Abstract
The mechanism(s) responsible for the breakdown (nanoporation) of cell plasma membranes after nanosecond pulse (nsEP) exposure remains poorly understood. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. However, the delivery of a high-voltage nsEP to cells by tungsten electrodes creates a multitude of biophysical phenomena, including electrohydraulic cavitation, electrochemical interactions, thermoelastic expansion, and others. To date, very limited research has investigated non-electric phenomena occurring during nsEP exposures and their potential effect on cell nanoporation. Of primary interest is the production of acoustic shock waves during nsEP exposure, as it is known that acoustic shock waves can cause membrane poration (sonoporation). Based on these observations, our group characterized the acoustic pressure transients generated by nsEP and determined if such transients played any role in nanoporation. In this paper, we show that nsEP exposures, equivalent to those used in cellular studies, are capable of generating high-frequency (2.5 MHz), high-intensity (>13 kPa) pressure transients. Using confocal microscopy to measure cell uptake of YO-PRO®-1 (indicator of nanoporation of the plasma membrane) and changing the electrode geometry, we determined that acoustic waves alone are not responsible for poration of the membrane.
Highlights
Nanoporation, a type of electroporation that generates very small (< 2 nm) holes in plasma membranes, is hypothesized to result from exposures of sub-microsecond electric pulses in the megavolt/meter range[1,2]
When the electrodes were placed in very close proximity (< 1 mm) to the probe beam, termed the near-field, substantial deflections of the probe beam were detected upon nanosecond pulse (nsEP) exposure
The nsEP exposure was administered with a pulse width of 600 ns and an applied voltage of 1000 V to generate an electrical field of approximately 13.1 kV/cm at 50 μ m from the electrodes
Summary
Nanoporation, a type of electroporation that generates very small (< 2 nm) holes in plasma membranes, is hypothesized to result from exposures of sub-microsecond electric pulses in the megavolt/meter range[1,2]. The biophysical interactions that occur with an nsEP exposure are complex; determination of the mechanism of nanoporation is quite difficult. Electrodeformation is an electrical-field-driven internal mechanical stress that causes the entire cell to deform, leading to a higher probability of pore formation[4] Another competing theory of poration, championed by Vernier, has suggested poration occurs “due to field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces”, presumably this reorganization of water molecules creates more energetically favorable situation for pore formation[5]. These theories of poration, plausible, are not all-inclusive and do not account for other non-electrical factors, such as external mechanical stress caused by interactions with pressure transients. The findings in this paper provide new insights as to the nature of the physical mechanisms that occur rapidly after the application of nsEP at the surface of the electrodes and how these events could potentially contribute to the breakdown of plasma membranes
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