Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment-electrochemotherapy. However, electrochemotherapy is associated with ...microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy.
The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently ...serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1–5 μs × 250 and 100 μs × 1–8 pulsing protocols in 1–2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin.
•D-luciferin oxidation can be used to detect cell permeabilization threshold.•Cell permeabilization results in several-fold increase of cell bioluminescence.•Bioluminescence can be used to define protocols with preserved cell viability.
Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and ...propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant
in 5⁻20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of
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Calcium electroporation is a modality of electrochemotherapy (ECT), which is based on intracellular electric field-mediated delivery of cytotoxic doses of calcium into the cells resulting in rapid ...cell death. In this work, we have developed a CHO-K1 luminescent cell line, which allowed the estimation of cell membrane permeabilization, ATP depletion and cytotoxicity evaluation without the use of additional markers and methodologies. We have shown the high efficiency of nanosecond pulses compressed into a MHz burst for application in calcium ECT treatments. The 5 kV/cm and 10 kV/cm nanosecond (100 and 600 ns) pulses were delivered in bursts of 10, 50 and 100 pulses (a total of 12 parametric protocols) and then compared to standard microsecond range sequences (100 µs × 8) of 0.4-1.4 kV/cm. The effects of calcium-free, 2 mM and 5 mM calcium electroporation treatments were characterized. It was shown that reversible electroporation is accompanied by ATP depletion associated with membrane damage, while during calcium ECT the ATP depletion is several-fold higher, which results in cell death. Finally, efficacy-wise equivalent pulse parameters from nanosecond and microsecond ranges were established, which can be used for calcium nano-ECT as a better alternative to ESOPE (European Standard Operating Procedures on Electrochemotherapy) protocols.
•Sub-microsecond range high frequency pulses trigger electro-transfection.•High frequency pulsing enables residual transmembrane voltage accumulation.•300–700 ns pulses can be used for transfection ...independently on plasmid size.
Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300–700 ns) high frequency pulsed electric fields of 2–15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2–5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 μs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.
Exposure of cells to pulsed electric fields (PEFs) induces a phenomenon known as electroporation, which leads to increase of membrane permeability. Electroporation is applied in biotechnology, food ...processing, and medicine, including cancer treatment. Recently, a contactless method based on pulsed magnetic fields (PMFs) for the permeabilization of biological cells has been proposed; however, the permeabilization mechanism of the PMF method is still hypothetical. In this paper, we have shown that it is possible to reversibly permeabilize Sp2/0 myeloma cells by sub-microsecond (450 ns) PMF in the range of 0-3.3 T. The PMF methodology was also combined with PEF treatment to evaluate additive effects. The 1.35 kV/cm 1 × 100 μs (PEF) and 3.3 T, 50 pulses, 0.25 Hz (PMF) protocols were applied. The cells were treated in the presence of fluorescence dye YO-PRO-1 and influx into the cells was evaluated by cytometry. Cell viability after the treatment was evaluated by CellEvent Caspase-3/7 assays. A significant (P <; 0.05) additive effect of the two pulsed power methodologies was detected, resulting in up to 12% increase of membrane permeabilization. The PMF method is an emerging technique and the results of the study can be used for the development of new effective protocols, while the determined additive effects with PEF are promising in the field of electrochemotherapy.
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.
Invasive infections caused by drug-resistant bacteria are frequently responsible for fatal sepsis, morbidity and mortality rates. In this work, we propose a new methodology based on nanosecond high ...frequency electric field bursts, which enables successful eradication of bacteria in vivo. High frequency (15 kHz) 15-25 kV/cm 300-900 ns pulsing bursts were used separately and in combination with acetic acid (0.1-1%) to treat Pseudomonas aeruginosa in a murine model. Acetic acid 1% alone was effective resulting in almost 10-fold reduction of bacteria viability, however combination of nanosecond electric field and acetic acid 1% treatment was the most successful showing almost full eradication (0.01% survival compared to control) of the bacteria in the contaminated area. The short duration of the pulses (sub-microsecond) and high frequency (kHz range) of the burst enabled reduction of the muscle contractions to barely detectable level while the proposed applicators ensured predominantly topical treatment, without electroporation of deeper tissues. The results of our study have direct application for treatment of wounds and ulcers when chemical treatment is no longer effective.
Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The severity and duration of this disease are associated with many different ...factors. Some of these factors may represent markers for monitoring disease activity and the individual response to an intervention.
We used a targeted metabolomics approach to find such factors in the serum of individuals with ACD. Metabolomics profiles were examined and compared in the acute phase of the disease and also in the absence of disease activity.
Our study identified a significant remission phase of ACD-associated systemic biochemical shifts in 2 metabolic pathways: tryptophan-kynurenine and phenylalanine-tyrosine.
Although the responsible mechanisms are unclear, these results suggest that the remission phase of ACD is linked to tryptophan metabolism via kynurenine and phenylalanine-tyrosine pathways. However, further replication studies with a larger number of subjects and their subgroups are necessary to validate our results. These studies may provide a new perspective with which to understand the mechanism of and find potential biomarkers of ACD, as well as a new reference for personalized treatment.
Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the ...microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds—acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid—were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.
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