Reversible and irreversible electroporation mechanisms: an in vitro study on two pancreatic cancer cell models

Electroporation (EP) is the increase of permeability of plasma membranes to ions and macromolecules, by intense and short pulsed electric fields (PEF), that induce an over-threshold increase of the physiological transmembrane voltage. Below a certain electric field threshold, the cell can restore the plasma membrane integrity (reversible EP, REP). Above the threshold, irreversible EP (IRE) occurs, leading to cell death due to extensive, non-repairable damage. The advantages of REP [1], and IRE [2] are widely recognized in biotechnology and medicine. Both REP and IRE applications rely on pulses with duration in the microsecond to millisecond time scale, whereas the main differences are in the number of applied pulses, and/or in the electric field strength. Despite the established applications, physical and biological mechanisms of EP have not been fully elucidated, which implies a poor control of PEF parameters, and definition of pulsing protocols by trials and errors. Indeed, a better insight into the role of pulse parameters in determining membrane permeabilization or cell death is crucial to control and optimize therapeutic protocols. Here we report on the preliminary results of the activities carried out in the framework of a national project (Digging into rEversible and irreversible ElectroPoration: in vitro and in silico multiphysical analyses on cEll modelS for cancer Treatment – DEEPEST, PRIN 2022, Italian Ministry of University and Research) which aims to develop and standardize a robust and sensitive methodology for a multi-physics (electromagnetic, thermal and biological), multi-level (single cells, 2D and 3D cell systems) analysis of EP mechanisms, under REP and IRE pulsing conditions, that can be tailored to different cell models, and used to improve outcome and optimization of pulsing protocols. First experiments were carried out on PANC-1 and MIA PaCa-2 pancreatic cancer cell lines to identify REP and IRE conditions to be further investigated for molecular characterization of the cellular stress mechanisms leading to damage and death. Cells in a low-conductivity pulsing buffer were exposed in 4 mm EP cuvettes by means of an ELECTROcell-B15 high voltage pulse generator (Leroy Biotech). Long (100 μs) voltage electric pulses, 1 Hz repetition rate, were applied with variable pulse number (1-50) and voltage-to-distance ratio (1 kV/cm – 2.5 kV/cm). A flow-cytometric method based on double-staining of samples with the fluorescent dyes calcein acetoxymethyl ester (CAM) and propidium iodide (PI) was used to quantify EP efficiency and cell death, and to identify REP and IRE pulsing conditions. Cell viability was assessed by the ability of cells to grow over a 24h and 48h period by means of MTT assay. The results of the ongoing activities will be presented at the conference.