[Show abstract][Hide abstract] ABSTRACT: Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.
Cellular and Molecular Life Sciences CMLS 04/2014; · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Positive and negative streamer discharges in atmospheric pressure air were generated in a shielded sliding discharge reactor at operating voltages as low as 5 kV for a gap length of 1.6 cm. In this reactor, electrodes are placed on top of a dielectric layer and one of the electrodes, generally the one on ground potential, is connected to a conductive layer on the opposite side of the dielectric. The energy per pulse, at the same applied voltage, was more than a factor of seven higher than that of pulsed corona discharges, and more than a factor of two higher than that of sliding discharges without a shield. It is explained on the basis of enhanced electric fields, particularly at the plasma emitting electrode. Specific input energy required for 50 % removal from ~1,000 ppm initial NO could be reduced to ~18 eV/molecule when ozone in the exhaust of negative streamers was utilized. For sliding discharges and pulsed corona discharges this value was ~25 eV/molecule and it was 35 eV/molecule for positive shielded sliding discharges. Also, the ozone energy yield from dry air was up to ~130 g/kW h and highest for negative streamer discharges in shielded sliding discharge reactors. The high energy density in negative streamer discharges in the shielded discharge reactor at the relatively low applied voltages might not only allow expansion of basic studies on negative streamers, but also open the path to industrial applications, which have so far been focused on positive streamer discharges.
Plasma Chemistry and Plasma Processing 01/2014; · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presence of increased temperature for gene electrotransfer has largely been considered negative. Many reports have published on the lack of heat from electrotransfer conditions to demonstrate that their effects are from the electrical pulses and not from a rise in temperature. Our hypothesis was to use low levels of maintained heat to aid in gene electrotransfer. The goal was to increase gene expression and/or reduce electric field. In our study we evaluated high and low electric field conditions from 90 V to 45 V which had been preheated to 40 °C, 43 °C, or 45 °C. Control groups of non-heated as well as DNA only were included for comparison in all experiments. Luciferase gene expression, viability, and percent cell distribution were measured. Our results indicated a 2–4 fold increase in gene expression that is temperature and field dependent. In addition levels of gene expression can be increased without significant decreases in cell death and in the case of high electric fields no additional cell death. Finally, in all conditions percent cell distribution was increased from the application of heat. From these results, we conclude that various methods may be employed depending on the end users desired goals. Electric field can be reduced 20-30% while maintaining or slightly increasing gene expression and increasing viability or overall gene expression and percent cell distribution can be increased with low viability.
[Show abstract][Hide abstract] ABSTRACT: Shielded sliding discharges are nanosecond streamer discharges which develop along a dielectric between metal foil electrodes, with one of the foils extended over the entire rear of the dielectric layer. The electrode configuration not only allowed rearranging discharges in parallel due to the decoupling effect of the metal layer, but also to modify the electric field distribution in such a way that components normal to the surface are enhanced, leading to an increased energy density in the discharge plasma. By varying the electrode gap, the applied voltage, and the repetition rate, it is shown that by keeping the average electric field constant, the discharge voltage can be reduced from tens of kV to values on the order of a few kV, but only at the expense of a reduced energy density of the plasma. Varying the repetition rate from 20 to 500 Hz resulted in a slightly reduced energy per pulse, likely caused by residual charges on the dielectric surface. Measurements of the NO conversion to NO2 and ozone synthesis in dry air showed that the conversion is only dependent on the energy density of the discharge plasma. Although reducing the pulse voltage from the tens of kV range to that of few kV, and possibly even lower, causes a reduction in energy density, this loss can be compensated for by increasing the electrode gap area. This and the possibility to form discharge arrays allows generating large volume discharge reactors for environmental applications, at modest pulsed voltages.
[Show abstract][Hide abstract] ABSTRACT: Experiments with CHO cells exposed to 60 and 300 ns pulsed electric fields with amplitudes in the range from several kV/cm to tens of kV/cm, showed a decrease of the uptake of calcium ions by more than an order of magnitude when, immediately after a first pulse, a second one of opposite polarity was applied. This effect is assumed to be due to the reversal of the electrophoretic transport of ions through the electroporated membrane during the second phase of the bipolar pulse. This assumption, however, is only valid if electrophoresis is the dominant transport mechanism, rather than diffusion. Comparison of calculated calcium ion currents with experimental results showed that for nanosecond pulses, electrophoresis is at least as important as diffusion. By delaying the second pulse with respect to the first one, the effect of reverse electrophoresis is reduced. Consequently, separating nanosecond pulses of opposite polarity by up to approximately hundred microseconds allows us to vary the uptake of ions from very small values to that obtained with two pulses of the same polarity. The measured calcium ion uptake obtained with bipolar pulses also allowed us to determine the membrane pore recovery time. The calculated recovery time constants are on the order of ten microseconds.
