Protein Adsorption on Biomaterials - American Chemical Society

17 Protein Adsorption on Biomaterials THOMAS A. HORBETT

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University of Washington, Department of Chemical Engineering, BF-10, Seattle, WA 98195 The problem of finding acceptable materials for use in contact with tissue is highly relevant to present day clinical practice. The difficulty of this problem reflects the complex nature of tissue-material interactions which are influenced by properties of the tissue, properties of the material, and by the transport of fluids around the implant. Implants designed with each of these aspects in mind may produce a "biocompatible environment," analogous to the nonthrombogenic environment thought to be required for blood compatibility (1). The foreign body reaction occurring around soft tissue implants and thrombosis on surfaces in contact with blood are the major reactions encountered with implants. Both reactions involve the interaction of cells with the implant, especially in the later stages, and much previous study has therefore emphasized cellular events in the biocompatibility process. However, cells encounter foreign polymer implants under conditions that ensure the prior adsorption of a layer of protein to the polymer interface. The properties of the adsorbed layer are therefore important in mediating cellular response to the material. Adsorbed proteins are important in a variety of biological processes, as illustrated in Table I. The length and diversity of this list suggest that all biological processes occurring at interfaces will be greatly affected by proteins. Unfortunately, many major questions concerning adsorbed proteins remain unanswered because of the difficulty of studying proteins at interfaces. For example, why are certain proteins present at interfaces rather than other proteins? What properties of proteins and surfaces regulate the localization of proteins at surfaces? Why are proteins at interfaces often more influential than they are in the bulk phase? The mediating layer of adsorbed protein is present on all materials, yet cellular responses are not the same— how does the implied difference in the organization of the adsorbed layer occur? The sensitivity of cellular interactions to interfacial proteins probably is due to the presence of cell surface receptors for specific proteins and to the enhancement of receptor-protein interaction by the concentration of proteins at interfaces. To illustrate the role of specific proteins at interfaces, 0065-2393/82/0199-0233$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.

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

Table I. Biological Processes Influenced by Proteins at Interfaces •

Biocompatibility of synthetic polymers — T h r o m b o s i s o n catheters a n d replacements, arteries, a n d heart valves — F o r e i g n b o d y reaction to soft tissue implants — O t h e r processes s u c h as contact lens f o u l i n g

•

Cell adhesion — T i s s u e cells are anchorage d e p e n d e n t for g r o w t h


— P l a t e l e t a n d w h i t e cells r e q u i r e adhesion for f u n c t i o n — B a c t e r i a l c e l l a d h e s i o n is i n v o l v e d i n tooth decay and m a r i n e f o u l i n g •

Blood coagulation — T h e i n t r i n s i c system: factor X I I activation o n surfaces — T h e extrinsic system: p h o s p h o l i p i d vesicles accelerate p r o t h r o m b i n activation — P l a t e l e t activation b y collagen a n d other proteins

•

Immunology — A n t i b o d y b i n d i n g to f o r e i g n cells ("opsonization") — C o m p l e m e n t p r o t e i n activation a n d attack o n foreign cells — P h a g o c y t i c processes a i d e d b y m e m b r a n e receptors for proteins

•

Other — M e m b r a n e receptors — T u m o r cells m a y differ i n surface proteins (?) — P r o t e i n separation b y h y d r o p h o b i c or affinity c h r o m a t o g r a p h y ; enzyme immobilization

Table II s u m m a r i z e s t h e effects o f five c o m m o n plasma proteins o n the response o f a variety o f cells to foreign materials. T h e examples i n this table show that c e l l u l a r response to materials is i n f l u e n c e d strongly b y adsorbed proteins. F o r e x a m p l e , a d s o r b e d c o l d i n s o l u b l e g l o b u l i n enhances the adhesion o f fibroblasts, t h e phagocytosis o f particles b y macrophages, and t h r o m bus f o r m a t i o n o n p o l y v i n y l c h l o r i d e ( P V C ) exposed to b l o o d . H o w e v e r , the response is specific, since not a l l cells react the same to a given p r o t e i n : f i b r i n o g e n enhances platelet adhesion b u t decreases r e d - c e l l adhesion. O n l y a few o f the proteins i n p l a s m a e l i c i t a positive response f r o m a given c e l l , p r o v i d i n g f u r t h e r specificity. T h e strong and f r e q u e n t l y specific influence of adsorbed proteins o n c e l l u l a r reactions w i t h materials is the major reason for s t u d y i n g the nature of p r o t e i n a d s o r p t i o n . T h i s chapter describes the general behavior of proteins at interfaces a n d the specific questions r e g a r d i n g p r o t e i n adsorption f r o m c o m p l e x m i x t u r e s w h i c h appear to be most important.

