Thermal Analysis of Infrared Irradiation-Assisted Nanosecond-Pulsed Tumor Ablation.

Nanosecond Pulsed Electric Fields (nsPEF) have the potential to treat a variety of cancer types including melanoma, pancreatic and lung squamous cancers. Recent studies show that nsPEF-based cancer therapy may be improved further with the assistance of moderate heating of the target. A feedback-looped heating system, utilizing a 980-nm fiber optic laser, was integrated into nsPEF electrodes for tumor ablation. The laser beam profile was determined to be Gaussian using a knife-edge technique. Thermal properties of the biological target were evaluated based on the treatment area, penetration depth and thermal distribution due to laser irradiation with or without nsPEF. Synergistic effects between nsPEF and the moderately elevated temperature at the target was observed, resulting in enhanced overall survival tumor regression up to 50% in the treatment of lung squamous cell cancer in mice.

From the basic science of biological effects of ultrashort electrical pulses to medical therapies.

This article is based on my presentation at the D'Arsonval Ceremony at the Joint Annual Meeting of the Bioelectromagnetics Society and the European BioElectromagnetics Association in Hangzhou, China, in June of 2017. It describes the pathway from the first studies on the effects of intense, nanosecond pulses on biological cells to the development of medical therapies based on these effects. The motivation for the initial studies of the effects of high voltage, nanosecond pulses on mammalian cells was based on a simple electrical circuit model, which predicted that such pulses allow us to affect not just the plasma membrane but also the subcellular structures. The first experimental study that confirmed this hypothesis was published in 2001 in the Bioelectromagnetics journal. It was followed by a large number of publications that showed that such ultrashort pulses affect cell functions, such as programmed cell death, and, at lower intensity, calcium mobilization from intracellular structures. These basic studies were leading to novel cancer treatments, treatments of cardiac arrhythmia, and advanced wound healing. Further, by reducing the pulse duration into the picosecond range, antenna-based neural stimulation seems to be possible. This manuscript gives an overview of the progress in this field of research in the decade after the initial bioelectric studies with high-voltage, nanosecond pulses, particularly the research performed at the Frank Reidy Research Center for Bioelectrics. It also tells you about my journey and that of my colleagues at the Center for Bioelectrics into and through this fascinating bioelectromagnetics research area. Bioelectromagnetics. 39:257-276, 2018. © 2018 Wiley Periodicals, Inc.

A subnanosecond electric pulse exposure system for biological cells.

An exposure system adapted for use on a microscope stage was constructed for studying the effects of high electric field, subnanosecond pulses on biological cells. The system has a bandpass of 3 GHz and is capable of delivering high-voltage electric pulses (6.2 kV) to the electrodes, which are two tungsten rods (100 µm in diameter) in parallel with a gap distance of 170 µm. Electric pulses are delivered to the electrodes through a π network, which serves as an attenuator as well as an impedance matching unit to absorb the reflection at the electrodes. By minimizing the inductance of the pulse delivery system, it was possible to generate electric fields of up to 200 kV/cm with a pulse duration of 500 ps at the surface of the cover slip under the microscope. The electric field at the cover slip was found to be homogenous over an area of 50-70 µm. Within this area, neuroblastoma cells placed on the cover slip were studied with respect to membrane potential changes caused by subnanosecond pulses. This allowed us, for the first time, to demonstrate depolarization of the cell membrane potential.

A Dielectric Rod Antenna for Picosecond Pulse Stimulation of Neurological Tissue.

A dielectrically loaded wideband rod antenna has been studied as a pulse delivery system to subcutaneous tissues. Simulation results applying 100 ps electrical pulse show that it allows us to generate critical electric field for biological effects, such as brain stimulation, in the range of several centimeters. In order to reach the critical electric field for biological effects, which is approximately 20 kV/cm, at a depth of 2 cm, the input voltage needs to be 175 kV. The electric field spot size in the brain at this position is approximately 1 cm2. Experimental studies in free space with a conical antenna (part of the antenna system) with aluminum nitride as the dielectric have confirmed the accuracy of the simulation. These results set the foundation for high voltage in situ experiments on the complete antenna system and the delivery of pulses to biological tissue.

