Synthetic Hydrogels for Biomedical Applications - ACS Publications

1 Synthetic Hydrogels for Biomedical Applications

Downloaded by UNIV OF SOUTH AUSTRALIA on October 6, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0031.ch001

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, properties, and biomedical applications of synthetic hydrogel materials. As the literature on this 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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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 e can be c i t e d f o r hydrogel m a t e r i a l s . With respect to the r e a l advantages, two i n p a r t i c u l a r stand out. F i r s t , the expanded nature of the hydrogel s t r u c t u r e and i t s 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



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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 P o t e n t i a l as w e l l as A c t u a l Biomedical A p p l i c a t i o n s of S y n t h e t i c Hydrogels 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



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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 l D e f i n i t i o n s . In d i s c u s s i n g the b i o c o m p a t i b i l i t y of polymers a number of problems are encountered 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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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 e or no adhering thrombus even a f t e r the two week i m p l a n t a t i o n p e r i o d . A v a r i a t i o n of t h i s t e s t u s i n g a 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


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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 types o f hydrogels w i l l be d i s c u s s e d 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 emâsis 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-HÔ) 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


Synthetic Hydrogels


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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

2

X

C0 "C H 2

2

4

Ν-R

(R,R' R" = - H , - C H , - C H ) t

3

VINYL PYRIDINE

-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

CH

9

= CH

2

3

—Ν — VINYL

P Y R R O L I DON Ε

C H = C H - Ν 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 described f o r prep aring g e l s composed p r i m a r i l y of polyacrylamide w i t h s m a l l e r f r a c t i o n s of p o l y ( 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 -



12


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 g three types of b i o m e d i c a l hydrogels (4). I n p a r t i c u l a r the pore s i z e and water content of PAAm i s 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

2

5



1.


Synthetic Hydrogels

13

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 i d e a l monomer f o r use i n covalent s u r f a c e g r a f t i n g systems. A number of g r a f t P-NVP copolymers are d i s 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


14



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 enthusiasm expressed f o r i t , and that other synt h e t i c s would be more s a t i s f a c t o r y i n s i m i l a r s i t u a t i o n s (91). 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



1.


Synthetic Hydrogeh

15

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 r a t s , f o r P-HEMA specimens c o n t a i n i n g 4% m e t h a c r y l i c a c i d , c a l c i f i c a t i o n (which was found i n n e u t r a l 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



16

HYDROGELS FOR

MEDICAL AND


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 s u b s t r a t e s w i t h h y d r o p h i l i c polymers or copolymers. (Table V). Aside from the c o n v e n t i o n a l technique of d i p p i n g i n a s o l u t i o n 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 -


1.


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

—

C o ^ , Electron Irrodiotion

V///// 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.



18

HYDROGELS FOR

MEDICAL AND


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 . These i n c l u d e polymer degradation, c r o s s l i n k i n g and the formation of unwanted chemical species (e.g., peroxides, a c i d s ) . However, degradation and c r o s s l i n k i n g can 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



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


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 thrombogenicity of the s i l i c o n e rubber as judged by the vena cava r i n g t e s t (108). 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.


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


22



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 occur between b i o l o g i c a l systems and the 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).



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 on the o r g a n i z a t i o n of water w i t h i n hydrog e l s are only p r e l i m i n a r y i n d i c a t i o n s ; furthermore, the e f f e c t s 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 .


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 Table X Biomedical A p p l i c a t i o n s of Immobilized Enzymes 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


1.


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



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

.COî^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.


Synthetic Hydrogels

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 contact with blood have, a t times, been made of such m a t e r i a l s as poly(methyl methacrylate) ( 1 3 6 ) , p o l y v i n y l 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


27


28


0C0NH

2

Carbamate (inert) ^-0-C=N

OH BrCNL

Activation step

ÔH

/ /

OH Imidocarbonate (reactive)

C = NH Intermediate cyanate structure

4-


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

Coupling step

H N-Protein

C = NH

N-substituted imidocarbonate

C = N - Protein

2

Imidocarbonate

Isourea derivative

^-O-C-NH-Protein N-substituted carbamate

'/ 0 ;-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



1.


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 s of m a t e r i a l s have shown great v e r s a t i l i t y and e x c e l l e n t performance when used i n b i o m a t e r i a l a p p l i 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.


30


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.

Literature Cited Kiraly, R.J. and Nosé, Y., Biomat., Med. Dev., Art. Org., (1974), 2, 207. Homsy, C.A., J. Biomed. Mat. Res., (1970), 4, 341. Hoffman, A.S., J. Biomed. Mat. Res. Symposium, (1974), No. 5 (Part 1), 77. Bruck, S.D., J. Biomed. Mat. Res., (1973), 7, 387. Bruck, S.D., Biomat. Med. Dev., Artif. Organs, (1973), 1,79. Mason, R.G., Bull. N.Y. Acad. Med., (1972), 48, 407. Gott, V.L. and Furuse, Α., Fed. Proc., (1971), 30, 1679. Daniels, A.U. and Mortensen, J.D., Biomat., Med. Dev., Artif. Organs, (1974), 2, 365. Kusserow, B.K., Larrow, R.W. and Nichols, J., "Analysis and Measurement of the Effects of Materials on Blood Leuko cytes, Erythrocytes and Platelets," Contract No. PH 43-681427, National Heart and Lung Institute, National Insti tutes of Health, Bethesda , Maryland, Annual Report, (Dec. 1, 1972), PB218-651. Autian, J., Critical Reviews of Toxicology, (1973), 2, 1. Coleman, D.L., King, R.N. and Andrade, J.D., J. Biomed. Mater. Res. Symposium, (1974), No. 5 (Part 1), 65. Wichterle, O., in "Encyl. Polym. Sci. and Technol.", (1971), ed. by H.F. Mark and N.G. Gaylord, Vol. 15, 273. Wichterle, O. and Lim, D., Nature, (1960),185, 117. Ind. and Chem. Eng., (1936), 28, 1160. Refojo, M.F. and Yasuda, H., J. Appl. Polymer Sci., (1965), 9, 2425. Wichterle, O. and Chromecek, R., J. Polymer Sci., (1969), Part C, 16, 4677. Gouda, J.H., Povodator, Κ., Warren, T.C., and Prins, W., Polymer Letters, (1970), 8, 225. Ratner, B., Ph.D. Thesis, (1972), Polytechnic Institute of Brooklyn. Bohdanecky, M. and Tuzar, Z., Coll. Czech Chem. Commun., (1969), 34, 3318. Halpern, B.D., McGonigal, P.J. and Blessing, H.W., "Polymer Studies Related to Prosthetic Cardiac Materials Which are Non-Clotting at a Blood Interface," Contract No. PH 43-661124, National Heart and Lung Institute, N.I.H., Bethesda, Maryland, Annual Report, (Sept. 28, 1972), PB212-724. Bruck, S.D., Personal Communication. Sevcik, S., Stamberg, J. and Schmidt, P., J. Polymer Sci., (1967), Part C, 16, 821. Stamberg, J. and Sevcik, S., Coll. Czech Chem. Commun., (1966), 31, 1009. Wichterle, O. and Lim, D., U.S. Patent 3,220,960, (1965). Ratner, B.D. and Miller, I.F., J. Polymer Sci., (1972), Part A-1, 10, 2425. Refojo, M., J. Polymer Sci., (1967), Part A-1, 5, 3103.



1.


