Jody A. White

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.

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca(2+)](i) response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca(2+)](i) was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca(2+)](i) response was due to the release of Ca(2+) from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca(2+)](i) was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca(2+)](i) exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca(2+)](i) response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca(2+) release may be due to nanopore formation in the endoplasmic reticulum.

Compact, Nanosecond, High Repetition Rate, Pulse Generator for Bioelectric Studies

The high dielectric strength and high permittivity of water allow for its use for energy storage and switching in compact pulse power systems. A 10-Omega pulse generator with flowing water as dielectric and as the switching medium is presented here. It can provide a 10-ns pulse with a risetime of approximately 2 ns and an amplitude of up to 35 kV into a matched load. The system was operated in burst mode with repetition rates of up to 400 Hz, limited by the charging power supply. For a switch with two pin electrodes, strong electrode erosion limits the use of the pulser to less than 1,000 pulses before electrode readjustment is necessary. A considerable reduction of the erosion effect on breakdown voltage was obtained with coaxial electrodes. The pulse generator was used to study the effect of the repetition rate (or the time between successive pulses) on the viability of B16 murine melanoma cells.

Real-time imaging of the membrane cirarging of mamalian cells exposed to nanosecond pulsed electric fields

For real-time imaging of the transmembrane voltage of Jurkat cells, exposed to nanosecond pulsed electric fields, the cells were stained with a voltage sensitive membrane dye (VSD) and illuminated with a 4.8 ns long dye-laser pulse at various time during the electric field pulse. The stained cells were located in a 100 mum stainless steel electrode arrangement mounted at the stage of an inverted microscope. Due to the weak fluorescence response from the membrane an intensified CCD camera was used for image acquisition. The camera was operated in an open-shutter mode. ANNINE-6, a recently developed ultra-fast VSD, and Di-8-ANEPPS were used for transient membrane voltage monitoring. Best results could be achieved with the ANNINE-6 dye. First results indicate a clear response of the VSDs attached to the membrane in case of exposing the cells to a 60 ns long electric field pulse

Nanosecond pulsed electric fields mimic natural cell signal transduction mechanisms

Applications of nanosecond pulsed electric fields (nsPEF) to human cells and mammalian tissues indicate that, as the pulse durations and/or the electric field intensities decrease, effects on the plasma membrane decrease and effects in intracellular signal transduction mechanisms increase. NsPEFs that are below the threshold for electroporation-like effects on the plasma membrane mimic cell-signaling mechanisms that determine cell fate, depending on the nsPEF conditions and the cell type. At relatively high electric fields, cell-signaling mechanisms are activated to induce death by apoptosis in cells and tumors. At electric fields below the threshold for apoptosis, nsPEFs induce calcium release from intracellular stores that mimic physiologic ligand effects on IP3-dependent calcium channels in the endoplasmic reticulum and subsequent capacitative calcium influx activated by store operated calcium channels (SOCC) in plasma membranes. In human platelets, nsPEF-induced calcium mobilization mimics thrombin-induced platelet activation and aggregation, a natural mechanism to clot blood and heal wounds. Thus, nsPEFs recruit intracellular signaling mechanisms, providing a new technology to modulate cell function for potential therapeutic and/or diagnostic applications in the future

Measurements of the Transmembrane Voltage in Biological Cells for Nanosecond Pulsed Electric Field Exposures

We studied the charging of cell membranes in response to ultrashort pulsed high electric fields with a temporal resolution on the same order as the electrical pulse, i.e. nanoseconds. The real-time resolution was achieved by using a pulsed laser (5 ns) as light source, together with a novel voltage-sensitive dye (Annine-6). The laser pulse was synchronized with the pulsed electric field to enable snapshots at different times before, during and after exposure. Electric fields were provided by a 50-Omega Blumlein pulse generator connected to a microreactor installed on a microscope. When Jurkat cells are exposed to a 60 ns, 100 kV/cm pulse, we observed changes in the transmembrane potential of up to 1.6 V at 15-20 ns after the electric field was applied. Within several tens of nanoseconds after the pulse, the transmembrane voltage returns to resting potential values, indicating that in spite of the extremely high transient electric fields in the membrane (3.2MV/cm), the cell membrane is not permanently damaged.

