First results on transient plasma-based remediation of nanoscale particulate matter in restaurant smoke emissions.

Recent studies have shown that nanoscale particulate matter produced in commercial charbroiling processes represents a serious health hazard and has been linked to various forms of cancer and cardiopulmonary disease. In this study, we propose a highly effective method for treating restaurant smoke emissions using a transient pulsed plasma reactor produced by nanosecond high voltage pulses. We measure the size and relative mass distributions of particulate matter (PM) produced in commercial charbroiling processes (e.g., cooking of hamburger meat) both with and without the plasma treatment. Here, the plasma discharge is produced in a 3" diameter cylindrical reactor with a 5-10 ns high voltage (17 kV) pulse generator. The distribution of untreated nanoparticle sizes is peaked around 125-150 nm in diameter, as measured using a scanning mobility particle sizer (SMPS) spectrometer. With plasma treatment, we observe up to a 55-fold reduction in relative particle mass and a significant reduction in the nanoparticle size distribution using this method. The effectiveness of the nanoscale PM remediation increases with both the pulse repetition rate and pulse voltage, demonstrating the scalability of this approach for treating particulate matter at higher flow rates and larger diameter reactors.

Water influx and cell swelling after nanosecond electropermeabilization.

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 10ns 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 10MV/m pulse of 5ns 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.

Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF).

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.

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells.

Nanosecond, high-voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP-induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators-rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt-quenched calcein-we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO-PRO-1 influx) was detected in addition to mitochondrial membrane permeabilization.

Microchamber setup characterization for nanosecond pulsed electric field exposure.

Intracellular structures of biological cells can be disturbed by exposure to nanosecond pulsed electric field (nsPEF). A microchamber-based delivery system mounted on a microscope setup for real-time exposure to nsPEF is studied in this paper. A numerical and experimental characterization of the delivery system is performed both in frequency and time domains. The microchamber delivery system presents a high impedance compared to classical 50 Ω loads. Its frequency behavior and limits are investigated using an in-house finite-difference time-domain (FDTD) simulator and through experimental measurements. High-voltage measurements for two nsPEF generators are carried out. The applied pulse voltage measured across the microchamber electrodes is ∼1 kV, corresponding to ∼10 MV/m electric fields in the microchamber. Depending on the nsPEF generator used, the measured pulse durations are equal to 3.0 and 4.2 ns, respectively. The voltage distribution provided by FDTD simulations indicates a good level of homogeneity across the microchamber electrodes. Experimental results include permeabilization of biological cells exposed to 3.0-ns, 10-MV/m PEFs.

Two-dimensional nanosecond electric field mapping based on cell electropermeabilization.

Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 mum from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration of electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. PACS Codes: 87.85.M-

Cardiac myocyte excitation by ultrashort high-field pulses.

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 <10 nsPEF vs. 80.6% for > or =10 nsPEF). Repetitive Ca2+ waves arising at the anodal side and Ca2+ destabilization occurred after repeated nsPEF (12/29) or during steady-state single nsPEF delivery at 2 Hz. Removing extracellular Ca2+ abolished responses to nsPEF. Verapamil did not affect nsPEF-induced Ca2+ transients, but decreased responses to CP. Tetrodotoxin and KB-R7943 increased the repetition threshold in response to nsPEF: 1-20 nsPEF caused local anodal Ca2+ waves without Ca2+ transients, and > or =20 nsPEF caused normal transients. Ryanodine-thapsigargin and caffeine protected against nsPEF-induced Ca2+ waves and showed less recovery of diastolic Ca2+ levels than CP. Voltage recordings demonstrated action potentials triggered by nsPEF, even in the presence of tetrodotoxin. nsPEF can mobilize intracellular Ca2+ in cardiac myocytes by inducing action potentials. Anodal Ca2+ waves and resistance to Na+ and Ca2+ channel blockade suggest nonselective ion channel transport via sarcolemmal nanopores as a triggering mechanism.

Nanosecond electric pulse-induced calcium entry into chromaffin cells.

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 field reduces the permeability of potato cell wall.

The effect of the application of pulsed electric fields to potato tissue on the diffusion of the fluorescent dye FM1-43 through the cell wall was studied. Potato tissue was subjected to field strengths ranging from 30 to 500 V/cm, with one 1 ms rectangular pulse, before application of FM1-43 and microscopic examination. Our results show a slower diffusion of FM1-43 in the electropulsed tissue when compared with that in the non-pulsed tissue, suggesting that the electric field decreased the cell wall permeability. This is a fast response that is already detected within 30 s after the delivery of the electric field. This response was mimicked by exogenous H2O2 and blocked by sodium azide, an inhibitor of the production of H2O2 by peroxidases.

Receptor-targeted quantum dots: fluorescent probes for brain tumor diagnosis.

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.

In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies.

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.

pH-sensitive Photoluminescence of CdSe/ZnSe/ZnS Quantum Dots in Human Ovarian Cancer Cells.

