Heparin Covalently Bonded to Polymer Surface - ACS Publications

16 Heparin Covalently Bonded to Polymer Downloaded by KTH ROYAL INST OF TECHNOLOGY on September 10, 2015 | http://pubs.acs.org Publication Date: June 1, 1968 | doi: 10.1021/ba-1968-0087.ch016

Surface B. DAVID HALPERN and RIICHIRO SHIBAKAWA Polysciences, Inc., Warrington, Pa. 18976

Cross-linked

polystyrene beads were para-nitrated

surface then reduced to the aminopolystyrene

on

the

and finally

converted by phosgenation to isocyanato-polystyrene.

Cou-

pling to sodium heparin was achieved at room temperature in formamide.

The covalently bound heparin beads were

non-clotting for twenty-four hours when tested by method.

Special antithrombogenic

hydrogel

Lee-White

tubes were

used for the tests since other surfaces induced clotting. similar

synthetic

sequence

to

surface

modify

A

injection

molded polystyrene rings for in v i v o testing in canine hearts yielded badly distorted pieces which were only moderately antithrombogenic.

The roughened surface was thought to

have contributed to clot formation. copolymers

of

N-vinylphthalimide

The use of cross-linked later reduced

to

the

amine was shown to be a route which allowed distortion-free polymers containing pendant isocyanate groups for heparin coupling.

T i J " u c h w o r k o n h e p a r i n - q u a t e r n a r y a m m o n i u m complexes ITX

has

been

r e p o r t e d as a n o u t g r o w t h of the o r i g i n a l p u b l i c a t i o n s b y G o t t

(2,

3 ) w h i c h s h o w e d t h e i r v a l u e as a n t i t h r o m b o g e n i c surfaces for prosthetic heart valves. T h e G B H surfaces ( g r a p h i t e - b e n z a l k o n i u m c h l o r i d e - h e p a r i n coating)

of G o t t h a v e s u p e r i o r a n t i t h r o m b o g e n i c properties w h e n

p a r e d w i t h glass, p o l y c a r b o n a t e s , a n d silicone. L e i n i n g e r et al. ( 8 ) M e r r i l l et al. ( 9 )

further developed

comand

this a p p r o a c h b y u s i n g c a t i o n i c

modifications of a n u m b e r of p o l y m e r surfaces i n c l u d i n g p o l y p r o p y l e n e , p o l y s t y r e n e , a n d p o l y e t h y l e n i m i n e grafted onto cellulose as substrates for c o m p l e x i n g the h e p a r i n . 197 In Interaction of Liquids at Solid Substrates; Alexander, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

198

INTERACTION

OF

LIQUIDS

AT

SOLID

SUBSTRATES

O n e of t h e most difficult p r o b l e m s yet to b e o v e r c o m e i n t h e d e v e l o p m e n t of a w o r k a b l e artificial heart is t h a t of t h r o m b o s i s o n t h e surface of t h e p r o s t h e t i c m a t e r i a l s ( 2 ) u s e d i n its c o n s t r u c t i o n .

A l t h o u g h the

G B H t y p e surfaces of G o t t a n d later w o r k e r s are of research p r o m i s e , there is c o n c e r n that f o r l o n g t e r m i m p l a n t a t i o n t h e h e p a r i n w h i c h is

Downloaded by KTH ROYAL INST OF TECHNOLOGY on September 10, 2015 | http://pubs.acs.org Publication Date: June 1, 1968 | doi: 10.1021/ba-1968-0087.ch016

electrostatically c o m p l e x e d

to t h e surface m a y u l t i m a t e l y b e c o m e d e -

p l e t e d o w i n g to d i s s o c i a t i o n ( 5 , 6 ) .

T h u s , the prosthetic device m a y

b e c o m e susceptible to c l o t t i n g . I f h e p a r i n w e r e to b e c o v a l e n t l y b o n d e d to a substrate i n a b i o l o g i c a l l y stable m a n n e r w h i c h d i d n o t cause a loss of its a n t i t h r o m b o g e n i c n a t u r e , w e w o u l d t h e n h a v e a n o n - m i g r a t a b l e f o r m of h e p a r i n .

