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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).
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.
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).
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).
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).