From Submicrosecond to Subnanosecond Pulses - Entering a New Domain of Electric Field-Cell Interactions

Summary form only given. By reducing the duration of electrical pulses from microseconds into the nanosecond range, the electric field-cell interactions shift increasingly from the plasma (cell) membrane to subcellular structures. Yet another domain of pulsed electric field interactions with cell structures and functions opens when the pulse duration is reduced to values such that membrane charging becomes negligible, and direct electric field-molecule effects determine the biological mechanisms. For mammalian cells, this holds for a pulse duration of less than one nanosecond. In addition to entering a new domain of electric field-cell interactions, entering the subnanosecond temporal range will allow us to use near-field-focusing, wideband antennas, rather than needle or plate electrodes, to generate large pulsed electric fields with reasonable spatial resolution in tissue. Modeling results indicate that electric field intensities of tens (up to perhaps hundreds) of kV/cm with a spatial resolution of a few mm can be generated with prolate-spheroidal reflectors with TEM wave-launching structures, and using state-of-the-art pulsed power technology. In order to study the biological effect of subnanosecond pulses, we have developed a sub-ns pulse generator capable of delivering 250 kV into a high impedance load. The pulse width is approximately 600 ps with a voltage rise of up to 1 MV/ns. The pulses have been applied to B16 (murine melanoma) cells, and the plasma membrane integrity was studied by means of trypan blue exclusion. The results show that temporary nanopores in the plasma membrane are generated, allowing the uptake of drugs or nanoparticles without affecting the viability of the cells.

Cell Membrane Charging in Intense Nanosecond Pulsed Electric Fields

In order to study membrane charging in mammalian cells during exposure to pulsed electric fields of 60 ns duration we measured transmembrane voltage changes during and after exposure in real-time, i.e. with a resolution that is short compared to the duration of the administered electrical pulse. The applied electric field was varied between 5 kV/cm and 90 kV/cm. Under all conditions the voltage at the hyperpolarized pole of the cell is changing by more than 1 V during the first 5 ns of the exposure. A further hyperpolarization of the membrane of up to 1.6 V depends on the strength of the applied field. A change of at least 1.4 V at the anode will cause pores to open and allow ion exchange. Immediately after this maximum is reached, potential differences start to readjust. In principle, voltages at the depolarized pole follow the same pattern. However, the change is, in general lower by 1 V, limiting the depolarization to a maximum of 0.6 V

Dynamic effects and applications for nanosecond pulsed electric fields in cells and tissues

Nanosecond, high intensity pulsed electric fields [nsPEFs] that are below the plasma membrane [PM] charging time constant have decreasing effects on the PM and increasing effects on intracellular structures and functions as the pulse duration decreases. When human cell suspensions were exposed to nsPEFs where the electric fields were sufficiently intense [10-300ns, ≤300 kV/cm.], apoptosis signaling pathways could be activated in several cell models. Multiple apoptosis markers were observed in Jurkat, HL-60, 3T3L1-preadipocytes, and isolated rat adipocytes including decreased cell size and number, caspase activation, DNA fragmentation, and/or cytochrome c release into the cytoplasm. Phosphatidylserine externalization was observed as a biological response to nsPEFs in 3T3-L1 preadipocytes and p53-wildtype and -null human colon carcinoma cells. B10.2 mouse fibrosarcoma tumors that were exposed to nsPEFs ex vivo and in vivo exhibited DNA fragmentation, elevated caspase activity, and reduced size and weight compared to contralateral sham-treated control tumors. When nsPEF conditions were below thresholds for apoptosis and classical PM electroporation, non-apoptotic responses were observed similar to those initiated through PM purinergic receptors in HL-60 cells and thrombin in human platelets. These included Ca2+ mobilization from intracellular stores [endoplasmic reticulum] and subsequently through store-operated Ca2+ channels in the PM. In addition, platelet activation measured as aggregation responses were observed in human platelets. Finally, when nsPEF conditions followed classical electroporation-mediated transfection, the expression intensity and number of GFP-expressing cells were enhanced above cells exposed to electroporation conditions alone. These studies demonstrate that application of nsPEFs to cells or tissues can modulate cell-signaling mechanisms with possible applications as a new basic science tool, cancer treatment, wound healing, and gene therapy.