Communication

Sulfated Glycopolymer Thin Films—Preparation, Characterization,and Biological Activity

Ringo Grombe, Marie F. Gouzy, Manfred F. Maitz, Uwe Freundenberg,Stefan Zschoche, Frank Simon, Tilo Pompe, Claudia Sperling,Carsten Werner*

The impact of heparinoid characteristics on model surfaces obtained from immobilization ofsole sulfate groups as well as sulfated glycosides, sulfated cellulose, and definite heparin hasbeen investigated. The obtained layers were physico-chemically characterized regarding filmthickness, chemical composition, wettability, andsurface morphology. Antithrombin adsorption, stu-died by fluorescence labeling, revealed a strongdependence on the presence of glycosidic structuresand on the molecular weight of the grafted saccha-ride. On contact with whole blood, the coatingsresulted in a diminished plasmatic and cellularcoagulation in vitro, which did not reflect well theantithrombin binding. Therefore, more complexactivating pathways are discussed.

Introduction

Blood coagulation is the result of an activation of

plasmatic coagulation factors such as thrombin (T) and

factor Xa (FXa). These factors are regulated by antithrom-

bin (AT) complexation catalyzed by heparin. Heparin

immobilization is an applied technique to reduce blood

coagulation; however, the structure–function relation is

R. Grombe, M. F. Gouzy, M. F. Matiz, U. Freundenberg, S. Zschoche,F. Simon, T. Pompe, C. Sperling, C. WernerLeibniz Institute of Polymer Research, Max Bergmann Center ofBiomaterials, Hohe Strasse 6, 01069 Dresden, GermanyC. WernerInstitute of Biomaterials and Biomedical Engineering, Universityof Toronto, 5, King’s College Road, M5S 3G8 Toronto, Ontario,CanadaFax: (þ1) 351 4658 533; E-mail: werner@ipfdd.de

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still unsolved. Heparin, bearing sulfate, and carboxylate

groups along the polysaccharide chain, belongs to the

glycosaminoglycans (GAGs). Variations in the chain length

and sulfation degree of GAGs determined their anti-

coagulant activity.[1–4]

The anticoagulant activity of sulfated glycopolymers

containing a linear hydrocarbon backbone varied with the

molecular weight, the degree of sulfation, and the density

of attached sulfated glycoside units.[5–7] Also synthe-

tic particles with randomly distributed carboxylates

and sulfonates showed enhanced antithrombic pro-

perties.[8–10]

We now report investigations on maleic anhydride

copolymer (MA) films being a versatile base for biomater-

ials.[11] A set of different molecules was immobilized to

explore the structure–function relation of the synthetic

polymer architectures as shown in Figure 1. Furthermore,

the heparinoid features to be mimicked are listed in Table 1.

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R. Grombe et al.

Figure 1. Schematic presentation of the MA copolymer modifications. The picture shows the grafting sites of the copolymer and theintroduced functionalities (bold). The comonomer units (ethylene-, propylene units) are not drawn.

196

The film thickness, chemical composition, wettability, and

surface morphology have been determined by ellipsometry,

X-ray photoelectron spectroscopy (XPS), static contact angle

measurement, and atomic force microscopy (AFM), respec-

tively. The impact of the sulfate groups, the glycoside, and

polysaccharide structures present at the thin films on AT

binding was analyzed. Samples were incubated in vitro with

whole blood to examine the activation of coagulation,

thrombocytes, and complement.

Experimental Part

AT Binding Studies by Confocal Laser Scanning

Microscopy (cLSM)

Two samples of each type were incubated with 0.1 U �mL �1 AT in

PBS for 30 min. As reference, a third sample was incubated with

Table 1. Listing of the functional groups, the heparinoid feature at t

Label Functional group

HYDRO COO�

AEHS OSO�3

GLC, MALT-sulf OH, OSO�3

CEL-sulf OH, OSO�3

HEPARIN OH, HNSO�3 , OSO�

3 , C

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PBS buffer. Non-specific binding places were blocked using a

60 mg �mL�1 normal donkey serum solution for 30 min, then the

adsorbed AT was labeled with a FITC-conjugated antibody (sheep

antihuman AT, Cedarlane Laboratories Ltd) for 1 h. The surfaces

were analyzed on a TCS SP microscope (Leica, Bensheim,

Germany). Averages of at least five measurements of six images

on each sample are reported.