[Show abstract][Hide abstract] ABSTRACT: Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, Propidium Iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (15min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.
Biochemical and Biophysical Research Communications 12/2013; · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A sliding surface discharge was formed on a dielectric layer in steam at ~100 °C and atmospheric pressure. The material properties and the thickness of the dielectric layer were found to strongly affect the energy deposition into the plasma. With a 0.32 cm thick dielectric the energy deposition was 1.4 times greater than with a 0.48 cm thick dielectric, and with window glass it was 1.3 times greater than with Macor of the same thickness. Product gases were H2 (73 ± 4%) and O2 (27 ± 1%), and H2O2 accumulated in the condensed water up to 0.4 g l−1. The energy yield for hydrogen was 1.2 ± 0.1 g H2 kWh−1 and independent of the input power and thickness or material of the dielectric. However, for hydrogen peroxide the energy yield, which varied between 0.61 and 3.2 g H2O2 kWh−1, was found to depend strongly on the thickness and material of the dielectric. The addition of benzene to the steam increased the energy efficiency of hydrogen to 2.3 g kWh−1, and decreased oxygen and hydrogen peroxide by about 3 and 6 times, respectively. It also caused the deposition of phenol and polymer-like layers on the dielectric. The results are explained on the basis of reactions of H and OH radicals adsorbed on the surface and/or in gas phase.
Journal of Physics D Applied Physics 03/2013; 46(14):145201. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have for the first time recorded action potentials in rat hippocampus
neurons when they were stimulated by subnanosecond electric pulses. The
preliminary results show that applying a series of pulses allowed the
accumulation of depolarization before activating the voltage gated
channels. The depolarization only occurred when the electric pulses were
applied. It is unclear whether the depolarization is caused by the
charge accumulation across the membrane or the cation influx due to the
membrane permeabilization. We have also conducted an electromagnetic
simulation of delivering subnanosecond pulses to tissues using an
impulse radiating antenna. The results show that the pulses can be
confined in the deep region in the brain but the amplitude is reduced
significantly due to the attenuation of the tissues. A partially lossy
dielectric lens may be used to reverse the decreasing trend of the
[Show abstract][Hide abstract] ABSTRACT: In the research of bioelectrics, bipolar pulses appear to enhance the membrane effects by creating anode on both sides of the cell sequentially. On the other hand, the membrane charging resulted from the first part of the pulse may be removed by the following pulse of opposite polarity. To explore the biological effects of bipolar pusles on cells, we have constructed several types of nanosecond bipolar pulse generators, which can drive low-impedance loads of, for example, 10 Ω. They have been used for loads of electroporation cuvettes. The pulses were generated by means of five, 50 Ω transmission lines connected in parallel which were then switched on by a single spark gap switch, or a RLC circuit charged by a Marx generator. The pulse waveforms are symmetrical for long pulses, such as 600 ns, but for short pulses, such as 10 ns, an apparent asymmetry was observed. We suggest several causes for the deviation of the theoretical pulse-duration prediction.
Pulsed Power Conference (PPC), 2013 19th IEEE; 01/2013
[Show abstract][Hide abstract] ABSTRACT: The rate of trypan blue uptake of liver cancer cells, indicating cell death, when exposed to subnanosecond high electric field pulses, increased strongly when the temperature was raised above 37 °C. The exposure of Hepa 1-6 cells to 2000 pulses of 200 picosecond duration and electric field amplitudes exceeding 80 kV/cm induced cell death in almost 30% of the cells when the temperature was increased to 47 °C for the time of the pulsing. For temperatures at 37 °C and below, the same exposure to pulsed electric fields did not show any measurable effect. Even for the maximum elevated temperature of 47 °C, thermal effects were not found to cause fatalities for the time of exposure, which was, for 2000 pulses at a repetition rate of 7-9 pulses per second, on the order of 5 min. The effect of temperature on the electrical properties of the cell was measured by means of dielectric spectroscopy. The membrane voltages derived from these values were found to be too low to cause electroporation at room temperature. However, the reduced viscosity of the membrane with temperature is likely to reduce the threshold for poration, and together with the effect of multiple pulses, is considered to be the cause for the observed high death rate of the cells. This argument is supported by molecular dynamics simulations which show an increased probability for pore formation with temperature.
[Show abstract][Hide abstract] ABSTRACT: Comparative studies revealed that surface plasmas developing along a solid-gas interface are significantly more effective and energy efficient for remediation of toxic pollutants in air than conventional plasmas propagating in air. Scaling of the surface plasma reactors to large volumes by operating them in parallel suffers from a serious problem of adverse effects of the space charges generated at the dielectric surfaces of the neighboring discharge chambers. This study revealed that a conductive foil on the cathode potential placed between the dielectric plates as a shield not only decoupled the discharges, but also increased the electrical power deposited in the reactor by a factor of about forty over the electrical power level obtained without shielding and without loss of efficiency for NO removal. The shield had no negative effect on efficiency, which is verified by the fact that the energy costs for 50% NO removal were about 60 eV/molecule and the energy constant, k(E), was about 0.02 L/J in both the shielded and unshielded cases.