T h e m e t h o d u s e d to b o n d h e p a r i n c o v a l e n t l y t o a

p o l y m e r substrate a n d a p r e l i m i n a r y d e m o n s t r a t i o n of t h e a n t i t h r o m b o g e n i c v a l u e in vitro of s u c h a surface constitute t h e basis f o r this p a p e r . W e also b o n d e d h e p a r i n i o n i c a l l y to a p o l y m e r substrate i n m u c h t h e same w a y as L e i n i n g e r d i d ( 8 ) since this surface is k n o w n to h a v e g o o d in vivo a n t i t h r o m b o g e n i c character a n d c o u l d thus serve as a g o o d in vitro c o n t r o l . Covaient Linking

Reaction

I n o r d e r that t h e h e p a r i n r e m a i n b i o l o g i c a l l y a c t i v e after fixation to a p o l y m e r substrate, t h e c o v a l e n t b o n d m u s t b e a c h i e v e d via f u n c t i o n a l groups o n t h e h e p a r i n m o l e c u l e , w h i c h are non-essential f o r its b i o l o g i c a l activity. F u r t h e r , the b i n d i n g reaction should be performed under react i o n c o n d i t i o n s w h i c h themselves d o n o t cause loss of t h e a n t i t h r o m b o g e n i c c h a r a c t e r of t h e h e p a r i n . T h e f u n c t i o n a l groups of h e p a r i n s u i t a b l e for f o r m a t i o n of a covalent b o n d i n c l u d e t h e a l c o h o l , c a r b o x y l i c a c i d , a n d a m i n o groups. T h e s t r u c t u r e of h e p a r i n is s h o w n i n F i g u r e 1. T h e a m i n o groups r e a d i l y react w i t h

H

NHSOg

H

OH

Figure 1.

H

NHSO3

H

0S0"

Proposed heparin molecule

Molecular weight: 12,000 Sulfate group: 4-5 per unit a c y l a t i n g a n d a l k y l a t i n g agents a n d a l d e h y d e s a n d isocyanates as w e l l as w i t h d i a z o n i u m salts. T h e h y d r o x y groups react w i t h a c y l a t i n g agents,

In Interaction of Liquids at Solid Substrates; Alexander, A.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

16.

HALPERN

AND

SHiBAKAWA

199

Heparin

isocyanates; a n d t h e y also c o u p l e w i t h d i a z o n i u m salts. C o v a l e n t b o n d s c a n also b e e s t a b l i s h e d b e t w e e n c a r b o x y l i c a c i d groups i n h e p a r i n a n d a p o l y m e r substrate c o n t a i n i n g a n u c l e o p h i l i c g r o u p s u c h as a n a m i n o g r o u p , b y e m p l o y i n g c r o s s - l i n k i n g agents. T h e d i c y c l o h e x y l c a r b o d i i m i d e a n d diisocyanates are t y p i c a l examples of c r o s s - l i n k i n g agents w h i c h c a n


react u n d e r m i l d e n v i r o n m e n t a l c o n d i t i o n s .

Sample

Standard

Sample

Phenol Phenol f

||Naphth I Naphth I

Figure 2.

Gas chromatographic identification for isocyanate group

I n one of the recent studies o n the c h e m i c a l c o u p l i n g r e a c t i o n , A x e n ( I ) s u c c e e d e d i n b o n d i n g a n e n z y m e c o v a l e n t l y onto a n i n s o l u b l e p o l y mer by

e m p l o y i n g the isothiocyanate as the c h e m i c a l coupler.

The

e n z y m e r e t a i n e d a l l its c a t a l y t i c activities. T h e m e t h o d u s e d b y us to a t t a c h h e p a r i n onto a p o l y m e r substrate m a d e use of a p e n d a n t isocyanate



