a b e l l e d w i t h ^ H - t h y m i d i n e by g r o w i n g c e l l s f o r 24 h o u r s i n media c o n t a i n i n g 0.1 y C i / m l o f H - t h y m i d i n e f o l l o w e d by 48 h o u r s o f g r o w t h i n n o n - l a b e l l e d media ( 6 ) . T h i s t e c h n i q u e p r o v i d e d a method f o r c o u n t i n g t h e c e l l s a n d d e t e r m i n i n g t h e r e l a t i v e a b i l i t y o f S w i s s 3T3 f i b r o b l a s t s t o a t t a c h t o s u b s t r a t e s o f v a r i o u s hydrogel compositions. The s u b s t r a t e s were p r e p a r e d f r o m washed h y d r o g e l membranes e q u i l i b r a t e d i n p h o s phate b u f f e r e d s a l i n e . D i s c s ( 1 5 mm d i a m e t e r ) o f t h e g e l s were p l a c e d i n t h e a t t a c h m e n t media f o r two h o u r s p r i o r t o t h e attachment assay. The a t t a c h m e n t media c o n t a i n e d p h o s p h a t e b u f f e r e d s a l i n e s u p p l e m e n t e d w i t h d i v a l e n t c a t i o n s ( 1 0 0 mg/1 M g S 0 £ . 7 H 0 and 100 mg/1 C a C l 2 ) and 10% d o n o r c a l f s e r u m . 3

2

The a t t a c h m e n t a s s a y c o n s i s t e d o f i n o c c u l a t i n g d i s h e s c o n t a i n i n g h y d r o g e l s u b s t r a t e s w i t h a known a l i q u o t ( 2 . 0 χ 10 c e l l s i n one m i l l i l i t e r o f a t t a c h m e n t m e d i a ) o f t h e l a b e l l e d cells. A t e a c h o f t h e t i m e p o i n t s , one d i s c was removed f r o m each d i s h ( t h e r e were two d i s h e s f o r e a c h g e l c o m p o s i t i o n ) , d i p p e d t h r e e t i m e s i n t h r e e s e p a r a t e washes o f p h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g d i v a l e n t c a t i o n s ( 1 0 0 mg/1 M g S O ^ r ^ O and 100 Mg/1 C a C l 2 ) and t h e n p l a c e d i n s c i n t i l l a t i o n v i a l s containing Bray's s c i n t i l l a t i o n solution. fi

5

R e s u l t s and D i s c u s s i o n The b i o l o g i c a l r e a c t i o n t o t h e p r e s e n c e o f an i m p l a n t e d m a t e r i a l , o f t e n r e f e r r e d t o as t h e " f o r e i g n body r e s p o n s e " , i s i n f l u e n c e d by a c o m p l e x s e t o f v a r i a b l e s . A recent review ( 7 j d i f f e r e n t i a t e d some m a j o r c l a s s e s o f t h e s e v a r i a b l e s a n d a t t e m p t e d t o d i s t i n g u i s h between t h o s e a s s o c i a t e d w i t h m a t e r i a l p r o p e r t i e s a n d t h o s e i n v o l v e d i n t h e " n o r m a l " wound h e a l i n g process.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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The l a r g e number o f p o t e n t i a l l y r e l e v a n t f a c t o r s has sometimes made p r e v i o u s i n v e s t i g a t i o n s d i f f i c u l t t o c o n t r o l and t h e i r r e s u l t s d i f f i c u l t t o i n t e r p r e t . The i s s u e o f what c o n s t i t u t e s a " b i o c o m p a t i b l e " m a t e r i a l i n t h e s o f t t i s s u e s has been f u r t h e r c o n f u s e d by a p e r s i s t a n t n o t i o n t h a t a t h i n f i b r o u s membrane a r o u n d an i m p l a n t i s a l w a y s a " g o o d " r e a c t i o n and a t h i c k c a p s u l e i s i n d i c a t i v e o f a s e v e r e o r " b a d " r e s p o n s e . T h i s c o n c e p t was f u r t h e r e d by L a i n g ' s e a r l y s t u d y ( 8 ) o f i m p l a n t e d m e t a l s w h i c h d e m o n s t r a t e d t h a t an i n c r e a s e i n m e t a l i o n c o n c e n t r a t i o n o r i o n t o x i c i t y c o r r e l a t e d w i t h an i n c r e a s e i n t h e s u r r o u n d i n g f i b r o u s membrane t h i c k n e s s . Although i t i s true that chronic inflammation, t i s s u e n e c r o s i s , o r c o n t i n u e d c e l l u l a r p r o l i f e r a t i o n w o u l d be c o n s i d e r e d u n d e s i r a b l e i n a l m o s t any c l i n i c a l a p p l i c a t i o n o f an i m p l a n t e d m a t e r i a l , i t doe f i b r o u s capsule per s e sequence o f t h e b i o l o g i c a l r e a c t i o n . F o r e x a m p l e , when m e c h a n i c a l s t a b i l i t y i s d e s i r e d t o l i m i t l a t e r a l movement, s u c h as i n a hydrocephalus shunt, a dense, well organized a c e l l u l a r , and n o n a d h e r e n t c a p s u l e w o u l d a l l o w f o r l o n g i t u d i n a l g r o w t h adjustments w h i l e p r o v i d i n g the necessary l a t e r a l s t a b i l i t y . In t h e same a p p l i c a t i o n , t h e f o r m a t i o n o f f i b r o u s t i s s u e i n o r a r o u n d t h e d r a i n s e c t i o n o f t e n l e a d s t o b l o c k a g e and e v e n t u a l d e v i c e f a i l u r e . A much b e t t e r u n d e r s t a n d i n g o f b a s i c i n t e r a c t i o n s between m a t e r i a l p a r a m e t e r s and b i o l o g i c a l m e d i a t o r s i s needed b e f o r e t h i s h i g h l e v e l o f c o n t r o l o v e r t h e r e a c t i o n can be a c h i e v e d . D u r i n g t h i s i n v e s t i g a t i o n o u r p r i m a r y o b j e c t i v e was t o determine i f the charge of the m a t e r i a l s i g n i f i c a n t l y a f f e c t e d t h e p r o c e s s o f f i b r o u s c a p s u l e f o r m a t i o n . The h y d r o g e l s y s t e m was s e l e c t e d as t h e m a t e r i a l model b e c a u s e t h e c h a r g e c o u l d be v a r i e d by c h a n g i n g t h e c h e m i c a l c o m p o s i t i o n o f t h e f u n c t i o n a l g r o u p s w i t h o u t c h a n g i n g t h e main c h a i n m e t h a c r y l a t e s t r u c t u r e . The i n i t i a l monomer r e a c t i o n s o l u t i o n s , as shown i n T a b l e I, a l l c o n t a i n e d t h e same p e r c e n t o f c r o s s l i n k i n g a g e n t and t h e same amount o f w a t e r . A f t e r s w e l l i n g t o e q u i l i b r i u m t h e h i g h l y a c i d i c g e l s c o n t a i n e d s l i g h t l y more w a t e r (47%) t h a n t h e most b a s i c g e l s ( 4 1 % ) . These d i f f e r e n c e s may have c o n t r i buted to the observed v a r i a t i o n s i n the b i o l o g i c a l response, but t h e y were c o n s i d e r e d t o be s m a l l when compared w i t h t h e w i d e r a n g e o f c h a r g e d e n s i t y and s i g n t h a t was p o s s i b l e w i t h t h i s system. Scanning microscopy o f the polymers r e v e a l e d f a i r l y u n i f o r m , smooth s u r f a c e s w i t h s i m i l a r m o r p h o l o g y f o r a l l compositions. I t i s now w e l l e s t a b l i s h e d (7) t h a t many o t h e r e x p e r i m e n t a l p a r a m e t e r s must a l s o be c o n s i d e r e d as p o s s i b l e sources of a r t i f a c t during the e v a l u a t i o n of implanted m a t e r i a l s . To m i n i m i z e t h e i n f l u e n c e o f i m p l a n t edge and shape e f f e c t s ( 9 ) , o n l y t h e t i s s u e a d j a c e n t t o t h e c e n t r a l r e g i o n o f i m p l a n t s was i n c l u d e d i n the a n a l y s i s . The i m p l a n t , i t s e l f , was p l a c e d i n

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

24.

ROSEN ET AL.