Synthetic Hydro gels

31

27. Jadwin, T.A., Hoffman, A.S. and Vieth, W.R., J. Appl. Poly mer Sci., (1970) 14, 1339. 28. Yasuda, H., Gochin, M. and Stone, Jr., W., J. Polymer Sci., (1966), Part A-1, 4, 2913. 29. Allen, L.F., A.C.S. Polymer Preprints, (1974), 15 (2), 395. 30. Ilavsky, M. and Prins, W., Macromolecules, (1970), 3, 415. 31. Dusek, K., Bohdanecky, M. and Prokopova, E., European Polymer J., (1974), 10, 239. 32. Janacek, J. and Hasa, J., Coll. Czech Chem. Commun., (1966), 31, 2186. 33. Janacek, J., J. Macromol. Sci., (1973), C9, 1. 34. Barvic, M., Kliment, K. and Zavadil, M., J. Biomed. Mater. Res., (1967), 1, 313. 35. Cerny, E., Chromecek, R., Opletal, Α., Papousek, F. and Otoupalova, J., Scripta Medica, (1970), 43, 63. 36. Sprincl, L., Kopecek, J. and Lim, D., J. Biomed. Mater. Res., (1971), 5, 447. 37. Barvic, Μ., Vacik, J., Lim, D. and Zavadil, M., J. Biomed. Mater. Res., (1971), 5, 225. 38. Sprincl, L., Kopecek, J. and Lim, D., Calc. Tissue Res., (1973), 13, 63. 39. Sprincl, L., Vacik, J. and Kopecek, J., J. Biomed. Mater. Res., (1973), 7, 123. 40. Winter, C.D. and Simpson, B.J., Nature, (1969), 223, 88. 41. Levowitz, B.S., LaGuerre, J.N., Calem, W.S., Gould, F.E., Scherrer, J. and Schoenfeld, Η., Trans. Amer. Soc. Artif. Int. Organs, (1968), 14, 82. 42. Tollar, M., Stol, M. and Kliment, K., J. Biomed. Mater. Res., (1969), 3, 305. 43. LaGuerre, J.N., Kay, Η., Lazarus, S.M., Calem, W.S., Weinberg, S.R. and Levowitz, B.S., Surg. Forum, (1968), 19, 522. 44. Lazarus, S.M., LaGuerre, J.N., Kay, Η., Weinberg, S. and Levowitz, B.S., J. Biomed. Mater. Res., (1971), 5, 129. 45. Majkus, V., Horakova, Z., Vymola, F. and Stol, M., J. Bio med. Mater. Res., (1969), 3, 443. 46. Drobnik, J., Spacek, P. and Wichterle, O., J. Biomed. Mater. Res., (1974), 8, 45. 47. Scott, H., Kronick, P.L., May, R.C., Davis, R.H. and Balin, H., Biomat.,Med. Dev., Art. Org., (1973), 1, 681. 48. Michnevic, I. and Kliment, II., K., J. Biomed. Mater. Res., (1971), 5, 17. 49. Warren, Α., Gould, F. E., Capulong, R., Glotfelty, E., Boley, S.J., Calem, W.S. and Levowitz, B.S., Surgical For um, (1967), 18, 183. 50. Singh, M.P. and Melrose, D.G., Biomedical Eng., (1971), 6, 157. 51. Singh, M.P., Biomedical Eng., (1969), 4, 68.



32


52. Halpern, B.D., Cheng, H., Kuo, S. and Greenberg, Η., in Artificial Heart Program Conference Proceedings, ed. by R.J. Hegyeli, U.S. Government Printing Office, Washington, D.C. (1969), 87. 53. Refojo, M.F., J. Biomed. Mater. Res., (1969), 3, 333. 54. Refojo, M.F., Survey of Ophthalmology, (1972), 16, 233. 55. Krejci, L. and Krejcova, Η., Brit. J. Ophthal., (1973), 57, 675. 56. Kocvara, S., Kliment, Ch., Kubat, J., Stol, M., Ott, Z. and Dvorak, J., J. Biomed. Mater. Res., (1967), 1, 325. 57. Kliment, K., Stol, M., Fahoun, K. and Stockar, B., J. Biomed. Mater. Res., (1968), 2, 237. 58. Molday, R.S., Dreyer, W.J., Rembaum, A. and Yen, S.P.S., J. Cell Biol., (1975), 64, 75. 59. Refojo, M.F., Preprints - Division of Organic Coatings and Plastics Chemistry, A.C.S., (1967), 27, (2), 136. 60. Andrade, J.D., Kunitomo, Κ., Van Wagenen, R., Kastigir, B., Gough, D. and Kolff, W.J., Trans. Amer. Soc. Artif. Int. Organs, (1971), 17, 222. 61. Spacek, P. and Kubin, M., J. Biomed. Mater. Res., (1973), 7, 201. 62. Ratner, B.D. and Miller, I.F., J. Biomed. Mater. Res.,(1973), 7, 353. 63. Rubin, R.M. and Marshall, J.L., J. Biomed. Mater. Res., (1975), 9, 375. 64. Winter, G.D., Proc. Roy. Soc. Med., (1970), 63, 1111. 65. Holly, F.J. and Refojo, M.F., J. Am. Optom. Assoc., (1972), 43, 1173. 66. Refojo, M.F., Contact and Intraocular Lens Med. J.,(1975), 1, 153. 67. Leibowitz, H.M., Laing, R.A. and Sandstrom, Μ., Arch.Opthal., (1973), 89, 306. 68. Friendly, D.S., Bruner, B.S., Frey, T., Lederman, M.E., Parks, M.M. and Oldt, Ν., Arch. Ophthal. (1973), 90, 344. 69. Refojo, M.F., J. Appl. Polymer Sci., (1965), 9, 3161. 70. Refojo, M.F., Anales de la Real Sociedad Española de Fisica y Quimica, (1972), 68, 697. 71. Nierzwicki, W. and Prins, W., J. Appl. Polym. Sci., (1975), 19, 1885. 72. Kudela, Κ., Stoy, A. and Urbanova, R., European Polymer J., (1974), 10, 905. 73. Moens, J. and Smets, G., J. Polymer Sci., (1957), 23, 931. 74. Nagase, K. and Sakaguchi, K., J. Polymer Sci., (1965), A3, 2475. 75. Pinner, S.H., J. Polymer Sci., (1953), 10, 379. 76. Kopecek, J., Sprincl., L., Bazilova, H. and Vacik, J., J. Biomed. Mater. Res., (1973), 7, 111. 77. Jenkins, L.B., Kredel, F.E. and McCord, W.M., A.M.A. Archives of Surgery, (1956), 72, 612. 78. Mohn, G., Acta Histochem., (1960), 9, 76.



