NBSIR 76-1017

Interaction of Blood Proteins

with Solid Surfaces

R. R. Stromberg, B. W. Morrissey, L. E. Smith, W. H. Grant, and R. E. Dehl

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

March 5, 1976

Annual Report for Period

November 1, 1974 - October 31, 1975

Prepared for

Biomaterials Program

Division of Blood Diseases and Resources

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement
NOTICE

This report was prepared as an account of work sponsored by

the National Heart and Lung Institute. It Is not to be construed

as a report, opinion, or recommendation of the National Heart

and Lung Institute. It will be available from the National

Technical Information Service, 5285 Port Royal Road, Springfield,

Virginia 22151.

Certain commercial equipment is identified in this report in order

to adequately specify the experimental procedure. In no case does

such identification imply recommendation or endorsement by the

National Bureau of Standards nor does it iin)ly that the equipment

identified is the best available.
NBSIR 76-1017

INTERACTION OF BLOOD PROTEINS

WITH SOLID SURFACES

R. R. Stromberg, B. W. Morrissey, L E. Smith, W. H. Grant, and R. E. Dehl

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

March 5, 1976

Annual Report for Period

November 1, 1974 - Octoer 31, 1975

Prepared for

Biomaterials Program

Division of Blood Diseases and Resources

National Hear and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

T

U.S. DEPARTMENT OF COMMERCE, Elliot L. Richardson, S9eretarY

James A. Baker, III, Under Secretary

Dr. Betsy Ancker-Johnson, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler. Acting Director

TABLE OF CONTENTS

Page

ABSTRACT i

SUMMARY il

INTRODUCTION 1

EXPERIMENTAL 2

Protein Purity 2

Fibrinogen 2

Albumin 2

Y-Globulin 2

6-Lactoglobulin 2

Ellipsometry 2

Radiotracers 3

RESULTS AND DISCUSSION 4

EXAMINATION OF NHLI BIOMATERIAL PROGRAM MATERIALS 4

Polyether urethane (Stanford Research Institute) 4

Ion-Plated carbon (General Atomics Corp.) 7

Poly (vinyl acetate-co-crotonic acid) (Union Carbide) ... 8

Polyacrylamide hydrogels (Franklin Research Institute) . . 8

Glow discharge coatings (Research Triangle Institute) . . 8

FACTORS INDUCING CONFORMATION CHANGES 9

Applied Potential 9

Surface Concentration 10

Adsorption Time 17

Denaturing Media 18

RADIOTRACER RATE STUDIES 21

Measurement 21

Desorption and Exchange 22

Silicone Rubber 27

REFERENCES 29

FIGURES

Page

Figure 1: Adsorbance of Human Fibrinogen on SRI Polyether

Urethane 3-2000-1-E 5

Figure 2: Extension of Adsorbed Hiiman Fibrinogen on SRI

Polyether Ure thane 3-2000-1-E 6

Figure 3: Stability of refractive index components of platinum

in 0.15N NaCJl with applied potentials 11

Figure 4: Instability of apparent optical density of germanium

in 0.15N NaCi, 12

Figure 5: Instability of apparent optical density of germanium

in 0.15N NaCA with applied potentials 13

Figure 6: Extension and adsorbance of bovine yglobulin on

fused silica 16

Figure 7: Effect of urea on adsorbance of human fibrinogen

adsorbed on polyethylene 19

Figure 8: Effect of urea on extension of human fibrinogen

adsorbed on polyethylene 20

Figure 9: Desorption and exchange of fibrinogen adsorbed on

polyethylene with fibrinogen 24

Figure 10: Desorption of human serum albumin from polyethylene

and chrome into buffer 25

Figure 11: Desorption of HSA-^^^I from polyethylene into 8M

buffered urea 26

Figure 12: Rate of Adsroption of HSA-'^-^-'-I on Silicone Rubber ... 28

TABLES

Page

Table I: yGlobulin Adsorbed on Silica 15

Table II: Apparent Adsorbance as a Function of Specific

Activity of HSA--^-^^! Solution 23

INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

ABSTRACT

The overall objective of this investigation is to help characterize

materials iised for implants in the cardiovascular system and to develop

test methods to aid in the selection of the most useful materials. The

focus of the work has been an investigation of the adsorption of blood

proteins on surfaces with special emphasis on changes in conformation

occurring upon adsorption. An important component has been an ellipsometric

examination of materials produced by contractors of the NHLI Biomaterials

Program that were considered to be of potential clinical value. In

contrast to the behavior of serum albumin and prothrombin, conformational

changes in yglobulin and 3-lactoglobulin occurring as a consequence of

changing surface population were previously reported and were confirmed

during this reporting period. An investigation of conformational changes

at vei^r short adsorption time was initiated. Adsorption, desorption and

exchange of adsorbed protein with protein in solution was studied. The

results indicate strong hydrophobic bonding between the protein and the

synthetic polymer surface.

i
INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

SUMMARY

The goal of this investigation has been to further characterize the

synthetic materials used for Implants in the cardiovascular system and to

develop test methods to aid in the selection of the most useful materials.

The focus of the work has been directed at changes that occur when certain

blood proteins interact with a foreign material.

The proteins studied have been the same as those used in earlier phases

of this investigation; fibrinogen, albumin, yglobulin and 6-lactoglobulln.

They have been selected because of their importance to the clotting process,

abundance in the blood plasma, or as a model to better understand a more

complex situation. Repurification or special labeling have been carried out

as necessary.

