Influence of Electroporation Medium on Delivery of Cell-Impermeable Small Molecules by Electrical Short-Circuiting via an Aqueous Droplet in Dielectric Oil: A Comparison of Different Fluorescent Tracers

Abstract

Membrane permeabilization stimulated by high-voltage electric pulses has been used to deliver cell-impermeable exogenous molecules. The electric field effect on the cells depends on various experimental parameters, such as electric field strength, the number of electric pulses, and the electroporation medium. In this study, we show the influence of the electroporation medium on membrane permeabilization stimulated by electrical short-circuiting via an aqueous droplet in dielectric oil, a novel methodology developed by our previous investigations. We investigated the membrane permeabilization by three methods, influx of calcium ions, uptake of nucleic acid-binding fluorophores (YO-PRO-1), and calcein leakage. We demonstrated that the external medium conductivity had a significant impact on the cells in all described experiments. The short-circuiting using a low-conductivity electroporation medium enhanced the formation of both transient and irreversible membrane pores. We also found that clathrin-mediated endocytosis contributed to YO-PRO-1 uptake when a cell culture medium was used as an electroporation medium.

Influence of DNA characteristics on cell membrane damage stimulated by electrical short-circuiting via a low-conductive aqueous droplet in dielectric oil

“…We have also investigated the mechanisms of gene electrotransfection using short-circuiting via an aqueous droplet (Fig 1) [17,18]. A previous investigation demonstrated that electrical stimulation increases transient membrane permeability to calcium ions and cellimpermeable nucleic acid-binding fluorescent dyes (YO-PRO-1), indicating transient membrane pore formation [17]. Short-circuiting using a low-conductivity electroporation medium enhanced the formation of both transient and irreversible membrane pores.…”

“…The droplet electroporation apparatus was fabricated as described in our previous papers [17,18]. The apparatus consisted of pin headers and a printed circuit board, and the gap between the electrodes was 5.08 mm.…”

“…The leakage of small intracellular molecules stimulated by droplet electroporation was monitored using a cell-impermeant fluorescent dye, calcein. This experiment was conducted as previously described [17] with some modifications. Jurkat cells were incubated with 0.5 μM calcein-AM (Dojindo, Kumamoto, Japan) in D-PBS (-) for 30 minutes at 37˚C.…”

Sucrose Treatment Enhances the Electrotransfer of DNA by Activating Phospholipase A2

“…Sucrose has also been used in drug and gene delivery [ 13 , 14 , 15 ]. One of its applications is in improving electrotransfer (ET) [ 14 , 16 , 17 ], which is a technology for the delivery of molecular cargo, including DNA, RNA, protein, and ribonucleoprotein (RNP) [ 18 , 19 , 20 ]. ET is cost-effective, versatile, multiplexable, and applicable to almost all cell types [ 21 , 22 ].…”

“…It is usually performed with the help of specially formulated buffers to achieve optimal cell viability and delivery efficiency [ 23 ]. Sucrose has been used as an important component in ET buffers as an osmotic balancing agent [ 14 , 16 , 17 ]. Recently, we discovered that ET efficiency could be enhanced through the pretreatment of cells with sucrose in vitro [ 24 ], partly due to the formation of large, amphisome-like bodies (ALBs) and enlargement of lysosomes.…”