Alginate/Polyethyleneimine-Based Nitric Oxide-Releasing Hydrogel As a Potential Platform to Study the Effects of NO on Carcinogenesis

Abstract

Numerous biological functions are affected by the functions of nitric oxide (NO), such as cell proliferation and programmed cell death. NOis a ubiquitous free radical gas that exerts a wide range of biological effects and acts as a signaling molecule in the body. Recent studies have indicated that nitric oxide regulates multiple cancer-related processes, such as angiogenesis, apoptosis, cell cycle, invasion, and metastasis. Alternatively, it is also emerging as a potential anti-oncogenic agent under other conditions. Nitric oxide is synthesized by a complex family of nitric oxide synthase (NOS) enzymes. There is encouraging interest in developing NO-releasing materials as potent tumoricidal agents in which high and localized concentrations of NO may be directly released in a sustained manner to the tumor site. The goal of this project is to develop a hydrogel that incorporates inducible nitric oxide synthase (iNOS) using a layer-by-layer building strategy to form layers of polyethyleneimine (PEI) and iNOSoxy as NO-releasing coatings on alginate hydrogel. When the hydrogel coated with PEI/iNOSoxy films are exposed to arginine, a source of reducing equivalents, and other required ingredients, nitric oxide is formed and released. In this work, FTIR spectroscopy was employed to characterize the functional groups of pristine sodium alginate (SA), polyethyleneimine (PEI) and SA/PEI composite hydrogels. We also used scanning electron microscopy (SEM) for surface characterization. Cyclic voltammetry was used to determine the amount of electroactive heme-enzyme adsorbed on the modified surfaces. We examine how the electroactive heme enzyme in the thin films correlates with the enzymatic NOS activity in terms of NO release fluxes from PEI/NOS-coated hydrogels. After the structural characterization of the NOS/hydrogel films using spectroscopy, we examined their function in terms of NO release profiles. We observed an initial “burst” of NO release during the first 4 hours of activity, followed by a decline and then stable NO release for up to 144 hours possibility to interrogate the role of NO on the balance of cell proliferation and cell death in these cell lines. The measured fluxes are higher than what have been reported in the literature for other inorganic NO-releasing systems. This data will allow us to build NOS-alginate hydrogels with defined NO release profiles for application in cell biology to test the effect of sustained NO release on cell proliferation and cell death on specific cancer cell lines.