ORGANIZATION

Polymer Science and Standards Division

Center for Materials Science

ADDRESS

National Bureau of Standard

REPORT TITLE NBSIR 79-1784

INTERACTION OF BLOOD PROTEINS WITH

SOLID SURFACES

Washington DC 20234

CONTRACT NO. 2 YOL-HV-30014 Reimbursable Agreement

AUTHORS

R, E. Dehl and W. H. Grant

REPORT NO. NBSIR 79-1784

TYPE OF REPORT

Annual (6th)

DATE OF REPORT

September 30, 1979

PERIOD COVERED

Augu£t__1^19^ 1978

ORGANIZATION

Polymer Science and Standards

Center for Materials Science

National Measurement Laboratory

National Bureau of Standards

Washington DC 20234

TOTAL REPLACEMENT

! PHYSIOLOGY

BIOt.UGY

I chemistry

1 REPORT SUBJECT INDEX

BLOOD PUMPS S

1^ MATERIALS

[ j v _ -V' ENERGY

1L

CONTROLS h 1

j INSTRUMENTATION

OXYGENATORS

_ EMERGENCY SYSTEM. ..

^

.

' ... —

j j TEMPORARY SYSTEM

PERMANENT SYSTEM 5

5

r=;

. .. PHYSICAL SCIENCE

ENGIN EERING

. " V.. Nb)|J

TEST AND EVALUATION

-« ‘ " SYS FEMS

RESPIRATORY DEVICES

PREPARED FOR biomaterials program

DEVICES UNO TECHNOIOGV BDINCH

miSIM IF HMI m VASCILU DISEASES

NATIONAL HEART, LUNG, AND BLOOD INSTITUTE

BETHESDA, MARYLAND 20014

U. S. DEPARTMENT OF

HEALTH, EDUCATION, AND WELFARE

PUBLIC HEALTH SERVICE

NATIONAL INSTITUTES OF HEALTH

nih- 15^6 (Kev. n-v-^1

AlllDD T6b3D3

^IMBS

PUBLICATIONS’^

NAT'L INST OF STAND & TECH R.I.C.

AlllOM T3TS7fl

NBSIR 79-1784

Interaction of Blood Proteins

With Solid Surfaces

R. E. Dehl and W. H. Grant

Polymer Science and Standards Division

Center for Materials Science

National Bureau of Standards

U S. Department of Commerce

Washington, DC 20234

Annual Report for Period

August 1, 1977 - September 30, 1978

Issued August 1979

Prepared for Biomaterials Program

Division of Heart and Vascular Diseases

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

QC—

100

U56

79-1784

eragency Reimbursable Agreement

JAN 3 1 191 oS r : -

nr'

Page

ABSimCT V

SUMMARY vi

INTRODUCTION . 1

EXPERIMENTAL 2

Protein Purity . 2

Fibrinogen 2 Y-Globulin 2 Human Serum Albumin - 3 Ellipsometry 4

Radiotracer Counting 4

Control Surfaces 5 NHLBI Contractor Biomaterials . . 5 Secondary Ion Mass Spectrometry 5 RESULTS ANT) DISCUSSION 7

PURIFICATION OF IY\BELED AND UNIABELED SERUM ALBUMIN 7

Separation of Aggregates by Fractionation 7 Electrophoretic Analysis of Fractionated and

Unfractionated HSA 19

Ion Exchange Purification 19

ADSORPTION OF HSA(^^^I) AND (^'*0 ON CONTROL SURFACES .... 21

Adsorption of HSA(^^^I) Fractions 21

Adsorption of HSA(^^^I) and (^“*0 on Polyethylene ... 21

Adsorption on Silicon Oxide and Platinum 23

CANDIDATE BIOMATERIAL SURFACES 28

Ethylcellulose Perfluorobutyrate and Glew-Discharge

Coatings 28

Polyacrylamide Hydrogel 28

Polyetherurethanes 30

Heparin-Coated Silicone Rubber 31

Biomer . . . 31

Carbon Coated Steel 31

ADSORPTION OF y-GLOBULIN Olj OXIDIZED SILICON 34

REFERENCES 37

U3 Z ud

' J ‘-y- v N

TABLE OF CONTENTS

1
FIGURES

Page

Figure 1: Gel permeation chromatogram of Standard Reference

Bovine Serum Albumin 8

Figure 2: Gel permeation chromatogram of unlabeled HSA 11

Figure 3: Gel permeation chromatogram of unlabeled HSA 12

Figure 4: Gel permeation chromatogram of unlabeled HSA ..... 13

125

Figure 5: Gel permeation chromatogram of I HSA 14

125

Figure 6: Gel permeation chromatogram of I HSA 15

125

Figure 7 : Gel permeation chromatogram of I HSA 16

131

Figure 8: Gel permeation chromatogram of I HSA 17

Figure 9: Gel permeation chromatogram and specific activity

of 125 j hsa 18

125

Figure 10: Adsorption isotherm for the monomer of I HSA

on polyethylene 22

125

Figure 11: Rate of adsorption of the monomer of I HSA

on polyethylene 24

14

Figure 12: Rate of desorption of C HSA from polyethylene

into buffer 25

14

Figure 13 : Rate of desorption of C HSA from Biomer into

buffer solution 32

Figure 14: Adsorbance vs pH for bovine y-globulin on hydrophobic and hydrophilic silicon oxide 35
TABLES