Journal of hazardous materials 03/2012; 209-210:293-8. · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals.
Technology in cancer research & treatment 02/2012; 11(1):83-93. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Energetic and scalable non-equilibrium plasma was formed in pure water vapour at atmospheric pressure between wire-to-strip electrodes on a dielectric surface with one of the electrodes extended forming a conductive plane on the back side of the dielectric surface. The energy deposition increased by an order of magnitude compared with the conventional pulsed corona discharges under the same conditions. The scalability was demonstrated by operating two electrode assemblies with a common conductive plane between two dielectric layers. The energy yields for hydrogen and hydrogen peroxide generation were measured as ∼1.2 g H2/kWh and ∼4 g H2O2/kWh.
Journal of Physics D Applied Physics 01/2012; 45(13). · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Delivery of subnanosecond pulses into biological tissue can be undertaken by an impulse radiating antenna (IRA). Previous analysis shows that it is important to add a dielectric lens, which reduces the abrupt change of dielectric constant from air to tissue and therefore increases the transmission of the pulses. As a proof of concept, we have simulated subnanosecond pulsed radiation focused into a tissue simulant which consists of homogeneous, hemisphere tissues using 3-D electromagnetic solver, CST Microwave Studio. The simulation of an IRA in conjunction of a lens indicates subnanosecond pulses can be focused 6 cm below tissue surface with a spot diameter approximately 1 cm. The focal point coincides with the geometric focus of the IRA. However, this result is only valid for a tissue with a low conductivity (σ< 0.3 S/m). For lossier tissues, the electric field decreases from the surface monotonically as the subnanosecond pulses penetrate in depth. Two approaches were proposed to solve this problem. One was to use an inhomogeneous dielectric lens, with lossy material partially filled, to attenuate the incident field in the small azimuthal angles but to spare the field in the larger azimuthal angels. A desirable focusing was observed. The second approach was to use a dipole antenna in conjunction with the impulse radiating antenna. The dipole antenna decreases the surface field intensity generated by the aperture antenna, but at the destination, the field will be mostly given by the aperture antenna, resulting in a focusing.
Power Modulator and High Voltage Conference (IPMHVC), 2012 IEEE International; 01/2012
[Show abstract][Hide abstract] ABSTRACT: It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.
PLoS ONE 01/2012; 7(12):e51349. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) conversion has been studied for two different types of atmospheric-pressure pulsed-corona discharges, one generates a surface-plasma and the other provides a volume-plasma. For both types of discharges the energy cost for NO removal increases with decreasing oxygen concentration and initial concentration of NO. However, the energy cost for volume plasmas for 50% NO removal, EC(50), from air was found to be 120 eV/molecule, whereas for the surface plasma, it was only 70 eV/molecule. A smaller difference in energy cost, but a higher efficiency for removal of NO was obtained in a pure nitrogen atmosphere, where NO formation is restricted due to the lack of oxygen. For the volume plasma, EC(50) in this case was measured at 50 eV/molecule, and for the surface plasma it was 40 eV/molecule. Besides the higher NO removal efficiency of surface plasmas compared to volume plasmas, the energy efficiency of surface-plasmas was found to be almost independent of the amount of electrical energy deposited in the discharge, whereas the efficiency for volume plasmas decreases considerably with increasing energy. This indicates the possibility of operating surface plasma discharges at high energy densities and in more compact reactors than conventional volume discharges.
Journal of hazardous materials 12/2011; 197:220-8. · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In-vivo porcine studies on the effect of nanosecond high-voltage pulses on liver tissue have shown that cell death can be induced in well-defined tissue volumes without damaging collagen-predominant structures. Comparison of the experimental results with the results of a 3-D finite element model allowed us to determine the threshold electric field for cell death. For 30, 100-ns-long pulses this was found to be in the range from 12 to 15 kV/cm. Modeling of the temperature distribution in the tissue using Pennes' bioheat equation showed that the lethal effect of nanosecond pulses on cells is nonthermal. Muscle contractions, generally caused by high-voltage pulses, were significantly reduced for the 100-ns pulses compared to microsecond-long pulses. The results of these studies indicate that high-voltage nanosecond pulses reliably kill normal liver cells in vivo, and therefore, may be useful for liver tumor treatments.
IEEE Transactions on Biomedical Engineering 09/2011; · 2.35 Impact Factor