Reversible-Irreversible Protein Adsorption and Polymer Surface

Chapter 28

Reversible-Irreversible Protein Adsorption and Polymer Surface Characterization Adam Baszkin, Michel Deyme, Eric Perez, and Jacques Emile Proust

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch028

Physico-Chimie des Surfaces et Innovation en Pharmacotechnie, UA Centre National de la Recherche Scientifique 1218, Université Paris—Sud, 5 rue J. B. Clément, 92296 Châtenay-Malabry, France

The present review paper describes the work on adsorption of proteins performed in the authors'labora tory. Behavior of two proteins of different type : collagen (rigid rod-like molecule) and mucin (flexible molecule) was investigated at the solid-solution inter faces using surface force and in situ adsorption/desor ption measurements. The in situ adsorption/desorption technique, based on the use of C labeled proteins, allows to distinguish between irreversibly and rever sibly adsorbed protein. Results obtained from these experiments provide direct data on the structure and orientation of adsorbed collagen or mucin molecules at the studied surfaces. It is shown that the degree of reversibility of the adsorbed protein depends on the type of protein and surface. Since the nature, distri bution and orientation of polymer functional groups have a direct influence upon the mechanism of protein adsorption, emphasis is given to the techniques of characterization of polymer surfaces developed in the laboratory. 14

Characterization of Polymers Quantification of functional sites on polymer surfaces. The p r i n c i p l e of the method developed in our laboratory for these purposes is based upon the use of radioactive isotopes emitting soft- $ radiation ( C , ^ C a ) . When a polymer film bearing functional s i t e s capable of adsorbing S C N " or C a ions i s placed in contact with a solution containing one of these ions, the measured r a d i o a c t i v i t y above the solution-polymer interface would come from the molecules adsorbed in excess at the interface plus that of a 1 4

5

14

4 5

2 +

0097-6156/87/0343-0451 $06.00/0 © 1987 American Chemical Society

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

452

PROTEINS AT INTERFACES

thin layer of solution. As the mean free path of these radiations in aqueous solution i s respectively equal to 0.16 mm and 0.59 mm, a l l radiation originating from the solution below this depth is attenuated. To allow for the r a d i o a c t i v i t y from the adjacent thin solution layer a separate experiment i s performed in which instead of a surface treated polymer film an untreated polymer film of the same thickness which does not adsorb SCN* or C a ions i s used. Figure 1 i l l u s t r a t e s the apparatus used for these measurements. Detailed description of a f - r a d i o t r a c e r adsorption method i s given in the paragraph dealing with mucin and collagen adsorption at interfaces. The cal ci um/thi ocyanate method was extensively used in the laboratory for quantification of functional groups on different surface modified polymers. Figure 2 exemplifies a series of such typical polymeric surfaces. While the calcium adsorption isotherms on poly(maleic acid) grafted polyethylene surfaces yielded the amounts of dissociated C00H groups, thiocyanate adsorption isotherms were used to determine the amounts of quaternized polyamine groups on surfaces. Depending on the grafting conditions, pH and ionic strength of the aqueous adjacent phase, the surface density of functional groups on these polymers varied in the range of 1 0 ^ 10 sites/cm . The calcium/thiocyanate isotherms, combined with contact angle measurements, reveal, on a molecular l e v e l , any rearrangement or reorientation of surface functional groups produced by the variation or alternation of the polymer adjacent phase. The main references to these studies are given in (1-5).


2 +

17

2

Thin wetting f i l m s . The Scheludko's technique of thin wetting films was adapted to study the behavior of proteins at various interfaces and in p a r t i c u l a r or, contact lenses. The p r i n c i p l e of the method, shown in Figure 3, i s the f o l l o wing : the sample is placed at the bottom of the c e l l made of optical glass which is f i l l e d with a protein solution of a given concentration. An a i r bubble i s formed in the solution by means of a c a p i l l a r y tube. The pressure of the a i r bubble i s maintained constant using a mercury pump adjusted with a manometer. The d i s tance, h, between the c a p i l l a r y tube and the sample may be adjusted so as to obtain a thin film of the solution between the a i r and the sample. The film thickness, h, varies between 20-150 nm, while i t s diameter i s about 300 m. The c e l l i s fixed on the table of a metallographic microscope and the kinetics of the f a i l u r e or of the formation of the l i q u i d film i s observed d i r e c t l y or photographed by means of a movie camera. Two main parameters are studied : (1) the break-up time which i s defined as the time which elapses from the moment when the radius of the thin film formed becomes constant (about 100 /cm) and the appearance of the f i r s t hole : and (2) the kinetics of dry spot



28.

