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: [email protected]
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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.
DOI: 10.1002/mabi.200600210 195
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
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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
DOI: 10.1002/mabi.200600210
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.
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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
www.mbs-journal.de 197
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.
198
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
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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.
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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.
www.mbs-journal.de 199
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.
Macromol. Biosci. 2007, 7, 195–200
� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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
[1] E. Holmer, K. Kurachi, G. Soderstrom, Biochem. J. 1981, 193,395.
[2] P. Vongchan, W. Sajomsang, D. Subyen, P. Kongtawelert,Carbohydr. Res. 2002, 337, 1233.
[3] C. Sissi, A. Naggi, G. Torri, M. Palumbo, Semin. Thromb.Hemost. 2001, 27, 483.
[4] R. M. Maaroufi, M. Jozefowicz, J. Tapon-Bretaudiere,A. M. Fischer, Carbohydr. Res. 2006, 341, 672.
[5] M. Akashi, N. Sakamoto, K. Suzuki, A. Kishida, Bioconjug.Chem. 1996, 7, 393.
[6] X. L. Sun, D. Grande, S. Baskaran, S. R. Hanson, E. L. Chaikof,Biomacromolecules 2002, 3, 1065.
[7] S. Baskaran, D. Grande, X. L. Sun, A. Yayon, E. L. Chaikof,Bioconjug. Chem. 2002, 13, 1309.
[8] M. Mauzac, N. Aubert, M. Jozefowicz, Biomaterials 1982, 3,221.
[9] F. M. Kanmangne, D. Labarre, H. Serne, M. Jozefowicz, Bio-materials 1985, 5, 297.
[10] C. Douzon, F. M. Kanmangne, H. Serne, D. Labarre,M. Jozefowicz, Biomaterials 1987, 8, 190.
[11] T. Pompe, S. Zschoche, N. Herold, K. Salchert, M.-F. Gouzy,C. Sperling, C. Werner, Biomacromolecules 2003, 4, 1072.
[12] C. Sperling, R. B. Schweiss, U. Streller, C. Werner, Biomaterials2005, 26, 6547.
[13] R. Grombe, M.-F. Gouzy, M. Nitschke, H. Komber, C. Werner,Colloids Surf. A: Physicochem. Eng. Aspects. 2005, 284–285,295.
[14] U. Freudenberg, S. Zschoche, F. Simon, A. Janke, K. Schmidt,S. H. Behrens, H. Auweter, C. Werner, Biomacromolecules2005, 6, 1628.
[15] in preparation.[16] T. Pompe, L. Renner, M. Grimmer, N. Herold, C. Werner,
Macromol. Biosci. 2005, 5, 890.[17] R. M. Hakim, Cardiovasc. Pathol. 1993, 2, 187S.[18] M. B. Gorbet, M. V. Sefton, Biomaterials 2004, 25, 5681.[19] B. Casu, M. Guerrini, G. Torri, Curr. Pharm. Des. 2004, 10, 939.[20] M. Petitou, C. A. A. van Boeckel,Angew. Chem. Int. Ed. 2004, 43,
3118.
DOI: 10.1002/mabi.200600210
Top Related