Nanosecond pulses have traditionally been employed for the permeabilization of intercellular organelle membranes, however, the application of this technique can be challenging. An alternative approach is the use of microsecond pulsed electric fields (μsPEF) which provide the capability to regulate cytosolic signaling molecules such as calcium (Ca2+) concentrations. The increase in cytosolic Ca2+ has been shown to alter gene expression through the activation of Ca2+-responsive transcription factors [1]. Thus, μsPEF may serve as an external gene expression inducer in gene therapy by inducing Ca2+ influx.
In this study, we aimed to achieve ion influx through electropermeabilization of the endoplasmic reticulum (ER). Previous literature has indicated that the ER can be permeabilized by 100 μs PEF without adverse effects on cell viability [2]. To facilitate electropermeabilization, the extracellular conductivity was altered by modifying the extracellular medium composition. 100 μs and 1 Hz electric pulses were generated to disrupt intracellular organelle membranes. Cytosolic Ca2+ concentration changes were used as a marker for ER permeabilization. The increase in cytosolic Ca2+ concentration was observed in the HEK 293 cell line using fluorescence microscopy with the fluorescent calcium detection reagent Fluo-4. Furthermore, live cell impermeant SYTOXTM Green, a fluorescent nucleic acid stain, indicated that 0.6 kV/cm μsPEF caused plasma membrane damage.
Our results demonstrate that μsPEF treatment leads to an increase in cytosolic Ca2+ concentration, however, further studies are required to confirm the source of this increase by blocking specific Ca2+ transport system components. This study highlights the potential of easily applied microsecond pulses as a modulator of signaling molecule concentrations in gene therapy.