Adsorption of Fibronectin to Polyurethane Surfaces - American

Chapter 21

Adsorption of Fibronectin to Polyurethane Surfaces: Fourier Transform Infrared Spectroscopic Studies W. G. Pitt, S. H. Spiegelberg, and S. L. Cooper

Downloaded by STANFORD UNIV GREEN LIBR on July 5, 2012 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch021

Department of Chemical Engineering, University of Wisconsin, Madison, WI 53706

The infrared spectra of plasma fibronectin adsorbed to three polyurethanes shows evidence of structural change upon adsorption. These block copolymers have identical hard segment chemistry, but they differ in soft segment composition and surface energy as measured by contact angle. On the more hydrophobic surfaces, the amount of adsobed fibronectin and the extent of spectral changes were greater than on the more hydrophilic surface. When compared to the spectrum of FN in solution, the spectra of the pro tein which adsorbs first appear to have more exten sive spectral changes than protein adsorbing at later times. On all surfaces, increasing the concentration of protein in solution increased the amount of adsorbed protein. Plasma f i b r o n e c t i n (FN) o r c o l d i n s o l u b l e g l o b u l i n i s a h i g h m o l e c u l a r - w e i g h t g l o b u l a r g l y c o p r o t e i n which i s thought t o mediate c e l l a d h e s i o n and growth p r o c e s s e s on a r t i f i c i a l s u r f a c e s . Recent r e v i e w s o f the s t r u c t u r e and f u n c t i o n o f FN have been p u b l i s h e d (L>1) · m e d i a t i n g p l a t e l e t a d h e s i o n and thrombus f o r m a t i o n on polymer s u r f a c e s exposed to n o n - a n t i c o a g u l a t e d whole b l o o d has been p r e v i o u s l y s t u d i e d i n t h i s l a b o r a t o r y u s i n g an ex v i v o A-V f e m o r a l shunt i n c a n i n e s (3^6)· These s t u d i e s have shown t h a t when FN i s p r e - a d s o r b e d t o v a r i o u s polymers p r i o r t o i m p l a n t a t i o n , the amount o f p l a t e l e t and f i b r i n o g e n d e p o s i t i o n on the polymers i s i n c r e a s e d by an o r d e r o f magnitude, s u g g e s t i n g that the adsorbed FN had r e t a i n e d i t s a b i l i t y to b i n d p l a t e l e t s . Other i n v i t r o s t u d i e s , however, have i n d i c a t e d t h a t when FN i s a d s o r b e d , i t s n a t i v e c o n f o r m a t i o n i s changed and i t l o s e s some b i o l o g i c a l a c t i v i t y toward a n t i b o d y b i n d i n g ( 7 - 1 0 ) . These types o f c o n f o r m a t i o n a l changes were o b s e r v e d t o a g r e a t e r e x t e n t on h y d r o p h o b i c s u r f a c e s than on h y d r o p h i l i c s u r f a c e s , s u g g e s t i n g t h a t the s u b s t r a t e s u r f a c e energy a f f e c t s the c o n f o r m a t i o n of the a d s o r b e d protein. A l s o , FN was o b s e r v e d t o a d s o r b t o a g r e a t e r e x t e n t on the h y d r o p h o b i c s u r f a c e s than on the more p o l a r s u r f a c e s T

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0097-6156/87/0343-0324$06.00/0 © 1987 American Chemical Society

In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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A l t h o u g h the s u r f a c e energy i s p r o b a b l y n o t the o n l y parameter w h i c h determines the s t a t e of adsorbed FN, i t does appear t o be important. An u n d e r s t a n d i n g of how s u r f a c e e n e r g e t i c s e f f e c t the i n t e r a c t i o n of FN w i t h polymer s u r f a c e s would be an i m p o r t a n t c o n t r i b u t i o n i n the a r e a s of c e l l growth and the i n t e r a c t i o n of b l o o d p r o t e i n s w i t h s y n t h e t i c polymers. T h i s study a d d r e s s e s the q u e s t i o n of how b u l k polymer che m i s t r y and s u r f a c e energy a f f e c t the amount and the c o n f o r m a t i o n of FN adsorbed to a s e r i e s of p o l y u r e t h a n e u r e a s . The technique of F o u r i e r t r a n s f o r m i n f r a r e d s p e c t r o s c o p y (FTIR) coupled w i t h a t t e nuated t o t a l r e f l e c t a n c e (ATR) o p t i c s was used to c o n t i n u o u s l y and n o n - i n v a s i v e l y measure the k i n e t i c s of FN a d s o r p t i o n , as w e l l as to m o n i t o r c o n f o r m a t i o n a l changes o c c u r i n g d u r i n g a d s o r p t i o n . M a t e r i a l s and Methods P r o t e i n P u r i f i c a t i o n . Canine plasma f i b r o n e c t i n was used i n t h i s study i n o r d e r to c o r r e l a t e these i n v i t r o s t u d i e s w i t h c a n i n e ex v i v o e x p e r i m e n t s i n v o l v i n g preadsorbed c a n i n e p r o t e i n s . Canine FN was i s o l a t e d from c i t r a t e d c a n i n e plasma u s i n g the methods of R u o s l a h t i ( 1 1 ) . The FN was suspended i n phosphate b u f f e r e d s a l i n e (PBS) c o n t a i n i n g 0.02% ΝβΝβ, and then s n a p f r o z e n and s t o r e d a t -70°C u n t i l l e s s than 24 hours b e f o r e use. The p r o t e i n was then snapthawed a t 40°C, f i l t e r e d (0.22 um M i l l e x GV, M i l l i p o r e , B e d f o r d , MA), and d i l u t e d to c o n c e n t r a t i o n s o f 0.07 o r 0.21 mg/ml as determined by UV absorbance The p u r i t y and homogeneity of the lamide g e l e l e c t r o p h o r e s i s i n t r a n s m i s s i o n FTIR spectrum o f windows w i t h a path l e n g t h of resolution.