An important component of this effort has been an investigation in our

laboratory of materials produced by contractors of the NHLI Biomaterials

Program and considered by the Project Officer to be of potential clinical

value. The objective is to attenqjt to find a direct correlation between the

adsorption behavior of blood proteins and thrombogenicity of a material. Such

a correlation could, of course, be developed into a useful test method.

Materials produced by five contractors were examined by means of ellipsometry.

Results for the adsorption of fibrinogen on a polyether urethane prepared by

Stanford Research Institute were quite promising in that the measured fibrinogen

adsorbances and extensions were in agreement with those measured on other

materials of similar surface energy.

The in situ quantitative ellipsometric investigation of the relationship

between surface potential and protein-surface interaction was extended to

germanium. This is a material of special interest because of its applicability

to internal reflection spectroscopy in the infrared regions. It was found

that the germanium is unstable compared to platinum, in the presence of NaCi.

both with and without applied potential. This appears to cast doubt regarding

its use in spectroscopic studies under these conditions.

Work on the effect of surface concentration on the conformation of

adsorbed proteins included some additional measurements of the extension and

number of carbonyl contacts for y-globulin and 3-lactoglobulin. The conclusions

were essentially the same as those previously reported, i.e. conformational

changes in these two proteins apparently occur as a result of increased surface

population, in contrast to no change for serum albumin or prothrombin.

Measurements of changes in conformation of the adsorbing molecule at very

short adsorption times (~28) has been initiated with an automatic ellipsometer.

Techniques are being developed and some preliminary results have been obtained.

The adsorption and desorption of proteins on polyethylene has been

investigated ellipsometrically and by use of radiotracers. In addition, a serious examination of artifacts resulting from the labeling of proteins with

radioactive iodine has been made. The polyethylene is quite interesting in

that no desorption into buffer or exchange with protein in solution could be

measured from this low energy surface, implying strong hydrophobic bonding

between the protein and the synthetic polymer.

ii
INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

INTRODUCTION

The adsorption of plasma proteins Is the first In a complex series of

events that occurs when a synthetic material Is placed In the cardiovascular

system. It Is known that this protein adsorption can alter the course of

subsequent reactions at the Implant surface but the mechanism by which this

occurs Is not well understood. If the detailed behavior of proteins adsorbed

at the solid surface were known and capable of control, the fate of artificial

materials placed in body could be predicted with greater certainty and more

successful materials could, perhaps, be designed. The investigation is

directed toward the identification of factors capable of affecting protein

adsorption and the development of a description of the changes that occur

in the protein upon Interaction with a surface.

This approach to the problem of design criteria and test methods for

blomaterlals has been developed more extensively in previous reports and

experimental techniques have also been described in detail. This report

discusses our studies of the effect of applied surface potential, population

of protein on the adsorbing surface, and denaturing media on the conformation

of the adsorbed protein. We have continued our Investigation into the use

of radiotracers to measure the adsorption behavior of proteins and discuss

a number of difficulties with their application. Several materials prepared

especially for the Blomaterlals Program have been examined.

1
EXPERIMENTAL

Protein Purity

Fibrinogen

Bovine and human fibrinogen were repurified in our laboratories by the

Laki (1) and Batt (2) methods, respectively, to yield a product that is greater

than 96.7% clottable. (The theoretical maximum is 97%).

Albijmin

The human and bovine serum albumin were obtained as four times recrystallized material with an electrophoretic purity of 100% as specified by the

supplier.

y- Globulin

This material is recrygtallized and has purity greater than 95% as reported by the supplier. ,

3-Lactoglobulin

Bovine g-lactoglobulin, three times recrystallized, was used as received.

Ellipsometry

The general experimental technique of ellipsometry and methods of data

analysis have been described in previous reports (3,4). The only changes in

these procedures were those necessitated by investigations designed to measure

conformation changes at very short adsorption times. The optical constants

of the surface are obtained in the usual manner, with measurements made under

buffers. In order to make measurements in the initial stages of the adsorption

process, it is necessary to expose the "bare" surface to the protein solution

in as short a time period as possible. At the same time the introduction of

artifacts such as denatured matetial must also be avoided. For the preliminary

adsorption experiments with the automatic ellipsometer described in this

report, a straightforward method of introducing the protein solutions was

adopted.

After determining the optical constants of the substrate under buffer

solution, the solution was withdrawn from the ellipsometer cell as completely

as possible with a syringe, leaving a thin film of solution on top of the

(hydrophilic) substrate. A premeasured quantity of filtered protein solution

was then quickly poured into the cell. Since the solution is introduced in a

highly turbulent manner, it should mix rapidly with any residual buffer

solution in the cell. This technique allowed measurement of the ellipsometric

parameters in less than 2 seconds after the solution pouring was begun. The

resultant changes in the parameters were smooth and monotonic throughout the

total observation times, which ranged up to 3 hours.

2
Radiotracers

Human serum albumin (HSA) labeled with radioactive iodine was secured

as follows

131 125

1. HSA- I and HSA- I, injection quality diagnostic grade from

Squibb Pharmaceutical Co., as a 1% solution in 1.6% NaH2P0^•H20 and 0.16%

Na2HP04 buffer.

125

2. HSA- I was labeled in our laboratory using the ICl method of

McFarlane (5).

The solvent for all experiments was O.IM phosphate buffer with a pH

of 7.4. Procedures for cleaning the substrates and determining the amount

of labeled material adsorbed or remaining after desorption were the same

as previously descirbed (3).