BASZKIN ET AL.

Reversible-irreversible Protein Adsorption

453

Figure 1. Schematic representation of adsorption measuring appa ratus. (1) gas flow counter ; (2) f l o a t i n g polymer film ; (3) teflon window ; (4) c i r c u l a r glass container ; (5) support.

Benzoyl peroxyde 90°C

POLYETHYLENE

Maleic anhydride Acetic anhydride

POLYMALEIC ANHYDRIDE

POLYETHYLENE

H2O

POLYETHYLENE POLYMALEIC ANHYDRIDE

PC I5

POLYETHYLENE POLYMALEIC ACID

POLYETHYLENE POLYACYL CHLORIDE

100°C

35°C 24h CCI 4

Ν,Ν-Dimethyltrimethylenediamine

POLYETHYLENE POLYMALEIC ACID

POLYETHYLENE POLYACYL CHLORIDE

POLYETHYLENE TERTIARY POLYAMINE

POLYETHYLENE QUATERNIZED POLYAMINE

POLYETHYLENE TERTIARY POLYAMINE

Figure 2. Outline of the surface reactions on poly(maleic anhy dride) grafted polyethylene. wetting film

polymer

Figure 3. Schematic drawing i l l u s t r a t i n g formation of film on a s o l i d substrate.

a


thin


454


formation. This l a t t e r parameter i s characterized as the variation of the mean diameter of a hole formed in the l i q u i d film with time. An example of the dry spot formation of mucin films on a s i l i cone contact lens is i l l u s t r a t e d in Figure 4. The s p e c i f i c i t y of the method r e l i e s on i t s dynamic character. Spectacular results may be obtained with i t s use in rrany situations were the s t a t i c w e t t a b i l i t y measurements are net sensitive enough. In p a r t i c u l a r , the method allows the detection of even minor surface modifications or changes in solution parameters. These alterations are evidenced by the s t a b i l i t y and thickness change of the wetting fi1ms. Various contact lenses were characterized by means of the wetting thin film technique and the results of these investigations are described in (6, 7).

Mucin and Collagen at

Interfaces

When biomaterials come into contact with various biological fluids (blood, s a l i v a , tears) protein adsorption at the s o l i d - l i q u i d i n t e r face is the f i r s t phenomenon which occurs. This primary adsorption process then exerts a profound influence over subsequent events and mi y give r i s e to such well recognized and undesired processes as thromtus formation, formation of dental plaque or dry spot formation in the case of contact lenses. Although the phenomenon of protein adsorption at the l i q u i d / a i r and s o l i d / l i q u i d interfaces has been the subject of a large number of investigations during the past several years, the answer to the key questions : what is the behavior of proteins at interfaces and why do proteins behave as they do at interfaces, remains unclear and further research effort has to be directed toward understanding of the mechanism of protein adsorption. In p a r t i c u l a r there i s s t i l l a lack of d i r e c t experimental evidence on the organisation of various adsorbed protein layers and on t h e i r composition when protein adsorption takes place from multicomponent mixtures. In our laboratory, two techniques have been extensively used for studying protein behavior at various interfaces. The f i r s t technique consists of in s i t u measurement of protein adsorption with 14c labeled proteins ; the second technique based on multiple-beam interferons try measures surface forces between two mica sheets with adsorbed proteins (Tabor-Israelachvi1i technique). While the in s i t u measurements enable quantitation of protein adsorption, forcedistance measurements provide d i r e c t experimental data on the exten sion of adsorbed protein layers towards the solution and on t h e i r conformation. Reported below are the adsorption and surface force experiments with two proteins of different type (mucin and collagen). Each of these proteins was isolated in the laboratory from animal organs.