thawed FN were v e r i f i e d by p o l y a c r y sodium d o d e c y l s u l f a t e (SDS-PAGE). A 5.02 mg/ml FN i n PBS between CaF2 3 pm was c o l l e c t e d a t 8 cm"l

Polymer S u r f a c e P r e p a r a t i o n and C h a r a c t e r i z a t i o n . Three p o l y u r e t h a n e ureas were prepared as p r e v i o u s l y d e s c r i b e d ( 1 2 ) . These c o n t a i n a methylene b i s ( p - p h e n y l d i i s o c y a n a t e ) (MDI) hard segment, an e t h y l e n e diamine c h a i n e x t e n d e r , and a p o l y e t h e r s o f t segment i n mole r a t i o s of 2/1/1 r e s p e c t i v e l y . The s o f t segment m a t e r i a l s were p o l y e t h y l e n e o x i d e (PEO) and polytetramethyleneoxide (PTMO), both of 1000 m o l e c u l a r w e i g h t , and p o l y d i m e t h y I s i l o x a n e (PDMS) of 2000 m o l e c u l a r w e i g h t . I n t h i s paper, these polymers w i l l be r e f e r e d to as PEO-PEUU, PTMO-PEUU and PDMS-PEUU r e s p e c t i v e l y . These were d i s s o l v e d i n Ν,Ν-dimethy1 acetamide (DMA) t o make a 0.1 wt.% s o l u t i o n . Germanium i n t e r n a l r e f l e c t i o n elements (IRE, 50x20x3, 45° a p e r t u r e ) were p o l i s h e d t w i c e w i t h 0.3 um a l u m i n a p o l i s h , r i n s e d w i t h d i s t i l l e d w a t e r , r i n s e d w i t h e t h a n o l , and then c l e a n e d i n a r a d i o frequency plasma d i s c h a r g e . The I R E s were p u l l e d v e r t i c a l l y ( d i p coated) from the polymer s o l u t i o n s a t 3mm/mln. Each coated IRE was d r i e d i n a vacuum oven a t 60°C f o r a t l e a s t 4 h o u r s , and was s t o r e d under vacuum u n t i l use. Underwater c o n t a c t a n g l e s of a i r and octane i n double d i s t i l l e d d e i o n i z e d w a t e r were determined as p r e v i o u s l y d e s c r i b e d ( 1 3 ) . The harmonic mean e q u a t i o n (14) was used to e s t i m a t e the s u r f a c e energy parameters of the polymer coated IRE s. 1

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The t h i c k n e s s o f the polymer c o a t i n g s was determined as f o l l o w s . Polymer f i l m s were s p i n c a s t (15) from a MDI/ED/PTMO p o l y u r e t h a n e u r e a o f known c o m p o s i t i o n . The f i l m t h i c k n e s s , measured by a micrometer, was found t o c o r r e l a t e l i n e a r l y w i t h the h e i g h t o f the 1600 cm" peak (v(C=C) benzene r i n g ) , thus a l l o w i n g d e t e r m i n a t i o n o f a Beer's law e x t i n c t i o n c o e f f e c i e n t f o r the mass f r a c t i o n o f benzene r i n g s i n these polymers. T r a n s m i s s i o n s p e c t r a of the d i p c o a t e d IRE* s were o b t a i n e d , and the polymer t h i c k n e s s determined assuming t h a t the Beer's law e x t i n c t i o n c o e f f i c i e n t was unchanged on these t h i n f i l m s . Downloaded by STANFORD UNIV GREEN LIBR on July 5, 2012 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch021