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

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Protein Adsorption on Biomaterials

General Properties of Adsorbed Proteins C e r t a i n general p r o p e r t i e s o f proteins a n d t h e i r behavior at interfaces are i m p o r t a n t to r e m e m b e r w h e n t r y i n g to c o m p r e h e n d the w i d e s p r e a d influence o f t h e interfacial p r o t e i n layer. M o s t basic is t h e fact that the proteins are i n t r i n s i c a l l y surface active a n d t e n d to concentrate at interfaces, due partly to t h e i r p o l y m e r i c structure a n d partly to their a m p h o t e r i c nature (15). T h u s , m u l t i p l e contact points w i t h the interface are possible for each p r o t e i n m o l e c u l e because o f its large size, an effect that greatly increases the tendency for the m o l e c u l e to r e m a i n at the interface. T h e presence of polar, Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch017

charged, a n d n o n p o l a r a m i n o acid side chains i n proteins provides the o p p o r tunity for m u l t i p l e m o d e s o f b i n d i n g w i t h m a n y different types o f surfaces. T h e general t e n d e n c y for n o n p o l a r residues to b e i n t e r n a l i z e d i n the native p r o t e i n (16) m a y r e q u i r e structural alterations u p o n adsorption i n o r d e r to m a x i m i z e the n u m b e r o f contacts w i t h the surface (17). F o r example, p r o t e i n adsorption o n a h y d r o p h o b i c surface c o u l d involve conformational changes to o p t i m i z e t h e various b o n d i n g interactions b e t w e e n the protein's h y d r o p h o b i c and h y d r o p h i l i c sites w i t h the surface a n d water phases, respectively. T h e a d s o r p t i o n o f proteins at interfaces can lead to enormous local ο

concentrations, far i n excess o f the b u l k phase value. A 100-A-thick a l b u m i n

Table II. Plasma Proteins Affecting Cellular Interactions with Foreign Materials

Protein

Plasma Concentration (mg/mL) Material

Complement C3

1.2

Immunoglobulin G

10-30

Cold insoluble globulin Fibrinogen

Albumin

0.1-0.3

3-5

40-60

Cell Type

Response

cellophane mineral oil S. aureus

granulocytes neutrophils neutrophils

acute leukopenia (2) phagocytosis enhanced (3) attachment enhanced (4)

glass

platelets

polyvinyl toluene PHEMA-PEMA

leucocytes fibroblasts and red cells

adhesion and release enhanced (5) phagocytosis enhanced (6) adhesion prevented (7)

falcon dishes RE-test emulsion PVC

adhesion enhanced (8) fibroblasts macrophages phagocytosis enhanced (9) thrombus enhanced (10) platelets

polystyrene

platelets

silicone rubber glass

fibroblasts red cells

glass

platelets

polyethylene

red cells

falcon dishes

fibroblasts

adhesion and release enhanced (11) adhesion enhanced (12) adhesion prevented (13) adhesion and release decreased (5) strength of adhesion decreased (14) adhesion prevented (8)


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layer at 1 μ ^ τ η

2

has a c o n c e n t r a t i o n at the surface of about 1000 m g / m L , not

far b e l o w the theoretical m a x i m u m of 1400 m g / m L c o r r e s p o n d i n g to the local concentration i n the d o m a i n of the p r o t e i n m o l e c u l e itself. Interfacial c o n centrations o f 1 μg/cm

2

are t y p i c a l f o r m a n y surfaces exposed to p r o t e i n

solutions that are 1 m g / m L o r less i n the b u l k phase. T h e consequences o f such extreme local c o n c e n t r a t i o n i n terms o f effects o n p r o t e i n properties have yet to b e d e t e r m i n e d clearly, b u t i n general, most c h e m i c a l reactions are affected strongly b y c o n c e n t r a t i o n o f the reactants. T h u s , m a n y of the reactions that proteins can u n d e r g o i n t h e b u l k phase m i g h t b e strongly