Figure lens.

4. Example of the k i n e t i c s of mucin f i l m Rate of camera m o t i o n 4 frames/s.

rupture

on a s i l i c o n e


contact

LA

4^

o

».

g^

3

I i

> Γ"

Τ*

Ν

C/î

>

DO

456


Mucin was extracted from bovine submaxillary glands and collagen was isolated from rat t a i l tendons. The experimental protocols d e s c r i bing e x t r a c t i o n , i s o l a t i o n and the C radiolabeling procedures of these proteins were reported in Refs.(8, 11). Mucin. Bovine submaxillary mucin (BSM) belongs to the class of glycoproteins. It i s a large f l e x i b l e macromolecule believed to exist in a bottle-brush form. It has a molecular weight of about 4 χ 10 and consists of a long polypeptide core with numerous disaccharide and oligosaccharide side chains. The oligosaccharides, mainly sialyl-N-acetylglucosamine, are linked to the peptide through glyco side bonds between N-acetyl-glucosamine and the hydroxyl group of serine or threonine. The length of the molecule i s about 800 nm and i t s radius of gyration measured by l i g h t scattering i s 140 nm. The main function of mucin from secretions of submaxillary glands, along with similar mucoproteins found in the r e s p i r a t o r y , gastrointestinal, reproductive tracts and also in the tear l i q u i d , i s to lubricate e p i t h e l i a l c e l l s and protect them from the external environment. The role of the mucous glycoproteins as a macromolecular surfactant i s therefore of great importance in the science and technology of biomaterials. Such different biosurfaces as dentures, contact lenses or intrauterine contraceptive devices, in spite of different functions, have one common feature, namely that a l l are placed on a mucosal surface. 1 4


6

Collagen. Different types of collagens have one common feature : t h e i r molecule i s composed of three continuous h e l i c a l polypeptide chains wound together over most of t h e i r length. The t r i p l e helix of this glycoprotein i s s t a b i l i z e d by intermolecular hydrogen bonds. The soluble collagen used in our experiments i s Type I collagen. Its molecular weight i s 300,000 and i t s molecule can be regarded as a r i g i d rod, about 300 nm long and 0.15 nm in diameter. Collagen molecules undergo self-assembly by l a t e r a l associa tions into f i b r i l s and fibers and are able, therefore, along with other biological functions, to ensure the mechanical support of the connective t i s s u e . Collagen also plays an important role in many bioadhesion processes. Collagen molecules bound to implant materials enhance adhesion of epidermal c e l l s to the surfaces of biomaterials and prevent implant f a i l u r e . In Situ Adsorption/Desorption Measurements. The techniques to study adsorption/desorption of proteins at interfaces are s i m i l a r to those i n i t i a l l y developed for quantification of functional groups at polymer surfaces and described above. To measure adsorption of proteins at the s o l u t i o n / a i r interface the apparatus shown in Figure 5A i s used. A c i r c u l a r glass container i s f i l l e d with a C protein and covered with a teflon window. The gas flow counter measures the r a d i o a c t i v i t y and continuously displays i t on a recorder as a function of time. To allow for the r a d i o a c t i v i t y originating from the solution (Afc) close to the 1 4



Β

F i g u r e 5. Adsorption measuring devices. (A) For a d s o r p t i o n a t s o l u t i o n - a i r i n t e r f a c e ; ( B ) f o r a d s o r p t i o n on p o l y m e r o r m i c a s u r f a c e s . ( 1 ) , (3) Supports e n s u r i n g r e p r o d u c i b i l i t y o f g e o m e t r i c a l c o n d i t i o n s and t i g h t n e s s ; ( 2 ) g l a s s c o n t a i n e r ; (4) polymer or mica f i l m .

