Nitric oxide synthase-based thin films as antithrombotic coatings

 产品说明：为了减轻心血管和其他疾病的并发症而进行的许多外科手术介入需要引入长期和短期血液接触装置，例如血管内导管和传感器、移植物以及冠状动脉和血管支架，仅举几例。然而，由于这些装置的表面的血栓形成性质，这样的过程可能导致受伤部位处的复发问题和更多并发症，包括凝块形成，这是一种称为血栓形成的过程，其通常在外来装置的表面处触发。80%的血管通路功能障碍是由移植物血栓形成引起的，仅移植物血栓形成就带来了超过10亿美元/年的相关医疗保健成本。一氧化氮（NO）是已知的，以抵消体内血栓形成。释放一氧化氮的生物聚合物有可能延长血管移植物和支架的效力，而不会产生不良的全身血管舒张。目前，基于NO的涂层的开发虽然有前途，但在需要能够持续和延长一氧化氮释放的稳定材料的背景下相对有限。这部分是由于可以负载在涂层中的NO当量的有限量。 该项目的目标是开发稳定的NO释放薄膜，作为短期和长期植入式医疗器械的生物相容性涂层，其中一氧化氮释放是由嵌入的一氧化氮合酶（NOS）酶促进的。这个特定的应用程序的目的是研究这些酶为基础的NO释放薄膜，通过开发的NOS为基础的聚乙烯亚胺（PEI）的聚合物涂层建立的逐层的方法。酶驱动的NO生成将使用血液基质中发现的内源性化合物在血液/聚合物-器械界面释放NO。我们手中的初步观察表明，纯化的重组NOS酶保留其结构和催化功能时，嵌入在表面上的薄膜。我们假设，这将允许血液中可用的内源性化合物在聚合物表面和血液之间的界面处启动和维持酶促反应，从而释放NO，从而增强材料的抗血栓性。为了验证这一假设，我们提出了以下三个具体目标：1）制备和表征具有嵌入的一氧化氮合酶酶的生物相容性聚合物涂层。2)评价在各种条件下形成的基于NOS的聚合物涂层在持续的NO产生和NO释放水平方面的性能。3)[通过体外血小板粘附测定对基于NOS的涂层的性能进行体外评价;该测定将告知我们基于NOS的膜在工作的后期阶段中的抗血栓性的潜力，这超出了本提案的范围];逐层方法将用于制备纳米结构的基于NOS的生物聚合物涂层。薄膜内NOS的结构和功能完整性将是 使用光谱和电化学工具进行研究。在目标#2下，将评估基于NOS的PEI薄膜的NO释放水平和可持续性。将针对各种膜配置和条件（例如优化的pH驱动的酶加载）评估NO通量。最后，在具体目标#3下，我们将在涂层表面的血小板粘附测定（对血栓形成至关重要）方面评估我们的基于NOS的膜的性能。还已知NO释放涂层可以抵消生物膜的形成。因此，我们将评估所提出的薄聚合物，NOS为基础的，涂层在防止细菌膜粘附和生物膜形成方面的性能。这种方法是创新的，因为它允许将负责体内NO产生的酶嵌入生物聚合物基质中，以在医疗装置上的涂层表面与周围血液基质之间的界面处产生NO，从而防止血栓形成和其他术后并发症。拟议的研究是显着的，因为它利用内源性底物存在于血液基质中启动的酶促反应，这使得连续和潜在的无限释放NO。因此，我们克服了固有的局限性，最近开发的NO释放涂层由于固有的有限负载。我们提出的基于NOS的生物相容性膜内NO的酶促生成不受限制，并且当该方法扩展到可植入医疗器械时，产生具有潜在延长的抗血栓性的涂层。

 DESCRIPTION: 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.