Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil

Abstract

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours’ incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

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

“…Figure 1 shows the experimental setup. The apparatus was fabricated with pin headers and a printed circuit board as described in the previous paper [ 35 ]. The electrodes’ gap was 5.08 mm.…”

“…The influx of calcium ions presenting in the cytoplasm at a low concentration was measured to demonstrate transient membrane permeabilization stimulated by short-circuiting. The measurement of intracellular calcium ions was conducted as previously described [ 35 ], with some modifications: 4.0 × 10 6 Jurkat cells suspended in Dulbecco’s phosphate-buffered saline without magnesium chloride and calcium chloride (D-PBS (-), FUJIFILM Wako Pure Chemical) were incubated with 4.5 μM Fluo-4 AM (Thermo Fisher Scientific) for 60 min at 37 °C. The Fluo-4-loaded cells were harvested by centrifugation and resuspended in HEPES-saline or HEPES-sucrose with and without 10 mM CaCl 2 .…”

“…Uptake of cell-impermeable nucleic acid-binding fluorophores YO-PRO-1 iodide can also indicate electroporation. The measurement of YO-PRO-1 uptake was conducted to demonstrate transient membrane permeabilization stimulated by short-circuiting as previously described [ 35 ], with some modifications. A 3.0 μL cell suspension containing 1.0 × 10 5 Jurkat cells and 1 μM YO-PRO-1 (Thermo Fisher Scientific) prepared with an appropriate electroporation medium was dispensed into the silicone oil, and the cells were treated with short-circuiting.…”

“…Endocytic pathways were considered to be different YO-PRO-1 uptake pathways from membrane permeabilization; therefore, the effect of endocytosis inhibition on YO-PRO-1 uptake was investigated. 4.0 × 10 6 Jurkat cells in D-PBS (-) were treated with endocytosis inhibitor Pitstop 2-100 (Abcam, Cambridge, UK) for 30 min at 37 °C, 5% CO 2 [ 35 , 36 ]. Following the Pitstop 2-100 treatment, the cell suspension was centrifuged and the cells were resuspended in RPMI-1640 without FBS.…”

“…Second, the droplet system is suitable for miniaturization using microfluidics. We have also elucidated the exogenous gene expression mechanism in mammalian cells [ 35 ]. The previous investigation demonstrated the following highlights: (1) the electrical stimulation increases transient membrane permeability to calcium ions and YO-PRO-1 fluorescent dyes, indicating transient membrane pore formation.…”