A Theory-Based Electroporation Method for Optimized Molecular Delivery

电穿孔是一种进入细胞细胞质以传递分子的方法，同时保持细胞活力和功能。在这种技术中，电场可以在体外或体内施加，瞬间渗透细胞膜，生物活性分子可以通过细胞膜进入细胞，如DNA， RNA和氨基酸。电穿孔的应用包括基因转染、癌症治疗和干细胞分化。尽管进行了广泛的研究，并且对孔隙形成机制的理解有所提高，但电穿孔方法仍然存在效率有限和细胞过度损伤的问题。我们认为，对电穿孔后控制分子运输的机制缺乏理解是这些不足的根本原因。我们提出，电穿孔中的分子运输是由电动力学机制控制的，电动力学机制增加了运输速率，导致细胞内分子种类的积累，而不仅仅是通过打开的孔扩散。基于电流体动力学理论和我们的数值模拟的尺度分析以及前人的实验结果支持了电动力学的重要作用。基于这些研究，我们相信在电穿孔过程中，通过增加进入细胞的运输，同时减少细胞渗透性，可以利用电动力学介导的运输来提高效率和细胞活力。在本提案中，我们建立在之前的工作基础上，设计了基于电动力学传输原理的协议和微设备，专门用于电穿孔细胞。因此，本提案的具体目标是：目标1：在电穿孔过程中合理地将外加电场分成两个阶段——“渗透”阶段和“运输”阶段——以最大限度地提高分子传递和细胞活力。在典型的电穿孔中，一个脉冲被传递，在细胞膜上形成孔，并驱动运输进入或离开细胞。然而，渗透所需的场强明显大于离子和大分子有效运输所需的场强。同样地，虽然电穿孔所需的电场强度下的长脉冲持续时间会显著损伤细胞，但在低电场强度下的相同脉冲持续时间可能会通过增加传输时间来增强传递。基于我们的分析，我们将构建一个两阶段的电穿孔装置，该装置为电穿孔和电动介导的运输提供单独的脉冲。我们将通过证明积累依赖于细胞内和细胞外电导率的比例，以及带正电荷和负电荷的物种的明显积累，来证实这种运输是通过电动介导的，并利用这些理论驱动的实验来优化参数空间，以实现最大的递送和细胞活力。目标2：小型化两级装置，以实现高效率和单细胞通量输送。在许多应用中，为了阐明信号机制，需要将单个基因或基因组合递送到单个或细胞群体中。递送到单个细胞通常是通过微移液管注射DNA来完成的，这种方法保持了高度的有效性，但存在吞吐量有限和自动化问题；相反，通过电穿孔给细胞的悬浮递送会使细胞暴露在不同的电场中，而电场的效力和活力也会发生变化。通过将理论驱动的协议与微流体相结合，我们将开发用于有效转染细胞群的高通量设备。我们将把我们的两阶段现场递送协议整合到微流控片上电穿孔装置中，以便快速有效地递送到单个细胞。我们将根据悬浮细胞的结果对效率和生存能力进行基准测试。所建议的工作的智力价值包括：1。这项工作将是第一个明确证明通过电穿孔在活细胞中进行的电动介导的运输。2. 基于我们的建模框架的具体方案将被设计用于大幅提高对活细胞的高效和有效递送。3. 将我们的定制协议与微流体相结合，增强了电穿孔技术的能力，并可作为多路复用高通量设备的原理验证设备。拟议工作的更广泛影响包括：基于合理的、基本的科学和工程原理，开发具有成本效益、可重复、安全和高效的电穿孔设备和方案；对于生物学研究和临床应用来说，这两个领域都有极大的潜力造福人类健康和福祉。2. 拟议的跨学科研究将整合到针对生物医学和机械工程学生的教育工作中，以及旨在鼓励代表性不足的学生学习科学、技术、工程和数学（STEM）学科的推广工作中。3. 工作成果将通过在专业会议（包括ASME， BMES和FASEB）上的报告，在工程和实验生物学社区广泛传播，并提交给知名期刊，如流体力学杂志，生物物理杂志，芯片实验室和生物技术与生物工程等。

0967598ShreiberElectroporation is a means to access the cytoplasm of a cell for delivery of molecules, while simultaneously maintaining viability and preserving functionality. In this technique, an electric field, which can be applied in vitro or in vivo, transiently permeabilizes the cell membrane, through which biologically active molecules can enter the cell, such as DNA, RNA, and amino acids. Applications of electroporation include gene transfection, cancer therapies, and stem cell differentiation. Despite extensive research, and an improved understanding of the mechanisms of pore formation, electroporation methods still suffer from limited efficiency and excessive cell damage. We believe that a fundamental lack of understanding of the mechanisms that govern molecular transport following electroporation is the root cause for these shortfalls. We propose that molecular transport in electroporation is controlled by electrokinetic mechanisms that increase transport rates and cause accumulation of molecular species within the cell, and not merely diffusion through opened pores. The important role of electrokinetics is supported by scaling analyses based on electrohydrodynamic theory and our numerical simulations, as well as experimental results by previous researchers. Based on these studies, we believe that electrokinetically-mediated transport during electroporation can be exploited to improve efficiency and cell viability by increasing transport into the cell while minimizing cell permeabilization. In this proposal, we build on our previous work to design protocols and microdevices based on principles of electrokinetic transport specifically for electroporating cells. Accordingly, the Specific Aims of this proposal are:Aim 1: To rationally split the applied electric field during electroporation into two phases - a 'permeabilizing' phase and a 'transport' phase - to maximize both molecular delivery and cell viability In typical electroporation, a single pulse is delivered