1.


Synthetic Hydrogels

33

79. Burnette, L.W., Proc. Sci. Section T.G.A., (Dec. 1962), 38,1. 80. Williams, K.E., Kidston, E.M., Beck, F. and Lloyd, J.B., J. Cell Biol. (1975), 64, 113. 81. Rivano, R., Lago, T.F. and Biancheri, V., Pathologica(Genoa), (May-June, 1957), 49, 301. 82. Richards, V. and Persidsky, H., J. Cardiovasc. Surg.,(1964), 5, 313. 83. PVP - An Annotated Bibliography, 1951-1966, General Aniline and Film Corporation, (1967). 84. Luttinger, M. and Cooper, C.W., J. Biomed. Mater. Res., (1967), 1, 67. 85. Davis, B.K., Proc. Nat. Acad. Sci. U.S.A., (1974), 71,3120. 86. Maroudas, N.G., Nature, (1975), 254, 695. 87. Markley, L.L., Bixler, H.J. and Cross, R.A., J. Biomed. Mater. Res., (1968), 2, 145. 88. Marshall, D.W., Cross, R.A. and Bixler, H.J., J. Biomed. Mater. Res., (1970), 4, 357. 89. Bixler, H.J., Cross, R.A. and Marshall, D.W., in Artificial Heart Program Conference Proceedings, ed. by R.J. Hegyeli, U.S. Government Printing Office, Washington, D.C. (1969), 79. 90. Bruck, S.D., J. Biomed. Mater. Res., (1971), 5, 139. 91. Alder, R.H. and Darby, C., U.S. Armed Forces Medical Journal, (1960), 11, 1349. 92. Merrill, E.W., Salzman, E.W., Wong, P.S.L., Ashford, T.P., Brown, A.D. and Austen, W.G., J. Appl. Physiol., (1970), 29, 723. 93. Merrill, E.W., Salzman, E.W., Wong, P.S.L. and Silliman, J., ACS Polymer Pre-Prints, (1972), 13(1), 511. 94. Bray, J.C. and Merrill, E.W., J. Biomed. Mater. Res.,(1973), 7, 431. 95. Lee, H., Stoffey, D.G. and Abroson, F., in Artificial Heart Program Conference Proceedings, ed. by R.J. Hegyeli, U.S. Government Printing Office, Washington, D. C., (1969), 143. 96. Sawyer, P.N. and Srinivasan, S., in Artificial Heart Program Conference Proceedings, ed. by R.J. Hegyeli, U.S. Government Printing Office, Washington, D.C., (1969), 243. 97. Leonard, F., Trans. Amer. Soc. Artific. Int. Organs, (1969), 15, 15. 98. Walter, C.W., Murphy, W.P., Jessiman, A.G., and Ahara, R.M., Surgical Forum, (1951), 2, 289. 99. Bray, J.C. and Merrill, E.W., J. Appl. Polymer Sci., (1973), 17, 3779. 100. Ratner, B.D. and Hoffman, A.S., in "Biomedical Applications of Polymers", (1975), ed. by H.P. Gregor, Plenum Press, N.Y., 159. 101. Ratner, B.D. and Hoffman, A.S., J. Appl. Polymer Sci.,(1974), 18, 3183. 102. Lee, H.B., Shim, H.S. and Andrade, J.D., ACS Polymer Preprints, (1972), 13(2), 729.



