Potential Anti-Thrombotic Nitric Oxide Generating Layer-By-Layer Assembly.

Abstract

Nitric oxide (NO) is a potent platelet inhibitor released by healthy endothelial cells (EC) lining the human vasculature. Polymeric materials capable of releasing NO from synthetic NO donors have exhibited enhanced hemocompatibility in vivo. Alternatively, NO can be catalytically generated from endogenous S-nitrosothiols (RSNO) by some organodiselenide species (RSe). In this dissertation research, a Layer-by-Layer (LbL) assembly has been developed to immobilize RSe catalysts on diverse biomedical devices/materials to achieve surface NO generation. Because the preparation of such coatings is based on pure electrostatic interaction, universal application of this approach is expected. The catalytic LbL was fabricated by manually depositing RSe linked to polyethyleneimine (SePEI) with sodium alginate (Alg) on quartz surfaces in an alternate fashion. This (SePEI/Alg)n multilayer demonstrated potent catalytic activity to decompose RSNO, to NO in presence of free thiols. Under exaggerated reaction conditions, such an LbL exhibited good stability. The NO generated from given concentrations of S-nitrosoglutathione and glutathione is proportional to the number of (SePEI/Alg) bilayers deposited. The LbL was applied to silicone rubber and polyurethane substrates to demonstrate its applicability on commercial biomedical grade materials. The fabrication of (SePEI/Alg)n LbL was ultimately automated using a commercial coating apparatus to achieve higher NO flux by increasing the number of (SePEI/Alg) bilayers. Under typical biological conditions, a (SePEI/Alg)100 was discovered to generate an NO flux of 1.5 x 10-10 mol cm-2 min-1 using physiological RSNO levels, which is comparable with that of human EC layer. Even after prolonged contact with blood, the multilayer still showed significant NO generation activity. These automatically prepared LbLs were then successfully applied on exemplary metal surfaces (nitinol, titanium and stainless steel). In addition, heparin was immobilized on the surface of (SePEI/Alg)n via either electrostatic interactions or covalently attaching the anti-coagulant on the aminated LbL surface. Both strategies lead to a surface with combined NO generation and anti-FXa activity. However, the covalent immobilization of heparin resulted in a decreased NO generation activity of the underlying catalytic LbL, due to decreased permeability of glutathione into the films.