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Measurement o f P r o t e i n A d s o r p t i o n U s i n g FTIR/ATR. The study o f p r o t e i n a d s o r p t i o n u s i n g FTIR/ATR i s p r e s e n t e d i n d e t a i l elsewhere (13,16-21), and a r e o n l y b r i e f l y reviewed here. I n f r a r e d r a d i a t i o n from the s p e c t r o m e t e r source e n t e r s and then r e f l e c t s i n t e r n a l l y a l o n g the l e n g t h o f an i n t e r n a l r e f l e c t i o n element (IRE) mounted i n a flow c e l l . Each r e f l e c t i o n o f the i n f r a r e d beam on the IRE s u r f a c e produces an " e v a n e s c e n t " wave w h i c h decays e x p o n e n t i a l l y as i t extends i n t o the p r o t e i n s o l u t i o n . Polymer, b u f f e r and p r o t e i n w i t h i n t h i s evanescent wave absorb some o f the i n f r a r e d energy and d e c r e a s e the i n t e n s i t y o f the i n f r a r e d r a d i a t i o n e x i t i n g the IRE. T h i s a t t e n u a t i o n i s d e t e c t e d and p r o c e s s e d by the FTIR, p r o d u c i n g an i n f r a r e d absorbance spectrum o f the m o l e c u l e s w i t h i n the eva n e s c e n t wave. By s u b t r a c t i n g the s p e c t r a o f polymer and b u f f e r (and water v a p o r , when p r e s e n t ) , one o b t a i n s the spectrum o f the p r o t e i n near the p o l y m e r - s o l u t i o n i n t e r f a c e . T h i s spectrum c o n t a i n s c o n t r i b u t i o n s from b o t h the adsorbed and the non-adsorbed ( o r " b u l k " s o l u t i o n ) p r o t e i n w i t h i n the evanescent wave ( 1 9 , 2 0 ) . The d e t a i l s o f p r o c e s s i n g the s p e c t r a have been reported p r e v i o u s l y (13,16). The i n f r a r e d spectrum o f a p r o t e i n p r o v i d e s i n f o r m a t i o n on b o t h the amount ( t h e amide I I band) and the c o n f o r m a t i o n ( t h e amide I and I I I bands) o f the p r o t e i n . P r e v i o u s s t u d i e s have used b o t h the h e i g h t (17) and a r e a (22) o f the amide I I band t o q u a n t i t a t e the amount of p r o t e i n on a s u r f a c e . The p o l y u r e t h a n e u r e a s were exposed t o the FN s o l u t i o n s i n a f l o w c e l l c o n t a i n e d i n s i d e a c o n s t a n t temperature (39°C) compart ment b u i l t i n t o a N i c o l e t 170SX FTIR equipped w i t h a MCT d e t e c t o r ( N i c o l e t , Madison, W l ) . The polymer s u r f a c e s were exposed t o PBS b u f f e r i n the f l o w c e l l f o r a t l e a s t 40 minutes p r i o r t o i n t r o d u c t i o n o f the p r o t e i n i n o r d e r to a t t a i n some e q u i l i b r a t i o n between the polymer and b u f f e r . The b u f f e r was d i s p l a c e d by i n j e c t i n g a t 1 ml/sec a t l e a s t 4.5 ml o f FN s o l u t i o n from a s y r i n g e . D u r i n g i n j e c t i o n the w a l l shear r a t e was 400 sec"*-, and a f t e r i n j e c t i o n the s o l u t i o n was s t a t i c u n t i l t e r m i n a t i o n o f the experiment. The volume o f s o l u t i o n i n j e c t e d was shown by r e s i d e n c e time d i s t r i b u t i o n experiments ( d a t a n o t shown) to remove 97% o f the b u f f e r . From the time o f i n j e c t i o n of the FN i n t o the f l o w c e l l , s p e c t r a were c o l l e c t e d c o n t i n u o u s l y a t 8 cm" r e s o l u t i o n f o r a t l e a s t 2 h r , a l t h o u g h a few experiments were c o n t i n u e d f o r up t o 18 h r . D u r i n g d a t a c o l l e c t i o n , the number o f coadded scans i n c r e a s e d from 4 a t 'he f i r s t time p o i n t t o 2000 f o r times a f t e r 30 min. Upon c o m p l e t i o n >f an a d s o r p t i o n e x p e r i m e n t , the non-adsorbed p r o t e i n was d i s p l a c e d by f l o w i n g b u f f e r g e n t l y through the f l o w c e l l , and a f i n a l s p e c t r a o f the r e m a i n i n g adsorbed p r o t e i n was c o l l e c t e d . Two 1


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a d s o r p t i o n experiments were done on each polymer a t each p r o t e i n concentration. Q u a n t i t a t i o n of Adsorbed F i b r o n e c t i n . G r i n n e l l has shown t h a t 1 2 5 j _ l a b e l i n g o f FN causes l o s s of some of i t s b i o l o g i c a l a c t i v i t y (7)· I n s t e a d of u s i n g r a d i o l a b e l e d FN, the area of the amide I I absorbance band (22) was used to q u a n t i t a t e the amount of adsorp t i o n i n t h i s s t u d y . Known amounts of FN were d r i e d from a 0.1 M NaCl s o l u t i o n onto bare I R E s w h i c h were then p l a c e d i n the same o p t i c a l c o n f i g u r a t i o n as i n the a d s o r p t i o n experiments. S p e c t r a of the d r i e d p r o t e i n were o b t a i n e d , and the areas of the amide I I from 1590 to 1474 cm"** band were measured. The a r e a of the amide I I a b s o r p t i o n d i d n o t change s i g n i f i c a n t l y as the p r o t e i n was d r i e d (±5% s t a n d a r d d e v i a t i o n ) . The l i n e a r c o r r e l a t i o n between the amide I I a r e a and the s u r f a c e c o n c e n t r a t i o n of FN, shown i n F i g u r e 1, was used to determine the amount of FN adsorbed i n the experiments.


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R e s u l t s and

Discussion

S u r f a c e C h a r a c t e r i z a t i o n . T a b l e I p r e s e n t s the c o n t a c t a n g l e s ( t h r o u g h the w a t e r p h a s e ) , i n t e r f a c i a l e n e r g i e s , and polymer t h i c k n e s s e s of the polymers coated onto the Germanium c r y s t a l . The c o n t a c t a n g l e s f o r the polymers decrease i n the o r d e r PTMO-PEUU, PDMS-PEUU and PEO-PEUU, i n d i c a t i n g t h a t the polymer s u r f a c e s become more p o l a r i n t h a t o r d e r . The y a l s o decreases i n t h i s o r d e r , a l t h o u g h these v a l u e s s h o u l d o n l y be taken as approximate v a l u e s c o n s i d e r i n g t h a t the r e l a t i o n s h i p between c o n t a c t a n g l e s and s u r f a c e energy on p o l a r s o l i d s i n not w e l l e s t a b l i s h e d ( 2 3 ) . T h a t these t h i n f i l m s have lower s u r f a c e energy than t h i c k e r f i l m s of the same polymers (12) may be a r e s u l t of t h e i r b e i n g a p p l i e d as a v e r y t h i n c o a t i n g which c o u l d d i s r u p t the b u l k polymer morphology that exists i n thicker films. Scanning e l e c t r o n m i c r o s c o p i c exami n a t i o n of the s u r f a c e s d i d n o t r e v e a l e v i d e n c e of h o l e s or b r e a k s i n the f i l m s . s w

