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，包括高能创伤。随着膜离子屏障功能的丧失，迅速的代谢能量耗竭，然后是急性细胞坏死。电击性组织损伤是这类细胞损伤的极佳模型，因为膜损伤是通过电穿孔、暴露在高温和可能的高功率声应力下发生的(附录I和II)。由于细胞的体积相对较大，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、实验和临床数据都表明，电穿孔造成的膜损伤是导致骨骼肌和神经损伤的一个重要原因(附录II)。我们的实验室和其他实验室已经表明，泊洛沙姆表面活性剂(泊洛沙姆188和泊洛沙明1107)减少由膜破裂介导的急性坏死(附录III，Sharma等人)。1996年，Merchant et al.1998年，Hannig et al.2000)。因此，我们推测这些表面活性物质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泊洛沙姆188在体内封闭电穿孔的骨骼肌细胞膜和改善功能恢复方面的有效性。我们建议使用定量实时功能分析测量技术(表面肌电图法和放射性药物成像)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，在已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，以增强其疗效，并提出镁ATP将增强对膜封闭的反应性。这项建议还涉及的一个基本需求是改进和校准实时表面肌电图仪和放射性药物成像，作为量化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，因为快速检测、识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在体膜封闭的实验模型，但这些结果和实验方法将直接关系到其他以膜通透性为特征的疾病，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.