# Efficient Gene Transfection by Electroporation--In Vitro and In Silico Study of Pulse Parameters
## 5 Conclusions  
In summary, we showed that GET can also be achieved with nanosecond pulses (with low pulse repetition rate, i.e., 10 Hz) in addition to widely used millisecond and microsecond pulses and previously shown HF-BP pulses. GET efficiency depends on pDNA concentration, cell line, and pulse parameters. In our experiments, smaller pDNA did not significantly improve GET efficiency, but it had an effect on the degree of transgene expression. We showed that the time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but differ greatly between the two cell lines. A simple mathematical model of the probability of pDNA and cell membrane contact during GET shows that pDNA migration for nanosecond (at low repetition frequency) and HF-BP pulses is dominated by diffusion, and for micro- and millisecond pulses, the process is dominated by electrophoresis. A Peclet (\(Pe\)) number has been defined that can be used to infer the role of diffusion and electrophoresis in a wide  
Figure 23: **(A–E)** Evolution of \(P(X,t)\) without free energy barrier (Equation (4)) for 100 pulses of 300 ns and 7 kV/cm applied at a high frequency of 1 MHz and for a pDNA and cell membrane distance of 170 nm (corresponding to 250 \(\upmu\)g/mL). The pulse numbers for each pulse and corresponding time are shown on top of **(A–E)**. Solid lines indicate the during pulse \(P(X,t)\) and dotted lines represent after pulse \(P(X,t)\). **(F)** Evolution of \(PSC(t)\) without free energy barrier. variety of pulsing conditions. However, for the process of pDNA molecules coming into contact with the cell membrane in the presence of a strong free energy barrier close to the cell membrane, the role of diffusion and electrophoresis cannot be isolated as effectively. Nevertheless, the migration of pDNA close to the cell membrane can still be inferred based on electrophoresis and diffusion. The developed model and modeling results can provide valuable guidance for further experiments and interpretations of results obtained by various pulse protocols. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12168237/s1](https://www.mdpi.com/article/10.3390/app12168237/s1)]([https://www.mdpi.com/article/10.3390/app12168237/s1](https://www.mdpi.com/article/10.3390/app12168237/s1)), Figure S1: Schematic of distribution of pDNA molecules and cells in suspension; Figure S2: Comparison of permeabilization and survival curves of C2C12 myoblast and 1306 fibroblasts with 200 ns pulses and 10 Hz pulse repetition rate; Figure S3: Comparison of permeabilization and survival curves of C2C12 myoblast and 1306 fibroblasts with 500 ns pulses and 10 Hz pulse repetition rate; Figure S4: Comparison of MFI of permeabilizedized C2C12 myoblast and 1306 fibroblasts; Figure S5: Comparison of permeabilization, survival curves, and MFI of C2C12 myoblast and 1306 fibroblasts with 8 \(\times\) 100 \(\upmu\)s and 8 \(\times\) 5 ms pulses; Figure S6: Comparison of permeabilization, survival curves, and MFI of C2C12 myoblast and 1306 fibroblasts with HF-BP pulses; Table S1: Various estimates of the distance between a pDNA molecule and the nearest cell membrane (\(R_{DNA}\)); Table S2: Various estimates of the distance between a DNA molecule and the nearest cell membrane and between two pDNA molecules; Table S3: Nanosecond, HF-BP, micro and millisecond pulse protocols with which the best overall GET with 500 \(\upmu\)g/mL of pEGFP-N1 was achieved in C2C12 myoblasts; Table S4: Nanosecond, HF-BP, micro and millisecond pulse protocols with which the best overall GET with 500 \(\upmu\)g/mL of pEGFP-N1 was achieved in 1306 fibroblasts; Table S5: Maximum temperature increases of the cell sample during pulse delivery. References [107,108] are cited in the supplementary materials. Conceptualization: T.P. ([PERSON]), [PERSON], [PERSON] and [PERSON]; experiments: [PERSON]. ([PERSON]) and [PERSON] ([PERSON]); model, S.S. and A.M.L. ; investigation: [PERSON]. ([PERSON]), [PERSON] and A.M.L.