200

INTERACTION

OF

LIQUIDS A T

SOLID

SUBSTRATES

g r o u p o n a p o l y m e r b a c k b o n e . W e find that t h e isocyanate c o u p l i n g r e a c t i o n for b o n d i n g t h e h e p a r i n ( w h i c h is i n its s o d i u m salt f o r m ) m u s t p r o c e e d u n d e r v e r y m i l d c o n d i t i o n s . E x t r e m e s of p H a n d h i g h r e a c t i o n temperatures m u s t b e a v o i d e d i n o r d e r to m i n i m i z e t h e h y d r o l y s i s of t h e n a t u r a l l y o c c u r r i n g sulfate a n d a m i d o s u l f a t e groups i n h e p a r i n . F o r m a m i d e w a s u n i q u e a m o n g m a n y solvents tested i n b e i n g able t o dissolve s o d i u m h e p a r i n at r o o m t e m p e r a t u r e . C r o s s - l i n k e d p o l y s t y r e n e beads w e r e u s e d as t h e starting p o i n t for t h e f o r m a t i o n of polystyrene-isocyanate. T h e a m o u n t of h e p a r i n w h i c h b o n d e d to this substrate w a s d e t e r m i n e d b y s u l f u r m i c r o a n a l y s i s of t h e final p r o d u c t after i t h a d b e e n w a s h e d exhaustively w i t h w a t e r . It is d e s i r a b l e to use c r o s s - l i n k e d p o l y s t y r e n e rather t h a n h o m o p o l y m e r styrene, as t h e latter has a t e n d e n c y to s w e l l a n d dissolve i n some of t h e reactions a n d a d i s t o r t e d surface m a y result. Materials and Methods. T h e stepwise sequence f o r t h e synthesis o f polystyrene-isocyanate a n d subsequent r e a c t i o n w i t h h e p a r i n is s h o w n below:

c

' (Cross-linked with 1% divinyl benzene)

-jCH-CFyn-

QV)

-(CH-CH )n2

(Where R is heparin attached through its hydroxy I group)

PREPARATION OF P-NITROPOLYSTYRENE (I). A p o l y s t y r e n e w h i c h is c r o s s - l i n k e d to t h e extent of 1 % w i t h d i v i n y l b e n z e n e a n d passed t h r o u g h a screen of 2 4 m e s h , a n d r e t a i n e d o n a screen size of 5 0 m e s h , w a s u s e d as a p o l y m e r substrate. T o a s o l u t i o n of 5 m l . of c o n c e n t r a t e d n i t r i c a c i d w a s a d d e d 1 5 grams of t h e c r o s s - l i n k e d p o l y s t y r e n e beads. T h e r e a c t i o n w a s c a r r i e d o u t at 6 0 ° C . f o r h a l f a n h o u r w i t h s t i r r i n g . T h e p r o d u c t w a s filtered a n d w a s h e d w i t h w a t e r several times a n d t h e n t h o r o u g h l y w a s h e d w i t h methanol on a Buchner funnel. T h e product was dried under vacu u m f o r 2 4 hours. M i c r o a n a l y s i s s h o w e d t h a t 0 . 7 % b y w e i g h t of n i t r o g e n


16.

