Nitric oxide synthase-based thin films as antithrombotic coatings

 描述：为了减少心血管和其他疾病的并发症而进行的许多外科干预措施都需要引入长期和短期的血液接触装置，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，由于这些设备表面的血栓形成性质，这样的过程可能会导致损伤部位反复出现问题和更多并发症，包括血栓形成，这一过程通常在异物设备的表面触发。80%的血管通路功能障碍是由移植物血栓引起的，仅此一项就伴随着每年超过10亿美元的相关医疗费用。众所周知，一氧化氮(NO)可以中和体内的血栓形成。一氧化氮释放生物聚合物有可能延长血管移植物和支架的效力，而不会对全身血管产生不利的扩张作用。目前，NO基涂层的发展前景看好，但在需要能够持续和长期释放一氧化氮的稳定材料的背景下，发展相对有限。这部分是由于涂层中可以负载的无等效物的数量有限。该项目的目标是开发稳定的NO释放薄膜，作为生物相容性涂层，用于短期和长期植入式医疗设备，其中嵌入的一氧化氮合酶(NOS)酶促进了NO的释放。这种特殊应用的目的是通过开发基于一氧化氮合酶(NOS)的聚乙烯亚胺(PEI)聚合物涂层来研究这些基于酶的NO释放薄膜。酶驱动的NO生成将使用血液基质中的内源性化合物在血液/聚合物-设备界面释放NO。我们手中的初步观察表明，纯化的重组NOS酶包埋在表面的薄膜中时，保持了其结构和催化功能。我们假设这将允许血液中的内源化合物启动和维持酶反应，从而在聚合物表面和血液之间的界面上释放NO，从而增强材料的抗凝血性。为了验证这一假设，我们提出了以下三个具体目标：1)制备和表征嵌入一氧化氮合酶酶的生物相容性聚合物涂层。2)评估在不同条件下形成的基于NOS的聚合物涂层的性能，包括持续的NO生成和NO释放水平。3))[通过体外血小板黏附试验对一氧化氮合酶涂层的性能进行体外评估；这一分析将在工作的后期阶段告知我们一氧化氮合酶薄膜在血栓抗性方面的潜力，这不在本提案的范围内]；将使用逐层方法制备纳米结构一氧化氮合酶生物聚合物涂层。薄膜内一氧化氮合酶的结构和功能完整性将是 使用光谱和电化学工具进行了研究。在AIM#2下，将对基于NOS的PEI薄膜的NO释放水平和可持续性进行评估。将评估各种膜结构和条件下的无助熔剂，如pH驱动的酶负荷优化。最后，在具体目标#3下，我们将通过涂层表面的血小板粘附性分析(血栓形成的关键)来评估我们的基于NOS的薄膜的性能。不释放涂层也被认为可以抑制生物膜的形成。因此，我们将评估拟议的基于NOS的薄聚合物涂层在防止细菌膜粘连和生物膜形成方面的性能。这种方法是创新的，因为它允许将体内负责产生NO的酶嵌入到生物聚合物基质中，以在医疗设备涂层表面与周围血液基质之间的界面产生NO，从而防止血栓形成和其他手术后并发症。这项拟议的研究意义重大，因为它利用血液基质中存在的内源性底物来启动酶反应，从而使NO能够连续且潜在地无限制地释放。因此，我们克服了最近开发的不释放涂料的固有限制，这是由于固有的有限载荷。我们提出的基于一氧化氮合酶的生物相容薄膜中NO的酶促生成是不受限制的，当该方法扩展到植入式医疗设备时，可以产生具有潜在延长血栓抗性的涂层。

 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.