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

电穿孔是一种利用脉冲电场在细胞膜上制造孔的技术。可逆性孔隙形成已被认为是将大分子引入细胞，同时保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）已被用于不良组织的消融。结果表明，由于IRE的非热性质，它保留了重要的组织成分，如细胞外基质、大血管和神经。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，但被诊断为GBM的患者的中位生存期只有15个月。生存率低的原因之一是胶质瘤细胞通常浸润超过可见肿瘤体积2厘米。IRE过程中电场的耗散会在消融区之外产生可逆电穿孔细胞区域，这些区域可能更容易被药物吸收。该提案将评估IRE与化疗药物联合治疗可逆电穿孔区域内浸润细胞的能力。由于血脑屏障（BBB）的存在，药物递送不足也限制了恶性胶质瘤的治疗。因此，该计划还将研究IRE是否可以用于介导血脑屏障破坏，以帮助化疗药物的递送。在分子、细胞和组织水平上结合实验和建模将用于确定IRE的机制，并加深对电场在大脑中的影响的科学理解。具体来说，将确定微电穿孔装置中的单个细胞、悬浮细胞和活体脑组织中可逆和不可逆电穿孔的电场强度-持续时间关系。对于每种制备，数值模型将计算实验中测量的电和热量，并将亚细胞水平、细胞水平和组织水平的反应联系起来。具体而言，本研究旨在：1)在细胞水平上定量了解电穿孔以及ire诱导的电穿孔细胞死亡和化疗介导的电穿孔细胞死亡的机制。2)建立血脑屏障破坏和IRE病灶消融区域以及可逆电穿孔区域的多尺度数值模型，用于预测治疗计划。3)利用大鼠模型量化IRE对GBM消融、血脑屏障破坏和浸润性细胞死亡的体内影响。这一建议的学术价值将是解决长期以来关于IRE诱导细胞死亡机制的争议。了解IRE在体外和体内的电场、温度和电导率分布将有助于我们确定膜破裂、通过毛孔的过度渗漏或细胞内热效应是否导致细胞死亡。这是第一次，温度将被实时测量，以评估和验证IRE协议产生细胞死亡而不引起热损伤的理论。此外，这些信息将极大地提高人们设计有效的IRE治疗计划模型的能力。捕获膜动态变化的多尺度模型将被构建，并最终可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用于消融脑内侵袭性、浸润性肿瘤的非热技术，并利用IRE介导的血脑屏障破坏改善肿瘤边缘以外抗癌药物的吸收。这项提议的更广泛的影响包括一种非热消融方法的发展，这将使我们不仅能够对抗多种类型的癌症，而且还能对抗其他病理，如心律失常。将IRE纳入主流医学将带来巨大的社会经济效益，并显著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、工业和生物防御方面的其他新兴应用。在教育方面，该项目将为奥克伍德大学（Oakwood University）的学生开设生物医学工程辅修课程，学生将花两个夏天在弗吉尼亚理工大学（Virginia Tech）上课并进行研究。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.