Nitric oxide synthase-based thin films as antithrombotic coatings

基于一氧化氮合酶的薄膜作为抗血栓涂层

• 批准号: 8958393
• 负责人: MEKKI BAYACHOU
• 金额: $ 42.13万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-21 至 2019-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8958393
• 关键词: Address; Adhesions; American; Arginine; Biochemical Reaction; Biocompatible; Biological Assay; Biopolymers; Blood; Blood Platelets; Blood Vessels; Bypass; Cardiovascular Diseases; Cardiovascular system; Catheters; Cessation of life; Chemicals; Coagulation Process; Coronary; Coronary artery; Development; Devices; Disease; Endothelial Cells; Enzymes; Evaluation; Failure; Film; Functional disorder; Generations; Goals; Health Care Costs; Heart; Implant; In Situ; In Vitro; Infection; Injury; Lead; Lower Extremity; Medical Device; Methodology; Methods; Microbial Biofilms; Nature; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Operative Surgical Procedures; Patients; Performance; Polyethyleneimine; Polymers; Preparation; Procedures; Process; Production; Public Health; Recombinants; Research; Resistance; Review Literature; Site; Source; Staging; Stenosis; Stents; Structure; Surface; Testing; Therapeutic; Thrombosis; Thrombus; Time; Vascular Graft; Vasodilation; Work; base; diazeniumdiolate; implantable device; implantation; in vivo; injured; innovation; nanostructured; novel strategies; operation; prevent; public health relevance; restenosis; sensor; surface coating; tool

 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.

 描述：许多为减轻心血管和其他疾病并发症而进行的外科手术都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。然而，由于这些装置表面的血栓形成性质，这样的过程可能会导致受伤部位反复出现问题和更多并发症，包括凝块形成，这一过程称为血栓形成，通常在表面触发。的80% 的血管通路功能障碍是由移植物血栓引起的，已知一氧化氮 (NO) 每年会产生超过 10 亿美元的相关医疗费用。生物聚合物有可能延长血管移植物和支架的效力，而不会产生不利的全身血管舒张。目前，基于NO的涂层的开发虽然很有前景，但在需要能够稳定的材料的情况下相对有限。这部分是由于涂层中可负载的一氧化氮当量有限，该项目的目标是开发稳定的一氧化氮释放薄膜作为短期和长期的生物相容性涂层。植入式医疗设备，其中嵌入的一氧化氮合成酶 (NOS) 酶促进一氧化氮的释放。这一特定应用的目的是通过开发基于 NOS 的酶来研究这些基于酶的 NO 释放薄膜。通过逐层方法构建的聚乙烯亚胺 (PEI) 聚合物涂层将利用血液基质中发现的内源性化合物在血液/聚合物-装置界面处释放 NO。纯化的重组 NOS 酶在嵌入表面薄膜时保留其结构和催化功能，我们发现这将使血液中的内源性化合物能够启动并维持酶反应，从而释放 NO。为了验证这一假设，我们提出了以下三个具体目标：1）嵌入一氧化氮合酶的生物相容性聚合物涂层的制备和表征。 2) 评估在不同条件下形成的基于 NOS 的聚合物涂层在持续 NO 产生和 NO 释放水平方面的性能。 3) ) [通过体外评估基于 NOS 的涂层的性能。体外血小板粘附测定；该测定将告诉我们基于 NOS 的薄膜在工作后期的抗血栓能力的潜力，这超出了本提案的范围]；制备基于NOS的纳米结构生物聚合物涂层，薄膜内NOS的结构和功能完整性将受到影响。 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，并将针对各种薄膜配置和条件（例如pH驱动的酶负载优化）进行评估。最后，在具体目标 3 下，我们将评估基于 NOS 的薄膜在涂层表面的血小板粘附测定（对血栓形成至关重要）方面的性能，众所周知，NO 释放涂层也具有抵消作用。因此，我们将评估所提出的基于 NOS 的薄聚合物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，因为它允许在体内嵌入负责产生 NO 的酶。 ，进入生物聚合物基质，在医疗器械涂层表面和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。这项研究意义重大，因为它利用了内源性底物。展示因此，我们克服了最近开发的 NO 释放涂层由于内在有限负载而产生的 NO 的固有限制。薄膜不受限制，并且当该方法扩展到可植入医疗设备时，可以产生具有潜在延长抗血栓性的涂层。