3
RESULTS AND DISCUSSION

EXAMINATION OF NHLI BIOMATERIAL PROGRAM MATERIALS

An important part of this investigation is the direct application of the

surface techniques used in our laboratory to materials produced by contractors

to the NHLI Biomaterials Program. This involves an examination of some of the

surface properties of a material and, when feasible, an investigation of the

interaction of blood proteins with it. Because of its importance in the

clotting sequence and because of the body of information already available,

fibrinogen was selected for inter- comparison purposes. The objective was to

attempt to find some direct correlation between the adsorption behavior of

blood proteins and the thrombogenic behavior of clinically promising materials.

In order to demonstrate such a relationship, it is necessary to examine material

with poor as well as those with desirable characteristics.

The results obtained on materials examined to date are described briefly

below. They were, in general, used "as received". It should be recognized

that for most of the materials this information given here does not represent

a completed investigation. In some cases our studies are still in progress.

In other cases, we are attempting to adapt our techniques to a "new" type of

surface or the contractor, who is developing a new material, still is in the

process or making significant changes to his product. The materials to be

investigated are selected collaboratively with the Project Officer.

Polyether Urethane. 3-2000- 1-E (Stanford Research Institute)

Samples of polyether urethane, 3-2000- 1-E were obtained from Dr. Brauman

of the Stanford Research Institute (SRI) for ellipsometric studies of protein

adsorption. These samples invariably turned white and opaque after a few

hours under water or buffer. Not withstanding this change in appearance, the

refractive index of the SRI polyurethane did not change significantly and the

material was stable for several days. In addition, good agreement was found

with the refractive index (1.48) determined in air on the same material.

The molecular extension and adsorbance of fibrinogen adsorbed on SRI

material 3-2000- 1-E were determined at pH 7.4 in situ . As shown in Figures 1

and 2, there are no changes in adsorbance and only a slight increase in

extension of purified human fibrinogen over the time interval of 5 to 150

minutes following the introduction of the protein solution at a concentration

of 3.0 mg/ml. The adsorbance of approximately 4 mg/m^ and an extension of

50 nm may be compared with the previous study (4) of fibrinogen adsorption

on a variety of materials ranging in surface energy from platinum to polyethylene. In all previous cases, the adsorbance was found to lie between

3.7 and 5.4 mg/m^. The molecular extension varied from 23 nm on the high

energy platinum surface, to 43 nm on carbon and 59 nm on the relatively low

energy polyethylene surface. The extension of fibrinogen on the polyether

urethane therefore lies between carbon and polyethylene, as one would predict

based on its critical surface tension for wetting.

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Ion-Plated Carbon (General Atomic Corp.)

The feasibility of performing quantitative ellipsometric studies of

protein deposition on thinly coated substrates depends, among other things,

on accurate knowledge of the refractive index of the coating. As discussed

in our previous annual report (4) , ion-beam deposited carbon films (Whittaker

Corp.) have a significant optical adsorbance at 632.8 nm, and for such films

it is necessary to determine both the real (n) and imaginary (k) parts of

the refractive index as well as the film thickness.

From the two measured ellipsometric parameters, it is possible to

determine only two of the three unknown properties of the carbon film. A

more involved strategy must be employed to estimate the third quantity.

One of the most sensitive of such methods is to measure the identical films

on two different substrates which doubles the number of independent measurements and maintains the same number of unknowns.

During this reporting period we have measured by ellipsometry the

optical constants of various samples of "ion-plated" carbon (manufacturer's

notation) on stainless steel and glass, obtained from Mr. Haubold of General

Atomic Corp. Two of the steel samples were reported to have 200 nm and

400 nm carbon coatings; two other samples were reported to have essentially

the same (but unknown) thickness of carbon deposited on stainless steel

and on glass.

We assumed that the thickness (d) values given by the supplier for two

of the stainless steel samples were approximately correct. For the "400 nm"

film, n and k were found to be constant for values of d in excess of about

350 nm. Thus, for a carbon film thicker than 350 nm, the light which enters

the film is all absorbed, and one obtains the same values for the optical

constants of the carbon film, whether or not the substrate optical constants

are included in the calculation. For this sample, n was found to be 1.75,

and k, 0.40. The optical parameters of the "200 nm" films were not substrateindependent, but the calculated values of n (1.68) and k (0.38) assuming

the film to be exactly 200 nm thick, were in fiirly close agreement with

those of the thicker film. No substantial improvement in the agreement

was found when the carbon film thickness was assumed to be somewhat greater

or less than 200 nm.

For the carbon films of unknown thickness on steel, the ellipsometric

parameters were considerably different from those of either ther 200 nm or

400 nm film, indicating that the film thickness must be less than 350 nm

and not very close to 200 nm. Calculations indicated that a number of

film thicknesses in the range 200-350 nm were consistent with the measured

ellipsometric parameters. (No solutions were found for d < 200 nm) . The

n and k values found for this range of thickness varied little; n was about

0.2 less than that found for the very thick film while k was about the same

as the thick film value. Thus, we find good agreement among the values of

k for the 3 coated steel samples, and fair agreement among the values of n.

Since the carbon on the glass substracte was also presumed to be

200-350 nm thick, we should have found n and k to be the same as for the

coated steel when the calculations were performed for this range of d values.

