Mechanisms and Implications of Nanoelectroporation in Living Cells

活细胞纳米电穿孔的机制和意义

• 批准号: 8099680
• 负责人: Andrei G Pakhomov
• 金额: $ 28.19万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099680
• 关键词: Action Potentials; Adverse effects; Affect; Behavior; Biological; Calcium; Caliber; Cell Shape; Cell Volumes; Cell membrane; Cell physiology; Cells; Chemicals; Colloids; Complement; Complex; Cytoplasmic Granules; DNA; Dependence; Detection; Development; Devices; Dimensions; Dyes; Electric Conductivity; Electroporation; Engineering; Gated Ion Channel; Ion Channel; Ion Channel Protein; Ion Transport; Ions; Life; Lipid Bilayers; Mediating; Medical; Medical Research; Medicine; Membrane; Metabolism; Mitochondria; Modification; Muscle; Muscle Cells; Nerve Tissue; Neurons; New Agents; Pathway interactions; Permeability; Physiologic pulse; Physiological; Physiology; Polymers; Procedures; Property; Propidium; Proteins; Reporting; Research; Research Proposals; Sampling; Shapes; Structure; Study models; Techniques; Technology; Tissues; Trypan Blue; Water; Work; base; design; electric impedance; improved; millisecond; molecular dynamics; nanometer; nanopore; nanosecond; novel; patch clamp; public health relevance; research study; response; tool; trafficking; uptake; voltage

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.

描述（由申请人提供）： 脉冲功率技术的最新进展最终导致新设备的工程设计能够向生物组织和细胞样本等低阻抗负载提供高压、纳秒持续时间的电脉冲 (nsEP)。我们发现 nsEP 可以作为一种独特的工具来改变活细胞质膜的生理学并改变细胞功能。 nsEP 最显着的效果是打开长寿命、电压和电流敏感、整流、离子选择性、纳米或亚纳米直径的不对称孔（“纳米孔”）。这些复杂的行为通常只有蛋白质离子通道等复杂的设备才能实现，并将纳米孔与传统（较大）电孔区分开来。一旦被诱导，纳米孔就会在开放和准开放（电静默）状态之间振荡几分钟，然后逐渐重新密封或突然分解成更大的孔，并立即丧失纳米孔特有的特性。纳米孔似乎具备足够的能力来实现传统上归因于经典离子通道的某些功能；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。 此前曾在合成箔和平面脂质双层中报道过纳米孔，但我们的工作是第一个记录纳米孔的形成及其在活细胞中特性的工作。此外，我们还建立了 nsEP 处理后内源性离子通道的抑制和促进反应，以及由于渗透压失衡导致的细胞生理学变化。该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；揭示纳米孔执行其复杂活动的机制；并阐明 nsEP 对质膜屏障功能和离子流量影响的机制：具体目标 1：探索纳米孔形成对电脉冲物理参数的依赖性，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）并揭示导致这些特性的机制。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以及神经和肌肉细胞兴奋和动作电位传播的影响。具体目标 4：探索纳米孔对质膜水渗透性和细胞体积控制影响的机制。 公共卫生相关性： 这项研究将重点关注纳米电穿孔的新现象，即暴露于纳秒持续时间的高压电脉冲（nsEP）的活细胞中形成稳定的、电压和电流敏感的纳米直径膜孔。我们将重点研究质膜纳米电穿孔和 nsEP 对内源离子通道和水代谢影响的基础和确定的物理化学和生理机制。预期结果将促进使用 nsEP 有意修改细胞功能（特别是神经和肌肉组织中的细胞功能）的新医学和研究应用的开发。