ELECTROPORATION MECHANISM, MICRODOSIMETRY AND INCREASINGLY REALISTIC CELL MODELS

描述（由申请人提供）：电穿孔应用在基础研究和医学中的数量和意义都在迅速增长。几乎任何大小的分子都可以通过合适的电脉冲被输送到活细胞中。最成熟的临床应用是电化学疗法（ECT），它将小的癌症药物递送到靶向肿瘤细胞中。人们对递送大分子也很感兴趣，特别是用于DNA疫苗接种。另外两种电穿孔介导的肿瘤消融治疗是无药物的，完全基于改变细胞膜的非热电相互作用。一种是不可逆电穿孔（IRE），它采用相对较大的脉冲来改变细胞的最外层膜。这导致肿瘤细胞通过坏死而死亡。另一种是基于纳秒脉冲电场（nsPEF），其涉及更大但更短的脉冲。这些引起细胞内变化，通过凋亡杀死靶细胞。除了影响肿瘤细胞外，附近的神经细胞（本质上更大）可能会受到损害，即使它们位于目标区域之外。尽管取得了令人兴奋的进展，但对预期结果和副作用的机制仍然知之甚少。我们已经成功地构建了越来越逼真的细胞计算机模型，描述了电穿孔的基本特征。这是一个困难的问题，其中细胞膜在纳秒到毫秒的时间尺度上改变其电特性，从而重新分配细胞内的电场。我们的一般机制假设是，电力创建的瞬态孔占非热细胞响应强电场脉冲的关键特征。孔隙产生，膨胀/收缩，然后消失。由此产生的通过临时孔的分子运输可以杀死细胞。在细胞膜上，孔的数量和大小是变化的。它们控制着不同大小和电荷的分子的传输。分子摄取和释放是所有这些过程在细胞内不同部位同时发生的结果。我们建议扩展我们成功的模型，包括几个不规则形状的细胞紧密结合在一起，代表在体内环境。这些模型应该提供有用的描述分子运输甚至细胞器膜，并可能导致“在硅片”标记细胞死亡。这将为基于计算机的筛选不同体内电极配置的电穿孔脉冲波形奠定基础。可能的体内电极配置和EP脉冲波形的数量基本上是无限的，仅通过实验探索或评估这些组合是不现实的。因此，我们希望我们日益逼真的模型可以与FDA/IT'IS最近开发的一套解剖学上正确的全身模型（“虚拟家庭”）一起使用，以指导科学理解并为医疗器械监管过程做出贡献。 公共卫生相关性：电穿孔在基础研究和医学领域的应用持续快速增长，但其基本机制仍知之甚少。我们建议扩展我们成功的模型，以代表在体内环境中的细胞，这将提供有用的描述细胞内的分子运输，并可能导致在硅片上的细胞死亡的标志物。先进的多单元模型可与最近开发的FDA/IT'IS解剖学上正确的虚拟系列模型一起使用，以指导科学理解并协助医疗器械监管。

DESCRIPTION (provided by applicant): Electroporation applications continue to grow rapidly in number and significance in both basic research and medicine. A molecule of almost any size can be delivered into living cells by suitable electrical pulses. The most established clinical application is electrochemotherapy (ECT), which delivers small cancer drugs into targeted tumor cells. There is also great interest in delivering large molecules, particularly for DNA vaccination. Two other electroporation-mediated tumor ablation treatments are drug-free, based entirely on non-thermal electrical interactions that alter cell membranes. One is irreversible electroporation (IRE), which employs relatively large pulses that change a cell's outermost membrane. This leads to tumor cell death by necrosis. The other is based on nanosecond pulsed electric fields (nsPEF), which involve much larger but shorter pulses. These cause intracellular changes that kill targeted cells by apoptosis. In addition to affecting tumor cells, nearby nerve cells (which are larger, by nature) may be damaged even if they lie outside the targeted region. In spite of this exciting progress, the mechanisms underlying the desired outcomes and side effects remain poorly understood. We have succeeded in constructing increasingly realistic computer models of cells that describe essential features of electroporation. This is a difficult problem in which the cell's membranes change their electrical properties on a time scale of nanoseconds to milliseconds, which redistributes the electric field within the cell. Our general mechanistic hypothesis is that electrically created transient pores account for key features of non-thermal cell responses to strong electric field pulses. Pores are created, expand/contract and later vanish. The resulting molecular transport through the temporary pores can kill cells. The changing number and size of pores varies across a cell membrane. These govern transport of molecules of different size and charge. Molecular uptake and release is the result of all these processes taking place simultaneously at different sites within a cell. We propose extending our successful models to include several irregularly shaped cells close together, representing in vivo environments. These models should provide useful descriptions of molecular transport across even organelle membranes, and may lead to "in silico" markers of cell death. This should set the stage for computer-based screening of electroporation pulse waveforms with different in vivo electrode configurations. The number of possible in vivo electrode configurations and EP pulse waveforms is essentially infinite and it is unrealistic to explore or evaluate these combinations by experiment alone. We thus expect that our increasingly realistic models can be used with the set of anatomically correct whole body models ("Virtual Family") recently developed FDA/IT'IS to guide scientific understanding and to contribute to the medical device regulatory process. PUBLIC HEALTH RELEVANCE: Electroporation applications continue to grow rapidly in both basic research and medicine, but the basic mechanisms remain poorly understood. We propose extending our successful models to represent cells within in vivo environments, which will provide useful descriptions of molecular transport within cells and may lead to in silico markers of cell death. Advanced multicell models can be used with the recently developed FDA/IT'IS anatomically correct Virtual Family models to guide scientific understanding and to assist medical device regulation.