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Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization.

ABSTRACT: 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.

SUBMITTER: Sozer EB

PROVIDER: S-EPMC5910485 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization.

Sözer Esin B EB Pocetti C Florencia CF Vernier P Thomas PT

The Journal of membrane biology 20170508 2

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) ...[more]

PMID: 28484798

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Project description:BackgroundApplications 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.ResultsInflux 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.ConclusionsThis 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.

Dataset Information

Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization.

Publications

Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization.

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