A Theory-Based Electroporation Method for Optimized Molecular Delivery

简而言之，电穿孔是一种进入细胞质以输送分子的手段，同时保持活性和保留功能。在这项技术中，一个可以在体外或体内施加的电场瞬间渗透细胞膜，生物活性分子可以通过它进入细胞，如DNA、RNA和氨基酸。电穿孔技术的应用包括基因导入、癌症治疗和干细胞分化。尽管进行了广泛的研究，并对孔形成的机制有了更深入的了解，但电穿孔方法仍然存在效率有限和细胞损伤过大的问题。我们认为，对电穿孔后分子传输机制的根本缺乏了解是这些不足的根本原因。我们认为，电穿孔中的分子传输是由电动机制控制的，这种机制增加了传输速率并导致分子物种在细胞内积累，而不仅仅是通过开放的孔扩散。电动力学的重要作用得到了基于电流体理论的标度分析和我们的数值模拟以及前人的实验结果的支持。基于这些研究，我们相信，电穿孔过程中的电动介导的运输可以通过增加进入细胞的运输来提高效率和细胞活力，同时最大限度地减少细胞的通透性。在这项提案中，我们在以前工作的基础上，专门为电穿孔细胞设计了基于电动传输原理的协议和微型设备。因此，这项提议的具体目标是：目标1：将电穿孔过程中的外加电场合理地分为两个阶段--渗透阶段和运输阶段--在典型的电穿孔中，为了最大化分子输送和细胞存活率，传递单个脉冲以在细胞膜上形成孔洞，并驱动进入或离开细胞的输送。然而，渗透所需的场强明显大于有效传输离子和大分子所需的场强。类似地，尽管电穿孔所需的场强下的长脉冲持续时间会显著损害细胞，但在低场强下的相同持续时间可以通过增加传输时间来增强传输。基于我们的分析，我们将建立一个两级电穿孔装置，提供用于电穿孔和电动中介传输的单独脉冲。我们将通过证明积累依赖于细胞内和细胞外电导率的比率以及不同的正负电荷物种的积累来证实这种运输是电动调节的，并利用这些理论驱动的实验来优化最大输送和细胞存活率的参数空间。目标2：将两级装置小型化，以实现高效和向单细胞的吞吐量输送。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传递到单个或多个细胞。对单个细胞的输送通常是通过微管注射DNA完成的，这种方法保持了高度的效力，但存在产量有限和自动化问题；相反，通过电穿孔将DNA输送到悬浮细胞会使细胞暴露在不同的电场中，相关的效力和活力也会发生变化。通过将理论驱动的方案与微流控技术相结合，我们将开发高通量的设备，用于高效地转染细胞群体。我们将把我们的两阶段现场输送协议集成到微流控芯片电穿孔设备中，以便快速高效地输送到单个电池。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。拟议工作的智力价值包括：1.这项工作将首次明确展示电动媒介通过电穿孔在活细胞中的运输。2.将根据我们的建模框架设计具体的协议，以大幅改进对活细胞的高效和有效的交付。3.将我们定制的协议与微流体相结合，增强了电穿孔技术的能力，并作为多路高通量设备的原理验证设备。拟议工作的更广泛影响包括：1.基于健全的、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协议；对于生物研究和临床应用，这两个领域都具有造福人类健康和福利的巨大潜力。2.拟议的跨学科研究将纳入针对生物医学和机械工程学生的教育工作，以及旨在鼓励代表不足的学生学习科学、技术、工程和数学(STEM)学科的外联工作。3.这项工作的成果将通过在ASME、BMES和FASE B等专业会议上的陈述和提交给著名期刊，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，通过工程和实验生物学社区广泛传播。

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.