Bovine Plasma Protein Adsorption onto Radiation-Grafted Hydrogels

11 Bovine Plasma Protein Adsorption onto

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 21, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch011

Radiation-Grafted Hydrogels Based on Hydroxyethyl Methacrylate and N-Vinylpyrrolidone T. A. HORBETT and A. S. HOFFMAN Department of Chemical Engineering and Center for Bioengineering, University of Washington, Seattle, Wash. 98195

Fibrinogen, γ-globulin, and albumin adsorption onto water -swollen synthetic polymers (hydrogels) were studied. These hydrogels were based on hydroxyethyl methacrylate (HEMA) and N-vinylpyrrolidone (NVP) and were radiation grafted onto silicone rubber. Adsorption from water, physiologic buffered saline, and blood plasma were studied. The intrin sically low protein adsorption onto poly(HEMA) can be overshadowed by adsorption due to low levels of ionic impurities. Fibrinogen adsorption isotherms at concentra tions up to physiologic levels show maximum adsorptions between ca. 0.2 and 0.8 μg/cm for the surfaces examined with adsorption onto poly(HEMA) being the least. These levels are five- to tenfold higher than those observed for fibrinogen adsorption from plasma. Albumin—fibrinogen and γ-globulin-fibrinogen competition experiments only partially explain the depression of plasmafibrinogenadsorption. 2

Τ T y d r o g e l s are a class of s y n t h e t i c p o l y m e r s of d i v e r s e c h e m i c a l n a t u r e A

A

d i s t i n g u i s h e d f r o m other p o l y m e r s b y the c a p a c i t y to i m b i b e r e l a

t i v e l y l a r g e amounts of w a t e r i n t h e i r s t r u c t u r e . T h e w a t e r content

of

these m a t e r i a l s varies f r o m a b o u t 30 to 90 w t % d e p e n d i n g o n b o t h the c h e m i c a l n a t u r e a n d p h y s i c a l structure of the p o l y m e r .

M a n y natural

or b i o c o m p a t i b l e p o l y m e r s are also h i g h l y h y d r a t e d , e.g. 3 0 - 5 0 w t

%

w a t e r is b o u n d b y g l o b u l a r proteins ( 1 ). P a r t l y for this reason, h y d r o g e l s 230 Baier; Applied Chemistry at Protein Interfaces Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

11. have

HORBETT AND H O F F M A N p o t e n t i a l usefulness

Adsorption

onto

231

Hydrogels

as b i o m a t e r i a l s c a p a b l e

of m i n i m i z i n g t h e

u n f a v o r a b l e reactions often i n d u c e d b y f o r e i g n m a t e r i a l s i n contact w i t h b l o o d o r tissue (2).

H o w e v e r , p r o t e i n a d s o r p t i o n onto materials i n c o n -

tact w i t h b l o o d q u i c k l y modifies t h e i r i n t e r f a c i a l p r o p e r t i e s , a n d i t is b e l i e v e d to b e one of t h e p r i m a r y events l e a d i n g to t h r o m b u s f o r m a t i o n at the b l o o d / m a t e r i a l interface (3, 4). hydrogels under development

T h u s , p r o t e i n a d s o r p t i o n onto t h e

i n this l a b o r a t o r y has b e e n one of t h e

p r i m a r y areas of s t u d y i n t h e assessment of this n e w class of p o t e n t i a l Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 21, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch011

biomaterials. H y d r o g e l s i n aqueous m e d i a are m e c h a n i c a l l y w e a k a n d , 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 , t h e y f r e q u e n t l y r e q u i r e b o n d i n g to stronger s u b strate m a t e r i a l s . A v a r i e t y of t e c h n i q u e s h a v e b e e n d e v e l o p e d

f o r this

p u r p o s e i n c l u d i n g e l e c t r o n b e a m r a d i a t i o n - i n i t i a t e d graft p o l y m e r i z a t i o n ( 5 ) , d i p c o a t i n g o n sutures (6), a c t i v e v a p o r o r p l a s m a i n i t i a t e d g r a f t i n g onto a r t e r i a l prostheses (7,8,9),

