Abstract
The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.
Highlights
Permeabilization of the cell membrane by intense electric field pulses, or electroporation, has been known for decades and has found numerous applications in gene and drug delivery, cancer ablation, membrane biophysics research, as well as in food processing and decontamination [1,2,3,4,5]
Electropermeabilization research has expanded to the nanosecond-range pulsed electric fields, revealing new and different bioeffects
NsPEFs are distinguished (1) by the ability to disrupt intracellular membranous structures, such as endoplasmic reticulum and mitochondria [6,7,8,9], (2) by the phenomenon of bipolar cancellation, which stands for the suppression of diverse bioeffects upon the nanosecond pulsed electric field (nsPEF) polarity reversal [10,11,12,13], and (3) by the predominant formation of smaller membrane defects, often referred to as “nanopores” or “nanoelectropores,” the exact nature of these defects remains uncertain [4,9,14,15,16,17,18]
Summary
Permeabilization of the cell membrane by intense electric field pulses, or electroporation, has been known for decades and has found numerous applications in gene and drug delivery, cancer ablation, membrane biophysics research, as well as in food processing and decontamination [1,2,3,4,5]. Nanopores may remain open for minutes, and demonstrate complex conductive properties unexpected from simple “holes” in a lipid bilayer, including inward rectification, current and voltage sensitivity, and ion selectivity [15,16]. Some of these properties are similar to those of funnel-shaped artificial nanopores in polymer films [19], but it is difficult to explain how any conical lipidic pores would form and persist, for a long time, in a cell membrane which is only a few nanometers thick. It is possible that there are several different types of electropores, which contribute differently to the permeabilized membrane state
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