In Vitro Hemocompatibility Assay

The experimental procedure for the whole blood incubation is

described elsewhere.[12] The analysis was performed in two

independent runs with freshly drawn and heparinized blood

(2 IU �mL�1) of the same donor. Altogether, six values were

obtained for the parameters in the blood phase whereas surface

bound parameters were derived from four measurements each.

Borosilicate glass slides were used as positive control.

he surface and the sample labels corresponding to Figure 1.

s Mimicked heparinoid feature

Blind

Sulfate charge

Glycoside structure

Polysaccharide backbone

OO� Heparinoid surface

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Sulfated Glycopolymer Thin Films—Preparation, Characterization, and Biological Activity

Synthesis

The molecules 2-[2-(2-aminoethyl)-ethyl]-ethyl-b-D-glucopyrano-

side (GLC) and 2-[2-(2-aminoethyl)-ethyl]-ethyl-4-(a-D-glucopy-

ranosyl)-b-D-glucopyranoside (MALT), displayed in Figure 2, were

synthesized following a reported procedure.[6,13]

Surface Modification

Silicon wafers and glass coverslips were spin-coated with

poly(ethylene-alt-maleic anhydride) copolymer (PE-MA, Mw ¼125 000, Sigma-Aldrich) and poly(propylene-alt-maleic anhydride)

copolymer (PP-MA, Mw ¼ 39 000, Leuna-Werke AG, Germany) as

described in detail elsewhere.[11] The samples obtained from

chemical modification of the MA supports are depicted in Figure 1

and were named as follows. The hydrolyzed MA layers were

defined as HYDRO whereas the surfaces obtained from immobi-

lization of 2-amino ethylene hydrogen sulfate were labeled AEHS.

Sulfation of the samples bearing glucoside GLC or maltoside MALT

(see Figure 2) resulted in GLC-sulf or MALT-sulf, respectively. AEHS,

GLC-sulf, and MALT-sulf had PP-MA as polymeric support.

Cellulose and heparin were grafted on PE-MA. The samples

gained from cellulose immobilization were termed CEL-sulf after

sulfation. The heparinized surfaces were labeled HEPARIN.

Regarding AEHS (Aldrich-Fluka Chemicals), the PP-MA samples

were immersed in 15 or 100� 10�3M solutions (pH 10, borate

buffer) at room temperature overnight. After rinsing with

Millipore water, an annealing step at 120 8C for 2 h was done.

Non-immobilized AEHS molecules were washed off with a 0.01 M

NaOH solution. Subsequently, a rinsing step was carried out with

water and the samples annealed at 120 8C for 2 h. The glycosides

GLC and MALT were immobilized from a 10�10�3M solution

(pH 8, borate buffer) as reported previously.[13]

PE-MA thin films were modified with heparin (Aldrich-Fluka

Chemicals) or cellulose ‘‘Avicel’’ (DP 215–240, Aldrich-Fluka

Chemicals). Concerning heparin, PE-MA surfaces were immersed

into a 0.1 M solution of 1,4-diaminobutane in isopropanol for

4 min. The samples were then immersed subsequently into

isopropanol (1 min), Millipore water (1 min), and 0.01 M HCl

solution (1 min). After rinsing twice in water for 1 min, the

samples were dried under N2 stream and annealed at 120 8C for

Figure 2. Structural formula of the aminoterminated glycosides.

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2 h. The aminated polymeric supports were immersed into a

borate buffered solution (0.1 M, pH 8) containing heparin

(5 mg �mL�1) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodi-

imide hydrochloride (EDC) (400�10�3M) for 4 h. Subsequent

rinsing with Millipore water was followed by autoclaving at

120 8C for 20 min. The samples were finally dried under N2 stream.

