Calcium electroporation causes ATP depletion in cells and is effective both in microsecond and nanosecond pulse range as a modality of electrochemotherapy.

Calcium electroporation is a modality of electrochemotherapy (ECT), which is based on intracellular electric field-mediated delivery of cytotoxic doses of calcium into the cells resulting in rapid cell death. In this work, we have developed a CHO-K1 luminescent cell line, which allowed the estimation of cell membrane permeabilization, ATP depletion and cytotoxicity evaluation without the use of additional markers and methodologies. We have shown the high efficiency of nanosecond pulses compressed into a MHz burst for application in calcium ECT treatments. The 5 kV/cm and 10 kV/cm nanosecond (100 and 600 ns) pulses were delivered in bursts of 10, 50 and 100 pulses (a total of 12 parametric protocols) and then compared to standard microsecond range sequences (100 µs × 8) of 0.4-1.4 kV/cm. The effects of calcium-free, 2 mM and 5 mM calcium electroporation treatments were characterized. It was shown that reversible electroporation is accompanied by ATP depletion associated with membrane damage, while during calcium ECT the ATP depletion is several-fold higher, which results in cell death. Finally, efficacy-wise equivalent pulse parameters from nanosecond and microsecond ranges were established, which can be used for calcium nano-ECT as a better alternative to ESOPE (European Standard Operating Procedures on Electrochemotherapy) protocols.