Microsecond Pulsed Electric Fields Research Articles

Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 μs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Scheduled dosage regimen by irreversible electroporation of loaded erythrocytes for cancer treatment.

Precise control of cargo release is essential but still a great challenge for any drug delivery system. Irreversible electroporation (IRE), utilizing short high-voltage pulsed electric fields to destabilize the biological membrane, has been recently approved as a non-thermal technique for tumor ablation without destroying the integrity of adjacent collagenous structures. Due to the electro-permeating membrane ability, IRE might also have great potential to realize the controlled drug release in response to various input IRE parameters, which were tested in a red blood cell (RBC) model in this work. According to the mathematical simulation model of a round biconcave disc-like cell based on RBC shape and dielectric characteristics, the permeability and the pore density of the RBC membrane were found to quantitatively depend on the pulse parameters. To further provide solid experimental evidence, indocyanine green (ICG) and doxorubicin (DOX) were both loaded inside RBCs (RBC@DOX&ICG) and the drug release rates were found to be tailorable by microsecond pulsed electric field (μsPEF). In addition, μsPEF could effectively modulate the tumor stroma to augment therapy efficacy by increasing micro-vessel density and permeability, softening extracellular matrix, and alleviating tumor hypoxia. Benefiting from these advantages, this IRE-responsive RBC@DOX&ICG achieved a remarkably synergistic anti-cancer effect by the combination of μsPEF and chemotherapy in the tumor-bearing mice model, with the survival time increasing above 90 days without tumor burden. Given that IRE is easily adaptable to different plasma membrane-based vehicles for delivering diverse drugs, this approach could offer a general applicability for cancer treatment.

Enhanced Cell Viability and Migration of Primary Bovine Annular Fibrosus Fibroblast-like Cells Induced by Microsecond Pulsed Electric Field Exposure.

This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (μsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 μsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 μs-duration pulses induce reversible electroporation, while 100 μs pulses induce irreversible electroporation of the cells. Additionally, μsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 μm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial-depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the μsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration.

Threshold Microsecond Pulsed Electric Field Exposures for Change in Spinach Quality.

Pulsed electric fields (PEFs) are often used to pretreat foods to enhance subsequent processes, such as drying, where maintaining food product quality is important for consumer satisfaction. This study aims to establish a threshold PEF exposure to determine the doses at which electroporation is viable for use on spinach leaves, wherein integrity is maintained postexposure. Three numbers of consecutive pulses (1, 5, 50) and two pulse durations (10 and 100 μs) have been examined herein at a constant pulse repetition of 10 Hz and 1.4 kV/cm field strength. The data indicate that pore formation in itself is not a cause for loss of spinach leaf food quality, i.e., significant changes in color and water content. Rather, cell death, or the rupture of the cell membrane from a high-intensity treatment, is necessary to significantly alter the exterior integrity of the plant tissue. PEF exposures thus can be used on leafy greens up until the point of inactivation before consumers would see any alterations, making reversible electroporation a viable treatment for consumer-intended products. These results open up future opportunities to use emerging technologies based on PEF exposures and provide useful information in setting parameters to avoid food quality diminishment.

An Overview of Subnanosecond Pulsed Electric Field Biological Effects: Toward Contactless Technologies for Cancer Treatment

During the last decades, millisecond, microsecond, and nanosecond electric pulses were all successfully used for both biological and medical applications. Among medical applications, irreversible electroporation (IRE) and electrochemotherapy (ECT) by microsecond pulsed electric fields (PEFs) are both minimally invasive established cancer treatments for superficial tumors, based on IRE or reversible electroporation combined with a therapeutic drug, respectively. The aim of this review is to summarize the present data, still rather scarce, on the biological effects induced by subnanosecond PEFs (sub-nsPEFs) following an approach “with contact.” A nonexhaustive overview of the effects of the nanosecond PEFs (nsPEFs) is also presented to foresee the possibilities offered by the application of sub-nsPEFs. Indeed, it is demonstrated that nsPEFs can indeed produce: permeabilization, followed by the transfer of molecules inside the cell; cell stimulation, with calcium mobilization through voltage-gated channels activation, and cell death. This review is extended to the medical applications of nsPEFs for cancer treatment in vivo, a procedure known as nanoablation. The long-term future for the PEF-based cancer treatment techniques implies a broadening of their applications to deep-seated tumors. This raises the challenge of overcoming the inherent invasiveness associated with the treatment of these tumors. A possible solution is the development of contactless ECT and IRE, noninvasive procedures that are possible only using highly sophisticated impulse antenna techniques. Thus, we also present existing contactless technologies recently developed worldwide and considered potential candidates for future biomedical applications. This review is therefore providing the community with a summary of the major “with contact” biomedical applications of sub-nsPEFs, while also exploring all the currently existing contactless technologies, by highlighting their importance in strengthening the role of PEFs in the fight against cancer.