Fibrous

Capsules

and

Fibroblasts

333

a " p o c k e t " opened by b l u n t d i s s e c t i o n away f r o m t h e s i t e o f t h e initial incision. V a r i a t i o n s due t o a n a t o m i c a l l o c a t i o n were r e d u c e d by a l w a y s p l a c i n g t h e i m p l a n t d i r e c t l y o v e r t h e m u s c l e f a c i a i n a p p r o x i m a t e l y i d e n t i c a l p o s i t i o n s f o r each a n i m a l . By c h o o s i n g t h i s p a r t i c u l a r p l a c e m e n t f o r t h e i m p l a n t , new c o l l a g e n formed a s p a r t o f t h e f i b r o u s c a p s u l e was e a s i l y d i f f e r e n t i a t e d from t h e " n o r m a l " t i s s u e s . F i g u r e 4 shows e x a m p l e s o f t h e i n t e r f a c e between t h e i m p l a n t (on t h e l e f t ) and t h e s u r r o u n d i n g t i s s u e s . Within the fibrous capsule, the collagen fibers f i l l e d only part o f the t i s s u e space. The r e m a i n d e r o f t h e c r o s s - s e c t i o n a l c a p s u l e a r e a was f i l l e d w i t h c e l l s , f l u i d , o r s m a l l v e s s e l s . The p e r centage o f the capsule area a c t u a l l y f i l l e d w i t h c o l l a g e n , r e f e r r e d to i n Tables I I III and IV a s " C o l l a g e n D e n s i t y " v a r i e d w i t h implant tim r e l a t e d t o t h e morpholog The t h i c k n e s s o f t h e c a p s u l e m u l t i p l i e d by t h e c o l l a g e n d e n s i t y was p r o p o r t i o n a l t o t h e t o t a l c o l t a g e n p r o d u c e d p e r u n i t a r e a along t h e capsule c r o s s - s e c t i o n . T h i s v a l u e was o b t a i n e d f r o m a r e a measurements made on 176 m i c r o m e t e r segments o f c a p s u l e f o r a l l samples and i s r e p o r t e d a s " R e l a t i v e Amount C o l l a g e n " i n Tables I I , I I I , IV. This a r b i t r a r y b u t c o n s t a n t u n i t o f c a p s u l e l e n g t h r e p r e s e n t e d a p p r o x i m a t e l y one m i c r o s c o p i c f i e l d a t 400x magnification. A p a r a l l e l measurement o f t o t a l c e l l u l a r a c t i v i t y i n t h e v i c i n i t y o f t h e i m p l a n t was d e t e r m i n e d by c o u n t i n g t h e n u c l e i o f a l l c e l l s w i t h i n 250 m i c r o m e t e r s o f t h e i m p l a n t s u r f a c e and normalizing t o a constant unit area. T h i s c o u n t was n o t l i m i t e d t o t h e c a p s u l e r e g i o n b e c a u s e s i g n i f i c a n t v e s s e l d e v e l o p m e n t and other c e l l a c t i v i t y often occurred outside the actual fibrous capsule. The number o f c e l l s p e r u n i t a r e a i n normal c o n n e c t i v e t i s s u e i s r e l a t i v e l y l o w ( a s i n d i c a t e d by t h e s u r g i c a l c o n t r o l ) and t h e r e f o r e , t h e v a l u e f o r t o t a l c e l l u l a r a c t i v i t y i s representative o f c e l l s that are d i r e c t l y involved i n the response t o the implant m a t e r i a l . The e n t i r e r a n g e o f t h e b i o l o g i c a l r e s p o n s e , f o r a l l m a t e r i a l s t e s t e d , was w i t h i n t h e l i m i t s o f what h a s been c o n s i d e r e d " m i l d " ( 8 ) . C o n t r a r y t o p r e v i o u s work ( 2 ) none o f t h e hydrogel m a t e r i a l s c o n s i s t a n t l y e l i c i t e d a "gross purulent exudate". O b v i o u s i n f e c t i o n s o f t h i s t y p e o c c u r r e d r a r e l y and did not correlate with the material compositions. The i n v i v o e x p e r i m e n t s o f t h i s s t u d y have d e m o n s t r a t e d s i g n i f i c a n t and r e p r o d u c i b l e c h a n g e s i n b o t h t h e amount o f c o l l a g e n p r o d u c e d and i t s o r g a n i z a t i o n a l m o r p h o l o g y a s a d i r e c t r e s u l t o f v a r i a t i o n s i n t h e charge o f t h e hydrogels. The b a r g r a p h s i n F i g u r e s 1 , 2 , a n d 3 show, f o r e x a m p l e , t h a t t h e c a p s u l e formed around p o s i t i v e l y charged hydrogels d u r i n g t h e f i r s t t h r e e weeks a f t e r i m p l a n t a t i o n i s t h i c k e r t h a n the capsules around o t h e r h y d r o g e l s . This r e s u l t agrees w i t h

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

TABLE I COMPOSITION OF HYDROGEL MONOMER SOLUTIONS COMPOSITION (Volu me Percent) DEAEMA HEMA MAA

HYDROGEL MONOMER (Mole Percent) 100 HEMA

100.0

95 HEMA, 5 MAA 80 HEMA,20 MAA 60 HEMA,40 MAA

96.0 84.0 67.5

95 HEMA, 5 DEAEMA 80 HEMA,20 DEAEMA 60 HEMA,40 DEAEMA

94.0 75.5 51.0

4.0 16.0 32.5 6.0 24.5 49.0

95 HEMA,2.5 MAA,2. 80 HEMA, 10 MAA, 60 HEMA, 20 MAA, HEMA= 2-Hydroxyethyl methacrylate, MAA= M e t h a c r y l i c a c i d , DEAEMA= Diethyl ami no ethyl methacrylate, Crosslinker= Tetraethylene g l y c o l dimethacrylate: 2.2 mole percent, D i s t i l l e d water: 33.0 volume percent, Initiator: (NH^nSOg: 0.5 mole percent Reducing Agent: m^^S 0.5 mole percent. 1

IN

TABLE I I VIVO RESPONSE TO HYDROGELS IMPLANTED FOR ONE DAY

HYDROGEL MONOMER (Mole Percent)

CAPSULE THICKNESS (Micrometers) MEAN (S.D.)

COLLAGEN DENSITY (Percent) MEAN (S.D.)

RELATIVE AMOUNT COLLAGEN MEAN (S.D.)

TOTAL CELLULAR ACTIVITY MEAN (S.D.)

100 HEMA

27.1 (11.9)

54.4 ( 8.4)

14.2 ( 6.2)

192.1 (35.6)

95 HEMA, 5 MAA 60 HEMA,40 MAA

27.8 ( 4.2) 41.8 ( 8.6)

48.9 (10.6) 60.9 ( 9.7)

14.9 ( 3.9) 24.0 ( 9.0)

190.0 (26.6) 133.9 (20.6)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA

63.8 (19.8) 77.1 ( 7.2)

68.4 ( 9.4) 55.9 (10.9)

42.4 (11.5) 36.7 (13.5)

207.3 (57.9) 361.7 (49.6)

95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA

37.8 (10.4) 19.2 ( 6.9)

48.9 (10.6) 60.9 ( 9.7)

17.1 (9.8) 11.5 ( 3.8)

223.1 (55.5) 227.4 (48.9)

31.7 (10.6)

34.1 (11.7)

67.5 (17.7)

SURGICAL SHAM 1. 2. 3. 4.

4

1

2

3

Percent o f f i b r o u s capsule composed o f c o l l a g e n f i b e r s . Area o f c o l l a g e n f i b e r s p r o p o r t i o n a l to new c o l l a g e n formation. Number o f c e l l s per u n i t area adjacent to implant. Values obtained from connective t i s s u e adjacent to s u r g i c a l l y prepared site.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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TABLE I I I IN VIVO RESPONSE TO HYDROGELS IMPLANTED FOR ONE WEEK HYDROGEL MONOMER (Mole Percent)

CAPSULE THICKNESS (Micrometers) MEAN (S.D.)

COLLAGEN DENSITY (Percent) MEAN (S.D.)

100 HEMA

84.8 (11.5)

81.1 ( 5.8) 59.6 (15.1)

130.6 (26.7)

95 HEMA, 5 MAA 60 HEMA,40 MAA

97.9 (16.3) 79.4 (20.5)

65.7 ( 7.8) 61.4 ( 8.4)

63.7 ( 8.4) 46.9 (13.2)

87.6 (14.3) 159.8 (22.7)

87.7 (16.6) 85.3 (18.1)

54.6 (14.8) 62.1 ( 5.1)

50.7 ( 8.9) 53.1 ( 7.3)

133.9 (25.5) 149.5 (26.2)

51.8 ( 7.1)

87.0 (11.9)

88.5 (17.8)

RELATIVE AMOUNT COLLAGEN MEAN (S.D.)

TOTAL CELLULAR ACTIVITY MEAN (S.D.)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA 95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA SURGICAL SHAM

TABLE IV IN VIVO RESPONSE TO HYDROGELS IMPLANTED FOR THREE WEEKS HYDROGEL MONOMER (Mole Percent)

CAPSULE THICKNESS (Micrometers) MEAN (S.D.)

COLLAGEN DENSITY (Percent) MEAN (S.D.)

RELATIVE AMOUNT COLLAGEN MEAN (S.D.)

TOTAL CELLULAR ACTIVITY MEAN (S.D.)

