# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
###### Abstract  
Gene electrotransfer (GET) is a widely used method for nucleic acids' delivery into cells. We explored, evaluated, and demonstrated the potential use of different pulse durations for introducing plasmid DNA (pDNA) into cells in vitro and compared the efficiency and dynamics of transgene expression after GET. We performed experiments on cell suspensions of 1306 fibroblasts and C2C12 myoblasts with four ranges of pulse durations (nanosecond, high frequency bipolar (HF-BP), and micro- and millisecond). Six different concentrations of pDNA encoding green fluorescent protein were used. We show that GET can be achieved with nanosecond pulses with a low pulse repetition rate (10 Hz). The GET's efficiency depends on the pDNA concentration and cell line. Time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but depend greatly on cell line. Lastly, based on the data obtained in the experiments of pDNA concentration effect on GET the model of the probability of pDNA and cell membrane contact during GET was developed. The model shows that pDNA migration is dominated by diffusion for nanosecond and HF-BP pulses and by electrophoresis for micro- and millisecond pulses. Modeling results can provide valuable guidance for further experiments and interpretations of the results obtained by various pulse protocols. g Date Date: 2019 2022 202  
of most electroporation protocols used for cell therapy is, however, the low transfection efficiency and cell death [13]. The efficiency of GET can be measured in different ways and may depend on the GET application, most often as percentage of transfected cells (referred as GET in our paper), which gives information about the percentage of cells that were successfully transfected among the cells that survived the treatment [14]. This could be useful in applications where cells which can rapidly multiply are used, such as bacteria. For applications of GET in gene therapy, it may be crucial that the majority of cells survive the treatment. In this case, it is important that the results of GET efficiency also include cell survival. GET efficiency representing the percentage of transfected cells based on the number of cells that were exposed to electric pulses is referred to as the overall GET in the paper [15]. Furthermore, GET efficiency, if cells are transfected with fluorescent proteins, can be measured with median fluorescence intensity (MFI), representing the quantity of produced transfected protein [16]. This is important in applications where the goal is the production of high levels of therapeutic proteins such as in DNA vaccination. In this paper, we present results of GET in vitro using all three types of GET efficiency determination, namely, GET, MFI, and overall GET. In GET applications, pulse parameters (amplitude, duration, repetition frequency, and number) are usually varied to achieve cell membrane permeabilization and at the same time prevent excessive cell damage [17]. The choice of appropriate parameters of electric pulses is one of the most relevant steps of GET. GET can be applied equally to all cell types and at all stages of the cell cycle, but GET efficiency depends on and varies greatly between different cell types. The origins of these differences are not yet well understood. Generally smaller cells require a higher electric field to permeabilize their cell membrane. This is an important consideration for ex vivo gene delivery, especially to hematopoietic cells. Electroporation thresholds for different cells in a heterogeneous tissue thus vary [18; 19]. Currently, gene transfer by electroporation is believed to rely on cell membrane permeabilization and electrophoresis of pDNA during pulse delivery. Electrophoresis, which is present during electric field delivery, acts on negatively charged pDNA molecules and enables a higher number of pDNA molecules to come in contact with the cell membrane than would do so solely by diffusion [20].