enhanced i n the surface phase. F o r example, m o d i f i c a t i o n b y traces of e n z y m e m i g h t o c c u r m u c h m o r e r e a d i l y i n the surface phase than i n the b u l k , and the n o r m a l l y weak t e n d e n c y f o r most proteins to aggregate o r f o r m complexes m i g h t b e greatly e n h a n c e d o n t h e surface. F i n a l l y and most i m p o r t a n t l y , the c o n c e n t r a t i o n and localization of proteins at interfaces may greatly facilitate the a b i l i t y o f cells to react w i t h the proteins, since inter action w i t h closely spaced p r o t e i n molecules b y t h e c e l l c o u l d enhance b i n d i n g . E x a m p l e s of the special role of interfaces i n the properties of p r o teins i n c l u d e activation of c l o t t i n g factor X I I (18,19), e n h a n c e m e n t of c o m p l e m e n t C 3 activation (20), a n d activation of the alternate pathway of c o m p l e m e n t by a d s o r b e d i m m u n o g l o b u l i n G (IgG) (21).

Organization of the Adsorbed Protein Layer T h e exposure of any m a t e r i a l to a m i x t u r e of proteins such as plasma should lead to e n r i c h m e n t of certain proteins at the surface relative to the b u l k phase. T h e r e f o r e , t h e c o m p o s i t i o n o f the surface phase s h o u l d differ from t h e b u l k phase. T h e c o m p o s i t i o n s h o u l d also b e different o n each material because the c h e m i c a l p r o p e r t i e s l e a d i n g to e n r i c h m e n t vary signifi cantly b e t w e e n various materials. F u r t h e r m o r e , t h e site density o f any p r o t e i n i n the layer (expressed as molecules p e r square centimeter), and the accessibility or reactivity of the p r o t e i n to larger probes such as cells s h o u l d vary on different surfaces. T h e w i d e range i n affinity of proteins for surfaces, w h i c h is the basis for most of these expectations, is e v i d e n c e d by the routine separation o f proteins b y c o l u m n c h r o m a t o g r a p h y u s i n g ionic a n d h y d r o p h o b i c matrices (22,23). C o m p o s i t i o n , site density, a n d reactivity o f the proteins are the key organizational aspects of the adsorbed layer f o r m e d f r o m c o m p l e x m e d i a s u c h as p l a s m a . S u r p r i s i n g l y , few studies of these aspects o f the adsorbed p r o t e i n layer have b e e n m a d e . T h e differences i n affinity of proteins for surfaces is not readily d i s c e r n e d f r o m the extensive l i t e r a t u r e o n a d s o r p t i o n of proteins to surfaces f r o m p u r e p r o t e i n solutions (24). T h e t y p i c a l result o f such studies is the adsorption i s o t h e r m , b u t d e t e r m i n a t i o n of the strength of interaction or surface affinity f r o m such m e a s u r e m e n t s is not possible because the adsorption is essentially


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irreversible. T h e surface activity of the plasma proteins has not b e e n s t u d i e d systematically as yet, nor have the structural properties of proteins that control this activity. H o w e v e r , an e m p i r i c a l study of the c o m p e t i t i o n of various p l a s m a proteins for surfaces f r o m s i m p l e mixtures does p r o v i d e information about the affinity of proteins for surfaces. F o r example, the adsorption of f i b r i n o g e n to several p o l y m e r s is r e d u c e d to half its o r i g i n a l value b y an approximately t e n f o l d w e i g h t excess of a l b u m i n or 7 - g l o b u l i n (25). H e m o globin (in the f e r r i c form) competes m u c h m o r e effectively than any other p r o t e i n yet tested; at o n e - t e n t h the f i b r i n o g e n concentration, h e m o g l o b i n Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch017

reduces the a d s o r p t i o n of f i b r i n o g e n to p o l y e t h y l e n e b y 5 0 % (26).

In a

three-way m i x t u r e s i m u l a t i n g the c o m p e t i t i o n of the three major plasma proteins, f i b r i n o g e n forms 55 to 7 0 % of the p r o t e i n adsorbed i n the first 2 m i n of exposure ( d e p e n d i n g on the p o l y m e r ) , but at steady state (2.5 h later), each m a t e r i a l h a d a p p r o x i m a t e l y the same c o m p o s i t i o n of adsorbed p r o t e i n : 4 3 - 4 5 % f i b r i n o g e n , 1 7 - 2 4 % 7 - g l o b u l i n , and 3 2 - 3 9 % a l b u m i n (27,28). A d s o r p t i o n studies u s i n g w h o l e p l a s m a p r o v i d e the most relevant i n vitro approach to the c o m p o s i t i o n of the adsorbed p r o t e i n layer. Several observations not p r e d i c t a b l e f r o m s i m p l e r systems have b e e n made i n the plasma studies. F o r e x a m p l e , surfaces exposed to plasma for 10 s or less b i n d f i b r i n o g e n antibodies, w h i l e l o n g e r exposure results i n loss of antifibrinogen b i n d i n g (29,30). T h i s process of " c o n v e r s i o n " apparently involves h i g h m o lecular w e i g h t k i n i n o g e n , since loss of a n t i f i b r i n o g e n b i n d i n g does not occur w i t h p l a s m a deficient i n this m a t e r i a l (31).

