DC coronation electroporation.

Abstract

Cells are surrounded by a semi-permeable bilayer lipid membrane that acts as a barrier against the entry of foreign molecules. In the fields of molecular biology, biotechnology, and medicine, the ability to breach the cell membrane and introduce molecules into cells for therapeutic purposes is often necessary. Molecules, which are considered foreign to the cell like drugs and extraneous genetic materials, are administered to cells for numerous applications including the treatment and prevention of diseases. There are many accepted methods of facilitating the delivery of molecules to cells. Of all these methods, one important and well-established physical method is electroporation which has been utilised for decades. Electroporation is a widely adopted procedure for the temporary permeabilization of cell membranes due to the application of short electrical pulses. It is a phenomenon resulting from the effects of pulsed electric fields, which induces biochemical and physiological changes to a cell membrane. As a result, some of the molecules that are ordinarily unable to pass through the membrane are thereafter able to gain access to the cell interior via pores that are formed in the membrane. Even though electroporation is fairly safe, there are some drawbacks associated with this method. The traditional method of electroporation requires direct contact of high voltage electrodes and fairly high currents are involved. As a result, the procedure can cause pain, muscle spasms, discomfort, burning and cell and tissue damage. Alternative methods of molecular delivery are therefore being researched, especially non-contact methods such as the use of high voltage plasma and high voltage corona discharge. Successful cell permeabilization with corona discharge ions and plasma has been previously demonstrated. These methods offer the advantage of contact-free treatment with low associated current. In this thesis, the research investigates the delivery of tracer molecules, SYTOX Green, into HeLa cells and the consequential cell destruction by the phenomenon of corona discharge. A high voltage DC, multipoint-to-plane atmospheric-air corona discharge apparatus was designed and constructed to investigate the conditions as well as the characteristics of the corona discharge current pulses that resulted in an acceptable balance between high cell permeabilization and low cell destruction. Firstly, the salient variables that affect molecular delivery and cell destruction were established. Secondly, the variables were optimized to allow for reliable molecular delivery to cells with acceptable levels of cell destruction. Thirdly, the nature and variation of the corona discharge current pulses and its effect on molecular delivery and cell destruction were investigated. Finally, a new method of assessing cell destruction, which combined the measurements of cell viability and cell lysis were used. The variables that were identified, over the course of many experiments, were exposure time to corona discharge, incubation time with SYTOX Green, volume of liquid during exposure, and inter-electrode distance. Further experiments show that when the variables of the experiment are set at optimal values, cell permeabilization is reliable with minimal damage to cells. Once these conditions were obtained and optimised, the effect of different applied voltages on the level of cell permeabilization and the short-term destructive effects on cells were investigated. The general trend is an increase in fluorescence and therefore, molecular delivery, with an increase in applied voltage. Cell destruction also tends to increase with increasing applied voltage. The characteristics of the corona current pulses that were analyzed include amplitudes, repetition rates, widths, and rise-times. The characteristic frequencies of single pulses, obtained from the application of a discrete fast Fourier transform, were also analyzed. For the corona-generating device constructed and the voltages tested, it was found that the only characteristic that varies appreciably with voltage is the pulse repetition rate. A higher pulse repetition rate relates to a greater number of pulses per unit time and therefore, a greater exposure of the cells to the applied electric field. This would, therefore, translate to a higher extent of molecular delivery and a higher accompanying level of cell destruction. This study shows that permeabilization of HeLa cells due to corona discharge can be reliably achieved and the results provide a greater understanding of cell permeabilization due to the influence of corona discharge. It therefore forms an important basis for future research on practical applications that would promote the establishment and acceptance of corona discharge as a procedure for molecular delivery to cells.