A perspective on modeling pore energy and pulsed electromagnetic field induced cell membrane perforation

Pulsed electric field-induced electroporation has been widely used, but its specific perforation theory has not been fully elucidated. Therefore, this Perspective paper takes as a clue the dynamic development relationship between the pore energy and the pore state in cell membranes. First, based on the contribution of line tension, surface tension, steric repulsion, and applied electric field to pore energy, the theoretical models of reversible electroporation of a microsecond pulsed electric field and irreversible electroporation of a high-frequency nanosecond pulsed electric field are reviewed. Then, the contribution of elastic strain energy to pore energy is increased, and the theoretical model of pulsed electric field electroporation considering the mechanical properties of cell membranes is further reviewed. Based on the contribution of magnetic stress generated by the magnetic field and the gradient magnetic field to pore energy, a theoretical model of cell membrane magnetoporation under the action of a pulsed magnetic field is proposed, which lays a theoretical foundation for the popularization and application of non-contact cell membrane perforation technology.

Combined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields.

In modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.

Histone deacetylase 4 and 5 translocation elicited by microsecond pulsed electric field exposure is mediated by kinase activity.

Electroporation-based technologies using microsecond pulsed electric field (µsPEF) exposures are established as laboratory and clinical tools that permeabilize cell membranes. We demonstrate a µsPEF bioeffect on nucleocytoplasmic import and export of enzymes that regulate genetic expression, histone deacetylases (HDAC) -4 and -5. Their μsPEF-induced nucleocytoplasmic transport depends on presence and absence of extracellular calcium ions (Ca2+) for both MCF7 and CHO-K1 cells. Exposure to 1, 10, 30 and 50 consecutive square wave pulses at 1Hz and of 100µs duration with 1.45kV/cm magnitude leads to translocation of endogenous HDAC4 and HDAC5. We posit that by eliciting a rise in intracellular Ca2+ concentration, a signaling pathway involving kinases, such as Ca2+/CaM-dependent protein kinase II (CaMKII), is activated. This cascade causes nuclear export and import of HDAC4 and HDAC5. The potential of µsPEF exposures to control nucleocytoplasmic transport unlocks future opportunities in epigenetic modification.

Pulse width and intensity effects of pulsed electric fields on cancerous and normal skin cells

Microsecond pulsed electric fields (PEF) have previously been used for various tumour therapies, such as gene therapy, electrochemotherapy and irreversible electroporation (IRE), due to its demonstrated ability. However, recently nanosecond pulsed electric fields (nsPEF) have also been used as a potential tumor therapy via inducing cell apoptosis or immunogenic cell death to prevent recurrence and metastasis by interacting with intracellular organelles. A large proportion of the existing in-vitro studies of nsPEF on cells also suggests cell necrosis and swelling/blebbing can be induced, but the replicability and potential for other effects on cells suggesting a complicated process which requires further investigation. Therefore, this study investigated the effects of pulse width and intensity of nsPEF on the murine melanoma cells (B16) and normal murine fibroblast cells (L929) through electromagnetic simulation and in-vitro experiments. Through examining the evolution patterns of potential difference and electric fields on the intracellular compartments, the simulation has shown a differential effect of nsPEF on normal and cancerous skin cells, which explains well the results observed in the reported experiments. In addition, the modelling has provided a clear evidence that a few hundreds of ns PEF may have caused a mixed mode of effects, i.e. a ‘cocktail effect’, including cell electroporation and IRE due to an over their threshold voltage induced on the plasma membrane, as well as cell apoptosis and other biological effects caused by its interaction with the intracellular compartments. The in-vitro experiments in the pulse range of the hundreds of nanoseconds showed a possible differential cytotoxicity threshold of electric field intensity between B16 cells and L929 cells.

