# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
###### Abstract
Nanosecond pulses widen the use of electroporation while also having an effect on intracellular structures. It has been shown that nanosecond pulses can penetrate the cell interior and permeabilize membranes of internal organelles, such as endosomal vesicles, endoplasmic reticulum, nuclear envelope, and cytoskeleton, leading to the disruption of intracellular vesicles, release of calcium from endoplasmic reticulum, and immediate and prolonged loss of mitochondrial membrane potential [44; 50; 51; 52; 53; 54]. Successful GET with shorter (i.e., less than 10 us) pulses in vitro is, however, mostly achieved with high pDNA concentrations, which might be challenging to reach in in vivo settings, at least over a larger volume [2]. Conventional GET, using micro- to millisecond-long pulses, generally relies on electrophoresis to bring the pDNA molecules to the cell membrane for successful transfection [55]. However, for nanosecond duration pulses, electric field/electrophoresis might not be present for sufficient time to ensure a pDNA cell membrane contact. Even with HF-BP pulses, net electrophoretic movement is balanced out due to the bipolar nature of pulses. For these pulses (nanosecond and HF-BP), pDNA molecules then have to rely on diffusion to reach the cell membrane. Moreover, there is only a limited window of time during which the cell membrane is permeable and competent enough to absorb pDNA molecules for successful GET [1; 56]. Therefore, depending on how far the pDNA molecule is from the cell membrane, or how concentrated the pDNA solution is, the efficiency of GET for the wide variety of pulse durations (nanosecond to millisecond) can be analyzed using the framework of how electrophoresis and diffusion influence the probability of pDNA molecule reaching the permeabilized cell membrane. A theoretical framework based on the drift-diffusion equation of probability density and the one-dimensional Fokker-Planck equation was used in this work to determine such probabilities [57]. A similar probabilistic framework has also been used earlier to evaluate probabilities of successful pDNA translocation through an electropore in the cell membrane, along with its qualitative comparison to experimental GET efficiencies [58], and has also been compared to experimental pDNA translocation efficiencies in model systems [59]. The purpose of this paper is to explore, evaluate, and demonstrate the potential use of different pulses for introducing pDNA into cells in vitro and to compare GET efficiency and dynamics of gene expression after GET. We performed experiments in vitro, on cell suspensions of immortalized human skin fibroblasts 1306 cell line and on C2C12 myoblasts, covering a range of pulse durations from 200 ns to 5 ms, monopolar and the recently used microsecond bipolar, so-called HFIRE pulses. To clarify the role of electrophoresis in the transport of pDNA in the electroporation medium during GET with different durations of electric pulses, we also developed a simple mathematical model that gives the probability of pDNA contact with the cell membrane during GET and compared experimental results with those calculated by the model.