Heparin Bearing Coatings or Surfaces: Quantitative Relations

15 Heparin Bearing Coatings or Surfaces: Downloaded via AUBURN UNIV on April 20, 2019 at 12:04:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Quantitative Relations L Y M A N F O U R T , A. M . SCHWARTZ, R. H . B E C K E R , and E I L A O. K A I R I N E N Harris Research Laboratories, Division of Gillette Research Institute, Inc., 1413 Research Boulevard, Rockville, M d . 20850

The amounts of heparin removed from solution,

and of

heparin adsorbed on cationic surfaces, were determined using

S labelled heparin.

35

The averages agree in order of

magnitudes but the surface shows large local variation, con firmed by autoradiographs.

Initial strike effects do not level

out in 48 hours exposure to aqueous heparin solution.

How

ever, uniform heparin deposit is obtained by dipping

the

cationic surface at a steady rate. Minimum heparin loading for

anticoagulant

effect (clotting

min.) approximates 0.3 μg./cm.

2

time longer than

100

for graphite-benzalkonium

-heparin and polyvinylpyridine-heparin

surfaces, and also for

cellulose nitrate lacquers containing

benzalkonium-heparin

complex.

Electrolyte

in the heparin solution reduces the

amount adsorbed, with greater effect on than on graphite-benzalkonium

polyvinylpyridine

surfaces.

' T p h e d i s c o v e r y b y G o t t , W h i f f en, a n d D u t t o n (4) t h a t h e p a r i n a d s o r b e d u p o n a s u i t a b l y r e c e p t i v e surface c o u l d p r e v e n t b l o o d f r o m c l o t t i n g has i n s p i r e d m u c h w o r k o n t h e p r e p a r a t i o n of h e p a r i n b e a r i n g p l a s t i c surfaces.

C o a g u l a t i o n tests, in vitro or in vivo, t e n d to b e "go-no g o "

tests a n d h a v e s e l d o m b e e n u s e d to d e t e r m i n e q u a n t i t a t i v e relations b e t w e e n the a m o u n t of h e p a r i n o n the surface, the m e c h a n i s m of b i n d i n g to t h e surface, a n d the a n t i c o a g u l a n t effectiveness.

Q u a n t i t a t i v e d a t a are

n e e d e d to g i v e e v i d e n c e o n t h e t h e o r e t i c a l q u e s t i o n , w h e t h e r h e p a r i n i n t r o d u c e d o n a surface acts there, or b y g o i n g i n t o s o l u t i o n ; a n d the p r a c t i c a l questions, of the most s u i t a b l e m o d e s of b i n d i n g h e p a r i n to a surface for l o n g t e r m in vivo effectiveness a n d for f a b r i c a t i o n of p r o s t h e t i c systems. 186 Alexander; Interaction of Liquids at Solid Substrates Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

15.

FOURT

ET

AL.

Heparin Bearing Coatings

187

E a r l i e r q u a n t i t a t i v e estimates of the a m o u n t of h e p a r i n i n i t i a l l y present o n a n t i c o a g u l a n t surfaces are s u m m a r i z e d i n T a b l e I.

These

estimates s h o u l d b e r e g a r d e d as i n d i c a t i n g orders of m a g n i t u d e r a t h e r t h a n p r e c i s e measurements. Table I.

Reported Amounts of Heparin on Anticoagulant Surfaces Method

Heparin lig'/cm*

Reference

S , direct Chemical, depletion H , direct H , direct S , direct S , direct

3 or less 0.24 2 3 toi 1.6 1 to 15.7

10,11,12 3 6,7 5 2 2

Chemical, depletion H , direct

3 4 to 700

3 6,7

Surface Graphite-benzalkonium

3 5

3

3

3 5

Cation grafted to silicone rubbers or polypropylene Polyvinylpyridine Cellulose, ethyleneimine

