Stephen J. Beebe

Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition

Pulse power technology using high intensity (up to 300 kV/cm) nanosecond pulsed electric fields (nsPEF) has been applied for decontamination and amelioration of biofouling, but until now effects on human cells have not been investigated. To analyze structural and functional changes in human cells and solid tumors following exposure to nsPEF we utilized flow cytometry and immunofluorescence microscopy. We provide further support for the hypothesis that as the pulse duration is decreased, there is a lower incidence of electric field interactions at the plasma membrane and a higher incidence of interactions with intracellular structures. The nsPEF effects are pulse duration/electric field intensity-dependent and energy density- or temperature-independent. We also show that nsPEF induces programmed cell death (apoptosis) in cultured cells as indicated by cell shrinkage, Annexin-V-FITC binding to phosphatidylserine on intact cells, and caspase activation. Mouse fibrosarcoma tumors exposed to nsPEF exhibit fragmented DNA and reduced tumor growth in a mouse model. These studies show that nsPEF effects are distinctly different than electroporation pulses and provide the first evidence for the potential application of nsPEF to induce apoptosis and inhibit tumor growth.

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.

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.

The effect of pulsed electric fields on biological cells: experiments and applications

The effect of pulsed electric fields with amplitudes in the range of 100 V/cm-100 kV/cm on bacteria and aquatic nuisance species has been explored. The pulse duration was so short that heating of the biological matter could be neglected. The electrical energy required for lysing of bacteria, or stunning of aquatic species, decreases when the pulse duration is reduced. For lysing of Eschericia coli, this tendency has been proven to hold for pulsewidths as short as 60 ns. For macroorganisms, however, it was found that for pulsewidths of less than 5 /spl mu/s, the tendency is reversed: the energy required to affect the macroorganisms increases again. This minimum in energy, or maximum in efficiency, respectively, can be understood by taking the time required for electrical charging of the cell membrane into account. Applications of the pulsed electric field technique (PEFT) are in biofouling prevention, debacterialization of liquids, and in the field of medicine. A series of field tests on biofouling prevention in a cooling system with untreated water as coolant has demonstrated the economic feasibility of the electro-technology.

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.

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.

The Effect of Intense Subnanosecond Electrical Pulses on Biological Cells

Nanosecond electrical pulses have been successfully used to treat melanoma tumors by using needle arrays as pulse delivery systems. Reducing the pulse duration of intense electric field pulses from nanoseconds into the subnanosecond range will allow us to use wideband antennas to deliver the electromagnetic fields into tissue with a spatial resolution in the centimeter range. To explore the biological effect of intense subnanosecond pulses, we have developed a generator that provides voltage pulses of 160 kV amplitude, 200 ps rise time, and 800 ps pulse width. The pulses are delivered to a cylindrical Teflon chamber with polished flat electrodes at either end. The distance between the electrodes is variable and allows us to generate electric fields of up to 1 MV/cm in cell suspensions. The pulses have been applied to B16 (murine melanoma) cells, and the plasma membrane integrity was studied by means of trypan blue exclusion. For pulse amplitudes of 550 kV/cm, approximately 50% of the cells took up trypan blue right after pulsing, whereas only 20% were taking it up after 1 h. This indicates that the plasma membrane in a majority of the cells affected by the pulses recovers with a time constant of about 1 h. The cells that show trypan blue uptake after this time suffer cell death through apoptosis. Evaluation of the experimental results and molecular dynamics modeling results indicate that with a pulse duration of 800 ps, membrane charging and nanopore formation are the dominant bioelectric effects on B16 cells. This information has been used in a continuum model to estimate the increase in membrane permeability and, consequently, the increase in pore size caused by repetitive pulsing.

Nanosecond pulse electric field (nanopulse): A novel non-ligand agonist for platelet activation

Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca2+) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca2+ ion concentration ([Ca2+]i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300ns pulse with an electric field of 30kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7kV/cm with maximum effects seen with approximately 30kV/cm. The increases in intracellular Ca2+ release and Ca2+ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30kV/cm. The increases in [Ca2+]i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca2+ to leak out of intracellular Ca2+ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca2+ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca2+]i, however by a different mechanism.