High Field Gradient Targeting Magnetic Nanoparticle Loaded Cells to Stents

描述（由申请人提供）：我们将研究一种新的方法来提高支架成形术的治疗效果。据推测，可生物降解的非聚合磁性纳米颗粒（MNP）可用于使体外培养的循环内皮祖细胞（EPC）具有磁性响应，从而能够将具有磁性靶向的治疗相关转基因传递到已部署的支架上，从而增强损伤和病变动脉的再生。目的1：制定和表征磁性靶向复合物的组成部分，包括：可生物降解的非聚合MNP， EPC和辅助腺病毒载体（HD-Ad）。在这些基因传递实验中使用的MNP可以通过在其组合物中包含磁铁矿而不影响其生物相容性来呈现磁响应。EPC将从外周血中采集，并在MNP加载条件下研究其磁响应性和活力。HD-Ad载体，导致持续的转基因表达而不需要基因组整合，将与贝勒医学院的Phillip Ng博士合作构建，使用两种报告基因（绿色荧光蛋白，GFP和荧光素酶，Luc）以及诱导型一氧化氮合酶（iNOS）作为我们的主要治疗基因。目的2：研究磁性细胞在体内的靶向性，建立最佳递送条件。将在大鼠颈动脉支架手术中研究预加载MNP的gfph - ad转导EPC，将EPC与304钢支架与对照组进行比较，以确定最佳递送条件和表达GFP的EPC在目标动脉段的分布。更长期的研究，长达28天，将跟踪304个用LucHD-Ad转导的EPC靶向支架中的Luc表达，并通过Luc光学成像跟踪转基因随时间的表达。目的3：研究iNOSHD-Ad介导的mnp修饰的EPC在体内的抗再狭窄作用。根据Aim 2的结果，我们已经建立了最佳的细胞制备和递送方案，我们将研究用iNOSHD-Ad修饰的磁靶向EPC在大鼠颈动脉支架模型和猪股动脉支架血管成像术中的抗再狭窄作用。

DESCRIPTION (provided by applicant): We will investigate a novel approach to improve the therapeutic efficacy of stent angioplasty. It is hypothesized that biodegradable nonpolymeric magnetic nanoparticles (MNP) can be used to render ex vivo cultured circulating endothelial progenitor cells (EPC) magnetically responsive and thus able to deliver a therapeutically relevant transgene with magnetic targeting to a deployed stent thereby enabling enhanced regeneration of injured and diseased arteries. Aim 1: To formulate and characterize the components of a magnetic targeting complex including: Biodegradable nonpolymeric MNP, EPC and Helper Dependent Adenoviral Vectors (HD-Ad). MNP used in these gene delivery experiments can be rendered magnetically responsive by inclusion of magnetite in their composition without compromising their biocompatibility. EPC will be harvested from peripheral blood and investigated under conditions of MNP loading for magnetic responsiveness and viability. HD-Ad vectors, that result in sustained transgene expression without genomic integration will be constructed in collaboration with Dr. Phillip Ng, Baylor College of Medicine, with both reporter constructs (green fluorescent protein, GFP, and luciferase, Luc), as well as inducible nitric oxide synthase (iNOS) as our lead therapeutic gene. Aim 2: To study magnetic cell targeting in vivo with reporter constructs to establish an optimal range of delivery conditions. GFPHD-Ad transduced EPC preloaded with MNP will be investigated in rat carotid stent procedures comparing EPC targeted to 304 steel stents versus controls to determine optimal delivery conditions and the distribution of the GFP expressing EPC in the targeted arterial segment. Longer term studies, up to 28 days, will follow Luc expression in 304 stents targeted with EPC transduced with LucHD-Ad, following transgene expression over time with Luc optical imaging. Aim 3: To investigate in vivo the antirestenotic efficacy of MNP-modified EPC transduced with iNOSHD-Ad. Having established protocols for optimal cell preparation and delivery per Aim 2 results, we will investigate the antirestenotic effect of magnetically targeted EPC modified with iNOSHD-Ad in both the rat carotid stenting model and pig femoral stent angioplasties.