Protein Adsorption on Polymers - Advances in Chemistry (ACS

21 Protein Adsorption on Polymers Visualization, Study of Fluid Shear and Roughness Effects, and Methods to Enhance Albumin Binding ROBERT C. EBERHART, M I C H A E L E. LYNCH , FERTAC H . BILGE, JOHN F. WISSINGER , MARK S. MUNRO, STEPHEN R. ELLSWORTH, and A L F R E D J. QUATTRONE

Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 2, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch021

1

2

3

University of Texas Health Science Center, Department of Surgery, Dallas TX 75235 and the University of Texas, Biomedical Engineering Program, Austin, TX 78712

We visualized protein adsorbates on Teflon, silicone rubber, and polyurethane by partial gold decoration transmission elec tron microscopy, and the results were verified by several meth ods.

Critical-point-dried

Cohn

I fibrinogen

adsorbates

formed extensive, reticulated networks; γ-globulin adsorbates resembled these networks protein-protein

but appeared to have weaker

bonds. In contrast, critical-point-dried

albu

min formed irregular, unconnected adsorbates with lower sur face coverage. Fibrinogen

preferentially adsorbed in surface

cracks but albumin did not, and albumin was desorbed by a wall shear rate greater than 1500 s

-1

but fibrinogen

was not.

Polymers treated by covalently binding C alkyl residues selec 18

tively enhanced albumin

affinity.

Simultaneous

albumin

-fibrinogen exposure to alkylated surfaces showed that fibrino gen adsorption was reduced in proportion to enhancement of albumin adsorption.

O

ne aspect of the question of blood-polymer compatibility that is receiving much attention is the interaction between plasma proteins and the poly mer substrate. A n increasing body of evidence suggests that these events, particularly the initial glycoprotein-polymer interaction, set the stage for

thrombogenesis, both in terms of platelet adhesion and aggregation (1-3) and in terms of contact-activated coagulation (4). Recent evidence also sug gests that a larger class of cell adhesion phenomena is governed by the nature of the protein adsorbate (5). Cell adhesion in this more general view may Current address: Westinghouse, Round Rock, TX 78664 Current address: U.T. Medical Branch, Galveston, TX 77550 Current address: Lab Procedures West, Woodland Hills, CA 91367

1 2 3

0065-2393/82/0199-0293$06.00/0 ©1982 American Chemical Society In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

294

BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS

govern the total response of b l o o d a n d tissue to p o l y m e r - b a s e d implants i n terms of pannus f o r m a t i o n (6) a n d , perhaps, calcification (7). U n d e r the i m p e t u s of c r i t i c a l reviews of the state of knowledge c o n c e r n i n g the p h y s i c o c h e m i c a l surface properties of i m p l a n t p o l y m e r s (8), and the detailed nature of the b l o o d - p o l y m e r interaction (9), a n u m b e r of methods for p h y s i c o c h e m i c a l a n d b i o l o g i c a l analysis have b e e n a p p l i e d to the b l o o d c o m p a t i b i l i t y p r o b l e m . W e d e v e l o p e d additional methods, v i s u a l i z i n g , by


partial g o l d decoration transmission electron microscopy ( P G D T E M ) ,

the

p o l y m e r surface a n d the p r o t e i n adsorbate. T h i s visualization m e t h o d may be useful for several tasks i n p r o t e i n - p o l y m e r sorption studies: to elucidate the adsorption patterns of p l a s m a proteins (10),

to quantify the surface free

energy d i s t r i b u t i o n (11 ), to evaluate the c o n t r i b u t i o n of surface roughness i n p r o t e i n a d s o r p t i o n and d e s o r p t i o n , to characterize the p r o t e i n species interacting w i t h the p o l y m e r i n c o m p e t i t i v e adsorption studies (4), and to d o c u ment the effects of e n v i r o n m e n t a l parameters such as fluid shear stress, temperature, etc., on p r o t e i n adsorption (12).

T h e results of some of these

studies are r e v i e w e d a n d e x t e n d e d i n this chapter. O n e of the m o r e significant results of these studies is the a b i l i t y of spontaneously adsorbed a l b u m i n to i n h i b i t a d s o r p t i o n of C o h n I fraction, c o m p o s e d of f i b r i n o g e n , 7-globulin, and f i b r o n e c t i n . T h e s e proteins are thought by many to play central roles i n cell attachment to surfaces a n d thrombogenesis

(2,3,5).

Unfortunately,

spontaneously b o u n d a l b u m i n is o n l y w e a k l y b o u n d to the surface,

and

l o n g - t e r m prospects for i n h i b i t i o n of adsorption of other plasma proteins are not good

(12).

W e , therefore, d e v e l o p e d a n e w m e t h o d for e n h a n c i n g a l b u m i n adsorption, a m e t h o d that may p r o v i d e i n d e f i n i t e protection against t h r o m bogenesis a n d c e l l a d h e s i o n . T h e m e t h o d takes advantage of the h y d r o p h o b i c affinity and reversible d y n a m i c b i n d i n g of a l b u m i n f r o m plasma to C

1 8

alkyl

residues that are, i n t u r n , covalently b o u n d onto various p o l y m e r surfaces.

Experimental Test polymers for visualization studies were polyurethane (Pellethane, 236380A, Upjohn), filler-free polydimethylsiloxane (Sil-Med Corporation), two forms of Teflon, sintered (TFE, DuPont) and Fluorofilm (Dilectrix Corporation), and polyurethane-silicone rubber copolymer (AVCOthane 51, AVCO). Samples of 1 cm or, for shear studies, 5 x 20 X 0.5-cm sheets, were washed in ionic detergent solution (Alconox) at 60°C for 1 h, rinsed in deionized water, and refluxed in absolute ethanol for 1 h. Materials were dried and stored in a desiccator until use. For the visualization studies the proteins were crystalline human albumin, 99% pure (Miles Laboratories and U.S. Biochemical Corporation), bovine 7-globulin, Cohn fraction II (U.S. Biochem.), human fibrinogen, Cohn fraction I, 65% clottable (U.S. Biochem.), and ferritin-conjugated rabbit antibovine albumin (immunoglobulin G, IgG) solution (Cappel Labs). For the albumin enhancement studies the proteins were crystalline (fraction V) fatty acid-free human albumin, and plasminogen-free fibrinogen (Pentex, Miles Laboratories). The major component of the Cohn I fibrinogen fraction is fibrinogen, but other proteins, notably 7-globulins and plasma fibronectin, are included. 2

In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


21.

EBERHART ET AL.