Α


458


solution/air interface, a separately run experiment i s performed. Instead of a l^C protein the glass container is f i l l e d with a solu tion of a non-adsorbing substance, containing the same radioactive element K^CNS , and i t s r a d i o a c t i v i t y i s measured in the same geometrical conditions as those in the experiments with proteins. The r a d i o a c t i v i t y can then be calculated from cp Ab

=

V


c'p' where c and c' are the concentrations of the protein solution and of the non-adsorbing solution respectively, ρ and p'are t h e i r respec tive specific a c t i v i t i e s , and A ' i s the r a d i o a c t i v i t y of the non adsorbing solution. Subtraction of A5 from the total measured r a d i o a c t i v i t y (At) gives the r a d i o a c t i v i t y of protein molecules adsorbed in excess at the interface (A j) for each of the protein concentrations studied (Figure 6). At low protein solution concentrations (< 0.005 mg/ml), Ab i s very small and At represents almost e n t i r e l y the adsorbed quantity (A j) ; for the adsorption at higher protein concentration (> 0.5 mg/ml), A5 represents about 50% of the adsorbed value. To measure protein adsorption on polymers two techniques are used. The f i r s t technique with the polymer films f l o a t i n g on the protein solution surface i s i l l u s t r a t e d in Figure 1. The second technique, which can be used with either a polymer or a mica thin film, i s shown in Figure 5b. Both techniques give identical results indicating that with the use of the measuring device as i l l u s t r a t e d in Figure 5b and under the conditions in which our adsorption expe riments were performed, neither mucin nor collagen p r e c i p i t a t i o n was observed. To measure the A value, the same procedure as above, with !4CNS- ions, i s used. In a d d i t i o n , the At value has to allow for the absorption of radiation by the sample. The magnitude of the correction for each sample i s determined with the help of a l^C methyl methacrylate s o l i d source placed above the polymer or mica window and in the same geometrical conditions as for the adsorption measurements. The necessary checks have been done to ascertain that protein labeling with C did not cause any change of t h e i r surface activities and different protein adsorbability on the surfaces studied (8, Π, 1_3). For both adsorption techniques, the radio a c t i v i t y measured, in counts/minute, i s converted to the amount in milligrams of protein per square meter. This i s dene by depositing, drying on mica surfaces known amounts of labeled protein which, when counted, yielded a c a l i b r a t i o n factor per unit amount of protein. Knowing the conversion f a c t o r , and the area of the sample exposed to protein s o l u t i o n , the amount of protein adsorbed in mg/m is obtained. D

ac

ac

D

1 4

2


28.

BASZKIN ET AL.

Reversible-Irreversible Protein Adsorption

The in s i t u desorption experiments on polymer or mica surfaces are performed using the apparatus shown in Figure 5b. The protein solution is pumped out of the c e l l and simultaneously replaced by water or a buffer solution. Multiple replacement operations lead to a negligible protein concentration in the c e l l . The A value being zero, the measured r a d i o a c t i v i t y after allowing for the absorption of radiation by a s o l i d sample is d i r e c t l y converted into the sur face concentration of the i r r e v e r s i b l y adsorbed protein. The loosely bound protein (reversibly adsorbed protein) as a fraction of the total adsorbed l a y e r , is thus obtained by subtraction of the irre versible adsorption from the total adsorption value. Mucin and collagen adsorption/desorption data on different interfaces have been reported previously. They include adsorption studies at the s o l u t i o n / a i r interface and adsorption/desorption data on hydrophobic and surface modified hydrophobic polymers (8-12). Adsorption of mucin was also studied on s i l i c o n e and poly(vinyl pyrrolidone)-grafted contact lenses (1Ό) and on mica surfaces (13, 14). It was shown that increasing solution concentration of these proteins tended to increase the i n i t i a l rate of adsorption as well as the amount adsorbed at l a t e r times. It was also shown that modi f i c a t i o n of hydrophobic polymers by different treatments capable of generating functional groups at t h e i r surfaces (oxidation, super f i c i a l grafting of polar monomers) enhances adsorption of mucin and of collagen. The adsorbed amounts of these proteins increase with increasing surface density of functional s i t e s on such polymers (8). However, in spite of these s i m i l a r i t i e s , the adsorbed amounts and the structure of the adsorbed mucin and collagen layers on the surfaces studied are e n t i r e l y d i f f e r e n t . The behavior of these proteins i s analyzed here on the hydrophobic polyethylene surface (water contact angle Θ Η 2 Ο 9 5 ° ) , on the surface modified polyethylenes : oxidized polyethylene (θμ Q = 7 4 ° ) and poly(maleic acid) grafted polyethylene ( Η 0 ) hydrophilic mica sur face ( θ Q = o ° ) . Acidic pH = 2.75 (for collagen) and s l i g h t l y alkaline pH = 7.2 (for mucin) were chosen in order to minimize the association of these proteins in solution and to make possible the analysis of t h e i r adsorbabilities in comparable conditions. Figure 7 shows typical adsorption kinetics of mucin and c o l l a gen on polyethylene and mica surfaces followed by displacement of the protein solution by a buffer solution. It may be noted that collagen adsorption on polyethylene i s five times higher than that of mucin and that the i n i t i a l adsorption rates are protein d i f f u s i v i t y dependent. The diffusion coefficients for mucin and collagen were reported by us previously. At a protein solution concentration of 0.05 mg/ml they are respectively equal to 1.5 χ 10"^ mZ.sec- and 1.0 χ 1 0 ~ mZ.sec" (11, 13). Desorption experiments, given also in Figure 7, c l e a r l y i n d i cate that the adsorbed mucin and collagen layers d i f f e r substan t i a l l y in t h e i r nature. While on polyethylene mucin i s entirely D