100 HEMA

94.1 (11.6)

77.4 ( 6.4)

68.0 (11.6)

143.3 (25.0)

95 HEMA, 5 MAA 60 HEMA,40 MAA

75.6 ( 7.6) 50.2 ( 7.9)

67.6 ( 6.2) 79.2 ( 6.5)

50.9 ( 5.6) 39.8 ( 7.7)

71.2 ( 6.7) 81.2 (16.3)

130.9 (28.0) 143.2 (17.9)

59.2 ( 4.9) 60.4 ( 5.5)

65.7 ( 7.1) 77.0 (15.4)

148.6 (19.5) 178.3 (14.2)

64.2 (14.6) 70.6 ( 5.8)

62.3 (10.8) 85.1 ( 7.3)

54.7 (11.2) 60.0 ( 6.4)

99.9 (16.9) 116.7 (21.9)

36.4 (10.1)

61.9 (17.2)

54.9 (16.0)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA 95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA SURGICAL SHAM

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

60 % HEMA

100 %-HEMA

4 0 % DEAEMA

6 0 % HEMA

60%

HEMA

4 0 % MA

Bar configurations used in Figures 2, 2, and 3 to represent the indicated hydrogel compositions

DAYS

IMPLANT

TIME

Figure 1. The effect of hydrogel composition and implant time on the mean thickness of the fibrous capsule

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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DAYS

TIME

Figure 2. The effect of hydrogel composition and implant time on the percent of the fibrous capsule composed of collagen fibers

21 DAYS

IMPLANT

TIME

Figure 3. The effect of hydrogel composition and implant time on the relative amount of collagen produced during the formation of the fibrous capsule

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figure 4. Fibrous capsule adjacent to hydrogel implants (at left) three weeks after implantation, 4a. 60% HEMA-40% MAA; 4b. 60% HEMA-40% DEAEMA; 4c. 100% HEMA. The tissues were stained with hematoxilin and eosin (340X ).

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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t h e q u a l i t a t i v e o b s e r v a t i o n s made by B a r v i c {2). Quantitative e v a l u a t i o n p r o v i d e d by t h e image a n a l y s i s t e c h n i q u e s w h i c h have been summarized h e r e and d e s c r i b e d i n d e t a i l ( 1 0 ) r e v e a l s t h a t although the t o t a l t h i c k n e s s i s g r e a t e r f o r p o s i t i v e l y charged g e l s , t h e d e n s i t y o f c o l l a g e n f i b e r s d e c r e a s e s and t h e a c t u a l amount o f c o l l a g e n p r o d u c e d r e m a i n s e s s e n t i a l l y c o n s t a n t d u r i n g t h e same t h r e e week p e r i o d . To b e g i n t o d i f f e r e n t i a t e between p o s s i b l e models t h a t c o u l d a c c o u n t f o r t h e s e c h a n g e s , f i b r o b l a s t - h y d r o g e l i n t e r a c t i o n s were s t u d i e d u n d e r more c o n t r o l l e d c o n d i t i o n s . C e l l c u l t u r e s were used t o i n v e s t i g a t e t h e k i n e t i c s o f f i b r o b l a s t a t t a c h m e n t t o hydrogel s u b s t r a t e s . S u b s t r a t e p o l y m e r c o m p o s i t i o n s were c h o s e n f r o m , and i d e n t i c a l t o t h o s e used f o r t h e i n v i v o e x p e r i m e n t s . O n l y some o f t h e s e c o m p o s i t i o n s have been i n c l u d e d i n t h i s p a p e r to i l l u s t r a t e t h e rang The r e s u l t s o f t h p r e s e n t e d i n F i g u r e s 5 , 6 , and 7 . The v a l u e s f o r a t t a c h m e n t o f c e l l s a r e r e p o r t e d as a percentage o f t h e t o t a l c e l l s a v a i l a b l e f o r attachment. The s u b s t r a t e s were e q u i l i b r a t e d i n p h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g d i v a l e n t c a t i o n s and c a l f serum (see Methods). Fresh s o l u t i o n s o f t h i s composition also served as t h e a t t a c h m e n t m e d i a . For the short duration o f these e x p e r i m e n t s , t h e a b s e n c e o f v i t a m i n s and amino a c i d s n o r m a l l y f o u n d i n g r o w t h media d i d n o t a f f e c t t h e r a t e o r e x t e n t o f c e l l attachment ( 1 1 ) . The s p e c i f i c r o l e o f t h e serum p r o t e i n s has n o t been e l a b o r a t e d d u r i n g t h i s phase o f t h e s t u d y , a l t h o u g h i t i s n o t u n r e a s o n a b l e t o assume t h a t t h e s e p r o t e i n s i n t e r a c t d i f f e r e n t l y w i t h hydrogel s u r f a c e s o f d i f f e r e n t charges. The p r e s e n c e o r a b s e n c e o f serum has been shown t o i n f l u e n c e c e l l a t t a c h m e n t i n o t h e r model s y s t e m s ( 1 2 ) a n d i s p r o b a b l y i n v o l v e d i n t h e mechanisms r e s p o n s i b l e f o r t h e o b s e r v e d v a r i a t i o n s i n t h e h y d r o gel system. The i n i t i a l o b j e c t i v e o f t h i s s t u d y has been t o determine i f f i b r o b l a s t s respond d i f f e r e n t l y t o s u b s t r a t e s o f d i f f e r e n t c h a r g e d e n s i t y a n d s i g n when t h e r e a r e no i n t e r a c t i o n s w i t h o t h e r c e l l t y p e s , complement o r c l o t t i n g p r o t e i n s . The g r a p h s i n F i g u r e s 5 , 6 , and 7 show t h a t f i b r o b l a s t s do a l t e r t h e i r a t t a c h m e n t b e h a v i o r when t h e c h a r g e o f t h e s u b s t r a t e is varied. These changes o c c u r d u r i n g t h r e e d i s t i n c t p h a s e s of t h e attachment process. The f i r s t phase b e g i n s when t h e c e l l s f i r s t come i n c o n t a c t w i t h t h e s u b s t r a t e m a t e r i a l and ends when c e l l s begin t o a t t a c h r a p i d l y . T h i s so c a l l e d " l a g " phase i s o f t e n i n t e r p r e t e d as a time o f c e l l "accomodation" o r " a d j u s t ment" t o t h e s u b s t r a t e . The s e c o n d phase i s a p e r i o d o f r a p i d a t t a c h m e n t c h a r a c t e r i z e d by a s t e e p e r s l o p e on t h e a t t a c h m e n t curve. The t h i r d phase i s t h e " p l a t e a u " o r " l e v e l i n g - o f f " phase when t h e r e a r e no f u r t h e r s i g n i f i c a n t i n c r e a s e s i n t h e total c e l l s attached. F o r t h e n e u t r a l g e l s t h a t c o n t a i n e d no c h a r g e g r o u p s , t h e l a g phase l a s t e d between f i v e and t e n m i n u t e s . The p e r i o d o f r a p i d a t t a c h m e n t was o v e r w i t h i n f o r t y - f i v e

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Figures 5, 6, and 7 are the results of an assay to determine the effect of hydrogel substrate composition on the attachment of Swiss 3T3 fibroblasts. The cells are labelled with H-thymidine and inocculated into dishes containing the hydrogel substrates. After times varying from 5 min to 2 hr, substrate discs are removed, washed, and counted using scintillation techniques to determine the percent of available cells that attached to the substrates. 3

Ο 60HEMA - 40 DEAEMA ο Θ0ΗΕΜΑ - 20-DEAEMA • 10 OHEMA

-o ι

TIME Figure 5.