Page

Table I: Adsorption of ^ hSA on control surfaces 27

Table II: Adsorption of ^ HSA and HSA on candidate

biomaterials 29

i i i
NOTICE

This is a report prepared as an account of progress

of work sponsored by the National Heart, Lung and

Blood Institute. It is not to be construed as a report,

opinion, or recommendation of the National Heart, Lung and

Blood Institute. When produced in final form it will be

available from the National Technical Information Service,

5285 Port Royal Road, Springfield, Virginia 22151.

Certain commercial materials and equipment is identified in

this report in order to adequately specify the experimental

procedure. In no case does such identification imply recommentation or endorsement by the National Bureau of Standards

nor does it imply that the equipment identified is the best

available

.

XV
ABSTRACT

The purpose of this investigation is to help develop methods of

characterizing the surface properties of implant materials for use in

the cardiovascular system. These properties are related to the ultimate

success or failure of an implant in vivo . The investigation during the

current reporting period has been chiefly concerned with the amount of

protein deposited on implant material surfaces . Ion exchange treatment

of radiolabeled proteins has been found to lead to much greater accuracy

and reliability of adsorption measurements. Results are reported for

the quantitative adsorption of labeled proteins thus purified onto well

characterized test surfaces, and onto the surfaces of materials supplied

by contractors to the NHLBI Biomaterials Program. Tlie effect of the

label vs. ^**0 on adsorption of proteins was studied. Hydrophobic and hydrophilic oxidized silicon surfaces were used to demionstrate

that the adsorption of y-globulin, measured by ellipsometry , and serum

albumin, measured by radiolabeled protein, both are significantly

affected by charge interactions between the surface and the protein.

V
SUMMARY

This investigation is designed to help develop surface characterization methods for implant materials to be used in the cardiovascular

system. Since surface properties play a major role in determining the

success or failure of implants, these studies can form the basis for

in vitro test methods used to design and select suitable implant

materials

.

The first in a complex series of events that occurs when blood

comes into contact with an implanted device is the adsorption of proteins on the surface. The detailed characteristics of this adsorbed

protein layer can affect subsequent events, such as attachment of

formed elements, which may lead to failure of the implant. Factors

which are potentially important in determining the response of a

protein-covered surface to formed elements in the blood are the amount

of each protein deposited from the mixture, the lateral distribution

of the protein on the surface, the depth of penetration of the protein

into the material, and any changes in the conformation of the proteins

resulting from their interaction with the surface

.

Accomplishments during the past year are described below.

Improved Protein Purification Method

Using human serum albumin (HSA) labeled with which has been

purified by ion exchange chromatography as well as our usual gel

permeation chromatographic separation, we have obtained results for- the

amount of protein absorbed from solution which are consistent with

VI
monolayer coverage.

Adsorption and Penetration of Proteins on NHLBI Contractor Biomaterials

Using HSA purified according to our new procedure, we have

obtained results for the adsorption of HSA from solution onto six

substrates obtained from NHLBI contractors . Hydrogel and heparin

coated samples have been found to adsorb much more protein than uncoated

surfaces, indicating possible penetration of protein into the coatings

Another HSA sample, labeled with ^**0, was also adsorbed onto six surfaces, including three which had been studied with the labeled

HSA. Generally good a^^eement was found between the adsorbance of the

two labeled proteins

.

Protein Adsoiption on Model Surfaces

The adsorption isotherm and rate of adsorption of purified HSA

onto a well characterized linear polyethylene surface, NBS

Standard Reference 1475, have been determined. Silicon oxide surfaces

having different surface charges have been found to adsorb greatly

different amounts of HSA Platinum as observed in previous

studies (1) e>diibits an unusually high adsorption of iodine-labeled

protein.

The effect of surface charge on the adsorption of yglobulin onto

silicon oxide has been investigated, and it has been found that both

surface charge and intermolecular repulsion contribute to the net

adsorption of protein.

