Nitric Oxide Releasing Alginate/Polyethyleneimine Hydrogel Based on Inducible Nitric Oxide Synthase for Antithrombotic Activity

Abstract

Although a tremendous number of Biomaterials are recently labelled 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), a small signaling molecule generated from natural endothelial cells has been known to exert anti thrombotic, anti-proliferative, and anti-inflammatory effects in the vasculature. In addition to, NO that is released by neutrophil/macrophages functions as a potent natural antimicrobial agent. Nowadays, there are several different polymers incorporating nitric oxide generating substances for improving 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 has confirmed to be challenging, and often requires the addition of other components to the polymer. The goal of this project is to develop a hydrogel that incorporates inducible nitric oxide synthase (iNOS) using a layer-by-layer thin film building strategy to form layers of polyethyleneimine (PEI) and iNOSoxy as NO-releasing coatings on alginate hydrogel. Here, the iNOSoxy enzyme protein used is negatively charged and adsorbed onto the positively charged matrix layer polyethyleneimine which is built on a negatively charged sodium alginate hydrogel. When hydrogel coated with PEI/iNOSoxy films are exposed to arginine, a source of reducing equivalent, and other required ingredients, nitric oxide is formed and released. We characterize the Sodium alginate/PEI/iNOSoxy hydrogel in terms of structure of iNOSoxy within the films as well as the amount of active concentration and NO release profile. Spectroscopic and electrochemical analysis show the structure-activity relationships of these NOS-containing thin films and how this relates to enzymatic activity and resulting NO release fluxes from sodium alginate/PEI/NOS-containing hydrogel. 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 hydrogel is affected by the amount of NO released from the PEI/NOS hydrogel.