a n d g a m m a - r a y r a d i a t i o n - i n i t i a t e d graft

p o l y m e r i z a t i o n onto several types of surfaces ( J O , 11, 12, 13). T h e h y d r o g e l s s t u d i e d here w e r e m a d e b y r a d i a t i o n g r a f t i n g H E M A or N V P onto silicone r u b b e r ( S i l a s t i c ) . T h e s i l i c o n e r u b b e r b a c k b o n e of these g r a f t e d h y d r o g e l s overcomes t h e i n t r i n s i c m e c h a n i c a l weakness of the h y d r a t e d p o l y ( H E M A ) a n d p o l y ( N V P ) h y d r o g e l s (11, 12, 13). T h e resultant p o l y ( H E M A ) / S i l a s t i c about 30-60 w t %

and poly ( N V P ) / S i l a s t i c

grafts r e t a i n

w a t e r , r e s p e c t i v e l y , i n contrast to t h e u n d e r l y i n g

s i l i c o n e r u b b e r w h i c h adsorbs less t h a n 1 % w a t e r (13).

These materials

h a v e s h o w n c o n s i d e r a b l e b l o o d c o m p a t i b i l i t y i n tests w i t h the in vivo v e n a c a v a r i n g test

(14).

P r o t e i n a d s o r p t i o n onto p o t e n t i a l b i o m a t e r i a l s has b e e n s t u d i e d i n t h e past u s i n g t w o b a s i c approaches, each w i t h its l i m i t a t i o n s . A d s o r p t i o n f r o m w h o l e , f l o w i n g b l o o d c l e a r l y represents t h e most r e l e v a n t s i t u a t i o n since g o o d b l o o d - c o m p a t i b i l i t y is a n i m p o r t a n t c h a r a c t e r i s t i c f o r b i o materials. Studies of this t y p e h a v e b e e n r e p o r t e d (15).

I t is e x t r e m e l y

difficult to assess s u c h studies, h o w e v e r , b e c a u s e t h e effects of

blood

flow rate, b l o o d c e l l a d h e s i o n , degree of a c t i v a t i o n of t h e c o a g u l a t i o n system, a n d specific p r o t e i n a d s o r b e d are n o t r e a d i l y e v a l u a t e d .

Thus

the studies h a v e l a r g e l y b e e n l i m i t e d to s h o w i n g t h a t a n y m a t e r i a l i n contact w i t h b l o o d becomes c o a t e d w i t h p r o t e i n i n a v e r y short t i m e . V r o m a n ' s g r o u p has d e v e l o p e d u s i n g specific

some p o t e n t i a l l y u s e f u l t e c h n i q u e s

antisera to i d e n t i f y i n d i v i d u a l proteins

p l a s m a onto o x i d i z e d m e t a l surfaces (16, 17, 18, 19, 20).

adsorbed

by

from

Another basic

a p p r o a c h i n v o l v e s m e a s u r i n g t h e a d s o r p t i o n of a single p u r i f i e d p r o t e i n onto t h e m a t e r i a l f r o m w a t e r o r buffer (21, 22). I n o u r studies, a p a r t i a l synthesis o f these approaches

has b e e n

a t t e m p t e d , o n t h e p r e m i s e t h a t o n l y a clear u n d e r s t a n d i n g of p r o t e i n a d s o r p t i o n onto h y d r o g e l s f r o m s i m p l e b u f f e r e d saline c o u l d l e a d to a n

Baier; Applied Chemistry at Protein Interfaces Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

232

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

i n t e r p r e t a t i o n of a p a r t i c u l a r protein's a d s o r p t i o n b e h a v i o r f r o m b l o o d or p l a s m a . T h u s , p r o t e i n a d s o r p t i o n o n t o h y d r o g e l s f r o m w a t e r , f r o m buff e r e d saline, f r o m p r o t e i n m i x t u r e s , a n d f r o m p l a s m a h a v e b e e n s t u d i e d . T h e l a r g e n u m b e r of experiments necessary i n s u c h a m u l t i - f a c e t e d a p p r o a c h cannot b e p r e s e n t e d here i n t h e i r entirety. I n s t e a d , s e v e r a l k e y e x p e r i m e n t s representative of e a c h f a c t of this r e s e a r c h are p r e s e n t e d to p r o v i d e a n o v e r v i e w of these studies a n d to p o i n t out some c r i t i c a l aspects of m e a s u r i n g p r o t e i n a d s o r p t i o n onto h y d r o g e l s . Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 21, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch011

