Electroporation is considered a nonthermal process, and when used in medical therapies great care is taken to ensure that temperature effects due to Joule heating are avoided by limiting the average electrical power of the electroporation pulse train. However, moderate temperature increases, far below those which are used in hyperthermia therapies, have shown to increase safety and efficacy of electroporation-based therapies. This chapter provides an introduction into the heating mechanisms by applied electrical pulses, Joule heating and heating due to dielectric relaxion, followed by a short introduction of external heat sources, with emphasis on infrared light sources. The second part describes basic thermal effects on cells, particularly on cell membranes. This is followed by an overview of recent in vitro and in vivo studies on the effects of thermal assistance in electrotherapies with emphasis on its application in cancer treatments. Moderate heating of tumors in combination with electrical pulse treatments was found, for a wide range of pulse durations, to cause a significantly higher rate of complete tumor regression compared to the use of pulsed electric fields alone.

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.

Efficient gene delivery and expression in the skin can be a promising minimally invasive technique for therapeutic clinical applications for immunotherapy, vaccinations, wound healing, cancer, and peripheral artery disease. One of the challenges for efficient gene electrotransfer (GET) to skin in vivo is confinement of expression to the epithelium. Another challenge involves tissue damage. Optimizing gene expression profiles, while minimizing tissue damage are necessary for therapeutic applications. Previously, we established that heating pretreatment to 43 °C enhances GET in vitro. We observed a similar trend in vivo, with an IR-pretreatment for skin heating prior to GET. Currently, we tested a range of GET conditions in vivo in guinea pigs with and without preheating the skin to ~43 °C. IR-laser heating and conduction heating were tested in conjunction with GET. In vivo electrotransfer to the skin by moderately elevating tissue temperature can lead to enhanced gene expression, as well as achieve gene transfer in epidermal, dermal, hypodermal and muscle tissue layers.

Electric pulse based technology has been developed and studied as a non-thermal ablation method for local control of pancreatic cancer. Irreversible electroporation (IRE) has shown a significant survival benefit for local advanced pancreatic cancer in clinical trials. However, incomplete ablation with local recurrence and major complications limit the potential of this new technology. We have developed an integrated moderate heating electric pulse delivery system which consists of controllable tumor heating, multi-parameter monitoring and electric pulse delivery. The impedance of tumor is greatly decreased after moderate heating at 42°C for 1–2 min, which does not cause any cell death. Moderate heating significantly enlarges the ablation zone of tumor treated with IRE. In contrast to IRE alone, moderate heating assisted IRE results in a high rate of complete tumor regression and a significant longer median survival. Another electric pulse technology, nanosecond electric pulses, has been assessed for the treatment of pancreatic cancer as well. Nanosecond electric pulse treatment achieves more survival benefit in animals with partial tumor ablation than those treated with IRE and leads to a vaccine-like protective effect in animals with complete local ablation. More studies are needed to demonstrate the advantages and translational feasibility of the enhanced electric pulse technologies.

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.

Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times larger than in cells exposed to nanosecond pulse stimulation at room temperature. Application of moderate heating alone did not cause cell death. A nanosecond pulse stimulation electrode with integrated controllable laser heating was developed to treat murine ectopic squamous cell carcinoma. With this innovative system, we were able to quickly heat and maintain the temperature of the target tumor at 43°C during nanosecond pulse stimulation. Nanosecond pulse stimulation with moderate heating was shown to significantly extend overall survival, delay tumor growth, and achieve a high rate of complete tumor regression. Moderate heating extended survival nearly 3-fold where median overall survival was 22 days for 9.8 kV without moderate heating and over 63 days for tumors pulsed with 600, 100 ns pulses at 5 Hz, at voltage of 9.8 kV with moderate heating. Median overall survival in the control groups was 24 and 31 days for mice with untreated tumors and tumors receiving moderate heat alone, respectively. Nearly 69% (11 of 16) of tumor-bearing mice treated with nanosecond pulse stimulation with moderate heating were tumor free at the completion of the study, whereas complete tumor regression was not observed in the control groups and in 9.8 kV without moderate heating. These results suggest moderate heating can reduce the necessary applied voltage for tumor ablation with nanosecond pulse stimulation.