HALPERN

A N D SHIBAKAWA

Heparin

201


was i n c o r p o r a t e d . T h e I R spectra of p - n i t r o p o l y s t y r e n e s h o w e d a m e d i u m a b s o r p t i o n at 7.5 m i c r o n s because o f t h e n i t r o g r o u p . A different p a t t e r n of a b s o r p t i o n peaks f r o m that of p o l y s t y r e n e w a s also o b s e r v e d i n t h e h i g h e r w a v e l e n g t h area. PREPARATION OF P-AMINOPOLYSTYRENE ( I I ) . R e d u c t i o n of t h e p n i t r o p o l y s t y r e n e w a s a c c o m p l i s h e d b y u s i n g s o d i u m d i t h i o n i t e or stannous c h l o r i d e as the r e d u c i n g agent. F i v e grams of p - n i t r o p o l y s t y r e n e ( I ) w a s s u s p e n d e d i n a s o l u t i o n c o n t a i n i n g 20 m l . of w a t e r , 2 5 m l . c o n c e n t r a t e d a m m o n i u m h y d r o x i d e , a n d a n excess a m o u n t of s o d i u m d i t h i o n i t e . T h e m i x t u r e w a s refluxed for 24 h o u r s w i t h s t i r r i n g . T h e p r o d u c t w a s filtered out a n d w a s h e d w i t h d i s t i l l e d w a t e r several times. T h e resultant p - a m i n o p o l y s t y r e n e w a s d i a z o t i z e d w i t h nitrous a c i d . W e w e r e a b l e to s h o w t h e presence of p - a m i n o p o l y s t y r e n e b y c o u p l i n g w i t h p h e n o l a n d a n i l i n e t o g i v e intense y e l l o w i s h a n d r e d colors r e s p e c t i v e l y . CONVERSION T O ISOCYANATE DERIVATIVES ( I I I ) . T h e a m i n o groups w e r e c o n v e r t e d to isocyanate groups b y treatment of t h e a m i n o - p o l y m e r w i t h phosgene gas i n hexane. I n t o the suspension of 5 grams of w e l l d r i e d p - a m i n o p o l y s t y r e n e ( I I ) i n 300 m l . hexane, a constant stream of p h o s g e n e gas w a s passed for 6 hours after w h i c h t h e m i x t u r e w a s refluxed. A f t e r c o o l i n g t o r o o m t e m p e r a t u r e , t h e p r o d u c t w a s filtered u n d e r n i t r o g e n a t m o s p h e r e a n d w a s h e d t h o r o u g h l y w i t h hexane. I t w a s u s e d d i r e c t l y f o r t h e c o u p l i n g r e a c t i o n . T h e isocyanate content w a s d e t e r m i n e d b y gas c h r o m a t o g r a p h y u s i n g n a p h t h a l e n e as a n o n - r e a c t i v e i n t e r n a l s t a n d a r d a n d p h e n o l as a c o u p l i n g agent b o t h d i s s o l v e d i n toluene. F i g u r e 2 shows t h e d u p l i c a t e s of t h e q u a n t i t a t i v e i d e n t i f i c a t i o n of t h e isocyanate groups. T h e " S t a n d a r d " consists of p h e n o l as a c o u p l i n g reagent, n a p h t h a l e n e as a n i n t e r n a l s t a n d a r d a n d t o l u e n e as a solvent. T h e " S a m p l e " consists of t h e s t a n d a r d s o l u t i o n a n d t h e isocyanate-polystyrene. I t w a s o b s e r v e d that t h e h e i g h t difference b e t w e e n n a p h t h a l e n e a n d p h e n o l i n the " S t a n d a r d " a p p r e c i a b l y d i m i n i s h e d c o m p a r e d to t h e " S a m p l e " i n w h i c h phenol a n d isocyanate-polystyrene reacted for a f e w minutes. T h e m i n i m u m content of t h e isocyanate g r o u p w a s c a l c u l a t e d f r o m t h e d e p l e t i o n of p h e n o l as b e i n g 244 p . p . m . b y w e i g h t of isocyanate p e r g r a m of p o l y m e r . F I X A T I O N O F H E P A R I N ( I V ) . H e p a r i n w a s c o u p l e d to p - i s o c y a n a t o p o l y s t y r e n e b y t h e f o l l o w i n g p r o c e d u r e . F i v e h u n d r e d m g . of h e p a r i n w a s d i s s o l v e d i n 50 m l . of f r e s h l y d i s t i l l e d f o r m a m i d e at r o o m t e m p e r a t u r e w i t h s t i r r i n g . I n t o this clear s o l u t i o n , 100 grams of f r e s h l y m a d e p - i s o c y a n a t o - p o l y s t y r e n e ( I I I ) w a s a d d e d a n d t h e r e s u l t i n g suspension s t i r r e d f o r 24 h o u r s at r o o m t e m p e r a t u r e u n d e r a n i t r o g e n atmosphere. A t t h e e n d of this p e r i o d , t h e r e a c t i o n p r o d u c t w a s c o l l e c t e d o n a filter a n d exhaust i v e l y w a s h e d w i t h w a t e r to r e m o v e a l l u n r e a c t e d h e p a r i n . F i n a l l y , i t w a s w a s h e d f u r t h e r w i t h m e t h a n o l a n d d r i e d u n d e r v a c u u m for 24 h o u r s . M i c r o a n a l y s i s i n d i c a t e d that t h e s u l f u r content of t h e final p r o d u c t w a s 0 . 1 9 % b y w e i g h t . T h i s corresponds to 1.7% of h e p a r i n fixed to t h e p o l y styrene surface. P a r a l l e l controls s h o w that w h e n isocyanate groups are absent, n o s u l f u r is f o u n d i n t h e p r o d u c t . T h e a b o v e result is i n t e r p r e t e d as e v i d e n c e of covalent b o n d i n g of t h e h e p a r i n t o t h e p o l y s t y r e n e backbone.


