Oxidative Effects of Nanosecond Pulsed Electric Field Exposure in Cells and Cell-Free Media

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H2O2 and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage

A Theoretical Analysis of the Feasibility of a Singularity-Induced Micro-Electroporation System

Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1) insulator thickness and (2) electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to regular, round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane-cytoskeleton interaction and cell motility

Diffusion-Weighted MRI for Verification of Electroporation-Based Treatments

Clinical electroporation (EP) is a rapidly advancing treatment modality that uses electric pulses to introduce drugs or genes into, e.g., cancer cells. The indication of successful EP is an instant plasma membrane permeabilization in the treated tissue. A noninvasive means of monitoring such a tissue reaction represents a great clinical benefit since, in case of target miss, retreatment can be performed immediately. We propose diffusion-weighted magnetic resonance imaging (DW-MRI) as a method to monitor EP tissue, using the concept of the apparent diffusion coefficient (ADC). We hypothesize that the plasma membrane permeabilization induced by EP changes the ADC, suggesting that DW-MRI constitutes a noninvasive and quick means of EP verification. In this study we performed in vivo EP in rat brains, followed by DW-MRI using a clinical MRI scanner. We found a pulse amplitude–dependent increase in the ADC following EP, indicating that (1) DW-MRI is sensitive to the EP-induced changes and (2) the observed changes in ADC are indeed due to the applied electric field

Comparison of Electroporation Threshold for Different Cell Lines in vitro

The electroporation threshold was compared at various electric pulse durations for three cell lines: two non-tumor cell lines (human erythrocytes and Chinese hamster ovary cells) and one tumor cell line (rat glioma C6 cells). First, the dependences of the fraction of electroporated cells on the pulse intensity were obtained for the cells exposed to single square-wave electric pulses with the durations of 0.02-2 ms. Then, the average cell radii were measured for each cell line and the transmembrane potential induced by the external electric field was calculated. The obtained values of the transmembrane potential were in the range of 480-930 mV and decreased with increasing pulse duration. The obtained dependences of the transmembrane potential required to electroporate 50% of cells on the pulse duration were close to each other for all cell lines studied

High-Voltage Pulses Potentiate Bleomycin Cytotoxicity Towards Cancer Cells in vitro

In this study the influence of high-voltage pulses on bleomycin cytotoxicity towards mouse hepatoma MH-22A and rat glioma C6 cells in vitro was determined. It was obtained that combining bleomycin with pulses of strong electric field significantly enhances the number of cells killed by bleomycin. Bleomycin alone was cytotoxic to both cell lines at concentrations above 1 μM. Treatment of cells by electric pulses in the presence of bleomycin greatly potentiated its cytotoxicity towards both cell lines tested - the bleomycin concentration required to reduce cell survival by 50% was 3.5-3.8 nM only. Exposure of cells to electric pulses only did not reduce cell viability

Changes of the Electrode Surface Roughness Induced by High-Voltage Electric Pulses as Revealed by AFM

The changes of the surface topography of stainless-steel and aluminium electrodes occurring due to the action of electric pulses which are utilized for cell electroporation, have been studied by using atomic force microscopy. The surfaces of the polished stainless-steel electrodes were smooth - the average roughness was 13-17 nm and the total roughness 140-180 nm. The total roughness of the aluminium electrodes was about 320 nm. After the treatment of the chambers filled with 154 mM NaCl solution by a series of short (20-40 μs), high-voltage (4 kV) pulses with the total dissolution charge of 0.20-0.26 A s/$cm^{2}$, the roughness of the surface of the electrodes has increased, depending on the total amount of the electric charge that has passed through the unit area of the electrode. Up to a two- and threefold increase of the surface roughness of the stainless-steel and aluminium anodes respectively was observed due to the dissolution of the anode material. Therefore, the use of high-voltage electric pulses leads to the increase of the inhomogeneity of the electric field at the electrode, which facilitates the occurrence of the electric breakdown of the liquid samples and causes non-equal treatment of each cell