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.

High frequency electroporation efficiency is under control of membrane capacitive charging and voltage potential relaxation.

The study presents the proof of concept for a possibility to achieve a better electroporation in the MHz pulse repetition frequency (PRF) region compared to the conventional low frequency protocols. The 200ns×10 pulses bursts of 10-14kV/cm have been used to permeabilize Chinese hamster ovary (CHO) cells in a wide range (1Hz-1MHz) of PRF. The permeabilization efficiency was evaluated using fluorescent dye assay (propidium iodide) and flow cytometry. It was determined that a threshold PRF exists when the relaxation of the cell transmembrane potential is longer than the delay between the consequent pulses, which results in accumulation of the charge on the membrane. For the CHO cells and 0.1S/m electroporation medium, this phenomenon is detectable in the 0.5-1MHz range. It was shown that the PRF is an important parameter that could be used for flexible control of electroporation efficiency in the high frequency range.

Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses.

BACKGROUND: Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. METHODS: The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. RESULTS: The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. DISCUSSION: The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study.

Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2-3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100-250 µs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

Controlled inactivation of Trichophyton rubrum using shaped electrical pulse bursts: Parametric analysis.

The dermatophytes infect the skin by adherence to the epidermis followed by germination, growth, and penetration of the fungal hyphae within the cells. The aim of this study was to investigate the efficacy of the pulsed electric fields (PEF) of controlled inactivation of Trichophyton rubrum (ATCC 28188). In this work, we have used bursts of the square wave PEF pulses of different intensity (10-30 kV/cm) to induce the irreversible inactivation in vitro. The electric field pulses of 50 µs and 100 µs have been generated in bursts of 5, 10, and 20 pulses with repetition frequency of 1 Hz. The dynamics of the inactivation using different treatment parameters were studied and the inactivation map for the T. rubrum has been defined. Further, the combined effect of PEF with the antifungal agents itraconazole, terbinafine, and naftifine HCl was investigated. It has been demonstrated that the combined effect results in the full inactivation of T. rubrum colony. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1056-1060, 2016.

Irreversible electropermeabilization of the human pathogen Candida albicans: an in-vitro experimental study.

Pathogenic fungi cause many life-threatening infections, especially among individuals with immune system dysfunction. The antifungal drugs commonly used to suppress fungal pathogens can result in long-lasting and toxic therapy. In this work, irreversible electropermeabilization was used to investigate the dynamics of the decrease in Candida albicans colony vitality after application of a pulsed electric field (PEF) and use of antifungal drugs. The fungi were subjected to single 250-µs to 2-ms (0.5-2.5 kV/cm) pulses or repeated short 5-µs pulses, and efficacy was compared. It was shown that electropermeabilization combined with antifungal agents results in rapid and more effective treatment, eliminating more than 90% of C. albicans colony-forming units in a single procedure, which is advantageous in biomedicine. It was also observed that, because of application of PEF and use of the antifungal agents, the Candida cells form cell aggregates and average live cell size is reduced by as much as 53%.

Irreversible magnetoporation of micro-organisms in high pulsed magnetic fields.

Electroporation is an appealing way of stimulating living cells, which causes permanent or temporary nanoporosities in the structure of the biological objects. However, the technique has a disadvantage such as a requirement of contact between the electrodes and the cell medium. In this review, a methodology of contactless treatment of the biological objects using pulsed magnetic fields is proposed. The eukaryotic micro-organisms Achlya americana and Saprolegnia diclina have been used in the study and magnetic fields up to 7 T were applied, which caused effects similar to irreversible electroporation resulting in the death of the species. The proposed technique is applicable for different types of the biological cells or micro-organisms and possibly can be used in the area of cancer, antifungal treatment and other biotechnological fields.

Contactless dielectrophoretic manipulation of biological cells using pulsed magnetic fields.

Contactless method for manipulation of polar or polarisable micro and nanoscale particles based on the dielectrophoresis force exerted by the induced electric field in high pulsed magnetic field is presented in this study. Finite element method analysis of the magnetic and resulting induced electric fields is carried out. The structure of the magnetic field generator that was based on a controlled frequency spark gap, and the structure of the coil that was used as a load are described. Experimental data showing positive dielectrophoresis on the Jurkat T-lymphoblasts is presented. The study discusses further developments of the technique, its limitations and possible applications.

Microsecond pulsed magnetic field improves efficacy of antifungal agents on pathogenic microorganisms.

Control and treatment of the emerging filamentous and yeast fungal diseases are of high priority in the biomedical field. This study investigated the influence of the pulsed magnetic field combined with common antifungal agents on the viability of various pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Trychophyton rubrum. Repetitive microsecond pulsed magnetic fields up to 6.1 T were applied in the study. The synergistic effect of co-applying drugs and magnetic treatment to different fungi species causing various human mycoses showed the potential for more effective and less toxic therapy.