EFFECTS OF NANOSECOND PULSES DELIVERED AT ULTRA-HIGH FREQUENCY ON MITOCHONDRIAL DEPOLARIZATION, ROS, CELL PERMEABILIZATION AND CELL VIABILITY

It is theoretically predicted that ultrashort nanosecond pulses could be utilized to permeabilize intracellular membranes, including mitochondria [1]. Such a phenomenon would have high applicability in cancer treatment context and could be employed for shifting toward immunogenic cell death as well as induction of immune response [2]. However, currently there is a lack of experimental evidence on the interaction of pulsed electric fields with mitochondria and their function. In this study, for the first time, we characterize the effects of ultra-high repetition frequency (1–5 MHz) nanosecond pulses (6–18 kV/cm, 100 and 300 ns) in the context of mitochondrial depolarization (Tetramethylrhodamine, methyl ester (TMRM)), ROS generation (MitoSOX), cell plasma membrane permeabilization (YO-PRO-1) and cell viability. As a reference, standard microsecond range parametric protocols were used (100 μs × 8 pulses). Our pilot data indicate that depending on the pulse repetition frequency, the efficiency of electroporation can be modulated, i.e., the higher is the frequency, the higher is the permeabilization. Also, the oxidation level has a dose-dependent scaling with pulse burst amplitude, while 100 ns pulses induce significantly lower oxidation than 300 ns pulses. In the context of TMRM fluorescence, our data indicate that treatment of cells with pulsed electric fields induces mitochondrial depolarization, however, the selectivity of the phenomenon is questionable and requires further research. Importantly, the results of our study show that boosting the pulse repetition frequency towards 5 MHz significantly improves the effects of 100 ns pulses, i.e., the protocols cause effective electroporation even in fields, which are considered sub-threshold for this pulse duration range. Therefore, derivation of new and more effective protocols for electroporation-based procedures can be performed without the increase of the energy input.