Cell stimulation and calcium mobilization by picosecond electric pulses.

We tested if picosecond electric pulses (psEP; 190 kV/cm, 500 ps at 50% height), which are much shorter than channel activation time, can activate voltage-gated (VG) channels. Cytosolic Ca(2+) was monitored by Fura-2 ratiometric imaging in GH3 and NG108 cells (which express multiple types of VG calcium channels, VGCC), and in CHO cells (which express no VGCC). Trains of up to 100 psEP at 1 kHz elicited no response in CHO cells. However, even a single psEP significantly increased Ca(2+) in both GH3 (by 114 ± 48 nM) and NG108 cells (by 6 ± 1.1 nM). Trains of 100 psEP amplified the response to 379 ± 33 nM and 719 ± 315 nM, respectively. Ca(2+) responses peaked within 2-15s and recovered for over 100 s; they were 80-100% inhibited by verapamil and ω-conotoxin, but not by the substitution of Na(+) with N-methyl-D-glucamine. There was no response to psEP in Ca(2+)-free medium, but adding external Ca(2+) even 10s later evoked Ca(2+) response. We conclude that electrical stimuli as short as 500 ps can cause long-lasting opening of VGCC by a mechanism which does not involve conventional electroporation, heating (which was under 0.06 K per psEP), or membrane depolarization by opening of VG Na(+) channels.

Ion transport into cells exposed to monopolar and bipolar nanosecond pulses.

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 those 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 10 µs.

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity.

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.

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses.

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 (at 15 min) 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.

Simulation study of delivery of subnanosecond pulses to biological tissues with an impulse radiating antenna.

We have numerically studied the delivery of subnanosecond pulsed radiation to biological tissues for bioelectric applications. The antenna fed by 200 ps pulses uses an elliptical reflector in conjunction with a dielectric lens. Two numerical targets were studied: one was a hemispherical tissue with a resistivity of 0.3-1 S/m and a relative permittivity of 9-70 and the other was a realistic human head model (HUGO). The electromagnetic simulation shows that despite tissue heterogeneity of the human head, the electric field converges to a spot 8 cm in depth and the spot volume is approximately 1 cm × 2 cm × 1 cm in both cases when using only the reflector and a lens as an addition. Rather than increasing as it approaches the converging point, the electric field decreases strongly with distance from the skin to the converging point due to tissue resistive loss. The electric field distribution, however, can be reversed by making the dielectric lens lossy with the two innermost layers being partially resistive. The lossy lens causes an attenuation of the electric field near the axis, but the electric field generated by the waves which pass the lens at a wider angles compensate for this loss. A local maximum electric field in a deeper region of the tissue may form with the lossy lens. The study shows that it is possible to generate the desired electric field distribution in the complex biological target by modifying the dielectric properties of the lens used in conjunction with the reflector antenna.

Scaling of surface-plasma reactors with a significantly increased energy density for NO conversion.

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.

Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability.

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.

Comparative study of NO removal in surface-plasma and volume-plasma reactors based on pulsed corona discharges.

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.

Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields.

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4-1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

Comparative study of long- and short-pulsed electric fields for treating melanoma in an in vivo mouse model.

A mouse melanoma model was set up with green fluorescent protein (GFP) expression in vivo. With the same energy, long- (1 ms) and short- (300 ns) pulsed electric fields were delivered to two melanomas injected into the same mouse. The tumor growth and green fluorescence were followed up to compare the different treatment efficacy of long and short pulses. After two days post treatment, short pulse-treated tumors showed a significantly lower tumor volume compared with long pulse-treated tumors (n=8, p<0.05). On 8 experimental animals, a short nanosecond pulsed electric field (nsPEF) had lesser or delayed effects on GFP quenching and greater effects in reducing tumor size. Short pulses produced by nsPEFs can cause melanoma regression with less effect on the plasma membrane.

Subnanosecond electric pulses cause membrane permeabilization and cell death.