L a r g e differences i n the uptake

of fluorescent antibodies against a l b u m i n , f i b r i n o g e n , and i m m u n o g l o b u l i n by various types of h e m o d i a l y s i s m e m b r a n e s after exposure to b l o o d have been a t t r i b u t e d to o v e r l a y i n g of antigenic p r o t e i n w i t h other plasma proteins (32). T h e u n e q u a l availability of the proteins i n the adsorbed layer suggested by b o t h studies may be e x t r e m e l y i m p o r t a n t i n u n d e r s t a n d i n g w h y some surfaces are able to e l i c i t m o r e intense c e l l u l a r reactions than others. E x a m i n a t i o n of the a d s o r b e d p r o t e i n layer f o r m e d f r o m plasma by electrophoretic separation of the proteins i n a detergent eluate of the surface has revealed the c o m p l e x i t y of the layer (33-35). A s m a n y as n i n e separate proteins have b e e n d e t e c t e d , i n c l u d i n g f i b r i n o g e n , I g G , a l b u m i n , and h e m o g l o b i n , as w e l l as several u n i d e n t i f i e d proteins present i n smaller amounts (35). T h e s e studies e m p h a s i z e the fact that the adsorbed layer is not d o m i nated b y any p a r t i c u l a r p r o t e i n . T h e c o m p o s i t i o n of the adsorbed layer appears to reflect differences i n surface activity and b u l k concentration of the various proteins i n the plasma. T h u s , proteins w i t h h i g h surface activity such as f i b r i n o g e n a n d h e m o g l o b i n are present i n the layer despite their relatively low b u l k - p h a s e c o n c e n t r a t i o n . C o n v e r s e l y , although a l b u m i n is not p a r t i c u larly surface active, its h i g h b u l k - p h a s e concentration gives a competitive edge to this p r o t e i n . T h e p r e s e n c e of the major p l a s m a proteins o n all p o l y m e r s exposed to plasma, a n d the lack of d o m i n a n c e b y f i b r i n o g e n or other proteins i n this


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layer, pose the q u e s t i o n of what differences i n the adsorbed layer are suf ficient to e l i c i t differences i n c e l l u l a r responses. Since a c e l l u l a r response may r e q u i r e the close spacing of s i m i l a r molecules on the interface to b i n d p r o p e r l y or o t h e r w i s e trigger the c e l l , the site density or molecules p e r unit area of a p a r t i c u l a r p r o t e i n may be a key parameter. Variations i n the site density of specific p r o t e i n s i n the a d s o r b e d layer f o r m e d from c o m p l e x m e d i a have b e e n d e m o n s t r a t e d i n several studies, u s i n g I - l a b e l e d proteins a d d e d to plasma. T h e amounts of f i b r i n o g e n , 7 - g l o b u l i n , and a l b u m i n adsorption from the p l a s m a seem to vary a great deal, d e p e n d i n g on both material and p r o t e i n . F o r e x a m p l e , a l b u m i n adsorption v a r i e d f r o m about 0.2 μg/cm on Teflon F E P to 3.0 μg/cm on B i o m e r , w h i l e f i b r i n o g e n adsorption was gener ally m u c h l o w e r : 0.03 μg/cm on Teflon to 0.06 μg/cm on B i o m e r (36). I n other studies, a l b u m i n adsorption f r o m plasma to p o l y e t h y l e n e , p o l y (2-hydroxyethyl methacrylate) ( P H E M A ) and p o l y ( e t h y l methacrylate) ( Ρ Ε Μ Α ) v a r i e d over a n a r r o w e r range (0.14-0.21 μg/cm ), w h i l e f i b r i n o g e n adsorption was s i m i l a r l y l o w e r (0.019-0.044 μg/cm ) (37, 38). 125

