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Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10–50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca2+ mobilization from Ca2+-storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3–4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr), in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at < 4 µM), and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60 ns, 13.2 kV/cm, 10 Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3 min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for the contact-forming and -breaking activity of the lipids in the bilayer. Our novel analysis of lipid interaction networks under periodic boundary conditions shows that the disruption of the bilayer, as measured by the breaking activity, is associated with the externally imposed pore formation. Moreover, the breaking activity can be used for statistically ranking the importance of individual lipids and solvent molecules through a bridging between fast and slow degrees of freedom. The heat map approach highlighted a small number of important lipids and solvent molecules, which allowed us to efficiently search the trajectories for any functionally relevant mechanisms. Our algorithms are freely disseminated with the open-source package TimeScapes.

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated transport and with molecular simulations of transport across electropores in a phospholipid bilayer. The results challenge the “drift and diffusion through a pore” model that dominates conventional explanatory schemes for the electroporative transfer of small molecules into cells and point to the necessity for a more complex model.

Pulsed electric fields are used to permeabilize cell membranes in biotechnology and the clinic. Although molecular and continuum models provide compelling representations of the mechanisms underlying this phenomenon, a clear structural link between the biomolecular transformations displayed in molecular dynamics (MD) simulations and the micro- and macroscale cellular responses observed in the laboratory has not been established. In this paper, plasma membrane electropermeabilization is characterized by exposing Jurkat T lymphoblasts to pulsed electric fields less than 10 ns long (including single pulse exposures), and by monitoring the resulting osmotically driven cell swelling as a function of pulse number and pulse repetition rate. In this way, we reduce the complexity of the experimental system and lay a foundation for gauging the correspondence between measured and simulated values for water and ion transport through electropermeabilized membranes. We find that a single 10 MV/m pulse of 5 ns duration produces measurable swelling of Jurkat T lymphoblasts in growth medium, and we estimate from the swelling kinetics the ion and water flux that follows the electropermeabilization of the membrane. From these observations we set boundaries on the net conductance of the permeabilized membrane, and we show how this is consistent with model predictions for the conductance and areal density of nanoelectropulse-induced lipid nanopores.

It is widely accepted that electroporation occurs when the cell transmembrane voltage induced by an external applied electric field reaches a threshold. Under this assumption, in order to trigger electroporation in a spherical cell, Schwan’s equation leads to an inversely proportional relationship between the cell radius and the minimum magnitude of the applied electric field. And, indeed, several publications report experimental evidences of an inverse relationship between the cell size and the field required to achieve electroporation. However, this dependence is not always observed or is not as steep as predicted by Schwan’s equation. The present numerical study attempts to explain these observations that do not fit Schwan’s equation on the basis of the interplay between cell membrane conductivity, permeability, and transmembrane voltage. For that, a single cell in suspension was modeled and the electric field necessary to achieve electroporation with a single pulse was determined according to two effectiveness criteria: a specific permeabilization level, understood as the relative area occupied by the pores during the pulse, and a final intracellular concentration of a molecule due to uptake by diffusion after the pulse, during membrane resealing. The results indicate that plausible model parameters can lead to divergent dependencies of the electric field threshold on the cell radius. These divergent dependencies were obtained through both criteria and using two different permeabilization models. This suggests that the interplay between cell membrane conductivity, permeability, and transmembrane voltage might be the cause of results which are noncompatible with the Schwan’s equation model. This work was supported by the Ministry of Economy and Competitiveness of Spain through Grant TEC2014-52383-C3-2-R. PTV received support from the Old Dominion University Frank Reidy Research Center for Bioelectrics and the Air Force Office of Scientific Research (FA9550-15-1-0517, FA9550-14-1-0123).

