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.

/pdf/efficient-gene-transfection-by-electroporation-in-vitro-and-s5qij2zm.pdf

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

Invasive infections caused by drug-resistant bacteria are a problem responsible for many fatal cases, especially in burn wound care centers, while bacterial resistance to antibiotics is growing dramatically worldwide. In this work, we utilize pulsed electric fields (up to 25 kV/cm × 750 ns) in combination with low-concentration (1%) acetic acid for the inactivation of P. aeruginosa. An in vivo superficial infection model is developed in BALB/C mice using a luminescent strain of P. aeruginosa. We show that an up to 25 kV/cm electric field (3 kV, 1.2 mm gap), when combined with acetic acid, induces a bacteriostatic effect, preventing further infection for up to 7 days after treatment. Additionally, we evaluate antibodies against surface and intracellular P. aeruginosa bacteria antigens following the treatment. It is shown that the levels of surface IgG and IgG1 antibodies are significantly lower in the murine serum of electric-field-treated mice compared to the bacterial-infection-bearing group of mice treated with acetic acid alone. The results of this work are useful as a proof of concept for the development of novel clinical procedures to fight drug-resistive microorganisms responsible for wound contamination and chronic wounds.

/pdf/killing-bacteria-using-acetic-acid-and-nanosecond-pulsed-15nzejxi.pdf

Killing Bacteria Using Acetic Acid and Nanosecond Pulsed Electric Fields—An In Vivo Superficial Infection Model Study and Immune Response

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.

/pdf/effect-of-experimental-electrical-and-biological-parameters-dedr3911.pdf

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

The distinguishing electrical properties of cancer cells.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

/pdf/improving-nonviral-gene-delivery-using-mhz-bursts-of-1wgf432l.pdf

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification

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.

/pdf/transfection-by-electroporation-of-cancer-and-primary-cells-25xddbqg.pdf

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

Nanosecond electrochemotherapy using bleomycin or doxorubicin: Influence of pulse amplitude, duration and burst frequency.

Simple Summary Standard microsecond electrochemotherapy (μsECT) is used in clinical trials for the elimination of tumours, while the first studies on nanosecond electrochemotherapy (nsECT) only started to appear recently. Nanosecond pulses enable more homogeneous treatment and better control of pulse burst energy, and thus the whole field of electroporation is moving towards the shorter pulse range. In order to ensure a full anticancer response and potentially prevent any metastases, the immunomodulatory effects should also be induced. Therefore, in this work, we used nsECT protocols based on kHz and MHz pulse bursts and characterized the response of the immune system to the novel modality of nano-electrochemotherapy. The results of this study are useful for the development of effective anticancer treatment strategies based on high frequency nanosecond electric fields. Abstract In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4+ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4+ Tr1 cells, increased the expansion of splenic CD8+ T, CD4+CD8+ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin− population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

https://www.mdpi.com/2072-6694/14/24/6254/pdf?version=1671604852

Veronika Malyško-Ptašinskė

Papers

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

Nanosecond electrochemotherapy using bleomycin or doxorubicin: Influence of pulse amplitude, duration and burst frequency.

Killing Bacteria Using Acetic Acid and Nanosecond Pulsed Electric Fields—An In Vivo Superficial Infection Model Study and Immune Response

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification

High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival