Transfection by Electroporation of Cancer and Primary Cells Using Nanosecond and Microsecond Electric Fields

Abstract

Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly transient permeability of the targeted cell membrane. In this work, we present the electrotransfection of CHO-K1, 4T1 cell lines, and primary murine DCs with detectable protein-encoding plasmids in the sub-microsecond range. Microsecond (µs)- and nanosecond (ns)-range pulsed electric field transfection protocols were used. The efficiency of electrotransfection was evaluated using green fluorescent protein (GFP)-encoding plasmids (4.7 kbp; p-EGFP-N1) and plasmids expressing a firefly luciferase and red fluorescent protein (tdTomato) (8.5 kbp; pcDNA3.1(+)/Luc2 = tdT)). It was shown that the used nsPEFs protocol (7 kV/cm × 300 ns × 100, 1 MHz) ensured a better transfection efficiency than µsPEFs (1.2 kV/cm × 100 µs × 8, 1 Hz). Plasmid size and concentration had a strong impact on the cell transfection efficiency too. We also showed that there were no significant differences in transfection efficiency between immature and mature DCs. Finally, the nsPEF protocols were successfully applied for the stable transfection of the CHO-K1 cell line with the linearized pcDNA3.1(+)/Luc2 = tdT plasmid. The results of the study are applicable in gene therapy and DNA vaccination studies for the derivation of optimal electrotransfection conditions.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols

“…NsCaEP, on average, resulted in a better treatment for tumor growth inhibition. The variation within a group could be attributed to non-homogeneous electric field distribution within the tumor due to the application of non-invasive plate electrodes [ 48 ].…”

“…As a result, the electrotransfer of drugs or genes is significantly improved, enabling derivation of equivalent efficiency or even superior to ESOPE protocols [ 21 , 47 ]. The high potential of MHz burst of pulses was confirmed in vivo using bleomycin electrochemotherapy [ 27 , 48 ].…”

Efficient Gene Transfection by Electroporation—In Vitro and In Silico Study of Pulse Parameters

“…Recently, 300 ns pulses have been successfully used for GET [106]. However, 100 pulses were used at a pulse repetition frequency of 1 MHz.…”

“…This high frequency of pulse repetition, or the corresponding short duration between successive pulses, could imply that the cumulative effect of 100 pulses at 1 MHz pulse repetition frequency is equivalent to a single pulse of duration 100 µs. To test this, Equation (4) was solved for 100 pulses of duration 300 ns and an electric field intensity of 7 kV/cm at a pulse repetition frequency of 1 MHz and a pDNA cell membrane distance of 170 nm (corresponding to 250 µg/mL, Supplementary Table S1), similar to the conditions used in [106]. The evolution of P(X, t) is shown in Figure 23 for pulse numbers 1, 5, 10, 20, and 30.…”

“…The process of high-frequency ns pulses can also be analyzed from the point of view of Peclet number defined as Pe = µEt p √ 2D/ f . For experimental conditions in [106], t p = 300 ns, E = 7 kV/cm, and f = 1 MHz, which gives Pe = 5.57, indicating that electrophoresis is dominating the transport of pDNA to the cell membrane, and multiple pulses at such a high pulse repetition frequency can be thought of as a single long pulse providing the electrophoresis. For the experimental conditions in this work, t p = 200 ns, E = 15.8 kV/cm, f = 10 Hz, and Pe = 0.03 (see Table 2), indicating that the pDNA molecule reaches the cell membrane predominantly by diffusion.…”