Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by 2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA–protein complexes

Nanosecond Pulsed Electric Fields: A New Stimulus to Activate Intracellular Signaling

When new technologies are introduced into the sci-entific community, controversy is expected and both ex-citement and disappointment enrich the lives of those who initiate the new ideas. It becomes the mission of the “inventors ” to embrace the burden of proof to estab-lish their ideas and convince the skeptics and disbeliev-ers who will undoubtedly temper their enthusiasm and test their patience. While open mindedness is generally a scientific motto, those who review patents, manuscripts, and grants do not always readily practice it, even when the evidence is convincingly presented; old ideas and concepts often die hard. So it has been and still is in many instances as engineers, physicists, biologists, and physicians pursue innovative ideas and novel technolo-gies. So what is “Bioelectrics”? It is the application of ultra-short pulsed electric fields to biological cells, tissues, and organs. More specifically, it is the analysis of how these bi-ological systems respond to high electric fields (10–100 s of kV/cm) when applied with nanosecond (1–300) dura-tions. Compressing electrical energy by means of pulsed power techniques allows the generation of ultrashort (bil-lionth of a second) electrical pulses [1]. Because the pulses are so short the energy density is quite low and there-fore nonthermal. However, the power is extremely high generating billions of watts. This can be compared to a coal power plant, which generates less than billion watts, but does it continuously. For example, for a 10 ns, 40 kV, 10Ω pulse generator, the power provided by the pulse is 160MW, however, the energy is only 1.6 J. Depositing thi

Quantification of propidium iodide delivery with millisecond electric pulses: A model study

A model study of propidium iodide delivery with millisecond electric pulses is presented; this work is a companion of the experimental efforts by Sadik et al. [1]. Both membrane permeabilization and delivery are examined with respect to six extra-cellular conductivities. The transmembrane potential of the permeabilized regions exhibits a consistent value, which corresponds to a bifurcation point in the pore-radius-potential relation. Both the pore area density and membrane conductance increase with an increasing extra-cellular conductivity. On the other hand, the inverse correlation between propidium iodide delivery and extra-cellular conductivity as observed in the experiments is quantitatively captured by the model. This agreement confirms that this behavior is primarily mediated by electrophoretic transport during the pulse. The results suggest that electrophoresis is important even for the delivery of small molecules such as propidium iodide. The direct comparison between model prediction and experimental data presented in this work helps validate the former as a robust predictive tool for the study of electroporation

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected

Synergistic Effects of Nanosecond Pulsed Electric Fields Combined with Low Concentration of Gemcitabine on Human Oral Squamous Cell Carcinoma In Vitro

Treatment of cancer often involves uses of multiple therapeutic strategies with different mechanisms of action. In this study we investigated combinations of nanosecond pulsed electric fields (nsPEF) with low concentrations of gemcitabine on human oral cancer cells. Cells (Cal-27) were treated with pulse parameters (20 pulses, 100 ns in duration, intensities of 10, 30 and 60 kV/cm) and then cultured in medium with 0.01 mu g/ml gemcitabine. Proliferation, apoptosis/necrosis, invasion and morphology of those cells were examined using MTT, flow cytometry, clonogenics, transwell migration and TEM assay. Results show that combination treatments of gemcitabine and nsPEFs exhibited significant synergistic activities versus individual treatments for inhibiting oral cancer cell proliferation and inducing apoptosis and necrosis. However, there was no apparent synergism for cell invasion. By this we demonstrated synergistic inhibition of Cal-27 cells in vitro by nsPEFs and gemcitabine. Synergistic behavior indicates that these two treatments have different sites of action and combination treatment allows reduced doses of gemcitabine and lower nsPEF conditions, which may provide better treatment for patients than either treatment alone while reducing systemic toxicities

Nanopulse Generators: Their Design and Application to Cancer Therapy Studies

Effective nanopulse generators have become critical in recent decades concerning the study of subcellular affects in response to nanosecond pulsed electric fields. It has been observed that nanosecond duration electric pulses can target intracellular organelles, ultimately leading to cell apoptosis, suggesting the possibility of a new, minimally invasive, low risk cancer therapy methodology. The standard topology for developing a medical nanopulser is the Blumlein “transmission line” approach. This approach relies on the nearly infinitesimal, yet finite amount of time required for an electromagnetic field to propagate down a short transmission line. Prior to design, requirements and constraints must be defined that are determined by the specific applications and experiments that the nanopulser will be used for. Special effort must be put into nanopulser design to prevent undesirable reflections and oscillations at the load. Critical design objectives common to most nanopulse generators include choosing effective switching elements that facilitate a minimal rise time, configuring the load electrodes to be compatible with experimental setups, and enabling a wide degree of versatility and adjustability concerning pulse parameters

Plasma Membrane Temperature Gradients and Multiple Cell Permeabilization Induced by Low Peak Power Density Femtosecond Lasers

Calculations indicate that selectively heating the extracellular media induces membrane temperature gradients that combine with electric fields and a temperature-induced reduction in the electro- permeabilization threshold to potentially facilitate exogenous molecular delivery. Experiments by a wide-field, pulsed femtosecond laser with peak power density far below typical single cell optical de- livery systems confirmed this hypothesis. Operating this laser in continuous wave mode at the same average power permeabilized many fewer cells, suggesting that bulk heating alone is insufficient and temperature gradients are crucial for permeabilization. This work suggests promising opportunities for a high throughput, low cost, contactless method for laser mediated exogenous molecule delivery without the complex optics of typical single cell optoinjection, for potential integration into microscope imaging and microfluidic systems