# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 4 Discussion
### Effect of pDNA Concentration on Overall Gene Electrotransfer  
The high concentration of pDNA can have a negative effect on cell survival [16] and consequently on GET efficiency, but it is also needed to obtain GET with nanosecond and HF-BP pulses [15; 49]. Pathogen-associated molecular patterns that are associated with pDNA, for example, unmethylated CpG motifs in pDNA, can be recognized by TLR9 and can induce an innate immune response in tissue, which can lead to apoptosis activation [70; 71]. pDNA concentrations used in our experiments were higher than concentrations usually used in in vitro experiments, which are most often between 10 and 100 mg/mL [14; 72; 73; 74; 75]. On the other hand, it has been shown that pDNA concentration affects GET efficiency and that, with higher pDNA concentration, efficient GET can also be achieved with shorter pulses [15; 16; 25; 76]. The effect of the pDNA concentration was observed in our experiments, where a decreasing pDNA concentration led to a significant decrease in the overall GET. This was especially observed in 1306 fibroblasts, where, at pDNA concentrations up to 100 mg/mL, the overall GET was higher with 8 \(\times\) 100 us, HF-BP, and 8 \(\times\) 5 ms pulses compared to 500 ns and 200 ns pulses, whereas, at 500 mg/mL of pDNA, there was no significant difference in the overall GET between different pulse protocols. Interestingly, this was not observed in C2C12 myoblasts where the overall GET rose with increasing pDNA concentration for all pulse protocols. At the highest pDNA concentration (500 mg/mL), the overall GET was, however, still significantly higher with longer pulse protocols (8 \(\times\) 100 us and 8 \(\times\) 5 ms), where electrophoresis acts for a longer period of time compared to shorter pulse protocols (HF-BP, 500 ns, and 200 ns) (Figure 5). Difference in overall GET between two cell lines confirms that the efficiency of GET does not depend solely on parameters of applied pulses. GET is a multistep process including various intracellular mechanisms (cell membrane repair mechanism, DNA sensors activation, endocytic pathways, cytoskeleton reorganization) which can differ between cell lines [77; 78].  
Contrary to our previous results on a CHO cell line [15], in C2C12 myoblasts and 1306 fibroblasts, we observed some decrease in cell survival with increasing pDNA concentration, however, not for all pulse protocols. With 8 \(\times\) 5 ms pulses, we did not observe a decrease in cell survival with increasing pDNA concentrations (in both cell lines). In addition, in C2C12 myoblasts, pDNA concentration did not have an effect on cell survival after GET with the HF-BP, 100 \(\times\) 500 ns, and 25 \(\times\) 200 ns pulse protocols. A decrease in cell survival with 250 mg/mL of pDNA was only observed in 1306 fibroblasts after the 8 \(\times\) 100 us pulse protocol; with other pulse protocols, decreased survival was observed only with the highest pDNA concentration, 500 mg/mL (Figure 4). Our results are similar to [79], where the authors observed decreased cell survival with pDNA concentrations higher than 400 mg/mL. However, the decrease in cell survival in our experiments was not high enough to cause a drop in the overall GET at 500 mg/mL of pDNA. These and our previous results [15] show that decreases in cell survival following GET with high pDNA concentration seem to be cell-line-dependent. pDNA by itself was not toxic to cells, as observed before [15; 80]. High pDNA concentration could trigger a decrease in cell survival in different steps of GET. We have shown previously that higher pDNA concentration leads to larger pDNA aggregates formed on cell membrane [15] during pulse delivery, which could slow down or obstruct cell membrane repair. Furthermore, high pDNA concentration in endosomes or cytoplasm could activate endosomal and/or cytoplasmic DNA sensors which can induce programmed cell death [81]. Finally, cell death could be triggered by high number of pDNA copies in nucleus or by the high number of transgenes being produced [82].