Inactivation of Escherichia coli Using Nanosecond Electric Fields and Nisin Nanoparticles: A Kinetics Study

Nisin is a recognized bacteriocin widely used in food processing, however, being ineffective against gram-negative bacteria and in complex food systems. As a result, the research of methods that have cell wall–permeabilizing activity is required. In this study, electroporation to trigger sensitization of gram-negative bacteria to nisin-loaded pectin nanoparticles was used. As a model microorganism, bioluminescent strain of E. coli was introduced. Inactivation kinetics using nanosecond pulsed electric fields (PEFs) and nisin nanoparticles have been studied in a broad range (100–900 ns, 10–30 kV/cm) of pulse parameters. As a reference, the microsecond range protocols (100 μs × 8) have been applied. It was determined that the 20–30 kV/cm electric field with pulse duration ranging from 500 to 900 ns was sufficient to cause significant permeabilization of E. coli to trigger a synergistic response with the nisin treatment. The kinetics of the inactivation was studied with a time resolution of 2.5 min, which provided experimental evidence that the efficacy of nisin-based treatment can be effectively controlled in time using PEF. The results and the proposed methodology for rapid detection of bacteria inactivation rate based on bioluminescence may be useful in the development and optimization of protocols for PEF-based treatments

Mielės atopinio dermatito etiologijoje

Isolation and identification of all yeast species found on skin affected by atopic dermatitis, evaluation of their influence to the synthesis of IgE antibodies, and assessment of the possible cross-reactivity between different yeast species was performed. It was shown that in 36.9 % of the cases of atopic dermatitis, the affected skin was colonized with yeast belonging to three genera: Candida, Malassezia and Rhodotorula. Systematic and phylogenetic analysis of sequences from atypical Malassezia restricta strain M8 indicated that this isolate could be a member of a new yeast species. Three atypical Malassezia isolates M47, M54 and M235 were identified as non-lipid-dependent variants of Malassezia furfur. It was shown that in atopic dermatitis, cutaneous colonization with yeast is two-fold higher in adults than in children. The sera of atopic dermatitis patients have specific IgE antibodies to cross-reactive intracellular yeast antigens. Candida pelliculosa and house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae might share some allergenic epitopes. The results of this study suggest that attention should be given to a cutaneous colonization by saprophytic yeast since the immune response to the allergens could further exacerbate allergic inflammation due to cross-reactive epitopes

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study

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.This article belongs to the Special Issue Novel Physical and Chemical Methods for Facilitated Drug DeliveryThis research was funded Research Council of Lithuania, grant number S-MIP-19-2

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

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

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation

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 Staphylococcus aureus 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 S. aureus.This article belongs to the Collection Antibiotics & Superbugs: New Strategies to Combat Antimicrobial ResistanceThis research was funded by Research Council of Lithuania grant number LAT-02/2016

Sub-microsecond electrotransfection using new modality of high frequency electroporation

Article no. 107594Micro-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 ls 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 pulsesBiologijos katedraValstybinis mokslinių tyrimų institutas. Inovatyvios medicinos centrasVilniaus Gedimino technikos universitetasVytauto Didžiojo universiteta

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 therapiesBiologijos katedraVilniaus Gedimino technikos universitetasVilnius Gediminas Technical University, LithuaniaVytauto Didžiojo universiteta

Antitumor Response and Immunomodulatory Effects of Sub-Microsecond Irreversible Electroporation and Its Combination with Calcium Electroporation

In this work, we have investigated the feasibility of sub-microsecond range irreversible electroporation (IRE) with and without calcium electroporation in vivo. As a model, BALB/C mice were used and bioluminescent SP2/0 myeloma tumor models were developed. Tumors were treated with two separate pulsed electric field (PEF) pulsing protocols PEF1: 12 kV/cm × 200 ns × 500 (0.006 J/pulse) and PEF2: 12 kV/cm × 500 ns × 500 (0.015 J/pulse), which were delivered with and without Ca2+ (168 mM) using parallel plate electrodes at a repetition frequency of 100 Hz. Both PEF1 and PEF2 treatments reduced tumor growth and prolonged the life span of the mice, however, the PEF2 protocol was more efficient. The delay in tumor renewal was the biggest when a combination of IRE with calcium electroporation was used, however, we did not obtain significant differences in the final mouse survival compared to PEF2 alone. Anti-tumor immune responses were also investigated after treatment with PEF2 and PEF2+Ca. In both cases the treated mice had enlarged spleens and increased spleen T cell numbers, lower percentages of suppressor cell subsets (conventional CD4+CD25+ Treg, CD4+CD25−DX5+ Tr1, CD8+DX5+, CD4+CD28−, CD8+CD28−), changed proportions of Tcm and Tef/Tem T cells in the spleen and increased amount of tumor cell specific antibodies in the sera. The treatment based on IRE was effective against primary tumors, destroyed the tumor microenvironment and induced an anti-tumor immune response, however, it was not sufficient for complete control of tumor metastasis.This article belongs to the Special Issue Electric Field Based Therapies in Cancer Treatment: a Selection of Studies Presented at the 3rd World Congress on ElectroporationThis research was funded by Research Council of Lithuania, grant number S-MIP-19-22