However, while n and k were fotind to be substrate-independent for d > 210 nm.
the calculated n (2.08) and k (0.49) were both significantly higher than

the values found for the very thick carbon coating on steel. Apparently,

the optical constants of carbon films are not constant over a range of

different sample thicknesses and different substrates. For an arbitrary

carbon-coated substrate, one would have to know the thickness of the film

by some independent means iii order to measure the optical constants of the

carbon and thus obtain the necessary information for quantitative measurements

of protein deposition. For very thick carbon films, however, the optical

constants can easily be determined. Therefore, such samples are suitable

for protein adsorption studies.

Poly (vinyl acetate-co-crotonic acid) (Union Carbide)

Polypropylene plaques coated with a polyelectrolyte hydrogel, poly (vinyl

acetate-co-2%-crotonic acid) 60% sodium ionomer were obtained from Dr. Lundell.

They were found by ellipsometry to give a diffuse reflection from the surface,

making accurate measurements possible. This problem could be due to surface

roughness of the substrate and/or non-uniform hydrogel coating. To eliminate

the first possibility. Dr. Lundell attempted to graft a hydrogel coating

onto a sheet of highly reflective polyethylene supplied by us. The graft

was not successful, however, apparently due to the lack of tertiary hydrogens

in polyethylene.

Polyacrylamide Hydrogels (Franklin Research Institute)

Samples of vapor active monomer polymerized acrylamide on SRI Urethane

(3-2000- 1-E) were obtained from Dr. Kronick. The first set of samples

received showed a tendency of the grafted film to "flake off" the surface.

An ellipsometric examination of a subsequent sample under buffer showed

almost no difference between the nominally "coated" and "uncoated" sides

of the substrate. Several explanations must be considered to explain the

inability to detect a film. First, a film would not be optically detected

if its refractive index were essentially identical to that of the substrate.

Second, if the film has coated both sides of the substrate in its preparation,

the sides would be indistinguishable from each other with no "base" surface

available to obtain substrate values. Third, as indicated by Dr. Kronick,

it is possible that no film existed, but rather a bulk property alteration

occurred as a result of acrylamide penetration during the grafting process.

Glow Discharge Coatings (Research Triangle Institute)

Glow discharge polymerized films deposited on germanium were supplied

by Dr. Yasuda. The systems chosen, acetylene/N2/H20 hexamethyldisiloxane,

acrylic acid, and tetrafluoroethylene were chosen to cover a range of biocompatibilities as indicated by a Lindholm clotting assay. The first set

of samples were standard germanium internal reflection elements with one side

coated. As these samples were too large for our cell, the films were measured

in air. Given below are average values for each film.

Hexamethyldisiloxane film

Thickness = 40 nm

Refractive index (n) = 1.49

8
Acety lene, N2 , H2O fl Im

t Thickness = 18 nm

Refractive Index (n) = 1.66

A second set of samples fit the ellipsometer cell and measurements were

made under buffer solution. For these specimens average values are given

below:

Acrylic acid film

Thickness = 49 nm

Refractive index (n) = 1.50

Hexamethyldisiloxane film

Thickness = 42 nm

Refractive index (n) = 1.43

Tetraf luoroethylene film

Data cannot be analyzed

The acrylic acid film "flaked off" the substrate and the data obtained

for the tetrafluoroethylene film could not be analyzed. Since only one

sample remained and comparisons were not possible, protein adsorption has

not yet been pursued.

RESULTS AND DISCUSSION

FACTORS INDUCING CONFORMATIONAL CHANGES

Applied Potential

The previous annual report (3) and a subsequent publication (6) have

detailed the results of our study of the effect of an applied potential

on the conformation of adsorbed blood proteins. The investigation of

three proteins showed the existence of an anodic potential at which enhanced

adsorption occurred and a cathodic potential at which a conformational

change occurred as evidenced by a large increase in extension. Data obtained

from a platinum substrate indicated that the potential of enhanced

adsorption was dependent on the particular protein studied, although the

9
data do not correlate simply with the net charge on the protein molecule.

In connection with this work, we have collected and evaluated data

characterizing the relative stability of both platinum and germanium when

used as substrates for In situ protein adsorption with applied potentials.

The procedures followed were as previously described (3,6) with the addition

that germanium internal reflection elements used as substrates were cleaned

with detergent, rinsed, dried, and given a two minute treatment with a glow

discharge plasma xmit. The immersion medium in all cases was 0.15N NaC£ at

pH 7.4.

As shown in Figure 3 the small drifts in the real (n) and imaginary (k)

components of the index of refraction of platinum which occurred with time

were independent of either exchange of the immersion medium or applied

potential. The instability of germanium, relative to platinum, was indicated

by much larger changes in the ellipsometer readings and a visible etching of

the surface upon removal from the system. If one ignores scattering due to

roughening, the ellipsometric data can be analyzed to determine the apparent

optical constants of germanium in the presence of 0.15N NaCil. The reflection

coefficients, r-*-*", can be used to calculate the optical density per

reflection, OD"*"*'* = -2 log r-*"*". Large changes in the apparent optical

density of germanium as a function of both the immersion time and the applied

potential are shown in Figures 4 and 5. The similar analysis in terms of

optical densities for the platinum data of Figure 3 yields a variation in OD-'^ and OD" of from 0.062 to 0.059 and 0.402 and 0.394, respectively. If errors

observed at visible wavelength carry over into infrared wavelengths, the

etching could also produce artifacts in internal reflection measurements of

protein adsorption on germanium. The observed ATR intensities would be

unpredictably increased by these artifacts. Unfortunately, these artifacts

cannot be accounted for in control experiments since the Drude model required

for data analysis is no longer valid. Obviously such artifacts preclude the

use of the Drude equations either for ellipsometry or internal reflection

spectroscopy in such a system.