459

=

θ

=

7 4 0

a n ? d

o

n

t

n

e

2

Η 2

1

12

1



460


concentration

Figure 6. Schematic representation of adsorption measuring technique. At = total r a d i o a c t i v i t y measured ; A = r a d i o a c t i v i t y measured at the s o l u t i o n / a i r or s o l i d / s o l u t i o n interface with a non-adsorbing substance ; A j = r a d i o a c t i v i t y corresponding to the amount adsorbed at the interface. D

ac

Figure 7. Adsorption kinetics for mucin (Mu) and collagen (Col) on polyethylene (PE) and mica (Mi). Protein solution concen tration 0.05 mg/ml. Temp. 20°C. Collagen adsorption from 0.2 M NaCl - 0.1 M CH3COOH buffer at pH = 2.75. Mucin adsorption from ΙΟ" M phosphate buffer with 0.15 M NaCl at pH = 7.2. Dotted lines and arrows indicate desorption and the amount after desorption. 3



28.


BASZKIN ET AL.

461

i r r e v e r s i b l y adsorbed, one fourth of the adsorbed collagen can be eluded from t h i s surface. A s i g n i f i c a n t l y higher adsorption of mucin in comparison to collagen i s observed on mica surfaces. The surface density/solution concentration isotherms, not shown in this paper, r e f l e c t also the differences in the behavior of mucin and collagen upon t h e i r adsorption at s o l i d interfaces. While the collagen isotherms on polyethylene and surface-grafted polyethylene show a plateau of adsorption at solution concentrations higher than 0.05 mg/ml, no plateau values for mucin adsorption are observed on polyethylene and surface oxidized polyethylene. The desorption-adsorption relationship for mucin and collagen on mica i s represented in Figure 8. This relationship for mucin i s l i n e a r and c l e a r l y indicates that half of the adsorbed quantities can be desorbed. On the contrary, for collagen, this relationship shows a threshold value (1.1 mg/m ) corresponding to the maximum i r r e v e r s i b l y adsorbed value. Above this value a l l adsorbed collagen can be desorbed (the slope of the straight line i s one). Finally, Figure 9 presents the desorption-adsorption r e l a t i o n ship for mucin and collagen on polyethylene and surface-modified polyethylene. The adsorption of mucin on untreated polyethylene i s t y p i c a l l y i r r e v e r s i b l e and the maximum adsorbed quantity i s equal to 2.2 mg/m . In contrast, low but continuous desorption of mucin with increasing adsorbed concentrations i s observed on surface oxidized polyethylene. Collagen desorption-adsorption l i n e a r functions on polyethylenes exhibit c l e a r l y defined t r a n s i t i o n points. A l l collagen which adsorbs in addition to the i r r e v e r s i b l y adsorbed layers (represented for polyethylene and grafted polyethylene by t h e i r abscissa values 3.0 and 3.7 mg/m ) can be e n t i r e l y desorbed. Above the t r a n s i t i o n points the desorption-adsorption slopes are equal to one. The contrasting features of mucin and collagen adsorption are summarized in Table I. To explain the mechanism of collagen adsorption on polyethylene surface, we have formulated a simple hypothesis according to which the reversible adsorption of collagen molecules i s realized by t h e i r attachment to the i r r e v e r s i b l y adsorbed layer. The intramolecular collagen-collagen bonds, much weaker than protein-polymer bonds, break during the washing out procedure causing the release of a fraction of the adsorbed protein. From a comparison of the