Vll
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INTRODUCTION

The adsorption of plasm proteins is the first in a complex series

of events that occurs when a synthetic mterial is placed in contact

with blood. It is known that this protein adsorption can alter the

course of subsequent reactions at the inplant surface but the mechanism

by which this occurs is not well understood. If the detailed beha,vi.or

of proteins absorbed at the solid surface were kncwn and capable of

control, the fate of artificial materials implanted in the body could

be predicted with greater certainty and more successful materials perhaps could be designed. This 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.

Using an ion exchange treatment which we have recently developed

for further purifying radiolabeled proteins, we have been able to

obtain accurate quantitative informtion about the adsorption of human

serum albumin onto a number of candidate synthetic implant materials

provided by contractors to the NHLBI Biomterials Program. Specially

prepared oxidized silicon surfaces, in the hydrophobic and hydrophilic

states, have also been used to test the effects of surface free energy

and electric charge interactions on y-globulin adsorption by ellipsometry and on radiolabeled humn serum albumin adsorption by radiotracer

counting

.

1
EXPERIMENTAL

Protein Purity

All proteins used in the experiments reported here were dissolved

in 0.15 M potassium phosphate buffer solution, at pH 7.4 unless otherwise noted. Gel permeation chromatography separations were performed

at room temperature on a Sephadex G-200 column, 2.5 cm in

diameter and 100 cm long. Flow rate through the column was approximately 0.1-0. 2 ml/min. The column packing was tested by passing blue

dextran (MW 40,000) solution through the column. Approximately 10 ml

of protein solution, at a concentration of about 0.5 mg/ml, were used

in each chromatographic separation. Specific activities of radiolabeled protein solutions used in the adsorption experiments were

typically of order 10^-10^ counts/min/mg protein.

Fibrinogen I

Four grams of purified human fibrinogen were obtained for use in

studying adsorption onto NHLBI contractor biomaterials. The fibrinogen,

distributed by AVCO Everett Research Laboratory, Inc., has been stored

at -80°C since it was received. According to the supplier, the protein

is 96 ± 0.5% clottable. It was supplied frozen in 0.3 M NaCl. Electrophoretic analysis of the reduced protein showed the characteristic

3 band patterns. Traces of Factor XIII and plasminogen were found,

together with 2.5% "cold-insoluble globulin".

y-Globulin

The purity of our bovine yglobulin (Cohn Fraction II) from ICN

Pharmaceuticals, Inc. has been tested by gel permeation chromatography

2
fractionation on a Sephadex G-200 column, as reported in our last

Annual Report (1977). Although the elution chromatogram showed the

presence of some high molecular weight aggregates, the amount of protein deposited on a test surface was the same from a solution of the

chromatographed monomer as from the unfractionated protein. Therefore,

we have concluded that it is not necessary to further purify our "asreceived” Y-globulin in order to obtain meaningful quantitative

adsorption data.

Human Serum Albumin

Human serum albumin (HSA) obtained from commercial sources, even

though stated to be of high purity, has usually been found by us to contain considerable amounts of dimer and higher molecular weight aggregates, as well as polypeptide fragments. Sodium dodecyl sulfate gel

electrophoresis and gel permeation chromatography both indicated that

commercial samples of HSA were quite impure. Each sample of labeled and unlabeled albumin which we have received has been chromatographically fractionated to separate the desired protein monomer from the

other components. Further details of the purification procedure and

their effect on the behavior of the albumin solutions in quantitative

adsorption experiments appear in a later section of this Report. The

labeled HSA was prepared by Dr. I. Miller at the Weizmann Institute,

RehoVOt , I srae1

The label was introduced by acetylation of cold, freeze-dried

HSA with labeled acetic anhydride. No change in enzymic activity of

other proteins, such as trypsin and RNAse was found by Dr. Miller after'

acetylation by this procedure. An electrophoretic analysis of the

3
labeled HSA, performed by Dr. Miller, revealed essentially pure HSA.

Ellipsometry -

- • -

All ellipsometry experiments reported here were performed with a

commercially available ellipsometer, which employs a He-Ne laser light

source (A = 632.8 nm) with an incident angle of 70°. Further details

of the ellipsometry instrumentation, the cells used to study adsorption

from solution, and experimental procedures may be found in previous

Annual Reports (1-5).

Analysis of the ellipsometry data to determine protein film

thickness, refractive index, and amount adsorbed on the substrate was

performed with a computer program developed at NBS by Dr. F.L.

McCrackin (6).

Radiotracer (lounting

A highly efficient y-counting system which we assembled with

commercially available components was described in our last Annual

Report (5). All radiotracer protein adsorption studies were carried

out at 37°C in pH 7.4 phosphate buffer solution under static conditions.