Surface Concentration

The adsorption of clotting factors and proteins on the surface of a

synthetic implant can modify both their biological activity (7-9) and the

subsequent interfaction of formed cellular elements (10) with the surface.

As has been noted (11), the amoimt adsorbed and composition of adsorbed

protein films do not account for the variation in biocompatibility observed

for some materials. Additional details, in particular the adsorbed

conformation, are apparently required to characterize materials and rationalize

the effect of blood-surface interactions. It has been assumed (12,13) that

adsorbing proteins initially arriving at a surface unfold to occupy a large

number of available surface adsorption sites resulting in a denaturation

at the solid/solution interface. The inapplicability of this assumption to

all cases has been demonstrated by recent studies of adsorbed serim albumin

and prothrombin (4,14) which indicated no change in conformation as the

surface concentration increased.

Two separate methods are available for changing surface population, time

and solution concentration. In order to directly determine the existence and

extent of conformational changes in adsorbed proteins due to mutual interaction

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at the interface, jLn situ Infrared bound fraction and elllpsometrlc studies

have been carried out to characterize the adsorption of yglo^^lii^ 6-lactoglobulln on silica. Through studies of the properties of native and crosslinked y-globulin, one can determine the correspondence between adsorbed and

solution conformation, and thereby directly obtain a measure of surface

"denaturation"

Although most of the results obtained in this study were presented in

our previous annual report (3), some additional work was carried out during

this reporting period and recent ellipsometric results for native yglobulin

adsorption have slightly modified those previously reported. To summarize

those reported results, the bound fraction measurements for both y-globulin

and 3-lactoglobulin showed that there was a significant decrease in the number

of surface attachments as the surface concentration increased. For y-globulin,

the change was from 165 to 30 carbonyl surface attachments as shown in Table I,

while for 3-lactoglobulin, the decrease in bound fraction corresponded to a

change of from 30 to approximately 2 attachments. Bound fraction results

obtained for serum albumin and prothrombin (4,14) indicated no change in

conformation for either protein, even though both have elliptical solution

conformations with large axial ratios. It is, therefore, unlikely that the

observed trend in the bound fraction of y-globulin or 3-lactoglobulin is

due merely to changes in orientation of the adsorbed molecules.

Ellipsometric results obtained for y-globulin adsorbed on planar fused

silica provide further evidence of conformational changes dependent on the

surface concentration. As shown in Figure 6, adsorbed y-globulln achieves

a steady state conformation rather quickly, although material continues to

be adsorbed over an extended time period. As Indicated in Table I, the

extension of adsorbed y-globulin is higher for adsorption from the higher

solution concentration. At concentrations of 0,3 and 10.0 mg/ml, eHtensions

of 14 ± 3 nm (calculated standard deviation) and 32 ± 11 nm were observed,

respectively.

The protein extension values reported in Table I were obtained from

the ellipsometric data by use of the Drude equations (4,15). The model upon

which the Drude equations are based is that of a homogeneous film of constant

refractive index with discrete boundaries. However, the adsorbed protein

film, consisting of a mixture of protein and solvent is not homogeneous,

and therefore will deviate from this model. The protein concentration in

the film, and hence the refractive index would be expected to vary with

distance from the surface, probably decreasing monotonically . The use of

the Drude equations will result, therefore, in an average extension value

which would be about 50% greater than the root-mean-square segment to surface

distance calculated using the actual concentration distribution. The technique

for relating the ellipsometric thickness to the root-mean-square extension

in any particular case has been described by McCrackin (16).

Both the infrared bound fraction and ellipsometric results presented

above indicate the presence of lateral interactions between adsorbed molecules

of y-globulin and 3-lactoglobulin. It is of interest to note, however, that

a Langmuir plot (C^j/Cg vs. Cb where = equilibrium solution concentration

and Cg = the surface concentration) of the adsorption data obtained for both

proteins adsorbed on divided silica, resulted in good linear correlations

for 2 0.1 mg/ml. Since lateral interactions have been demonstrated

14
TABLE I

Y-Globulin Adsorbed on Silica

Cone. Adsorbance Bound No. Carbonyl

mg/ml mg/m^ Fraction Attachments

0.30 1.8 ± 0.8 0.12 165

10.0 3.4 ± 0.6 0.02 30

DEM Cross linked

0.30 2.2 ± 0.6 0.06

10.5 2.1 ± 0.6 0.01

15
ADSORBANCE. mq/m'

o

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16
experimentally, the consistency with the prediction based on the Langmuir

model must be considered fortuitious. The use of the Langmuir equation

to determine the presence or absence of lateral interactions should be

viewed with skepticism.

Previous bound fraction and ellipsometric studies (3) of the adsorbed

conformation of y-globulin cross-linked with diethyl malonimidate hydrochloride indicated that the adsorbed conformation corresponding to the

"native" conformation in solution was intermediate between high and low

surface concentration. The adsorption isotherm for native y-globulin lies

above that for the cross-linked material and rises more steeply, even

though both isotherms have approximately the same plateau adsorbance. It,

therefore, appears that adsorbed native molecules can apparently rearrange

on the surface to minimize interactions which results initially in a

higher adsorbance and a sharper break in the isotherm as it approaches the

plateau value.

Thus, comparison of the bound fraction and ellipsometric results for

native and cross-linked y-globulin (Table I) indicates that there are

distortions of the native conformation at both low and high adsorbance.

