CAREER: An experimental and numerical investigation of the biophysical processes of pulsed electric field induced irreversible electroporation for Glioblastoma Multiforme

Davalos 1055913电穿孔是一种采用脉冲电场在细胞膜上产生孔的技术。可逆孔形成已被认为是将大分子引入细胞同时维持细胞活力的有力手段。最近，导致细胞死亡的不可逆电穿孔（IRE）已被用于消融不需要的组织。结果表明，由于其非热性质，IRE保留了重要的组织成分，如细胞外基质，主要血管和神经。本项目将研究IRE方案是否适用于多形性胶质母细胞瘤（GBM）肿瘤的消融。尽管最近脑癌治疗取得了进展，但被诊断患有GBM的人的中位生存期仅为15个月。存活率差的原因之一是胶质瘤细胞通常浸润超过可见肿瘤体积达2cm。在IRE期间电场的消散引起消融区外部的可逆电穿孔细胞区域，其可能更容易吸收药物。该建议将评估IRE与化疗药物联合治疗可逆电穿孔区内浸润细胞的能力。恶性神经胶质瘤的治疗也受到血脑屏障（BBB）导致的药物递送不足的限制。因此，本研究将从分子、细胞和组织水平探讨IRE介导血脑屏障破坏的机制，加深对电场在脑内作用的科学认识。具体地，将针对微电穿孔装置中的单个细胞、悬浮液中的细胞和体内脑组织确定可逆和不可逆电穿孔的电场的强度-持续时间关系。对于每种制备，数值模型将计算实验期间测量的电学和热学量，并将在亚细胞水平，细胞水平和组织水平反应之间建立联系。具体而言，该提案旨在：1）在细胞水平上对电穿孔以及电穿孔细胞中IRE诱导的细胞死亡和化疗介导的细胞死亡的机制进行定量理解。2)创建BBB破坏和IRE局灶性消融区域沿着可逆电穿孔区域的多尺度数值模型，用于预测性治疗计划。3)使用大鼠模型定量IRE对GBM消融、BBB破坏和浸润性细胞死亡的体内作用。这一建议的智力价值将是关于IRE诱导细胞死亡机制的长期争议的解决方案。获得的电场，温度和电导率分布在整个IRE在体外和体内的知识将帮助我们查明是否膜破裂，通过孔隙过度泄漏，或细胞内的热效应是负责细胞死亡。这是第一次，温度将被实时测量，以评估和验证IRE协议在不诱导热损伤的情况下产生细胞死亡的理论。此外，这些信息将大大提高设计有效的IRE治疗计划模型的能力。将构建捕获膜中动态变化的多尺度模型，并最终将其转化为涉及脉冲电场的任何类型的治疗。这项研究将最终发展一种非热消融技术，用于消融大脑中的侵袭性、浸润性肿瘤，并利用IRE介导的BBB破坏改善肿瘤边缘以外的抗癌药物的摄取。这项提议的更广泛影响包括发展一种非热消融方法，使我们不仅能够对抗多种类型的癌症，而且能够对抗其他病理，例如心律失常。将IRE整合到主流医学中将产生巨大的社会经济效益，并显着提高数百万患者的生活质量和寿命。它还将影响电穿孔在组织工程、基因治疗、DNA疫苗接种、医学、工业和生物防御中的其他新兴应用。在教育方面，该项目将为奥克伍德大学的学生创建一个生物医学工程辅修专业，学生将在弗吉尼亚理工大学花两个夏天上课和进行研究。学生将有机会从事拟议研究的要素以及各种其他生物工程项目。

Davalos1055913Electroporation is a technique, which employs pulsed electric fields to create pores across cell membranes. Reversible pore formation has been recognized as a powerful means to introduce macromolecules into cells, while maintaining cell viability. Recently, irreversible electroporation (IRE), which results in cell death, has been used for the ablation of undesirable tissue. Results indicate that due to its non-thermal nature, IRE preserves important tissue components, such as the extracellular matrix, major blood vessels, and nerves. This project will investigate whether IRE protocols can be adapted for the ablation of Glioblastoma Multiforme (GBM) tumors. Despite recent advancements in brain cancer therapies, the median survival for people diagnosed with GBM is only 15 months. One of the reasons for poor survival is that glioma cells typically infiltrate up to 2 cm beyond the volume of the visible tumor. Dissipation of the electric field during IRE gives rise to regions of reversibly electroporated cells outside the ablation zone that may be more susceptible to the uptake of drugs. This proposal will assess IRE's capacity to treat infiltrative cells within the zone of reversible electroporation when combined with chemotherapeutic agents. Treatment of malignant gliomas is also limited by insufficient delivery of drugs due to the blood-brain-barrier (BBB). Therefore, this plan will also investigate whether IRE can be applied to mediate BBB disruption to aid in the delivery of chemotherapeutic agents.A combination of experiments and modeling on the molecular, cellular, and tissue levels will be used to determine the mechanism of IRE and deepen scientific understanding of the effects of electric fields in the brain. Specifically, strength-duration relationships of the electric field for reversible and irreversible electroporation will be determined for individual cells in a micro-electroporation device, cells in suspension, and in vivo brain tissue. For each preparation, numerical models will compute electrical and thermal quantities measured during the experiments, and they will make the connection between subcellular-level, cellular-level, and tissue-level responses. Specifically, this proposal aims to: 1) Develop a quantitative understanding of electroporation at the cellular level and the mechanism of both IRE-induced cell death and chemotherapy mediated cell death in electroporated cells. 2) Create a multi-scale numerical model of BBB disruption and IRE focal ablation regions along with regions of reversible electroporation for predictive treatment planning. 3) Quantify the in vivo effects of IRE for GBM ablation, BBB disruption, and infiltrative cell death using a rat model. The intellectual merit of this proposal will be the resolution of the longstanding controversy regarding the mechanism of cell death induced by IRE. Gaining knowledge of the electric field, temperature, and conductivity distributions throughout IRE both in vitro and in vivo will help us pinpoint whether membrane rupture, excessive leakage through pores, or intracellular thermal effects are responsible for cell death. For the first time, temperature will be measured in real-time in order to assess and validate the theory that IRE protocols generate cell death without inducing thermal damage. Additionally, this information will tremendously improve one's ability to design effective IRE treatment planning models. A multi-scale model that captures the dynamic changes in the membrane will be constructed and can ultimately be translated to any type of therapy involving pulsed electric fields. This research will culminate in the development of a non-thermal technique for ablating aggressive, infiltrative tumors in the brain, and the improvement of uptake of anti-cancer agents beyond the tumor margin utilizing IRE mediated BBB disruption.The broader impacts of this proposal include the development of a method of non-thermal ablation that will allow us not only to fight numerous types of cancer, but other pathologies, such as cardiac arrhythmias. Integration of IRE into mainstream medicine will have enormous socioeconomic benefits and dramatically enhance the quality and length of life for millions of patients. It will also impact other emerging applications of electroporation in tissue engineering, gene therapy, DNA vaccination, medicine, industry, and biodefense. In education, this project will create a Biomedical Engineering Minor for students at Oakwood University, in which the students would spend two summers taking classes and conducting research at Virginia Tech. The students would have the opportunity to work on elements of the proposed research as well as a variety of other bioengineering projects.