IN

MINUTES

Attachment of Swiss 3T3 fibroblasts to hydrogel substrates

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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m i n u t e s and t h e p l a t e a u a t t a c h m e n t v a l u e was a p p r o x i m a t e l y t w e n t y - f i v e p e r c e n t o f t h e a v a i l a b l e c e l l s . As n e g a t i v e c h a r g e s a r e added t o t h e g e l n e t w o r k , t h e l a g phase r e m a i n s e s s e n t i a l l y u n c h a n g e d , b u t t h e s l o p e o f r a p i d a t t a c h m e n t and t h e p l a t e a u value decrease. If positive c h a r g e s a r e a d d e d , t h e l a g phase goes t o w a r d z e r o and t h e s l o p e o f r a p i d a t t a c h m e n t i n c r e a s e s dramatically. The p l a t e a u v a l u e i n c r e a s e s , b u t i s n o t a t t h e maximum p e r c e n t a t t a c h e d due t o some c e l l s r e l e a s i n g f r o m t h e s u b s t r a t e a t around t h i r t y minutes. The i o n o g e n i c n e u t r a l g e l s w h i c h c o n t a i n e q u i m o l a r amounts o f p o s i t i v e and n e g a t i v e c h a r g e g r o u p s , have p r o p o r t i o n a t e l y l o w e r v a l u e s f o r e a c h phase t h a n any o f t h e o t h e r m a t e r i a l s . Conclusions The i o n o g e n i c h y d r o g e l e v a l u a t i n g the e f f e c t o s o f t - t i s s u e response. Q u a n t i t a t i v e e v a l u a t i o n and c o m p a r i s o n o f t h e c a p s u l e t h i c k n e s s , c o l l a g e n d e n s i t y and t h e amount o f c o l l a g e n p r o d u c e d , and t h e r e l a t i v e d e g r e e o f c e l l u l a r a c t i v i t y were made p o s s i b l e by t h e image a n a l y s i s o f s p e c i a l l y p r e p a r e d h i s t o l o g y s e c t i o n s o f the i n v i v o response to the implanted hydrogels. The n e u t r a l , homopolymer o f h y d r o x y e t h y l m e t h a c r y l a t e i s p r o g r e s s i v e l y e n c a p s u l a t e d d u r i n g the f i r s t t h r e e weeks. The c a p s u l e c o n t a i n s i n c r e a s i n g amounts o f c o l l a g e n w h i c h pack more e f f i c i e n t l y w i t h time t o produce a moderate, d e n s e l y packed structure. As p o s i t i v e c h a r g e s a r e added by c o - p o l y m e r i z i n g with d i e t h y l aminoethyl m e t h a c r y l a t e , s i g n i f i c a n t q u a n t i t i e s of c o l l a g e n a r e p r o d u c e d i m m e d i a t e l y a f t e r i m p l a n t a t i o n and a r e a c c o m p a n i e d by v e r y h i g h c e l l u l a r a c t i v i t y . A l t h o u g h new c o l l a g e n c o n t i n u e s t o be p r o d u c e d , e f f i c i e n t p a c k i n g i s h i n d e r e d ( p e r h a p s by t h e h i g h c e l l a c t i v i t y ) , and v e r y t h i c k , l o o s e , capsules r e s u l t . The f a c t t h a t t h e c a p s u l e does n o t o r g a n i z e e f f i c i e n t l y w i t h time i s c h a r a c t e r i s t i c of the response to these m a t e r i a l s and i s i n d i c a t e d by t h e c o n s i s t a n t l y l o w v a l u e f o r t h e collagen density. F i g u r e 4b i s t y p i c a l o f t h e c a p s u l e formed around p o s i t i v e l y charged g e l s . The a c i d i c g r o u p s a l s o have i n i t i a l l y high c e l l a c t i v i t y , but t h i s l e v e l decreases d u r i n g t h e s e c o n d week t o a l l o w f o r dense c a p s u l e f o r m a t i o n w i t h o u t l a r g e q u a n t i t i e s of c o l l a g e n being produced. When e q u i m o l a r q u a n t i t i e s o f a c i d i c and b a s i c g r o u p s a r e present i n the g e l , the e a r l y c e l l a c t i v i t y decreases w i t h i n a f e w d a y s and r e m a i n s s i g n i f i c a n t l y l o w e r t h a n t h e n e u t r a l g e l throughout the response. The c o l l a g e n p r o d u c t i o n a r o u n d t h e s e g e l s d e v e l o p s g r a d u a l l y and r e m a i n s c o n s t a n t a t v a l u e s l e s s t h a n o r equal to the n e u t r a l g e l . The c e l l c u l t u r e a t t a c h m e n t a s s a y showed t h a t as t h e g e l s a r e changed f r o m h i g h l y b a s i c t h r o u g h n e u t r a l t o h i g h l y acidic: 1) the time f o r c e l l accomodation or " l a g phase" i s

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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increased, 2 ) t h e s l o p e o f t h e r a p i d a t t a c h m e n t phase d e c r e a s e s s h a r p l y , and 3 ) t h e maximum p e r c e n t o f a t t a c h e d c e l l s d e c r e a s e s . C e l l attachment t o ionogenic, e l e c t r o n e u t r a l gels i s p r o p o r t i o n a t e l y l o w e r t h a n f o r any o t h e r g e l s . The i m p l i c a t i o n s o f t h e c e l l c u l t u r e e x p e r i m e n t s c a n n o t be f u l l y a p p r e c i a t e d u n t i l f u r t h e r s t u d i e s w h i c h a r e d e s i g n e d t o e l a b o r a t e t h e mechanisms o f a d h e s i o n t o h y d r o g e l substrates a r e c o m p l e t e d . A l t h o u g h d i r e c t c o r r e l a t i o n between c e l l c u l t u r e * i n v i v o experiments i s premature a t t h i s t i m e , c e r t a i n i n t e r e s t i n g trends are apparent. For example, t h e p o s i t i v e l y c h a r g e d g e l s have a h i g h e r l e v e l o f c e l l a c t i v i t y a r o u n d i m p l a n t s and a much more r a p i d and e x t e n s i v e a t t a c h m e n t o f fibroblasts in culture. I f t h e subsequent r e l e a s e o f c e l l s f r o m p o s i t i v e l y c h a r g e d s u b s t r a t e s r e p r e s e n t s c e l l damage o r d e a t h and t h i s a l s o o c c u r density o f collagen i n numbers o f c e l l s and c e l l d e b r i s a s s o c i a t e d w i t h t h e i m p l a n t interface. a n (

The combined i n f o r m a t i o n o b t a i n e d f r o m f u r t h e r c e l l c u l t u r e and q u a n t i t a t i v e a n a l y s i s o f i n v i v o r e a c t i o n s w i l l be u s e f u l i n e v a l u a t i n g t h e r o l e o f s p e c i f i c mechanisms and b i o l o g i c a l pathways t h a t c o n t r o l t h e f o r m a t i o n o f f i b r o u s c a p s u l e s a r o u n d implanted m a t e r i a l s .

Literature Cited 1.

Kellermeyer, R.W., and K.S. Warren, J . of Exp. Med., (1970), 131, (1) 21-39. 2. Barvic, M., J . Vacik, D. Lim, and M. Zavadil, J . of Biomed. Mat. Res. (1971) 5, 225-238. 3. Grinnell, F., M. Milam, and P.A. Srere, Arch. of Biochem. and Biophys., (1972), 153, 193-198. 4. Rappaport, C., in "The Chemistry of Biosurfaces", M.L. Hair ed., Marcel Dekker, Inc., New York (1972). 5. Weiss, L., Exper. Cell Res., (1974), 83, 311. 6. Culp, L.A., J . of Cell Biol. (1974), 63, 71-83. 7. Coleman, D.L., R.N. King, and J.D. Andrade, J . of Biomed. Mat. Res. (1974), 8, (5) 199. 8. Laing, P.G., A.E. Ferguson, and E.S. Hodge, J . of Biomed. Mat. Res. (1967), 1, 135. 9. Wood, N.K., E.J. Kaminski, and R.J. Oglesby, J . of Biomed. Mat. Res., (1970), 4, 1. 10. Rosen, J.J., and D.F. Gibbons, (submitted for publication). 11. Mapstone, R. and L.A. Culp, (submitted for publication). 12. Witkowski, J.A. and W.D. Brighton, Exper. Cell Res., (1972) 70, 41.

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

INDEX

Acid catalyzed process 244 Acid-impregnated support 110 Acrylamide 70 Acrylic acid -containing polymers, vena cava implantation of 320 /ethylene (EAA) copolymers and ionomers 309,312 /ethylene polyelectrolytes 312 /N-vinyl pyrrolidone ( NVP/A copolymers and ionomers 309,312,320 /A/-vinyl pyrrolidone polyelectro lytes 318 copolymers 181,182 hydrogels 181,182,252,261 Acrylonitrile 11 N-Aeryloi para-phenylene diamine ... 27 Activated-state model, Ree-Eyring's .. 122 Adhesion cell 206 promoters, silane 242,311 tension 260,269 of aqueous polymer solutions to gels and solids 273 effect of synthetic and biopoly mers on 268 Wolfram plot of 277,278 Adsorption 2 equation, Gibbs 270 of polymers 217 plasma protein 217 Agar 300 Agarose 238,243, 255,258 gel 253 AIBN ( azobisisobutyronitrile) 91,127,129,130,132,136 Albumin 27 adsorbed on PHEMA gel and polyethylene 272 bovine serum (BSA) 268,273,274 film pressure of 272 serum 53, 55 Allyltrimethylammonium bromide 16 Ammonium persulfate ...126,129,130,132,136,152 Anionic hydrogels 14 initiators 91 polymerization 140

Antibiotics Application Aqueous environments polymer solutions solutions of synthetic and bio polymers and surfactant Asparaginase Atmosphere, non-saturated Attachment media Azo initiators Azobisisobutyrate esters

24,168 112 200 273 269 25 106 331 91 94

Azobismethoxydiethoxyethyl isobutyrate ... 91,128,129,131,132,136 Azobismethoxyethyl isobutyrate 129,131,132,136 Azobismethyl isobuty rate 81, 91,127,129,130,132,136 Β

Bartenev's empirical method 120,132 Base catalyzed process 244 Basophilic granules 154 Biocompatible material 332 Biocompatibility-operational defini tions 4 Biological compatibility testing 307 Biological mediators 332 Biomaterials 2 biologically active 23 Biomedical applications of immobilized 24 enzymes 1 applications, synthetic hydrogels for studies utilizing PHEMA hydro 9 gels Biomedically important hydrogels 6 Biomolecules to gels containing carboxyl groups, coupling 26 Biopolymers on adhesion tension, effect of 268 film pressure of 271 surface tension of 269,275,279 wettability of 272 Blood compatibility 206 of ethylene/vinyl sulfonic acid copolymers and ionomers 319, 320 of vinyl acetate/crotonate co polymers and ionomers 321

347

American Chcniicai Society Lib sry 1155 16th St.

Washington, D. C. 20036 In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

348

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Blood (continued) continuous glucose analysis of 164 glucose 27,162,165 vessel walls 14 Bond, hydrophobic 299 Bonding, hydrogen 299 Bone formation 9,151 Bone induction studies 151 Borosilicate glass particles 233,234 Bovine serum albumin (BSA) 268,273,274 Bovine submaxillary mucin (BSM) .. 268 Breast augmentation 9 Brinolase 27 BSA (bovine serum albumin) .268, 273, 274 BSM (bovine submaxillary mucin) .... 268 Buckling procedure, scleral 40 Bueche-Harding method 121,128,129 Bueche's relaxation time 12 Bulk to gel network, transfer of water from 62 -grafting 254 and surface structure 204 i-Butylaminoethyl methacrylate 15 Byring's model 136 C C-HEMA gel 92-94 Calcification 15,151,156,159 Calcium determination 156 Calcium hydroxyapatite 152 Calibration 165 Calibration graph 162 Calorimetry, differential scanning (DSC) 22,142 Canine implantation studies 308 Capillaries, gel-coated glass 241 Capsule, fibrous 329,337 Capsule thickness 342 Captive-bubble technique 254 Carbonyl triads 144 Carboxymethyl cellulose 300 Carboxyl-containing copolymers 305 Carboxyl groups 26 Cartilage 14 Catalysis, intramolecular 25 Catheter 9 Cell adhesion 29,206 attachment 329 culture 329 line, Swiss 3T3 331 Cellophane 14 Cellular activity 333,342 Cellulose acetate 21 Chain ends, dangling 203 Chain transfer 92, 99 Charge surface 232,305,306,312 14

Charged groups introduced into HEMA polymer 248 Chemical potential, solvent 201 Chick embryo, hypophysectomized ... 174 Chorioallantoic membrane 176 Chromatograpy column 110 gas 105 thin-layer (tic) 105,106,111,117 Chymotrypsin 57 Clustered regions 75 Clustering functions 299,301 Co radiation 324 Coagulation time assays 4 Coating(s) 3 agents 231 copolymer 247 60

materials 229,237 parameters 325 of polyelectrolyte hydrogels onto polypropylene 311 silane and polysaccharide 245 surface 225 Coiling 201 Collagen 52,300,301 density 342 fibers 333,337 Column chromatography 110 Compatibility blood 206 testing, biological 5,307 tissue 5,206 Compression, mechanical 45 Conductivity, specific 22 Conformation, changes in 200 Conformational distribution 203 Connective tissue 52, 53, 59 Contact angle(s) dynamic 215 hysteresis 257,258 limiting advancing 259 methods 209 receding 275 test 246 two-phase 263 water 254,255 Contact lens ( es ) ... .9,38,43,46,48,49,267 Copolymer(s) (see also specific compound names)

97

blood compatibility of 319,321 coatings 247 grafts, EMA/HEMA 291 matrix 187 ratios 88 thromboresistance of 320,395 Copolymerization, radiation graft 295 Cores for trilaminate devices 187 Cornea 50

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

349

INDEX

Corneal epithelium 50 Corneal surgery 9 Creep 64 Critical surface tension of acrylic hydrogels 261 of adsorbed biopolymers 275 of biopolymers adsorbed on PHEMA and polyethylene 279 determination 260 of the hydrogels 259 ofPE 276 of PHEMA 258,276 Crosslink(s) 59 density 74,75,200 effective 200,204 region, effective 79 Crosslinker 210,245 Crosslinking 172 agents 80,9 irradiation 31 Crotonate/vinyL acetate copolymers and ionomers 321 Crotonic acid/vinyl acetate (VA/CA) copolymers and ionomers 310, 312, 321

Diffusion (continued) quasi-steady state 175 through hydrogel membranes 80 Diffusivities of water in grafted hydrogel/silastic films 302 2,3-Dihydroxypropyl methacrylate (DHPMA) 60,88 Dilatometry 22 Dimethacrylate esters 90 Dimethylaminoethyl methacrylate (DMAEMA) 12,91,247 Dip-coat 16 Dissection, blunt 333 DMAEMA ( dimethylaminoethyl methacrylate) 12,91,247 copolymers, HEMA100,101 Dog blood glucose 165 Dogs with intraoral fluoride-releasing

DEAE-methylcellulose (diethylaminoethyl-methylcellulose) .229,234 Dehydrated state 59 Dehydration of two PHEMA hydrogels 44 Density of PGMA hydrogels 42,44 Dental applications 180 Detection 109 limits of HEMA 108 Device(s) 3 design 185 evaluation of 189,194 fabrication of 189 intraoral, controlled-release 180 monolithic controlled-release 187 trilaminate 187 Dextran 238 solutions 40,43 Dextrin 243 DHPMA (2,3-dihydroxypropyl meth acrylate) 60,88 Diagnostic liquids 275 Diethyl ether 110 Diethylaminoethyl methacrylate 330 Diethylaminoethyl-methylcellulose ( DEAE-methylcellulose ) 229,234 Diethyleneglycol methacrylate 106 Differential scanning calorimetry (DSC) 22,142 Diffusion 2 coefficient 82, 83,86,181,184,300 constant, D, apparent 175 free volume 173,175

Ε

Drug delivery 9 Drug release from a polymeric chain 25 DSC ( differential scanning calo rimetry) 22,142 thermograms 145,148 Dynamic contact angles 215

EAA (ethylene/acrylic acid) 309,312 Echtblauslaz Β 109 EDM A (ethylene dimethacrylate) 105,111 EG (ethylene glycol) 8,106, 111 EGDMA (ethylene glycol dimeth acrylate ) 6, 81, 86 Elastin 53 Electrical double layer 217 Electrocapillary maximum 270 Electrodes, enzymatic 29 Electrode,fluoridespecific-ion 184 Electron microscopy 57 Electron spectroscopy for chemical analysis (ESCA) 218 Electroosmosis 216,225,241 Electroosmotic fluid flow 225,226 Electroosmotic mobility 228,234 Electrophoresis 216, 241 analytical particle 225 chambers 236 particle 225 Electrophoretic mobility 227,233,234 Electrophoretic velocity 227,235 Ellipsometry 218 Elongation curve, stress— 65,66 EMA/HEMA copolymer grafts 285,287,288,291 Embolus tests, renal 325 Encapsulation 154 Endocrine system 167 Energy effects which control stability 199

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

350

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Entrapment of biologically active molecules 23 Entrapment, enzyme 25 Entropy of sorption, standard differ ential 298 Enzymatic activity in developing muscle 176 Enzymatic electrodes 29 Enzyme(s) entrapment 25 immobilized 23, 24 microsomal 25 proteolytic 54 Eosin stain 153 Epichlorohydrin 230 Epithelium, corneal 50 Epoxy-silane surfaces 244 Equilibrium hydration 25 relative humidity ( %ERH) 45 swelling 37,38, 201,332 in dextran solution 43 linear 96 of pure homopolymers 89 under mechanical compression .... 45 volume 54 water content for HEMA/MMA copolymers 190 Erythrocytes 313 ESCA ( electron spectroscopy for chemical analysis) 218 Ethyl methacrylate 283 Ethylene /acrylic acid (EAA) 309,312 dimethacrylate (EDMA) 105,111 glycol (EG) 8,106,111 dimethacrylate (EGDMA) 6,81,86 crossliner 210 /vinyl sulfonate (EVS) 312 polyelectrolytes 318 /vinyl sulfonic acid copolymers and ionomers 309,319,320 Evaluation, quantitative 339 EVS (ethylene/vinyl sulfonate) 312 Eyring's theory 130 Eyring's zero shear viscosity 135

Film(s) hydrogel/silastic 296,302 pressure 271-273 silicone rubber 283,295 ungrafted silastic 297 Flory-Huggins polymer solution theory 61,199 Flow curves of HEMA-water mixtures 126-128 parameters for HEMA-water mixtures 129-133 past a gel 204 properties 119 resistance 69,204 system, constant-temperature 192 Fluidflow,electroosmotic 226 Fluids, receiving 191 Fluoride levels, saliva 196 partition coefficient of 181 prophylaxis 180 -releasing device ... 180,190,192,195,196 saturation solubility of 181 specific-ion electrode 184 topical application of 180 Flux 73 Force-free state 59 Foreign body response 331 Free energy of sorption, standard differ ential 301 radical polymerization 140 volume diffusion 175 Freeze etch SEM examination 211 -etching 207 PHEMA water opaque gel 212, 213 Friction coefficient 73, 75 Fugacity 45 G

β-Galactosidase 25 Gas chromatography 105 Gel(s) adhesion tension of aqueous poly mer solutions to 273 F agarose 253 aqueous polyacrylamide 76 Feedback control circuit 72 blocks 72 Fiber, collagen 33,337 -coated glass capillaries 241 Fiber network 57 containing carboxyl groups 26 Fibrinogen adsorption 21 entrapped glucose oxidase 162 Fibroblast(s) 153 flow past a 204 attachment 339,340 formation 119 interactions at charged hydrogel freely and partially draining 76 interfaces 329 freeze, PHEMA water opaque ... 212, 213 mouse 331 HEMA 98 Fibrous capsule 329,337 macroporous 15 Fick's law 181,187

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

351

INDEX

Gel(s) (continued) membranes 92 microporous 15 modifiers 91, 94 networks 60, 62 poly ( hydroxyethyl methacrylate ) PHEMA 8, 60,93,94, 96, 210,212,213,272 pMEEMA 98 -solvent interface 200 structure 69, 200 substance, immobile 75 surface 201 Gelatin 252,300 foam 153 Gibb's adsorption equation 270 Gibb's surface excess concentration .... 260 Glass capillaries 24 particles 233-235 transition 142 Glaucoma symptoms 168 Glucoamylase 25 Glucose blood 27,162,164,165 oxidase, gel entrapped 162 -6-phosphate dehydrogenase 174 Glutaraldehyde 14,57 Glyceryl methacrylate (GMA) 38,182 Glycidyl methacrylate 89 Glycosaminoglycans 53 GMA (glyceryl methacrylate) 38,182 Graft copolymerization, radiation 295 EMA/HEMA copolymer 291 HEMA 208 polymerization 18,312 water content 286,289 Grafted hydrogel ( s ) 19 Grafted hydrogel/silastic films 302 Grafting of HEMA or EMA 288 Grafting techniques 16 Granylocytes 153 Ground substance components 53 H H-thymidine 331, 340 Haematoxylin 153 HDEEMA (hydroxydiethoxyethyl methacrylate ) 88 Heat of sorption, standard differential 298 HEEMA (hydroxyethoxyethyl methacrylate ) 88 HEMA ( hydroxyethyl methacrylate ) 83, 88,105,182, 243,295,330 analysis and purification of 105 C -labeled 92 dependence of n on water content of 117

3

14

D

20

HEMA (continued) detection limits of 108 -DMAEMA copolymers 100 /EMA copolymer grafts 285,287,291 and ethyl methacrylate, radiationinduced co-graft polymerization of 283 gel 98 grafting of 288 grafts on polypropylene 208 hydrogels 21 implants, hydrocortisone succinate .. 176 -ionic methacrylate copolymers 99 -MAA copolymers 100 -MAA-DMAEMA 101 -MEEMA comonomers 100 -MEMA comonomers 98 /MMA copolymers 190 polymer, charged groups introduced into 248 R data for 113 tic of 105, 111, 117 -water mixtures 126-133 Hemodialysis 9 Heparin 14 N-Hexane 110 Homogeneous materials 3 Homopolymers 89,285 Humidity, equilibrium relative ( % ERH ) 45 Hyaluronic acid 53 Hyaluronidase 56 testicular 54 -treated tissue 55 Hydrated homogeneous membranes .. 80 Hydration degree of 184 equilibrium 96,252 of the gel 44 of hydrogels 38 Hydrocortisone sodium succinate release 167 Hydrocortisone-succinate discs 173 HEMA implants 176 release 172,175 Hydrodynamic effects 204 interactions 69 radius 77 Hydrogels acrylic 181,252,261 anionic and cationic 14 for biomedical applications, synthetic 1 biomedically important 6 coatings 16 composition, diffusion coefficients, permeabilities and 83 F

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

352

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Hydrogels (continued) contact lens 43,46, 48, 49 converted polymers used as 7 critical surface tension of the 259, 261 for dental applications 180 grafted 19 glyceryl methacrylate 38 heterogeneous phosphorylated 151 hydration of 38 implants 334,335,338 interfaces,fibrouscapsule formation andfibroblastinteractions at.... 329 ionogenic 329 membranes 80 methacrylate 88 monomer solutions 334 monomers used in 7 PGMA 39,41,42,44 PHEMA 9,43-46,8 polyelectrolyte 311 radiation grafted HEMA 21 /silastic films 296,302 substrates, attachment of fibroblasts to 340 surface 258 surface coated 16 vapor pressure and swelling pres sure of 37 water activity in 45 interface 198,206,267 sorption in radiation-grafted 295 wettability of 252 Hydrogen bonding 8,299 Hydrolysis, acid 8 Hydron polymer-Type Ν 169 Hydroquinone 106 Hydrophilic methacrylate coatings 246 methacrylate monomers 88,90,119 sites 284 Hydrophilicity of polyelectrolytes 306 Hydrophilicity using two-liquid sys tems 263 Hydrophobic and hydrogen bonding interactions 8 bond 299 sites 284 Hypophysectomy 174 Hydroxydiethoxyethyl methacrylate (HDEEMA) 88 Hydroxyethoxyethyl methacrylate (HEEMA) 88 Hydroxyethyl methacrylate (see HEMA) Hydroxyl groups 28 2-Hydroxy-3-methacryloloxypropyltrimethylammonium chloride 15 Hydroxypropylmethylcellulose 243 Hygrodynamics 48

Hygrometer 47 Hygrosensor 47 Hysteresis, relative contact angle (H ) 257,258 R

I

Image analysis techniques 329 Immobilization of biologically active molecules 23 of enzymes 23,162 of a protein 26,28 Implant(s) of hydrocortisone succinate HEMA 176 hydrogel 338 phosphorylated polyHEMA 155 rejection 153 tests, vena cava 326 Implantation of acrylic acid-containing polymers, vena cava 320 studies, canine 308 subcutaneous 8 Implanted hydrogels, in vivo response to 334,335 Inflammation 153 Infrared spectroscopy 116 Ingrowth of tissue 152 Inhibition pressure 48 Inhibitor 107 Initiator(s) 91,129,130,132 ammonium persulfate as 152 anionic 91 azo 91 organometallic 139 Insulin delivery system 29 Intercrosslink distance 201, 204 Interface(s) 201 charged hydrogel 329 film pressure of biopolymers ad sorbed at the 271 film pressure of bovine serum albu min at 273 gel-solvent 200 hydrogel/water 198,206,267 potentials 216 segment distribution in the 203 stereochemical changes at 256 Interfacial tension 2 Interstitial fluid 59 Intraoral, controlled-release device ... 180 Intraoral orthodontic appliance with device attached 195 Intrauterine device 9 Invertase 25 Ion electrode,fluoridespecific184 Ion partitioning 217 Ionic methacrylates 102 Ionogenic hydrogels 329

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

353

INDEX

Ionomers ethylene/acrylic acid 309 ethylene/vinyl sulfonic acid 309, 319, 320 vinyl acetate/crotonate 321 vinyl acetate/crontonic acid (VA/CA) 310 N-vinyl pyrrolidone/acrylic acid 309, 320 Irradiation crosslinking 311 Isopropylidineglyceryl methacrylate .. 89 Isotactic content 139 Isotonic solution 38 J

Jellies

42 Κ

K (permeation coefficient) Kinetics offibroblastattachment s

73,7 339

MEMA (continued) -HEMA comonomers 98 -HEMA copolymers 98 Membrane(s) analysis 73 choroallantoic 176 diffusion through hydrogel 80 hydrated homogeneous 80 materials 182 partition type 82 poly ( 2-hydroxyethyl methacrylate ) (pHEMA) 80,95,167 -polymer solution 202 preparation of gel 92 rate-controlling 187 water swelling of pHEMA and pMEEMA 95 Methacrylate

copolymers, HEMA-ionic 99 hydrogels, chemistry of 88 ionic 102 L monomers, hydrophilic 88,90,119 stereoregular 139 Lag phase 339 Laser scattering 204 Methacrylic acid (MAA) 6,91,105, 111, 151,247,330 Lee White test 4 Ligament prosthesis 9 Methoxydiethoxyethyl methacrylate (MDEEMA) 88 Limiting advancing contact angle 259 Linear swelling (Is) 95 Methoxyethoxyethyl methacrylate (MEEMA) 83,88,100,243 Liquids diagnostic 275 Methoxyethyl methacrylate (MEMA) 83, 88, 98,139,243 systems, two263 p-Methoxyphenol 106 used in critical surface tension de termination, organic 260 Methyl methacrylate Lysozyme (LYZ) 268,272 (MMA) 43,45,106,182 2-Methyl-5-vinyl pyridine 15 Methylcellulose 229,238,242 M Ν,Ν-Methylenebisacrylarnide 11 Methylisobutyl ketone 110 MAA (methacrylic acid 6,91,105, 111, 151,247,330 Microautoradiography 217 copolymers, HEMA100 Microcirculation 53 -DMAEMA, HEMA101 Microporous 82 Macromolecular fuzz 201 gels 15 Macromolecule, isolated 203 network 77 Macroporous 302 Microscopy, optical 207 gels 15 Microsomal enzymes 25 network 77 Mixing energies 200 Mass spectrometry 114 MMA (methyl meth Material parameters 332 acrylate) 45,106,182,190 MDEEMA ( methoxydiethoxyethyl Mobile phase 110 methacrylate ) 88 Mobilities, electroosmotic 234 Mechanical pressure 37, 44 Mechanical spring 57 Modifiers, commercially available gel 94 43 Mediators, biological 332 Molecular diameter Molecular weight 74 MEEMA ( methoxyethoxyethyl distribution 119 methacrylate) 83,88,243 Molecules, biologically active 23 -HEMA comonomers, water solu 330 bility of 100 Monomers composition on the graft water con -HEMA copolymers 100 tent, effect of 289 MEMA (methoxyethyl meth preparation 140 acrylate) 83,88,139,243