At low surface concentration, lack of competition for surface adsorption

sites results in a flatter adsorbed conformation, while at high surface

concentration, repulsions cause a more extended conformation with fewer

surface attachments. Adsorbed yglo^ulin would, therefore, appear to be

rather pliant, underscoring the importance of defining the adsorbed

conformation when carrying out macroscopic measurements of surface-cellular

interactions in the))pre8ence of proteins.

Adsorption Time

A second means of changing surface population, time of adsorption,

although more difficult to achieve, should provide considerable additional

insight into the adsorption mechanism. Because of instrumental limitations,

our previous ellipsometric studies of protein adsorption have been limited

by the inability to observe these early steps in the adsorption process.

It is impossible to make meaningful measurements if the optical properties

of the film are changing on the time scale of a single measurement, which

is likely to be the case for the early stages of protein adsorption.

We have recently obtained an automatic ellipsometer with which one

can simultaneously measure at very short time intervals two parameters

which define the state of the elliptically polarized light reflected from

a surface. These parameters, aximuth (a) and ellipticity (e) , are easily

converted into the more familar reflection parameters A and \\). By use of

the Drude equations (3,15) we can calculate the thickness, refractive index,

and adsorbance of a protein film. Since the instrximent is capable of

measuring a and e 54 times per second, it is possible to observe very

rapid changes in the parameters.

Several protein adsorption experiments using platinum as the substrate

have been performed to date with the automatic ellipsometer. As yet, none

of the experiments has been repeated and the results must be regarded as preliminary and tentative. Human fibrinogen (2.5 and 0.42 mg/ml) and bovine

y-globulin (12.2, 0.22 and 0.044 mg/ml) were separately adsorbed onto clean

17
platinum slides according to the method described in the Experimental section.

For the most concentrated yglobulin solution, essentially all of the change

in the ellipsometric parameters occurred by the time of the first observation,

approximately 2 s following the introduction of the solution. For each of

the other protein solutions, however, significant changes in a and e occurred

during the first minute of observation, and smaller changes occurred during

the next hour.

Selected points along the recorder tracings of a and e vs. time were

used to compute the film thickness and adsorbance. Almost all of the data

points could be analyzed to give reasonable values of molecular extension

and adsorbance. In each case, the calculated protein adsorbance increased

during the total time of observation, with a large fraction of the total

increase occurring during the first minute. In all cases but one, the

calculated film thickness (molecular extension) remained essentially constant

throughout the run. The one exception to this, the 0.42 mg/ml fibrinogen

solution, indicated a large increase in the film thickness during the first

minute, and an equally large further increase during the next 2 hours. Since

there are experimental artifacts which could cause this type of behavior,

this result must be regarded as highly questionable, pending replicate experiment

Nevertheless, our initial experiments with the automatic ellipsometer indicate

that the technique can provide much information about the kinetics of polymer

film deposition on surfaces.

Denaturing Media

As observed in our radiotracer studies, adsorbed protein desorbs very

slowly, if at all, from polyethylene. This "irreversible" behavior on the

hydrophobic polymer surface contrasts strongly with the highly reversible

adsorption on silica, a typical high energy surface. These findings are

somewhat surprising since previous ellipsometric studies (4) have shown that

the extension of an adsorbed protein is greater on low energy than on high

energy surfaces. If one assumes that the extension and bound fraction are

inversely related, as observed for some synthetic polymers, then a protein

adsorbed on polyethylene should have fewer attachments and desorb more rapidly.

This irreversible adsorption on polyethylene suggests, however, the existence

of a larger number of surface attachments or a higher segment-surface interaction energy. To clarify the cause of the lack of desorption, as well as

to gain an insight into protein adsorption mechanisms, an ellipsometric study

of the effect of a strong denaturing agent, urea, on yglobulin and fibrinogen

adsorbed on polyethylene was undertaken. Measurements were carried out using

O.IM phosphate buffer at pH 7.4 and buffered 8M urea.

The results given in Figures 7 and 8 for the adsorbance and extension

of fibrinogen on polyethylene are typical. Adsorption is fast and virtually

complete at the time of the first measurement and an extension of 30 nm is

attained in each case. Exchange of the protein solution for buffer induces

no significant changes in the adsorbance or conformation of either fibrinogen

or Y~glot)ulin. The further exchange of buffer for buffered 8M urea, however,

results in a large increase in extension of both Y~globulin and fibrinogen

as shown in Figure 8 for fibrinogen. Some of this increase may result from

the large refractive index difference between the urea and buffer solutions.

However, a significant increase in extension does occur even after taking

this into account.

18
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20
The high protein adaorbance on polyethylene, even somewhat higher

than that on silica, and especially the "irreversible" nature of the

adsorption, may be important considerations when designing polymeric

implant devices. The higher adsorbance of Y~glot)ulin when adsorbed on

polyethylene compared to silica is not inconsistent with the finding that

the extension in general increases with decreasing surface energy. If

increased extension correlates with fewer surface attachments, then the

reduced area per adsorbed molecule could result in a higher adsorbance.

The inability to desorb fibrinogen and yglobulin from polyethylene into

buffer or 8M urea indicates a strong interaction with the surface. The

large increase in extension, and presumably the large decrease in the

number of surface attachments, which occurs during attempted desorption

into urea solution implies that strong hydrophobic bonding, and not a large

number of surface attachments, is the cause of the "irreversible"

adsorption on polyethylene.

