Nanosecond range electric pulse application as a non-viral gene delivery method: proof of concept

Paulius Ruzgys,Jurij Novickij,Saulius Šatkauskas,Vitalij Novickij

doi:10.1038/s41598-018-33912-y

Paulius Ruzgys, Jurij Novickij + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-33912-y

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Abstract

Current electrotransfection protocols are well-established for decades and, as a rule, employ long micro-millisecond range electric field pulses to facilitate DNA transfer while application of nanosecond range pulses is limited. The purpose of this paper is to show that the transfection using ultrashort pulses is possible by regulating the pulse repetition frequency. We have used 200 ns pulses (10–18 kV/cm) in bursts of ten with varied repetition frequency (1 Hz–1 MHz). The Chinese Hamster Ovary (CHO) cells were used as a cell model. Experiments were performed using green fluorescent protein (GFP) and luciferase (LUC) coding plasmids. Transfection expression levels were evaluated using flow cytometry or luminometer. It was shown that with the increase of frequency from 100 kHz to 1 MHz, the transfection expression levels increased up to 17% with minimal decrease in cell viability. The LUC coding plasmid was transferred more efficiently using high frequency bursts compared to single pulses of equivalent energy. The first proof of concept for frequency-controlled nanosecond electrotransfection was shown, which can find application as a new non-viral gene delivery method.

Highlights

Electroporation is a common method to facilitate intracellular delivery of membrane-impermeable molecules[1,2,3]
We have shown that it is possible to generate a high frequency pulse burst to achieve a threshold pulse repetition frequency (PRF), when the discharging time of the membrane is longer than the delay time between the pulses[49]
We have evaluated the transfection expression levels of green fluorescent protein (GFP) coding plasmid after application of 200 ns × 10 pulses bursts using different PRF protocols and compared the results to a single pulse (2 μs) of identical energy and to the standard 100 μs pulses (2 × 1.4 kV/cm × 100 μs), the latter serving as a positive control

Summary

Introduction

Electroporation is a common method to facilitate intracellular delivery of membrane-impermeable molecules[1,2,3]. The array of applications includes cancer treatment[4,5,6,7], drug delivery[8,9], gene transfer[10,11,12], food processing[13] and biotechnology[14,15] Such procedure requires specific pulse parameters (amplitude, duration, number of pulses, etc.) to trigger the desired electroporation effect, which varies between different cell types[16,17,18,19,20]. Low frequency protocols offer the possibility to induce the phenomenon of cell sensitization[44], while higher frequency range allows to counter bioimpedance problems and reduce muscle contractions[45,46]. Taking into the account the membrane charge relaxation phenomenon (in high PRF region) and the capability to induce a more uniform exposure (Fig. 1), we have speculated that it is possible to achieve successful transfection using nsPEF, which has not been shown so far

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