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International audience; Pulsed electric fields of sufficient magnitude and duration trigger functional responses and modifications in biological cells. Transient nanometer-sized pores are believed to form within nanoseconds in cell membranes exposed to high-intensity (MV/m) nanosecond pulsed electric fields (nsPEFs), and while it is clear that polar water molecules play a key role in electroporation, no signature for pore initiation has yet been identified. To address this, we combine molecular dynamics simulations and quasi-static 3-D finite-difference analysis to investigate the electrostatic interactions that drive pore formation in homogenous lipid bilayers exposed to intense nsPEFs. The developed methodology uniquely enables the extraction of 3-D spatiotemporal profiles of electric potentials, electric fields, and electric field gradients in biological membranes with atomistic detail and sub-nanosecond resolution. As a result, this study captures and elucidates several dynamic phenomena observed experimentally and provides a fundamental framework for further development.

International audience

Nanosecond, megavolt-per-meter electric pulses applied to biological cells can target subcellular structures with minimal loss of plasma membrane integrity, opening up new perspectives for intracellular manipulations. Experimentally observed effects of intense nanopulses include intracellular calcium release, externalization of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, and non-thermal cell death by apoptosis. Molecular dynamics (MD) simulations have shown that PS re-distribution occurs after the electric-field-driven formation of nanometer-sized pores in the plasma membrane and is facilitated by electrophoresis of PS along the pore walls. Nanopulse-induced pore creation occurs on a nanosecond time scale, but the underlying molecular mechanisms are not yet clear. Experimental observations of the process of pore formation are challenging because of the time and spatial scales required.In this study, we combine MD simulations and a quasi-static approach using a custom implementation of 3D finite-difference analysis to investigate the physical mechanisms of electropore creation. First, MD simulations of pore formation in phospholipid bilayers in external electric fields are performed at nanoscopic scale. From these simulations we extract the charge densities across the electroporated bilayer. Second, the charge densities are injected into a new, custom, quasi-static algorithm based on the Poisson equation. The software computes 3D nanoscopic profiles of the transmembrane potential, electric field, and electric field gradient. The goal of the two-step simulation is to establish whether and how electric field gradients, water and phospholipid head group dipole moments, and the site of initial water intrusion in pore initiation are correlated.

International audience; In this paper, we propose and characterize a setup based on a Transverse ElectroMagnetic (TEM) cell to expose a Petri dish filled with a biological suspension to nanosecond high-voltage pulsed electric fields. Monopolar and bipolar pulses of 1.2 ns duration and 1.6 kV amplitude are delivered to the TEM cell. Time domain measurements and numerical results show that the system is well suited to deliver high-intensity pulsed electric fields with 1.2 ns duration and amplitudes of at least 100 kV/m.

International audience; 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.