Subnanosecond electric pulses (200 ps) at electric field intensities on the order of 20 kV/cm cause the death of B16.F10 murine melanoma cells when applied for minutes with a pulse repetition rate of 10 kHz. The lethal effect of the ultrashort pulses is found to be caused by a combination of thermal effects and electrical effects. Studies on the cellular level show increased transport across the membrane at much lower exposure times or number of pulses. Exposed to 2000 pulses, NG108 cells exhibit an increase in membrane conductance, but only allow transmembrane currents to flow, if the medium is positively biased with respect to the cell interior. This means that the cell membrane behaves like a rectifying diode. This increase in membrane conductance is a nonthermal process, since the temperature rise due to the pulsing is negligible.

Targeted tissue ablation with nanosecond pulses.

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 three-dimensional finite element model allowed us to determine the threshold electric field for cell death. For 30, 100 nanosecond long pulses this was found to be in the range from 12 to 15 kV/cm. Modelling of the temperature distribution in the tissue using Pennes' bioheat equation showed that the lethal effect of nanosecond pulses on cells is non-thermal. Muscle contractions, generally caused by high voltage pulses, were significantly reduced for the 100 nanosecond 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.

Histopathological follow-up by tissue micro-array in a survival study after melanoma treated by nanosecond pulsed electric fields (nsPEF).

A recent study has shown that nanosecond pulsed electric fields (nsPEF) can affect the intracellular structures of melanoma within weeks. nsPEF is a non-drug, non-thermal treatment using ultrashort, intense pulsed electric fields with nanosecond durations. In the current study we followed up melanoma histopathology and metastasis with tissue micro-array 5 months post-nsPEF. After nsPEF treatment, tumor growth, tumor histology, metastasis, peri-tumor vessel and micro-vessel density were examined for the effect of nsPEF treatment on melanoma in vivo. The 17 nsPEF-treated mice were tumor-free for 169 days, significantly longer than those 19 control mice bearing melanoma without nsPEF. Histopathology follow-up showed that melanoma did not recur to the primary injection place after complete elimination. Compared with the control tumor, nsPEF-treated tumors present decreased micro-vessel density in a time-course manner in this survival study. Treatment with nsPEF caused continuous histopathological changes in melanomas, eliminated melanoma without recurrence at the primary site and prolonged animal survival time by inhibiting tumor blood supply and leading to tumor infarction. Thus, nsPEF could be applied in a non-ionizing therapeutic approach, without other agents, to locally treat tumors within a defined boundary.

Nanosecond pulsed electric field ablation of hepatocellular carcinoma.

Hepatocellular carcinoma often evades effective therapy and recurrences are frequent. Recently, nanosecond pulsed electric field (nsPEF) ablation using pulse power technology has emerged as a local-regional, non-thermal, and non-drug therapy for skin cancers. In the studies reported here we use nsPEFs to ablate murine, rat and human HCCs in vitro and an ectopic murine Hepa 1-6 HCC in vivo. Using pulses with 60 or 300 ns and electric fields as high as 60 kV/cm, murine Hepa 1-6, rat N1S1 and human HepG2 HCC are readily eliminated with changes in caspase-3 activity. Interestingly caspase activities increase in the mouse and human model and decrease in the rat model as electric field strengths are increased. In vivo, while sham treated control mice survived an average of 15 days after injection and before humane euthanasia, Hepa 1-6 tumors were eliminated for longer than 50 days with 3 treatments using one hundred pulses with 100 ns at 55 kV/cm. Survival was 40% in mice treated with 30 ns pulses at 55 kV/cm. This study demonstrates that nsPEF ablation is not limited to effectively treating skin cancers and provides a rationale for treating orthotopic hepatocellular carcinoma in pre-clinical applications and ultimately in clinical trials.

Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields.

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large DNA fragments, but not 180 bp fragmentation ladders, were observed, suggesting incomplete apoptosis. Nevertheless, tumor weight and volume decreased and tumors disappeared. One week after treatment, vessel numbers, vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor (PD-ECGF), CD31, CD35 and CD105 were decreased, indicating anti-angiogenesis. The nsPEFs activate multiple melanoma therapeutic targets, which is consistent with successes of nsPEF applications for tumor treatment in vivo as a new cancer therapeutic modality.

Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma.

Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.