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

Cell Responses Without Receptors and Ligands, Using Nanosecond Pulsed Electric Fields (nsPEFs)

Stephen J Beebe Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk VA, USAThe plasma membrane is a lipid bilayer that surrounds and shelters the living structural and metabolic systems within cells. That membrane is replete with transmembrane proteins with and without ligand binding sites, oligosaccharides, and glycolipids on the cell exterior. Information transfer across this structure is closely controlled to maintain homeostasis and regulate cell responses to external stimuli. The plasma membrane is contiguous with the endoplasmic reticulum (ER) and nuclear membranes. A number of proteins form ER–mitochondria junctions, allowing interorganelle communications, especially for calcium transport. Transport mechanisms across these membranes include nongated channels or pores; single-gated channels for ion transport; carrier molecules for facilitated diffusion; and pumps for active transport of ions and macromolecules. During the activation of these transport systems, "pores" are formed through protein structures that transiently connect the intracellular and extracellular milieu. These pores are nanoscale structures with diameters of 0.2−4.0 nm. However, there can also be maligned movements of molecules across the plasma membranes. Staphylococcus aureus protein α-toxin and Streptococcus pyogenes protein streptolysin O both create pores that allow unsolicited molecular transfer across membranes that disrupts vital functions. Cytotoxic T-cells permeabilize the invading cell membranes with perforin, creating pores through which granzymes can induce apoptosis. These pores have a lumen of 5–30 nm with the majority at 13–20 nm.

Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0-1000 V/cm for a selected duration in the range 10-1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible 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

Nano- and Micro-Second Electrical Pulsing of B16-F10 Mouse Melanoma Cells: Plasma Membrane and Sub-Cellular Organelle Changes

High electric field-treated cells are permeable to molecular dye through either opening of pores in the plasma membrane or other unknown processes which can disturb the membrane in an organized way. However, direct morphological evidence is lacking and responses of intracellular organelles are not clear. We used traditional chemical fixatives and biochemical techniques to capture cell membrane and organelle changes immediately after pulsing with high voltage electric field application. Different pulse durations, nanosecond (ns) and microsecond (µs), and field magnitudes, 60 kV/cm and 1.2 kV/cm, were applied to mouse melanoma B16-F10 cells. Two different ns durations (60 and 300 ns) with an electric field of 60 kV/cm and microsecond duration (100 µs) at 1.2 kV/cm were used in this study. Morphological changes on plasma membranes and cell organelles were analyzed with transmission electron microscopy (TEM) immediately after one to six applied pluses. TEM micrographs demonstrated morphological changes in plasma membrane and mitochondrial structure for treated cells under certain pulse conditions. Additionally, B16-F10 cells were: I) assessed post-pulse for membrane permeability and live/dead ratio using trypan blue; 2) monitored for mitochondrial membrane potential (Δψm) changes with JC-1, a voltage-sensitive mitochondrial dye; and 3) cultured for 24 hrs post-pulse to determine long-term viability. Detailed cellular responses were evaluated based on the different electric fields, pulse duration, and number of pulses. Cell membranes appeared to be unperturbed while mitochondrial membranes were negatively affected after the defined ns pulse treatments. Increasing the number of ns pulses introduced more mitochondrial abnormalities and led to decreased cellular viability. With fewer pulse numbers (1-2 pulses), mitochondrial morphology and Δψm were similar to controls. With µs pulse duration, intracellular organelles were less disturbed than the cell membranes. Under high electric field (60 kV/cm), changes in cell membrane permeability and irregularity increased, while cell viability and mitochondrial potential decrease, both with the longer duration (300 ns vs. 60 ns) and with higher pulse numbers under the same duration. The low electric field (1.2 kV/cm) caused fewer changes to the cell membrane and intracellular organelles even though the pulse durations (100 µs vs. 300 or 60 ns) were longer

Production of radiotracers for medical imaging using laser-acceleration techniques

The objective of the doctoral thesis is to study whether the acceleration of laser-induced particles can be a competitive option for the production of positron-emitting radiotracers. To do this, an experimental work is proposed using the laser of the Laboratorio Láser de Aceleración y Aplicaciones (L2A2) of the Universidad de Santiago de Compostela. This work will consist of putting into operation the necessary technology to accelerate a sufficient and continuous amount of protons or deuterons with energies of up to 10 MeV