202

INTERACTION

Synthesis of Τrimethylaminopolystyrene Its Reaction with

OF

LIQUIDS A T

SOLID

SUBSTRATES

Iodide and

Heparin

Reaction Scheme: (VT)

(V) Downloaded by KTH ROYAL INST OF TECHNOLOGY on September 10, 2015 | http://pubs.acs.org Publication Date: June 1, 1968 | doi: 10.1021/ba-1968-0087.ch016

-(CH-CHjn-

9

-(CH-CH )n-

-(CH-CHJn-

2

5%CH

I 3 — in C H , OH

( C H , ) , N Heparin

PREPARATION PROCEDURES. F i v e grams of p - a m i n o p o l y s t y r e n e ( I I ) p r e p a r e d as before f r o m c r o s s - l i n k e d p o l y s t y r e n e w a s refluxed w i t h 5 % CH3I i n C H 3 O H for 1 0 hours. T h e p r o d u c t was filtered, w a s h e d w i t h water and methanol, and then dried under vacuum. This quaternary a m m o n i u m p o l y m e r f u r t h e r r e a c t e d w i t h 5 0 m g m . of h e p a r i n i n w a t e r s o l u t i o n to f o r m the c o m p l e x ( V I ) . T h e s u l f u r m i c r o a n a l y s i s s h o w e d 0 . 9 % by weight.

Determination Covalently

of Anti-Clotting

Bound Heparin

Activities

of

Beads

T h e a b o v e samples w e r e

sent to the U n i v e r s i t y of P e n n s y l v a n i a

H o s p i t a l for in vitro testing of the a n t i t h r o m b o g e n i c c h a r a c t e r of

the

c o v a l e n t l y b o u n d h e p a r i n a n d the i o n i c a l l y b o u n d h e p a r i n o n p o l y s t y r e n e . T h e p r o c e d u r e u s e d for d e t e r m i n a t i o n of c l o t t i n g t i m e is a m o d i f i c a t i o n of a L e e - W h i t e C l o t t i n g Test. B e f o r e e a c h test w a s u n d e r t a k e n , the surface of e a c h s a m p l e w a s t h o r o u g h l y w a s h e d w i t h d i s t i l l e d w a t e r a n d o v e n d r i e d . B l o o d u s e d i n these tests w a s o b t a i n e d f r o m a n o r m a l h u m a n v o l u n t e e r a n d w a s u s e d as d r a w n w i t h o u t c i t r a t i o n . T h e t w e n t y cc. of b l o o d u s e d for e a c h test w e r e d r a w n f r o m a n a n t e c u b i t a l v e i n . I n o r d e r to ensure that the b l o o d w h i c h w a s u s e d i n e a c h test w a s of a l o w tissue thromboplastin concentration, a two syringe technique was used a n d o n l y the last 6 cc. of b l o o d t a k e n w e r e u s e d i n the test; the first 1 4 cc. were discarded.

T i m e m e a s u r e m e n t w a s started as soon as the

blood

entered the t u b e a n d s t o p p e d u p o n the onset of clot f o r m a t i o n .

Occa

s i o n a l l y , w h e n the s a m p l e was o b s e r v e d not to h a v e c l o t t e d w i t h i n t h i r t y m i n u t e s , a p o r t i o n of the b l o o d w a s r e m o v e d f r o m the t u b e a n d p l a c e d o n a p i e c e of gauze a n d c a r e f u l l y e x a m i n e d for s l i g h t e v i d e n c e of clot formation.

T h e samples w h i c h w e r e u s e d for the a b o v e test w e r e as

follows:


16.

H A L P E R N A N D SHiBAKAWA

(1) Polystyrene benzene. (2)

beads

203

Heparin

(50

mesh),

1%

cross-linked b y d i v i n y l -

A m i n o p o l y s t y r e n e s y n t h e s i z e d f r o m 1.

(3) Trimethylaminopolystyrene-heparin ionically bonded beads s y n t h e s i z e d f r o m 1.

complex


( 4 ) P o l y - p - i s o c y a n a t o s t y r e n e - h e p a r i n c o v a l e n t l y b o n d e d beads s y n t h e s i z e d f r o m 1. E a c h different test w a s c o n d u c t e d b o t h i n glass tubes a n d i n h y d r o g e l tubes ( 4 ) . I t w a s a p p a r e n t that a glass t u b e w a s n o t s u i t a b l e f o r c o n d u c t i n g t h e b l o o d c l o t t i n g test f o r e v a l u a t i n g t h e p o l y s t y r e n e beads, since t h e glass t u b e itself i n d u c e s c l o t t i n g . I n o r d e r to o b v i a t e t h e c l o t t i n g effect b y glass, w e chose a h y d r o g e l t u b e w h i c h w a s s h o w n to b e n o n - c l o t t i n g to w h o l e b l o o d after 24 hours of contact ( 4 ) . T h e h y d r o g e l u s e d f o r t h e a b o v e test w a s a c o p o l y m e r

hydrogel of 1 6 . 4 % acrylamide a n d 5 %

dimethylaminoethyl methacrylate, cross-linked b y 0 . 1 % methylenebisacrylamide. T o p r e v e n t h e m o l y s i s of t h e b l o o d i n t h e h y d r o g e l tubes, i t w a s necessary to k e e p t h e g e l p H a n d s a l i n i t y e q u i v a l e n t t o that of b l o o d . T h i s w a s d o n e b y a d j u s t i n g t h e p H of t h e m o n o m e r s o l u t i o n to 7.38 b y p h o s p h a t e buffer s o l u t i o n a n d 0 . 8 5 % of s o d i u m c h l o r i d e w a s a d d e d . F i v e m g . a n d fifty m g . of e a c h p o l y m e r s a m p l e w e r e p l a c e d i n t h e h y d r o g e l tubes a n d t h e c l o t t i n g tests c o n d u c t e d i n d u p l i c a t e . T h e results are presented i n T a b l e I . Table I.

Lee-White Clotting

Test

In Glass Tube (minutes) Polystyrene beads (5.0 mg.) Polystyrene beads (50 mg.) Aminopolystyrene (50 mg.) Aminopolystyrene ( 50 mg. ) Trimethylaminopolystyrene-heparin ionic complex (5 mg.) Trimethylaminopolystyrene-heparin ionic complex (50 mg.) Isocyanate-polystyrene-heparin covalent (5mg.) Isocyanate-polystyrene-heparin covalent (50 mg.) Hydrogel-control

11; 11; 11; 11;

11 11 11 11

11; 11 11; 11 11; 11 11; 11

In Hydrogel* (minutes) 26; 16; 38; 21;

36 16 34-1/2 19-1/2

2811/2; 24 21; 16-1/2 720; 41 (owing to air bubbles) 16; 16 720; 720

16.4% Acrylamide, 5% Dimethylaminoethyl methacrylate, and 0.1% Methylenebisacrylamide copolymer hydrogel. 0.85% NaCl added and pH 7.38 buffered. α

W h e n glass tubes w e r e u s e d as containers f o r t h e beads, a l l of t h e samples c l o t t e d w i t h i n eleven m i n u t e s .

T h e r a p i d c l o t t i n g is a s c r i b e d


204

INTERACTION

OF

LIQUIDS

AT

SOLID

SUBSTRATES

to t h e o v e r r i d i n g effect of t h e glass surface of t h e tube. H o w e v e r , w h e n these same b e a d samples w e r e p l a c e d i n t h e h y d r o g e l tubes, significant differences

i n clotting time were obtained.

It was observed

that t h e

p o l y s t y r e n e beads, t h e p o l y a m i n o s t y r e n e beads, a n d t h e p o l y t r i m e t h y l a m i n o s t y r e n e - h e p a r i n i o n i c a l l y b o n d e d beads c a u s e d c l o t t i n g i n less t h a n


35 m i n u t e s .

H o w e v e r , one of t h e p o l y - p - i s o c y a n a t o s t y r e n e - h e p a r i n co-

v a l e n t l y b o n d e d b e a d samples gave a greater t h a n 720 m i n u t e s c l o t t i n g t i m e , w h i c h is a p p r e c i a b l y longer t h a n t h e i o n i c a l l y b o n d e d h e p a r i n i z e d surface a n d c o m p a r a b l e to t h e n o n - c l o t t i n g b e h a v i o r of t h e g e l surface itself. T h e s a m p l e of b l o o d w a s n o t c l o t t e d after 24 hours, a l t h o u g h t h e a c t u a l L e e - W h i t e test w a s c a r r i e d o u t for o n l y 12 hours ( 7 2 0 m i n . ) . O n e of t h e samples w h i c h c o n t a i n e d 5 m g . of c o v a l e n t l y

bonded

h e p a r i n i z e d beads h a d o n l y 41 m i n u t e s c l o t t i n g t i m e w h i c h is v e r y m u c h shorter t h a n t h e other s a m p l e , b u t still l o n g e r t h a n t h e i o n i c a l l y b o n d e d h e p a r i n c o m p l e x beads.

