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最终导致了能够向生物组织和细胞样本等低阻抗负载输送高压、纳秒持续时间电脉冲（nsEP）的独特设备的工程化。与较长的脉冲（例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（每秒1012-1014 V/cm）和极高的高峰电场（103-106 V/cm），而沉积到暴露组织中的总能量保持较低，焦耳加热不超过几度。由于极端的电场值，nsEP可引起独特的生物效应，如Ca 2+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”、坏死和凋亡性细胞死亡。结合仅影响有限体积的组织的容易性，nsEP是用于组织消融和实体瘤破坏的有前景的新治疗模式。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知之甚少，这阻碍了nsEP在医学应用中的进展。尚未解释不同细胞类型的显著不同的nsEP敏感性，并且不知道哪些nsEP参数（例如，电场、脉冲频率、吸收剂量）决定细胞毒性效应。我们的初步实验建立了nsEP效应与稀疏电离辐射（SIR）和引起氧化应激的化学试剂的已知效应的意外相似性。对于这两种方式，细胞死亡的主要机制是由自由基损伤，我们假设这也是nsEP暴露的情况。该研究包括四个特定目的，旨在量化不同细胞和不同生理条件下的nsEP细胞毒性效应，以验证自由基损伤假说，并探索nsEP诱导细胞死亡的机制和途径。具体目标1：细胞死亡依赖于nsEP治疗的物理参数的大规模定量分析，包括脉冲持续时间、电压、剂量、脉冲数量及其重复率。具体目标二：探索细胞培养物的生理条件（细胞周期阶段、生长阶段和分化）对nsEP暴露敏感性的作用。具体目标3：分析自由基损伤机制在nsEP暴露引起的细胞死亡中的可能参与。具体目标4：分析nsEP长期破坏质膜离子电导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。公共卫生相关性本研究将侧重于物理化学和生理机制，这些机制是决定哺乳动物细胞对纳秒级高压电脉冲（nsEP）敏感性的基础。预期结果将有助于量化，预测和有目的地修改nsEP敏感性，帮助理解nsEP生物效应的机制，并促进nsEP医疗应用的发展，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.