The very important rinsing procedures required to remove radioactive

solution carry-out from adsorbates have been described in previous

Annual Reports (1-3). Also described in the earlier reports are the

method of determining specific activity of the labeled protein and

the tests for self-absorption of radiation by the solution and its

container. Most experiments were carried out in duplicate, to test for

reproducibility. Our current procedures usually permit us to replicate

a given adsorption result within ± 2%.

4
Control Surfaces

For coirparison of results obtained by radiotracer methods and

ellipsometry 5 three control surfaces were used. Linear polyethylene,

a well characterized polymer (Standard Reference 1475) was obtained

from the Office of Standard Reference Materials at NBS. It was molded

into 1 mm thick sheets, fipom which discs were punched for the adsorption experiments . Oxidized silicon surfaces , obtained fron the

semiconductor processing group at NBS, were prepared by controlled

oxidation of polished silicon wafers. Freshly prepared surfaces, which

were hydrophobic, could easily be hydrolyzed by refluxing in boiling

water overnight. Thus, the effect of surface free energy on protein

adsorption could be studied, with minimal variation of surface chemistry

NHLBI Contractor Biomaterials

Most of the NHLBI contractor samples were shipped to us in

sterile packages. All were packaged dry, except for the Polysciences

hydrogel-coated polyetherurethane , which was packed under water.

Samples were rinsed in distilled water before each protein adsorption

experiment

Secondary Ion Mass Spectrometry (SIMS)

The SIMS technique was described in detail in our last Annual

Report ( 5 )

At that time , we reported that instrumental breakdowns

had prevented us from exploiting the technique in a productive way

These difficulties have been resolved, and spectra are currently being

obtained. Following a recent discussion with Dr. Bale Newbury (NBS),

5
we have prepared platinum and chromium slides coated with human serum

albumin for exploratory studies of protein SIMS spectra. The information thus obtained will enable us to proceed with SIMS investigations

of protein adsorbed on NHLBI contractors ’ samples

.

6
RESULTS AND DISCUSSIONS

PURIFICATION OF y\BELED AND UNLABELED SERUM ALBUMIN

Separation of Aggregates by Fractionation

A major problem in the application of radiotracer methods to the

study of protein adsorption is the presence of high and low molecular

weight impurities. These impurities compete with the monomer for

adsorption sites. Most of the available commercial suppliers of

radiolabeled and unlabeled human serum albumin (HSA) quote a high

degree of purity for their samples, in some cases as high as 100% as

measured by electrophoresis. After using some of these samples for our'

adsorption studies, we have found that the commercially supplied proteins needed additional purification. In Figures 1-9 we show GPC

separations of both labeled and unlabeled HSA obtained from commercial

sources. Most of these commercial samples are either contaminated

with low molecular weight polypeptides or high molecular weight

aggregates (mostly dimer) or in some cases both. We have therefore

continued to use a gel permeation chromatography (GPC) column packed

with Sephadex G-200 to separate the various molecular weight components

which might affect the adsorption of the monomeric protein. As

discussed in a later section under Ion Exchange Purification, we have

now shown that the GPC separation does not remove all of the impurities

which can affect the adsorption measurement.

Figure 1 is a Standard Reference Material sample of bovine serum

albumin, obtained from the Office of Standard Reference Materials at

the National Bureau of Standards. This sample with its small amount ol

7
’lO •H

r-Tf

eoueqiosqv

GcL

permeation

chromatogram

of

Standard

Reference

Bovine

Serum

Albumin.
dimer provides us with a reference point for determining which

fractions correspond to the HSA monomer. In this way, we discovered

that one commercial sample of HSA (^H) had a large amount of dimer but

little or no monomer was present. Without the reference material GPC,

we might have mistakenly used the dimer for our adsorption studies. In

Figures 2-4, we observe that the unlabeled HSA preparations contain a

large amount of dimer and higher aggregates as well as monomer. No

low molecular weight polypetides were found in the unlabeled protein

,

indicating that these impurities in the labeled HSA probably resulted

from the labeling procedure.

In Figures 5-9, we show GPC chromatograms of four different

samples of ^^^I labeled HSA and one sample of ^^^I labeled HSA, all

obtained from the same commercial labeling laboratory. Samples shown

in Figures 5 and 6 display good molecular weight separations, with a

small amount of both dimer and polypeptides in one sample, and no

observable dimer in the other. However, Figure 7 shows another HSA

sample which is clearly quite contaminated with dimer and

polypeptides. Figure 8 is a sample of HSA labeled with ^^^I, which

shows a small amount of dimer and no polypeptides.

Figure 9 is a plot of the radioactivity of each GPC fraction, for

another laj^eled HSA sample. The specific activities of the

aggregated (dimer and higher) molecular weight fractions are shown to

be essentially the same as the monomer fractions, while the polypeptide

fractions appear to have a much lower specific activity. Assuming

that the concentration of protein in these fractions was correctly

determined, using the same UV extinction coefficient as the whole

9
protein, we conclude that a high percentage of the low molecular

weight fractions is unlabeled.

