Yu-Hsuan Wu

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.

Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane

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.

A linear, single-stage, nanosecond pulse generator for delivering intense electric fields to biological loads

A compact pulse generator capable of producing high voltage pulses with halfmaximum widths as short as 2.5 ns and amplitudes as high as 5 kV has been developed to enable current and future in vivo and in vitro research into the effects of ultra-short, intense electric fields on biological matter. This pulse generator is small, simple, and free of saturable magnetic cores, which frequently introduce amplitude jitter and an undesirable correlation between amplitude and pulse width. In place of a non-linear pulse-forming network is a single-stage resonant network that drives a bank of junction recovery diodes. The diodes function as an opening switch that commutes current from an inductor to a resistive load. The use of air-core inductors in the resonant network results in a stable output pulse with an amplitude that scales linearly with input voltage and a pulse width that is independent of amplitude. The ability to scale the output amplitude independently of the pulse width simplifies the setup for experiments that require pulses with different electric field strengths but the same rise time and duration. Jurkat T lymphoblast cells exposed to 2.5 ns fields produced by this pulse generator showed an increasing degree of electropermeabilization with increasing pulse dosage and electric field intensity.

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.

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.

Moveable Wire Electrode Microchamber for Nanosecond Pulsed Electric-Field Delivery

In this paper, an electromagnetic characterization of a moveable wire electrode microchamber for nanosecond pulse delivery is proposed. The characterization of the exposure system was carried out through experimental measurements and numerical simulations. The frequency and time domain analyses demonstrate the utility of the proposed assembly for delivering pulses as short as 2.5 ns. High-voltage measurements (~1.2 kV) were also performed using pulse generators based on two different technologies with applied pulse durations of 5.0 and 2.5 ns. Validation of the delivery system was accomplished with biological experiments involving cell electroporation with 2.5 and 5.0 ns, 10-MV/m pulsed electric fields. A dose-dependent area increase (osmotic swelling) of the Jurkat cells was observed with pulses as short as 2.5 ns.

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 μm 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-

Surface chemical immobilization of parylene C with thermosensitive block copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide: Synthesis, characterization, and cell adhesion/detachment†

Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert-butylacrylamide) (pNTBAM), and their copolymer brushes were covalently immobilized onto parylene C (PC) surfaces via surface initiated atom transfer radical polymerization (ATRP). Contact angle measurement between 13 and 40°C showed that the hydrophobicity of the modified PC surfaces was thermally sensitive. Among these samples, PC grafted with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and PC grafted with copolymer brushes containing pNTBAM and pNIPAM (PC-NT-NI) exhibited the lower critical solution temperature (LCST) at 29, 22, and 24°C, respectively. Cytocompatibility study for the modified surfaces was performed by 5 days human skin fibroblast culture at 37°C. Data showed that only a very small amount of cells adhered on the PC and PC-NI surfaces, while a significantly higher amount of cell adhesion and growth was observed on PC-NT and PC-NT-NI surfaces. Furthermore, cell detachment at the temperatures of 24 and 6°C were studied after the substrates were cultured with cells at 37°C for 24 h. The results showed that the cells on PC-NI formed the aggregations and loosely attached on the substrate after 30-min culture at 24°C, while no significant cell detachment was observed for PC-NT and PC-NT-NI samples at this temperature. By continuing the cell culture for additional 100 min at 6°C for PC-NT and PC-NT-NI, about 10 and 35% of the cells were found detached respectively, and the unattached cells aggregated on the substrate. In comparison, cells cultured on the tissue culture petri dish (TCP) exhibited no quantity and morphology changes at the culture temperatures of 37, 24, and 6°C. This study showed that: (1) immobilization of PC with nonthermal sensitive pNTBAM could provide PC surface thermal sensitive hydrophilicity; (2) the chlorines on the polymer brushes of PC-NT could be used to further initiate the ATRP pNIPAM and form block copolymer brushes; (3) the incorporation of pNTBAM into pNIPAM on PC-NT-NI could change the surface thermal hydrophilicity property, and be further applied to decrease the LCST of the modified PC surface; (4) grafted pNIPAM brushes on PC-NI by ATRP showed very low cell adhesion and proliferation in 5 days fibroblast culture at 37°C, and cell detached at 24°C; (5) the incorporation of pNTBAM into pNIPAM on PC-NT-NI decreased the thermal sensitivity of cell adhesion/detachment, cell detached at 6°C, but the cell adhesion and proliferation were significantly improved at a wide temperature range.

Single-electrode He microplasma jets driven by nanosecond voltage pulses

Excited by 5 ns, 8 kV voltage pulses, a 260 μm-diameter, 8 mm long helium plasma jet was generated with a single-electrode configuration in ambient air. Application of fast high voltage pulses (≥1012 V s−1) resulted in rapid acceleration of the microplasma plumes; within 5 ns the plume velocity reached 8 × 105 m/s, almost three times higher than that of the plasma jet generated with the pulsed voltage of the same amplitude but with a lower increase rate (1011 V s−1). Importantly, the ultrashort electric pulses were able to efficiently deposit energy in the plasma during the initiation process, which may be responsible for the rapid acceleration of the ionization wavefronts during the streamer onset, as well as efficient production of reactive plasma species including O(5P) and N2+(B2Σu+) via electron-induced processes. Emission spectral comparison between the plasma jets excited with 5 ns voltage pulses and with 140 ns voltage pulses showed enhanced O(5P) and N2+(B2Σu+) emission by the shorter pulses than the longer ones, while the vibrational and rotational temperature for both plasma jets are at 3000 K and 300 K, respectively.

Scalable, compact, nanosecond pulse generator with a high repetition rate for biomedical applications requiring intense electric fields

A high repetition rate, high voltage pulse generator has been developed that scales up the output voltage of a recently reported compact, nanosecond pulse generator that is currently being used in various biomedical applications, including experiments into the mechanisms that drive cellular electropermeabilization and plasma generation for an endodontic disinfection tool [1, 2]. This single-stage, nanosecond architecture is based is composed of a bank of power MOSFETs, a linear network of inductors and capacitors, and a bank of junction recovery diodes; it was reported to feature an output pulse amplitude voltage to input voltage ratio between 5 and 6 [3, 4]. Since commercially available power MOSFETs tend to be limited to 1 kV, the output amplitude of the single-stage pulse generator does not exceed 5 or 6 kV. To combat this limitation, two different architectures have been developed that enable scaling of the output voltage. The first of these increases the voltage input to the pulse-forming network by means of a solid-state Marx bank that employs power MOSFETs arranged in a series-parallel arrangement to handle the high voltage and high current requirements of the switching stage. The second architecture employs a saturating transformer to handle the high current. Each of these has its own advantages: the first architecture has a shorter trigger-to-output delay time and is capable of producing low-jitter pulses with a linear input-output voltage relationship; whereas, the architecture with a saturating core features fewer components and reduced complexity. Prototypes of both architectures have been designed, built, tested, and are currently being used.