Membrane Sealing:Biopolymers for Tissue Electroporation

细胞膜脂质双层结构的破坏是许多疾病中组织坏死的常见原因，包括高能量创伤。 膜离子屏障功能丧失后，代谢能迅速耗尽，然后是急性细胞坏死。 电击诱导的组织损伤是这种类型细胞损伤的极好模型，因为电穿孔、暴露于高温和可能的高功率声应力会发生膜损伤（附录I和II）。由于细胞相对较大，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大部分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实验室已经证明，泊洛沙姆表面活性剂（泊洛沙姆188和泊洛沙胺1107）可减少膜破裂介导的急性坏死（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。我们建议确定如何有效地静脉注射泊洛沙姆188有和没有辅因子是在体内密封电穿孔骨骼肌细胞膜和改善功能恢复。 我们建议使用定量真实的时间功能测定测量技术（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，在完成的实验的基础上，我们假设抗氧化剂（即抗坏血酸）可以保护泊洛沙姆免受氧化降解，以提高其疗效，并提出MgATP将提高膜密封的响应性。本提案中还提出了一个基本需求，即实时表面肌电图和放射性药物成像的完善和校准，作为量化对膜封闭治疗的治疗反应和临床研究中实时评估的工具。 这种诊断工具将具有巨大的临床价值，因为组织损伤的快速检测、辨别和定位将加速并指导临床治疗。 虽然我们选择电穿孔作为实验模型来测试在体膜密封性，但这些结果和实验方法将直接相关于以膜透化为特征的其他疾病，例如缺血-再灌注、冻融和机械创伤。

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.