10
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Volume
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9.

Gel

permeation

chromatogram

and

specific

activity

of

HSA.

Solid

line,

elution

chromatogram;

broken

line,

specific

activity.
Electrophoretic Analysis of Fractionated and Unfmctionated HSA

SDS gel electrophoresis was performed upon a number of labeled

and unlabeled HSA solutions, including fractionated and unfractionated

samples. In each case, the results were consistent with the GPC data

for that sample. Unfractionated HSA displayed several bands in its

electrophoretic pattern, corresponding to monomer, dimer, and higher

aggregates. Monomer and dimer fractions each displayed a single band,

with the dimer trailing behind the monomer, as in the unfractionated

HSA. Autoradiographs of unfractionated labeled HSA indicated the

presence of activity in each fraction.

Ion Exchange Purification

The commercially labeled and nonlabeled proteins were further

purified by passing these materials through an ion exchange column

packed with 50-100 mesh Dcwex 1-X4 resin (7). This additional yjrification was necessary to prevent any confusing and potentially misleading experimental artifacts due to the presence of impurities still

remaining in the fractionated monomeric protein after passage through

the GPC column. Two passes of the protein through 2 cc of the resin,

which had been previously washed with 1 N HCL and with 20% and 0.85%

NaCl, were sufficient to give reproducible results for the adsorbance

of HSA on polyethylene. A third pass through the column gave

essentially the same result as the second, indicating that the column

had removed the interfering impurities. Following the above exp c'-

ments, we subjected an unchromatognaphed labeled protein solution i.m

the same ion exchange treatment and fouiid that the r- v'ultini': ''olut: n

19
w

gave adsorbance values on polyethylene which were identical to those

using ion exchange treated protein monomer fractions . These results

suggest that fractionation may not be necessary in order to obtain

adsorbance results consistent with monolayer surface coverage of monomeric protein. Rather, it may only be Important to remove unreacted

iodine and/or iodide ion, species which could be preferentially

adsorbed to the substrate and yield high apparent adsorbances of

labeled protein.

20
ADSORPTION OF HSA AND (^'*0) ON CONTROL SURFACES

Adsorption of HSA Fractions

Various molecular weight components of HSA indicated as

I, II, III, IV on the elution pattern in Fig. 6, were used in adsorbance measurements on polyethylene. The results were obtained before

the ion exchange treatment of the protein, described above, was found

to lower the adsorbance of HSA to a value consistent with monolayer

surface coverage. Neverdiheless , the relative values obtained from

fractions I- IV may be indicative of the adsorption behavior expected

for fractionated HSA. The dimer fraction (I) showed an adsorbance

slightly smaller than did the monomer fractions (II and III) which

were both about the same. If small quantities of dimer were present

in monomer fraction II, they apparently had little or no effect on the

measured adsorbance, as compared with monomer fraction III, which is

assumed to have less dimer content than II. The apparent adsorbance of

the polypeptide fraction (IV) was much less than that of the higher

molecular weight components, which is consistent with the observation

that the adsorbance of proteins increases with molecular weight ( 1 )

.

Adsorption of HSA (^ ^^I) and (^**0 on Polyethylene

Figure 10 shows the adsorbance from different concentrations of

monomeric HSA (^^^I), purified by ion exchange treatment, on polyethylene . Each point is an average of two samples . The overall

isotherm shape suggests Langmuir adsorption and is consistent wi_th the

formation of a close-packed monomolecular layer based on nominal

surface area. This general behavior has been observed (1) with othr^r

21
^uj/6uj 'eoueqjospv

<

c/3

22

Fig.

10.

Adsorption

isotherm

for

the

monomer

of

on

polyethylene.
types of mcromolecules at the solid-solution interface and on hydrophobic polymer surfaces (2,3).

Figure 11 is a plot of the adsorbance of HSA on polyethylene

as a function of time. As observed, the adsorbed protein concentration

on the surface increases with time until a plateau value of approximately

5 mg/m^ is reached. A little more than five minutes is required for

the plateau value to be achieved.

Adsorption of labeled HSA on polyethylene was found to yield

5.44 mg/m^, from a solution concentration of 4 mg/ml. This agrees well

with the adsorbance of the iodine labeled compound and is consistent

with monolayer adsorption.

The rate of desorption of the ^“*0 labeled HSA is shown in Figure

12. About one third of the protein is desorbed within the first hour,

and the amount remaining after 17 h was found to be about half of that

initially adsorbed. These results are compared with desorption from

a polyetherurethane substrate, Biomer, in the following section.