Substantially Improved Electrofusion Efficiency of Hybridoma Cells: Based on the Combination of Nanosecond and Microsecond Pulses.

As the initial antibody technology, the preparation of hybridoma cells has been widely used in discovering antibody drugs and is still in use. Various antibody drugs obtained through this technology have been approved for treating human diseases. However, the key to producing hybridoma cells is efficient cell fusion. High-voltage microsecond pulsed electric fields (μsHVPEFs) are currently one of the most common methods used for cell electrofusion. Nevertheless, the membrane potential induced by the external microsecond pulse is proportional to the diameter of the cell, making it difficult to fuse cells of different sizes. Although nanosecond pulsed electric fields (nsPEFs) can achieve the fusion of cells of different sizes, due to the limitation of pore size, deoxyribonucleic acid (DNA) cannot efficiently pass through the cell pores produced by nsPEFs. This directly causes the significant loss of the target gene and reduces the proportion of positive cells after fusion. To achieve an electric field environment independent of cell size and enable efficient cell fusion, we propose a combination of nanosecond pulsed electric fields and low-voltage microsecond pulsed electric fields (ns/μsLVPEFs) to balance the advantages and disadvantages of the two techniques. The results of fluorescence experiments and hybridoma culture experiments showed that after lymphocytes and myeloma cells were stimulated by a pulse (ns/μsLVPEF, μsHVPEF, and control), compared with μsHVPEF, applying ns/μsLVPEF at the same energy could increase the cell fusion efficiency by 1.5–3.0 times. Thus far, we have combined nanosecond and microsecond pulses and provided a practical solution that can significantly increase cell fusion efficiency. This efficient cell fusion method may contribute to the further development of hybridoma technology in electrofusion.

Abstract 3314: Glioblastoma treatment using epigenetic modification induced by microsecond pulsed electric field (µsPEF) exposure

Abstract Epigenetic modifications derived from changes in sub-cellular localization and activity of the post-translational histone modifying enzymes, histone deacetylases (HDACs) are one of the strategies to treat various types of cancer. Aberrant HDAC activity is an indicator of glioblastoma multiforme (GBM), generally leading to hypoacetylation of histones and a transcriptionally repressed chromatin state. Most noticeably, HDAC4 expression increases 61,000% in brain tumors!1 Upregulation of HDAC4 in human glioma cells (U87-MG) stimulates proliferation and invasiveness, making HDAC4 a therapeutic target. The effectiveness of pharmacological HDAC inhibitors is hampered by non-specific targeting and low selectivity, especially in solid tumors like GBM, and by side-effects with systemic delivery, e.g. cardiac toxicity.2 Existing electroporation-based approaches to GBM aim to induce cell death either directly3 or in combination with adjuvants such as excess extracellular calcium4 or chemotherapy drugs5.A cytostatic approach is introduced in which the initial electroporation from non-thermal, localized µsPEF exposure elicits downstream epigenetic responses that decrease cell proliferation, circumventing obstacles experienced by pharmacological agents. Accumulation of HDAC4 in the nucleus of U87-MG cells following exposure to µsPEF is hypothesized to lead in a dose-dependent manner to cell death via apoptosis and decreased proliferation.A BTX GeminiX2 electroporator delivered square-wave µsPEF of 100 µs and 1.45 kV/cm, while the number of pulses (0, 1, 10, 20 pulses, delivered at 1 Hz) were varied to determine thresholds for HDAC4 translocation and proliferation reduction of U87-MG cells. For immunofluorescence assay to track HDAC4 location at 3 h after exposure, cells were cultured and exposed on glass-bottom dishes. For MTT and ATP kinase assays, cells were exposed in electroporation cuvettes and transferred to 96-well plates to measure proliferation at 6, 24 and 72 h after exposure. The ratio of HDAC4 in the nucleus compared to the cytoplasm (N/C ratio) more than doubles after 20 pulses, compared to sham control with no µsPEF exposure. Cell concentration relative to control drops over 90% given 20 pulses. Our data reflect the inhibition of cell proliferation and HDAC4 accumulation in nuclei. Although electroporation-based therapies for GBM have been studied pre-clinically, this study delves further into fundamental mechanisms and optimization of the energy delivered by µsPEF exposure that induces cell death with respect to epigenetic modification. 1. Lee P. et al. Anticancer Res, 35:615, 2015.2. Slingerland M. et al. Anticancer Drugs, 25:140, 2014.3. Rossmeisl J.H. et al. J Neurosurg, 123:1008, 2015.4. Wasson E.M. et al. Ann Biomed Eng, 45:2535, 2017.5. Sharabi S. et al. Sci Rep, 10:2178, 2020. Citation Format: Zahra Safaei, Gary L Thompson. Glioblastoma treatment using epigenetic modification induced by microsecond pulsed electric field (µsPEF) exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3314.