dimen sions of collagen molecules and the i r r e v e r s i b l y adsorbed quan tities, it i s most unlikely that these molecules are adsorbed in t h e i r flattened "side-on" o r i e n t a t i o n . The most r e a l i s t i c picture of the i r r e v e r s i b l e adsorption would imply binding of r i g i d r o d - l i k e collagen molecules in a t i l t e d "end-on" p o s i t i o n . We believe that they are bound to polyethylene e s s e n t i a l l y via hydrophobic i n t e r actions. The angle which they form with the surface may vary between 0° and 90° and would to a large extent depend upon the type and number of protein-surface bonds. Thus for the poly(maleic acid) 2

2

2



462


•

ADSORPTION

( mg/m ) 2

Figure 8 . Desorption-adsorption relationships on mica surfaces, (t) mucin ; ( â ) collagen. Adsorption time 20 hrs ; temp. 20°C. Adsorption conditions as indicated in Figure 7 .

0

1

2

3 ADSORPTION

5

I

6

(mg/m ) 7

Figure 9 . Desorption-adsorption relationships (Δ) polyethylenecollagen ; (A) poly(maleic acid) grafted polyethylene-collagen ; (o) polyethylene-mucin ; (t) surface oxidized polyethylene-mucin. Adsorption time 20 hrs ; temp. 20°C. Adsorption conditions as indicated in Figure 7 .


28.

BASZKIN E T A L .


463

Table I. Features of mucin and collagen adsorption on polyethylene, surface treated polyethylene and mica surfaces

Protein Surface


Mucin

Collagen

Polyethylene

low e n t i r e l y irreversible adsorption

high i r r e v e r s i b l e adsorption followed by complete rever s i b l e adsorption

Surface treated polyethylene

continuous, very high i r r e v e r s i b l e adsorption (90% of total adsorption)

high i r r e v e r s i b l e adsorption followed by complete rever s i b l e adsorption

Mica

continuous equi valent i r r e v e r s i b l e and rever s i b l e adsorption

low i r r e v e r s i b l e adsorption followed by complete rever s i b l e adsorption

grafted polyethylene which bears functional sites on i t s surface, in addition to hydrophobic interactions via d i r e c t hydrogen bond forma tion may occur. The presence of these interactions would increase the amounts of i r r e v e r s i b l y adsorbed collagen molecules and "tighten" the interactions with the polymer. This seems to be the case, since the i r r e v e r s i b l e adsorption i s higher (3.7 mg/m instead of 3.0 mg/m for the polyethylene) and less collagen desorbs (the slope of the lower part of the collagen curve in Figure 9 i s 0.05 while that of polyethylene i s 0.2). The increase of i r r e v e r s i b l e adsorption by 0.7 mg/m between the surface grafted and untreated polyethylene may be attributed to the appearance of additional bonds, most probably hydrogen bonds between the polymer surface and adsorbed collagen. The amount of collagen adsorbed i r r e v e r s i b l y to mica i s about one third of that measured on polyethylenes (1.1 mg/m ). Since mica i s an e n t i r e l y hydrophilic surface i t may be considered that hydro gen bonds between the hydrated ions present on i t s surface and collagen chains would account for the major part of the binding mechanism. Otherwise this quantity (1.1 mg/m ) i s comparable with the adsorption increase (0.7 mg/m ) due to the introduction of hydrophilic sites on a polyethylene surface. Mucin may be regarded as a f l e x i b l e macromolecule. In contrast to collagen i t can assume a much greater number of different surface 2

