Mitochondrial depolarization and ATP loss during high frequency nanosecond and microsecond electroporation.

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca2+ electrochemotherapy, ensuring excellent cytotoxic efficacy.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (µsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

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

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.

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy.

OBJECTIVE: this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses. METHODS: As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 µL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 µs x 8 protocol was used. RESULTS: It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses. SIGNIFICANCE: the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 µs pulses.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

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.

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.

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.

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.

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.

Ras/PKA signal transduction pathway participates in the regulation of Saccharomyces cerevisiae cell apoptosis in an acidic environment.

The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast Saccharomyces cerevisiae. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2(Val19) allele or deletion of PDE2 gene increases cell death, displaying the markers of apoptosis in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha-ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions.