The equivalence of different types of electric pulses for electrochemotherapy with cisplatin - an in vitro study

Abstract

Background: Electrochemotherapy (ECT) is a treatment involving the administration of chemotherapeutics drugs followed by the application of 8 square monopolar pulses of 100 μs duration at a repetition frequency of 1 Hz or 5000 Hz. However, there is increasing interest in using alternative types of pulses for ECT. The use of high-frequency short bipolar pulses has been shown to mitigate pain and muscle contractions. Conversely, the use of millisecond pulses is interesting when combining ECT with gene electrotransfer for the uptake of DNA-encoding proteins that stimulate the immune response with the aim of converting ECT from a local to systemic treatment. Therefore, the aim of this study was to investigate how alternative types of pulses affect the efficiency of the ECT.

Materials and methods: We performed in vitro experiments, exposing Chinese hamster ovary (CHO) cells to conventional ECT pulses, high-frequency bipolar pulses, and millisecond pulses in the presence of different concentrations of cisplatin. We determined cisplatin uptake by inductively coupled plasma mass spectrometry and cisplatin cytotoxicity by the clonogenic assay.

Results: We observed that the three tested types of pulses potentiate the uptake and cytotoxicity of cisplatin in an equivalent manner, provided that the electric field is properly adjusted for each pulse type. Furthermore, we quantified that the number of cisplatin molecules, resulting in the eradication of most cells, was 2-7 × 10⁷ per cell.

Conclusions: High-frequency bipolar pulses and millisecond pulses can potentially be used in ECT to reduce pain and muscle contraction and increase the effect of the immune response in combination with gene electrotransfer, respectively.

Keywords: cisplatin uptake; electrochemotherapy; electroporation; equivalent pulse parameters; phenomenological model.

FIGURE 1.

(A) 50 × 50 HF pulses. From left to right: 50 bursts were applied with a repetition frequency of 1 Hz; one burst with 200 μs total pulse on time and consisted of 50 bipolar pulses; one bipolar pulse of amplitude U consisted of a 2 μs long positive pulse, and a 2 μs long negative pulse (both of voltage U) with a 2 μs long interpulse delay. (B) 8 × 100 μs or 8 × 5 ms monopolar pulse of amplitude U and pulse duration of 100 μs or 5 ms were applied with a repetition frequency of 1 Hz

FIGURE 2.

Schematic of the model that describes electroporation and molecular transport. (A) The equivalent circuit, which considers electroporation (membrane pore/defect formation) to be a two-step process, as depicted in (B). The blue capacitance and resistance represent the intact cell membrane. When the electric field is applied, the cell membrane becomes permeable first to small ions, indicating the first porous state (N) of the membrane represented by green resistance. Then the membrane becomes permeable to small molecules, indicating the second porous state (M) of the membrane represented by magenta resistance. Reproduced from Sweeney et al.56 with permission.

FIGURE 3.

Cell survival (solid) and cell membrane permeability (dashed) as a function of the electric field when (A) 50 × 50 HF pulses; (B) 8 × 100 μs pulses; (C) 8 × 5 ms pulses are used. The chosen optimal electric fields are encircled. Each data point presents the mean ± standard deviation from 3–4 experiments. ^* = statistically significant differences from control (p < 0.05) performing one-way ANOVA if the normality test passed or otherwise ANOVA on ranks. The light blue, red, and green asterisks are related to survival experiments.

FIGURE 4.

Cytotoxicity of cisplatin (A) and cisplatin molecules per cell (B) at different concentrations of cisplatin at a fixed electric field: 1.4 kV/cm for 50 × 50 HF pulses, 1.2 kV/cm for 8 × 100 μs pulses and 0.6 kV/cm for 8 × 5 ms pulses. Each data point presents the mean ± standard deviation from 3–4 experiments. ^*= statistically significant differences from control (p < 0.05) performing twoway ANOVA test. The color of the asterisk corresponds to the line color for a specific type of tested pulse. Cell survival as a function of cisplatin molecules per cell in combination with electroporation (C) our experimental data and (D) experimental data replotted from Vižintin et al. with permission.

FIGURE 5.

Comparison between the number of cisplatin molecules obtained experimentally (asterisks) and using the model (solid line) for (A) 50 × 50 HF pulses, (B) 8 × 100 μs pulses, (C) 8 × 5 ms pulses, (D) 1 × 200 ns pulses, and (E) 25 × 400 ns pulses. We used three different extracellular concentrations of cisplatin: 0 μM, 10 μM, 30 μM, and 50 μM.