Magnetic targeting of stents with endothelial cells for prevention of restenosis

描述(申请人提供)：在过去的十年里，支架血管成形术的出现和最近药物洗脱支架的使用导致了血管疾病护理的范式转变。血管介入治疗的有害后遗症是不可避免的血管壁机械损伤的结果。内皮单层的破坏暴露了底层介质，并诱导了一系列细胞和生物事件，导致血管壁功能异常。损伤后增加血管壁内皮细胞数量的策略可能通过重建管腔屏障和细胞分泌旁分泌因子来限制并发症，如血栓形成、血管痉挛和新的内膜形成。以前的快速“内皮化”植入设备的策略并没有显示出临床应用所需的期望的有效性、安全性和易用性。在这里，我们提出了一种将内皮细胞磁靶向支架的方法，该方法基于适度的均匀磁场，既最大限度地磁化负载磁性纳米颗粒的内皮前体细胞，又在钢支架金属丝网内产生大的局部磁场梯度。这种机制将允许实现最大的磁力，这将导致血管损伤部位内皮细胞的有效定位。我们在大鼠颈动脉支架植入模型中使用牛主动脉内皮细胞的初步数据表明了这种方法的可行性。在这个项目中，我们计划研究更多与治疗相关的细胞，这些细胞能够分化为具有内皮表型的细胞(即同种异体血生长内皮细胞，BOEC和内皮祖细胞，EPC)，这些细胞来自将接受磁细胞治疗的相同物种(大鼠)。拟议研究的具体目标1将侧重于开发分离、培养和鉴定内皮前体细胞的方案，以用于进一步的磁靶向研究。具体目标2将集中于生物可降解磁性纳米颗粒细胞负载方案的优化，评估细胞负载对细胞形态、生长和功能完整性的影响。这个目标还将解决一些机械方面的磁性负载和操纵的EC细胞，评估其基因表达谱，粘附性和血栓形成。具体目标3将致力于利用合适的动物模型定量评价体内磁性细胞靶向的效率，研究非靶向内皮细胞的器官生物分布，以及检测磁性定位的同种异体内皮细胞对损伤血管壁的有益治疗作用。此外，还将评估其长期疗效。我们真诚地相信，由此产生的数据，加上同时进行的磁性植入物开发的临床前工作，将提供一种在血管和其他应用中广泛实施的磁性细胞靶向策略。 公共卫生相关性：建议的方法有可能成为目前使用的药物洗脱支架(DES)的重要替代品，尽管DES已被证明可以减少支架植入后再狭窄的发生率，但也会导致延迟的内皮化，导致后来的血管并发症，需要长时间使用抗血小板治疗。我们的方法可以通过有效地将内皮细胞定位于支架血管来加速血管组织的再生，从而在长期范围内减少与再狭窄相关的并发症。此外，这项研究的结果将对在许多治疗环境中使用可磁化钢植入物作为有序沉积基于细胞的治疗的靶向设备的靶向细胞输送具有广泛的普遍意义。

DESCRIPTION (provided by applicant): For the past decade the advent of stent angioplasty and the even more recent use of drug eluting stents have resulted in a paradigm shift in the care of vascular disease. Deleterious sequelae of vascular interventions are the result of unavoidable mechanical damage to the vessel wall. Disruption of the endothelial monolayer exposes the underlying media and induces a cascade of cellular and biological events, resulting in abnormal vascular wall function. Strategies that enhance the number of endothelial cells in the vessel wall following injury may limit complications such as thrombosis, vasospasm, and neointimal formation, through reconstitution of a luminal barrier and cellular secretion of paracrine factors. Previous strategies to rapidly "endothelialize" implanted devices did not show desired efficacy, safety, and ease of use required for clinical applications. Herein we propose a method for magnetic targeting of endothelial cells to stents based on use of a modest uniform magnetic field to both maximally magnetize the magnetic nanoparticle-loaded endothelial precursor cells and produce large local magnetic field gradients within the steel stent wire network. This mechanism will allow achieving maximized magnetic force that will result in efficient localization of endothelial cells at the blood vessel injured site. Our preliminary data using bovine aortic endothelial cells in the rat carotid-stenting model indicate on feasibility of this approach. In this project we plan to study more therapeutically relevant cells that are capable of differentiating into cells with endothelial phenotype (i.e. allogeneic blood outgrowth endothelial cells, BOEC and endothelial progenitor cells, EPC) derived from same species (rats) that will receive magnetic cell therapy. Specific Aim 1 of the proposed research will focus on the development of protocols for isolation, culture and characterization of endothelial precursor cells to be used for further magnetic targeting studies. Specific Aim 2 will concentrate on the optimization of protocols for cell loading by biodegradable magnetic nanoparticles, evaluating the effects of cell loading on cell's morphology, growth, and preservation of functional integrity. This aim will also address some mechanistic aspects of magnetically loaded and manipulated EC cells, evaluating their gene expression profiles, adhesion and thrombogenicity. Specific Aim 3 will be dedicated to quantitatively evaluate the efficiency of magnetic cell targeting in vivo using appropriate animal model, to investigate the organ biodistribution of the off-targeted endothelial cells as well as to examine the beneficial therapeutic effect on the injured vessel wall by magnetically localized allogeneic endothelial cells. The long term therapeutic effect will also be assessed. We sincerely believe that resulting data, coupled with concurrent pre-clinical work on magnetic implant development will provide a widely implementable strategy of magnetic cell targeting in vascular and other applications. PUBLIC HEALTH RELEVANCE: The proposed approach has the potential to be a vital alternative to the currently used drug eluting stents (DES) which although have been shown to reduce the incidence of restenosis after stenting, but also result in delayed endothelialization leading to a later vascular complications requiring prolonged use of antiplatelet therapy. Our approach can provide the capability for an accelerated regeneration of vascular tissue via the efficient localization of endothelial cells to stented blood vessels, resulting in fewer restenosis-related complications on the long term scale. Additionally, the outcomes of this study will have broad general implications for targeted cell delivery in a number of therapeutic settings using magnetizable steel implants as targeting devices for the ordered deposition of cell-based therapies.