to form pores in the cell membrane and to drive transport into or out of the cell. However, the field strength necessary for permeabilization is significantly greater than that required for effective transport of ions and macromolecules. Similarly, whereas a long pulse duration at field strengths necessary for electroporation can significantly damage cells, the same duration at low field strengths may enhance delivery by increasing transport time. Based on our analyses,we will build a two-stage electroporation device that delivers separate pulses for electroporation and electrokinetically- mediated transport. We will confirm that the transport is mediated electrokinetically by demonstrating dependence of accumulation on the ratio of intracellular to extracellular conductivity and distinct accumulation of positively and negatively charged species, and use these theory-driven experiments to optimize a parameter space for maximum delivery and cell viability.Aim 2: To miniaturize the two-stage device for high efficiency and throughput delivery to single cells. In many applications, delivery of single genes or combinations of genes to individual or populations of cells is desired for elucidation of signaling mechanisms. Delivery to individual cells is typically done with micropipette injection of DNA, which maintains a high degree of efficacy, but suffers from limited throughput and automation problems; conversely, delivery to cells in suspension via electroporation exposes thecells to a varying electric field with associated variability in efficacy and viability. By combining the theorydriven protocols with microfluidics, we will develop high-throughput devices for efficient transfection of cell populations. We will integrate our two-stage field delivery protocols into a microfluidic on-chip electroporation device for fast and efficient delivery to single cells. We will benchmark efficiency and viability capabilities against results from cells in suspension.The intellectual merit of the proposed work includes: 1. This work will be the first to definitively demonstrate electrokinetic-mediated transport via electroporation in living cells. 2. Specific protocols based on our modeling framework will be designed for substantial improvement in efficient and effective delivery to living cells. 3. Combining our customized protocols with microfluidics enhances the capabilities of electroporation technology and serves as a proof-of-principle device for mutli-plexed high-throughput devices.The broader impact of the proposed work includes: 1. The development of cost effective, reproducible, safe, and efficient electroporation devices and protocols, built on sound, fundamental scientific and engineering principles; for both biological research and clinical applications both arenas have great potential to benefit human health and welfare. 2. The proposed interdisciplinary research will be integrated into an educational effort directed toward students in Biomedical and Mechanical Engineering, as well as an outreach effort aimed at encouraging under-represented students to the study of the Science, Technology, Engineering, and Mathematics (STEM) disciplines. 3. The results of the work will be broadly disseminated through the Engineering and Experimental Biology communities via presentations at professional meetings, including ASME, BMES, and FASEB, and submission to prestigious journals, such as the Journal of Fluid Mechanics, Biophysical Journal, Lab-on-a-chip, and Biotechnology & Bioengineering, among others.