Table I . Polymer

P r o p e r t i e s of D i p Coated P o l y u r e t h a n e

air-water contact angle ( d e g r e e s )

PEO-PEUU PDMS-PEUU PTMO-PEUU

35 ± 3 38 ± 3 59 ± 5

Surfaces

thickness octane w a t e r c o n t a c t sw (dyn/cm) (Â) a n g l e (degrees) Y

59 ± 1 68 ± 1 87 ± 2

8.7 17.8 19.6

173 137

The t h i c k n e s s of the d i p coated PEO-PEUU c o u l d n o t be d e t e r mined. The polymer t h i c k n e s s e s r e p o r t e d here s h o u l d be taken as o n l y approximate v a l u e s , due to the c u m u l a t i v e e r r o r s i n the s p e c t r a l measurements a s s o c i a t e d w i t h the very low s i g n a l / n o i s e of these t h i n f i l m s . The t h i c k n e s s e s of these polymers a r e about 1/20 of the depth of p e n e t r a t i o n of the i n f r a r e d evanescent wave (about 400 nm a t 1550 cm*" ) ( 2 1 ) . Thus t h e r e i s adequate space w i t h i n the e v a n e s c e n t wave f o r a d s o r p t i o n of the 4x60 nm FN m o l e c u l e s to be observed (24). 1


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A d s o r p t i o n K i n e t i c s . F i g u r e s 2A and 2B show the FN a d s o r p t i o n k i n e t i c s on the t h r e e s u r f a c e s from 0.07 and 0.21 mg/ml FN s o l u t i o n s r e s p e c t i v e l y . Each l i n e i s the average of two e x p e r i m e n t s on a g i v e n polymer. A t each p r o t e i n c o n c e n t r a t i o n , the i n i t i a l r a t e of a d s o r p t i o n i s independent o f the type of polymer s u b s t r a t e , and a d s o r p t i o n from 0.21 mg/ml i s n e a r l y 3 times f a s t e r than from 0.07 mg/ml FN. The i n i t i a l a d s o r p t i o n r a t e s a r e l i n e a r i n t i m e / until a d s o r p t i o n exceeds 0.06 ug/cm on PEO-PEUU and 0.10 ug/cm on the o t h e r polymers ( d a t a n o t shown). T h i s suggests t h a t the a d s o r p t i o n i s d i f f u s i o n c o n t r o l l e d up t o the above s u r f a c e c o n c e n t r a t i o n s , a f t e r w h i c h p o i n t the a d s o r p t i o n r a t e d e c r e a s e s and becomes depen dent upon the polymer s u r f a c e c h e m i s t r y . The amount of FN adsorbed does n o t reach a p l a t e a u w i t h i n 120 m i n u t e s , n o r does i t reach a p l a t e a u when a d s o r p t i o n c o n t i n u e s f o r 18 hours ( d a t a n o t shown). A t both c o n c e n t r a t i o n s , the l o w e s t a d s o r p t i o n o c c u r s on PEO-PEUU, s u p p o r t i n g p r e v i o u s o b s e r v a t i o n s t h a t h y d r o p h i l i c s u r f a c e s adsorb l e s s FN than more h y d r o p h o b i c s u r f a c e s (7j-9). A l s o a t b o t h con c e n t r a t i o n s , PDMS-PEUU adsorbs s l i g h t l y more FN than the PTMO-PEUU, even though the c o n t a c t angle data i n d i c a t e t h a t PDMS-PEUU i s s l i g h t l y more p o l a r , s u g g e s t i n g t h a t the s u r f a c e c h e m i s t r y as w e l l as s u r f a c e energy i n f l u e n c e the amount o f a d s o r p t i o n . 1


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C o n f o r m a t i o n a l Changes i n Adsorbed FN. One of the major advantages of FTIR s p e c t r o s c o p y i s i t s p o t e n t i a l f o r e l u c i d a t i n g p r o t e i n s t r u c t u r a l changes. I n t h i s s t u d y , changes i n the FN s p e c t r a were o b s e r v e d . The t r a n s m i s s i o n FTIR spectrum o f FN i n PBS i s shown i n F i g u r e 3 w i t h the amide I I I r e g i o n expanded by a f a c t o r o f 4. The amide I , I I and I I I bands a r e c e n t e r e d a t 1642, 1549, and 1247 cm*" r e s p e c t i v e l y , c h a r a c t e r i s t i c o f a p r o t e i n c o n t a i n i n g some 8s h e e t s t r u c t u r e . F i g u r e 3 a l s o shows t y p i c a l FN s p e c t r a a f t e r 2 h r o f a d s o r p t i o n onto the t h r e e polymers from the 0.07 mg/ml s o l u t i o n . S e v e r a l d i f f e r e n c e s between the s o l u t i o n and adsorbed s p e c t r a are obvious. F i r s t there i s a change i n the s m a l l absorbance band i n the 1740-1720 cm" r e g i o n . On PEO-PEUU, t h i s band i s a t the same f r e q u e n c y , and i s about the same magnitude as observed f o r FN i n s o l u t i o n . However, on the PDMS-PEUU and PTMO-PEUU, the band has s h i f t e d 20 cm" and has i n c r e a s e d i n magnitude. T h i s band c o u l d be a s s i g n e d t o the c a r b o n y l s t r e t c h i n g v i b r a t i o n of the COOH group, the p r o t o n a t e d form o f the c a r b o x y l i c a c i d w h i c h produces the band a t 1400 cm" . I n FN, c a r b o x y l i c a c i d s a r e found i n some p r o t e i n r e s i d u e s (Asp and G l u ) , a t the c a r b o x y l end o f the p o l y p e p t i d e , and i n the a c i d i c c a r b o h y d r a t e s ( s i a l i c a c i d ) . These have COOH v i b r a t i o n s a t 1735-1720 cm" , 1755-1720 cm" and 1748-1724 cm" r e s p e c t i v e l y (25). A s i m i l a r peak a t 1735 cm" was observed i n a study of lysozyme a d s o r p t i o n on c o n t a c t l e n s e s , and was a s s i g n e d t o i n t e r a c t i o n o f G l u o r Asp r e s i d u e s w i t h the polymer s u r f a c e (26). F o r m a t i o n of a p r o t o n a t e d c a r b o x y l i c a c i d o c c u r s when the pK o f the p a r t i c u l a r COOH group i s near o r g r e a t e r than the l o c a l pH of the s o l u t i o n , which i s n o t o f t e n the case f o r p r o t e i n s a t p h y s i o l o g i c pH. The p K o f the α-carboxyl ranges about 3.1-3.5 (27) and t h a t o f s i a l i c a c i d i s near 2.6 (28). The p K o f the a c i d i c p r o t e i n r e s i d u e s i s u s u a l l y between 3 and 5, a l t h o u g h a pK o f 6.5 has been r e p o r t e d f o r a Asp r e s i d u e i n a n o n p o l a r r e g i o n of lysozyme (29). The polymer s u r f a c e c h e m i s t r y appears t o i n f l u e n c e the 1

