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.

Keywords: Asymmetric molecular transport pattern; Electroporation; Nanosecond electropermeabilization.

Fig. 1

Typical 6 ns and 220 µs waveforms recorded during application to experimental samples

Fig. 2

Pattern analysis of a single cell. The cell area is divided into rows of pixels (represented schematically by the regions of different colors; in the actual analysis, there are roughly 50 rows of pixels per cell) parallel to the electrodes. The rows are grouped into cathode- and anode-side and middle regions and the average of the mean intensities of the rows is reported as the intensity of the region (Color figure online)

Fig. 3

Calibration curves for a YO-PRO-1 and propidium and b calcein with Leica TCS SP8 confocal microscope. Error bars are SD

Fig. 4

Small molecule concentration changes in cathode, middle, and anode sections of cells exposed to a single 220 µs, 250 kV m⁻¹ pulse. a YO-PRO-1 (n = 30); b propidium (n = 34); and c calcein (n = 30). YO-PRO-1 and propidium entry into the cell is asymmetrical and quickly cathode-dominant; calcein enters equally from every direction. Data points are at 1 s intervals (300 ms in inset). Curves smoothed as described in methods

Fig. 5

Localization of small molecule uptake in U-937 cells exposed to a single 220 µs, 250 kV m⁻¹ pulse. a Uptake differences between anode and cathode regions of cells for YO-PRO-1, propidium, and calcein. Note the brief, initial, anode-side influx dominance that quickly switches to the cathode side. Decaying exponential functions fit to the data have time constants of 40 s for both YO-PRO-1 and propidium. No preferential uptake is observed for calcein. The early positive peak (arrow) indicates the initially greater anode-side influx switched to the cathode-side. Inset data points every 200 ms. b Differences in YO-PRO-1 uptake between anode and middle regions and cathode and middle regions of cells. After 25 s YO-PRO-1 concentration is greater in the middle region than in the anode or cathode regions. Curves smoothed as described in methods. c Differences in propidium uptake between anode and middle regions and cathode and middle regions of cells. Slower intracellular diffusion of propidium slows the decline of cathode and anode dominance. Curves smoothed as described in the methods

Fig. 6

Fluorescence images of YO-PRO-1 and propidium transport into U-937 cells at 0.2, 0.4, 1, 5, 10, and 20 s after exposure to a single 220 µs, 250 kV m⁻¹ pulse. Circles mark the circumference of the cells, which can be seen in the transmitted light images at the left, along with plus and minus symbols indicating the anode and cathode directions

Fig. 7

Small molecule concentration changes in cathode, middle, and anode sections of cells exposed to 10, 6 ns, 20 MV m⁻¹ pulses at 1 kHz. a YO-PRO-1 (n = 34); b propidium (n = 30); and c calcein (n = 30). YO-PRO-1 and propidium entry into the cell is asymmetrical and initially anode-dominant; calcein enters equally from every direction. Data points are at 1 s intervals (300 ms in inset)

Fig. 8

a Differences in small molecule uptake between anode and cathode regions of cells exposed to 10, 6 ns, 20 MV m⁻¹ pulses at 1 kHz. Decaying exponential functions fit to the data have time constants of 20 and 140 s for YO-PRO-1 and propidium, respectively. b Relative absence of cathode-side influx for the nanosecond compared to microsecond pulse exposures for YO-PRO-1 and c propidium

Fig. 9

a YO-PRO-1 and b propidium transport asymmetry at different 6 ns nanosecond pulse repetition rates. (Note: pulse delivery begins at 5 s.) Anode-side dominance is sharply greater at 1 kHz, but still apparent over time even at 1 Hz. Data points are at 300 ms intervals

Fig. 10

Fluorescence images of YO-PRO-1 and propidium transport into U-937 cells at 0.2, 0.4, 1, 5, 10, and 20 s after exposure to 10, 6 ns, 20 MV m⁻¹ pulses delivered at 1 kHz. Circles mark the circumference of the cells, which can be seen in the transmitted light images at the left, along with plus and minus symbols indicating the anode and cathode directions

Fig. 11

Fluorescence images of YO-PRO-1 uptake after pulse delivery. a 5 s after a single 220 µs, 250 kV m⁻¹ pulse; b 5 s after 10, 6 ns, 20 MV m⁻¹ pulses at 1 kHz; c 10 s after a single 6 ns, 20 MV m⁻¹ (d) 115 s after a single 6 ns, 20 MV m⁻¹ pulse. Intensity in panels c and d is increased to equalize the relative brightness of the images in the four panels. d Time course of YO-PRO-1 uptake in the cathode, middle, and anode regions of the cells after a single 6 ns, 20 MV m⁻¹ pulse (n = 12). Extracellular YO-PRO-1 concentration for panels c, d, and e is 10 µM