295

Protein Adsorption on Polymers

Phosphate-buffered saline (PBS) at pH 7.4 was prepared from a stock concentrate. Following dilution, the solution was placed in a vacuumflaskand degassed for 1 h. The solution was retained under vacuum and used subsequently for all solution preparations and wash steps. Crystalline protein was weighed and placed in a dry 500-mL flask, and degassed PBS was transferred to theflaskto make the following concentrations: 2500 mg/dL albumin and 300 mg/dL Cohn I and II fractions. The flask was swirled gently and allowed to sit until all proteins had dissolved (30-40 min). Other solutions were prepared from the stock solutions by serial dilution under vacuum. The stock was discarded after 48 h. The static exposure studies were performed in a closed system consisting of roller-pump (Med Science Electronics) vacuum flasks containing the protein, PBS wash solutions, and four chambers to hold the samples. The samples were initially exposed to PBS to remove any adsorbed gases, followed by 1-h static protein solution exposure (sixfold volume replacement). After the protein exposure, the surfaces were rinsed with degassed PBS (sixfold volume replacement) for 1 h, circulating at a flow rate of 100 mL/min. The shear rate at the sample surface was less than 2 s . Sequential protein exposures (albumin-fibrinogen) were straightforward extensions of this regimen; however, they did not include an intervening PBS wash step. At the end of the hour-long rinse the coated surfaces were either air dried in a desiccator or critical-point dried. Alcohol dehydration was carried out according to the following schedule: 20% ethanol for 10 min; 50% for 5 min; 95% for 5 min; and 100% for 10 min. The samples were placed in the critical-point drying bomb with an ethanol-liquid C 0 mixture, pressurized to 1500 psi and decompressed at a pressure bleed-off rate of 100 psi/min. The shear experiments were carried out with a carefully constructed planeparallel flow cell. Details of the shear circuit have been reported previously (12). The recirculation and roller pump sections, accounting for much of the circulation duty cycle, had 3-5 times the test section wall shear rate. The shear system loading, exposure, and wash steps were analogous to those for the static exposure studies. Test surfaces were exposed to one of the following calculated wall shear rates: 0, 100, 500, 800, and 1500 s , for 1 h. Wash steps were carried out at a calculated wall shear rate at the test section of 25 s . The exposed surfaces were critical-point dried, as described for the static exposure studies. The wall shear rate calculation assumed a steady, plane-parallel flow with no edge effects, and a parabolic velocity profile. Following the drying steps, samples were prepared for P G D T E M as described previously (10,13). The primary difference between this technique and conventional biological sample preparation for electron microscopy is the development of a partial gold coat, covering 10-25% of the surface, backed with a carbon film for strength and contrast. P G D T E M has been a sensitive tool in metallurgical analysis for many years (14). Samples prepared this way were examined in a Jeolco JEM 150 transmission electron microscope (TEM) at an accelerating potential of 80 kV. Over 300 grids were examined and analyzed by at least two of us. Photographs of typical and noteworthy regions were taken. For three-dimensional representations, stereo pair photographs were taken on a modified Jeolco stage at a total angle of 20°, rather than the customary 12° angle, in order to enhance the depth effect. Gold nuclei preferentially formed on protein instead of on the polymers used in this study. Thus the image of the protein could be identified, and the area of the substrate covered with protein could be measured. This was done by tracking the outline of protein deposits and calculating the area inside the closed loops by a modified Simpsons Rule algorithm, using a Tracor Northern NS-800 digitizer computer. Samples for high-angle scanning electron microscopy (SEM) (to verify P G D T E M images) were fastened onto metal stubs with conductive glue, placed in a Denton DV-502 vacuum evaporator, and pumped down to less than 10 ~ Torr. The surfaces -1

2

_1

_1

6



296


were sputter coated (20 ma, 60 s) with gold-palladium to obtain a complete conductive film. The samples were then placed in a Jeol JSM-35 scanning electron microscope and were studied at tilt angles from 5° to 30°. Photographs of typical areas were taken. A modified negative-staining technique also was used to verify the P G D T E M images of the protein deposits, following the method of Brash and Lyman (15). The protein-exposed surfaces were coated with a 5% poly(vinyl alcohol) solution and allowed to dry. The dried films were stripped from the surface and carbon coated in the vacuum evaporator. After dissolving the p o l y v i n y l alcohol) in hot water and capturing the protein-carbon films on copper grids, the protein on the exposed side of the carbon films was stained with 2% phosphotungstic acid solution (pH adjusted to 7.4 with NaOH). These grids were examined in the T E M . A ferritin labeling technique also was used to verify protein adsorbate images. Bovine serum albumin (BSA)-coated Fluorofilm Teflon surfaces were placed in a 1% ferritin conjugated rabbit anti-BSA solution. The samples were exposed to 10% rabbit serum before the antibody treatment to prevent nonspecific binding. The protein was stripped off the surface and placed on a copper grid with carbon support, using polyvinyl alcohol) as described for the negative-staining technique. Samples prepared this way were examined by T E M . Because ferritin is electron dense, the absorbed protein deposits could be visualized without metal-atom image enhancement. The following formulations were used for the chemical derivatization of polyurethane: sheet Pellethane 2363-80A (Upjohn), tubing Biomer (Ethicon), and a sample tube extruded by Cordis from raw material provided by Mobay. Cleaning agents were redistilled toluene (Fisher, 99% estimated purity), ethanol, dried over molecular seives to 99% purity, and redistilled trimethylpentane, spectro grade (Aldrich, 99% estimated purity). Chemicals were 1-bromooctadecane, reagent grade (Aldrich), octadecyl isocyanate, technical grade (Aldrich), and zinc stéarate, reagent grade (MCB). Sodium ethoxide was prepared with pure sodium (Aldrich). In the two-step derivatization (Figure 1), 10-cm samples were soaked in toluene for 3 min to remove surface impurities. Samples were transferred in 25 mL of 0.04M sodium ethoxide in toluene under dry nitrogen, and were agitated at room temperature for 15 min. In the same vessel, 25 mL of 2.0M 1-bromooctadecane in toluene were added under nitrogen with 15 min mixing at room temperature. The chemically derivatized sheet was removed and soaked consecutively for 30 s at room temperature as follows: toluene, ethanol twice, deionized water twice, 0.LV hydrochloric acid, deionized water twice, followed by air drying for 24 h. A polyurethane was obtained that had random surface N-octadecylamine and urethane substitutions. The one-step derivatization of polyurethane began with soaking the sample in toluene for 10 min and in ethanol for 20 min. After vacuum drying overnight, the sample was placed in 50 mL of 0.25M η-octadecyl isocyanate in trimethylpentane under dry nitrogen, and was incubated, with mixing, for 1 h at 80°C. The sample was removed, soaked twice for 1 min in ethanol, twice in deionized water, and redried to yield η -octadecyl-derivatized polyurethane. Albumin was radiolabeled in the following manner. Defatted human serum albumin, 10 mg, was dissolved in 1 mL of PBS. From this solution, 100 was placed in an Iodogen-coated reaction vessel (prepared by P. Kulkarni). Α 25-μΙ-, aliquot of 0.25M phosphate buffer solution (pH 7.52) was added, and the vessel was chilled over ice for 10 min. Α 5-μ1 aliquot of Na I, 1-1.5 mCi, was then added, and the vessel was rotated slowly several times. The vessel was again placed in an ice bath for 2 min and then incubated for an additional 15 min at room temperature. At the end of the incubation period, 100 μL· of PBS was added to the reaction vessel, and the solution 2

125


297


EBERHART ET AL.