RESULTS AND DISCUSSION

RADIOTRACER RATE STUDIES

Measurement

Some of the factors which contribute to measurement errors associated

with the use of radiotracers to measure protein adsorption have been

identified (17) , We have observed that the incorporation of a radioactive

element into a protein for labeling purposes may result in artifacts

that are not immediately apparent. The use of such a label for the

measurement of adsorption is based on the assumption that the labeled

material is essentially identical in its adsorption behavior to the nonlabeled material. This assvnnption may be in error for several reasons.

First, adsorption of the label itself may dominate the adsorption process

and this effect may vary from one surface to another. Secondly, the

presence of the label in the protein or the method employed to incorporate

the label may significantly alter the structure of the protein. Thirdly,

the effect of any impurities in the small volume of labeled material

may be multiplied many times if there is a large dilution with non-labeled

materials. Our data indicate that all three of these factors are important

for some systems.

We have noted a marked difference among the adsorption values of HSA

depending on the method of labeling. The use of ^^^I, introduced by the ICl

method in our laboratory, resulted in adsorbance values on polyethylene

considerably higher than observed for HSA commercially labeled with -'^•^I.

When undiluted ^^^I labeled HSA tagged by the above method was adsorbed on

silica or polyethylene, an adsorbance of approximately 5 mg/m^ was observed.

However, when the same solution is diluted by a factor of 1:200 with nonlabeled HSA, an adsorbance of approximately 30 mg/m^ was observed. This

21
dilution effect (higher adsorbance for the diluted-labeled system) seems to

be responsible for the higher adsorbance found by others (18) using McFarlane's

ICl method to label fibrinogen and subsequently diluting it with non-labeled

fibrinogen by factors of 1:100 and 1:50.

Commercially labeled HSA with 125i as the labeled atom also gave

adsorption values several times higher than those reported for '•^^I, but lower

than that obtained for the ICJl labeled sample. Although the adsorbances

were different, reproducibility for each sample was excellent, both for run

to run using the same batch of labeled protein as well as for batch to batch.

The commercial materials may be quite suitable for the diagnostic purposes

for which they were prepared, but the protein may be altered from the native

material in a way that affects its adsorptive behavior. There are several

possible causes for these differences, but the most likely is aggregation of

the protein during the labeling procedure followed by incomplete purification.

We have previously shown (3) that HSA labeled with I and adsorbed on

platinum produced high apparent adsorbance values for that particular system

which resulted from the specific adsorption of the labeled species. This

artifact was first suggested by the fact that the measured adsorbance was not

independent of the concentration of the labeled species even when the total

protein concentration was not changed. Such a study was conducted for HSA

labeled with ^^^I and adsorbed on polyethylene. Table II gives the measured

adsorbance of HSA as a function of the amount of radio-labeled protein

present. In the absence of specific adsorption, or if the specific adsorption

is always proportional to the concentration of that species alone, there

would be no change in adsorbance with activity. This surface is, therefore,

free of this artifact.

Desorption and Exchange

In spite of some uncertainty in the adsorbance values estimated using

different radiolabeled species, desorption and exchange rates might be

valid when expressed as functions of the plateau adsorbance measured for

that particular system. This indeed seems to be the case for HSA adsorbed

on polyethylene as the desorption and exchange results are independent of

the labeled species or the activity of the label. As shown in Figure 9,

there is virtually no desorption or exchange of adsorbed protein with protein

in solution in this system, even after 25 hours. This finding can be

contrasted to previous results for the displacement of serum albiunin from

chromium as shown in Figure 10, which showed marked decreases in the amoimt

of adsorbed HSA as a function of time. These results suggest, as discussed

earlier in this report, that there is either a more uniform distribution of

energy sites on the low energy surface which results in a larger nvimber of

surface attachments per adsorbed molecule, or that the segment interaction

energy resulting from hydrophobic bonding is considerably greater than that

resulting from hydrogen bonding as envisioned for protein adsorption to a

chromium surface.

In contrast to the ellipsometric results on fibrinogen and Y~globulin

described earlier, desorption of HSA 125 j from polyethylene into 8M urea

resulted in the removal of approximately one-half of the protein as shown

in Figure 11.

22
TABLE II

Apparent Adsorbance as a Function of

Specific Activity of

HSA-125]; Solution

Polyethylene

Surface

Relative Activity mg/m^

1.00 32

0.67 33

0.50 33

0.33 41

0.25 34

0.20 31

23
in

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24
25
26
Silicone Rubber

Measurements of HSA--'-"^-'-! adsorbance on silicone rubber (non- reinforced

medical grade Silastic, Dow Corning) shown in Figure 12 indicates that the

time required to attain apparent equilibrium adsorbance is considerably slower

than the ten seconds required for chrome (3). The differences in rates to

plateau values may be related to the surface free energy, i.e., as the

surface energy increases, the times to plateau decrease. An increase in

the extension with decreasing surface energy has been noted for several

blood proteins (4).

DISCLAIMER

This report was prepared as an account of work sponsored by the

National Heart and Lung Institute. It is not to be construed as a report,

opinion, or recommendation of the National Heart and Lung Institute. It

will be available from the National Technical Information Service, 5285

Port Royal Road, Springfield, Virginia 22151.

27
28
REFERENCES

1. Laki, K. , Arch. Biochem. Biophys., 32, 317 (1951).

2. Batt, C. W. , Mikulka, T. W. , Mann, K. G. , Guarracino, C. L. , Altiere,

R. J., Graham, R. G. , Quigley, J. P., Wolf, J. W. , and Zafonte, C. W.

,

J. Biol. Chem. , 245, 4857 (1970).

3. Stromberg, R. R. , Morrissey, B. W. , Smith, L. E. , Grant, W. H. , and

Fenstermaker, C. A. , "Interaction of Blood Proteins with Solid

Surfaces", PB 241 267/4SL (Available from the National Technical

Information Service) , Annual Report prepared for the Biomaterials

Program, National Heart and Lung Institute, NIH, Bethesda, Md.

