A THEORY-BASED ELECTROPORATION METHOD FOR OPTIMIZED MOLECULAR DELIVERY

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.