A dielectric rod antenna was constructed for stimulating biological tissues. The antenna consists of three sections (a conical wave launcher, a cylindrical waveguide, and a conical emitting section). The simulation of the antenna in free space was confirmed with measurements of the return loss (S11) and the transient electric field. To reach the electric field threshold for tissue stimulation, a high voltage pulsed power system was designed and constructed to drive the antenna. Central to the pulsed power system is a transformer and an oil peaking switch. The transformer is an ultrafast, dual resonance pulse transformer with a linearly integrated primary (LIP) stage. It is capable of generating pulses with rise time < 50 ns. The load for the transformer was an oil-filled transmission line that was connected to the antenna. An oil peaking switch was used to switch the line to produce high voltage picosecond transients. The electric field was measured in the near field at a distance of 4 cm. A 500 ps pulse with an amplitude of 600 V/cm was recorded for a 25 kV input from the pulse transformer. These results demonstrate the feasibility of the system and so future work will be focused on increasing the voltage and the electric field in order to stimulate biological tissues.

Nanosecond pulsed non-thermal atmospheric-pressure plasmas are promising for numerous applications including air and water purification, ozone synthesis, surface sterilization, material processing, and biomedical care. However, the high cost of the nanosecond pulsed power sources has hindered adaptation of the plasma-based technologies for clinical and industrial use. This paper presents a low cost (<100US$) nanosecond pulsed plasma system that consists of a Cockcroft–Walton high voltage charging circuit, a compact nanosecond pulse generator using a spark gap as switch, and a plasma reactor. The nanosecond pulse power source requires only a 12 V DC input, hence is battery operable. Through the optimization of the experimental parameters, pulses with a peak voltage >10 kV, a 3 ns rise time (10 to 90 %), and a 10 ns pulse duration (full width at half maximum) at a pulse repetition rate of up to 500 Hz were achieved in the present study. It has been successfully tested to power three different plasma reactors to form pulsed corona discharges, dielectric barrier discharges, and sliding discharges. The energy efficiency of such a nanosecond pulsed sliding discharge system was assessed in the context of ozone synthesis using air or oxygen as the feed gas, and was found comparable to a previously reported non-thermal plasma system that used commercial high voltage pulsed power sources. This study demonstrated that this low-cost nanosecond pulsed power source can prove to be an energy efficient and simple supply to drive various non-thermal atmospheric-pressure plasma reactors for environmental, medical and other applications.

Irreversible electroporation (IRE) as a non-thermal tumor ablation technology has been studied for the treatment of pancreatic carcinoma and has shown a significant survival benefit. We discovered that moderate heating (MH) at 43 °C for 1-2 minutes significantly enhanced ex vivo IRE tumor ablation of Pan02 cells by 5.67-fold at 750 V/cm and by 1.67-fold at 1500 V/cm. This amount of heating alone did not cause cell death. An integrated IRE system with controllable laser heating and tumor impedance monitoring was developed to treat mouse ectopic pancreatic cancer. With this novel IRE system, we were able to heat and maintain the temperature of a targeted tumor area at 42 °C during IRE treatment. Pre-heating the tumor greatly reduced the impedance of tumor and its fluctuation. Most importantly, MHIRE has been demonstrated to significantly extend median survival and achieve a high rate of complete tumor regression. Median survival was 43, 46 and 84 days, for control, IRE with 100 μs, 1 Hz, 90 pulses and electric fields 2000–2500 V/cm and MHIRE treatment respectively. 55.6% of tumor-bearing mice treated with MHIRE were tumor-free, whereas complete tumor regression was not observed in the control and IRE treatment groups.

