Membrane Sealing:Biopolymers for Tissue Electroporation

细胞膜脂质双分子层结构的破坏是许多疾病中组织坏死的常见原因，包括高能创伤。膜离子屏障功能丧失后，代谢能量迅速耗竭，然后急性细胞坏死。电击引起的组织损伤是这类细胞损伤的极好模型，因为膜损伤是通过电穿孔、暴露于高温和可能的高功率声应力发生的（附录I和II）。由于细胞体积较大，骨骼肌和神经特别容易受到细胞膜损伤的直接电机制（电穿孔和电构象蛋白变性）的影响。理论、实验和临床数据均表明，电穿孔引起的膜损伤是导致骨骼肌和神经损伤的重要原因（附录II）。我们的实验室和其他人已经证明，poloxamer表面活性剂（poloxamer 188和Poloxamine 1107）可以减少由膜破坏介导的急性坏死（附录III， Sharma等人，1996年，Merchant等人，1998年，Hannig等人，2000年）。因此，我们假设这些表面活性剂可用于显著减少电穿孔后的组织坏死，从而显著改善组织存活和功能。我们建议确定静脉注射波洛沙姆188在体内封闭电穿孔骨骼肌细胞膜和改善功能恢复方面有多有效。我们建议使用定量实时功能分析测量技术（表面肌电图和放射性药物成像）以及反映膜完整性和组织坏死的标准组织学和生化标志物来评估结果。此外，基于已完成的实验，我们假设抗氧化剂（即抗坏血酸）可能保护poloxamers免于氧化降解以增强其功效，并提出MgATP会增强膜密封的响应性。本提案还解决了一个基本需求，即改进和校准实时表面肌电图和放射性药物成像，作为定量膜密封治疗反应和临床研究实时评估的工具。这种诊断工具将具有巨大的临床价值，因为快速检测、鉴别和定位组织损伤将加速和指导临床治疗。虽然我们选择电穿孔作为实验模型来测试体内膜的密封，但这些结果和实验方法将与其他以膜渗透性为特征的疾病直接相关，如缺血再灌注、冻融和机械损伤。

Disruption of the cell membrane lipid bilayer structure is a common cause of tissue necrosis in many illnesses, including high-energy trauma. Loss of membrane ionic barrier function is followed by rapid metabolic energy exhaustion and then acute cellular necrosis. Electrical shock induced tissue injury is superb model for this type of cell injury because membrane damage occurs by electroporation, exposure to high temperatures and possibly high-power acoustic stresses (Appendices I and II). Because of the relatively large size of the cells, skeletal muscle and nerve are especially vulnerable to the direct electrical mechanisms of cellular membrane damage (electroporation and electroconformational protein denaturation). Theoretical, experimental and clinical data all indicate that membrane damage by electroporation is a significant cause of much of the skeletal muscle and nerve injury that results (Appendix II). Our lab and others have shown that poloxamer surfactants (Poloxamer 188 and Poloxamine 1107) reduce acute necrosis mediated by membrane disruption (Appendix III, Sharma et al. 1996, Merchant et al. 1998, Hannig et al. 2000). Thus, we postulate that these surfactants can be used to substantially reduce tissue necrosis following electroporation to result in significantly improved tissue survival and function. We propose to determine how effective intravenous Poloxamer 188 with and without cofactors are in sealing electroporated skeletal muscle cell membranes in vivo and in improving functional recovery. We propose to assess outcomes using quantitative real- time functional assay measurement techniques (surface electromyography and radiopharmaceutical imaging) as well as by standard histological and biochemical markers reflective of membrane integrity and tissue necrosis. Furthermore, on the basis of completed experiments, we postulate that antioxidants (i.e. ascorbate) may protect poloxamers from oxidative degradation to enhance its efficacy, and propose that MgATP will enhance responsiveness to membrane sealing. A basic need also addressed in this proposal is the refinement and calibration of real-time surface electromyography and radiopharmaceutical imaging as tools for quantifying therapeutic responses to membrane sealing therapy and for real-time assessment in clinical studies. Such diagnostic tools would be of tremendous clinical value because rapid detection, discrimination, and localization of tissue injury would accelerate and guide clinical therapy. Although we choose electroporation as the experimental model to test in vivo membrane sealing, these results and experimental methods will be directly relevant to other diseases characterized by membrane permeabilization, e.g. ischemia-reperfusion, freeze-thaw and mechanical trauma.