Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis.

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.

High-Intensity Pulsed Electromagnetic Field-Mediated Gene Electrotransfection In Vitro.

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 into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate.

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.

Revisiting the role of pulsed electric fields in overcoming the barriers to in vivo gene electrotransfer.

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.

Gene transfer by electroporation with high frequency bipolar pulses in vitro.

High-frequency bipolar pulses (HF-BP) have been demonstrated to be efficient for membrane permeabilization and irreversible electroporation. Since membrane permeabilization has been achieved using HF-BP pulses we hypothesized that with these pulses we can also achieve successful gene electrotransfer (GET). Three variations of bursts of 2 µs bipolar pulses with 2 µs interphase delay were applied in HF-BP protocols. We compared transfection efficiency of monopolar micro and millisecond pulses and HF-BP protocols at various plasmid DNA (pDNA) concentrations on CHO - K1 cells. GET efficiency increased with increasing pDNA concentration. Overall GET obtained by HF-BP pulse protocols was comparable to overall GET obtained by longer monopolar pulse protocols. Our results, however, suggest that although we were able to achieve similar percent of transfected cells, the number of pDNA copies that were successfully transferred into cells seemed to be higher when longer monopolar pulses were used. Interestingly, we did not observe any direct correlation between fluorescence intensity of pDNA aggregates formed on cell membrane and transfection efficiency. The results of our study confirmed that we can achieve successful GET with bipolar microsecond i. e. HF-BP pulses, although at the expense of higher pDNA concentrations.

Effect of electroporation and recovery medium pH on cell membrane permeabilization, cell survival and gene transfer efficiency in vitro.

Electroporation is a method which uses an adequate number of electric pulses of enough amplitude, duration and number applied to cells, thus inducing transient permeabilization of the cell membrane. Due to possibility that microenvironment in applications of in vivo electroporation is slightly acidic, we studied the effects of slightly acidic electroporation and recovery medium. We observed no difference in the permeabilization threshold, detected by propidium iodide, of cells which were electroporated and allowed to recover in growth (pH 7.8) or acidic (pH 6.5) medium. In contrast, statistically significant difference was observed in survival of cells that were exposed to pulse amplitudes greater than permeabilization threshold. Survival of cells was greater if acidic electroporation and recovery medium were used, but acidic extracellular pH decreased gene electrotransfer efficiency. We also observed differences in morphology between cells that were electroporated and left to recover in growth medium and cells that were electroporated and left to recover in acidic medium. Our results imply that slightly acidic extracellular pH allows more efficient repair of damage that is induced on cell membrane during electroporation with high pulse amplitudes.

Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors.

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 cells in vitro and 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 vemurafenib in vitro implies on clinical applicability of ECT in melanoma patients with BRAF mutation and/or during the treatment with BRAF inhibitors.