Background: The aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cellsin vitroand in combination with BRAF inhibitors.Materials and methods: ECT with bleomycin was performed on two human melanoma cell lines, with (SK-MEL-28) or without (CHL-1) BRAF V600E mutation. Cell survival was determined using clonogenic assay to determine the effectiveness of ECT in melanoma cells of different mutation status. Furthermore, the effectiveness of ECT in concomitant treatment with BRAF inhibitor vemurafenib was also determined in BRAF mutated cells SK-MEL-28 with clonogenic assay.Results: The survival of BRAF V600E mutated melanoma cells was even lower than non-mutated cells, indicating that ECT is effective regardless of the mutational status of melanoma cells. Furthermore, the synergistic interaction between vemurafenib and ECT with bleomycin was demonstrated in the BRAF V600E mutated melanoma cells.Conclusions: The effectiveness of ECT in BRAF mutated melanoma cells as well as potentiation of its effectiveness during the treatment with vemurafenibin vitroimplies on clinical applicability of ECT in melanoma patients with BRAF mutation and/or during the treatment with BRAF inhibitors.

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.

Gene electrotransfer (GET) is a widely used method for nucleic acids’ delivery into cells. We explored, evaluated, and demonstrated the potential use of different pulse durations for introducing plasmid DNA (pDNA) into cells in vitro and compared the efficiency and dynamics of transgene expression after GET. We performed experiments on cell suspensions of 1306 fibroblasts and C2C12 myoblasts with four ranges of pulse durations (nanosecond, high frequency bipolar (HF-BP), and micro- and millisecond). Six different concentrations of pDNA encoding green fluorescent protein were used. We show that GET can be achieved with nanosecond pulses with a low pulse repetition rate (10 Hz). The GET’s efficiency depends on the pDNA concentration and cell line. Time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but depend greatly on cell line. Lastly, based on the data obtained in the experiments of pDNA concentration effect on GET the model of the probability of pDNA and cell membrane contact during GET was developed. The model shows that pDNA migration is dominated by diffusion for nanosecond and HF-BP pulses and by electrophoresis for micro- and millisecond pulses. Modeling results can provide valuable guidance for further experiments and interpretations of the results obtained by various pulse protocols.

A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.