Novel Nitric Oxide Releasing Films Based on Inducible Nitric Oxide Synthase and Their Biomedical Applications

Abstract

Although a tremendous number of biomaterials are labeled as biocompatible for blood-contacting medical devices, such as vascular grafts, stents, heart valves, and catheters, the thrombogenic nature of these materials can cause serious complications in patients, and eventually functional failure. There are two major limiting factors to clinical application of these materials: 1) platelet activation and thrombus formation, and 2) infection. Nitric oxide (NO) has been known to exert anti-thrombotic, anti-proliferative, and anti-inflammatory effects in the vasculature. Several polymers incorporate NO-generating substances in order to improve biocompatibility of blood-contacting medical devices. However, an important drawback for these NO-release materials is the finite stoichiometric amount of embedded NO that they contain, which could impact their function for long-term or semi-permanent types of implants. In addition, controlling the release rates of NO flux that can be emitted at the polymer/blood interface proved to be challenging, and often requires the addition of other components to the polymer. Therefore, we hypothesized that incorporating the enzyme nitric oxide synthase into polymeric films as functional catalytic units that generate NO would result its antithrombotic coatings with potentially unlimited amounts of NO released.In this project, we use layer-by-layer thin film building strategy to form layers of polyethyleneimine (PEI) and recombinant NOS enzymes as NO-releasing coatings. Charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films with defined thickness and catalytic properties. When surfaces coated with PEI/NOS multilayered films are exposed to substrate arginine, a source of reducing equivalents, and other ingredients of the NOS reaction, nitric oxide is formed and released. In this work, we characterize the PEI/NOS thin films in terms of structure of NOS within the films as well as the amount of active NOS. Fourier transform infrared (FTIR) spectroscopic analysis was used to characterize structure-activity relationships of these NOS-containing thin films. We used cyclic voltammetry to determine the active enzyme concentration on the modified surfaces. We also examined how this activity relates to enzymatic activity in terms of NO released fluxes from the thin films. Finally, we assessed the functional performance of these films in terms of the extent to which they counteract platelet adhesion on the coated surfaces. To this end, we used platelet adhesion assays to determine how the number of platelets adsorbed on the PEI/NOS films is affected by the amount of NO released from the PEI/NOS thin films.