3 5

3

Materials Heparin Bearing Surfaces. G B H . G r a p h i t e - b e n z a l k o n i u m surfaces ( G B ) h a v e b e e n t r e a t e d w i t h s o d i u m h e p a r i n to m a k e the g r a p h i t e b e n z a l k o n i u m - h e p a r i n ( G B H ) surfaces d e s c r i b e d b y G o t t ( 4 ) . The g r a p h i t e ( D a g 154, A c h e s o n C o l l o i d s , 1 p a r t , p l u s 3 parts e t h y l a l c o h o l ) w a s a p p l i e d to films of polyester ( M y l a r ) b y p a i n t i n g , s p r a y i n g , o r d i p ping and draining. P V P Y R H Poly-4-vinylpyridine ( P V P y r ) prepared by conventional persulfate c a t a l y z e d p o l y m e r i z a t i o n w a s d i s s o l v e d i n e t h y l a l c o h o l o r m e t h y l C e l l o s o l v e ( 2 to 3 % s o l u t i o n ) for a p p l i c a t i o n to polyester film substances. T h e s e films adsorb h e p a r i n f r o m solutions of s o d i u m h e p a r i n to p r o d u c e a n t i c o a g u l a n t surfaces ( P V P y r H ) . F i l m s w e r e p a i n t e d o n , or s p r e a d b y k n i f e c o a t i n g . C B H . T h e s p a r i n g l y w a t e r s o l u b l e c o m p o u n d of h e p a r i n w i t h a n o r g a n i c base ( B H ) , s u c h as b e n z a l k o n i u m — e . g . , " Z e p h i r a n " ( W i n t h r o p ) or c e t y l t r i m e t h y l a m m o n i u m b r o m i d e or o c t a d e c y l a m i n e c a n b e d i s s o l v e d i n e t h y l a l c o h o l a n d m i x e d w i t h a n o r g a n i c p o l y m e r matrix—e.g., c o l l o d i o n i n ether a n d a l c o h o l (4). S u c h l a c q u e r m i x t u r e s are d e s i g n a t e d CBH. Methods Adsorption Studies. S A C K S . G B or P V P y r surfaces w e r e m a d e i n t o flat sacks, sealed o n three sides b y a heat s e a l i n g polyester film c o a t e d w i t h a p o l y o l e f i n i c adhesive ( S e a l - L a m i n , S e a l Inc., S h e l t o n , C o n n . ) . T h e f o u r t h side w a s s i m i l a r l y sealed after i n t r o d u c i n g the s o l u t i o n c o n t a i n i n g h e p a r i n l e a v i n g a n a i r b u b b l e enclosed. S u c h flat sacks, w i t h t w o c o a t e d faces e a c h 12 X 12 c m . (288 c m . for a d s o r p t i o n ) a n d o v e r a l l d i m e n s i o n s of 14 X 14 c m . w e r e h e l d b e t w e e n flat discs at a s p a c i n g s u c h t h a t the 2

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

188

INTERACTION

OF

LIQUIDS A T

SOLID

SUBSTRATES

enclosed a i r b u b b l e w o u l d t r a v e l a r o u n d the i n s i d e edge, t a k i n g u p a b o u t 1 / 3 of the e n c l o s e d v o l u m e , thus s t i r r i n g the contents w h e n the d i s c a s s e m b l y w a s r o t a t e d . A n assembly of sacks a n d discs w a s r o t a t e d i n a thermostat for 20 hours or longer. T h e sacks w e r e p a r t i a l l y o p e n e d b y s n i p p i n g off one corner, d r a i n e d , a n d r i n s e d , t h e n o p e n e d b y s l i t t i n g a l o n g the sealed edges. D I P P E D F I L M S . Short t e r m a d s o r p t i o n effects w e r e e x a m i n e d b y d i p p i n g films, c o a t e d onto one or b o t h sides of a polyester sheet, i n t o h e p a r i n solutions. I n s e r t i o n a n d r e m o v a l w a s either b y h a n d or i n s p e c i a l cases b y a constant rate m e c h a n i s m . T h e films w e r e let h a n g w i t h o u t s t i r r i n g i n the s o l u t i o n for a n i n t e r v a l after d i p p i n g . N a r r o w containers w e r e u s e d to m i n i m i z e v o l u m e of s o l u t i o n r e q u i r e d . Determination of Heparin. C H E M I C A L . M o d i f i c a t i o n s of the m e t h o d s of W a r r e n a n d W y s o c k i ( 9 ) a n d B a s s i o u n i ( I ) w e r e u s e d . W h e r e p l a s m a or b l o o d w a s not i n v o l v e d , a d i r e c t t i t r a t i o n of the h e p a r i n w i t h a z u r e A , either m e a s u r i n g the d y e r e m o v a l or r e d i s s o l v i n g the p r e c i p i t a t e , w a s u s e d . RADIO TRACER. H e p a r i n l a b e l l e d o n the N - s u l f a t e w i t h S was o b t a i n e d f r o m C a l b i o c h e m C o . Specific a c t i v i t y w a s 0.65 m c . / g r a m s , 140 U h e p a r i n / m g . T h i s c o u l d be c o u n t e d i n a l i q u i d s c i n t i l l a t i o n system, to d e t e r m i n e d e p l e t i o n f r o m s o l u t i o n . I n t e r n a l standards of C comp o u n d s w e r e u s e d to d e t e r m i n e efficiency. T h e l a b e l l e d h e p a r i n a d s o r b e d o n a surface c o u l d b e c o u n t e d o n a 2 X 2 c m . or 1 i n c h d i a m e t e r p o r t i o n s u p p o r t e d o n a p l a n c h e t i n a gas flow G e i g e r counter. A c a l i b r a t i o n c u r v e was p r e p a r e d b y e v a p o r a t i n g k n o w n amounts of l a b e l l e d h e p a r i n o n a s i m i l a r area of the a d s o r b i n g surface. T h e c a l i b r a t i o n c u r v e w a s l i n e a r over a greater r a n g e , a n d w i t h a h i g h e r c o u n t p e r m i c r o g r a m of l a b e l l e d h e p a r i n , for P V P y r surfaces t h a n for G B surfaces, w h i c h i n d i cates m o r e a b s o r p t i o n of r a d i a t i o n b y the g r a p h i t e layer. A U T O R A D I O G R A P H S . T h e surfaces b e a r i n g a d s o r b e d l a b e l l e d h e p a r i n w e r e d r i e d , t h e n p l a c e d i n d i r e c t contact w i t h " N o S c r e e n " x - r a y film ( K o d a k N S - 2 ) . T h e a d s o r p t i o n sacks c o u l d b e o p e n e d o n three sides, t h e n t w o film sheets i n s e r t e d so that a u t o r a d i o g r a p h s of b o t h surfaces c o u l d b e o b t a i n e d . " R e g i s t e r " of the t w o films w a s e s t a b l i s h e d b y m a k i n g i d e n t i f y i n g notches after assembly. D a r k e n i n g w a s seen o n l y o n t h e side i n contact w i t h h e p a r i n , i n d i c a t i n g t h a t a l l the S b e t a r a d i a t i o n w a s a b s o r b e d i n t h e film a n d its cellulose acetate base, t o t a l thickness 9 m i l s . T r i a l s w i t h the p l a n c h e t c o u n t e r s h o w that a 5 m i l polyester film reduces the c o u n t to 6 to 7 % of that w i t h d i r e c t exposure; w i t h a representative P V P y r film o n polyester the count falls to 4 % , a n d w i t h a G B film o n polyester, to 1.6%. H e n c e , e v e n i n a u t o r a d i o g r a p h s w i t h a d s o r p t i o n of h e p a r i n o n b o t h surfaces, the effect o b s e r v e d is m o r e t h a n 9 0 % f r o m the adjacent surface. A u t o r a d i o g r a p h s s h o w n o effect, w i t h i n the t i m e of exposure u s e d , of l a b e l l e d h e p a r i n a d s o r b e d o n a polyester c o n t r o l , although a small adsorption can be detected b y direct planchet counting. T h e a u t o r a d i o g r a p h s w e r e m a d e at different stages i n the d e c a y of the S ; t h e exposure times therefore v a r i e d , i n order to o b t a i n t h e p a t t e r n , f r o m 3 to 45 days. D Y E I N G O F H E P A R I N O N P V P Y R . T h e transparent films of p o l y v i n y l p y r i d i n e p e r m i t d e m o n s t r a t i o n of the presence of h e p a r i n , a n d some j u d g m e n t of the u n i f o r m i t y of a d s o r p t i o n , b y s t a i n i n g w i t h a b a s i c d y e s u c h as t o l u i d i n e b l u e o r a z u r e A , w h i c h c o m b i n e w i t h h e p a r i n to f o r m 3 5

