# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 4 Discussion
### Modeling the Probability of pDNA Cell Membrane Contact during GET
Also shown as dots are Overall GET (Figure 5) and GET (Figure 3) for C2C12 cells plotted against \(t_{competent}/l^{2}\) or against \(t_{competent}/l\), where \(l\) for the corresponding concentration was taken as \(R_{DNA}\) from Supplementary Table S1 and \(t_{competent}\) was determined by fitting Overall GET/GET to \(PSC(t)\) for \(l=77\) nm. Also shown in Figure 21A-J are the results for overall GET and GET for the respective pulse parameters. Overall GET (Figure 5) and GET (Figure 3) for C2C12 myoblasts are plotted against \(t_{competent}/l\) or against \(t_{competent}/l^{2}\). Values of \(l\) for the respective concentrations are taken from \(R_{DNA}\) from Supplementary Table S1. \(t_{competent}\) is taken to be the time for which the cell membrane remains competent to accept/absorb the incoming pDNA molecule. \(t_{competent}\) was determined by varying \(t_{competent}\) and minimizing the root mean squared error (RMSE) between Overall GET/GET vs. \(t_{competent}/l^{2}\) and \(PSC(t)\) vs. \(t/l^{2}\) (at \(l=77\) nm) in Figure 21A,C,E. For Figure 21B,D,F,G-J, \(t_{competent}\) was determined by varying \(t_{competent}\) and minimizing the root mean squared error (RMSE) between Overall GET/GET vs. \(t_{competent}/l\) and \(PSC(t)\) vs. \(t/l\) (at \(l=77\) nm). \(l=77\) nm was chosen since the curves are collapsed for various values of \(l\), and fitting to \(l=77\) nm would imply fitting to all the collapsed curves. The best fit values are given in the legends in Figure 21A-J against their respective Overall GET and GET. It should be noted that this exercise of plotting Overall GET/GET vs. \(t_{competent}/l^{2}\) or \(t_{competent}/l\) along with \(PSC(t)\) vs. \(t/l^{2}\) or \(t/l\) should not be considered as a direct one-to-one comparison between the model and the experiment. Such an exercise is only performed to place the experiments within the context of the model. Overall GET and GET cannot be directly compared to \(PSC(t)\), as overall GET and GET represent efficiencies which also include downstream processes of pDNA translocation across the cell membrane, migration through the cytoplasm, entering the nucleus, transcription, translation, and protein expression. \(PSC(t)\), on the other hand, only represents the probability of pDNA establishing contact with the cell membrane. However, in a first approximation, only those cells on which pDNA molecules have established contact with the cell membrane can be expected to be transfected. Therefore, \(PSC(t)\) can be considered as an upper limit of transfection efficiency. Moreover, different durations of pulses (short nanosecond pulses, HF-BP, or long micro- and millisecond pulses) can permeabilize the cell membrane in different ways. This can in turn result in the cell membrane being competent in different ways to absorb the pDNA molecule for different durations of pulses. Different durations of pulses can also alter the free energy barrier between the pDNA and the cell membrane, further influencing the results. Different types of cells themselves can be sensitive in different ways to permeabilization by the pulses, yielding different transfection efficiencies, as seen in Figure 3 for C2C12 myoblasts and 1306 fibroblasts. The models based on \(PSC(t)\) do not account for such parameters and differences. An experimental observable that can perhaps be better compared to the \(PSC(t)\) could therefore be the fraction of cells forming pDNA aggregates on the cell membrane [21; 56]. From the fitting parameters of \(t_{competent}\) in Figure 21A-J, it can be observed that \(t_{competent}\) for 200 ns, 500 ns, and HF-BP, where the process is diffusive, is much larger than \(t_{competent}\) for 100 us and 5 ms pulses, where the process is mainly electrophoretic. Absolute values \(t_{competent}\) cannot be considered, as \(t_{competent}/l\) or \(t_{competent}/l^{2}\) are not non-dimensional; however, from fitting, \(t_{competent}\) for 200 ns, 500 ns, and HF-BP is around two orders of magnitude larger than \(t_{competent}\) for 100 us and 5 ms pulses.