The cellulose was immobilized from a 0.5 wt.-% dimethyl

sulfoxide/4-methylmorpholine-4-oxide monohydrate (DMSO/

NMMO, 2/3 wt.-%) solution following a described procedure.[14,15]

The gained samples were immersed into Millipore water and

air-dried overnight. Annealing (120 8C, 2 h) was followed by

submerging the samples twice in water for 1 h, drying under N2

stream, and placing into a vacuo oven at 35 8C for 2 h.

Sulfation of the attached glycosides and cellulose was

performed using a 5 wt.-% SO3�NMe3/DMF solution at 908Cfor 3 h. After cooling down, the surfaces were washed thoroughly

with DMF and submerged into 0.01 N NaOH solution for 10–

15 min. Afterwards, they were kept in Millipore water overnight,

dried under N2 stream, and heated to 120 8C for 2 h.

Results and Discussion

Characterization of the Modified Surfaces

The film thickness of the PP-MA and PE-MA layers as well

as the film growth after sulfation are listed in Table 2. The

insignificant differences in the values prior to sulfation

(data not presented) were taken as proof of the stability of

the films.

The film growth of AEHS may result from electrostatic

repulsion of the carboxylate and sulfate groups as well as

from rearrangement of the polymeric chains. Pompe et al.

reported the swelling behavior of MA layers in aqueous

media.[16] Upon this swelling glycosides penetrate into the

layer as shown in earlier investigations and the alcoholic

groups may form inter- and intramolecular cross-links

with the MA chains.[13] Cross-linkage causes a more rigid

glycopolymer layer resulting in the film growth of GLC-sulf

and MALT-sulf.

Heparin and cellulose were covalently attached to

PE-MA supports. Sulfation of the grafted cellulose (CEL-

sulf) resulted in 8.4 nm of film growth. The reported value

for the heparinized sample is a rough orientation for the

film growth as still adsorbed water—due to the grafting

procedure—may cause a swollen state.

The elemental composition of the sulfated surfaces was

analyzed by XPS and the sulfur/carbon (S/C) ratio was

calculated (Table 2). Immobilization of a 15� 10�3M AEHS

solution resulted in still non-grafted anhydride function-

alities as the S/C ratio increased for the 100� 10�3M.

solution. The different S/C ratios for the glycopolymer,

GLC-sulf, and MALT-sulf, were due to the size of the

glycone moieties. Although cellulose is known for its low

solubility in many solvents, a high S/C ratio was found.

This suggests a greater OH quantity at the outermost layer

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R. Grombe et al.

Table 2. Data obtained from surface characterization of modified PP-MA and PE-MA samples after sulfation (5 wt.-% SO3 �NMe3/DMF,90 8C, 3 h).

Label Grafting condition Dddryc) S/Cd) ratio Q(H2O)e)

nm 8

HYDROPP-MAa) – 2.8W 0.3 – 71W 2

AEHSb) 15T 10S3M, pH 10 2.3W 0.2 0.042 33W 6

(3.0W 0.1) (0.06) n.d.f)

GLC-sulf 10T 10S3M, pH 8 2.6W 0.2 0.02 63W 3

MALT-sulf 10T 10S3M, pH 8 3.6W 0.6 0.032 58W 1

HYDROPE-MAa) – 6.3W 1.1 – 72W 3

CEL-sulf 0.5% NMMO, DMSO 8.4W 0.05 0.047 43W 3

spin coating

HEPARIN 5 mg �mLS1 5.4W 0.2 0.003 n.d.

EDC, sulfo-NHS, pH 8

a)HYDRO sample in anhydride state; b)Values in brackets derived from samples using a 100T 10S3M AEHS solution later

taken for the biological tests (AT adsorption, in vitro whole blood incubation); c)D ddry: Film growth as determined by

ellipsometry in dry state (mean value of three measurements); d)S/C ratio: sulfur/carbon ratio as calculated from XPS;e)Q(H2O): static water contact angle; f)n.d., Not determined.

Table 3. Grafting yields and ratio of sulfate/copolymer unit (sulf/copo) of the modified polymeric MA layers.