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F i g u r e 1. C o r r e l a t i o n o f the amide I I a r e a w i t h the mass o f FN d r i e d on a Germanium IRE.

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F i g u r e 2. FN a d s o r p t i o n onto PEO-PEUU ( # ) , PTMO-PEUU ( • ) and PDMS-PEUU ( • ) from 0.07 mg/ml (A) and from 0.21 mg/ml ( B ) .




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v i b r a t i o n o f the COOH group s i n c e the band appears a t d i f f e r e n t f r e q u e n c i e s and a d s o r p t i o n times on each polymer s u r f a c e . On PEO-PEUU, the band begins t o appear a t 1740 cm" a f t e r about 20 minutes o f a d s o r p t i o n and then i n c r e a s e s w i t h time as F i g u r e 4 i l l u s t r a t e s . On the PDMS-PEUU s u r f a c e ( F i g u r e 5 ) , a l a r g e r and b r o a d e r band appears a t 1725 cm" from t h e time o f f i r s t c o n t a c t o f the p r o t e i n w i t h the polymer. I t i n c r e a s e s f o r about 5 minutes and then remains c o n s t a n t a l t h o u g h the o t h e r peaks i n the p r o t e i n s p e c t r a I n c r e a s e i n magnitude. On the PTMO-PEUU s u r f a c e ( F i g u r e 6 ) , a band a l s o appears a t 1720 cm" from the time o f f i r s t c o n t a c t and i n c r e a s e s w i t h time f o r about 15 m i n u t e s , a f t e r w h i c h time i t remains c o n s t a n t . On a l l t h r e e polymers, the COOH band i s r e l a t i v e l y l a r g e r ( w i t h r e s p e c t t o t h e 1550 cm" band) f o r a d s o r p t i o n from the 0.07 mg/ml s o l u t i o n than from the 0.21 mg/ml s o l u t i o n ( d a t a n o t shown). The appearance o f the COOH peak a t 1720 cm" on the more h y d r o p h o b i c polymers s u g g e s t s t h a t these polymers may i n t e r a c t more s t r o n g l y w i t h the adsorbed FN. The decrease i n v i b r a t i o n a l f r e quency on PTMO-PEUU and PDMS-PEUU c o u l d be a t t r i b u t e d t o many f a c t o r s , one o f w h i c h c o u l d be hydrogen bonding o f the c a r b o n y l group i n COOH w h i c h would decrease i t s v i b r a t i o n a l frequency ( 2 5 ) . The i n c r e a s e d magnitude o f the COOH v i b r a t i o n on the h y d r o p h o b i c p o l y mers i n d i c a t e s t h a t more COO" groups have been p r o t o n a t e d t o the COOH form. A l t h o u g h one cannot r u l e o u t the p o s s i b i l i t y o f a major pK change due t o p r o t e i n d e n a t u r a t i o n , i t i s more l i k e l y t h a t t h e c a r b o x y l groups become p r o t o n a t e d as they approach ( o n the o r d e r o f Angstroms) the polymer s u r f a c e . Here the p r o x i m i t y o f a s u r f a c e w i t h a low d i e l e c t r i c c o n s t a n t would s h i f t the c a r b o x y l group d i s s o c i a t i o n e q u i l i b r i u m (COOH » COO" + H+) toward the n e u t r a l pro t o n a t e d s p e c i e s ( 3 0 ) . Whether p r o t o n a t i o n o c c u r s from d e n a t u r a t i o n o r from the p r o x i m i t y o f the s u r f a c e , t h i s e f f e c t i s g r e a t e r on t h e PDMS-PEUU and PTMO-PEUU s u r f a c e s than on the PEO-PEUU s u r f a c e . 1