M o n o m e r p u r i t y has a l a r g e effect o n p r o t e i n a d s o r p t i o n onto h y d r o gels. T h e i n t r i n s i c a l l y l o w p r o t e i n a d s o r p t i o n onto p o l y ( H E M A )

hydro-

gels c a n b e o v e r s h a d o w e d b y a d s o r p t i o n caused b y these i m p u r i t i e s . H o w ever, r a d i a t i o n g r a f t i n g of p o l y ( H E M A ) onto a h y d r o p h o b i c s u p p o r t does not affect

the a d s o r p t i o n of

poly(HEMA).

fibrinogen

i n comparison w i t h

P r o t e i n a d s o r p t i o n isotherms for

fibrinogen

ungrafted onto p o l y -

( H E M A ) / S i l a s t i c , p o l y ( N V P ) / S i l a s t i c , a n d S i l a s t i c alone at c o n c e n t r a tions u p to p h y s i o l o g i c a l levels s h o w a b a s i c s i m i l a r i t y i n shape, b u t t h e m a x i m u m a d s o r p t i o n levels v a r y b y a factor of four, w i t h

fibrinogen

a d s o r p t i o n onto p o l y ( H E M A ) / S i l a s t i c b e i n g the least. T h e s e m a x i m u m levels differ f r o m those o b s e r v e d f o r

fibrinogen

adsorption from plasma

onto these same m a t e r i a l s . T h e different a d s o r p t i o n b e h a v i o r o b s e r v e d i n buffer a n d p l a s m a are not e n t i r e l y e x p l a i n e d b y c o m p e t i t i o n curves p e r f o r m e d w i t h a l b u m i n - f i b r i n o g e n or γ-globulin-fibrinogen m i x t u r e s . T h e s e results e m p h a s i z e the i m p o r t a n c e of as yet u n k n o w n p l a s m a factors i n modifying Materials

fibrinogen

a d s o r p t i o n onto some of these surfaces.

and Methods

M a t e r i a l s . M e d i c a l g r a d e n o n - r e i n f o r c e d s i l i c o n e r u b b e r sheeting, S i l a s t i c b r a n d , w a s o b t a i n e d f r o m D o w C o r n i n g . H E M A m o n o m e r was obtained from Borden C h e m i c a l C o . commercial quality, and from H y d r o n L a b o r a t o r i e s , Inc., h i g h l y p u r i f i e d . M e t h a c r y l i c a c i d ( M A A c ) , N V P , and ethyleneglycol dimethacrylate ( E G D M A ) were purchased from B o r d e n C h e m i c a l C o . F i b r i n o g e n ( 9 0 % clottable, b o v i n e ) , Pentex b r a n d , was p u r c h a s e d f r o m M i l e s L a b o r a t o r i e s , K a n k a k e e , 111. A l b u m i n ( c r y s t a l l i n e , b o v i n e ) a n d γ-globulin ( f r a c t i o n I I , b o v i n e ) w e r e o b t a i n e d from Nutritional Biochemicals C o r p . N-2-hydroxyethyl piperazine-IV'-2e t h a n e s u l f o n i c a c i d ( H E P E S ) , A grade, a n d l a c t o p e r o x i d a s e , l y o p h i l i z e d Β grade, were purchased from C a l b i o c h e m , San Diego. Plasma, citrated, platelet poor, was prepared from freshly d r a w n bovine blood. Reagent grade n i n h y d r i n a n d m e t h y l Cellosolve (peroxide free) were obtained f r o m P i e r c e C h e m i c a l s C o . R e a g e n t g r a d e S n C l · 2 H 0 , acetic a c i d , a n d H 0 w e r e p r o d u c t s of M a l l i n c k r o d t C h e m i c a l W o r k s , St. L o u i s . R e a g e n t c i t r i c a n d b o r i c acids w e r e p u r c h a s e d f r o m M e r c k & C o . , R a h w a y , N . J . N a I was obtained from I C N C h e m i c a l and Radioisotope D i v i s i o n , Irvine, Calif, and from N e w E n g l a n d Nuclear, Boston, Mass. G-25 Sephadex was purchased from P h a r m a c i a C h e m i c a l Co., Piscataway, N . J . 2

2

2

2

1 2 5


11.