Label Grafting yield Sulf/copo ratio

% %

AEHSa) 42 (57) 42 (57)

GLC-sulf 46 25

MALT-sulf 46 45

CEL-sulf 61 50

HEPARIN n.d.b) n.d.

a)Values in brackets derived from samples using a 100 mM

AEHS solution later taken for the biological tests (AT

binding, in vitro whole blood incubation); b)n.d., Not deter-

mined.

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for the cellulose sample compared to the glycopolymers.

The S/C ratio for the heparinized PE-MA sample was the

lowest among all samples indicating a low heparin density

at the surface.

The static contact angles (listed in Table 2) of the PE-MA

and PP-MA films were the highest ones. The angles

decreased upon immobilization and sulfation. The differ-

ences between the angles of AEHS and those of the sulfated

glycopolymers (GLC-sulf, MALT-sulf) support the hypoth-

esis that the glycosides act as a cross-linker with the

produced ester bonds enhancing the hydrophobicity of the

polymer layers. The static contact angle of CEL-sulf

revealed a hydrophilic surface.

The alteration in surface morphology upon sulfation

was investigated. No significant changes were found even

after a prolonged reaction time of 24 h. The calculated

surface roughness (Ra) of the glycopolymer films was

similar; 0.4 nm before and 0.2 nm after the sulfation.

Immobilized cellulose caused a surface roughness of 2.9 nm

which changed slightly to 2.4 nm upon sulfation.

The grafting yields were estimated by linear equation

analysis from the elemental composition of thin films. The

atomic concentrations of carbon—CXHY (aliphatic), C–OH

(alcoholic), C––O (carboxylic)—nitrogen, sulfur, and silicon

oxide were determined by XPS. The theoretical sample

contained layers of SiO2 aminosilane, and MA copolymer

with the incorporated derivative. The grafting yields are

listed in Table 3.

The AEHS grafting yields were dependent on the

concentration of the used solution. GLC-sulf and MALT-sulf

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had the same grafting yields showing the size of the

glycoside to have no influence. The grafting yield for the

cellulose immobilization corresponded well to 58% found

by ellipsometry, where the PE-MA layer was 6.3 nm and

the CEL-sulf layer 8.4 nm (see Table 2).

The sulfate/copolymer unit (sulf/copo) ratio is also

shown in Table 3. In case of AEHS, grafting yield and sulf/

copo ratio are equal as a matter of principle. For other

samples the sulf/copo ratio was calculated from the

grafting densities and the sulfur atomic-%. The higher ratio

for MALT-sulf compared to GLC-sulf can be explained by

the molecular weight of the saccharide. Comparison of the

DOI: 10.1002/mabi.200600210

Sulfated Glycopolymer Thin Films—Preparation, Characterization, and Biological Activity

cellulose grafting yield and the sulf/copo ratio indicates

partially substituted OH functions at the cellulose.

Surface Binding of AT

Antithrombin binding to the surfaces was investigated by

measuring the fluorescence intensities of FITC-labeled

samples (Figure 3). All surfaces showed AT adsorption over

the blank (black bars). There was no obvious difference

between the hydrolyzed unmodified PE-MA and PP-MA

surface (HYDRO) (data not presented). The samples AEHS

and HYDRO had the lowest AT binding capacities. This

demonstrates that the presence of carboxylate groups or

solely the introduction of sulfates is not sufficient to

enhance the affinity of the surface towards AT. The

combination of pyranoside structures and sulfate groups

(GLC-sulf, MALT-sulf) led to a stronger AT binding. A

dependence of AT adsorption on the molecular weight of

the grafted units was observed. This was in agreement

with the polysaccharidic samples HEPARIN and CEL-sulf.

The high binding to HEPARIN, despite the low heparin

density in our samples, indicates that the sulf/copo ratio is

of minor importance compared to the saccharide chain

length.

Whole Blood Incubation Assays

The plasmatic and cellular coagulation was investigated

by means of the thrombin–antithrombin complex (TAT)

and platelet factor 4 (PF4), respectively [Figure 4(A)]. The

TAT values were ranked by size and the rank-values were

averaged. Sample MALT-sulf induced most of the TAT

formation among the modified surfaces. The other samples

Figure 3. Fluorescence intensities of FITC-labeled samples inves-tigated by cLSM; grey bars—ATþ FITC labeled anti-AT, blackbars—FITC-labeled anti-AT (blank), HYDRO corresponds to thehydrolyzed PP-MA surface.