1 4

3 5

3 5


15.

FOURT

ET

AL.


189

r e l a t i v e l y i n s o l u b l e c o m p o u n d s . A P V P y r film s t a i n e d w i t h either d y e is f a i n t l y b l u e ; i f h e p a r i n has b e e n a b s o r b e d , a r e d d i s h color appears i n m u c h greater strength. T h e redness ( m e t a c h r o m a t i c effect) i n d i c a t e s that this is the c o m p o u n d of the d y e w i t h h e p a r i n . S o m e h e p a r i n also leaves the surface a n d forms a p r e c i p i t a t e i n the s o l u t i o n . C O A G U L A T I O N T E S T S . F r e s h , c i t r a t e d n o r m a l "non-contact a c t i v a t e d " h u m a n p l a s m a , u s e d w i t h i n 6 hours of d r a w i n g the b l o o d , w a s u s e d t h r o u g h o u t unless s p e c i a l l y i n d i c a t e d . T h i s w a s p l a t e l e t r i c h p l a s m a . T h e p l a s m a w a s k e p t i n a n ice b a t h u n t i l just before use, w h e n i t w a s w a r m e d i n s m a l l lots to r o o m t e m p e r a t u r e . A l l h a n d l i n g a n d tests w e r e c o n d u c t e d i n p l a s t i c ( p o l y s t y r e n e , p o l y e t h y l e n e , p o l y ( v i n y l c h l o r i d e ) ) or i n s i l i c o n i z e d glass unless specifically n o t e d . T h e first 5 m i l of b l o o d w a s d i s c a r d e d , to m i n i m i z e tissue or t r a u m a t i c influence. T y p i c a l r e c a l c i f i c a t i o n c l o t t i n g t i m e i n p l a s t i c w a s 20 m i n u t e s ; i n glass, 8 m i n u t e s ; i n glass w i t h 5 m i n u t e s of d e l i b e r a t e a c t i v a t i o n b y r o l l i n g as a film o n the glass w a l l b e f o r e r e c a l c i f i c a t i o n , 3 m i n u t e s , s h o w i n g that the contact a c t i v a t i o n system ( F a c t o r s X I I , X I ) w a s present r e a d y for a c t i v a t i o n .

T w o types of surfaces w e r e m a d e : " C o n e s " — a p o r t i o n of film w a s f o l d e d t w i c e , as i n p r e p a r i n g filter p a p e r for a f u n n e l , t h e n o p e n e d o u t to f o r m a cone. " F l a t s u r f a c e s " — a c o n v e n i e n t area of film, u s u a l l y the 2 X 2 c m . s p e c i m e n o n w h i c h the a d s o r b e d h e p a r i n h a d b e e n d e t e r m i n e d b y the p l a n c h e d counter, w a s p l a c e d i n a P e t r i d i s h , a l o n g w i t h a separate p o r t i o n of cotton or filter p a p e r saturated w i t h w a t e r . C i t r a t e d p l a s m a (0.2 m l . ) w a s p i p e t t e d onto the center of the s p e c i m e n ; t h e n 0 . 0 5 M C a C l s o l u t i o n (0.2 m l . ) w a s a d d e d , a n d the m i x t u r e w a s s t i r r e d w i t h a T e f l o n s t i r r i n g r o d w i t h o u t t o u c h i n g the film a n d w i t h the least p o s s i b l e s p r e a d i n g of the large d r o p . T h e P e t r i d i s h c o v e r w a s t h e n u s e d to p r e v e n t e v a p o r a t i o n or a i r - b o r n e c o n t a m i n a t i o n . D i r e c t v i s u a l o b s e r v a t i o n , or o b s e r v a t i o n w i t h a l o w p o w e r , l o n g w o r k i n g d i s t a n c e stereo m i c r o s c o p e p e r m i t t e d d e t e r m i n a t i o n of the first a p p e a r a n c e of fibrin. T h e l i q u i d o r gel c o n d i t i o n c o u l d b e c h e c k e d b y t i p p i n g the P e t r i d i s h . I n some w o r k , t h e p l a s m a w a s r e c a l c i f i e d i n a p l a s t i c test t u b e , t h e n q u i c k l y t r a n s f e r r e d to the film, w i t h g e n e r a l l y s i m i l a r results, b u t shorter c l o t t i n g times. 2

Results U n i f o r m i t y of A d s o r p t i o n . R e g r e t t a b l y , a u t o r a d i o g r a p h i c testing of u n i f o r m i t y of a d s o r p t i o n was b e g u n s o m e w h a t late i n the w o r k , after the necessity f o r this w a s r e c o g n i z e d f r o m i r r e g u l a r i t i e s of d y e i n g , of c o a g u l a t i o n tests, a n d of p l a n c h e t counts o n s m a l l areas. P l a n c h e t counts s h o w ratios as h i g h as 3 0 : 1 b e t w e e n areas of strong adsorption a n d weak adsorption.

T h e average of a l a r g e n u m b e r

of

2 χ 2 c m . areas, h o w e v e r , shows a g e n e r a l c o r r e s p o n d e n c e to t h e o v e r - a l l average as d e t e r m i n e d b y d e p l e t i o n , as s h o w n i n T a b l e I I . H e n c e , some general guidance on adsorption can be obtained from depletion measure ments, a l t h o u g h for a n y great degree of p r e c i s i o n , u n i f o r m a d s o r p t i o n is required.


190

INTERACTION

OF

LIQUIDS

AT

SOLID

SUBSTRATES

Table II. Comparison of Adsorption of Heparin on P V P y r - M y l a r Surface, by Depletion (Indirect, Average for 288 cm. ) and by Planchet Count (Direct, for 4 cm. ) 4

2

2

Initial Heparin Available in Solution pg./cm. 0

2

B

Adsorbed Heparin on 2 X 2 cm. Squares

Adsorbed Heparin by Depletion ^g. /cm.

3.4

1.87

0.34

0.34

0.17

0.17

0.034

0.034

Average ILg./cm.

Individual fig./cm.

2

2

2

2.28,1.96, 0.92 1.26,1.90 0.07, 0.46, 0.53 0.10, 0.49, 0.61 0.76, 0.45, 0.05 0.06, 0.07 0.29, 0.21, 0.24 0.30, 0.23 0.050, 0.060, 0.020 0.087, 0.082

1.66 0.29

0.25 0.060

Amount in original solution divided by area. A u t o r a d i o g r a p h s of G B H surfaces sometimes s h o w a " s t r i k e " w h e r e

the a q u e o u s s o l u t i o n of h e p a r i n w a s d e l i v e r e d b y a p i p e t t e , a n d r a n d o w n t h e film, as seen i n F i g u r e 1, w h i c h shows the t w o sides of a n a d s o r p t i o n sack w i t h g r a p h i t e - b e n z a l k o n i u m surface.

T h e m a t c h of the a d s o r p t i o n

p a t t e r n o n the t w o surfaces indicates t h a t i t w a s a m a t t e r of i n i t i a l contact w i t h s o l u t i o n , not a p r o p e r t y of the surfaces. o n a n i n i t i a l l y d r y G B surface. tagged

T h i s pattern was

produced

T h e solution was 9 0 % cold heparin, 1 0 %

h e p a r i n , at 67 μ%. t a g g e d

heparin/ml.

a d s o r p t i o n , c a l c u l a t e d as t o t a l ( 1 0 χ

The

average

radioactive heparin

f r o m d e p l e t i o n of the s o l u t i o n , was 2.7 / x g . / c m .

2

heparin

adsorption)

S a c k w a s r o t a t e d 20

h o u r s , w i t h a b u b b l e , at 3 7 ° C . F i g u r e 2 shows t w o sides of a G B H sack w h i c h w a s w e t w i t h w a t e r b e f o r e i n t r o d u c i n g the h e p a r i n s o l u t i o n . T e n m l . of h e p a r i n s o l u t i o n w e r e i n t r o d u c e d i n t o 5 m l . of w a t e r , a v o i d i n g i n i t i a l contact w i t h the w a l l s . T h e p a t t e r n appears to b e r e l a t e d to the c i r c u l a r r o t a t i o n of t h e b u b b l e . O t h e r i n i t i a l l y w e t or d r y sacks s h o w e d s i m i l a r r o t a t i o n - s u g g e s t i n g terns, not t h e

flow-strike

patterns of F i g u r e 1.

pat

H e p a r i n concentration

w a s 648 / x g . / m l . i n the i n i t i a l 15 m l . m i x t u r e ; sacks w e r e r o t a t e d 48 h o u r s w i t h b u b b l e at 37 ° C .

A v e r a g e a d s o r p t i o n of h e p a r i n , b y d e p l e t i o n

of

s o l u t i o n , is 4.0 / x g . / c m . . 2

F i g u r e 3 shows one side of a p o l y v i n y l p y r i d i n e sack, i n i t i a l l y d r y . T e n m l . of h e p a r i n s o l u t i o n w e r e i n t r o d u c e d i n t o the d r y sack, f o l l o w e d b y 5 m l . w a t e r , a v o i d i n g contact w i t h the w a l l s . same e x p e r i m e n t as F i g u r e 2 ) .

( T h i s was p a r t of the

T h e p a t t e r n was m a t c h e d o n the other

side a n d appears to be r e l a t e d to the b u b b l e , its l o c a t i o n i n e a r l y stages


15.

FOURT

ET

AL.

191


Figure 1. Autoradio graphs showing uneven adsorption of heparin on an initially dry graphite-benzalkonium surface. A and Β are the two sides of a flat adsorption sack. Flow from the point of initial contact of the solution is indicated with an initial "strike" which did not level in 20 hours rotation, with a bubble, at 37°C. Average heparin adsorption 2.7 μg./cm. 2

Figure 2. Autoradio graphs showing uneven adsorption on an initially wet graphite-benzalkonium surface. A and Β are two sides of a flat adsorption sack, 10 ml. heparin solution intro duced into 5 ml. water, avoiding contact with the walls. Sack rotated 48 hours with bubble at 37°C; initial heparin concen tration 638 ^g./ml. in 15 ml.; average heparin adsorption 4.0 μg./cm. ; autoradiograph exposure 72 hours 2

of exposure p r i o r to r o t a t i o n , a n d its p a t h d u r i n g r o t a t i o n (48 h o u r s at 37°C).

A f o l d or c r a c k i n the h e p a r i n a d s o r b i n g film c a n also b e seen

i n the l o w e r t h i r d . T h e g e n e r a l l y d a r k e r tone, for the same s o l u t i o n c o n c e n t r a t i o n , age of h e p a r i n , a n d t i m e of a u t o r a d i o g r a p h exposure agrees w i t h the d e p l e t i o n a n d p l a n c h e t c o u n t measurements i n i n d i c a t i n g m o r e h e p a r i n a d s o r b e d p e r u n i t area o n the P V P y r films t h a n o n the G B

films,

at least as w e p r e p a r e t h e m . T h i s m a y i n d i c a t e m o r e c a t i o n i c sites a v a i l a b l e o n P V P y r , b u t w e l a c k i n f o r m a t i o n to c o m p a r e t o t a l cations a v a i l a b l e or the specific surface areas a n d d e p t h of p e n e t r a t i o n , so this o b s e r v a t i o n Alexander; Interaction of Liquids at Solid Substrates Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

192

INTERACTION

OF

LIQUIDS

AT

SOLID

SUBSTRATES

is p u r e l y e m p i r i c a l r e l a t i n g o n l y to the p a r t i c u l a r w a y i n w h i c h these films have been prepared.