A novel coaxial tubular device capable of generating a 2.5 cm long pencil-like plasma plume in ambient atmosphere has recently been developed to disinfect root canal systems during endodontic treatment. Powered with short (≈100 ns), intense (6 kV) electric pulses at 1 kHz, the plasma dental probe is safe for operation, electromagnetic noise-free, with low power consumption (an average power of ≈1 W) and minimal heating of materials under treatment. It thus has the essential features required for oral and dental disinfection. In this communication, we present the design of the device and evidence that the plasma dental probe is effective for tooth surface disinfection. Scanning electron microscopy shows complete destruction of endodontic biofilms for a depth of 1 mm inside a root canal after plasma treatment for 5 min. Plasma emission spectroscopy identifies atomic oxygen as one of the likely active agents for the bactericidal effect.

Electrically excitable bovine adrenal chromaffin cells were exposed to nanosecond duration electric pulses at field intensities ranging from 2 MV/m to 8 MV/m and intracellular calcium levels ([Ca(2+)](i)) monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca(2+)](i), with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca(2+)](i) could be triggered. The pulse-induced rise in [Ca(2+)](i) was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co(2+), or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca(2+)](i) that involves entry of calcium via L-type calcium channels.

Pulsed electric fields can induce transport of normally impermeant ions and small molecules across the cell membrane, a process called electroporation or electropermeabilization. This phenomenon is often studied by using microscopic imaging to monitor changes in the intracellular concentration of fluorescent dyes. Most of these studies, however, report their findings in relative or arbitrary units. To produce truly quantitative measurements of electroporative transport we employ calibrated confocal microscopy to track YO-PRO-1 entry into cells after a single 6 ns, 20 MV/m pulse, and we correlate these observations with molecular dynamics simulations of YO-PRO-1 transport through lipid electropores. These baseline studies have been extended to quantitative observations of transport of calcein (an anionic fluorescent small molecule) and propidium (a cationic dye like YO-PRO-1 that is fluorescent only after binding to polynucleotides). Our results challenge the common assumption that small molecule transport through electropermeabilized membranes is dominated by simple diffusion through electropores. Ongoing experimental studies into the potential role of the post-pulse transmembrane voltage in the migration of charged small molecules across permeabilized cell membranes will also be discussed.

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Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10 μ m), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative “long” and “short” pulses. The long pulse—1.5 kV/cm, 100 μ s—evolves two pore subpopulations with a valley at ∼ 5 nm, which separates the subpopulations that have peaks at ∼ 1.5 and ∼ 12 nm radius. Such pulses are widely used in biological research, biotechnology, and medicine, including cancer therapy by drug delivery and nonthermal physical tumor ablation by causing necrosis. The short pulse—40 kV/cm, 10 ns—creates 80-fold more pores, all small ( < 3 nm; ∼ 1 nm peak). These nanosecond pulses ablate tumors by apoptosis. We demonstrate the model’s responses by illustrative electrical and poration behavior, and transport of calcein and propidium. We then identify extensions for expanding modeling capability. Structure-function results from MD can allow extrapolations that bring response specificity to cell membranes based on their lipid composition. After a pulse, changes in pore energy landscape can be included over seconds to minutes, by mechanisms such as cell swelling and pulse-induced chemical reactions that slowly alter pore behavior., Harvard University--MIT Division of Health Sciences and Technology (Fellowship), National Science Foundation (U.S.) (Fellowship), National Institutes of Health (U.S.) (NIH grant GM063857), United States. Air Force Office of Scientific Research, University of Southern California. Center for High Performance Computing and Communications

High intensity, nanosecond duration electric pulses (NEPs) permeabilize plasma membranes causing osmotic cell swelling that can elicit a wide variety of cellular effects. This study examined the possibility that cell swelling is the mechanism by which 5 ns NEPs trigger the release of catecholamines from neuroendocrine adrenal chromaffin cells. Swelling was assessed by comparing measurements of cell area obtained from bright field images of the cells before and at 10 s intervals following exposure of the cells to 5 ns pulses at a field intensity of 5–6 MV/m. The results indicated that chromaffin cells do not swell in response to a single pulse or a train of ten pulses delivered at repetition frequencies of 10 Hz and 1 kHz. Swelling was also not observed in response to a train of 50 pulses whereas Jurkat T lymphoblast cell area increased 15% on average under the same NEP exposure conditions. These results demonstrating that chromaffin cells do not undergo swelling when exposed to 5 ns NEPs have important implications regarding the mechanism by which these pulses stimulate the release of catecholamines from these cells, namely that catecholamine secretion is most likely not caused by cell swelling.