Adsorption on Silicon Oxide and Platinum

HSA (^^^1) monomer fraction, purified by 2 passes through an ion

exchange column as described above, was used to measure the adsorption

from solution onto three different test surfaces, which we have used in

[revious studies of protein adsorption. TJ-ie results are tabuiatoci In

Table I. ITie theoretical monolayer coverage for HSA, assuming closepacked side-on ellipsoids (4 nm in diajiBter and 12 nm long), is about

2.5 mg/m^ , and for end on ellipsoids the value rises to 8.2 mg/m^ . One

cf the values in Table I lies within these limits, while the ni'c

23
14.0r

^uj/Buj 'aoueqjospv

24

Fig.

11.

Rate

of

adsorption

of

the

monomer

of

I

H.S.A.

on

polyethylene.

Solution

concentration

0.560

mg/ml.
$2

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oa

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0) IZ

0) EaMB II—

I

tiD iH

[i

Rate

of

desorption

of

H.S.A.

fror

polyethylene

into

buffer

solution.
well outside the monolayer limits.

The large difference in adsorbance on the hydrophobic and hydrophilic silicon oxide surfaces is qualitatively in accord with the

results of MacRitchie (8) for y-globulin on powdered silica surfaces

of high and low free energy, and with our results for y 'globulin on

the same two surfaces, as reported herein. At pH 7.4, albumin has a

high negative charge (isoelectric point 4,9), and the hydrophilic surface is also negatively charged. Charge repulsion between protein and

surface therefore leads to a relatively low amount adsorbed. On the

hydrophobic surface, where the charge is very small, the adsorbance is

within the approximate limits for monolayer coverage.

One adsorption experiment was performed using ^“*0 labeled H3A on

the hydrophobic Si02 surface. The adsorbance, from a solution concentration of 2.5 mg/ml, was 5.3 mg/m^ , which agrees fairly well with the

3.2 mg/m^ measured using ^^^l labeled HSA.

We have previously reported (9) a very high adsorbance on platinum

for HSA labeled with ^ which was attributed to a specific effect of

the labeling atom. The value which we report here is even higher than

that of the earlier report. Autoradiographs of platinum slides showing

a very non-uniform distribution of adsorbed HSA were reported in

our last Annual Report (5). It is not surprising therefore that large

differences in protein adsorption on platinum between different samples

are found by radiotracer counting.

26
TABLE I

ADSORPTION OF HSA ON CONTROL SURFACES

Sample Adsorbance (mg/m^)

Oxidized Silicon 3.2

(Hydrophobic

)

Oxidized Silicon 0.69

(Hydrophilic)

Platinum 150

27
CANDIDATE BIOMATERIAL SURFACES

HSA (^^®I) purified by the ion exchange method described above was

used to measure the adsorption from solution onto six different NHLBI

contractor sairples, as shown in Table II. In each case, the adsorption

time was one hour. The protein solution concentration was 0.56 mg/mi

for ail samples except for the heparin on silastic, for which it was

1.9 mg/ml.

HSA (^“*0 was adsorbed for one hour, at a solution concentration

of 4 mg/mi.

Ethylcellulose Perfluorobutyrate and 6low-Pis charge Coatings

The two hydrophobic substrates , ethylcellulose perfluorobutyrate

and glow-discharge tetramethyldisiloxane coated polyethylene, both

adsorb essentially the same amount of HSA as does polyethylene from the

same solution concentration. In our last Annual Report (5), we reported

the quantitative adsorption of fibrinogen onto these same surfaces

,

measured by ellipsometry . The adsorption of fibrinogen on both surfaces was essentially the same as on polyethylene, whic±i is in agreement with our present findings for serum albumin.

Polyacrylamide Hydrogel

The large apparent adsorbance of HSA on the Polysciences hydrogelgrafted polyethylene substrate is probably due to entrainment of

protein solution by the hydrogel. When the sample reported in Table

II vyas soaked in water for 18 minutes, the apparmt adsorbance was

28
TABLE II

ADSORPTION OF ^^^I HSA AND ^‘*0 HSA ON CANDIDATE BIOMATERIALS

Sample Adsorbance (mg/m^)

125j 14c

Ethylcellulose

Perfluorobutyrate

(Midwest Research Institute)

5.03

Glow-Discharge

Tetramethyldisiloxane

Coated Polyethylene

(Research Triangle Institute)

5.04

Polyacrylamide

Hydrogel Grafted to

Polyetherurethane

( Polysciences , Inc .

)

35.52

Polyetherurethane

3-2000-1-E

(Stanford Research Institute)

8.49 7.64

Polyetherurethane

3-1000/425 (7/3)-l-X

(Stanford Research Institute)

7.11 4.32

Heparin Bonded to

Silastic

(Carnegie-Mellon University)

11.50

Biomer

(Ethicon, Inc.)