HORBETT A N D H O F F M A N

Adsorption

onto

233

Hydrogels

I C I w a s a gift of D . L a g u n o f f . S o d i u m a z i d e w a s a p r o d u c t of J . T . B a k e r C h e m i c a l C o . A l l other c o m p o u n d s w e r e reagent g r a d e or the p u r e s t available commercially. Film Preparation. H e m a a n d N V P w e r e r a d i a t i o n - g r a f t e d onto Silastic to m a k e p o l y ( H E M A ) / S i l a s t i c a n d p o l y ( N V P ) / S i l a s t i c h y d r o g e l s as p r e v i o u s l y d e s c r i b e d (13). I Fibrogen. T w o m e t h o d s w e r e u s e d to p r e p a r e I-labeled fibrinogen. In both methods, unincorporated iodide was removed i m m e d i a t e l y after the r e a c t i o n b y g e l filtration o n a G - 2 5 S e p h a d e x c o l u m n p r e e q u i l i b r a t e d i n 0 . 0 1 M H E P E S , 0 . 1 4 7 M N a C l , 0 . 0 2 % a z i d e , p H 7.4. T h e first m e t h o d uses catalysis b y l a c t o p e r o x i d a s e a n d is b a s e d o n studies b y M a r c h a l o n i s ( 2 3 ) , a n d the s e c o n d m e t h o d uses i o d i n e m o n o c h l o r i d e ( I C I ) a n d is b a s e d o n w o r k b y H e l m k a m p et al. (24).


1 2 5

1 2 5

Protein Adsorption. F i l m s w e r e k e p t s u b m e r g e d i n buffer i n i n d i v i d u a l bottles. C o n c e n t r a t e d p r o t e i n s o l u t i o n i n the same buffer ( a n d at the same t e m p e r a t u r e a n d p H ) was a d d e d w i t h a p i p e t t e to a v o i d expos i n g the films to the a i r / w a t e r interface. T h e solutions w e r e m i x e d b y s w i r l i n g the films. I n studies at 37 ° C , the solutions w e r e k e p t i n a w a t e r b a t h r e g u l a t e d to ± 1 ° C . A f t e r e q u i l i b r a t i o n , the films w e r e first r i n s e d q u i c k l y b y a d i l u t i o n a n d d i s p l a c e m e n t t e c h n i q u e w h i c h insures that the films are not exposed to the p r o t e i n s o l u t i o n / a i r i n t e r f a c e . T h e d i l u t i o n - d i s p l a c e m e n t r i n s e was d o n e b y r u n n i n g solvent t h r o u g h the e q u i l i b r a t i o n b o t t l e at a b o u t 400 m l / m i n for a p p r o x i m a t e l y 1 m i n u s i n g a t w o - h o l e r u b b e r stopper fitted w i t h t w o glass tubes, one for entrance a n d one for exit of buffer. Fibrinogen Adsorption from Plasma. F i l m s w e r e s u b m e r g e d i n 2 m l of 0 . 0 1 M H E P E S , 0 . 1 4 7 M N a C l , 0 . 0 2 % a z i d e , p H 7.4. E i g h t m l of c i t r a t e d p l a s m a ( p H 7.6) c o n t a i n i n g I - f i b r i n o g e n was a d d e d , a n d the solutions w e r e m i x e d b y s w i r l i n g the films. 125

Fibrinogen—Albumin and Fibrinogen—γ-Globulin Competition E x periments. F i b r i n o g e n solutions c o n t a i n i n g I-fibrinogen were mixed w i t h a l b u m i n or γ-globulin solutions a n d buffer to g i v e a final fibrinogen c o n c e n t r a t i o n of 0.02 m g / m l a n d final a l b u m i n or γ-globulin c o n c e n t r a tions of b e t w e e n 0 a n d 20 m g / m l . F i l m s w e r e s u b m e r g e d i n 5 m l of buffer, a n d 5 m l of the d e s i r e d p r o t e i n s o l u t i o n w a s a d d e d . 125