2

2

2

2

2



464


induced conformations. Its a f f i n i t y for a p a r t i c u l a r surface with respect to others, would involve formation of a greater number of surface bonds or a greater mean energy per bond or both. The degree of r e v e r s i b i l i t y of i t s adsorption would depend upon the type of configuration which a p a r t i c u l a r surface may induce. The untreated, hydrophobic polyethylene would be a representative example of a surface inducing mucin configurations leading to a strong and i r r e versible adsorption. Increasing the number of polar anchoring s i t e s on polyethylene y i e l d s high i r r e v e r s i b l e levels of adsorption. The occurrence of configuration different from that on polyethylene would most probably involve formation of multiple hydrophobic and short range hydrogen bonding interactions between mucin and the surface. When the short range hydrogen bond interactions increase, as in the case of mica, the r e v e r s i b i l i t y of mucin adsorption increases. Surface Force Measurements. This technique enables the measurement of the force (10 mN accuracy) versus distance (0.1-0.2 nm accuracy) between two curved mica surfaces. The forces between two s o l i d surfaces across an aqueous solution are highly sensitive to the structure of the s o l i d / l i q u i d interfaces. When such surfaces are covered with adsorbed protein layers, then, the analysis of the force/distance p r o f i l e s may reveal the formation of protein bridges between the two surfaces, the occurrence of s t e r i c i n t e r a c t i o n s , or any possible protein conformation change. Figure 10 shows force/distance p r o f i l e s between mica surfaces bearing collagen or mucin adsorbed layers. The experiments were performed by f i r s t measuring forces between bare mica surfaces across a pure e l e c t r o l y t e solution and then injecting protein solu tion into the measuring c e l l . After allowing 3 hours for the protein to adsorb, the forces were measured against distance. The experi mental conditions were chosen in order to ensure the same protein (mucin or collagen) surface concentration at the mica/aqueous solu tion interface (2 mg/m ) and to allow therefore the comparison of protein effects on the surface forces. The collagen force/distance p r o f i l e s c l e a r l y indicate that on approaching the mica surfaces, no forces are present down to the 320 nm separation distance. On decreasing the distance, repulsive forces increase smoothly. An e n t i r e l y different type of behavior i s exhibited by adsorbed mucin layers. Forces are observed beginning at 450 nm (attractive interactions, inset in Figure 10). These are followed by a steep repulsion beginning at 100 nm. The a t t r a c t i v e interactions with mucin have been explained by bridging mechanisms (14, 1 5 ) . The repulsive regime can be interpreted in terms of s t e r i c interactions due to the confinement of adsorbed mucin molecules between two walls (reduced number of configurations). Other experiments, not reported in this paper, show that the solution ionic strength may profoundly 2



3

60

90

120

130

D (nm)

180

2

210

240

270

300

Figure 10. Forces (logarithmic scale) between mica surfaces covered with adsorbed collagen (1) and mucin (2). Inset: linear representation of forces. The forces are plotted as F / R where R is the radius of curvature of surfaces. Surface concentration of both proteins after adsorp tion in the force-measuring cell for 3 h was 2.0 mg/m . Mucin solution concentration: 0.05 mg/ml + 0.15 M NaCl; pH = 5.5. Collagen solution concentration: 0.05 mg/ml in 0.1 M NaCl + 0.05 M CH COOH; pH = 2.7.

30


466



influence mucin configurations. A l s o , bridging interactions are highly dependent on mucin surface density (15). For collagen, repulsive forces start at a distance which is smaller than twice the length of the collagen stiff rod. The force/distance p r o f i l e s for collagen confirm therefore the inter pretation of the adsorption-desorption data. Collagen molecules are attached to the mica surface in a t i l t e d end-on o r i e n t a t i o n . This orientation varies as a function of compression of the surfaces. The repulsive forces result from the head-to-head contacts of collagen rods or from the i n t e r p é n é t r a t i o n of the layers.

