Modulation of Biological Responses to 2 ns Electrical Stimuli by Field Reversal

Nanosecond bipolar pulse cancellation, a recently discovered Phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (\u3e 100 mu s) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results

Effects of Pulse Width on He Plasma Jets in Contact with Water Evaluated by OH(A-X) Emission and OHaq Production

Nanosecond pulsed helium plasma jets impinging on water produce hydroxyl radicals both in gas- and liquid-phase. In this study, the effects of pulse width on a repetitively pulsed plasma jet in contact with water are evaluated via OH(A-X) emission and OHaq production in water for various pulse widths ranging from 200 to 5000 ns. The maximal energy efficiency of OH(A-X) emission is obtained for pulse widths of 600-800 ns whereas the maximal efficiency of OHaq production is at 200 ns. Temporally-resolved emission spectroscopy shows that more than 40% of OH(A-X) emission is produced during the first 200 ns of the voltage pulse regardless of the pulse width. An equivalent circuit model of the plasma jet impinging on water is compiled to understand the charge transfer process, which is important for OHaq production via charge exchange reactions

Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization

Imaging of fluorescent small molecule transport into electropermeabilized cells reveals polarized patterns of entry, which must reflect in some way the mechanisms of the migration of these molecules across the compromised membrane barrier. In some reports, transport occurs primarily across the areas of the membrane nearest the positive electrode (anode), but in others cathode-facing entry dominates. Here we compare YO-PRO-1, propidium, and calcein uptake into U-937 cells after nanosecond (6 ns) and microsecond (220 µs) electric pulse exposures. Each of the three dyes exhibits a different pattern. Calcein shows no preference for anode- or cathode-facing entry that is detectable with our measurement system. Immediately after a microsecond pulse, YO-PRO-1 and propidium enter the cell roughly equally from the positive and negative poles, but transport through the cathode-facing side dominates in less than 1 s. After nanosecond pulse permeabilization, YO-PRO-1 and propidium enter primarily on the anode-facing side of the cell

Quantitative Limits on Small Molecule Transport via the Electropermeome - Measuring and Modeling Single Nanosecond Perturbations

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

Transport of Charged Small Molecules after Electropermeabilization - Drift and Diffusion

Background: Applications of electric-field-induced permeabilization of cells range from cancer therapy to wastewater treatment. A unified understanding of the underlying mechanisms of membrane electropermeabilization, however, has not been achieved. Protocols are empirical, and models are descriptive rather than predictive, which hampers the optimization and expansion of electroporation-based technologies. A common feature of existing models is the assumption that the permeabilized membrane is passive, and that transport through it is entirely diffusive. To demonstrate the necessity to go beyond that assumption, we present here a quantitative analysis of the post-permeabilization transport of three small molecules commonly used in electroporation research-YO-PRO-1, propidium, and calcein-after exposure of cells to minimally perturbing, 6 ns electric pulses. Results: Influx of YO-PRO-1 from the external medium into the cell exceeds that of propidium, consistent with many published studies. Both are much greater than the influx of calcein. In contrast, the normalized molar efflux of calcein from pre-loaded cells into the medium after electropermeabilization is roughly equivalent to the influx of YO-PRO-1 and propidium. These relative transport rates are correlated not with molecular size or cross-section, but rather with molecular charge polarity. Conclusions: This comparison of the kinetics of molecular transport of three small, charged molecules across electropermeabilized cell membranes reveals a component of the mechanism of electroporation that is customarily taken into account only for the time during electric pulse delivery. The large differences between the influx rates of propidium and YO-PRO-1 (cations) and calcein (anion), and between the influx and efflux of calcein, suggest a significant role for the post-pulse transmembrane potential in the migration of ions and charged small molecules across permeabilized cell membranes, which has been largely neglected in models of electroporation

Growth in a Biofilm Sensitizes \u3ci\u3eCutibacterium acnes\u3c/i\u3e to Nanosecond Pulsed Electric Fields

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells

2-ns Electrostimulation of Ca\u3csup\u3e2+\u3c/sup\u3e Influx Into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2 ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2 ns bipolar pulse with positive and negative phases of equal duration and amplitude, and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation

Microsecond Kinetics of Ion Transport and Membrane Interface Binding Before, During, and After Lipid Electropore Formation

Molecular dynamics simulations of lipid membranes reveal the nanoscale evolution of biophysical systems, including complex processes that are not observable with conventional experimental methods. Among these processes, electroporation, also called electropermeabilization, is used in medicine and biology to introduce drugs, nucleic acids, and other normally impermeant material into cells. It is known that the application of strong transmembrane electric fields causes the formation of bilayer-spanning water bridges and conductive lipid pores, and that material otherwise not able to go through the cell membrane can enter or exit the cell through these breaches of the membrane barrier. Knowledge about how specific ions and molecules are transported through electropermeabilized membranes, however, is very limited. Our simulations focus on the dynamics of ion-membrane interactions during electroporation. We describe the previously unexplored microsecond kinetics of ion binding to phospholipid bilayers and transport through lipid electropores in double bilayer systems containing K+, Ca2+, and Cl-. A double bilayer system allows us to simulate the different ion concentrations inside and outside the cell and to study their dynamics before, during and after the pulse. In particular, the intracellular distribution of Ca2+ is a key component in the operation of numerous regulatory and signaling pathways. Little is known about the evolution of the threedimensional [Ca2+] profile during the nanoseconds and microseconds after a porating electric pulse. Does Ca2+ diffuse freely into the cytoplasmor is it bound quickly to the intracellular interface of the lipid bilayer? Molecular simulations allow us to explore this nanoscale world in search of answers to these questions.https://digitalcommons.odu.edu/engineering_batten/1007/thumbnail.jp