Electroporation is the formation of permeabilizing structures in the cell membrane under the influence of an externally imposed electric field. The resulting increased permeability of the membrane enables a wide range of biological applications, including the delivery of normally excluded substances into cells. While electroporation is used extensively in biology, biotechnology, and medicine, its molecular mechanism is not well understood. This lack of knowledge limits the ability to control and fine-tune the process. In this article we propose a novel molecular mechanism for the electroporation of a lipid bilayer based on energetics analysis. Using molecular dynamics simulations we demonstrate that pore formation is driven by the reorganization of the interfacial water molecules. Our energetics analysis and comparisons of simulations with and without the lipid bilayer show that the process of poration is driven by field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces into more energetically favorable configurations, with their molecular dipoles oriented in the external field. Although the contributing role of water in electroporation has been noted previously, here we propose that interfacial water molecules are the main players in the process, its initiators and drivers. The role of the lipid layer, to a first-order approximation, is then reduced to a relatively passive barrier. This new view of electroporation simplifies the study of the problem, and opens up new opportunities in both theoretical modeling of the process and experimental research to better control or to use it in new, innovative ways.

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7-30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.

Electropermeabilization (electroporation) occurs when a supraphysiological electric field is applied across the cell membrane, but this phenomenon is not yet accessible to direct experimental observation. Molecular dynamics simulations suggest that interfacial waters located at the water:lipid interface are key determinants of electric field-driven pore formation in phospholipid bilayers [1]. We compared poration dynamics in water:vacuum:water and water:lipid:water systems at varying external electric fields. Both systems form water columns bridging the vacuum or lipid-filled gaps that are similar in structure and energetic signature. We identify these columns as electropores and study their structure and dynamics at the emerging and mature stages in their evolution. The electropores in both systems appear to originate from structurally similar water intrusions. We report pore formation times for each system as a function of electric field, and dipole-dipole interaction energies between interfacial water molecules.[1] Tieleman, D.P., The molecular basis of electroporation. BMC Biochemistry, 5:10, 2004.

When a cell is exposed to a sufficiently large electric field, even for a very short time, its plasma membrane is changed so that molecules that normally cross the membrane only in minute amounts can more readily pass through the barrier. This phenomenon was first described by Neumann and Rosenheck (1972) in this journal. A decade later gene transfer was achieved by means of electroporation (Neumann et al. 1982). This technique is now widely used in laboratories around the world and is gaining even more attention in recent years as a method for introducing foreign genes into cells in vivo (Mir 2009), with good prospects for use in a clinical setting (Daud et al. 2008). Electroporation has been used in the last decade also for improving cancer drug delivery to cells. Preclinical investigations in the late 1980s (Orlowski et al. 1988; Mir et al. 1991) were followed by the first clinical trial in 1991 (Mir et al. 1991; Belehradek et al. 1993). Electrochemotherapy as an effective and safe local treatment for cutaneous and subcutaneous tumors (Marty et al. 2006; Sersa and Miklavcic 2008) is now accepted in a number of countries, is routinely employed in more than 60 cancer treatment centers and is being further developed for treating more deep-seated tumors (Miklavcic et al. 2010). Electroporation can be used in all kinds of isolated cells as well as in tissues. The electric field to which one exposes the target cell has to be of sufficient strength and the exposure of sufficient duration. The magnitude of the electric field to be used depends on cell type, size, orientation and density; pulse duration; and number of pulses. The selection of pulse parameters is influenced also by the size and type of molecule to be internalized. Depending on the location and size of the targeted tissue, electric pulses will be delivered via appropriate electrodes chosen among a number of different types. Geometry and positioning of electrodes affect electric field distribution, which is important for effective in vivo electropermeabilization (Miklavcic et al. 1998, 2000). Strategies for the treatment of malignancies based on direct effects of pulsed electric fields on cells, independent of the induced influx of pharmacological or genetic material, are also under development. These include tumor ablation by irreversible electroporation (Al-Sakere et al. 2007) and by the application of intense (MV/m), nanosecond-duration pulses, which induce apoptosis and destruction of the tumor vascularization (Nuccitelli et al. 2006). This special issue of the Journal of Membrane Biology features selected peer-reviewed reports from investigators currently active in electroporation-related research who attended the Fourth International Scientific Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments in Ljubljana, Slovenia, in November 2009. This unique working conference at the University of Ljubljana has been organized every 2 years since the first one in 2003. More than 200 participants from 21 countries D. Miklavcic Faculty of Electrical Engineering, University of Ljubljana, Tržaska 25, 1000 Ljubljana, Slovenia e-mail: damijan.miklavcic@fe.uni-lj.si

