Electroporation 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 the cells 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.
The objective of this project was to develop a quantitative, theory-based understanding of how molecules are delivered to cells during electroporation. In electroporation, an electric field is applied to cells to temporarily permeabilize the cell membrane so that biologically active molecules can enter the cell, such as DNA, RNA, proteins, and drugs. Applications of electroporation include gene transfection, cancer therapies, and stem cell differentiation. However, despite extensive research and an improved understanding of the mechanisms of pore formation, electroporation methods still suffer from limited efficiency and excessive cell damage, primarily because scientists must empirically identify the appropriate electric pulses to apply. If the applied field is too strong, too many cells will die, but if it is too weak, not enough cells are permeabilized and insufficient delivery occurs. We believed that a fundamental lack of understanding of the mechanisms that govern how molecules are transported into the permeabilized cells was a root cause for these shortfalls. We believed 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 physical principles and electrohydrodynamic theory. In the project, we tracked the entry of different fluorescent molecules into cells during electroporation under defined conditions and compared the results to computer simulations that we developed. An example is shown in the attached figure, where the force of the electric field induces propidum iodide fluorescence to accumulate asymmetrically within the cell, as is predicted by our mathematical models. The accumulation is also rapid, with significant accumulation occurring in under 100ms. If transport were strictly diffusive, accumulation would be uniform and much slower. With these and other experiments, we proved that charged molecules are delivered into the cell by the electric field, and that this driving force can be much lower that what is required to permeabilize a cell. Therefore, electroporation protocols can be rationally designed to first permeabilize a cell with a very brief but higher strength field, followed by a safer, lower field that is still strong enough to push molecules into the cell electrokinetically but will not damage the cell. We also began to leverage this new, quantitative understanding towards miniaturized, lab-on-a-chip devices. The intellectual merit of the project derives from applying quantitative electrohydrodynamic theory and modeling to an aspect that has otherwise been examined empirically. Given the prevalent use of electroporation in research and in industry, our project and results have broad impact, as they will not only accelerate electroporation pulse protocol design, but also can lead to advanced devices that co-optimize cell viability and molecular delivery. The project has also served as the vehicle for training 3 PhD students and one MS student, and has provided opportunities for several undergraduates to perform cutting edge, interdisciplinary research at the intersection of engineering and biology.