N i n h y d r i n Assay for Adsorbed Proteins. M e a s u r e m e n t s w e r e m a d e b y a c o l o r i m e t r i c p r o c e d u r e b a s e d o n the r e a c t i o n of n i n h y d r i n w i t h a m i n o acids ( 2 5 ) . T h e films w e r e h y d r o l y z e d i n 5 m l of 2.5N N a O H for 2 hrs i n c a p p e d p l a s t i c tubes i n a b o i l i n g w a t e r b a t h . T h e n 1.5 m l of g l a c i a l acetic a c i d w a s a d d e d a n d m i x e d ; next 1 m l of n i n h y d r i n reagent was a d d e d a n d m i x e d . [ T h e reagent w a s t h r e e times m o r e concentrated i n n i n h y d r i n , S n C l , a n d citrate t h a n p r e s c r i b e d b y M o o r e a n d S t e i n ( 2 5 ) ] . T h e tubes w e r e c a p p e d a n d b o i l e d 20 m i n s m o r e . T h e s o l u t i o n w a s c l a r i f i e d b y c e n t r i f u g a t i o n , a n d the absorbance r e a d i m m e d i a t e l y at 570 n m o n a B e c k m a n D B spectrophotometer. I f necessary, t h e s a m p l e w a s d i l u t e d w i t h 5 0 - 5 0 2 - p r o p a n o l - w a t e r . C a l i b r a t i o n curves ( a b s o r b a n c e vs. /Ag of p r o t e i n ) w e r e c o n s t r u c t e d i n t h e 0 - 3 0 a n d 0 - 1 0 0 μg r a n g e w i t h k n o w n amounts of e a c h t y p e of p r o t e i n s u b j e c t e d to this same analysis procedure. 2


234

APPLIED

CHEMISTRY

A T PROTEIN

INTERFACES

F i l m a n d reagent b l a n k determinations w e r e always made a n d used to c o r r e c t t h e d a t a a p p r o p r i a t e l y . T h e assay w a s l i n e a r t h r o u g h o u t t h e r a n g e e n c o u n t e r e d here. Other Methods. P r o t e i n c o n c e n t r a t i o n s w e r e c a l c u l a t e d f r o m t h e 280 n m a b s o r b a n c e m e a s u r e d w i t h a B e c k m a n D B s p e c t r o p h o t o m e t e r . T h e p H of t h e p r o t e i n s o l u t i o n w a s a d j u s t e d as necessary w i t h a R a d i o m e ter G K 2 3 0 3 c p H e l e c t r o d e a n d a n O r i o n m o d e l 401 I o n a l y z e r . I was m e a s u r e d w i t h a p u l s e - h e i g h t , a n a l y z e r - c o u n t e r m o d u l a r system consist i n g o f m o d e l s 4 0 - 1 2 B , 49-25, 33-10, 30-19, 29-1, a n d 10-8 m a n u f a c t u r e d b y R a d i a t i o n I n s t r u m e n t D e v e l o p m e n t L a b . , D e s P l a i n e s , 111.


1 2 5

Results and Discussion The Effect of Monomer P u r i t y on Protein Adsorption onto Poly(HEMA).

T h e importance

of relatively minor contamination of the

monomers used i n formulating hydrogels to be used i n b i o m e d i c a l a p p l i cations has n o t b e e n r e c o g n i z e d w i d e l y as y e t , a l t h o u g h B r u c k h a s r e f e r r e d to this p r o b l e m i n c o n n e c t i o n

w i t h t h e soft c o n t a c t lens

P r o t e i n a d s o r p t i o n studies p e r f o r m e d w i t h h y d r o g e l s m a d e w i t h mers

of t y p i c a l commercial

Table I.

q u a l i t y i l l u s t r a t e this p o t e n t i a l

(26). mono

problem.

