Mechanisms and Implications of Nanoelectroporation in Living Cells

描述(由申请人提供)： 脉冲功率技术的最新进展最终导致了能够向生物组织和细胞样本等低阻抗负载提供高电压、纳秒持续时间的电脉冲(NsEP)的新设备的设计。我们发现，nsEP可以作为一种独特的工具来改变活细胞的质膜生理和改变细胞功能。NsEP最显著的作用是打开了长寿命、电压和电流敏感、整流、离子选择、不对称的纳米或亚纳米直径的孔(纳米孔)。这些复杂的行为通常只在蛋白质离子通道等复杂设备中出现，并将纳米孔与传统(更大)的电孔区分开来。一旦被诱导，纳米孔在开放和准开放(电学上无声)之间振荡几分钟，然后逐渐重新密封或突然破裂成更大的孔，立即失去纳米孔特有的特性。纳米孔似乎为传统上归因于经典离子通道的某些功能提供了足够的装备；我们假设纳米孔可能在生理和病理条件下形成，以补充离子通道作为额外的离子传输途径。纳米孔以前已经在合成膜和平面脂质双层中被报道，但我们的工作是第一次在活细胞中记录纳米孔的形成和它们的性质。此外，我们已经建立了内源性离子通道在nsEP治疗后的抑制和促进反应，以及由于渗透失衡引起的细胞生理学变化。这项研究应用旨在探索活细胞中的纳米电穿孔现象，并评估这项新技术在研究和医学中的潜在应用。拟议的研究包括四个具体目标，旨在表征和改进纳米电穿孔程序；揭示允许纳米孔执行其复杂活动的机制；以及阐明nsEP对质膜屏障功能和离子交通的影响机制：具体目标1：探索纳米孔形成对电脉冲物理参数的依赖，优化纳米电穿孔程序和纳米孔检测技术。具体目标2：分析纳米孔的结构和功能特性(孔寿命、开口直径、离子选择性、电压和电流敏感性)，并揭示影响这些特性的机理。具体目标3：探索纳米电穿孔对经典电压门控离子通道功能的影响，以及对神经和肌肉细胞的兴奋和动作电位传播的影响。具体目标4：探索纳米粒子对质膜透水性和细胞体积控制的潜在作用机制。 公共卫生相关性： 这项研究将集中在纳米电穿孔的新现象上，即在纳秒持续的高压电脉冲(NsEP)下，活细胞中形成稳定的、电压和电流敏感的纳米直径的膜孔。我们将集中讨论和确定质膜纳米电穿孔和nsEP对内源离子通道和水代谢的影响的物理化学和生理机制。预期的结果将促进利用nsEP刻意改变细胞功能，特别是神经和肌肉组织中细胞功能的新的医疗和研究应用的开发。

DESCRIPTION (provided by applicant): Recent advances in pulsed power technology culminated in engineering of new devices capable of delivering high-voltage, nanosecond-duration electric pulses (nsEP) to low-impedance loads such as biological tissues and cell samples. We found that nsEP can be employed as a unique tool to modify physiology of the plasma membrane in living cells and alter cell function. The most remarkable effect of nsEP was opening of long-lived, voltage- and current-sensitive, rectifying, ion-selective, asymmetrical pores of nano- or sub- nanometer diameter ("nanopores"). These complex behaviors are normally expected only from sophisticated devices like protein ion channels and distinguish nanopores from conventional (larger) electropores. Once induced, nanopores oscillated between open and quasi-open (electrically silent) states for minutes, followed by either gradual resealing or abrupt breakdown into larger pores, with immediate loss of nanopore-specific properties. Nanopores appeared adequately equipped for certain functions that are traditionally ascribed to classic ion channels; we hypothesize that nanopores may form under physiological and pathological conditions to supplement ion channels as an additional ion transport pathway. Nanopores have previously been reported in synthetic foils and planar lipid bilayers, but our work is the first one to document the formation of nanopores and their properties in living cells. Furthermore, we have established both inhibitory and facilitatory responses of endogenous ion channels after nsEP treatment, as well as cytophysiological changes due to the osmotic imbalance. This Research Application is designed to explore the phenomenon of nanoelectroporation in living cells and to evaluate potential applications of this novel technique in research and medicine. The proposed study consists of four Specific Aims intended to characterize and improve the nanoelectroporation procedure; to reveal mechanisms that allow nanopores to perform their complex activities; and to elucidate mechanisms that underlie nsEP effects on plasma membrane barrier function and ion traffic: Specific Aim 1: Explore the dependence of nanopore formation on the physical parameters of electric pulses, optimize nanoelectroporation procedures and nanopore detection techniques. Specific Aim 2: Analyze structural and functional properties of nanopores (pore lifetime, opening diameter, ion selectivity, voltage and current sensitivity) and reveal mechanisms responsible for these properties. Specific Aim 3: Explore the impact of nanoelectroporation on the function of classic voltage-gated ion channels, and on the excitation and action potential propagation in nerve and muscle cells. Specific Aim 4: Explore mechanisms underlying nanoporation effect on plasma membrane water permeability and cell volume control. PUBLIC HEALTH RELEVANCE: This study will be focused on the new phenomenon of nanoelectroporation, which is the formation of stable, voltage- and current-sensitive, nanometer-diameter membrane pores in living cells exposed to nanosecond- duration, high-voltage electric pulses (nsEP). We will focus on physico-chemical and physiological mechanisms that underlie and determine plasma membrane nanoelectroporation and nsEP effects on endogenous ion channels and water metabolism. Anticipated results will promote the development of new medical and research applications using nsEP for deliberate modification of cell functions, particularly in nerve and muscle tissues.