,

January 1975.

4. Fenstermaker, C. A., Grant, W. H. , Morrissey, B. W. , Smith, L. E. , and

Stromberg, R. R. , "Interaction of Plasma Proteins with Surfaces",

PB 232 629/6 (Available from the National Technical Information Service)

Annual Report prepared for the Biomaterials Program, National Heart

and Lung Institute, NIH, Bethesda, Md. , March 1974.

5. McFarlane, A. S., Nature, 182, 53 (1958).

6. Morrissey, B. W., Smith, L. E. , Stromberg, R. R. , Fenstermaker, C. A.,

to be published in J. Colloid and Interface Sci.

7. Gitel, S. N. , Owne, W. G. , Esman, C. T. , and Jackson, C. M. , Proc.

Nat. Acad. Sci. USA, 70_, 1344 (1973). '

8. Waugh, D. , Anthony, L. J., and Ng, H. , J. Biomed. Mater. Res., 9^, 511

(1975).

9. Margolis, J., Aust. J. Exp. Biol., 39, 249 (1961).

10. Mohandas, N. , Hochmuth, R. M. , and Spaeth, E. E. , J. Biomed. Mater.

Res., 8, 119 (1974).

11. Horbett, T. A., and Hoffman, A. S. in "Applied Chemistry at Protein

Interfaces", (R. E. Baier, Ed.) Advances in Chemistry Series No. 145,

American Chemical Society, Washington, D. C. (1975).

12. Baier, R. E., Loeb, G. I., and Wallace, G. T., Fed. Proc, 30,, 1523 (1971)

13. MacRichie, F. , J. Colloid Interface Sci., 38, 484 (1972).

14. Morrissey, B. W. , and Stromberg, R. R. , J. Colloid Interface Sci., 46 , 152 (1974).

15. Drude, P., Ann. Physik, 222. 532 (1889); 272 . 865 (1889); 275, 481 (1890).

16. McCrackin, F. L. , and Colson, J. P. , in "Ellipsometry in the Measurement

of Surfaces and Thin Films", Symposium Proceedings, NBS Misc. Publ.

256, Washington, D. C. 20234, p. 61 (1964).

29
17. Grant, W. H. , Smith, L. E. , and Stromberg, R. R. , to be published.

18. Leonard, E. F. , Kochwa, S. , Litwak, R. S., and Rosenfield, R. E.

"Coordinated Studies of Platelet and Protein Reactions on Artificai

Materials", PB 241 333/4SL (Available from the National Technical

Information Service) , Annual Report prepared for the Biomaterials

Program, National Heart and Lung Institute, NIH, Bethesda, Md. 20014

October 1974.

use OMM-NBS-DC 30
bTbLIOGRAPHIC DATA 1. Report No. 2. ^HEET NBSIR 76-1017

3. Recipient's Accession No.

1, Title and Subtitle

INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

5. Report Date

March 5, 1976

6.

7. Author(s) R. R. Stromberg, B. W. Morrissey, L. E. Smith,

tj ' H (Irant flnd R. E Dehl

8. Performing Organization Rept.

NBSIR 76-1017

), Performing Orfjanizacion Name and Address

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

10. Project/Task/Work Unit No.

11. Contract /Gran' Nr.. Interagency Reimburable Agreement 72-19

12, Sponsoring Organization Name and Address Biomaterlals Program

Devices and Technology Branch

Division of Heart and Vascular Diseases

NATIONAL HEART AND LUNG INSTITUTE

Bethesda, Maryland 20014

13. Type of Report & Peiiod

Coveted jjQ^^ 1974 - October 31, 1975

14.

15. Supplementary Notes

16. Abstracts

The overall objective of this investigation is to help characterize materials

used for implants in the cardiovascular system and to develop test methods to aid

I in the selection of the most useful materials •* The focus of the work has been an

i investigation of tTie adsorption of blood proteins on surfaces with special emphasis

on changes in conformation occurring upon adsorption. An important component has

been an ellipsometric examination of materials produced by contractors of the NHLI

Biomaterials Program that were considered to be of potential clinical value. In

j contrast to the behavior of serum albumin and prothrombin, conformational changes

I in y-globulin and 3-lactoglobulin occurring as a consequence of changing surface

population were previously reported and were confirmed during this reporting period.

An investigation of conformational changes at very short adsorption time was

initiated. Adsorption, desorption and exchange .of adsorbed protein in solution was

studied. The results indicate strong hydrophobic bonding between the protein and the

17. Key Words and Document Analysis. 17a. Descriptors Synthetic polymer SUrface.

Adsorption

Blood protein

Bound fraction

Ellipsometry

Implants

Polymer adsorption

Protein adsorption

Synthetic implants

I|7b. Identifiers/Open-Ended Terms

jVc. COSATI Field/Group

8. Availability Statement Roleasablc to the public.

Order from: National Technical Information Service

5285 Port Royal Road

- Spri ngfield, Virginia 22151 .

19. Security Class (This

Report)

IINCLASSIFIF.D

21. No. ot Pages

36

20. Security Class (I his

Page

UNCLASSIFIFD

22. Price

$ 4.00

ORM NT1S.35 (REV. 10-73) END()l<Si;D LiY ANSI AND UNT-SCO.