# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
###### Abstract
Small molecules (equal to or smaller than 15 bp [21]) can enter the cell with electrophoresis; however, large pDNA molecules form aggregates on cell membrane during electric field delivery, which later enter the cell with endocytosis [1; 22; 23; 24]. Because of its role in the formation of pDNA aggregates during electric field delivery, the lack of electrophoresis could represent an important weakness for the use of short pulses in GET [25; 26]. The presence of electrophoretic migration of pDNA is the reason why, usually, long millisecond pulses are used for GET. Although leading to efficient GET, these pulses are associated with triggering muscle contractions, pH changes, and electrolysis at the electrode electrolyte interface causing electrochemical reactions [27; 28; 29; 30; 31; 32; 33]. Recently suggested high-frequency bipolar pulses (HF-BP), first predominantly used for irreversible electroporation applications-HFIRE [34]--were demonstrated to also enable GET in vitro [15]. One of the characteristics of HF-BP pulses are reduced electrochemical reactions and less metal ions released from the electrodes compared to longer pulses [29; 35]. Furthermore, HF-BP pulses induce less muscle contractions and pain during pulse delivery [36; 37; 38; 39]. In addition, nanosecond pulses have already been used for GET. When applying 200 ns pulses (10-18 kV/cm) in bursts of ten with a varied pulse repetition rate (up to 1 MHz), it was shown that with the increase in the pulse repetition rate from 100 kHz to 1 MHz, GET increased, resulting in up to 17% GET with a minimal decrease in cell survival [40]. Furthermore, it was shown that with 100 \(\times\) 300 ns pulses delivered at 1 MHz and at 5 kV/cm, up to 40% GET can be achieved, which is comparable to an efficiency of 4 \(\times\) 100 us, 1 Hz, 1.5 kV/cm pulse protocol [41]. Interestingly, nanosecond pulses have been suggested to disrupt nuclear envelope which could facilitate delivery of pDNA to the nucleus and thus increase GET [42; 43; 44]. Several papers have described using different combinations of nanosecond and milli- or microsecond pulses with the attempt to improve GET. Firstly, it was shown that GET could be increased about four times when cells were exposed to 3.5 ms pulse with 0.3 kV/cm followed by 60 ns pulse at 60 kV/cm after 30 min [45]. However, these results were not reproduced successfully. Later, no effects of nanosecond pulses on GET were observed by applying 8 \(\times\) 5 ms or 4 \(\times\) 200 us pulses delivered at 1 Hz followed by 10, 12, or 15 ns pulses delivered at 10 Hz [46]. However, when the order of pulse delivery was reversed, a more pronounced effect of nanosecond pulses on GET was observed. Using the electroporation approach with a combination of nanosecond (600 ns, 100 kHz, 50 kV/cm) followed immediately by millisecond pulses (1 \(\times\) 10 ms, 500 V/cm) more than doubled transgene expression; at the same time, cell survival levels were similar as in standard millisecond electroporation [42]. The combination of nanosecond pulses (23 \(\times\) 60 ns, 1 Hz, 24 kV/cm) and millisecond electric pulses (1 \(\times\) 5 ms, 50 V) led to a 40-fold increase in GET if nanosecond pulses were applied first followed by millisecond pulse, but not in the reverse order. This effect of nanosecond pulses was time restricted, with the highest efficiency occurring when nanosecond pulses were delivered 5 min before millisecond pulses [47]. Nanosecond pulses can potentially offer a solution to limitations that are present in GET with longer milli- and microsecond pulses. Namely, some cells exposed to GET may lose viability due to excessive heat, pH changes, and ionic imbalance [3]. Nanosecond pulses decrease electrochemical reactions during pulse delivery and in in vivo settings offer more patient-friendly treatment, with reduced muscle contractions [40; 48]. Heating is also decreased during nanosecond pulses application, which lowers the thermal effects of treatment [48; 49].