Electrophoretic Component of Electric Pulses Determines the Efficacy of In Vivo DNA Electrotransfer

Abstract

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long lowvoltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery

“…Prior studies have shown that the combination of a short-duration, high-field-strength first pulse, together with a longer-duration, lowerfield-strength second pulse (denoted by 'first pulse' and 'second pulse', respectively) can increase electroporation efficiency and preserve cell viability, especially when delivering larger molecules (MW N 4 kDa [47]) or DNA [33,34,37,39,42,[57][58][59][60][61][62][63][64][65][66]. Pulsing parameters have been well studied, including the strength of the first pulse [33,37,58], the strength and duration of the second pulse [34,39,[57][58][59], and the delay between pulses [34,42,57,58,66,67].…”

“…Pulsing parameters have been well studied, including the strength of the first pulse [33,37,58], the strength and duration of the second pulse [34,39,[57][58][59], and the delay between pulses [34,42,57,58,66,67]. The two-pulse protocol has been reported to significantly increase delivery efficiency both in vitro in cell suspensions as well as in vivo in a variety of tissues [33,57] as compared to a single electroporation pulse.…”

New Insights into the Mechanisms of Gene Electrotransfer – Experimental and Theoretical Analysis

“…In contrast to viral vector transfer, the use of electric pulses for gene delivery represents a safer method, which is not hampered by concerns of immunogenicity and pathogenicity13. Different protocols where designed for in vitro and in vivo applications, either employing short hundreds-of-microsecond pulses4, long millisecond pulses7814 or combining high-voltage (10–1000 μs) and low-voltage (10–400 ms) pulses121516171819. We have recently shown in vitro that2021 longer electric pulses are optimal for high transfection efficiency but reduce viability, while shorter pulses enable moderate transfection efficiency and preserve viability.…”

The effects ofpprIgene ofDeinococcus radioduransR1 on acute radiation injury of mice exposed to 60Co γ-ray radiation

“…Nowadays, it has been commonly used for introducing genetic material to cells [27], and for delivering drugs across the cell membrane. However, there are also a lot of factors which can influence the transfection efficiency of this method, such as electric pulses [30], reactive oxygen species [31], inhibitors of endocytosis [32], and so forth. In this study, we confirmed successful transfer of the plasmid encoding pCMV-HA- pprI and found expression in mice 1 day after radiation, but not subsequently.…”