3.08

Carbon Coated Stainless Steel

(General Atomic Co.)

7.87

29
reduced to 25.4 ing/m^. Thus, although the protein my not be tightly

bound to the hydrogel, it does not quickly diffuse out of the hydrogel.

These results are highly tentative, and further experiments would be

required to quantitate the rate of diffusion into and out of the hydrogel coating.

Polyetherurethanes

In our last Annual Report ( 5 ) , we reported very high apparent

adsorbances of HSA on the two polyetherurethanes from Stanford

Research Institute from a solution concentration of 7 mg/ml, corresponding to many monolayers of protein, while polyethylene adsorbed

about the same (5 mg/m^) as it does from a much Icwer solution concentration. In our present experiments, reported in Table II, the

adsorbances on the polyethehurethanes are only 40-50% higher than on

polyethylene from a solution concentration of 0.56 mg/ml. Since the

earlier experiments were performed with labeled HSA which had not been

purified by the ion exchange method, it seems likely that the high

results were due to adsorption of labeled contaminants (possibly

iodide ions ) by the substrates

While we did not have enou^ purified labeled protein to

repeat the adsorption experiments at higher solution concentrations

,

we have adsorbed labeled HSA on the polyurethanes, at a solution

concentration of 4 mg/ml. The adsorbances, as shown in Table II, agree

fairly well with the labeled HSA. For both labeled proteins the

adsorbance is somewhat higher on the 3-2000-1-E sample. Since this

sample is also more permeable to water, the results may indicate sane

30
penetration of protein solution into the more porous substrate

.

Heparin-Coated Silicone Rubber

Ellipsometry measurements on the heparin-coated silicone

rubber samples from Carnegie-Mellon University were attempted,

as reported in our last Annual Report (5). However, they were unsuccessful due to light scattering from the rough surface. The HSA

adsorbance reported in Table II is higher than expected for monolayer

coverage, which suggests some permeation of solution into the heparin

coating.

Biomer

The adsorbance of labeled HSA on Biomer was similar to the

other polyetherurethanes reported in Table II . While the adsorbance

was somewhat lower than on polyethylene (5.44 mg/m^), there was no

significant desorption of the protein from Biomer into buffer solution

after 96 h, as shown in Figure 13. The amount retained on the two

surfaces, however, was about the same after prolonged desorption.

Carbon Coated Steel

Samples of "ion plated" carbon on stainless steel were obtained

from General Atomic Co. The adsorbance of ^**C labeled HSA on this substrate, as shown in Table II, was somewhat higher than on polyethylene

or the polyetherurethane substrates

.

Ellipsometry was used to determine the adsorbance of fibrinogen

on the carbon coated steel. The value thus obtained, 5 ng/m^ , is

31
(0

$

o

ir>

o

o

c4

Fig.

13.

Rate

of

desorption

of

H.S.A.

from

Biomer

into

buffer

solution.
conparable to the adsorbance of fibrinogen on polyethylene and polyetherurethanes

.

the ^ **0 labeled

Thus, the reason for the somewhat high adsorbance of

HSA is not readily apparent.

33
ADSORPTION OF y-GLOBULIN ON OXIDIZED SILICON

In our last Annual Report (5), we discussed the ellipometric

measurement of adsorption of bovine y-globulin on a specially prepared

oxidized silicon surface in its "native" (hydrophobic) and hydrolyzed

(hydrophilic) states, as a function of surface concentration. Preliminary results were also given for the adsorbance and molecular extension

of the protein at three different pH’s. These studies have been extended to a wider range of pH, as shown in Figure 14. At the highest

pH used in this study (9.2), the adsorbance on hydrophobic silicon oxide

is about four times that on the hydrophilic substrate. Near the isoelectric point (IP) of the y-globulin (6.4), the adsorbance is about

the same on the two surfaces, and at lower pH both are somewhat lower

than at the IP.

Qualitatively, these phenomena may be explained in the following

way. The hydrophilic surface has ionizable SiOH groups, which cause

the surface to become progressively more negative above the point of

zero charge. This point is unknown for the particular silicon oxide

used in this study, but from studies of other forms of SiOa it probably

lies between pH 1 and 4 (10). On the hydrophobic surface, the decrease

in adsorbance above and below the IP is probably due to charge repulsion between the adsorbed protein molecules. On the hydrophilic surface, this effect is added to charge repulsion between the protein and

the surface at high pH. At low pH, where the surface charge of both

substrates is very small, the adsorbance on both surfaces is about the

same.

34
LU

Cn <r

E ^

^

CO

ZD -z: CD o

o o

_J

o

CO

^ Q

u LUM

> Q O X

DD O

O• —

JD

O

_C

Q.

