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Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses

Angelika Vizintin
Angelika Vizintin
, 
Stefan Markovic
Stefan Markovic
, 
Janez Scancar
Janez Scancar
, 
Jerneja Kladnik
Jerneja Kladnik
, 
Iztok Turel
Iztok Turel
 and 
Damijan Miklavcic
Damijan Miklavcic
   | Aug 14, 2022
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Article Category: research article
Published Online: Aug 14, 2022
Page range: 326 - 335
Received: Jun 07, 2022
Accepted: Jun 19, 2022
DOI: https://doi.org/10.2478/raon-2022-0028
© 2022 Angelika Vizintin, Stefan Markovic, Janez Scancar, Jerneja Kladnik, Iztok Turel, Damijan Miklavcic, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Cell survival of (A) CHO and (B) B16F1 cells at different cisplatin concentrations determined by the clonogenic assay for non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the survival of non-electroporated cells without cisplatin. Survival data were combined from the previous8 (for non-electroporated cells and cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse).
Cell survival of (A) CHO and (B) B16F1 cells at different cisplatin concentrations determined by the clonogenic assay for non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the survival of non-electroporated cells without cisplatin. Survival data were combined from the previous8 (for non-electroporated cells and cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse).

Figure 2

Pt amount in cell pellets of (A) CHO and (B) B16F1 cells after 25 min incubation at different extracellular cisplatin concentrations in non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/ cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the measured number of cisplatin molecules in non-electroporated cells at the same extracellular cisplatin concentration.
Pt amount in cell pellets of (A) CHO and (B) B16F1 cells after 25 min incubation at different extracellular cisplatin concentrations in non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/ cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the measured number of cisplatin molecules in non-electroporated cells at the same extracellular cisplatin concentration.

Figure 3

Cell survival as a function of the number of cisplatin molecules per cell for (A) CHO cells and (B) B16F1 cells in non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation. Survival data were combined from the previous8 (for non-electroporated CHO cells and CHO cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse).
Cell survival as a function of the number of cisplatin molecules per cell for (A) CHO cells and (B) B16F1 cells in non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation. Survival data were combined from the previous8 (for non-electroporated CHO cells and CHO cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse).

Figure 4

1H NMR spectra of cisplatin, showing the signals for hydrogens of NH3 ligands labeled with asterisks (*). Spectra were recorded in a) D2O, b) D2O containing 154 mM NaCl, c) 90% H2O/10% D2O and d) 90% H2O/10% D2O containing 154 mM NaCl treated with 25 × 400 ns pulses (blue), 8 × 100 μs pulses (green) or no pulses (red).
1H NMR spectra of cisplatin, showing the signals for hydrogens of NH3 ligands labeled with asterisks (*). Spectra were recorded in a) D2O, b) D2O containing 154 mM NaCl, c) 90% H2O/10% D2O and d) 90% H2O/10% D2O containing 154 mM NaCl treated with 25 × 400 ns pulses (blue), 8 × 100 μs pulses (green) or no pulses (red).

Figure 5

The mechanism of cisplatin uptake into cells is not completely elucidated. In non-electroporated cells, cisplatin enters partially through passive diffusion and facilitated diffusion through ion channels including LRRC8 volume-regulated anion channels (VRAC) and membrane transporters like copper transporter 1 (CTR1) and organic cation transporters (OCTs). In electroporated cells, more cisplatin can enter through the permeabilized cell membrane (pore is a symbolic presentation of increased membrane permeability even though the mechanisms behind electroporation are more complex – refer to34).
The mechanism of cisplatin uptake into cells is not completely elucidated. In non-electroporated cells, cisplatin enters partially through passive diffusion and facilitated diffusion through ion channels including LRRC8 volume-regulated anion channels (VRAC) and membrane transporters like copper transporter 1 (CTR1) and organic cation transporters (OCTs). In electroporated cells, more cisplatin can enter through the permeabilized cell membrane (pore is a symbolic presentation of increased membrane permeability even though the mechanisms behind electroporation are more complex – refer to34).
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