Uniform field controlled magnetic cell targeting to stents

描述（由申请人提供）：拟定研究将探索以下假设：磁性引导内皮细胞（EC）靶向植入支架的动脉可减少支架血管成形术后的血管再闭塞（再狭窄）。以再内皮化为导向的策略有可能预防再狭窄，同时避免目前使用的治疗的副作用。然而，为了充分实现这种潜力，需要显著提高细胞递送效率。本研究将探讨两个新的想法：1）应用 磁性纳米颗粒（MNP），用于为EC提供足以将其靶向递送到支架动脉的磁响应性;和2）使用EC到可逆磁化支架的均匀场控制磁靶向，用于增强再内皮化和抑制支架植入后的再狭窄。目前的研究将解决以下具体目标：目的1：MNP表征方面的物理性质，细胞相容性，摄取和降解动力学。可生物降解的荧光标记的MNP将使用聚合物沉淀方法的修改来配制。将表征MNP的大小、组成和磁性，其细胞相容性、内化和降解动力学将是目标1实验的主要终点。用交变梯度磁强计测量MNP和经MNP处理的EC的磁响应性。动力学研究将使用荧光测定法和全局共振能量转移测量在培养细胞中进行。MNP的细胞相容性将使用荧光细胞毒性测定法测定。目的2：内皮细胞靶向和再内皮化研究。磁性与非磁性输送后的动脉定位和组织分布 在大鼠颈动脉支架模型中，将分别通过体内生物发光和光度法研究MNP浸渍的稳定表达萤火虫荧光素酶的EC。将在治疗后1、7和28天比较支架靶向细胞的局部动力学特征，并使用定量免疫组织化学策略确定内皮化。目的3：靶向EC的抗再狭窄疗效。在磁性条件下用负载MNP的EC处理的动物中的再狭窄程度将通过计算机化形态测定法来确定，并在递送后4周与非磁性递送和“仅支架植入”对照中的再狭窄程度进行比较。

DESCRIPTION (provided by applicant): The proposed research will explore the hypothesis that magnetically guided targeting of endothelial cells (EC) to stented arteries can reduce vessel reocclusion (restenosis) following stent angioplasty. Re-endothelialization oriented strategies have potential to prevent restenosis while avoiding the side effects of the currently used therapies. However, in order to fully realize this potential a significant improvement in cell delivery efficiency is required. This study will investigate two novel ideas: 1) the application of magnetic nanoparticles (MNP) for providing EC with magnetic responsiveness adequate for their targeted delivery to stented arteries; and 2) using a uniform field-controlled magnetic targeting of EC to reversibly magnetizable stents for enhanced reendothelialization and inhibition of restenosis post stenting. The present studies will address the following specific aims: AIM 1: MNP characterization with respect to their physical properties, cell compatibility, uptake and degradation kinetics. Biodegradable fluorescent-labeled MNP will be formulated using a modification of the polymer precipitation approach. The size, composition and magnetic properties of MNP will be characterized, and their cell compatibility, internalization and degradation kinetics will be the main endpoints of the Aim 1 experiments. The magnetic responsiveness of MNP and EC treated with MNP will be measured by alternating gradient magnetometer. The kinetic studies will be performed in cultured cells using fluorimetry and global FQrster Resonance Energy Transfer measurements. Cell compatibility of MNP will be determined using fluorimetric cell toxicity assays. AIM 2: EC targeting and reendothelialization studies. Arterial localization and tissue distribution after magnetic vs. non- magnetic delivery of MNP-impregnated EC stably expressing firefly luciferase will be studied by in vivo bioluminescence and luminometry, respectively, in the rat carotid stenting model. The local kinetic profiles of stent-targeted cells will be compared 1, 7 and 28 days post treatment with endothelialization determined using a quantitative immunohistochemical strategy. AIM 3: antirestenotic efficacy of targeted EC. The extent of restenosis in animals treated under magnetic conditions with MNP-loaded EC will be determined by computerized morphometry and compared that in non-magnetic delivery and 'stenting only' controls four weeks post delivery.