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Temporal Changes i n Amide I Absorbance. The absorbance o f t h e amide I band was observed t o change on a l l three s u r f a c e s d u r i n g the f i r s t few minutes o f a d s o r p t i o n as F i g u r e s 4-6 i l l u s t r a t e . On a l l polymer s u r f a c e s and a t b o t h p r o t e i n c o n c e n t r a t i o n s , t h e f i r s t seconds o f p r o t e i n a d s o r p t i o n showed an amide I peak growing n e a r 1669 cm" . Very q u i c k l y , u s u a l l y w i t h i n two minutes o f a d s o r p t i o n , the 1669 cm" peak ceased i n c r e a s i n g , and an absorbance c e n t e r e d a t 1638 cm" began growing. The 1638 cm" band c o n t i n u e d t o grow as the amount o f adsorbed p r o t e i n i n c r e a s e d , e n v e l o p i n g the 1669 cm" band w h i c h was o b s e r v a b l e o n l y as a s h o u l d e r a f t e r about t h r e e minutes. I n p r o t e i n s o l u t i o n s the absorbance o f a band n e a r 1669 cm" i s u s u a l l y a s s i g n e d t o the a n t i p a r a l i e 1 - 8 - s h e e t c o n f o r m a t i o n . However, b o t h e x p e r i m e n t a l o b s e r v a t i o n s and t h e o r e t i c a l c a l c u l a t i o n s i n d i c a t e t h a t 8-sheet amide I v i b r a t i o n s have a t l e a s t two bands, a s t r o n g a b s o r p t i o n n e a r 1635 cm" , and a s m a l l e r one n e a r 1680 cm" (25,31-33). Thus the assignment of t h i s i n i t i a l 1669 cm" band i n the absence o f a 1635 cm" band t o an 8-sheet c o n f o r m a t i o n may be i n c o r r e c t . O t h e r p o s s i b i l i t i e s f o r t h i s band a s s i g n ment are an unordered c o n f o r m a t i o n (1656-1658 cm" ) (25) o r a 8 - t u r n c o n f o r m a t i o n (1680 cm" ) ( 3 1 ) , a l t h o u g h the l a t t e r I s l e s s l i k e l y s i n c e t h i s 8-turn v i b r a t i o n has been observed t o d i s a p p e a r 1

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WAVENUMBERS F i g u r e 4. Absorbance s p e c t r a o f FN a d s o r b i n g t o the PEO-PEUU polymer from a 0.07 mg/ml s o l u t i o n . The a d s o r p t i o n time i n minutes i s i n d i c a t e d on each spectrum.

I 1800

i 1700

1600

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1500

14Ό0

1300

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WAVENUMBERS F i g u r e 5. Absorbance s p e c t r a o f FN a d s o r b i n g to the PDMS-PEUU polymer from a 0.07 mg/ml s o l u t i o n . The a d s o r p t i o n time i n minutes i s i n d i c a t e d on each spectrum.


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upon d e n a t u r a t i o n . A t p r e s e n t the 1669 cm** band w h i c h appears a t s h o r t a d s o r p t i o n times remains unassigned. A s i m i l a r amide I absorbance has been observed f o r lysozyme a d s o r p t i o n on c o n t a c t l e n s m a t e r i a l ( 2 6 ) . I n t h a t s t u d y , C a s t i l l o o b s e r v e d an a b s o r p t i o n a t 1672 cm"* w i t h o u t a concommitant band near 1635 cm" o n l y a t the s h o r t e s t a d s o r p t i o n time measured. The amide I v i b r a t i o n s p r o d u c i n g the 1638 cm** peak and the 1669 cm** s h o u l d e r ( o r peak a t e a r l y times) were s t u d i e d by f i t t i n g the amide I r e g i o n w i t h g a u s s i a n curves u s i n g a n o n - l i n e a r l e a s t squares curve f i t t i n g r o u t i n e . The spectrum o f FN i s s o l u t i o n was f i t by 2 curves a t 1638 and 1672 cm*" w i t h f u l l w i d t h s a t h a l f h e i g h t (FWHH) o f 40 and 37 cm*" r e s p e c t i v e l y . A l l the s p e c t r a o f adsorbed FN were f i t very w e l l by two g a u s s i a n peaks a t 1634±1 and 1669±3 cm" w i t h FWHH"s o f 40 and 45 cm" r e s p e c t i v e l y . T h i s curve f i t t i n g a n a l y s i s i n d i c a t e d t h a t a t e a r l y t i m e s , the 1669 cm" band i s much l a r g e r than the 1634 cm" band. B u t t h i s l a t t e r band b e g i n s growing a t l a t e r times and e v e n t u a l l y s u r p a s s e s the 1669 cm" band. As the amount o f adsorbed FN i n c r e a s e d , the r a t i o o f the 1634 to 1669 cm" bands i n c r e a s e d from near z e r o t o a p l a t e a u v a l u e n e a r e r t o the same r a t i o determined from curve f i t t i n g the s p e c t r a of FN i n s o l u t i o n ( s e e F i g u r e 7 ) . S i m i l a r l y , C a s t i l l o observed t h a t the FTIR s p e c t r a o f adsorbed lysozyme, mucin, a l b u m i n , and γ-globulin become more l i k e the s o l u t i o n s p e c t r a as the a d s o r p t i o n time i n c r e a s e s (22,26,32,33). T h i s does n o t n e c e s s a r i l y imply t h a t the p r o t e i n w h i c h c o n t a c t s the s u r f a c e f i r s t i s denatured b u t then r e g a i n s i t s n a t i v e c o n f o r m a t i o n w i t h time. R a t h e r , the p r o t e i n adsorbed f i r s t , and i n most d i r e c t con t a c t w i t h the s u r f a c e may r e t a i n i t s denatured form, b u t p r o t e i n w i t h a more n a t i v e c o n f o r m a t i o n (and s p e c t r a ) c o n t i n u e to adsorb. U s i n g the dimensions o f 4x60 nm f o r the FN m o l e c u l e , the mass o f a random packed s i d e - o n adsorbed monolayer i s l e s s than 0.36 pg/cm . Thus the p r o t e i n which a d s o r b s f i r s t and produces the s t r o n g 1669 cm" band would be l e s s than a monolayer coverage of randomly o r i e n t e d s i d e - o n adsorbed m o l e c u l e s . A l s o o f note i n F i g u r e 7 i s t h a t the s p e c t r a o f FN adsorbed on the PEO-PEUU polymer a t t a i n s a 1634 cm" /1669 cm" r a t i o s i m i l a r t o s o l u t i o n FN (dashed l i n e i n F i g u r e 7) a t lower t o t a l s u r f a c e c o n c e n t r a t i o n s than does PTMO-PEUU and PDMS-PEUU. T h i s suggests t h a t the PEO-PEUU s u r f a c e has adsorbed l e s s p r o t e i n w i t h a c o n f o r m a t i o n which produces the s t r o n g 1669 cm" amide I band o b s e r v e d a t e a r l y times. I t i s noteworthy t h a t the amide I absorbance a t 1669 cm" as w e l l as the COOH peak a t 1720 cm" a r e o f g r e a t e r magnitude on the PDMS-PEUU and PTMO-PEUU polymers than on the PEO-PEUU polymer. I n b o t h s p e c t r a l f e a t u r e s , p r o t e i n adsorbed on the PEO-PEUU s u r f a c e appears most l i k e the n a t i v e FN i n s o l u t i o n . T h i s s u p p o r t s the o b s e r v a t i o n o f o t h e r s t h a t the FN-polymer i n t e r a c t i o n s a r e l e s s on the more h y d r o p h i l i c s u r f a c e than on the more h y d r o p h o b i c s u r f a c e s . F o r example, Iwamoto observed changes i n the f l u o r e s c e n c e s p e c t r a of adsorbed FN w h i c h i n d i c a t e d t h a t the p r o t e i n i s denatured more on a h y d r o p h o b i c than on a h y d r o p h i l i c s u r f a c e (7). G r i n n e l l and o t h e r s have shown t h a t the i n t e r a c t i o n o f a n t i b o d i e s w i t h FN adsorbed on h y d r o p h i l i c s u r f a c e s i s g r e a t e r than w i t h FN adsorbed on h y d r o p h o b i c s u r f a c e s , s u g g e s t i n g t h a t the FN adsorbs i n a more n a t i v e c o n f o r m a t i o n on h y d r o p h i l i c s u r f a c e s ( 8 - 1 0 ) . 1