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.

In unexcitable, noncardiac cells, ultrashort (nanosecond) high-voltage (megavolt-per-meter) pulsed electrical fields (nsPEF) can mobilize intracellular Ca2+ and create transient nanopores in the plasmalemma. We studied Ca2+ responses to nsPEF in cardiac cells. Fluorescent Ca2+ or voltage signals were recorded from isolated adult rat ventricular myocytes deposited in an electrode microchamber and stimulated with conventional pulses (CPs; 0.5–2.4 kV/cm, 1 ms) or nsPEF (10–80 kV/cm, 4 ns). nsPEF induced Ca2+ transients in 68/104 cells. Repeating nsPEF increased the likelihood of Ca2+ transient induction (61.8% for

The intraoperative diagnosis of brain tumors and the timely evaluation of biomarkers that can guide therapy are hindered by the paucity of rapid adjunctive studies. This study evaluates the feasibility and specificity of using quantum dot-labeled antibodies for rapid visualization of epidermal growth factor receptor (EGFR) expression in human brain tumor cells and in surgical frozen section slides of glioma tissue. Streptavidin-coated quantum dots (QDs) were conjugated to anti-EGFR antibodies and incubated with target cultured tumor cells and tissues. The experiments were conducted first in human glioma tumor cell lines with elevated levels of EGFR expression (SKMG-3, U87) and then in frozen tissue sections of glioblastoma multiforme and of oligodendroglioma. The bioconjugated QDs used in the study were found to bind selectively to brain tumor cells expressing EGFR. QD complexed quickly to the cell membrane (less than 15 min), and binding was highly specific and depended on the expression level of EGFR on the cell membrane. Tissue experiments showed that only tumor specimens expressing EGFR were labeled in less than 30 min by QD complexes. These findings demonstrate that QD-labeled antibodies can provide a quick and accurate method for characterizing the presence or absence of a specific predictive biomarker.

When delivered to cells, very short duration, high electric field pulses (nanoelectropulses) induce primarily intracellular events. We present evidence that this emerging modality may have a role as a local cancer therapy. Five hematologic and 16 solid tumor cell lines were pulsed in vitro. Hematologic cells proved particularly sensitive to nanoelectropulses, with more than a 60% decrease in viable cells measured by MTT assay 96 hr after pulsing in 4 of 5 cell lines. In solid tumor cell lines, 10 out of 16 cell lines had more than a 10% decrease in viable cells. AsPC-1, a pancreatic cancer cell line, demonstrated the greatest in vitro sensitivity among solid tumor cell lines, with a 64% decrease in viable cells. When nanoelectropulse therapy was applied to AsPC-1 tumors in athymic nude mice, responses were seen in 4 of 6 tumors, including clinical complete responses in 3 of 6 animals. A single human subject applied nanoelectropulse therapy to his own basal cell carcinoma and had a complete pathologic response. In summary, we demonstrate that electric pulses 20 ns or less kill a wide variety of human cancer cells in vitro, induce tumor regression in vivo, and show efficacy in a single human patient. Therefore, nanoelectropulse therapy deserves further study as a potentially effective cancer therapy.

