Karl H. Schoenbach

Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells

Electroporation by using pulsed electric fields with long durations compared with the charging time of the plasma membrane can induce cell fusion or introduce xenomolecules into cells. Nanosecond pulse power technology generates pulses with high‐intensity electric fields, but with such short durations that the charging time of the plasma membrane is not reached, but intracellular membranes are affected. To determine more specifically their effects on cell structure and function, human cells were exposed to high intensity (up to 300 kV/cm) nanosecond (10–300 ns) pulsed electric fields (nsPEF) and were analyzed at the cellular and molecular levels. As the pulse duration decreased, plasma membrane electroporation decreased and appearances of apoptosis markers were delayed. NsPEF induced apoptosis within tens of minutes, depending on the pulse duration. Annexin‐V binding, caspase activation, decreased forward light scatter, and cytochrome c release into the cytoplasm were coincident. Apoptosis was caspase‐ and mitochondria‐dependent but independent of plasma membrane electroporation and thermal changes. The results suggest that with decreasing pulse durations, nsPEF modulate cell signaling from the plasma membrane to intracellular structures and functions. NsPEF technology provides a unique, high‐power, energy‐independent tool to recruit plasma membrane and/or intracellular signaling mechanisms that can delete aberrant cells by apoptosis.

Ultrashort electrical pulses open a new gateway into biological cells

An electrical model for biological cells predicts that for pulses with durations shorter than the charging time of the outer membrane, there is an increasing probability of electric field interactions with intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300-kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, and enhanced expression of genes. At increased electric fields, the application of submicrosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffect are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors.

Cold atmospheric pressure air plasma jet for medical applications

By flowing atmospheric pressure air through a direct current powered microhollow cathode discharge, we were able to generate a 2cm long plasma jet. With increasing flow rate, the flow becomes turbulent and temperatures of the jet are reduced to values close to room temperature. Utilizing the jet, yeast grown on agar can be eradicated with a treatment of only a few seconds. Conversely, animal studies show no skin damage even with exposures ten times longer than needed for pathogen extermination. This cold plasma jet provides an effective mode of treatment for yeast infections of the skin.

The Effects of Intense Submicrosecond Electrical Pulses on Cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

Bioelectric Effects of Intense Nanosecond Pulses

Electrical models for biological cells predict that reducing the duration of applied electrical pulses to values below the charging time of the outer cell membrane (which is on the order of 100 ns for mammalian cells) causes a strong increase in the probability of electric field interactions with intracellular structures due to displacement currents. For electric field amplitudes exceeding MV/m, such pulses are also expected to allow access to the cell interior through conduction currents flowing through the permeabilized plasma membrane. In both cases, limiting the duration of the electrical pulses to nanoseconds ensures only nonthermal interactions of the electric field with subcellular structures. This intracellular access allows the manipulation of cell functions. Experimental studies, in which human cells were exposed to pulsed electric fields of up to 300 kV/cm amplitude with durations as short as 3 ns, have confirmed this hypothesis and have shown that it is possible to selectively alter the behavior and/or survival of cells. Observed nanosecond pulsed effects at moderate electric fields include intracellular release of calcium and enhanced gene expression, which could have long term implications on cell behavior and function. At increased electric fields, the application of nanosecond pulses induces a type of programmed cell death, apoptosis, in biological cells. Cell survival studies with 10 ns pulses have shown that the viability of the cells scales inversely with the electrical energy density, which is similar to the dose effect caused by ionizing radiation. On the other hand, there is experimental evidence that, for pulses of varying durations, the onset of a range of observed biological effects is determined by the electrical charge that is transferred to the cell membrane during pulsing. This leads to an empirical similarity law for nanosecond pulse effects, with the product of electric field intensity, pulse duration, and the square root of the number of pulses as the similarity parameter. The similarity law allows one not only to predict cell viability based on pulse parameters, but has also been shown to be applicable for inducing platelet aggregation, an effect which is triggered by internal calcium release. Applications for nanosecond pulse effects cover a wide range: from a rather simple use as preventing biofouling in cooling water systems, to advanced medical applications, such as gene therapy and tumor treatment. Results of this continuing research are leading to the development of wound healing and skin cancer treatments, which are discussed in some detail.

Diverse Effects of Nanosecond Pulsed Electric Fields on Cells and Tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane

Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is approximately 1nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable conventional pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms

These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 μsec. The application of 300 high‐voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4‐month period after the initial melanoma had disappeared. These pulses generate small, long‐lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to small molecules and ions, as well as an increase in intracellular Ca2+, DNA fragmentation, disruption of the tumors blood supply and the initiation of apoptosis. Apoptosis was indicated by a 3‐fold increase in Bad labeling and a 72% decrease in Bcl‐2 labeling. In addition, microvessel density within the treated tumors fell by 93%. This new therapy utilizing nanosecond pulsed electric fields has the advantages of highly localized targeting of tumor cells and a total exposure time of only 180 μsec. These pulses penetrate into the interior of every tumor cell and initiate DNA fragmentation and apoptosis while at the same time reducing blood flow to the tumor. This new physical tumor therapy is drug free, highly localized, uses low energy, has no significant side effects and results in very little scarring.

Streamers in water and other dielectric liquids

Experimental results on the inception and propagation of streamers in water generated under the application of high electric fields are reviewed. Characteristic parameters, such as breakdown voltage, polarity of the applied voltage, propagation velocities and other phenomenological features, are compared with similar phenomena in other dielectric liquids and in gases. Consequently, parameters that are expected to influence the development of streamers in water are discussed with respect to the analogous well-established models and theories for the related mechanisms in gases. Most of the data support the notion that an initial low-density nucleation site or gas-filled bubble assists the initiation of a streamer. Details of this theory are laid out explaining the observed differences in the breakdown originating from the anode versus the cathode locations. The mechanisms can also be applied to streamer propagation, although some observations cannot be satisfactorily explained.