X

o

’_E

CL

—I o o

“D “D X X

X

X

- O)

- OD

c o o "d•H

- CD

LD

CL

OO

C\J

30 CD LD OO C\J O

UJ/6UJ ‘30NVay0SaV

35

Fig.

l4.

Ad^'crbance

vs

pH

for

bovine

y-Globulin

hydrophobic

and

hydrophilic

silicon

ox
A qualitatively similar effect of pH on the adsorption of bovine

serum albumin on high surface area silica powders was reported by

MacRitchie (13), and by us (1) using an IR solution depletion method.

The present ellipsometry stucfy, performed on a flat surface and without

appreciable solution depletion of protein, is a more realistic model of

the behavior of an implant material in contact with blood proteins

.

36
REFERENCES

1. Fenstemiaker, 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,

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

Fernstermaker, C.A., "Interaction of Blood Protein 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.

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

Dehl, R.E., "Interaction of Blood Proteins with Solid Srufaces,:

NBSIR 76-1017, Annual Report prepared for the Biomaterials Program,

National Heart and Lung Institute, NIH, Bethesda, Md., March 1976.

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

B.W., "Interaction of Blood Proteins with Solid Surfaces," NBSIR

76-1128, Annual Report prepared for the Biomaterials Program,

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

August 1976.

5. Smith, L.E., Grant, W.H., and Dehl, R.E., "Interaction of Blood

Proteins with Solid Surfaces," NBSIR 77-1392, Annual Report prepared for the Biomaterials Program, National Heart, Lung and

Blood Institute, NIH, Bethesda, Md., July 1977.

6. McCrackin, F.L., "A Fortran Program for Analysis of Ellipsometric

Measurements," NBS Technical Note 479, Washington, D.C. 2C234

(1969).

7. Helmkamp, R.W., Goodland, R.L.

,

Bale, W.F., Spar, I.L., Mutschler,

L.E., Cancer Research 20 , 1495 (1960).

8. MacRitchie, F., J. Colloid Interface Sci., 38 , 484 (1972).

9. Grant, W.H., Smith, L.E., Stromberg, R.R., J. Biomed. Mater. Res.

Syirp., 8_, 33 (1977).

10.

Tadros, Th.F, and Lyklema, J., J. Electroanal. Chem., 17 , 267 (1968).

37
BIBLIOGRAPHIC DATA

SHEET

1 . Report No. 2. 6

4. Title and Subtitle INTEPACTION OF BLOOD PROTEINS WIIH SOLID SURFACES

7. Author(s)

R.E. Dehl, W.H. Grant

9. Performing Org.anization Name and Address

Polymer Science and Standards Division

Center for Materials Science

National Bureau of Standards

Washington, D.C. 20234

12. Sponsoring Organization Name and Address

Devices and Technology Branch

Division of Heart and Vascular Diseases

National Heart, Lung, and Blood Institute

Bethesda, Maryland 20014

15. Supplementary Notes

16. Abstracts

3. Recipi ent’s Accession No.

5. Report Date August 1979

8. Performing Organization Rept.

, NBSIR 79-1784

10. Project/Task/Work Unit No.

11. Contract/Grant No.

2 YOl-FW- 30014

13. Type of Report & Period

Covered

Annual

14.

The purpose of this investigation is to help develop methods of characterizing the

surface properties of implant materials for use in the cardiovascular system. These

properties are related to the ultimate success or failure of an implant in vivo. The

investigation during the current reporting period has been chiefly concerned with tlie

amount of protein deposited on implant material surfaces . Ion exchange treatment of

radiolabeled proteins has been found to lead to much greater accuracy and reliabili ty

of adsorption measurements . Results are reported for the quantitative adsorption of

labeled proteins thus purified onto well characterized test surfaces, and onto the

surfaces of materials supplied by contractors to the NHLBI Biomaterials Program. The

effect of the label (^^^I vs. ^“*0 on adsorption of proteins was studied. Hydrophobic

and hydrophilic oxidized silicon surfaces were used to demonstrate that the adsorption

of Y-glot)ulin, measured by ellipsometry, and serum albumin, measured by radiolabeled

protein, both are significantly affected by charge interactions between the surface

17. Key Words and Document Analysis. 17a. Descriptors and the protein.

Adsorption

Biomaterials

Blood Rrotei.ns

Ellipsometry

Polymer Adsorption

Protein Adsorption

Ihsotein Chromatogr^aphy

Radiolabeled Proteins

17b. Identifiers/Open-Ended Terms

17c. COSATI Field/Group

18. Availability Statement 19. Security Class (This 21 . No. of Pages

Report

)

UNCLASSIFIED 45

26. Security Class (Thii 22. Price Page

UNCLASSIFIED $4.50

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