γ - G l o b u l i n Adsorption onto Poly ( H E M A ) / S i l a s t i c " Amount Adsorbed ^g/cm )

Protein Solvent

Material E x p e r i m e n t A ( c o m m e r c i a l grade H E M A ) untreated Silastic p o l y ( H E M A ) / S i l a s t i c ( 6 % graft) p o l y ( H E M A ) / S i l a s t i c ( 2 8 % graft) E x p e r i m e n t Β ( c o m m e r c i a l grade H E M A ) untreated Silastic untreated Silastic p o l y ( H E M A ) / S i l a s t i c ( 1 8 % graft) p o l y ( H E M A ) / S i l a s t i c ( 1 8 % graft) E x p e r i m e n t C (purified H E M A ) p o l y ( H E M A ) / S i l a s t i c ( 3 3 % graft) p o l y ( H E M A ) / S i l a s t i c ( 3 3 % graft)

2

0.8 12.7 22.9

H 0 H 0 H 0 2

2

2

H 0 buffered saline H 0 buffered saline

0.8 0.8

2

6

2

c

12.6 1.9

d

H 0 buffered saline

1.54 0.11

2

6

A l l a d s o r p t i o n experiments were done i n 0.5 m g / m l 7 - g l o b u l i n solutions a t r o o m t e m p e r a t u r e for a t least 40 hrs, followed b y a t least 6 hrs of rinsing i n the e q u i l i b r a t i o n solvent. A d s o r b e d p r o t e i n was d e t e r m i n e d u s i n g the n i n h y d r i n assay. Buffered saline: 0.01M H E P E S , 0.147M N a C l , p H 7.4. Buffered saline: 0.05M I m i d a z o l e , 0.112M N a C l , p H 7.4. H y d r o n H E M A is the purified H E M A referred t o . a

b

c

d


11.

HORBETT AND H O F F M A N

Adsorption

onto

235

Hydrogels

T a b l e I s u m m a r i z e s these early studies i n the f o r m of three k e y e x p e r i ments. T h e v e r y first measurements of p r o t e i n a d s o r p t i o n onto these h y d r o gels r e v e a l e d m a r k e d l y greater a d s o r p t i o n onto these m a t e r i a l s t h a n onto the u n t r e a t e d S i l a s t i c a n d t h e y s h o w e d t h a t p r o t e i n a d s o r p t i o n i n c r e a s e d as t h e a m o u n t of p o l y ( H E M A ) g r a f t e d onto the Silastic i n c r e a s e d ( e x p e r i ment A , Table I ) .

T h e s e results w e r e s u r p r i s i n g because the a p p a r e n t

s t r o n g i n t e r a c t i o n of

poly ( H E M A )

w i t h proteins e v i d e n c e d

by

such


g r e a t l y e n h a n c e d a d s o r p t i o n d i d not agree w i t h the e x p e c t e d l o w

free

e n e r g y at the h y d r o g e l - s o l u t i o n interface. T h e p r o t e i n h a d b e e n d i s s o l v e d i n d i s t i l l e d w a t e r i n these

initial

a d s o r p t i o n studies i n o r d e r to c o m p a r e the results w i t h the extensive d a t a of B r a s h a n d L y m a n (21 ) o n p r o t e i n a d s o r p t i o n f r o m w a t e r onto a v a r i e t y of m a t e r i a l s — i n c l u d i n g Silastic, the s t a n d a r d u s e d i n these studies.

The

r e l a t i v e l y close agreement of o u r d a t a w i t h those of B r a s h a n d L y m a n (21)

o n γ-globulin a n d

fibrinogen

a d s o r p t i o n f r o m w a t e r onto S i l a s t i c

s u p p o r t e d the v a l i d i t y of the a d s o r p t i o n a n d assay p r o c e d u r e s u s e d here. T h e m a x i m u m a d s o r p t i o n levels o b s e r v e d

i n b o t h studies for

plasma

p r o t e i n a d s o r p t i o n onto S i l a s t i c ( 0 . 8 - 1 . 8 / x g / c m ) are c o n s i d e r a b l y h i g h e r 2

t h a n the levels c o r r e s p o n d i n g to m o n o l a y e r f o r m a t i o n o n a flat, s m o o t h surface

( c a l c u l a t e d to b e a b o u t 0.2 / * g / c m

2

for these p r o t e i n s ) .

result suggested t h a t m u l t i l a y e r s of p r o t e i n w e r e b e i n g f o r m e d .

