Picosecond and Terahertz Perturbation of Interfacial Water and Electropermeabilization of Biological Membranes

Abstract

Non-thermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which membrane permeabilization occurs in less than 1 ns. Water dipoles in the interior of these model membranes respond in less than 1 ps to permeabilizing electric potentials by aligning in the direction of the field, and they re-orient at terahertz frequencies to field reversals. The mechanism for subnanosecond lipid electropore formation is similar to that observed on longer time scales-energy-minimizing intrusions of interfacial water into the membrane interior and subsequent reorganization of the bilayer into hydrophilic, conductive structures.

Fig. 1

Micro-coaxial cable pulse delivery system for neurons on microscope stage. The tip of the micro-coaxial cable (the inner conductor) is 20 μm above the cover slip. Regions A and B are projections of the micro-coaxial cable. Cells to be analyzed are within the two off-axis dashed ellipses. The color map shows the electric field distribution at the surface of the coverslip for a 1 kV pulse voltage, in which case the electric field in the elliptical areas is about 3 MV/m.

Fig. 2

Picosecond pulse waveform at the output of the pulse generator. 320 ps (full-width, half-maximum), 5.5 kV.

Fig. 3

Action potentials in a rat hippocampal neuron stimulated by 320 ps electric pulses. Membrane voltage in whole-cell configuration measured in current clamp mode with current stepped in 20 pA increments from the holding level of -100 pA, vertically separated for clarity. Current protocol shown below voltage traces. 100 ms after each current step (arrow), 100 pulses, 5 MV/m, 500 Hz) were delivered over a period of 200 ms. Pulse-induced depolarization and action potentials occurred at the -100 and -80 pA steps. Further depolarization by reducing the injected current immediately caused an action potential followed by a refractory state, so the picosecond pulses produced some additional depolarization without eliciting an action potential. The inset shows the neuron with a recording pipette attached in the vicinity of the pulse-delivering electrode (shadow).

Fig. 4

Ca²⁺ transients in NG108 rat neuroblastoma-glioma hybrid cells evoked by a single 500 ps, 19 MV/m pulse and a train of 5 pulses at 1 kHz. Measured with Fura-2 ratiometric imaging. Traces from 5 cells superimposed in each panel.

Fig. 5

Lipid electropore in POPC bilayer forms in less than 1 ns. In this high applied electric field (E_vacuum = 2.5 GV/m; E_effective = 12.5 MV/m), pore initiation is rapid and a hydrophilic pore is constructed in less than 1 ns. Water molecules (small red and white structures) penetrate the membrane interface and bridge the membrane interior, followed by the phospholipid head groups (blue – nitrogen; gold – phosphorus; red – backbone acyl oxygen). Gray strands are the lipid hydrocarbon tails.

Fig. 6

Permeabilization of POPC bilayer in 500 GHz alternating electric field. Water, then phospholipid head groups bridge the membrane interior in a very high porating electric field with polarity reversals every picosecond. Multiple water bridges appear, followed by head groups, in a few tens of picoseconds.

Fig. 7

Dipole reversal for water molecules in the phospholipid bilayer interior. Note that the oxygen (red) end of each of the bridging water molecules in the middle of the membrane is positioned toward the bottom of the left image and toward the top of the right image. The electric field direction is opposite in these two frames, which are 1 ps apart. Within that 1 ps the water dipoles in the membrane interior align in the new field direction. Dipole relaxation time for water in the membrane interior is much less than in bulk water, so that field-stabilized intruding water columns remain the lower energy configuration for interfacial water.