Electroporation (EP) is a method used to permeate cell plasma membrane (PM) by exploiting short electric pulsation to allow movement of extracellular and intracellular molecules across the PM. The technique may be used to deliver molecules such as oligo- and polynucleotides or small hydrophilic compounds into cells, and this is researched for application in cancer electrochemotherapy, gene therapy for treatment of neurological and congenital diseases as well as, in the case of oligonucleotide electrotransfection, development of cancer vaccines [1]. However, it is known that character of electrotransfer is dependent on mass, shape and charge of the compound, and different EP parameters must be used to effectively deliver substances of diverse characteristics [2,3]. Thus, the purpose of this investigation was to identify the nature of electrotransfer of molecules of different size: plasmid DNA (pDNA), oligonucleotides and propidium iodide (PI), in order to evaluate the influence of treatment conditions on electrotransfer efficiency and to conclude the most advantageous electroporation parameters – pulse voltage, duration and number.
Electroporation was performed on Chinese hamster ovary cells. The cells were suspended in low conductivity medium (0.1 S/m) and placed within a 2 mm gap between stainless steel electrodes. Propidium iodide and pDNA electrotransfer was performed by applying either 1, 5 or 10 LV (500 V/cm, 5 ms) pulses or 1 HV (1400 V/cm, 100 μs) pulse, and electrotransfer efficiency was determined via flow cytometry (in the case of pDNA transfection, GFP expression was evaluated 24 h after EP procedure). PI electrotransfer timelapse was further visualised via fluorescence microscopy using copper electrodes in coplanar configuration to deliver pulsation. Oligonucleotide transfection was performed upon the cells by applying a single pulse of varying duration and voltage (1500 V/cm 100 μs, 1200 V/cm 250 μs, 500 V/cm 5 ms), which allowed to identify optimum parameters that were subsequently further analysed by treating the cells with a different number of up to 10 pulses.
The results indicate that electrophoretic force caused by electrical pulses had considerable effect on oligonucleotide electrotransfer but had no significant impact on PI transfer. Conversely, PI electrotransfer appears not to be affected by total pulse duration and continues to occur after the external electric pulsation has ceased, which was not observed for oligo- and polynucleotide electrotransfection. Majority of the cells treated with 10 LV pulses remain permeable 15 minutes after the treatment was conducted, and this diminishes viability of the cells to up to 10 % of control viability. Furthermore, maximum pDNA expression was observed upon applying one HV pulse, whereas longer LV pulses had a negative impact on cell viability and negligible transfection rate. This, compared to oligonucleotide electrotransfer, indicates that the underlying phenomenon behind the uptake of these molecules into electropermeabilized cells is distinct, and additional research is required in order to establish optimum parameters to achieve effective nonconcurrent or simultaneous electrotransfer of nucleotides of varying size.