A review on bioelectrical effects of cellular organelles by high voltage nanosecond pulsed electric fields

The biomedical application of high-voltage nanosecond pulsed electric fields (nsPEFs) has become an emerging interdisciplinary research field in recent years. Compared with microsecond and millisecond pulsed electric fields, high-voltage nsPEFs can not only lead the cell membrane structure to polarize and dielectric break down the cell membrane structure, i.e. membrane electroporation, but also penetrate into the cell, triggering off organelle bioelectrical effects such as cytoskeleton depolymerization, intracellular calcium ion release, and mitochondrial membrane potential dissipation. Extensive attention has been attracted from related academic communities. In this article, the following aspects are involved. First, the physical model of high-voltage nsPEFs and its bioelectrical effects on cellular organelles are introduced. Then, the existing researches of the interactions of high-voltage nsPEFs with cytoskeleton, mitochondria, endoplasmic reticulum, cell nucleus and other subcellular structure are reviewed and summarized; the relationship between the influence on cellular organelles by high-voltage nsPEFs and the biological effects such as cell death and intercellular communication is highlighted. Finally, the key technical challenges to high-voltage nsPEFs in biomedical research are condensed, followed by the prospects of future research directions.

Pulsed Electric Fields Induce Extracellular Matrix Remodeling through Matrix Metalloproteinases Activation and Decreased Collagen Production

Impairment of extracellular matrix remodeling is observed in the tumor microenvironment or fibrosis and results in excessive collagen production and/or decreased degradation by matrix metalloproteinases (MMPs). Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel the extracellular matrix. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated invitro tissue-engineered human dermal substitute was used to show that microsecond and millisecond pulsed electric fields induced (i) a rapid modulation (4 hours after electrostimulation) of mRNA genes composing the matrisome, particularly a downregulation of procollagens and extracellular matrix maturation enzymes such as transglutaminase 2 and lysyl oxidase like; (ii) a transient decrease in procollagens production and hydroxyproline tissue content within a week after electrostimulation; (iii) a long-lasting ROS-dependent overactivation of matrix metalloproteinases for at least 48 hours; and (iv) a downregulation of TGFβ1. These observations underpin that pulsed electric fields, a technology already approved for clinical use combined with anticancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix.

Effects of microsecond pulsed electric field (μsPEF) and modular micro reaction system (MMRS) treatments on whey protein aggregation

Electric field (EF) processing has been suggested to be a promising technology in the valorisation of side-streams, such as whey, by enhancing the functionality of biomacromolecules. Shifting from batch to continuous processes and utilising more industry-relevant matrices are of great interest, yet studies thereof are lacking. A continuous plate–plate microsecond pulsed electric field (μsPEF) setup was optimised and whey protein concentrate (10%, w/w) was treated at 3 kV cm−1, 10 μs, 40–50 Hz. A diffuser inlet/outlet improved the process stability considerably. Average outlet temperatures between 64.1 °C and 73.2 °C were investigated and aggregate formation between 0.04 μm and 2000 μm was quantified. μsPEF-triggered particles were compared with those obtained in thermal treatments, conducted in a modular micro reaction system (MMRS) with similar temperature and residence times. A comparison of the Sauter mean diameter revealed that in the given EF process window, mainly thermal effects were responsible for aggregation shifts.

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells

Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. We analyzed invitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated invivo in a heterotopic mouse xenograft model. Our invitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 μs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. Invivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation invivo to enable complete inhibition of tumor growth. Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells.

Cell Electropermeabilisation Enhancement by Non-Thermal-Plasma-Treated PBS.

The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects.