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

354

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Monomers (continued) solutions, hydrogel 334 structure and nomenclature 143 used in hydrogels 7 Mooney-Rivlin equation 64 Mouse fibroblasts 331 Mouth,fluoride-releasingdevice in a beagle's 195 Mucin 272,275 Muscle differentiation, skeletal 167 facia 333 hydrocortisone succinate HEMA implants in 176 Multiple internal reflection infrared spectroscopy 219 Myoblast proliferation 174 Ν

n

on water content of HEMA, dependence of 117 Nakagima's theory 137 Narcotic antagonist 168 Network 75 bulk to gel 62 ideal, three-dimensional 200 macroporous 77 microporous 77 segments 200, 202, 203 water in the osmotic and viscoelastic behavior of gel 60 NMR techniques 22 Nomenclature, monomer 143 Non-Newtonian viscosity 120 NVP (N-vinyl pyrrolidone) 295 NVP/AA (N-vinyl pyrrolidone/ acrylic acid) 309,312 D

20

Ο

N-Octanol 110 Ophthalmic applications 38 Ophthalmology 38 Optical microscopy 207 Organometallic initiators 139 Organic liquids in critical surface ten sion determination 260 Orthodontic appliance, intraoral 195 Osmotic behavior of gel networks, water in the 60 effects due to the aqueous phase of a hydrogel contact lens 48 pressure 37,44,61,202 Outermost zone 218 Ρ PAA (polyacrylamide)

11,70,169, 253,255,258

PNVP (poly(N-vinyl-2-pyrrolidone).. 12 Pancreas, artificial 27 Particle electrophoresis 225 Partition coefficient (K ) 81,181,184 Partition type membrane 82 Partitioning, ion 217 PE (polyethylene) 263,268,272, 274,276,279 Permeability 83 apparatus 71 calculated 185 as a means to study the structure of gels 69 data 78 specific (K c) 76 parameters 183 Permeation coefficients (K ) 73 f

s

rates 77 studies 200 of water through poly(2-hydroxyethyl methacrylate) 80 PGMA ( polyglyceryl meth acrylate) 6, 253, 255, 258, 267 hydrogel 39, 41, 42, 44 Ρ (GM A/MM A) 255 PHEA ( polyhydroxyethyl acrylate ) 25, 253,255,258,267 PHEMA ( poly hydroxyethyl meth acrylate) 6, 43,105,139,142,146, 147,148,151,252, 255, 258,267 critical surface tension of ...258,276,279 gel 60, 96,119 albumin adsorbed on 272 containing ethylene glycol di methacrylate crosslinker 210 swelling behavior of 8 water in 23 hydrogels biomedical studies utilizing 9 dehydration of 44 EGDMA and TEGDMA in 86 swelling pressure of a 43,45,56 implant 155,156,158 membranes 80,95,167 permeation of water through 80 radiation-grafted 207 water opaque gel freeze 212,213 water uptake of 96 wettability of biopolymers adsorbed onto 272 Phosphoglucomutase 174 Phosphorus pentoxide 152 Phosphorylase 174 Phosphorylated hydrogels, hetero geneous 151 Phosphorylated PHEMA implants in the back of a rat 155

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

355

INDEX

Phosphate group 152 PHPMA (poly(hydroxypropyl methacrylate ) ) 6 Pilocarpine 168 Plasma expander 12 Plasma protein adsorption 217 Platelets 14,21,314 pMEEMA ( poly ( methoxyethyethylmethacrylate)) 87,95,98 PMMA ( polymethyl methacrylate) 27,143,145,268 Polyacrylamide (PAA) 11,70,169,253,255,258 gels, aqueous 76 Poly (acrylic acid) 11 Poly ( N-acrylyl morpholine ) 11 Polycarbonate 27 Polycarboxylic surfaces 26 Poly(N,N-diethyl acrylamide) Poly(diethyleneglycol methacrylate) 8 Polydimethyl siloxane 254 Polyelectrolytes anionic 305 biological and physical characteristics of 305 complexes 13 ethylene/acrylic acid 312 ethylene/vinyl sulfonate 318 hydrogels onto polypropylene, coating of 311 surface charge density and hydrophilicity of 306 vinyl acetate/crotonic acid 321 N-vinyl pyrrolidone/acrylic acid .... 318 Poly(N-ethyl acrylamide) 11 Polyethylene (PE) 263,268,272, 274, 276,279 Poly (glyceryl methacrylate) ( PGMA ) 6, 253,255,258,267 Poly(hydroxyalkyl methacrylate) 6 Polyhydroxyethyl acrylate (PHEA) 25,253,255,258,267 Poly (hydroxyethyl methacrylate) (see PHEMA) Poly ( N- ( 2-hydroxypropyl ) methacrylamide) 11 Poly ( hydroxypropyl methacrylate (PHPMA) 6 Poly(L-lysine) 57,58 PolyMEEMA ( poly( methoxyethoxyethyl methacrylate) ) 87,95,98 Polymer(s) adsorption of 217, 247 immobilization of a protein to a 28 solution(s) adhesion tension of aqueous 273 Flory-Huggins theory of 61,199 membrane202 synthesis 309

Polymer(s) (continued) synthetic or modified natural 268 used as hydrogels, converted 7 viscosity 136 Polymeric chain, drug release from a .. 25 Polymeric solutions, multicomponent 271 Polymerization anionic 140 conditions 141 direct graft 312 free radical 140 graft 18 of HEMA-EMA, co-graft 285,287 of hydrophilic methacrylate monomers 119 of 2-hydroxyethyl methacrylate and ethyl methacrylate 283 time 129-133,136 Poly(methacrylamide) 11 Polymethacrylate esters 139 Poly ( methoxyethoxyethyl methacrylate (polyMEEMA) 87,95,98 Polymethyl methacrylate (PMMA) 27,143,145,268 Polypropylene 208, 311,312 Polysaccharides 238,244,245 Poly (vinyl acetate) 14 -c-2% crotonic acid 20 Poly (vinyl alcohol) (PVA) 14 Poly ( vinyl benzyltrimethylammonium chloride ) 13 Polyvinyl chloride 27 Polyvinyl pyrrolidone 12,169, 254 Pore diameter 40, 43 Pore size 25,151 Potential, interface 216 Potential, streaming 216, 241 Precipitation of homopolymer 285 Pressure imbibition 48 mechanical 37, 44 osmotic 37, 44,61,202 swelling 37,38,41,43,45, 46, 61,202 transducer 70 vapor 37,43 Prigogine theory 61 Pronase 57 Prophylaxis, fluoride 180 Protein adsorption, plasma 217 immobilization 26,28 serum 53,339 water wettability of 267 Proteoglycans 52 Proteolytic enzymes 54 PVA ( polyvinyl alcohol ) 14 PVP grafted silicone 259