-o-c-r ^

C-(0-CH -CH )-

i/

2

2

H 'H Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 2, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch021

N

+ NaOEt or

or NaH

NaO Bu t

or

any other proton-abstracting —-~" base

Ο

.

.

Ο

-O-C-N-A .V-N-C- (0-CH -CH )2

H

2

^-^ Γ Na

+

N a , Y" +

Ο - ° -

C

,

- | A \

H

N

.

Ο

/VV-C-(0-CH -CH ^ 2

'

2

R

yields alkyl-substituted urethane groups,

î

-O-C-N H

NH

+

R-(0-CH -CH -)^ 2

2

R

and possibly alkyl-substituted secondary amine groups on the surface of these polyurethanes. Figure la. Reaction scheme between polyurethanes and alkyl halides. The haliae may be a bromide, tosylate, methylsulfonate, etc., and the alkyl residue may vary from C to C o8

3



Ο II

Ο ^)-rj4-C-(0-CH -CH )^0-CH -CH -OH

-C-f.

2

2

2

2


H R—N=C=0

2

H

^

2

2

2

C=0

J

R—N-H

and at the exposed primary alcohol residues:

Ο

Ο /V-N-C-iO-C^-CH^O-C^-CH^OH

-C-N-^x H

Ni

'

Η 0=C=N—R

X

-CH -CH )^0-CH -CH 2

OH

-C-N-/ \-N-C-(0-CH -CH )^0-CH -CH -

2

2

2

ο II , -N-C-(0Η Ο

/ 0=C-N-R Η

Figure Jfo. Reaction scheme between polyurethanes and alkyl isocyanates.


21.

EBERHART ET AL.


299

was transferred into a Dowex 1 X 8-50 anion-exchange column. PBS, 10 mL, was used to flush the I-labeled albumin through the column, giving a final volume of 10 mL with a specific activity of 3.91 μΟί/π^. A radio chromatogram was run with 70% methanol, and showed less than 5% free iodide in the solution. Human fibrino gen (Pentex) was radio labeled by a similar technique. The general technique is a modification of a globulin labeling procedure (16). Exposure of I-labeled protein solution to the derivatized and untreated poly mer samples was performed by methods different than those described previously. The protein solutions were degassed, but an air-liquid interface existed above the sample in the test chamber. Then, 0.1-mL aliquots of labeled protein were intro duced by pipet. Exposures of 30 s to 20 min were carried out with this technique, and a twofold wash with PBS was conducted at the end of the exposure period. In most series, care was taken to ensure that the sample remained well below the air-solution interface. In some series, the sample was introduced through that interface. Either single protein (albumin or fibrinogen) or simultaneous albumin-fibrinogen infusions from separate pipets were performed. Following the wash step, samples with bound radioactivity were transferred to counting vials and were counted for 5 min in a well-type scintillation counter (Tracor Analytic, Model 1191). 125


125

Results T y p i c a l g o l d nucleus d i s t r i b u t i o n s o n the smooth, and r o u g h F l u o r o f i l m Teflon surfaces are s h o w n i n F i g u r e s 2a and b, respectively. A range of gold nucleus d i m e n s i o n s w e r e o b s e r v e d , v a r y i n g f r o m less than 100 A to 600 A i n size. T h e larger n u c l e i , o b s e r v e d for the r o u g h Teflon, possibly r e s i d e d i n grooves of the Teflon surface ( F i g u r e 2c). S i m i l a r partial coverage gold n u c l e i were o b s e r v e d for the other p o l y m e r s , silicone r u b b e r , p o l y u r e t h a n e , and A V C O t h a n e 51, w i t h a p p a r e n t l y p o l y m e r - d e p e n d e n t variations i n the n u c l e us size and spacing d i s t r i b u t i o n s . T h e g o l d nucleus d e n s i t y o n the 7 - g l o b u l i n adsorbate was considerably h i g h e r than that o n the p o l y m e r substrate

( F i g u r e 3a). C o m p a r i s o n of

negative-stain t e c h n i q u e 7 - g l o b u l i n and ferritin-conjugated I g G adsorbates, obtained i n s i m i l a r e x p e r i m e n t s shown i n F i g u r e s 3b and c, respectively, verifies the r e p l i c a t i o n - b a s e d g o l d decoration t e c h n i q u e . T h e adsorbates i n F i g u r e 3 w e r e all a i r - d r i e d , a process that c o n s i d erably disrupts the p r o t e i n f i l m . Results for c r i t i c a l - p o i n t - d r i e d 7 - g l o b u l i n at a lower solution c o n c e n t r a t i o n are shown i n F i g u r e 4 to illustrate the point. T h e surface f i l m was m o r e extensive w i t h critical-point d r y i n g , yet e x h i b i t e d pores o n the o r d e r of m a g n i t u d e of 500 A i n diameter. T h e pores i n these more gently treated f i l m s may still be a processing artifact. W i t h this gentler treatment, essentially no variation i n surface coverage o c c u r r e d for 15-100 mg/dL solution c o n c e n t r a t i o n . F i g u r e 5 depicts s i m i l a r results for C o h n I f i b r i n o g e n . A i r d r y i n g was shown to have a m a r k e d d i s r u p t i v e effect on the adsorbate ( F i g u r e 5a). T h e m o r p h o l o g y of the adsorbate d i f f e r e d f r o m that of the a i r - d r i e d 7 - g l o b u l i n ; however, the m o r p h o l o g y and pore size of the c r i t i c a l - p o i n t - d r i e d adsor bate ( F i g u r e 5b) w e r e s i m i l a r to those for 7 - g l o b u l i n . C o m p a r i s o n of the


300



a

b

C

Figure 2. PGDTEM patterns on (a) sintered, 56% crystalline Teflon, (b) Fluorofilm Teflon, and(c) SEM continuous gold-palladium coat on Fluorofilm Teflon. Linear arrays of gold nuclei in (a) are typical, and may reside in surface imperfections. Aggregates of gold nuclei (b) collect at fiber crossovers as shown in (c). Key: bars equal 1 μτη.



21.

EBERHART ET AL.

Protein Adsomtion on Polymers

301

Figure 3a. The y-globulin adsorbed on the sintered Teflon sample of Figure 2a (air-dry technique). Bovine Cohn II, 100 mg/dL, partial gold decoration. Key: bar equals 1 μ/η.

Figure 3b. The y-globulin adsorbed on the sintered Teflon sample of Figure 2a (air-dry technique). Bovine Cohn II, 100 mg/dL, negative stain. Key: bar equals 1 ^m. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


302


Figure 3c. The -y-globulin adsorbed on the sintered Teflon sample of Figure 2a (dry air technique). Rabbit anti-BSA (IgG), 250 mg/dL, ferritin label, TEM, higher magnification. Key: bar equals 1 \Lm.

Figure 4. Bovine y -globulin on sintered Teflon (3 mg/dL solution; critical-point dried, partialgold decoration technique). Key: bar equals 0.1 \x,m.




EBERHART ET AL.

C

Figure 5. Cohn I fibrinogen, 3 mg/dL, adsorbed on Teflon (partial gold decoration technique). Key: a, air dried, sintered Teflon; b, critical-point dried, sintered Teflon; and c, critical-point dried, Fluorofilm Teflon. Bar equals 0.1 ^mfor b and c.


304



c r i t i c a l - p o i n t - d r i e d p r o t e i n o n the F l u o r o f i l m Teflon ( F i g u r e 5c) w i t h that of smoother Teflon ( F i g u r e 5b) i n d i c a t e d that no d i s t i n g u i s h i n g surface features d i s c r i m i n a t e d b e t w e e n the adsorbates. T h i s result suggests, i n t u r n , that a p r o t e i n " b l a n k e t " may s m o o t h the r o u g h e r surface, u t i l i z i n g p r o t e i n - p r o t e i n bonds to o b t a i n the h i g h surface coverage. A d d i t i o n a l e v i d e n c e s u p p o r t i n g this concept was obtained b y stereo pair T E M of c r i t i c a l - p o i n t - d r i e d C o h n I f i b r i n o g e n . In surface cracks of lengths d o w n to 0.1 μηι, C o h n I f i b r i n o g e n created a blanket layer, i n the crack v o i d , in c o m b i n a t i o n w i t h m u l t i p l e b i f u r c a t i n g strands of a material that accepts dense g o l d d e c o r a t i o n , a n d may be f i b r i n . T h e strands elaborated from the p r o t e i n f i l m adjacent to the v o i d also may be d e r i v e d from the v o i d f i l m . T h e films existed i n several layers i n the crack, i n c l u d i n g a p o l y m e r surfaceadherent layer. T h e surface flats e x h i b i t e d the same reticulated, extensive surface coverage seen i n F i g u r e s 5b a n d c. A i r - d r i e d a l b u m i n i n t w o - d i m e n s i o n a l v i e w e x h i b i t e d an irregular, par tially c o n n e c t e d m o r p h o l o g y , w i t h b l u n t e d ends a n d an aggregated appear ance ( F i g u r e 6a), s i m i l a r to that of a i r - d r i e d 7-globulin. A n o n r e p l i c a t e d sample, h i g h - a n g l e , g o l d - p a l l a d i u m S E M image from the same surface treat m e n t v e r i f i e d the results ( F i g u r e 6b). I n contrast to 7-globulin a n d C o h n I f i b r i n o g e n , c r i t i c a l - p o i n t - d r i e d a l b u m i n e x h i b i t e d an i r r e g u l a r , u n c o n n e c t e d adsorbate, w i t h a characteristic d i m e n s i o n of200 A , w h i c h appeared to follow the details of surface structure ( F i g u r e s 7a a n d b). T h e a l b u m i n adsorbate was characterized b y l o w surface coverage for all p o l y m e r s s t u d i e d , i n contrast to 7-globulin a n d C o h n I f i b r i n o g e n adsorbates.

Figure 6. Albumin, 2500 mg/dL solution adsorbed on sintered Teflon (air-dry technique). Key: a, PGDTEM and b, high incidence angle SEM. Bar equals 1 ^m.


EBERHART ET AL.


305


21.

Figure 7. Albumin adsorption by critical-point dry technique. Key: a, sintered Teflon, 250 mg/dL solution and b, Fluorofilm Teflon, 25 mg/dL solution. Bar equals 0.1 μτη.


306


Stereo p a i r T E M showed that the separation of a l b u m i n microadsorbates was p r e s e r v e d i n surface holes a n d cracks, i n contrast to t h e C o h n I f i b r i n o gen results. F i g u r e 8 s u m m a r i z e s the effects of f l u i d shear o n p r o t e i n a d s o r b e d to the r o u g h , F l u o r o f i l m T e f l o n . C o h n I f i b r i n o g e n , at 3 a n d 300 mg/dL, e x h i b i t e d a weak shear d e p e n d e n c e out to a w a l l shear rate of 1500 s " . T h e m o r p h o l o g y 1

of the adsorbates d i d not change; rather, the n u m b e r of pores i n t h e surface Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 2, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch021

film increased. A l b u m i n was also r e m o v e d , w i t h a weak d e p e n d e n c e o n wall shear

rate.

H o w e v e r , because

a l b u m i n exposure p r o d u c e d ,

character-

istically, o n l y 2 0 - 4 0 % surface coverage, t h e adsorbate was r e m o v e d essentially b y t h e 1 5 0 0 - s

1

w a l l shear rate. T h e gold-decorated adsorbate was

difficult to differentiate f r o m t h e g o l d n u c l e i o n the substrate for a w a l l shear rate greater than 8 0 0 s " . O u r i m p r e s s i o n o f this result is stronger than can 1

be s h o w n i n F i g u r e 8, o w i n g to t h e artifacts i n substrate d e c o r a t i o n . P r e e x p o s u r e o f the F l o u r o f i l m Teflon surface to a l b u m i n , followed b y C o h n I f i b r i n o g e n (in an air-free, degassed solution e n v i r o n m e n t ) a n d

100 Extended Linear Regression:

I

75 k α> σ> ο

! Î

.

^ Cohn I, 3mg/dl • •j Cohn I, 300mg/dl

λ

α*

§ Ο α> ο ο

Albumin, 2500 mg/dl

Albumin, 25mg/dl

ι 1500

500

2000

Wall Shear Rate, sec •1 Figure 8. Summary offluidshear dependence of protein adsorption on Fluorofilm Teflon, using critical-point dry technique for sample preparation and PGDTEM. Paired data represent high and low area estimates at one set of operating conditions. Lines to the right represent linear regression through all samples at one solution concen tration. Key: —, average gold nucleus coverage of the Teflon control substrate; •, r = 0.08; Μ, r = 0.95; ·, r = 0.75; and O, r = 0.79. 2

2

2

2


21.

EBERHART ET AL.


