Date of Award

2014

Degree Type

Dissertation

Department

Chemistry

First Advisor

Bayachou, Mekki

Subject Headings

Nitric-oxide synthase, Implants, Artificial, Biomedical materials, Polymers in medicine, Nanostructured materials, Chemistry

Abstract

Nitric oxide synthase enzyme (NOS) embedded in thin films and scaffolds, when exposed to a solution of its substrate arginine, a source of reducing equivalents, and other required ingredients of the NOS reaction, can release fluxes of nitric oxide (NO). The latter is a molecule known to counteract platelet aggregation, and thus can prevent the thrombosis cascade on the surfaces of implantable medical devices. Therefore NO antithrombogenic regimens such as active coatings and embedded scaffolds have the potential to increase the lifespan of implantable biomaterials. Layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/polyelectrolytes nanostructured films. Electrospun fiber matrices may embed proteins in aqueous pockets that allow the formation of functional scaffolds. Such functional coatings and polymer scaffolds have potential applications as antithrombotic surfaces. In this project, inducible nitric oxide synthase is proposed as a functional component in active thin films and electrospun scaffolds for nitric oxide release under physiologic conditions. Atomic force microscopic (AFM) imaging confirms the presence of enzyme in adsorbed thin films. Fourier transform infrared (FTIR) spectroscopic analysis was used to characterize structure-function relationships of NOS-containing thin films. Voltammetry was used to characterize the active catalyst concentration on adsorbed surfaces and activity of NOS-containing thin films. Further, analysis of cyclic voltammetric data enabled the study of Michaelis-Menten kinetics of NOS-containing thin films. Other spectrophotometric and spectrofluorometric assays were used to monitor nitric oxide release for the NOS-based thin films and scaffolds. Three polymers were characterized for their ability to embed iNOSoxy and show enzyme activity in electrospun scaffolds. For the LbL method, during protein adsorption, an adjusted pH of 8.6 or the use of a branched matrix immobilizes more enzyme units in thin layers compared to pH 7.0 or a li

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