A Theory-Based Electroporation Method for Optimized Molecular Delivery

基于理论的电穿孔方法优化分子传递

• 批准号: 0967598
• 负责人: David Shreiber
• 金额: $ 40.75万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0967598&HistoricalAwards=false
• 关键词: Theory; Based; Electroporation; Method; Optimized

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.

0967598 ShreiberElectroporation是一种进入细胞细胞质以递送分子的方法，同时保持活力并保留功能。在该技术中，可以在体外或体内施加的电场使细胞膜瞬时透化，生物活性分子可以通过其进入细胞，例如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，并且对孔形成的机制有了更好的理解，但电穿孔方法仍然存在效率有限和过度细胞损伤的问题。我们认为，从根本上缺乏对电穿孔后分子转运机制的理解是这些不足的根本原因。 我们提出，在电穿孔的分子运输是由电动机制，增加运输速率，并导致细胞内的分子物种的积累，而不仅仅是通过开放的孔扩散控制。基于电流体力学理论的标度分析和我们的数值模拟，以及以前的研究人员的实验结果支持电动力学的重要作用。基于这些研究，我们认为，可以利用电穿孔过程中的动电介导的转运，通过增加进入细胞的转运，同时最大限度地减少细胞透化，来提高效率和细胞活力。在这个建议中，我们建立在我们以前的工作，设计协议和微型设备的电动运输的原则，特别是电穿孔细胞的基础上。因此，本发明的具体目的是：目的1：在电穿孔过程中合理地将施加的电场分成两个阶段-"透化“阶段和”转运“阶段-以使分子递送和细胞活力最大化在典型的电穿孔中，递送单个脉冲以在细胞膜中形成孔并驱动转运进入或离开细胞。然而，透化所需的场强明显大于离子和大分子有效运输所需的场强。类似地，尽管电穿孔所需的场强下的长脉冲持续时间可显著损伤细胞，但低场强下的相同持续时间可通过增加运输时间来增强递送。基于我们的分析，我们将建立一个两阶段的电穿孔装置，提供单独的脉冲电穿孔和电动介导的运输。我们将证实，运输介导的电动证明依赖于细胞内的细胞外的电导率和不同的积累的正电荷和负电荷的物种的比例上的积累，并使用这些理论驱动的实验，以优化最大的交付和细胞violence.Aim 2的参数空间：为了实现高效率和高吞吐量交付给单细胞的两阶段设备。在许多应用中，需要将单个基因或基因组合递送至细胞个体或群体以阐明信号传导机制。递送至单个细胞通常通过微量移液管注射DNA来完成，其保持高度的功效，但遭受有限的通量和自动化问题;相反，通过电穿孔递送至悬浮液中的细胞使细胞暴露于变化的电场，其具有功效和活力的相关可变性。通过将理论驱动的协议与微流体相结合，我们将开发高通量设备，用于有效转染细胞群。我们将我们的两阶段现场交付协议集成到一个微流控芯片上的电穿孔设备快速和有效地交付到单细胞。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。这项工作将是第一个明确证明电动介导的运输通过电穿孔在活细胞中。2.基于我们的建模框架的特定协议将被设计用于实质性地改善向活细胞的高效和有效递送。3.将我们的定制方案与微流体技术相结合，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。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，建立在健全的基本科学和工程原则之上;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祉的巨大潜力。2.拟议的跨学科研究将被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。3.工作的结果将通过工程和实验生物学社区通过在专业会议上的演讲广泛传播，包括ASME，BMES和FASEB，并提交给着名期刊，如流体力学杂志，生物物理杂志，芯片实验室和生物技术生物工程等。