Many surgical interventions that are performed to mitigate the complications of cardiovascular and other diseases entail the introduction of long-term and short-term blood-contacting devices such as intravascular catheters and sensors, grafts, and coronary artery and vascular stents, to cite a few. However, due to the thrombogenic nature of the surface of these devices, such a process may lead to recurring problems and more complications at the injured site, including clot formation, a process known as thrombosis, which is often triggered at the surface of the foreign device. 80% of vascular access dysfunction is caused by graft thrombosis, which alone comes with an associated health-care cost of over $1 billion/year. Nitric oxide (NO) is known to counteract thrombosis in the body. Nitric oxide releasing biopolymers have the potential to prolong vascular graft and stent potency without adverse systemic vasodilation. Currently, the development of NO-based coatings, while promising, is relatively limited in the context of the need of stable materials that are capable of sustained and prolonged nitric oxide release. This is partly due to the finite amounts of NO equivalents that can be loaded in the coating. The goal of this project is to develop stable NO-releasing thin films as biocompatible coatings for short- and long-term implantable medical devices where nitric oxide release is facilitated by embedded Nitric Oxide Synthase (NOS) enzymes. The objective of this particular application is to study these enzyme-based NO-releasing thin films through the development of NOS-based polyethyleneimine (PEI) polymeric coatings built by the layer-by-layer methodology. The enzyme- driven NO generation will use endogenous compounds found in the blood matrix to release NO at the blood/polymer-device interface. Preliminary observations in our hands indicate that purified recombinant NOS enzymes retain their structure and catalytic functions when embedded in thin films on surfaces. We hypothesize that this will allow the endogenous compounds available in blood to initiate and sustain the enzymatic reaction, and thus NO release, at the interface between the polymeric surface and blood, leading to enhanced thrombo-resistance of the materials. In order to test this hypothesis, we propose the following three specific aims: 1) Preparation and characterization of biocompatible polymeric coatings with embedded Nitric Oxide Synthase enzymes. 2) Evaluation of the performance of the NOS-based polymeric coatings formed under various conditions in terms of sustained NO production and levels of NO-release. 3) ) [In vitro evaluation of performance of NOS- based coatings through in vitro platelet adhesion assay; this assay would inform us about the potential of the NOS-based film for thromboresistivity in a later stage of the work, which is outside the scope of this proposal]; The Layer-By-Layer method will be used to prepare nanostructured NOS-based bio-polymeric coatings. The structural and functional integrity of NOS within the thin film will be investigated using spectroscopic and electrochemical tools.
Under aim#2, the NOS-based PEI thin films will be evaluated for levels and sustainability of NO release. NO-fluxes will be evaluated for various film configurations and conditions such as pH-driven enzyme loading optimized. Finally, under specific aim #3, we will evaluate the performance of our NOS-based films in terms of platelet adhesion assays (critical to thrombus formation) at the surface of the coatings. NO-release coatings are also known to counteract biofilm formation. We will therefore evaluate the performance of the proposed thin polymeric, NOS-based, coatings in terms of preventing bacterial film adhesion and biofilm formation. This approach is innovative, because it allows the embedding of the enzyme responsible for NO production in vivo, into a bio-polymeric matrix to produce NO at the interface between the surface of the coating on a medical device and the surrounding blood matrix, thus preventing thrombosis and other post-operation complications. The proposed research is significant, because it utilizes endogenous substrates present in the blood matrix to initiate the enzymatic reaction, which enables continuous and potentially unlimited release of NO. We therefore overcome the inherent limitation of recently developed NO-release coatings due to intrinsic finite loadings. Our proposed NOS-based enzymatic generation of NO within biocompatible films is not limited, and yields coatings with potentially prolonged thromboresistance when the approach is extended to implantable medical devices.

Public Health Relevance

The number of revascularization procedures has increased dramatically, and is estimated at 15% after initial coronary revascularization, and approximately 50% after lower extremity bypass. The thrombogenic nature of various polymeric materials utilized to prepare various blood-contacting and implantable medical devices (intravascular catheters and sensors, stents, etc.), causes serious complications in patients. The proposed work, which aims at developing NO-releasing biocompatible coatings to counteract thrombosis and other complications, is at the heart of public health and challenges of the 21st century, including cardiovascular disease, which claims the lives of nearly 2,150 Americans each day, i.e. an average of 1 death every 40 seconds.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1-BST-F (80))
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Erim, Zeynep
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Cleveland State University
Schools of Arts and Sciences
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Haque, Mohammad Mahfuzul; Tejero, Jesús; Bayachou, Mekki et al. (2018) A cross-domain charge interaction governs the activity of NO synthase. J Biol Chem 293:4545-4554
AlTawallbeh, Ghaith; Haque, Mohammad M; Streletzky, Kiril A et al. (2017) Endothelial nitric oxide synthase oxygenase on lipid nanodiscs: A nano-assembly reflecting native-like function of eNOS. Biochem Biophys Res Commun 493:1438-1442