This Since

proteins are often less stable i n p u r e d i s t i l l e d w a t e r t h a n i n solutions of physiologic ionic strength and p H , multilayer adsorption could f r o m d e n a t u r a t i o n of the proteins.

Experiments were

begun

solvent m o r e closely r e l a t e d to the p h y s i o l o g i c a l s i t u a t i o n w i t h

occur

using a respect

to i o n i c strength a n d p H . E x p e r i m e n t B , T a b l e I shows that a d s o r p t i o n onto S i l a s t i c w a s l i t t l e affected b y b u f f e r e d saline, b u t a d r a m a t i c decrease i n a d s o r p t i o n onto poly ( H E M A ) / S i l a s t i c occurred.

T h i s l a r g e effect of i o n i c strength s u g

gested the presence of i o n i c i m p u r i t i e s i n the H E M A m o n o m e r u s e d to m a k e the p o l y ( H E M A ) a n d stressed the p o t e n t i a l b i o l o g i c a l i m p o r t a n c e of a n y t y p e of c o n t a m i n a t i o n of the

monomer.

Fortunately, highly purified H E M A became available (from H y d r o M e d S c i e n c e s ) a b o u t this t i m e , a n d other m o n o m e r s w e r e r e a d i l y p u r i f i e d b y v a c u u m d i s t i l l a t i o n . T h e p o l y ( H E M A ) h y d r o g e l s m a d e w i t h the p u r i fied

HEMA

buffered HEMA

s h o w e d f a r l o w e r p r o t e i n a d s o r p t i o n f r o m either w a t e r or

saline t h a n h y d r o g e l s

m a d e w i t h the c o m m e r c i a l l y a v a i l a b l e

as e x p e r i m e n t C , T a b l e I shows.

biological importance

T h e s e results e m p h a s i z e

of h y d r o g e l c o m p o s i t i o n

in particular and

the bio-

m a t e r i a l c o m p o s i t i o n a n d p u r i t y i n general. A c o m m o n c o n t a m i n a n t i n c o m m e r c i a l H E M A is M A A c , e.g., i t w a s present at a b o u t the 2 % l e v e l i n the u n p u r i f i e d H E M A u s e d i n the i n i t i a l


236

APPLIED CHEMISTRY A T PROTEIN

experiments HEMA

d i s c u s s e d above.

(0.02%

MAAc

level)

INTERFACES

T h e availability of the highly purified a l l o w e d us t o investigate t h e effect o f

M A A c levels i n the r a n g e u s u a l l y e n c o u n t e r e d i n the use o f u n p u r i f i e d H E M A b y s i m p l y a d d i n g k n o w n amounts o f M A A c t o the p u r e H E M A . T h e a m o u n t o f p r o t e i n a d s o r p t i o n onto t h e resultant

MAAo-HEMA/

Silastic h y d r o g e l s is s h o w n i n F i g u r e s 1 a n d 2 a n d T a b l e I I .


Adsorption (pg/cm ) 50

H

0

1

2

3

4

%MAAc in HEMA Figure 1. The effect of MAAc on protein adsorption onto poly(HEMA)/ Silastic at low ionic strength. The solvent was 0.005M HEPES, pH 7.4. Protein concentration was 0.5 mg/ml. See Table II for other details. T h e expected measurements

effect o f t h e M A A c

of protein

adsorption

is shown most clearly b y t h e

at l o w i o n i c

strength

(0.005M

H E P E S ) l i s t e d i n T a b l e I I a n d i l l u s t r a t e d i n F i g u r e 1 since i t is clear f r o m these that the p r o t e i n w i t h the most p o s i t i v e charge, i.e., γ-globulin, is a d s o r b e d most s t r o n g l y b y the n e g a t i v e l y c h a r g e d M A A c groups. T h e isoelectric p H ' s o f γ-globulin,

fibrinogen,

a n d a l b u m i n are ca. 6.8, 5.5,


11.

HORBETT AND H O F F M A N

Adsorption

onto

Hydrogels

Adsorption (yg/cm ) 2

i.

Bovine Plasma Protein Adsorption onto Radiation-Grafted Hydrogels

Recommend Documents