Elsevier

Biomaterials

Volume 26, Issue 35, December 2005, Pages 7418-7424
Biomaterials

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer

https://doi.org/10.1016/j.biomaterials.2005.05.053Get rights and content

Abstract

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 μg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.

Introduction

Silicones have a long history of use as biomaterials in applications ranging from intraocular lenses to catheters as a result of their good bulk properties, reasonable biocompatibility, processability and low cost. However, the relatively high surface hydrophobicity of these materials, which results in the adsorption of significant amounts of protein [1], constrains their use in biomedical applications. While potentially desirable in some applications [2], [3], the non-specific adsorption of proteins is generally considered detrimental to performance, as it has been widely demonstrated that the initially adsorbed protein layer is responsible for mediating subsequent biological effects [4]. In blood contacting applications, the non-specific adsorption of plasma proteins to an artificial surface is known to mediate subsequent thrombotic and immunogenic effects with potentially catastrophic results. Therefore, it is necessary to alter the surface properties of PDMS in order to render the material more biocompatible.

Heparin, a heterogeneous extended polymer of repeating sugar units with molecular weight ranging from 3000 to 30,000 and an average molecular weight of 15,000, and related compounds are among the most frequently used therapeutic agents for thrombin regulation. The utility of heparin-modified surfaces including heparin-modified silicone surfaces, however, has been mixed [5], [6]. Heparin contains a key pentasaccharide sequence that binds to the inhibitor antithrombin; this complex has a significantly increased reaction with serine proteases including thrombin compared with free heparin [7]. The maintenance of an intact three-dimensional structure, particularly one in which the accessibility of the key active pentasaccharide is maintained, is necessary for activity of the heparin molecule. One method to stabilize biomolecules at interfaces is to tether them through a poly(ethylene glycol) (PEG) spacer [8], which presumably has the additional advantage of reducing the non-specific adsorption of proteins [9]. There have been a number of studies where heparin has been immobilized to a variety of surfaces using a PEG spacer [10]. Heparin immobilization via a PEG spacer has been demonstrated to enhance its bioactivity relative to direct immobilization [11], [12], [13], [14], [15], [16].

Most PEG derivatives used for linking of biomolecules are limited to homobifunctional polymers and polymers with one reactive terminus and one unreactive terminus [17]. However, tethering of biological molecules via a homobifunctional PEG can result in a lower surface density due to the potential for multiple reactions between the PEG termini and the surface. The ability to tether biological molecules via a heterobifunctional polymer is therefore expected to result in more homogeneous, higher density surfaces.

We have previously reported on a method for generating surfaces with a high PEG density that show significant reductions in the non-specific adsorption of plasma proteins [18]. Herein we report on the surface first heparinization of PDMS surfaces via a heterobifunctional PEG spacer and the subsequent biological properties of the heparinized material.

Section snippets

Reagents

PEG monoallylether (average MW 500) was obtained as a gift from JuTian Chemical Co. (Nanjing, China). It was dried by azeotropic distillation with toluene before use. N,N′-Disuccinimidyl carbonate, o-xylene (97%, anhydrous), triethylamine (99%), acetonitrile (99%, anhydrous), Karstedt's Pt catalyst (2–3 wt% in xylene, [(Pt)2(H2Cdouble bondCH–SiMe2OSiMe2CHdouble bondCH2)3]), 2-mercaptoethanol, CF3SO3H were purchased from Aldrich Chemical Co. The synthesis of α-allyl-ω-N-succinimidyl carbonate-poly(ethylene glycol) has

Results

Advancing, receding and captive bubble water contact angles, measured on the control PDMS, and PDMS surfaces modified with PEG and PEG-heparin, respectively, are summarized in Fig. 1. Unmodified silicones showed characteristically high advancing, receding and captive bubble water contact angles (∼100°), as expected. Little hysteresis was observed on this surface, likely the result of its hydrophobicity and smoothness. The water contact angle for the control surface obtained by the captive

Discussion

Heparin, a well-known and widely used anticoagulant, has been extensively studied as a potential surface modifier for increasing the thromboresistance of biomaterials. It acts by catalyzing the reaction between thrombin and/or Factor Xa and ATIII. Thrombin and Factor Xa inhibition by ATIII occurs at a rate approximately 100 times higher in the presence of heparin than with free AT [25]. The acceleration results from heparin binding to AT, which induces an allosteric modification in the AT

Conclusions

Heparin was immobilized on polydimethylsiloxane elastomer surfaces through a heterobifunctional PEG spacer. Heparin immobilization was confirmed by XPS and by a toluidine blue assay, with the heparin density found to be 0.68 μg/cm2. There was a significant decrease in thrombin activation as measured using a chromogenic assay. While plateau values occurred with the control surfaces in 20–40 min, the heparin-conjugated surfaces did not show a plateau in the more than 3 h duration of the assay. While

Acknowledgements

The technical assistance and valuable insights of Rena Cornelius, Glenn McClung and Dr. John Brash are gratefully acknowledged. We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support.

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    Current address: School of Material Science and Engineering, Wuhan University of Technology, 133 Luoshird, 430070 Wuhan, PRChina.

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