34


103. Yasuda, H. and Refojo, M.F., J. Polymer Sci., (1964), Part A, 2, 5093. 104. Leininger, R.E., Cooper, C.W., Falb, R.D. and Grode, G.A., (1966), Science, 152, 1625. 105. Laizier, J. and Wajs, G., in "Large Radiation Sources for Industrial Processes", IAEA, Vienna, (1969), 205. 106. Miller, M.L., Postal, R.H., Sawyer, P.N., Martin, J.G. and Kaplit, M.J., J. Appl. Polym. Sci., (1970), 14, 257. 107. a. Hoffman, A.S. and Kraft, W.G., ACS Polymer Preprints, (1972), 13(2), 723. b. Hoffman, A.S. and Harris, C., ACS Polymer Preprints, (1972), 13(2), 740. 108. Ratner, B.D., Hoffman, A.S. and Whiffen, J.D., Biomat., Med. Dev., Artifi. Organs, (1975), 3, 115. 109. Horbett, T.A. and Hoffman, A.S., ACS Advances in Chemistry Series, (1975), 145, 230. 110. Ratner, B.D., Horbett, T., Hoffman, A.S. and Hauschka, S.D., J. Biomed. Mater. Res., (1975), 9, 407. 111. Kearney, J.J., Amara, I. and McDevitt, M.B., in "Biomedical Applications of Polymers," (1975), ed. by H.P. Gregor, Plenum Press, N.Y., 75. 112. Scott, H. and Hillman, E.E., "Active - Vapor Grafting of Hydrogels in Medical Prosthesis," Contract No. NIH-HHL171-2017, Natrional Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, (Feb. 1, 1973), PB-221-846. 113. Gott, V. and Baier, R.E., "Evaluation of Materials by Vena Cava Rings in Dogs (Vol. 1)", Contract No. PH 43-68-84, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, (Sept.1972), PB-213-109. 114. 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. NO1-HL3-2950 T, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, (July 1, 1972-June 30, 1973), PB-225-636. 115. Gott, V.L. and Baier, R.E., "Twelve Month Progress Report on Contract No. PH 43-68-84, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, (Apr. 30, 1970), PB-197-622. 116. Halpern, B.D. and Blessings, H.W., "Polymer Studies Related to Prosthetic Cardiac Materials which are Non-Clotting at a Blood Interface", Contract No. PH 43-66-1124, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland, Annual Report, (Feb. 2, 1973), PB-215-886. 117. Muzykewicz, K.J., Crowell, Jr., E.B., Hart, A.P., Schultz,M., Hill, Jr., C.G., and Cooper, S.L., J. Biomed. Mater. Res., (1975), 9, 487.



1.

RATNER

AND HOFFMAN

Synthetic Hydrogels

35

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 H.D. Brown, Academic Press, N.Y., (1971), 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.



36


141. Guilbault, G.G. and Lubrano, G., Anal. Chim. Acta., (1974), 69, 189. 142. Kirwan, D.J., "Removal of Airborn Infections," (1974), pre sented at Purdue University, Conference on Enzyme and Antibody Engineering - Economics and Directions, Jan. 2324, 1974, Lafayette, Indiana. 143. Baker, R.W. and Lonsdale, H.K., in "Controlled Release of Biologically Active Agents", Tanquary, A.C. and Lacey, R.E., Eds., Plenum Press, N.Y., (1974), pp. 15-71. 144. Abrahams, R.A. and Ronel, S.H., J. Biomed. Mater. Res., (1975), 9, 355. 145. Morawetz, Η., in "Advances in Catalysis and Related Subjects" Vol. 20, (1969), ed. by D.D. Eley, H. Pines and P.B. Weisz, Academic Press, N.Y., 341. 146. Gutcho, S.J., "Immobilized Enzymes - Preparation and Engi neering Techniques", Noyes Data Corporation, Park Ridge, N.J., (1974), 141. 147. Refojo, M.F., J. Appl. Polymer Sci., (1965), 9, 3417. 148. White, M.L., J. Phys. Chem., (1960), 64, 1563. 149. Hinberg, I., Kapoulas, Α., Korus, R. and O'Driscoll, Κ., Biotech. Bioeng., (1974), 16, 159. 150. Maeda, Η., Suzuki, H., Yamauchi, A. and Sakimae, Α., Bio tech. Bioeng., (1975), 17, 119. 151. Denti, E., Biomat., Med. Dev., Artif. Organs, (1974), 2,293. 152. Zaborsky, O.R., "Immobilized Enzymes", CRC Press, Cleveland, Ohio, (1973). 153. Brown, H.D. and Hasselberger, F.X., in "Chemistry of the Cell Interface", Part B, ed. by H.D. Brown, Academic Press, N.Y., (1971), p. 185. 154. Axen, R., Porath, J. and Ernback, S., Nature, (1967), 214, 1302. 155. Axen, R., Vretblad, P. and Porath, J., Acta. Chem. Scand., (1971), 25, 1129. 156. Ternynck, T. and Avrameas, S., FEBS Letters, (1972), 23,24. 157. Gough, D.A. and Andrade, J.D., Science, (1973), 180, 380. 158. Guilbault, G.G. and Hrabankova, E., Anal. Chim. Acta., (1971), 56, 285.


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