307

critical-point d r y i n g y i e l d e d an adsorbate r e s e m b l i n g a l b u m i n m o r e than C o h n I f i b r i n o g e n ( F i g u r e 9). Stereo pair T E M o f sequential a l b u m i n - C o h n I f i b r i n o g e n a d s o r p t i o n v e r i f i e d these observations. T h e s e results w e r e s u p p o r t e d f u r t h e r i n a larger study o f several p o l y mers, one i n w h i c h a i r d r y i n g was u s e d as the final step. T h e morphologies of the sequential exposure p r o t e i n adsorbate r e s e m b l e d a l b u m i n , not f i b r i n


ogen, a n d t h e extent o f surface coverage was r e d u c e d m a r k e d l y ( F i g u r e 10). T h u s , the a b i l i t y of p r e a d s o r b e d a l b u m i n to reduce C o h n I f i b r i n o g e n adsorp tion suggested a p o t e n t i a l m e t h o d for c o n f e r r i n g i m p r o v e d thromboresistance o n p o l y m e r s . H o w e v e r , the fragility o f the adsorbate, as e x e m p l i f i e d b y the l o w extent o f surface coverage a n d weak resistance to shear erosion, suggested that e n h a n c e m e n t o f p o l y m e r affinity for a l b u m i n w o u l d be neces sary to take advantage,

i n a c l i n i c a l e n v i r o n m e n t , of albumin's

apparent

thromboresistive p r o p e r t y . T h u s , w e t u r n e d to alkylation to m o d i f y a l b u m i n ' s affinity for p o l y m e r surfaces. C h e m i c a l d e r i v a t i z a t i o n o f the p o l y m e r surface was evaluated b y its ability to adsorb a l b u m i n . W e v e r i f i e d b y optical microscopy that the surfaces

Figure 9. Albumin, 2500 mg/dL solution, preadsorped on sintered Teflon followed by Cohn I fibrinogen, 300 mg/dL, lh each (critical-point dry and PGDTEM techniques). Key: bar equals 0.1 μ/η.


308


Teflon

Avcothcme _ Unfilled Silicone Rubber

-

•

I

-


•

i ι

F

Polyurethane

• •

·

-

• t

S

I

I

A:F

F

•

•

t

A:F

• • 1

F

t

t 1

A:F

ι

I

F

A: F

Figure 10. Summary of albumin preadsorption results on a number of polymers. Air drying and PGDTEM were used for all samples. Key: F, 30 mg % fibrinogen (Cohn I) and A:F, 2500 mg % albumin followed by 30 mg % fibrinogen (Conn I).

w e r e not s i m p l y e x t e n d e d b y r e s i d u a l solvent s w e l l i n g o r c r a c k i n g . T h e effectiveness of the octadecyl residue i n b i n d i n g a l b u m i n is shown i n F i g u r e 11. A l b u m i n was m o r e r a p i d l y b o u n d to a n u m b e r of alkylated polyurethanes than to the u n m o d i f i e d surfaces. T h e extent o f n o r m a l i z e d but not absolute a l b u m i n adsorption was t h e same after 2 0 - 3 0 m i n o f exposure, insofar as surface concentration is c o n c e r n e d . A series of 5-60-s exposures of the c o n trol p o l y u r e t h a n e surfaces to showed n o change i n

1 2 5

1 2 5

I a l b u m i n solutions o f e q u a l concentration

I uptake for 5 - 1 5 s, at values equivalent to those

o b t a i n e d b y d i p p i n g t h e sample t h r o u g h t h e a i r interface f i l m . T h i s result suggests that the ordinate i n t e r c e p t for the c o n t r o l samples represents n o n specific adsorption o f a i r - d e n a t u r e d a l b u m i n , a n d w o u l d have o c c u r r e d for the d e r i v a t i z e d samples as w e l l . T h u s , we have a d o p t e d a conservative inter pretation o f a l b u m i n a t i o n e n h a n c e m e n t b y a l k y l a t i o n . T h e e n h a n c e m e n t of a l b u m i n b i n d i n g , and its potential effectiveness i n i m p r o v i n g t h r o m b o r e s i s t a n c e , is f u r t h e r demonstrated i n t h e results o f si multaneous f i b r i n o g e n - a l b u m i n exposure, shown i n F i g u r e 12. R e d u c t i o n o f f i b r i n o g e n a d s o r p t i o n is p r o p o r t i o n a l to a d d - o n of a l b u m i n , p r e s u m e d to b e b o u n d to octadecyl residues. V a r y i n g y i e l d may d e p e n d o n the manufactur er's p o l y m e r process variables, t h e derivatization process variables, and the p r o t e i n solution exposure t e c h n i q u e .

Discussion Partial g o l d d e c o r a t i o n enhances t h e p r o t e i n adsorbate image. G o l d vapor atoms condense o n the surface to b e i m a g e d and migrate u n t i l a stable In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


21.

EBERHART ET AL.


Ο

5

10

309

15

Exposure Time (min) Figure 11. Enhancement of defatted albumin adsorption on Pellethane 2363-80A by binding to octadecyl residues. Symbols represent different derivatization runs.

l£

0

I

I

I

I

20

40

60

80

I

I

I

100 120 /deriv.-control \

I-Albumin Enhancement, counts \

control

/xlOO

Figure 12. Effect of enhanced binding of defatted albumin to octadecyl residues on reduction of fibrinogen adsorption. The ordinate gives reduction of fibrinogen adsorption in simultaneous albuminfihrinogen exposure to the treated samples. The abscissa gives enhancement of albumin adsorption on a duplicate treated sample, using the same albumin solution and identical exposure methodology except for the fibrinogen solution addition. Controls are underivatized samples exposed in the same manner. Both one-step and two-step derivatizations are represented. Distorted Biomer data were corrected by referencing to solvent-treated, but nonderivatized controls. Key: (polyurethanes) o, Biomer; ·, Biomer (distorted, corrected); •, Pellethane 1; 0, Pellethane 2; and Δ , Mobay; y =7.6+ 0.57x; r = 0.905; and S = 6.9. 2

