Evaluating in Vitro Cell Transfection Via Oscillatory Bipolar Electric Pulses

Recent pandemic and immunogenicity issues with carrier-based delivery such as lipid nanoparticle encapsulated mRNA-based vaccines has highlighted the need for a safer and effective alternative drug delivery method. Electroporation is a simple yet effective physical mechanism for intracellular delivery of nucleic acids (mRNA, plasmid DNA, etc.). It involves temporary permeabilization of cell membrane through micro-to-milliseconds electric pulses. However, conventional electroporators have several limitations such as low transfection efficiency and poor cell viability. These electroporator typically employ square wave and exponential decay electric pulses for intracellular delivery of nucleic acids, while oscillatory bipolar waveforms remain less explored. Moreover, the bulkiness and cost of conventional electroporators limit its applications. An ultra-low-cost handheld electroporator, ePatch generates oscillatory bipolar electric pulses and has shown enhanced delivery of both mRNA and plasmid DNA in rodents. Additionally, ePatch has shown excellent tolerance in humans and similar or better immune response against mRNA and DNA-based SARS-CoV-2 vaccines compared to lipid nanoparticles in mice. Cellular transfection via oscillatory electric pulses can be further enhanced by identifying a range of optimal electroporation parameters.

In this study, we conducted in vitro cell transfection of mouse macrophages (RAW 246.7 cells) via electroporation with ePatch. In the seeded plate, cells adhered to an electrically conductive indium tin oxide (ITO) glass surface in the well as a monolayer on glass. We generate bipolar oscillatory electric pulses at different electric field strengths with varying number of pulses. We applied 0 - 20 electric pulses for ePatch. Cell transfection and cell viability were evaluated using cell-impermeable dyes. Propidium iodide was used to identify non-viable cells and SYTOX Green was used to identify electroporated cells. Fluorescence microscopy showed an increase in number of electroporated cells with increasing numbers of pulses and tradeoff between viability and electroporation. For a constant electric field strength, 10 electric pulses via an ePatch achieved higher cell transfection with lower cell death compared to conventional electroporators, while 20 pulses resulted in higher cell death with comparable percentage of electroporated cells. We show that higher electric field strength resulted in irreversible electroporation (i.e., loss of cell viability). These results are based on monolayers of cells adhered on ITO glass slides and in the future, multilayers of cells could be used to further improve the understanding of cell electroporation. We will also present our results from ongoing work on the effect of electric field strength on cell transfection of cell-impermeable dyes and cell death. Our in vitro study provides a foundation for optimizing in vivo nucleic acid delivery using electroporation via ePatch.