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1638

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WAVENUMBERS F i g u r e 6. Absorbance s p e c t r a o f FN a d s o r b i n g t o the PTMO-PEUU polymer from a 0.21 mg/ml s o l u t i o n . The a d s o r p t i o n time I n minutes i s i n d i c a t e d on each spectrum.

0.0

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1

F i g u r e 7. R a t i o of the 1634 cm" peak t o the 1669 cm" peak from the curve f i t t i n g a n a l y s i s . The polymer s u b s t r a t e s a r e PEO-PEUU ( # ) , PTMO-PEUU ( • ) and PDMS-PEUU ( A ) . The dashed l i n e i s the same r a t i o f o r FN i n PBS b u f f e r . T h i s data i s the average o f a l l a d s o r p t i o n experiments a t 0.07 and 0.21 mg/ml c o n c e n t r a t i o n s on a g i v e n polymer.


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Changes i n the Amide I I I Region. I n a d d i t i o n t o the amide I band, the amide I I I v i b r a t i o n s a r e s e n s i t i v e t o the p r o t e i n c o n f o r m a t i o n . However, the low s i g n a l / n o i s e r a t i o o f the amide I I I r e g i o n makes spectral interpretation d i f f i c u l t . I n these experiments, the amide I I I r e g i o n was n o t i d e n t i c a l i n each experiment due t o random n o i s e and f l u c t u a t i o n s i n the b a s e l i n e . However, l a r g e changes i n s p e c t r a l f e a t u r e s were c o n s i s t e n t on a g i v e n polymer. I n F i g u r e 8, the amide I I I s p e c t r a f o r 4 s p e c t r a a t 2 h r o f a d s o r p t i o n on a g i v e n polymer have been added, thus enhancing the c o n s i s t e n t s p e c t r a l f e a t u r e s , and m i n i m i z i n g random n o i s e and b a s e l i n e f l u c t u a t i o n s . The amide I I I r e g i o n of the s o l u t i o n FN has peaks a t 1247, 1275 and 1290 cm" , a l l o f w h i c h a r e a l t e r e d i n the s p e c t r a of the adsorbed FN. F o r example, the s o l u t i o n FN peak a t 1247 cm" i s absent i n the adsorbed s p e c t r a , and t h e r e i s a new b r o a d e r peak o f lower frequency c e n t e r e d n e a r 1240 cm*" . Where the s o l u t i o n FN has peaks a t 1275 and 1290 cm" , the adsorbed s p e c t r a have a broad peak c e n t e r e d n e a r 1280 cm" on the PTMO-PEUU and PDMS-PEUU p o l y m e r s , w h i l e on the PEO-PEUU s u r f a c e , peaks i n t h i s r e g i o n a r e a b s e n t o r very s m a l l . These changes f u r t h e r i n d i c a t e t h a t the con f o r m a t i o n o f FN i s a l t e r e d as i t adsorbs t o these polymers. Previous infrared studies i n d i c a t e that proteins with a large amount of 3-sheet s t r u c t u r e absorb n e a r 1240 cm" and those w i t h α-helix s t u c t u r e absorb n e a r 1280 cm" (22,32). Absorbances f o r denatured a l b u m i n , r e p o r t e d l y c o n t a i n i n g random and 3-sheet con f o r m a t i o n s , a r e found a t 1240 and 1260 cm" ( 2 2 ) . These a s s i g n ments c o r r e l a t e w i t h the more s t u d i e d Raman s p e c t r o s c o p y o f the amide I I I r e g i o n w h i c h has v i b r a t i o n s a t 1230-1250 cm" f o r 3-sheet s t r u c t u r e , a t 1260-1290 cm" f o r α-helix s t r u c t u r e , and a t 1240-1265 cm" f o r u n s t r u c t u r e d p o l y p e p t i d e (34,35). A p p l y i n g these v i b r a t i o n a l assignments t o the s p e c t r a of F i g u r e 8 would suggest t h a t s o l u t i o n FN c o n t a i n s some α-helix s t r u c t u r e . However, t h i s i s p r o b a b l y an i n c o r r e c t assignment s i n c e the amide I band does n o t have a s t r o n g absorbance a t 1646-1650 cm" c h a r a c t e r i s t i c o f α-helix, and c i r c u l a r d i c r o i s m (CD) s t u d i e s i n d i c a t e l i t t l e o r no α-helical c o n