The field of microplasmas gained recognition as a well-defined area of research and application within the larger field of plasma science and technology about 20 years ago. Since then, the activity in microplasma research and applications has continuously increased. A survey of peer reviewed papers on microplasmas published annually shows a steady increase from fewer than 20 papers in 1995 to about 75 in 2005 and more than 150 in 2014. This count excludes papers that deal exclusively with technological applications where the microplasma is used solely as a tool. This topical review aims to provide a snap shot of the current state of microplasma research and applications. Given the rapid proliferation of microplasma applications, the topical review will focus primarily on the status of microplasma science and our understanding of the physics principles that enable microplasma operation. Where appropriate, we will also address microplasma applications, however, we will limit the discussion of microplasma applications to examples where the application is closely tied to the plasma science. No attempt is made to provide a comprehensive and in-depth review of the diverse range of all microplasma applications, except for the inclusion of a few key references to recent reviews of microplasma applications.

Modeling results and an increasing number of experimental results indicate that pulses with durations on the order or less than the charging time constant of the plasma membrane (which is on the order of 100 ns for cells in solution) affect the plasma membrane in a different way than classical electroporation pulses. Such nanosecond Pulsed Electric Fields (nsPEF) have been shown to also target subcellular structures and possibly proteins. When the pulse duration is shortened into the picosecond range (psPEF) the dielectric, rather than the resistive, properties of the outer medium and cytoplasm begin to determine the coupling between electric field and cell membranes. Besides entering a new field of electric field - cell interactions, the use of picosecond pulses allows the application of wideband antennas, rather than invasive electrodes, for the delivery of psPEF to 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 a 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 ~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 ~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 a biological tissue.

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.

Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expression and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer.

The energy deposition and the ozone generation in a shielded sliding discharge were compared with those of a simple surface discharge and a pulsed corona discharge in atmospheric pressure air and oxygen. All discharges were generated by applying 160 ns, high voltage pulses. Under the same conditions, the highest energy deposition per pulse was obtained with the shielded sliding discharge. The energy deposition was lower by a factor of four for the surface discharge plasma and by an order of magnitude for the pulsed corona discharge plasma. Replacing air with the more electronegative oxygen caused a decrease in the deposited energy due to electron attachment. The threshold voltage for plasma formation in oxygen in a shielded sliding discharge was approximately 5 kV, three times less than that of the surface discharge (≥15 kV) and four times less than that of the pulsed corona discharge (≥20 kV). A new finding of this study is that, whereas the decrease in energy in the pulsed corona discharge was ≥50 %, and that of the simple surface discharge ≥40 %, it was negligible in the shielded sliding discharge. Since the ozone generation scales with energy density, the results show that plasma reactors based on the nanosecond sliding discharge principle have major advantages in compactness, ignition voltage, and in the use of oxygen, rather than air, compared to surface discharges and corona discharges.

Effects of temperature on nitric oxide (NO) production and on byproduct nitrogen dioxide (NO2) and ozone (O-3) in a non-equilibrium sliding discharge in atmospheric pressure air are described. Heating the electrodes! dielectric surfaces in contact with the discharge plasma from 20 degrees C to 420 degrees C caused an increase in energy per pulse by more than an order of magnitude. More importantly, heating to a few hundred degrees above room temperature caused the destruction of ozone (O-3) and the reduction of nitrogen dioxide (NO2) but promoted NO formation. At 20 degrees C, 400-4500 ppm O-3 with an energy cost of 2-4 MJ/mol, and 12-180 ppm NO2 with an energy cost of 75-140 MJ/mol were generated from dry air flowing at 1 lpm. At 220 degrees C, however, NO and NO2 were produced in about equal proportion while O-3 was below detection limits. The efficiency of NO generation increased with an increase of temperature. At the maximum temperature used in this study, i.e., 420 degrees C, 160-1040 ppm NO was produced from dry air at an energy cost of 24-67 MJ/mol, with a NO/NO2 ratio of more than 4 and free of O-3. (C) 2015 Elsevier B.V. All rights reserved.

Experiments with CHO cells exposed to 60 and 300ns 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 10μs.