The photoluminescence of mercaptoacetic acid (MAA)-capped CdSe/ZnSe/ZnS semiconductor nanocrystal quantum dots (QDs) in SKOV-3 human ovarian cancer cells is pH-dependent, suggesting applications in which QDs serve as intracellular pH sensors. In both fixed and living cells the fluorescence intensity of intracellular MAA-capped QDs (MAA QDs) increases monotonically with increasing pH. The electrophoretic mobility of MAA QDs also increases with pH, indicating an association between surface charging and fluorescence emission. MAA dissociates from the ZnS outer shell at low pH, resulting in aggregation and loss of solubility, and this may also contribute to the MAA QD fluorescence changes observed in the intracellular environment.

Nanoelectropulse-driven membrane perturbation and small molecule permeabilization.

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.

Nanopore formation and phosphatidylserine externalization in a phospholipid bilayer at high transmembrane potential.

Atomic-resolution molecular dynamics simulations of lipid bilayers containing 7% phosphatidylserine (PS) on one leaflet are consistent with experimental observations of membrane poration and PS externalization in living cells exposed to nanosecond, megavolt-per-meter electric pulses. Nanometer-diameter aqueous pores develop within nanoseconds after application of an electric field of 450 mV/nm, and electrophoretic transport of the anionic PS headgroup along the newly constructed hydrophilic pore surface commences even while pore formation is still in progress.

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers--in cells and in silico.

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.

Selective functionalization of In2O3 nanowire mat devices for biosensing applications.

A strategy to covalently attach biological molecules to the electrochemically active surface of indium oxide nanowire (In2O3 NW) mat devices is presented. A self-assembled monolayer (SAM) of 4-(1,4-dihydroxybenzene)butyl phosphonic acid (HQ-PA) was generated on an indium tin oxide (ITO)-coated glass and In2O3 NWs surface. The chemical steps required for surface derivatization were optimized on an ITO surface prior to modifying the In2O3 NWs. The hydroquinone group contained in the HQ-PA SAM was electrochemically oxidized to quinone (Q-PA) at +330 mV. The monolayer of Q-PA was allowed to react with a thiol-terminated DNA. The DNA was paired to its complementary strand tagged with a fluorescence dye. Attachment of DNA was verified using fluorescence microscopy. A device was subsequently prepared on a SiO2-supported mat of In2O3 NWs by depositing gold electrodes on the mat surface. The reaction strategy optimized on ITO was applied to this In2O3 NW-based device. Arrays of In2O3 NWs on a single substrate were electrochemically activated in a selective manner to Q-PA. Activated In2O3 NWs underwent reaction with HS-DNA and gave a positive fluorescence response after pairing with the dye-DNA. The unactivated In2O3 NWs gave no response, thus demonstrating selective functionalization of an In2O3 NW array. This can be considered a key step for the future fabrication of large-scale, inexpensive, nanoscale biosensors.

Nanoelectropulse intracellular perturbation and electropermeabilization technology: phospholipid translocation, calcium bursts, chromatin rearrangement, cardiomyocyte activation, and tumor cell sensitivity.

Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in the plasma membrane, release intracellular calcium, trigger cardiomyocyte activity, and induce apoptosis in mammalian cancer cells, without the permeabilizing effects associated with longer, lower-field pulses. Dose dependencies with respect to pulse width, amplitude, and repetition rate, and total pulse count are observed for all of these phenomena. Sensitivities vary among cell types; cells of lymphoid origin growing in suspension are more susceptible to nanoelectropulse exposure than solid tumor lines. Simple electrical models of the cell are useful for first-order explanations, but more sophisticated treatments will be required for analysis and prediction at both biomolecular and tissue levels.

Electrode microchamber for noninvasive perturbation of mammalian cells with nanosecond pulsed electric fields.

Nanosecond pulsed electric fields can pass through the external membrane of biological cells and disturb fast-responding intracellular structures and processes. To enable real-time imaging and investigation of these phenomena, a microchamber with integral electrodes and optical path for observing individual cells exposed to ultrashort electric pulses was designed and fabricated utilizing photolithographic and microelectronic methods. SU-8 photoresist was patterned to form straight sidewalls from 10 to 30 microm in height, with gold film deposited on the top and sidewalls for conductive, nonreactive electrodes and a uniform electric field. Channel dimensions (10-30 microm x 100 microm x 12 000 microm) are suitable for observations of mammalian cells during nanosecond, megavolt-per-meter pulsed electric field exposure. Experimental studies utilizing the electrode microchamber include live-cell imaging of nanoelectropulse-induced intracellular calcium bursts and membrane phospholipid translocation.

Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts.

Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in cell membranes without the permeabilization associated with longer, lower-field pulses. A single 30 ns, 2.5 MV/m pulse produces perturbations consistent with phosphatidylserine (PS) externalization in Jurkat T lymphoblasts within milliseconds, polarized in the direction of the applied field, indicating an immediate interaction between membrane components and the electric field. This disturbance occurs only at the anode pole of the cell, supporting the hypothesis that the pulsed field drives the negatively charged PS head group toward the positive electrode, directly providing the energy for crossing the membrane dielectric barrier.

Nanoelectropulse-induced phosphatidylserine translocation.

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 2000 Hz 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.