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: 8500364
• 负责人: Andrei G Pakhomov
• 金额: $ 27.82万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500364
• 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.

描述（由申请人提供）： 脉冲电力技术的最新进展最终导致了能够输送高压，纳秒耐用的电脉冲（NSEP）的新设备的工程，以至于生物组织和细胞样品等低阻抗载荷。我们发现，NSEP可以用作独特的工具来改变活细胞中质膜的生理学并改变细胞功能。 NSEP的最显着效果是开放纳米或亚纳米直径的长寿命，电压和电流敏感，离子选择性，不对称的孔（“纳米孔”）。这些复杂的行为通常只能从蛋白质离子通道等复杂设备等复杂的设备中预期，并将纳米孔与常规（较大）电孔区分开。一旦诱导，纳米孔在开放式和准打开状态之间振荡了几分钟，然后逐渐重新密封或突然分解为较大的毛孔，并立即损失纳米孔特异性特性。纳米孔似乎适用于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。 纳米孔以前已经报道了合成箔和平面脂质双层，但我们的工作是第一个记录纳米孔形成及其在活细胞中特性的工作。此外，我们已经建立了NSEP处理后内源离子通道的抑制和促进反应，以及由于渗透失衡而引起的细胞生理变化。该研究应用程序旨在探索活细胞中纳米电化的现象，并评估这种新技术在研究和医学中的潜在应用。拟议的研究包括四个旨在表征和改善纳米电脑化程序的特定目的。揭示允许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产生影响的机制：特定目标1：探索纳米孔形成对电脉冲物理参数的依赖性，优化纳米载掺杂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，开放直径，离子选择性，电压和电流灵敏度），并揭示了负责这些特性的机制。特定目标3：探索纳米电脑对经典电压门控离子通道功能的影响，以及神经和肌肉细胞中的激发和动作电位传播。特定目的4：探索纳米构成对质膜渗透性和细胞体积控制的机制。

项目成果

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses.

• DOI: 10.1016/j.bbrc.2013.12.004
• 发表时间: 2014-01-10
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Ibey, Bennett L.;Ullery, Jody C.;Pakhomova, Olga N.;Roth, Caleb C.;Semenov, Iurii;Beier, Hope T.;Tarango, Melissa;Xiao, Shu;Schoenbach, Karl H.;Pakhomov, Andrei G.
• 通讯作者: Pakhomov, Andrei G.

Electrosensitization assists cell ablation by nanosecond pulsed electric field in 3D cultures.

• DOI: 10.1038/srep23225
• 发表时间: 2016-03-18
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Muratori C;Pakhomov AG;Xiao S;Pakhomova ON
• 通讯作者: Pakhomova ON

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity.

• DOI: 10.1007/s00018-014-1626-z
• 发表时间: 2014-11
• 期刊: CELLULAR AND MOLECULAR LIFE SCIENCES
• 影响因子: 8
• 作者: Pakhomov, Andrei G.;Semenov, Iurii;Xiao, Shu;Pakhomova, Olga N.;Gregory, Betsy;Schoenbach, Karl H.;Ullery, Jody C.;Beier, Hope T.;Rajulapati, Sambasiva R.;Ibey, Bennett L.
• 通讯作者: Ibey, Bennett L.

Cell stimulation and calcium mobilization by picosecond electric pulses.

• DOI: 10.1016/j.bioelechem.2015.05.013
• 发表时间: 2015-10
• 期刊: BIOELECTROCHEMISTRY
• 影响因子: 5
• 作者: Semenov, Iurii;Xiao, Shu;Kang, Dongkoo;Schoenbach, Karl H.;Pakhomov, Andrei G.
• 通讯作者: Pakhomov, Andrei G.

Diffuse, non-polar electropermeabilization and reduced propidium uptake distinguish the effect of nanosecond electric pulses.

• DOI: 10.1016/j.bbamem.2015.06.018
• 发表时间: 2015-10
• 期刊: Biochimica et biophysica acta
• 作者: Semenov I;Zemlin C;Pakhomova ON;Xiao S;Pakhomov AG
• 通讯作者: Pakhomov AG