yx



310


gold nucleus is f o r m e d . T h e d r i v i n g force for the transformation f r o m m i grating g o l d atom to stable nucleus is the free energy difference between the atom and stable n u c l e u s . T h e g o l d nucleus density may be d e s c r i b e d i n terms of the i m p i n g e m e n t rate of g o l d atoms, t h e i r surface residence t i m e , critical nucleus surface d e n s i t y , a n d rate of g r o w t h of n u c l e i to supercritical d i m e n sions. T h i s value may be expressed by X , the mean distance between stable n u c l e i , w h i c h can be m e a s u r e d e x p e r i m e n t a l l y and d e s c r i b e d analytically (11). T h e substrate ( p o l y m e r or p r o t e i n molecule) modifies the a t o m - n u c l e u s free e n e r g y difference, b o t h i n terms of the heat of desorption f r o m the substrate a n d the heat of diffusion on the substrate surface. B o t h p h y s i c a l and c h e m i c a l features of the p r o t e i n - p o l y m e r c o m p l e x can m o d i f y these parameters. T h e X for all p r o t e i n adsorbates s t u d i e d was significantly less than that for the p o l y m e r substrates of interest, for partial surface coverage less than 50%. A t h i g h e r g o l d coverage fractions, the differences i n surface features are obliterated as the g o l d n u c l e i grow, coalesce, and f o r m crystals. E m p i r i c a l observations indicate that a 1 0 - 2 5 % coverage of the substrate p o l y m e r is best to identify p h y s i c a l a n d c h e m i c a l features of the surface. C o m p a r i s o n of the m o r e gently treated, c r i t i c a l - p o i n t - d r i e d 7-globulin and C o h n I f i b r i n o g e n adsorbates w i t h a i r - d r i e d material suggests that p r o t e i n - p r o t e i n b i n d i n g , i n c o m b i n a t i o n w i t h p r o t e i n - p o l y m e r b i n d i n g , creates a t e t h e r e d n e t w o r k that is i n tension d u r i n g d e h y d r a t i o n . T h i s tension is resolved b y the c i r c u l a r , r e t i c u l a t e d pattern of pores i n the f i l m (criticalpoint d r y i n g ) , w h i c h m i n i m i z e s a i r - f i l m - s u r f a c e interfacial tension. S i m i l a r patterns have b e e n seen for c r i t i c a l - p o i n t d r i e d , p u r i f i e d plasma f i b r o n e c t i n on Teflon (17). A i r d r y i n g differentiates the 7-globulin a n d C o h n I f i b r i n o gen f i l m s : the 7-globulin f i l m is d i s r u p t e d a n d the adsorbate aggregates into e x t e n d e d b u t u n c o n n e c t e d deposits, whereas the C o h n I f i b r i n o g e n f i l m always maintains the r e t i c u l a t e d pattern. T h i s difference suggests greater strength of the p r o t e i n - p r o t e i n bonds for the f i b r i n o g e n - 7 - g l o b u l i n f i b r o n e c t i n c o m p l e x of C o h n I fraction i n the face of d e h y d r a t i o n a n d denaturation forces. T h e dramatic stereo p a i r images of e x t e n d e d f i b r i n o g e n films over surface defects p r o v i d e f u r t h e r a n d c o n v i n c i n g evidence of the strength of the p r o t e i n - p r o t e i n b o n d i n this instance. T h e existence of f i l m pores on surface flats, o b s e r v e d i n a l l cases for C o h n I and II fraction adsorbates, may relate to variations i n the c o n f o r m a t i o n of the adsorbed p r o t e i n , w i t h resultant alteration of b i n d i n g strength. I n the cracks and fissures of the rougher surfaces, the pores w e r e not as prevalent. T h i s result may be due to p h e n o m ena that m i g h t alter the force balance i n the f i l m , for example, concentration of dissolved p r o t e i n i n the defect, p r o t e c t i o n f r o m shear forces d u r i n g wash steps, interaction w i t h u n r e m o v e d m i c r o b u b b l e s l o d g e d i n cracks, or f i l m support b y elaborated f i b r i n strands.


21.

EBERHART ET AL.


311

O b s e r v a t i o n of strands of f i b r i n - l i k e material e m a n a t i n g f r o m m u l t i p l e films of C o h n I f i b r i n o g e n i n s m a l l surface cracks supports the macroscopic, c l i n i c a l observation of crack-propagated t h r o m b u s i n extracoporeal c i r c u l a tion a n d atherosclerotic vascular lesions. T h i s observation also supports f i n d ings b y V r o m a n et a l . (18) based o n plasma exposure to a c o n t r o l l e d fissure at a s p h e r e - p l a n e - p l a s m a interface. It suggests that surface s m o o t h i n g tech-


niques m u s t be c a r r i e d to a h i g h degree of p e r f e c t i o n i f thromboresistance is to be o b t a i n e d b y this m e t h o d alone. Identification of the f i l m c o m p o s i t i o n and strand structure may perhaps be c a r r i e d out b y x-ray crystallographic a n d f e r r i t i n - l a b e l e d a n t i b o d y T E M techniques. A i r - e x p o s e d a l b u m i n aggregates m o r e extensively than the c r i t i c a l p o i n t - d r i e d a l b u m i n adsorbate. Yet i n b o t h cases, no e v i d e n c e of the a b i l i t y (similar to that of f i b r i n o g e n , f i b r o n e c t i n , a n d 7-globulin) to support extensive films i n tension d u r i n g the d e h y d r a t i o n process exists. Identification

o f the

p r o t e i n adsorbate

b y the

ferritin-conjugated

a n t i b o d y - T E M t e c h n i q u e removes one major objection to acceptance of the partial decoration m e t h o d o l o g y i n biomaterials research. decoration a n d f e r r i t i n - l a b e l e d T E M techniques

are

However, gold

stop-and-interfere

methods, w h i c h m u s t be c o m p l e m e n t e d b y continuous (19) or s e m i c o n tinuous (20) t e c h n i q u e s i n o r d e r to u n d e r s t a n d the events i n b l o o d - m a t e r i a l interactions. T h e p a r t i a l g o l d decoration m e t h o d o l o g y has p r o v i d e d us w i t h superior r e s o l u t i o n of all e x a m i n e d p r o t e i n adsorbates, o n surfaces w i t h b o t h physical and c h e m i c a l i n h o m o g e n e i t y . If, as theory suggests, the metal n u cleus decoration m e t h o d can quantify the c h e m i c a l i n h o m o g e n e i t y of the p o l y m e r surface, it s h o u l d e m e r g e as a vital tool i n biomaterials research. T h e weak shear rate d e p e n d e n c e of a l b u m i n o n the F l u o r o f i l m Teflon substrate suggests that the p r o t e i n - p o l y m e r bonds are not strong e n o u g h to p r o v i d e e n d u r i n g a l b u m i n films for this " p h y s i o l o g i c a l " range of flow rates. These results are s u p p o r t e d b y the findings of B r a s h et al. (21 ). W e a k b i n d i n g also suggests w h y the surface coverage was i n c o m p l e t e i n "static" exposure. M o r e o v e r , the a p p a r e n t l y c o m p l e t e d e s o r p t i o n of a l b u m i n above 1500

s

- 1

supports the i n f e r r e d weak b i n d i n g f r o m static exposure studies, and casts doubts o n the effectiveness of spontaneous a l b u m i n a t i o n as a practical treatm e n t to enhance t h r o m b o r e s i s t a n c e . Yet p r e a d s o r b e d a l b u m i n , i n b o t h aird r i e d and c r i t i c a l - p o i n t - d r i e d settings, gave c o n v i n c i n g evidence of i n h i b i tion of C o h n I f i b r i n o g e n a d s o r p t i o n . O u r p r o t e i n v i s u a l i z a t i o n studies suggest that f i b r i n o g e n and g l o b u l i n fraction proteins r a p i d l y f o r m a t i g h t l y b o u n d , extensive coat that is relatively weakly attached to the surface. T h e coat is w e a k l y , but sufficiently sensitive to f l u i d shear that shear erosion of the coat w i l l occur at physiological flow rates over e x t e n d e d t i m e p e r i o d s . Shear erosion w o u l d p r o b a b l y increase for smoother surfaces. T h e " l o o s e l y adherent l a y e r " i n f e r r e d by total i n t e r n a l