Figure 4. Whole blood incubation (2 h) of GLASS and differentlymodified MA copolymer thin films using heparinized freshlydrawn blood (2 IU �mL�1); formation of (A) TAT and PF4, (B)C5a and C5b-9; HYDRO corresponds to the hydrolyzed PP-MAsurface.

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had mainly equal TAT concentrations apart from the

heparinized sample activating the smallest quantities of

TAT. Even small amounts of heparin at the surface resulted

in T deactivation. A further correlation to the AT binding

was not observed.

Highest PF4 amounts were detected for GLASS and

MALT-sulf. At the other surfaces, less but not different

platelet activation was found. No relation to chemical or

physical surface properties could be established. The

correlation of PF4 and TAT, however, indicates an

interaction of the two hemostatic systems. Thrombocyte

activation, also leading to T activation, may superpose the

inhibiting effects of bound AT.

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R. Grombe et al.

200

Solvable complement C5a and surface complement

C5b-9 were determined as markers of non-specific

immune response [Figure 4(B)].

Nucleophilic groups at surfaces generally cause en-

hanced complement activation due to the formation of

ester bonds with the complement factor C3b catalyzing the

cleavage of C5 in C5a and C5b.[12,17] In this study, low C5a

values were found for HYDRO (absence of alcoholic groups)

and GLC-sulf (few OH functions at the surface due to low

grafting yield). The higher C5a production at MALT-sulf

may have been derived from a higher extent of alcoholic

groups (less hydrophobic character). AEHS showed about

nearly same C5a concentration as MALT-sulf although no

alcoholic groups are present at the sample. The samples

CEL-sulf and HEPARIN caused the highest C5a values.

According to the literature, heparin should diminish

the complement activation at surfaces by binding the

complement inhibiting factor H.[18] Presumably, due to the

small amounts of immobilized heparin or to its sterical

arrangement at the surface, the inhibition in the present

study was negligible, compared to the activation potential

of the OH groups at CEL-sulf.

On the surfaces, low C5b-9 formation was observed at

the hydrolyzed copolymer films whereas GLASS and the

heparinized sample showed high values [see Figure 4(B)].

Otherwise the correlation with the soluble C5a was low. As

C5b-9 is not covalently bound to the substrate, adsorption–

desorption processes may affect the obtained values.

Conclusion

To express heparinoid characteristics at model surfaces,

sulfate groups, or sulfated saccharidic structures were

tested for their AT binding and complex interaction with

blood in order to evaluate structure–function relations.

Antithrombin binding could be well targeted by these

properties. Sole but densely packed sulfate groups had only

minor influence on AT binding. The affinity increased with

the combination of sulfates and pyranose structures. The

saccharide chain length and/or steric arrangement of the

sulfate groups appeared to have a superior influence on AT

binding over the sulfate group density. Thus, molecular

requirements improving AT binding to surfaces seem to be

similar to those found in solution.[19,20]

By in vitro incubation of the modified MA films on

whole blood coagulation activity was found to be

independent of AT binding capacity. Superposing effects

from direct activation of other procoagulant pathways

such as blood platelet activation are influential.

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Acknowledgements: This work was funded by the GermanFederal Ministry of Science and Education (grant No. 03N4022:‘‘BMBF Kompetenzzentrum fu Materialien im Blut- und Gewebe-kontakt’’). Dr. H. Komber, Dr. M. Nitschke, R. Schulze, and D. Voigt(all from the Leibniz Institute of Polymer Research Dresden e.V.)are gratefully acknowledged for the NMR, XPS, ellipsometry, andMALDI-TOF measurements, respectively. The authors are alsothankful to Mrs. G. Eberth for preparing the heparinized samples.

Received: September 22, 2006; Accepted: November 13, 2006;DOI: 10.1002/mabi.200600210

Keywords: adsorption; anticoagulation; antithrombin adsorp-tion; cellulose growth; mono-/diglycosides; serine protease;sulfate groups

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