Negative effects of cancellation during nanosecond range High-Frequency calcium based electrochemotherapy in vitro.

Drug delivery using nanosecond pulsed electric fields is a new branch of electroporation-based treatments, which potentially can substitute European standard operating procedures for electrochemotherapy. In this work, for the first time, we characterize the effects of ultra-fast repetition frequency (1-2.5 MHz) nanosecond pulses (5-9 kV/cm, 200 and 400 ns) in the context of nano-electrochemotherapy with calcium. Additionally, we investigate the feasibility of bipolar symmetric (↑200 ns + ↓200 ns) and asymmetric (↑200 ns + ↓400 ns) nanosecond protocols for calcium delivery. The effects of bipolar cancellation and the influence of interphase delay (200 ns) are overviewed. Human lung cancer cell lines A549 and H69AR were used as a model. It was shown that unipolar pulses delivered at high frequency are effective for electrochemotherapy with a significant improvement in efficiency when the delay between separate pulses is reduced. Bipolar symmetric pulses trigger the cancellation phenomenon limiting applications for drug delivery and can be compensated by the asymmetry of the pulse (↑200 ns + ↓400 ns or ↑400 ns + ↓200 ns). The results of this study can be successfully used to derive a new generation of nsPEF protocols for successful electrochemotherapy treatments.