2


2

2

2

2

2

A n o t h e r u n e x p e c t e d aspect of plasma p r o t e i n adsorption revealed by studies w i t h c o m p l e x m e d i a has b e e n the r a p i d rearrangement of the ad sorbed layer early i n the adsorption process. T h e kinetics of adsorption of proteins f r o m p l a s m a e i t h e r i n vivo or i n v i t r o were s t u d i e d recently i n three separate laboratories, a n d , s u r p r i s i n g l y , each observed the same major re sult: f i b r i n o g e n a d s o r p t i o n was i n i t i a l l y h i g h on some surfaces but then decreased w i t h i n an h o u r to lower, "steady-state" values (39-41). T h i s effect appears to d e p e n d on the nature of the surface: i n one study, the initially h i g h f i b r i n o g e n value has b e e n o b s e r v e d on P V C but not on B i o m e r (38), w h i l e i n another study f i b r i n o g e n adsorption was initially h i g h on Ρ Ε Μ Α but not on P H E M A (41). S i m i l a r transient adsorption m a x i m a for a l b u m i n w h i c h varied w i t h p o l y m e r type also w e r e o b s e r v e d i n two of these studies (39,41 ). H e m o g l o b i n a n d I g G a d s o r p t i o n f r o m plasma increased r a p i d l y and regularly to the saturation value o n Ρ Ε Μ Α a n d P H E M A , so the h i g h initial adsorption is specific to f i b r i n o g e n a n d a l b u m i n (41). T h e transient, initial m a x i m u m i n f i b r i n o g e n adsorption may be related to W o m a n ' s earlier observation of r a p i d conversion of a d s o r b e d f i b r i n o g e n to a f o r m unreactive w i t h antifibrinogen. T h e c o n v e r s i o n also appears to occur m o r e on some surfaces than on others (42). I o d i n a t i o n of f i b r i n o g e n i n the adsorbed state on surfaces exposed to plasma for various times also is u n u s u a l (43). O n h y d r o p h o b i c surfaces ad sorbed for 150 m i n a n d t h e n i o d i n a t e d , little i o d i n e uptake into f i b r i n o g e n was o b s e r v e d . A f t e r a 0 . 5 - m i n plasma exposure, however, most of the iodine i n c o r p o r a t e d into a d s o r b e d proteins was i n f i b r i n o g e n . O n h y d r o p h i l i c sur faces, surfaces exposed to p l a s m a showed increasing amounts of i o d i n e i n f i b r i n o g e n as adsorption t i m e increased. R e m o v a l of p r e a d s o r b e d f i b r i n o g e n on exposure to p l a s m a has b e e n o b s e r v e d (44). Taken together, the various kinetic studies of the structure of the adsorbed p r o t e i n f i l m f o r m e d f r o m plasma to surfaces suggest a r a p i d rearrangement of the adsorbed layer,


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d e p e n d i n g o n t h e nature o f the substrate, and p o i n t to the existence of a u n i q u e organization o n each type of m a t e r i a l . F u r t h e r m o r e , the major differences i n the kinetics of f i b r i n o g e n adsorption and i o d i n e uptake b y f i b r i n o g e n adsorbed f r o m p l a s m a to different p o l y m e r s suggest

t h e possibility o f

differences i n t h e structure o r accessibility o f adsorbed f i b r i n o g e n o n t h e polymers.

Structure of Adsorbed Proteins


Proteins are k n o w n to b e c o m e extensively " d e n a t u r e d " o r u n f o l d e d at the air/water interface (15). S i m i l a r b u t perhaps less extensive p e r t u r b a t i o n of a protein's structure b y t h e aqueous/solid interface is therefore often a reasonable b u t u n p r o v e n a s s u m p t i o n . T h e i d e a that proteins u n f o l d to different extents o n different p o l y m e r s , thus e l i c i t i n g differences i n cellular response b y t h e p o l y m e r s , is a major alternative hypothesis to t h e possible compositional variation i n t h e a d s o r b e d layer. T h e r e f o r e , t h e structure o f proteins at solid interfaces has b e e n the subject of many studies. T h e degree o f d e n a t u r a t i o n o f a d s o r b e d proteins is c u r r e n t l y o p e n to question. T h e fraction of the c a r b o n y l groups i n proteins adsorbed to silica surfaces that are i n contact w i t h t h e silica, d e t e r m i n e d w i t h differential infrared spectroscopy,