Conclusions Various surface chemistry techniques described in this paper and used in our laboratory can be used to characterize protein adsorb a b i l i t y at surfaces of different types. The in s i t u adsorption/desorption technique can be used to d i s tinguish between i r r e v e r s i b l y and reversibly adsorbed protein. The desorption/adsorption ratio depends on the nature of protein and surface. Further development of this technique would involve the design of a new type of measuring c e l l for adsorption/desorption measurements in flow conditions and extension of protein adsorption experiments to competitive protein adsorption measurement. Studies of albumin and fibrinogen competitive adsorption versus collagen (12) have recently been i n i t i a t e d . The surface force measurements between two adsorbed protein layers on mica are now being investigated on polymer surfaces. The coverage of mica surfaces by polymers may be achieved by successive dipping of these supports through a polymer monolayer (LangmuirBlodgett technique) or by a d i r e c t polymer plasma polymerization on mica sheets. Also studies of interactions between two surfaces each bearing a different adsorbed protein are anticipated. Studies on polymer monolayers spread at the air-water interface are now in progress in our laboratory. Biocompatible and biodegra dable polymers used as nanoparticles carrying b i o l o g i c a l l y active substances are characterized using the surface balance, surface potential and protein adsorption/desorption measurements. The combined data of a l l these measurements provide information on drug and protein penetration/delivery with these polymers.

Literature Cited 1. 2.

Baszkin, Α.; Ter-Minassian-Saraga, L. J. Polymer Sci. 1971, 34, 243-252. Baszkin, Α.; Deyme, M.; Nishino, M.; Ter-Minassian-Saraga, L. Prog. Colloid & Polymer Sci. 1976, 61, 97-108.


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Baszkin, Α.; Nishino, M.; Ter-Minassian-Saraga, L. J . Colloid Interface Sci. 1976, 54, 317-328. Leclercq, B.; Sotton, M.; Baszkin, Α.; Ter-Minassian-Saraga, L. Polymer 1977, 18, 675-680. Eriksson, J . C . ; Gölander, C.G.; Baszkin, Α.; Ter-Minassian -Saraga, L. J. Colloid Interface Sci. 1984, 100, 381-392. Baszkin, Α.; Boissonnade, M.M.; Proust, J.E.; Tchaliovska, S.D.; Ter-Minassian-Saraga, L . ; Wajs, G. J. Bioengineering 1978, 2, 527-537. Proust, J.E. Proc. International Tear Film Symposium, Lubbock Texas, 1984, in press. Proust, J.E.; Baszkin, Α.; Boissonnade, M.M. J. Colloid Inter face Sci. 1983, 94, 421-429. Baszkin, Α.; Proust, J.E.; Boissonnade, M.M. In Biomaterials & Biomechanics 1983; Ducheyne, P.; van der Perre, G.; Aubert, A.E., Eds.; Elsevier: Amsterdam, 1984; p 379. Baszkin, Α.; Proust, J.E.; Boissonnade, M.M. Biomaterials 1984, 5, 175-179. Deyme, M.; Baszkin, Α.; Proust, J.E.; Perez, E . ; Boissonnade, M.M. J. Biomed. Mater. Res. 1986, 20, 951-962. Deyme, M.; Baszkin, Α.; Proust, J.E.; Perez, E . ; Albrecht, G.; Boissonnade, M.M. J. Biomed. Mater. Res. in press. Perez, E . ; Proust, J . E . ; Baszkin, Α.; Boissonnade, M.M. Colloids & Surfaces 1984, 9, 297-306. Proust, J . E . ; Baszkin, Α.; Perez, E . ; Boissonnade, M.M. Colloids & Surfaces 1984, 10, 43-52. Perez, E . ; Proust, J.E. J. Colloid Interface Sci. in press.

RECEIVED January 28, 1987


Reversible-Irreversible Protein Adsorption and Polymer Surface

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