Richard Nuccitelli

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.

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.

Nanosecond Pulsed Electric Fields Cause Melanomas to Self-Destruct

Nanosecond pulsed electric fields (nsPEF) have been shown to penetrate into living cells to permeabilize intracellular organelles and release Ca2+ from the endoplasmic reticulum. They provide a new approach for physically targeting intracellular organelles with many applications, including initiation of apoptosis, enhancement of gene transfection efficiency and inhibiting tumor growth. We have been working with the murine melanoma model system and here we show that 40 kV/cm electric field pulses 300 nanoseconds in duration can rapidly stimulate pyknosis, reduce blood flow and fragment DNA in murine melanoma tumors in vivo with a total field exposure time of 1.8 microseconds. Three treatments of 100 pulses each results in a mean tumor size regression of 90% within two weeks. Another round of treatments at this time can completely eliminate the melanoma. This new therapy is the first to simultaneously trigger pyknosis and reduce tumor blood flow.

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana–Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

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.

Plasma Membrane Charging of Jurkat Cells by Nanosecond Pulsed Electric Fields

With the application of pulsed electric fields of only nanosecond duration but field strengths of several megavolts per centimeter, apoptosis can be induced in tumor cells. The detailed mechanisms of this process are not yet completely understood. The accumulation of charges along the membranes in the applied electric field is likely the primary trigger. 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 timescale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to a nanosecond pulsed electric fields (nsPEF) of 60 ns and amplitudes from 5 to 90 kV/cm in real time, i.e. with a temporal resolution of 5 ns* After an initial sudden increase to 1 V, the potential differences at the anodic pole continue to rise at a more moderate rate to ~1.6 V for applied field strengths equal to, or greater than, 50 kV/cm. The subsequent drop in voltage even while the electric field is still applied suggest the formation of pores.