The In Situ Synthesis Of Heparin-like Polysaccharide On The Surface Of Polyurethane

At present, the surface blood compatibility of material is one of the most important characteristics in the research of biomaterial. Many researchers have fixed heparin onto the surface of material through physical or chemical means. To some extent the anticoagulant activity of these materials has been improved. But some problems exist in these anticoagulated material modified by heparin, mainly focus on low anticoagulated activity, easily degradation by enzyme in vivo, uncontrollable activity. These materials will induce other immune response and side effects such as heparin induced thrombocytopenia. Thus, in order to enhance the blood compatibility of the material itself it is necessary to improve the way of modification on the surface of material.The structure of K5 polysaccharide is similar to that of desulfo-heparin. We can obtain heparin-like anticoagulant polysaccharide through the modification of sulfurtransferases (STs). In this paper an anticoagulant heparin-like substance biomaterial was in situ enzymatically synthesized on the surface of polyurethane in the purpose of improving the anticoagulant activity of polyurethane. The study including:(1) the isolation and purification of K5 polysaccharide and preparation of K5 polysaccharide derivatives with terminal aldehyde group, (2) the immobilization of K5 polysaccharide derivatives with terminal aldehyde group, (3) obtaining anticoagulant heparin-like polysaccharide derivatives on the surface of polyurethane through the combinatorial enzymatic method.Firstly, K5 polysaccharide was harvested from Eshcherichia coli Bi 8337/41 (O10:K5:H4) and then isolated from capsule of this strain. K5 polysaccharide derivative with teminal aldehyde group was prepared by a series of chemical methods, such as N-deactylation, N-sulfation and nitrous acid degradation. K5 derivatives was characterized by FT-IR, it indicated that K5 polysaccharide derivative with teminal aldehyde group was successfully prepared.K5 polysaccharide derivate was immobilized onto the surface of ammonited polyurethane through the reaction of reductive amiation. The density of immobilized K5 polysaccharide derivate was disccused by pH, tempreture and time. We found that it was benefit for immobilization under acid condition of pH 3.We analyzed the constitution of elements on the surface of material by X-ray photoelectron spectroscopy. We have confirmed that this end point attachment was successfully immobilized polysaccharide derivate on the surface of polyurethane because of the presence of aborbance of S2p at 168.8eV. An improved toluidin blue assay was applied to determining the density of immobilization K5 polysaccharide derivate, and the density of K5 polysaccharide derivate immobilized on the surface of polyurethane was 2.49μg/cm2.In Eshcherichia coli BL21 (DE3) host cells, four sulfurtansferases of AST-IV, 3-OST-1,2-OST and 6-OST-1 were successfully expressed. SDS-PAGE was used to verify the molecular weight of fusion protein product. Through Ni-NTA affinity chromatography resin,10.25mg/L of his-tagged fusion protein AST-IV and 4.4 mg/L of his-tagged fusion protein 3-OST-1 was isolated and purified separately. Through amylose resin,6.38 mg/L of MBP fusion protein 2-OST and 7.48 mg/L of MBP fusion protein 6-OST-lwas isolated and purified separately. Then enzyme activity unit of these four sulfurtransferases was determined. Finally, combinatorial emzymatic method was employed to modify K5 polysaccharide derivate on the surface of polyurethane, a heparin-like anticoagulant substance was emzymatic synthesized on the surface. Through a chromogenic substrate method, the activity of anti-Xa factor was 0.039U/cm2 on the surface.