has b e e n u s e d to detect conformational

changes

(45,46). A l b u m i n a n d p r o t h r o m b i n adsorbed to silica appear to retain t h e i r native structure, w h i l e a d s o r b e d 7 - g l o b u l i n appears to u n f o l d at l o w e r surface coverage (45,46). U n f o l d i n g of 7 - g l o b u l i n d u r i n g adsorption to polystyrene also was i n d i c a t e d b y the release of protons o c c u r r i n g d u r i n g adsorption (47). In the 7 - g l o b u l i n glass system, v e r y h i g h molar heats of adsorption indicative of substantial c o n f o r m a t i o n changes occur at lower degrees of adsorption, but the heat of a d s o r p t i o n (per m o l e of adsorbed species) decreases m a r k e d l y , and m u l t i l a y e r a d s o r p t i o n occurs at h i g h e r b u l k concentrations (48). T h e c a l o r i m e t r y results suggest differences i n the degree of denaturation as the distance f r o m t h e surface increases. T h e results o f the infrared and calo r m e t r i c studies suggest that i n i t i a l l y a r r i v i n g platelets w o u l d

encounter

proteins d e n a t u r e d to q u i t e different extents, because a m o d e l w i t h denat u r e d proteins u n d e r l y i n g m o r e loosely h e l d native molecules is p r e s u m a b l y quite different f r o m a u n i f o r m layer of d e n a t u r e d molecules (49).

Structural

changes i n factor X I I u p o n a d s o r p t i o n to quartz have b e e n detected w i t h circular d i c h r o i s m (18). S u c h p e r t u r b a t i o n may facilitate the proteolytic activation of factor X I I o n the surface (19), a m e c h a n i s m possibly i m p o r t a n t for all the surface-bound p r o t e i n s . A variety of o t h e r t e c h n i q u e s has b e e n used to examine the structure o f proteins at surfaces, i n c l u d i n g e l e c t r o n microscopy (50,51), e l l i p s o m e t r y (52), e l e c t r o p h o r e t i c m o b i l i t y (53), a n d total i n t e r n a l reflection fluorescence (TIRF) (54). Several n e w techniques are b e i n g a p p l i e d at present, i n c l u d i n g F o u r i e r transform i n f r a r e d spectroscopy ( F T I R ) and T I R F (see next section),


240


and electron spectroscopy for c h e m i c a l analysis ( E S C A ) (55). T h e s e techniques p r o m i s e to e n h a n c e greatly o u r p r e s e n t l y rather meager stock of k n o w l e d g e about the structure of proteins at interfaces.

Current Research T h e p r o t e i n a d s o r p t i o n studies d e s c r i b e d i n this v o l u m e p r o v i d e a representative cross section of the c u r r e n t research i n this area. T h e c o m positional a n d s t r u c t u r a l aspects of proteins at interfaces continue to receive


major attention. B o t h n e w a n d p r e v i o u s l y d e v e l o p e d techniques are b e i n g a p p l i e d to these p r o b l e m s . T h e use of F T I R (56) a n d the d e v e l o p m e n t of an i n t e r n a l reflection t e c h n i q u e capable of d e t e c t i n g i n t r i n s i c fluorescence f r o m tryptophane residues i n p r o t e i n (57) r e p r e s e n t p o w e r f u l n e w approaches to the structure of proteins a d s o r b e d to interfaces. T h e speed, sensitivity, a n d h i g h frequency resolution of the F T I R t e c h n i q u e have p e r m i t t e d studies at v e r y short adsorption intervals a n d d i s c r i m i n a t i o n a m o n g

