Extracellular-Ca2+-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses

Abstract

Electroporation-a transient electric-field-induced increase in cell membrane permeability-can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca²⁺ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl₂ effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl₂ concentration is increased.

Keywords: calcium; calcium electroporation; membrane repair; microsecond electroporation; nanosecond electroporation; pore resealing.

Figure 1

Flow cytometry gating strategies. Panel (A) represents cell distinction from the debris (FSC-A::SSC-A) and single cell (FSC-A::FSC-H) gating strategies. Panel (B) represents a gating strategy of cell fluorescence before (red) and 15 min after (blue) cell electroporation with HV pulse (1400 V/cm, 100 µs) in the presence of EtBr, YO-PRO-1 and PI molecules in electroporation medium containing 1 mM of CaCl₂.

Figure 2

Dependence of PI electrotransfer efficiency (A) and total cell fluorescence (B) of the treated cells on the applied electric field strength at various CaCl₂ concentrations. Cells were treated using 1 square HV pulse at a 100-μs pulse duration. PI fluorescence was measured 15 min after electric field application. The statistical differences between PI uptake (PI positive cells and total fluorescence) between control (0 mM CaCl₂ concentration) and 0.25, 0.5, 1 mM CaCl₂ concentrations are denoted by *, # and ^, respectively. One symbol denotes p < 0.05, two symbols—p < 0.01, three symbols—p < 0.001. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 3

Dependence of PI electrotransfer efficiency (A) and total cell fluorescence (B) of the treated cells on the applied electric field strength at various CaCl₂ concentrations. Cells were treated using 10 square HV pulses at a 200-ns pulse duration. PI fluorescence was measured 15 min after electric field application. The statistical differences between PI uptake (PI positive cells and total fluorescence) between control (0 mM CaCl₂ concentration) and 0.25, 0.5, 1 mM CaCl₂ concentrations are denoted by *, # and ^, respectively. One symbol denotes p < 0.05, two symbols—p < 0.01, three symbols—p < 0.001. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 4

Dependence of PI electrotransfer efficiency (A,B) and the total cell fluorescence (C,D) after treatment with 1 × 1400 V/cm strength and a 100-µs duration electric pulse (A,C) or 10 × 1.4 kV/cm strength and a 200-ns electric pulses at 1 Hz of repetition frequency (B,D) at various CaCl₂ concentrations; the results are represented in linear scale. The inserts in each figure represent the corresponding results in the range of low 0.0001–0.1 mM CaCl₂ concentrations on the logarithmic scale. PI fluorescence was measured 15 min after electric field application. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 5

Dependence of PI, EtBr and YO-PRO-1 electrotransfer efficiency on the presence of extracellular calcium after treatment with 1 × 1400 V/cm strength and a 100-µs duration electric pulse (A) on the presence of extracellular calcium after treatment with 10 × 14 kV/cm strength and 200-ns duration electric pulses at 1 Hz of repetition frequency (B). Fluorescence was measured 15 min after the applied electric field. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 6

Visualization of PI electrotransfer into cells in media with different CaCl₂ concentrations. The cells were electroporated with single 1400 V/cm (A) or 1800 V/cm (B) strength, 100 µs duration electric pulses. Electroporation was performed at time ‘0 s’. The fluorescent images in panels (C) and (D) represent key points images from the (A) and (B) panels, respectively. Statistical differences (A and B) of PI uptake between control (0 mM CaCl₂) and 0.25; 0.5; 1 mM CaCl₂ are denoted by ***—p < 0.001. The error represents the mean ± standard error of mean of n = 20 experimental replicates.

Figure 7

Membrane resealing dynamics after the treatment with a single 1400- (A) or 2800-V/cm (B) strength, 100-µs duration HV pulse, monitored by the entry of PI (40 µM) added 15–600 s after electroporation. Statistical differences between the control (0 mM CaCl₂ concentration) and either of the 0.25, 0.5 or 1 mM CaCl₂ groups are denoted as *, and between 0.25 mM CaCl₂ and either 0.5 or 1 mM CaCl₂ groups are denoted as #. A single symbol denotes two-tailed p < 0.05, double symbol—p < 0.01, and triple symbol—p < 0.001, PI fluorescence was measured 15 min after electric field application. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 8

Modelling of transmembrane potential distribution on cell surface (A) and the peak transmembrane potential at the electrode-facing poles after microsecond (B) and nanosecond (C) electric pulse treatment.

Figure 9

Dependence of PI electrotransfer efficiency and total cell fluorescence of the treated cells on the medium conductivity. Cells were treated with 1 × 1400 V/cm strength and a 100-µs duration electric pulse. The conductivity of the CaCl₂-free medium was adjusted by adding MgCl₂. The control represents untreated cells. PI fluorescence was measured 15 min after the applied electric field. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.

Figure 10

Cell viability after cell treatment using 1 HV pulse at a 100-μs pulse duration evaluated by flow cytometry assay (FCA, dark bars) and MTT (light bars) in dependence of the CaCl₂ concentration in the medium. Symbols denote statistical differences between the control (untreated cells) and electroporation groups at 0, 0.25, 0.5, 1 mM CaCl₂ concentration. Two symbols denote p < 0.01, three symbols—p < 0.001. The error bars represent the mean ± standard error of mean of n = 6 experimental replicates.