Cell Death Induction by High-Voltage, Nanosecond-duration Electric Pulses

描述（由申请人提供）：脉冲功率技术的最新进展在能够向低阻抗负载（如生物组织和细胞样本）提供高电压，纳秒持续电脉冲（nsEP）的独特设备的工程中达到顶峰。与较长的脉冲（如常规用于电刺激和电穿孔的脉冲）相比，nsEP的特点是电压急剧增加（1012-1014 V/cm /秒）和极高的峰值电场（103-106 V/cm），而暴露组织中的总能量沉积仍然很低，焦耳加热不超过几摄氏度。由于极端的电场值，nsEP可以引起独特的生物效应，如Ca2+爆发，电压门控离子通道的持续失活。细胞肿胀和起泡，膜的“纳米电穿孔”，坏死和凋亡细胞死亡。结合仅影响有限体积的组织，nsEP是一种很有前途的组织消融和实体瘤破坏的新治疗方式。首次动物试验证明了nsEP治疗接种肿瘤的有效性。然而，nsEP暴露后导致细胞死亡的物理和生理机制尚不清楚，这阻碍了nsEP在医学应用中的进展。不同细胞类型的nsEP敏感性有显著差异尚未得到解释，也不知道哪些nsEP参数（如电场、脉冲速率、吸收剂量）决定了细胞毒性作用。我们的初步实验发现，nsEP效应与已知的稀疏电离辐射（SIRs）和引起氧化应激的化学试剂的效应出人意料地相似。对于这两种方式，细胞死亡的主要机制是自由基的损伤，我们假设nsEP暴露也是如此。本研究包括四个具体目标，旨在量化nsEP在不同细胞和不同生理条件下的细胞毒性作用，验证自由基损伤假说，探索nsEP诱导细胞死亡的机制和途径。具体目标1：大规模定量分析细胞死亡对nsEP治疗物理参数的依赖，包括脉冲持续时间、电压、剂量、脉冲数及其重复率。专项目的2：探讨细胞培养的生理条件（细胞周期、生长阶段和分化）对nsEP暴露敏感性的影响。特异性目的3：分析nsEP暴露引起的细胞死亡中可能涉及的自由基损伤机制。具体目标4：分析nsEP长期破坏质膜离子电导率的机制及其作为导致nsEP诱导细胞死亡的主要生理事件的可能作用。本研究将重点关注哺乳动物细胞对纳秒级高压电脉冲（nsEP）敏感性的物理化学和生理机制。预期的结果将有助于量化、预测和有目的地修改nsEP的敏感性，有助于理解nsEP生物效应的机制，并促进nsEP医学应用的发展，如组织消融和肿瘤破坏。

DESCRIPTION (provided by applicant): Recent advances in pulsed power technology culminated in engineering of unique devices capable of delivering high-voltage, nanosecond-duration electrical pulses (nsEP) to low-impedance loads such as biological tissues and cell samples. Compared to longer pulses (such as those routinely used for electrostimulation and electroporation), nsEP are distinguished by a steep voltage increase (1012-1014 V/cm per second) and extremely high peak E-field (103-106 V/cm), whereas the total energy deposition into exposed tissue remains low and Joule heating does not exceed a few degrees C. Due to extreme E-field values, nsEP can cause unique bioeffects, such as Ca2+ bursts, lasting inactivation of voltage-gated ion channels, cell swelling and blebbing, "nanoelectroporation" of membranes, necrotic and apoptotic cell death. Combined with the ease of affecting only a limited volume of tissue, nsEP are a promising new therapeutic modality for tissue ablation and solid tumors destruction. First animal trials demonstrated the efficiency of nsEP treatment of inoculated tumors. However, physical and physiological mechanisms leading to cell death after nsEP exposure have been poorly understood, which hinders progress in medical applications of nsEP. Remarkably different nsEP sensitivity of different cell types has not been explained, and it is not known which nsEP parameters (e.g., E- field, pulse rate, absorbed dose) determine the cytotoxic effect. Our preliminary experiments established unexpected similarities of nsEP effects with known effects of both sparsely ionizing radiations (SIRs) and chemical agents that cause oxidative stress. For both these modalities, the principal mechanism of cell death is damage by free radicals, and we hypothesize that this is also the case for nsEP exposure. The proposed study consists of four Specific Aims intended to quantify nsEP cytotoxic effects in different cells and under different physiological conditions, to test the free radical damage hypothesis, and explore the mechanisms and pathways responsible for nsEP-induced cell death. Specific Aim 1: Wide-scale quantitative analysis of cell death dependence on the physical parameters of nsEP treatment, including pulse duration, voltage, dose, the number of pulses, and their repetition rate. Specific Aim 2: Explore the role of physiological conditions of the cell culture (cell cycle phase, growth stage, and differentiation) on the sensitivity to nsEP exposure. Specific Aim 3: Analyze possible involvement of free radical damage mechanism in cell death caused by nsEP exposure. Specific Aim 4: Analyze mechanisms of long-term disruption of plasma membrane ionic conductance by nsEP and its possible role as a primary physiological event that leads to nsEP-induced cell death. PUBLIC HEALTH RELEVANCE This study will be focused on physico-chemical and physiological mechanisms that underlie and determine mammalian cells sensitivity to nanosecond-duration, high-voltage electric pulses (nsEP). Anticipated results will help to quantify, predict, and purposefully modify nsEP sensitivity, assist understanding of mechanisms of nsEP bioeffects, and promote the development of nsEP medical applications, such as tissue ablation and destruction of tumors.