Date of Award
Master of Science (MS)
The application of short, pulsed electric fields to eukaryotic cells and tissues has been shown to permeabilize cells. This phenomenon has been used for clinical applications for irreversible electroporation of cancer cells or for molecule delivery for drug or gene therapies. Typically, a monophasic (monopolar) pulse train is used; however, recent studies have explored the possibility of using biphasic (often referred to as bipolar) pulses, primarily for irreversible electroporation (IRE), which report reduced muscle contraction during pulse train application compared to monophasic pulses. Additional studies show improved transfection efficiency using biphasic pulses, conversely, with low cell viability. The purpose of this current study is to evaluate parameters of biphasic pulses for improving gene transfer in vitro. B16-F10, mouse melanoma cells were cultured, suspended, and treated with microsecond pulsed electric fields in a 1mm cuvette. Various pulsing parameters were used to deliver either propidium iodide (PI) or plasmid DNA encoding green fluorescent protein (GFP) to observe cell permeabilization and transfection. Cell viability was evaluated via PrestoBlue assay. Increasing pulse trains to 8 and increasing positive pulse width to 100 us at low voltage of 40 V, both resulted in significant changes in transfection efficiency with reduced viability. On the other hand, increasing voltage to 120 V shows significantly enhanced transfection efficiency with low viability. Lastly, reducing positive pulse width to 20 us at 120 V applied, resulted in high transfection efficiency at 43% with high cell viability at 84%. This study shows that biphasic pulses enhance gene delivery of plasmid encoding GFP into B16-F10 and maintain high cell viability in vitro. These results are consistent with earlier studies that gene delivery enhancement is feasible with biphasic pulses. Additional, future studies will evaluate whether such gene delivery enhancement can be maintained in excitable cells without actional potential activation.
"Biphasic Gene Electrotransfer Enhances Gene Delivery In Vitro"
(2020). Master of Science (MS), Thesis, Electrical/Computer Engineering, Old Dominion University, DOI: 10.25777/cy9q-w007