proteins i n mixtures due to

their slightly different i n f r a r e d spectra. T h e T I R F t e c h n i q u e monitors t r y p tophane residues, w h i c h are often b u r i e d i n the p r o t e i n structure, a n d thus p r o v i d e a natural m a r k e r for changes i n p r o t e i n structure at interfaces. H o w ever, the signal u t i l i z e d i n b o t h techniques is not solely due to p r o t e i n molecules at the interface, because the l i g h t emanating f r o m the substrate penetrates m u c h f u r t h e r than the d i a m e t e r of a t y p i c a l p r o t e i n . Some degree of averaging of events away f r o m the interface is therefore i n t r i n s i c i n these methods. S t r u c t u r a l changes i n a d s o r b e d proteins u s i n g i m p r o v e d circular d i c h r o i s m m e t h o d o l o g y indicate relatively slow rearrangements (58). M i c r o c a l o r i m e t r i c studies of a d s o r b e d proteins indicate differences i n the denaturation b e h a v i o r of p r o t e i n s o n various p o l y m e r s (59). U s e of p r e w e t t e d p o l y m e r - c o a t e d a l u m i n a particles i n this study represents a technical advance in the use of m i c r o c a l o r i m e t r y , w h i c h is l i k e l y to make this t e c h n i q u e of greater interest i n the near f u t u r e . T h e r a p i d c o n v e r s i o n of surface-bound f i b r i n o g e n to a f o r m unreactive w i t h a n t i f i b r i n o g e n was r e p o r t e d o r i g i n a l l y some t i m e ago (29,30). From more recent studies, h i g h m o l e c u l a r w e i g h t k i n i n o g e n may be i n v o l v e d i n the f i b r i n o g e n c o n v e r s i o n reaction, perhaps b y direct replacement of f i b r i n o g e n by the k i n i n o g e n (31). Because h i g h m o l e c u l a r weight k i n i n o g e n m i g h t be necessary for the contact activation of coagulation (60), these results are potentially v e r y i m p o r t a n t . T h e a b i l i t y of narrow spaces b e t w e e n adjacent surfaces to s o m e h o w m o d u l a t e the c o n v e r s i o n reaction, as r e p o r t e d i n this v o l u m e , may be i m p o r t a n t i n u n d e r s t a n d i n g the influence of surface g e o m etry i n b l o o d coagulation (e.g., the i m p o r t a n c e of surface imperfections). T h e ability of r e d cells to i n f l u e n c e the c o m p o s i t i o n of the layer b y r e d u c i n g f i b r i n o g e n a d s o r p t i o n f r o m p l a s m a (61 ) is an u n e x p e c t e d p h e n o m e n o n , and is difficult to rationalize i n v i e w of the r a p i d transport of proteins to surfaces


17.

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relative to cells. If the c o m p o s i t i o n of the a d s o r b e d layer reflects b o t h c e l l u l a r and p r o t e i n interactions w i t h the surface, a m u c h m o r e c o m p l e x series o f events than heretofore i m a g i n e d m u s t b e i n v o l v e d i n the response o f b l o o d to foreign materials. T h e v i s u a l i z a t i o n o f proteins o n surfaces w i t h g o l d nucleation e l e c t r o n m i c r o s c o p y also appears to indicate behavior different than p r e v i o u s l y e x p e c t e d ; p r o t e i n d e p o s i t i o n often was i r r e g u l a r and r e t i c u lated instead o f b e i n g a regular m o n o l a y e r often e n v i s i o n e d for proteins at interfaces (62). It w o u l d seem i m p o s s i b l e , however, to e l i m i n a t e the possi b i l i t y o f fixation o r d r y i n g artifacts i n the p r e p a r a t i o n of samples for electron


microscopy. Results o f E S C A analysis o f f r o z e n , u n d r i e d h e m o g l o b i n o n Teflon also suggest i n c o m p l e t e coverage (55). T h e i n f l u e n c e o f specific proteins i n the c o m p l e x m i x t u r e i n the a d sorbed layer o n c e l l u l a r reactions has yet to b e demonstrated d i r e c t l y , b u t p u r i f i e d proteins p r e a d s o r b e d to surfaces a n d t h e n exposed to b l o o d p r o v i d e information o n the p o t e n t i a l o f various proteins to influence c e l l u l a r events. A m u c h m o r e c o m p r e h e n s i v e study o f this type w h i c h includes proteins such as v o n W i l l e b r a n d factor a n d a - m a c r o g l o b u l i n is p r e s e n t e d i n this v o l u m e 2

(63). T h e a b i l i t y o f p r o t e i n s to enhance or repress c e l l u l a r reactions w h e n used as p u r e preadsorbates does not indicate the m a g n i t u d e o f the role played b y the p r o t e i n w h e n present i n the c o m p l e x layer adsorbed f r o m plasma. T h e p r o t e i n m a y be present i n v e r y s m a l l amounts i n the plasmad e r i v e d adsorbate relative to the p u r i f i e d p r e a d s o r b e d layer. F o r example, von W i l l e b r a n d factor is present at v e r y l o w concentrations i n plasma (10-15 μg/mL) a n d is therefore p r o b a b l y present at v e r y l o w levels i n the absorbed layer f o r m e d o n p o l y m e r s exposed to plasma. T h u s , although v o n W i l l e b r a n d factor u n d o u b t e d l y c o u l d b e an i m p o r t a n t factor i n surface thrombosis, its influence at the v e r y l o w a d s o r p t i o n levels l i k e l y to exist i n vivo must be d e m o n s t r a t e d . H o w e v e r , the k n o w l e d g e o f w h i c h proteins are potentially important, d e r i v e d f r o m p r e a d s o r p t i o n studies, is e x t r e m e l y useful i n focus i n g attention o n specific proteins to study i n the actual adsorbed layer.