T h i s shortened c l o t t i n g t i m e w a s a t t r i b u t e d to

a n a i r b u b b l e w h i c h w a s t r a p p e d b y a m e c h a n i c a l error w h i l e t h e t e c h nician was a d d i n g the blood into the tube dropwise. T h e technician pred i c t e d a priori that this p a r t i c u l a r s a m p l e w o u l d h a v e a l o w v a l u e f o r clotting time. I n trials w h e r e w e g r e a t l y e x t e n d e d t h e surface area of t h e p o l y m e r beads c o n t a i n i n g c o v a l e n t l y b o n d e d h e p a r i n , w e f o u n d s i g n i f i c a n t l y l o w e r c l o t t i n g t i m e . W i t h t h e 50 m g . samples w h i c h h a d t e n times t h e surface area, r a p i d c l o t t i n g w a s o b s e r v e d i n a l l cases.

T h i s is a n i n d i c a t i o n that

either t o t a l surface o r excess h e p a r i n at t h e b l o o d i n t e r f a c e is of significance. W e h a v e h a d several occasions i n other tests w h e r e r e l a t i v e l y l a r g e amounts of h e p a r i n p o l y m e r i z e d i n t o h y d r o g e l s s h o w e d shorter c l o t t i n g times t h a n s i m i l a r gels w i t h lesser amounts of h e p a r i n .

Summary A m e t h o d of c o v a l e n t l y b o n d i n g h e p a r i n t o a p o l y m e r substrate is presented.

T h e synthetic r o u t e consists of c o u p l i n g h e p a r i n c o v a l e n t l y

w i t h p o l y i s o c y a n a t o s t y r e n e . T h i s r e a c t i o n w a s m a d e possible b y t h e fact that f o r m a m i d e is a r o o m t e m p e r a t u r e solvent f o r s o d i u m h e p a r i n a n d this a l l o w e d a l i q u i d - s o l i d interface r e a c t i o n to take place.

Lee-White

c l o t t i n g tests in vitro ( i n h y d r o g e l t u b e s ) s h o w e d these surfaces to b e n o n - c l o t t i n g after 24 hours whereas u n t r e a t e d controls a n d surfaces of G B H type clotted i n approximately 25-35 minutes. Acknowledgment W e g r a t e f u l l y a c k n o w l e d g e t h e s u p p o r t p r o v i d e d us b y t h e N a t i o n a l H e a r t Institute of t h e N a t i o n a l Institutes of H e a l t h , t h r o u g h C o n t r a c t N o .


16.

HALPERN

A N D SHIBAKAWA

PH-43-66-1124.

Heparin

205

T h e t e c h n i c a l assistance o f H s i u n g C h e n g a n d C o r n e l i u s

C a i n is also a p p r e c i a t e d .


Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9)

Axen, R., Porath, J., Nature (London) 210, 367 (1966). Gott, V. L., J. Surg. Res. 6, 274 (1966). Gott, V. L., Whiffen, J. D., Dutton, R. C., Science 142, 1297 (1963). Halpern, B. D., Shibakawa, R., Cheng, H., Am. Inst. of Chem. Eng. Sym. Biochem. Eng. Materials, Phila. Pa. (April 1, 1968). Kramer, R. S., Vasko, J. S., Morrow, A. G., J. Thorac Cardiov. Surg. 53, 130 (1967). Kramer, R. S., Vasko, J. S., Morrow, A. G., Surg. Forum 17, 136 (1966). Leininger, R. I., Cooper, C. W., Falb, R. D. et al., Science 152, 1625 (1966). Leininger, R. I., Epstein, M. M., Falb, R. D., Grode, G. Α., Trans. Am. Soc. Artif. Intern. Organs 12, 151 (1966). Merrill, E. W., Salzman, E. W., Lipps, B. J., Jr., Gilliland, E. R., Austen, W. G., Joison, J., Am. Soc. Artif. Int. Organs, 12, 139 (1966).

RECEIVED October 28, 1968.


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SUBSTRATES


INTERACTION


Heparin Covalently Bonded to Polymer Surface - ACS Publications

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