Effect of frequency of microsecond pulsed electric field on orientation of boron nitride nanosheets and thermal conductivity of epoxy resin-based composites

Nanosecond pulsed electric fields over 200 kV/mm have been used for filler alignment in polymer-based thin film (120–250 μm) composites to improve their thermal conductivity, but little research has been performed on bulk composites because of the requirement of an extremely high nanosecond pulsed voltage. In this paper, a microsecond pulsed voltage with a pulse width of 1 μs and an electric field of only 11.76 kV/mm was adopted for the first time to prepare bulk composites of 1.7 mm. The effects of frequency on nanosheets’ orientation and the thermal conductivity of epoxy composites were studied. The orientation degree of BN nanosheets was determined based on cross-sectional scanning electron microscopy images and X-ray diffraction peaks. The orientation degree and thermal conductivity of epoxy composites increased with the increase of the frequency, but the increase rate was lower under higher frequencies. The thermal conductivity of composites containing 10 wt. % BN nanosheets prepared under 100 Hz was 0.588 W/mK, which was more than twice that of composites without an applied electric field. Additionally, an effective medium theory model was adopted for thermal conductivity modeling. In addition, thermogravimetric and differential scanning calorimetry analyses were performed to evaluate the thermal properties of the composites. The results showed that after BN orientation in epoxy resin, the composites had higher glass transition temperature and better thermal stability. Microsecond pulsed electric fields are promising for preparing bulk insulation composites with higher thermal conductivity.

Multiparametric Finite-Element Simulation and Experiment on Thermal Effects in Skin Tumor Exposed to High-Frequency Nanosecond Pulse Bursts

This paper introduces unipolar high-frequency nanosecond pulse bursts, which combine the advantages of microsecond pulsed electric fields and nanosecond pulsed electric fields, for the treatment of tumors. To take full advantage of this nonthermal pulsed electric field treatment, it is necessary to study the thermal effects of the high-electric-field nanosecond pulse bursts inside tumor tissue. A multiparametric analysis is performed based on a finite-element model of skin with a melanoma tumor that is nipped using the Tweezertrodes. The electric field ranges from 1 to 10 kV/cm, the pulsewidth ranges from 50 to 500 ns, and the frequency in the pulse bursts ranges between 100 kHz and 1 MHz. The total pulselength is 100 μs, and the frequency of the pulse bursts is 1 Hz. The Pennes bioheat transfer equation and the Arrhenius equation are used to calculate the temperature and the thermal damage. For pulse parameters of 5 kV/cm, 500 ns, and 1 MHz, the simulated temperature of the skin tumor is only 37.48 °C after 1 s, and the thermal damage is negligible. The temperature and thermal damage to the melanoma tumor in the skin are simulated and calculated for various electric fields, pulse widths, and frequencies. The relationship between the thermal effects on the tumor and the pulse parameters is researched, and pulse parameters for a tumor without thermal damage are ascertained. Temperature measurements of melanoma tumors in nude mice in vivo show that the change in experimental temperature in the tumors is consistent with the simulated average tumor temperature.

Cell electrofusion based on nanosecond/microsecond pulsed electric fields.

Traditionally, microsecond pulsed electric field was widely used in cell electrofusion technology. However, it was difficult to fuse the cells with different sizes. Because the effect of electroporation based on microsecond pulses was greatly influenced by cell sizes. It had been reported that the differences between cell sizes can be ignored when cells were exposed to nanosecond pulses. However, pores induced by those short nanosecond pulses tended to be very small (0.9 nm) and the pores were more easy to recover. In this work, a finite element method was used to simulate the distribution, radius and density of the pores. The innovative idea of “cell electrofusion based on nanosecond/microsecond pulses” was proposed in order to combine the advantages of nanosecond pulses and microsecond pulses. The model consisted of two contact cells with different sizes. Three kinds of pulsed electric fields were made up of two 100-ns, 10-kV/cm pulses; two 10-μs, 1-kV/cm pulses; and a sequence of a 100-ns, 10-kV/cm pulse, followed by a 10-μs, 1-kV/cm pulse. Some obvious advantageous can be found when nanosecond/microsecond pulses were considered. The pore radius was large enough (70nm) and density was high (5×1013m-2) in the cell junction area. Moreover, pores in the non-contact area of the cell membrane were small (1–10 nm) and sparse (109-1012m-2). Areas where the transmembrane voltage was higher than 1V were only concentrated in the cell junction. The transmembrane voltage of other areas were at most 0.6V when we tested the rest of the cell membrane. Cell fusion efficiency can be improved remarkably because electroporation was concentrated in the cell contact area.

Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions

Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.