t e n t f o r FN (1,36-39). I t i s a l s o d o u b t f u l t h a t the peak a t 1280 cm" i n the adsorbed FN i s due t o α-helix s t r u c t u r e s i n c e the amide I band does n o t suggest s i g n i f i c a n t α-helix s t r u c t u r e . A t p r e s e n t , the peaks i n the r e g i o n from 1290 t o 1275 cm" remained u n n a s s i g n e d . Both the amide I peak a t 1638 cm" and t h e amide I I I peak a t 1247 cm" s u p p o r t CD o b s e r v a t i o n s o f the presence o f some 3-sheet s t r u c t u r e . The s h i f t o f the l a t t e r peak t o a lower frequency around 1240 cm" i n the adsorbed s t a t e suggest t h a t an i n c r e a s e i n 8-sheet s t r u c t u r e may o c c u r upon a d s o r p t i o n . A l s o the s h i f t i n the amide I band from 1642 cm" i n s o l u t i o n t o 1638 cm" when adsorbed f u r t h e r s u b s t a n t i a t e s the h y p o t h e s i s t h a t the 3-sheet c o n t e n t o f FN i n c r e a s e s upon a d s o r p t i o n . An i n c r e a s e i n 3-sheet s t r u c t u r e upon a d s o r p t i o n has been p r e v i o u s l y r e p o r t e d i n FTIR/ATR s t u d i e s o f pro t e i n a d s o r p t i o n on c o n t a c t l e n s m a t e r i a l s . S p e c i f i c a l l y , an ah e l i x t o random and 3-sheet t r a n s i t i o n has been observed f o r adsorbed a l b u m i n and lysozyme, as w e l l as a random t o 3-sheet t r a n s i t i o n f o r mucin (22,26,32). However, a decrease i n 3-sheet s t r u c t u r e has a l s o been observed f o r adsorbed γ-globulin w h i c h c o n t a i n s a h i g h c o n t e n t of 3-sheet s t r u c t u r e i n i t s n a t i v e form ( 3 3 ) . 1


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1275 1250 1225 WAVENUMBERS

1

1

1200

1175

1

1150

F i g u r e 8. Absorbance s p e c t r a o f the amide I I I r e g i o n o f s o l u t i o n FN ( A ) , and FN adsorbed on PEO-PEUU ( B ) , PDMS-PEUU (C) and PTMO-PEUU ( D ) . Each spectrum i s n o t from a s i n g l e e x p e r i m e n t , b u t i s the c o - a d d i t i o n o f 4 s p e c t r a on a g i v e n polymer a t 2 hours o f a d s o r p t i o n .


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Conclusions The FTIR/ATR s t u d i e s o f FN a d s o r p t i o n i n d i c a t e t h a t the polymer s u r f a c e p l a y s an i m p o r t a n t r o l e i n d e t e r m i n i n g b o t h the amount and the c o n f o r m a t i o n o f adsorbed FN. Comparison o f the PEO-PEUU s u r f a c e w i t h the PDMS-PEUU and PTMO-PEUU s u r f a c e s show t h a t on the more h y d r o p h i l i c PEO-PEUU polymer, l e s s p r o t e i n adsorbs, and t h e I n t e r a c t i o n s between the p r o t e i n and polymer take p l a c e more s l o w l y and a r e l e s s i n t e n s e as shown by the l a t e appearance o f the COOH v i b r a t i o n and the s m a l l e r 1669 cm" amide I peak observed a t e a r l y a d s o r p t i o n times. The p r o t e i n w h i c h adsorbs f i r s t appears t o i n t e r a c t most s t r o n g l y w i t h the s u r f a c e . On a l l p o l y m e r s , changes i n the amide I and the amide I I I r e g i o n o f adsorbed FN suggests t h a t the amount of β-sheet s t r u c t u r e i n FN i n c r e a s e s upon adsorp tion.


1

Acknowledgments The a u t h o r s w i s h t o acknowledge p a r t i a l support o f t h i s work t h r o u g h the N a t i o n a l I n s t u t u t e s o f H e a l t h g r a n t s HL-21001 and HL-24046, and f e l l o w s h i p s u p p o r t f o r WGP from the W. R. Grace Company. The h e l p f u l d i s c u s s i o n s w i t h Dr. Frank Wasacs o f M a t t s o n I n s t r u m e n t s and Dr. Deane F. Mosher o f the U n i v e r s i t y o f W i s c o n s i n , Department of M e d i c i n e were g r e a t l y a p p r e c i a t e d .

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