312

BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS


reflection fluorescence analysis (19) may desorb u n d e r the influence of flow and/or c o n f o r m a t i o n a l change to expose the f i l m pores r e p o r t e d above. T h e f i l m may t h i c k e n , elaborate into f i b r i n - l i k e strands, or adopt other i r r e v e r sible conformational changes w i t h t i m e . A l b u m i n may m o d i f y the conforma tion of the f i b r i n o g e n adsorbate as d e m o n s t r a t e d for a l b u m i n - f i b r o n e c t i n adsorption (22). P r e a d s o r b e d a l b u m i n a p p a r e n t l y occupies the relatively few surface b i n d i n g sites o t h e r w i s e available to f i b r i n o g e n a n d 7 - g l o b u l i n , such that a fibrinogen/globulin fraction f i l m may not d e v e l o p . A l b u m i n p r e v i o u s l y has b e e n strongly but i r r e v e r s i b l y b o u n d to p o l y mers b y a n u m b e r of m e t h o d s (23-25). T h e nature of these b i n d i n g tech niques suggests the i n a b i l i t y to desorb the material, once the functional biological activity of the surface b o u n d m o l e c u l e has b e e n lost. W e e x p l o i t e d a l b u m i n ' s h i g h affinity for the h y d r o p h o b i c f u n c t i o n of long-chain fatty acids i n the a l k y l r e s i d u e derivatization. W e u t i l i z e d selective high-affinity b i n d i n g sites on p o l y m e r surfaces to b o n d C _ i carbon aliphatic chains covalently to the surface. U s i n g the 18-carbon c h a i n as the surface substrate p r o v i d e d the advantages of (1) h a v i n g a n e a r - m a x i m u m affinity coefficient (near that of oleate K = 2.6 X 1 0 M ) a n d (2) b e i n g inaccessible to e n z y m a t i c degradation (aliphatic chains cannot be d e g r a d e d v i a β-oxidation w i t h o u t the p r e s e n c e of a t e r m i n a l acid function). In the follow i n g , we describe two m e t h o d s of b i n d i n g aliphatic chains to p o l y m e r surfaces. T h e first consists of a two-step substitution reaction i n w h i c h p o l y m e r s w i t h active surface h y d r o g e n s (i.e., p o l y u r e t h a n e , p o l y a m i d e s , polyesters, etc.) are d e p r o t o n a t e d b y an aprotonous base (sodium ethoxide) f o r m i n g an a m i d e ion i n t e r m e d i a t e . S u b s e q u e n t treatment w i t h an aliphatic halide w h i c h at tracts the i n t e r m e d i a t e i n an S 2 type reaction gives a substituted tertiary a m i n e . T h e second m e t h o d of surface substitution is the reaction b e t w e e n an aliphatic isocyanate a n d active sites of the p o l y m e r surface i n the presence of a z i n c catalyst. In a d d i t i o n to successful a n d p r o m i s i n g results for b o t h methods w i t h p o l y u r e t h a n e , these a n d other reactions also have b e e n c a r r i e d out successfully o n N y l o n , p o l y a c r y l a m i d e , a n d other p o l y m e r s . D a c r o n has b e e n substituted v i a transamination a n d F r i e d e l - C r a f t s reactions. 1 6

A

8

8

_1

N

B y establishing an endogenous a l b u m i n adsorbate t h r o u g h selectively increasing the affinity o f the surface for a l b u m i n , the i n h e r e n t p r o b l e m s of covalently b i n d i n g a l b u m i n m i g h t be e l i m i n a t e d . D e n a t u r e d and d e s o r b e d endogenous a l b u m i n m i g h t be r e p l a c e d b y a d d i t i o n a l endogenous a l b u m i n w h i c h c o u l d favorably c o m p e t e for the o p e n a l k y l residues, c o n t i n u i n g to maintain the u n a v a i l a b i l i t y of the surface to those glycoproteins i m p l i c a t e d i n c e l l adhesion a n d coagulation. T h u s , the h i g h a l b u m i n affinity substrate, w i t h its b i o l o g i c a l l y f u n c t i o n a l , renewable endogenous a l b u m i n coat may maintain the thromboresistance of a prosthetic device i n d e f i n i t e l y . W e p r o d u c e d two types of p r e l i m i n a r y evidence to support this h y p o t h esis. F i r s t , i n r a d i o l a b e l e d a l b u m i n studies, a l k y l - d e r i v a t i z e d p o l y u r e t h a n e samples h a d consistently greater a l b u m i n coverage than control samples,


21.

EBERHART ET AL.

313


with maximal enhancement

at short exposure times ( F i g u r e 11). S i m i l a r

results w e r e o b t a i n e d for o t h e r p o l y m e r s ( N y l o n , D a c r o n , polyacrylamide). In a d d i t i o n , m o d i f i e d p o l y u r e t h a n e samples e x h i b i t e d a distinctive two-rate k i n e t i c c u r v e . B a s e d o n the s i m i l a r i t y b e t w e e n k i n e t i c curves n o r m a l l y seen for high-affinity p r o t e i n - s u b s t r a t e c o m p e t i t i v e systems a n d those of the m o d ified p o l y m e r surface, w e h y p o t h e s i z e that the initial fast rate represents b i n d i n g of p r o t e i n to the available a l k y l residues, w h i c h is essentially c o m Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 2, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch021

plete w i t h i n 60 s of exposure. F u r t h e r m o r e , since the second, slower adsorption rate of the m o d i f i e d sample k i n e t i c c u r v e does not reassume that of the u n m o d i f i e d s a m p l e , w e h y p o t h e s i z e that nonspecific b i n d i n g sites of the original surface have e i t h e r b e e n r e p l a c e d b y specific sites t h r o u g h the modification p r o c e d u r e , or have b e c o m e masked b y the specifically b o u n d a l b u m i n . N e i t h e r hypothesis has b e e n substantiated at this t i m e . Second, i n simultaneous a l b u m i n - f i b r i n o g e n adsorption studies ( F i g u r e 12), i n h i b i t i o n of f i b r i n o g e n a d s o r p t i o n was p r o p o r t i o n a l to enhancement of a l b u m i n adsorption, r e f e r e n c e d to u n d e r i v a t i z e d controls. T h e effect was observed for a n u m b e r of p o l y u r e t h a n e f o r m u l a t i o n s . F u r t h e r m o r e , this effect represents substantial i m p r o v e m e n t for some o f the most b l o o d - c o m p a t i b l e p o l y m e r s c u r r e n t l y u n d e r investigation (26). W o r k remains to be done to verify the postulated d e s o r p t i o n of a l b u m i n at the h y d r o p h o b i c b i n d i n g site (27). T h e results, favorable i n the short t e r m , m u s t be validated i n the l o n g t e r m , a n d also i n the b l o o d e n v i r o n m e n t . N e v e r t h e l e s s , a m e t h o d of p r o m i s e apparently has b e e n d e v e l o p e d i n the search for materials of i m p r o v e d b l o o d compatibility.