P V P y r sacks w h i c h w e r e i n i t i a l l y w e t s h o w s i m i l a r

patterns. I n i t i a l m i x t u r e c o n c e n t r a t i o n w a s 648 jug. h e p a r i n / m l . ; average a d s o r p t i o n , b y d e p l e t i o n , 7.6 / x g . / c m . . 2

F i g u r e 4 shows t h e r e l a t i v e l y e v e n a d s o r p t i o n o n p o l y v i n y l p y r i d i n e surfaces s p r e a d b y k n i f e c o a t i n g a n d d i p p e d i n t o r a d i o a c t i v e h e p a r i n s o l u t i o n (500 / x g . / m l . ) for 6 m i n u t e s . S i x discs e a c h 1 i n c h i n d i a m e t e r w e r e d i e c u t f r o m e a c h of t w o d u p l i c a t e p r e p a r a t i o n s w h i c h w e r e s i m i l a r i n v i s u a l evenness to the ones s h o w n .

P l a n c h e t counts g a v e t h e results

shown below: Λ lig./cm.

Β iig./cm*

1.88 1.64 1.78 1.58 1.45 1.43 1.63 0.17 12%

1.52 1.43 1.47 1.44 1.45 1.27 1.43 0.08 6%

2

Average Standard Deviation Coefficient of Variation Influence of Electrolytes.

Presence of electrolyte of l o w m o l e c u l a r

w e i g h t i n the s o l u t i o n w i t h the h e p a r i n reduces the a m o u n t of h e p a r i n a d s o r b e d , a n d promotes

l e v e l i n g , as w o u l d b e e x p e c t e d

f r o m the fact

t h a t t h e r e l a t i v e l y w a t e r - i n s o l u b l e c o m p l e x e s or c o m p o u n d s of h e p a r i n and

other

mucopolysaccharides

are

increasingly dissociated

and

m u c o p o l y s a c c h a r i d e b r o u g h t i n t o s o l u t i o n b y i n c r e a s i n g electrolyte centrations ( 8 ) .

Since Gott (4)

the con

a n d most other surgeons h a v e a p p l i e d

h e p a r i n f r o m s o l u t i o n i n 0 . 1 5 M N a C l , the p r a c t i c a l f o r m a t i o n of G B H a n t i c o a g u l a n t surfaces o n s u r g i c a l i m p l a n t s has p r o b a b l y i n v o l v e d m u c h less n o n - u n i f o r m i t y t h a n m a n y of o u r experiments. T h e t w o types of surface, G B a n d P V P y r , differ i n the effect of elec trolyte on heparin adsorption.

O n G B , the h e p a r i n a d s o r p t i o n is u n

c h a n g e d b e t w e e n 0 . 1 5 M a n d 0 . 5 M N a C l , b u t d r o p s b y h a l f at 1 . 0 M ; o n P V P y r t h e a d s o r p t i o n is u n c h a n g e d b e t w e e n 0 . 0 5 M a n d 0 . 1 5 M , b u t d r o p s to close to zero at 0 . 5 M . T h e s e results w e r e a l l o b t a i n e d i n a d s o r p t i o n sacks. A u t o r a d i o g r a p h s i n d i c a t e d that w e h a d not f o u n d the c o n d i t i o n s for u n i f o r m a d s o r p t i o n , a l t h o u g h l o g i c a l l y a d s o r p t i o n c a n b e e x p e c t e d b e m o r e u n i f o r m u n d e r c o n d i t i o n s i n w h i c h i n i t i a l strike c a n b e v e n t e d , or l e v e l e d

by

equilibrium adsorption

and desorption

to

pre

during

l o n g e r exposure. We

are e n g a g e d i n o b t a i n i n g m o r e u n i f o r m surfaces f r o m

m o r e precise a n d accurate a d s o r p t i o n isotherms c a n b e o b t a i n e d .


which Results

15.

FOURT

ET

AL.


Figure 3. Autoradiograph showing uneven adsorption on an ini tially dry polyvinylpyridine surface. Rotated with bubble 48 hours at 37°C. Heparin concentration 638 μg./ml., average heparin adsorp tion 7.6 ^g./cm. . Autoradiograph exposure 72 hours (comparable with Figure 2). In the lower third there is a fold or crack line in the film, probably introduced before the sack was made 2

Figure 4. Rehtively even autoradiograph of two portions of polyvinylpyridine film, A and B. There is some gradation from top to bot tom, but the uniformity is high compared with Figure 1 through 3. This was dipped into heparin solution, held immersed 6 minutes, then withdrawn and rinsed. Planchet counts of die cut one inch circles taken from a pair of surfaces similar in visual evenness are given in the text, showing coefficients of variation of 12 and 6% for duplicates


193

194

INTERACTION

OF

LIQUIDS

AT

SOLID

SUBSTRATES

o b t a i n e d b y d e p l e t i o n analysis c a n o n l y i n d i c a t e the average a d s o r p t i o n over the w h o l e area. H o w e v e r , s u c h analyses i n d i c a t e that a l l the h e p a r i n a v a i l a b l e , u p to a n average of a b o u t 3 / x g / c m . , w i l l b e a d s o r b e d o n G B 2

surfaces.

T h e l e v e l for c o m p l e t e e x h a u s t i o n b y P V P y r films appears to

b e h i g h e r , ca. 10 / x g . / e m . . 2

H o w e v e r , the a u t o r a d i o g r a p h s s h o w t h a t the

true m a x i m u m m a y be m u c h higher. Effect of Thickness of Adsorbing Film.

E a r l i e r studies of effect of

film thickness ( 3 ) i n d i c a t e d t h a t the t r e n d b e t w e e n g r a p h i t e film t h i c k n e s s a n d a m o u n t of b e n z a l k o n i u m a d s o r b e d was s m a l l , f r o m a b o u t 2 to 4 /xg. benzalkonium per c m .

2

for v a r i a t i o n f r o m 200 to 1000 / x g . g r a p h i t e p e r

c m . . T h e analyses b y c h e m i c a l m e t h o d s for h e p a r i n a d s o r b e d w e r e not 2

precise e n o u g h to s h o w a n y t r e n d . C h e m i c a l analysis d i d s h o w that f r o m 1.5 to 7.5 /xg. of h e p a r i n w a s a b s o r b e d o n f r o m 400 to 1000 /xg. of P V P y r p e r c m . , b u t the range of h e p a r i n w a s so great at e a c h P V P y r l e v e l t h a t 2

n o clear t r e n d c o u l d b e seen.

M o r e recent a u t o r a d i o g r a p h i c studies w i t h

e a c h k i n d of film i n d i c a t e t h a t for b r i e f exposures to h e p a r i n s o l u t i o n i n d i s t i l l e d w a t e r there is i n d e e d a t r e n d for m o r e h e p a r i n to be

adsorbed

o n t h i c k e r films of either k i n d . U n i f o r m Adsorption by U n i f o r m Mechanical Exposure.

If an a d -

s o r b i n g film is i n t r o d u c e d i n t o the s o l u t i o n at a u n i f o r m rate, the " s t r i k e " effect c a n b e m a d e u n i f o r m . A u t o r a d i o g r a p h s s u c h as F i g u r e 4 s h o w t h a t this c a n b e a c c o m p l i s h e d , or at least that m u c h m o r e u n i f o r m a d s o r p t i o n c a n b e o b t a i n e d b y d i p p i n g t h a n b y exposure i n sacks.

Whether

the

h e p a r i n so a d s o r b e d is the same i n its e q u i l i b r i u m relations to solutions is yet to b e d e t e r m i n e d , b u t i t appears s i m i l a r i n a n t i c o a g u l a n t effect. Minimum Heparin Binding for Anticoagulant Effect.

U s i n g auto-

r a d i o g r a p h s as g u i d e s , selection of r e l a t i v e l y u n i f o r m areas o n w h i c h the h e p a r i n c a n b e d e t e r m i n e d b y p l a n c h e t c o u n t i n g has p e r m i t t e d estimates of r e l a t i o n to the a n t i c o a g u l a n t effect.

T a b l e I I I shows

some results,

w h i c h i n d i c a t e that for G B H surfaces, the l i m i t lies b e t w e e n 0.2 a n d 0.3 /xg. h e p a r i n / c m . . 2

T a b l e I I I also shows results o n P V P y r H

films

prepared by

d i p p i n g a n d d e m o n s t r a t e d to b e u n i f o r m b y a u t o r a d i o g r a p h s .

brief

H e r e the

effect of i n c r e a s i n g a m o u n t of h e p a r i n p e r u n i t area appears m o r e g r a d e d , b u t a g a i n close to 0.3 /xg. h e p a r i n / c m . is n e e d e d to d e l a y c l o t t i n g longer 2

t h a n 100 m i n u t e s . Minimum Heparin in a Lacquer. E a r l i e r w o r k ( 3 ) u s i n g c o a g u l a t i o n tests i n cones s h o w e d that the a n t i c o a g u l a n t effect of a l a c q u e r c o n t a i n i n g h e p a r i n - b e n z a l k o n i u m c o m p o u n d ( G B H ) d e p e n d e d not o n l y o n the t o t a l a m o u n t of h e p a r i n i n t h e l a c q u e r , b u t also r e q u i r e d a m i n i m u m c o n c e n t r a t i o n . V a r i a t i o n of h e p a r i n content at constant film thickness ( 5 m i c r o n s ) i n d i c a t e s a c r i t i c a l l e v e l b e t w e e n 0.3 a n d 0.5 / x g . / c m . ; v a r i a t i o n of 2

thickness at 0.3 or 0 . 6 %

film

B H i n the film i n d i c a t e s a s i m i l a r c r i t i c a l l e v e l ,


15.

FOURT

ET

AL.

195


b u t at 0 . 0 7 5 % B H i n the G B H film, n o a m o u n t of film or h e p a r i n , u p to 1.5 / A g . / c m . or 50 m i c r o n s thickness s h o w e d a n t i c o a g u l a t i o n . 2

Table III.

M i n i m u m H e p a r i n B i n d i n g for A n t i c o a g u l a n t Effect

Tests: C O A G U L A T I O N T I M E : 0.2 ml. citrated plasma placed on flat surface, recalcified with 0.1 ml. M / 2 0 C a C l , stirred with a Teflon strip. Controls are Mylar polyester surfaces. H E P A R I N : determined on each test portion by planchet count. A. Relatively uniform G B H areas from long-time adsorption sacks, selected with guidance of autoradiographs 2

Coagulation Time, min.

Heparin tig./cm.

(Darkening of Film)

Fibrin

Clot

0.0 0.17 0.51 1.2

control light medium heavier

25 25 100+ 100+

25 35

2

B.

Uniform areas produced by dipping, checked for uniformity by autoradiographs Surface Control GBH

Control PVPyrH

Heparin fig./cm.

Coagulation Time, min. Fibrin

0 0.05 0.09 0.27 0 0.08 0.9 0.13 0.25 0.29 0.31

27 37 63 100+ 26 50 48 47 68 84 100+

2

Discussion E a r l i e r d a t a ( 3 ) o b t a i n e d w i t h tests i n cones a n d c h e m i c a l d e t e r m i n a t i o n suggested that the a n t i c o a g u l a n t effect of h e p a r i n i n t r o d u c e d o n the w a l l was greater t h a n that to b e e x p e c t e d if a l l the h e p a r i n w e r e d i s s o l v e d i n t h e p l a s m a . T h i s c o n c l u s i o n requires c h e c k i n g w i t h

more

u n i f o r m h e p a r i n layers a n d i m p r o v e d c o a g u l a t i o n test t e c h n i q u e s o n flat surfaces.

It is k n o w n that some h e p a r i n goes i n t o s o l u t i o n i n p l a s m a

f r o m G B H or P V P y r H surfaces, as p r e p a r e d b y us, a n d i t seems p r o b a b l e that this is g e n e r a l l y t r u e for a n y a d s o r b e d h e p a r i n system i n w h i c h the


196

INTERACTION

O F LIQUIDS A T SOLID

h e p a r i n is h e l d t o the surface b y i o n i c b o n d s .

SUBSTRATES

H e n c e , the effect o f the

d i s s o l v i n g h e p a r i n c o u l d b e greater i n the diffusion l a y e r near the surface t h a n i f u n i f o r m l y d i s t r i b u t e d t h r o u g h the s o l u t i o n . W e b e l i e v e t h a t the a n s w e r t o the q u e s t i o n w h e t h e r the h e p a r i n is a c t i n g w h i l e a d s o r b e d m a y b e a p p r o a c h e d either b y m o r e precise q u a n t i t a t i v e studies, the r o a d t o w h i c h n o w seems o p e n , o r b y c o n c l u s i v e s h o w i n g of effectiveness of h e p a r i n c o v a l e n t l y o r c o m p l e t e l y b o u n d to the surface. F r o m a p r a c t i c a l p o i n t o f v i e w , t h e a n t i c o a g u l a n t effectiveness

of heparin b o u n d to a

surface b y different basic g r o u p s s h o u l d b e of i m p o r t a n c e w i t h respect to d u r a t i o n o f effectiveness, a n d a m o u n t o f h e p a r i n r e q u i r e d .

Literature Cited

(1) Bassiouni, M., J. Clin. Path. 7, 330 (1954). (2) Falb, R. D., Takahashi, M. T., Grode, G. Α., Leininger, R. I.,J.Biomed. Materials Res. 1, 239 (1967). (3) Fourt, L., Schwartz, A. M., Quasius, Α., Bowman, R. L., Trans. Am. Soc. Artif. Int. Organs 12, 155 (1966). (4) Gott, V. L., Whiffen, J. D., Dutton, R. C., Science 142, 1297 (1963). (5) Kramer, R. S., Vasko, J. S., Morrow, A. G., J. Thor. Cardiovasc. Surg. 53, 130 (1967). (6) Merrill, E. W., Salzman, E. W., Lipps, B. J., Gilliland, E. R., Austen, W. G., Joison, F., Trans. Am. Soc. Artif. Int. Organs 12, 139 (1966). (7) Salzman, E. W., Austen, W. G., Lipps, B. J., Jr., Merrill, E. W., Gilliland, E. R., Joison, F., J. Surgery 61, 1 (1967). (8) Scott, J. E., "Methods of Biochemical Analysis," Vol. VIII, p. 145, D. Glick, Ed., Interscience, Ν. Y. 1960. (9) Warren, R., Wysocki, Α., Surgery 44, 435 (1958). (10) Whiffen, J. D., Gott, V. L., J. Surg. Res. 5, 51 (1965). (11) Whiffen, J. D., Trans. Am. Soc. Artif. Int. Organs 12, 164 (1966). (12) Whiffen, J. D., Beekler, D. C., J. Thor. Cardiovasc. Surg. 52, 121 (1966). RECEIVED September 25, 1968. This work is part of Contract PH 43-64-498 of the Artificial Heart Program, National Heart Institute, National Institutes of Health.


Heparin Bearing Coatings or Surfaces: Quantitative Relations

Recommend Documents