Extracellular-Ca²⁺-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses

Electroporation-a transient electric-field-induced increase in cell membrane permeability-can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca²⁺ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl₂ effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl₂ concentration is increased.