Conclusions T h e P G D T E M t e c h n i q u e p r o d u c e d s u p e r i o r p r o t e i n adsorbate images, w h i c h have b e e n v e r i f i e d b y i n d e p e n d e n t methods. G o l d decoration images may not d i s c r i m i n a t e b e t w e e n p r o t e i n species adsorbates, however, f e r r i t i n conjugated antibodies may be u s e d for this p u r p o s e . Visualization is difficult for p r o t e i n deposits at l o w surface concentration. C o h n I f i b r i n o g e n adsorbates f o l l o w i n g a gentle wash ( c r i t i c a l - p o i n t - d r i e d ) , are reticulated and c o n n e c t e d , w i t h a p o r e d i m e n s i o n o f 400 A a n d h i g h surface coverage. C o h n I f i b r i n o g e n adsorbates o n a F l u o r o f i l m Teflon surface have a weak negative shear d e p e n d e n c e at w a l l shear rates less than 1500 s" . A d s o r p t i o n and 1

conversion of the C o h n I f i b r i n o g e n may be e n h a n c e d i n surface defects, a n d adsorption of C o h n I f i b r i n o g e n is i n h i b i t e d b y a l b u m i n p r e a d s o r p t i o n . T h e results suggest that p r o t e i n - p r o t e i n b i n d i n g is i n v o l v e d i n the extension of surface coverage b y C o h n I f i b r i n o g e n a n d b y 7-globulin. A l b u m i n adsorbates ( c r i t i c a l - p o i n t - d r i e d ) are i r r e g u l a r a n d u n c o n n e c t e d , w i t h a characteristic d i m e n s i o n of 200 A , a n d a l o w surface coverage is observed following gentle wash. A d s o r b e d a l b u m i n is essentially r e m o v e d b y a wall shear rate greater than 1500 s" , a n d the adsorbate is u n c h a n g e d b y surface structural 1

detail d o w n to 1000 A .


314


P o l y m e r s o f b i o m e d i c a l interest c a n b e treated i n o r d e r to covalently b i n d Cis analogs o f aliphatic chains. T h e treatment enhances b i n d i n g of defatted a l b u m i n i n s h o r t - t e r m exposure, a n d reduces f i b r i n o g e n adsorption, in p r o p o r t i o n to e n h a n c e m e n t o f a l b u m i n adsorption. T h e treatment appears p r o m i s i n g for g e n e r a l i m p r o v e m e n t o f b l o o d c o m p a t i b i l i t y of a n u m b e r o f


polymers.

Acknowledgments W e gratefully a c k n o w l e d g e the c o n t r i b u t i o n s of m a n y s k i l l e d colleagues: i n p r o t e i n r a d i o l a b e l i n g , P . K u l k a r n i ; i n e l e c t r o n microscopy t e c h n i q u e s , H . H a g l e r a n d W . S c h u l z ; a n d i n fluoroescence t e c h n i q u e , F . G r i n n e l l . I n a d d i t i o n , the h e l p f u l discussions w i t h M . Prager, B . B r i n k , J . L o S p a l l u t o , M . A . W i l k o v , J . W i l s o n , a n d F . G r i n n e l l , a n d the efficient manuscript t y p i n g by K a t h e r i n e R h o d e s , are also gratefully a c k n o w l e d g e d .

Literature Cited 1. Packham, Μ. Α.; Evans, G . ; Glynn, M . F.; Mustard, J. F. J. Lab. Clin. Med. 1969, 73, 686. 2. Lee, E . S.; Kim, S. W. ASAIO J. 1980, 3, 50. 3. Jamieson, G. Α.; Urban, C. L . ; Barber, A. J. Nature (London), New Biol. 1971, 234, 5. 4. Eberhart, R. C.; Wissinger, J. F.; Wilkov, M . A. Trans. Soc. Biomaterials 1978, 11, 136. 5. Grinnell, F. Int. Rev. Cytol. 1978, 53, 65. 6. Harasaki, H . ; Kambic, H . ; Whalen, R.; Murray, J.; Snow, J.; Murabayashi, S.; Hillegrass, D . ; Ozawa, K.; Kiraly, R.; Nose, Y. Trans. Am. Soc.Artif.Intern. Organs 1980, 26, 470. 7. Pierce, W. J.; Donachy, J. H.; Rosenberg, G . ; Baier, R. E. Science 1980, 208, 601. 8. Keller, K. H . , Ed. "Guidelines for Physicochemical Characterization of Bio materials," NIH Publication 80-2186, U. S. Dept. Health and Human Ser vices, September 1980. 9. Mason, R. G . , Ed. "Guidelines for Blood-Material Interactions," NIH Publica tion 80-2185, U. S. Dept. Health and Human Services, September 1980. 10. Eberhart, R. C.; Prokop, L. D . ; Wissinger, J. F.; Wilkov, M . A. Trans. Am. Soc. Artif. Intern. Organs 1977, 23, 134. 11. Bilge, F. H . , unpublished data. 12. Eberhart, R. C.; Lynch, M . E . ; Bilge, F. H . ; Arts, H. A. Trans. Am. Soc. Artif. Intern. Organs 1980, 26, 185. 13. Wissinger, J., M . S. Thesis, Univ. of Texas, Austin, 1977. 14. Neugebauer, C. A. In "Handbook of Thin Film Technology"; Maissel, L. I.; Glang, R.; Eds.; McGraw Hill: New York, 1970; Chap. 8. 15. Brash, J. L . ; Lyman, D. J. In "The Chemistry of Biosurfaces"; Hair, M . L., Ed.; Marcel Dekker: New York, 1971, Vol. 1. 16. Fraker, P. J.; Speck, J. C., Jr. Biochem. Biophys. Res. Commun. 1978, 80, 849. 17. Eberhart, R. C., unpublished data. 18. Vroman, L. et al. Chap. 19 in this book. 19. Watkins, R. W.; Robertson, C. R. J. Biomed. Mater. Res. 1977, 11, 915. 20. Ihlenfeld, J. V.; Mathis, T. R.; Barber, Τ. Α.; Mosher, D. F.; Updike, S. J.; Cooper, S. L. Trans. Am. Soc. Artif. Intern. Organs. 1978, 24, 727. 21. Brash, J. L.; Uniyal, S.; Samak, Q. Trans. Am. Soc.Artif.Intern. Organs 1974, 20, 69.


21.

EBERHART ET AL.

315



22. Grinnell, F.; Feld, M . K. J. Biomed. Mater. Res., in press. 23. Hoffman, A. S.; Schmer, G . ; Harris, C.; Kraft, W. G. Trans. Am. Soc. Artif. Intern. Organs 1972, 18, 10. 24. Platé, P. Α., Matrosovich, M . N. Doklady Akad. Nauk SSSR 1976, 229, 496. 25. Imai, Y.; von Bally, K. Nose, Y. Trans. Am. Soc. Artif. Intern. Organs 1970, 26, 17. 26. Schultz, J. S.; Barenburg, S.; Ciarkowski, Α. Α.; Lindenauer, S. M . ; Penner, J. A. "Evaluation of Compatibility of Biomaterials, Devices and Technology Branch Contractors Meeting," NHLBI, U. S. Dept. H . E . W . 1979, 121. 27. Tanford, C. Science 1978, 200, 1012 R E C E I V E D for review January 16, 1981. A C C E P T E D March 2 3 ,

1981.


Protein Adsorption on Polymers - Advances in Chemistry (ACS

Recommend Documents