Conclusions A d s o r b e d p r o t e i n s can greatly i n f l u e n c e c e l l u l a r reactions w i t h synthetic materials. T h e s e n s i t i v i t y to a d s o r b e d proteins, the variation i n cellular r e sponse to specific p r o t e i n s , a n d the r a p i d adsorption of proteins to all surfaces exposed to the b i o l o g i c a l e n v i r o n m e n t , have l e d to the idea that the c e l l u l a r response to i m p l a n t e d p o l y m e r s is the result o f specific interactions b e t w e e n components o f the a d s o r b e d p r o t e i n layer o n the p o l y m e r a n d the c e l l p e r i p h e r y . T h e s e observations, i n t u r n , have l e d to the hypothesis that c e l l u l a r interactions w i t h f o r e i g n materials are c o n t r o l l e d b y the presence at the surface o f specific p r o t e i n s at sufficiently h i g h surface density a n d degree o f reactivity to e l i c i t a response. E a c h o f these factors constitutes an i m p o r t a n t aspect o f the o r g a n i z a t i o n o f the a d s o r b e d p r o t e i n layer.


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O f the large n u m b e r o f proteins i n plasma, p r o b a b l y o n l y those w i t h specific c e l l u l a r receptors c a n elicit intense and specific responses f r o m a given c e l l type (e.g., f i b r i n o g e n platelets), w h i l e the other proteins (e.g., albumin) i n h i b i t c e l l u l a r reactions. T h e p r o p o r t i o n o f activating a n d passivating proteins i n p l a s m a for a g i v e n c e l l type is not k n o w n at present, b u t most plasma proteins p r o b a b l y are passive i n this sense. T h e particular balance b e t w e e n " r e a c t i v e " a n d " n o n r e a c t i v e " proteins is d e t e r m i n e d by the b i o m a t e r i a l surface c h e m i s t r y , and is d u e m a i n l y to affinity differences a m o n g the proteins i n m u c h t h e same way that it occurs i n affinity chromatography


of proteins. T h e a b i l i t y o f m a t e r i a l c o m p o s i t i o n to influence the p r o p o r t i o n of various proteins o n t h e surface has b e e n demonstrated clearly i n several studies. T h e materials b y themselves may elicit little o r no active cellular response, b u t they b e c o m e reactive b y selecting and interfacially concen trating specific proteins f r o m t h e b u l k phase onto t h e surface phase. T h e cellular reactivity o f the proteins i n t h e layer also may b e i n f l u e n c e d b y the accessibility a n d configurational state of the adsorbed proteins. T h e s e factors also may b e i n f l u e n c e d b y surface c h e m i c a l properties of the p o l y m e r s u n d e r l y i n g the p r o t e i n s . T h u s , b o t h t h e specific c o m p o s i t i o n of the adsorbed layer and the reactivity of the proteins i n t h e layer may affect the cellular response e l i c i t e d w h e n materials are u s e d as i m p l a n t s . T h i s concept constitutes a major hypothesis about b i o c o m p a t i b i l i t y a n d merits further testing.

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54. Watkins, R. W.; Robertson, C. R. J. Biomed. Mater. Res. 1977, 11, 915-938. 55. Ratner, B. D.; Horbett, Τ. Α.; Shuttleworth, D.; Thomas, H. R.J.Colloid Interface Sci. 1981, 83, 630-642. 56. Gendreau, R. M.; Leininger, R. I.; Winters, S.; Jakobsen, R. J. Chap. 24 in this book. 57. Van Wagenen, R. Α.; Rockhold, S.; Andrade, J. D. Chap. 23 in this book. 58. Walton, A. G.; Koltisko, B. Chap. 18 in this book. 59. Filisko, F. E.; Reichert, W. M.; Barenberg, S. A. Chap 13 in this book. 60. Griffin, J. H.; Cochrane, C. G. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2554-2558. 61. Uniyal, S.; Brash, J. L.; Degterev, I. A. Chap. 20 in this book. 62. Eberhart, R. C.; Lynch, M. F.; Bidge, F. H.; Wissinger, J. F.; Munro, M. S.; Ellsworth, S. R.; Qualtrone, A. J. Chap. 21 in this book. 63. Young, B. R.; Lambrecht, L. K.; Cooper, S. L.; Mosher, D. F. Chap. 22 in this book. RECEIVED for review April 6, 1981. ACCEPTED July 14, 1981.


Protein Adsorption on Biomaterials - American Chemical Society

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