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

356

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Scleral buckling procedure 40 Second virial coefficient 203 Quantitative evaluation 339 Secondary ion mass spectroscopy Quasi-steady state diffusion 175 (SIMS) 218 Sedimentation coefficient 69 Sedimentation constant 73 R Segment distribution 77,203 257 R data for HEMA 113 Segment mobility SEM ( scanning electron microRadiation scope) 207,211 dose 18 -surfactant 325 Sensor, blood glucose 27 graft copolymerization 283, 295 Serum graft polymerization 27 albumin 53-55,274 -grafted hydrogels in 295 bovine (BSA) 268,273,274 -grafted poly (hydroxyethyl methprotein(s) 53,339 acrylate) 207 samples 164 Radicals on surfaces, generating 17 SIL-g-PVP ( polydimethyl siloxane Radioactivity extracted for CHEMA gel 93,9 Raman spectroscopy 219 Silane Rat, phosphorylated polyHEMA imadhesion promoter(s) 242,311 plants in 155 coatings 245 Rate-controlling membrane 187 surfaces, epoxy244 Rate processes, theory of 122 291 Ratio, swelling 42 Silastic films, hydrogel/ 296,302 Ree-Eyring's activated-state model.... 122 films, ungrafted 297 Reference state 203 106 Reference substances 107 Silica gel 259 Refractive correction 10 Silicone, PVP grafted 18,21 Refractive index 114 Silicone rubber films 283,295 Relative contact angle hysteresis (H ) 257 Relaxation time, Bueche's 122 SIMS ( secondary ion mass spectroscopy) 218 Release 167 offluoridefrom a core 192 Skeletal muscle differentiation Sodium fluoride 181 offluoridefrom hydrogels for dental applications, controlled 180 Sodium poly( styrene sulfonate) 13 of hydrocortisone sodium succinate 167 Soft contact lens (es) 9,168 hydrocortisone succinate 172,175 Soft tissue compatibility tests 5 in vitro and in vivo 167 Solids, adhesion tension of aqueous Renal embolusringtest 5,325 polymer solutions to 273 Resistance, point of 77 Solubility theory 67 Retinal detachment surgery 40 Solution(s) Ring test, renal embolus 5,325 upon the hydration of hydrogels, Ring test, vena cava 5,290 effect of the external 38 Rontgenogram 154 isotonic 38 Rotational viscometry studies 119 polymerization 330 Rubberfilms,silicone 283, 295 surface tension 269 Rubber, silicone 18,21 tonicity 38 Solvent(s) S chemical potential 201 interface, gel200 Saliva non-polar 200 fluoride levels of dogs with intraoral 110 fluoride-releasing devices 196 systems human 194 Sorption behavior, water 295 synthetic 184,194 isotherms for water vapor 297 Salt concentration 58 of water vapor, standard differential Saturation solubility (C.) 181,185 entropy of 298 Scanning electron microscope free energy of 301 (SEM) 207,211 heat of 298 Scintillation 331 Q

F

14

R

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

357

INDEX

Spectroscopy for chemical analysis electron (ESCA) 218 multiple internal reflection infrared 219 raman 219 secondary ion mass (SIMS) 218 Spin casting 183 Spring, mechanical 57 Stability, energy effects which control 199 Stain, haematoxylin and eosin 153 Standard differential entropy of sorption of water vapor .. 298 free energy of sorption of water vapor 301 heat of sorption of water vapor 298 Starch 300 Stationary level 225 Steady state 54 approach, quasi17 diffusion, quasi175 Stereochemical changes at interface .. 256 Stereoregular methacrylates 139 Sterilization 10 Stokes radius 75 Streaming potentials 216,241,314 Streptokinase 27 Stress—elongation curves 65, 66 Stress relaxation 64 Stypven time 307 Subcutaneous implantation 8, 330 Submaxillary mucin, bovine (BSM) .. 268 Subsurface zone 219 Sulfanilic acid 109 2-Sulfoethylmethacrylate sodium salt.. 15 Surface(s) charge(s) 14,232,305 density 306,312 coated hydrogels 16 coatings 225 epoxy—silane 244 excess concentration, Gibb's 260,270 forces, range of 280 free-water layer 253 gel 201 generating radicals on 17 hydrogel 258 layer orientation 258 modifiers 237 structure 204 tension of acrylic hydrogels 261 of adsorbed biopolymers 275 of aqueous solutions of synthetic and biopolymers and surfac tant 269 of biopolymers adsorbed on PHEMA and polyethylene ... 279 determination 260 of the hydrogels 259 of PE 276

Surface tension (continued) of PHEMA solution of surfactant thromboresistant topography Surfactant, radiation doseSurfactant, surface tension of Surgery, corneal Surgery, retinal detachment Suspending medium Suture, coated Swelling agent, active behavior of PHEMA gels of connective tissue degree of equilibrium (see Equilibrium

258,276 269 269 284 207 325 269 9 40 75 9 57 8 52 40,55,58

linear (Is) 95 of pHEMA and pMEEMA mem branes, water 95 pressure 38,61,202 of high water-content glyceryl methacrylate hydrogels 38 of hydrogel contact lens materials 43 of hydrogels 37 of PGMA hydrogels 41 of a PHEMA hydrogel 43,45, 46 of pure homopolymers, equilibrium water 89 ratio 40-42 tendency of tissue 59 ultimate degree of 58 Swiss 3T3 cell line 331 Syndiotactic content 139 Τ

Tacticity 92,139,141 Tailing 110 Tear tonicity 50 TEGDMA (tetraethylene glycol dimethacrylate ) 81, 86,330 TEM (transmission electron micro scope) 207 Tension adhesion 260,268, 269,273 interfacial 2 surface (see Surface tension) Test methods, in vitro 307 Test methods, in vivo 308 Testicular hyaluronidase 54 Tetraethylene glycol dimethaceylate (TEGDMA) 81,86,330 Thermal analysis 142 Thermal behavior of various polymethacrylate esters 139 Thermodynamics of water sorption in radiation-grafted hydrogels 295

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

358

HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

Thermograms, DSC 145 Thin-layer chromatography (tic) 105,106,111,117 Thromboadherance 20 Thrombogenicity 283 Thromboplastin time, partial 307 Thromboresistance 305, 320 Thromboresistant surfaces 284 Tissue compatibility 206 connective 52, 59 hyaluronidase-treated 55 ingrowth of 152 loose connective 53 slices incubated in serum albumin .. 54 soft 332 swelling tendency of 59 volume 5 Tonicity, solution 3 Tonicity, tear 50 Topography, surface 207 Transmission electron microscope (TEM) 207 Triads, a-CH and carbonyl 144 Trilaminate devices 187 Trommsdorff effect 290 Trypsin 54, 56, 57 Tyrode solution 152 3

U

UGI reaction Ultracentrifuge Umbilical cord Uncoiling

25,28 73 53 201 V

VA/CA (vinyl acetate/crotonic acid) 310,312 Van de Graff 311 Vapor pressure of hydrogel contact lens materials 43 Vapor pressure of hydrogels 37 Vena cava implantation 320,326 Vena cavaringtest 5, 290,291 Vinyl acetate/crotonic acid (VA/CA) 310,312 polyelectrolytes 321 Vinyl acetate/crotonate copolymers and ionomers 321 2-Vinyl pyridine 15 4-Vinyl pyridine 15 Vinyl pyrrolidone (VP) 43,45 N-Vinyl pyrrolidone (NVP) 295 /acrylic acid (NVP/AA) ...309,312,320 polyelectrolytes 318 Vinyl sulfonate, ethylene/ ( EVS ) 312 polyelectrolytes 318

Vinyl sulfonic acid copolymers and ionomers, ethylene/ 309, 319 Vinyl sulfonic acid sodium ionomers, ethylene/ 320 Virial coefficient, second 203 Viscometry studies, rotational 119 Viscoelastic behavior of gel networks 60 Viscoelastic properties 64 Viscosity 73 Eyring's zero shear 135 non-Newtonian 120 polymerization time ( Byring's model) 136 Vitreous substitute 38 Volume, equilibrium 54 Von Kossa 154,156,159 VP (vinyl pyrrolidone) 43,45

Water activity in hydrogels 45, 47 bound 22,48,49 bulk 22 from bulk to gel network, transfer of 62 clustering functions for 301 concentrations, equilibrium 86 contact angles 254 content diffusion coefficients and 82 equilibrium 80 glyceryl methacrylate hydrogels, high 38 graft 286,289 of HEMA, dependence of n on 117 for HEMA/MMA copolymers, equilibrium 190 high 2 fraction (W ) 89,95 free 48,49 in grafted hydrogel/silastic films, diffusivities of 302 on the grafting of HEMA or EMA, effect of 228 on hydrogels, contact angle of 255 imbibed 22 interface, hydrogel198,206,267 interfacial 22,48 mixtures, HEMA126-133 opaque gel freeze, PHEMA 212,213 in the osmotic and viscoelastic behavior of gel networks 60 in PHEMA gels 23 through poly(2-hydroxyethyl methacrylate), permeation of 80 solubility of MEEMA-HEMA comonomers 100 solubility of MEMA-HEMA comonomers 98 D

f

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

20

359

INDEX

Water (continued) sorption 22 behavior 295 isotherms of hydrogel contact lens materials 46 in radiation-grafted hydrogels 295 structure 22, 60,65,295 swelling of pHEMA and pMEEMA membranes 95 transport 82 uptake of pHEMA 96 vapor 297,298,301 weight fraction of MEEMA-HEMA copolymers, equilbrium 100 weight fraction of MEMA-HEMA copolymers, equilbrium 98 wettability of hydrogels 252 wettability of proteins 267 Wettability of biopolymers 272 of bovine serum albumin 274

Wettability (continued) of hydrogels, water measurement of of proteins, water Wolfram plot Wound healing

252 254 267 262,277,278 331

X Xero-gels

42 Y

Young-Dupree equation

214

Ζ

Zero shear viscosity, Eyring's Zisman method Zisman plot

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

135 262 277, 278