Predicting electrotransfer in ultra-high frequency sub-microsecond square wave electric fields.

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (≥1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10-50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapies.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 µs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.

Invasive and non-invasive electrodes for successful drug and gene delivery in electroporation-based treatments.

Electroporation is an effective physical method for irreversible or reversible permeabilization of plasma membranes of biological cells and is typically used for tissue ablation or targeted drug/DNA delivery into living cells. In the context of cancer treatment, full recovery from an electroporation-based procedure is frequently dependent on the spatial distribution/homogeneity of the electric field in the tissue; therefore, the structure of electrodes/applicators plays an important role. This review focuses on the analysis of electrodes and in silico models used for electroporation in cancer treatment and gene therapy. We have reviewed various invasive and non-invasive electrodes; analyzed the spatial electric field distribution using finite element method analysis; evaluated parametric compatibility, and the pros and cons of application; and summarized options for improvement. Additionally, this review highlights the importance of tissue bioimpedance for accurate treatment planning using numerical modeling and the effects of pulse frequency on tissue conductivity and relative permittivity values.

Effects of extracellular medium conductivity on cell response in the context of sub-microsecond range calcium electroporation.

In the present study, we report the effects of extracellular medium conductivity on cell response in the context of sub-microsecond range (100 ns-900 ns) electroporation, calcium electroporation and cell size. The effects of 25 ns and microsecond range (100 µs) pulses were also covered. As a model, three different cancer cell lines of various size (C32, MCF-7/DX and MC38/0) were used and the results indicated different size-dependent susceptibility patterns to the treatment. The applied pulsed electric field (PEF) protocols revealed a significant decrease of cell viability when calcium electroporation was used. The dependence of calcium ion transport and finally the anticancer effect on the external medium conductivity was determined. It was shown that small differences in conductivity do not alter viability significantly, however, mostly affect the permeabilization. At the same, MC38/0 cell line was the least susceptible to calcium electroporation, while the C32 line the most. In all cases calcium electroporation was mostly dependent on the sensitivity of cells to electroporation and could not be effectively improved by the increase of CaCl2 concentration from 2 mM to 5 mM. Lastly, sub-microsecond PEF stimulated aquaporin-4 and VDAC1/Porin immunoreactions in all treated cells lines, which indicated that cell water balance is affected, ions exchange is increased and release of mitochondrial products is occurrent.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.