Background Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization. Results In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X7 receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts. Conclusion Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane.

In vivo fluorescent spectroscopy and imaging using endogenous and exogenous sources of contrast can provide new approaches for enhanced demarcation of brain tumor margins and infiltration. Quantum dots (QDs), nanometer-size fluorescent probes, represent excellent contrast agents for biomedical imaging due to their broader excitation spectrum, narrower emission spectra, and higher sensitivity and stability. The epidermal growth factor receptor (EGFR) is implicated in the development and progression of a number of human solid tumors including brain tumors and thus a potential target for brain tumor diagnosis. In this study, we investigate the up-take of ODs by brain tumor cells and the potential use of EGFR-targeted QDs for enhanced optical imaging of brain tumors. We conducted fluorescence microscopy studies of the up-take mechanism of the anti-EGFR-ODs complexes by Human U87, and SKMG-3 glioblastoma cells. Our preliminary results show that QDs can enter into glioma cells through anti-EGFR mediated endocytosis, suggesting that these nano-size particles can tag brain tumor cells.

Electroporation/electropermeabilization is a non-viral technique for gene transfection and drug delivery. Here, the transfer mechanisms were studied with fluorescent nanocrystals (quantum dots, QDs) in mammalian cells. Interactions of the cell membrane and nanoscale particles were visualized after electric pulse treatment. Responses of human multiple myeloma cells to nanocrystals were tracked for periods up to 7 days. Large particles do not cross the membrane directly after pulsing, even if the membrane is permeabilized to small molecules. Large QDs were trapped on the cell membrane for hours after electroporation and were gradually either excluded or internalized by cells. QD uptake efficiency depended on both particle size and membrane transport activity. These results, consistent with an electropermeabilization model, suggest that enhancing the interactions between the cell membrane and macromolecules may improve the transfer efficiency.

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis—caspase activation and loss of mitochondrial membrane potential—appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.

Monovalent ion concentration gradients, regulated by ion pumps and leak channels, are critical components of many cellular functions. This dynamic balance is disturbed by the electroporative permeabilization of the cell membrane, which bypasses the normal membrane barriers to transmembrane ion flux. A better understanding of ion transport during and after electroporation will enable more efficient and more effective utilization of this method in biomedicine and biotechnology.Molecular dynamics (MD) simulations provide a view of the behavior of ions and biomolecular structures at the molecular level. Previous MD studies have demonstrated some of the effects of monovalent ions on phospholipid bilayers, including decreased area per lipid, higher ordering in head group vertical orientations, and decreased lateral lipid mobility [1-4]. MD simulations of porated membranes have also shown that the binding of monovalent cations to phospholipids can increase pore line tension, which leads to a decrease in pore lifetime [5]. In this work we employ MD simulations to systematically study the effects of varying the concentration of Na+, K+, and Cl− in POPC lipid bilayer systems during different stages of electropore formation.1.Gurtovenko, A.A. and I. Vattulainen, J Phys Chem B, 2008. 112(7): p. 1953-62.2.Vacha, R., et al., Journal of Physical Chemistry A, 2009. 113(26): p. 7235-7243.3.Vacha, R., et al., J Phys Chem B, 2010. 114(29): p. 9504-9.4.Jurkiewicz, P., et al., Biochim Biophys Acta, 2012. 1818(3): p. 609-16.5.Leontiadou, H., A.E. Mark, and S.J. Marrink, Biophys J, 2007. 92(12): p. 4209-15.

Nanosecond, megavolt-per meter, pulsed electric field (nanoelectropulse) technology—a low-energy, nondestructive means for transiently electropermeabilizing biological cell membranes—is used in cancer therapy, genetic engineering, and cell biology. The effects of nanoelectropulses on the plasma membrane have been widely investigated. Opening of stable, long-lasting, lipid nanopores, selective uptake of fluorescence dyes, phosphatidylserine externalization, and cell volume change (swelling) due to water molecules uptake have been observed and reported. In particular, cell swelling has been recently introduced as a sensitive method for characterizing plasma membrane permeabilization. In this work, a systematic description and analysis of the cell swelling phenomenon is presented. Human Jurkat T lymphoblasts were exposed to 3 ns and 5 ns pulses from two different pulse generators, with different electrical characteristics. These pulse widths are much shorter than those reported previously. Different pulse counts (0, 1, 3, 5, 10, 20, 30, 50) and repetition rates (1 Hz or 1 kHz) were tested. In addition, to understand the types of pores formed by nanoelectropulses, swelling was induced in the presence of lanthanide ions (Gd3+ and La3+) and Hg2+, which are known to act on specific membrane channels. Mechanisms of nanoelectropulse-induced swelling will be discussed, and an empirical equation will be developed to describe the effects of different pulse parameters on cell membrane electropermeabilization.

Mitochondria, which play a crucial role in apoptosis, release several apoptosis-inducing factors into the cytoplasm, presumably through the mitochondrial permeability transition pore (MTP). Under certain conditions, nanoelectropulses can manipulate mitochondrial structure and permeabilize the mitochondrial membrane without permanent damage to the plasma membrane. In this work we investigate the effects of nanoelectropulses on mitochondrial membrane permeabilization, assess changes in mitochondrial transmembrane potential, and monitor plasma membrane integrity under the same pulse conditions. 4 ns electrical pulses were applied to living human Jurkat T lymphoblasts in an electrode microchamber on a microscope slide. Changes in mitochondrial transmembrane potential were evaluated with rhodamine 123 (R123), a lipophilic cationic fluorescent dye that is accumulated within mitochondria. For assessing MTP opening, a calcein-cobalt quenching method was used. Calcein-AM is an anionic fluorochrome that enters cells freely and labels cytoplasmic as well as mitochondrial regions following esterase removal of the acetoxymethyl group. Because cobalt ions do not readily pass through mitochondrial membrane, mitochondria can be specifically identified by the cobalt quenching of cytoplasmic, not mitochondrial, calcein fluorescence, and MTP opening can be recognized by the decrease of calcein fluorescence within mitochondria. Finally, cell membrane integrity was evaluated with propidium iodide (PI), which is excluded from the cell interior by intact cell membranes. When the cell membrane is permeabilized, PI enters the cell, binds to double-stranded nucleic-acid molecules, and exhibits red fluorescence. The effects of different pulse amplitudes and pulse numbers on mitochondrial membrane permeability will be reported, providing a framework for an analysis of pulse doses and exposure conditions which lead to mitochondrial modifications while minimizing effects on the plasma membrane. We will also discuss the interpretation of data from fluorescence microscopic imaging analysis using R123 and cobalt-quenched intracellular calcein fluorescence intensity and the influx of PI.

Intense (5-30 MV/m) ultra-short (10-300 ns) pulsed electric fields (US-PEFs) have been demonstrated to interact with sub-cellular membranes and structures [Schoenbach et al., IEEE Trans. Diel. El. Ins., 14 (5): 1088-1109, 2007]. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of US-PEFs, including cancer treatment, and b) better understanding of the mechanisms underlying such bioelectrical effects. T-lymphoblast Jurkat cells were exposed to a single pulse of 60 ns duration and 2.5 MV/m amplitude, without affecting cell growth and viability, to give insight into effects underlying interaction mechanisms. A cuvette-based exposure system, fed by a classical Blumlein pulse-forming network, was employed. A statistically significant reduction in DNA migration was detected immediately after US-PEF exposure by means of the alkaline comet assay [Zeni et al., Sensors, 8: 485-496, 2008]. This effect, associated with plasma membrane poration (YO-PRO-1 uptake) has been demonstrated to recover within 2 hours after pulse delivery (Figure 1), indicating that our exposure protocol targets the nucleus, affecting DNA structure. In our experimental conditions, a transient conformational change in DNA molecule can thus be hypothesized.View Large Image | View Hi-Res Image | Download PowerPoint Slide