Delivery and expression of plasmid DNA into cells by a novel non-thermal plasma source.

Medical applications such as plasma assisted gene transfer is a minimally invasive approach that can substantially reduce potential discomfort of treated area. Atmospheric pressure plasma discharge is an effective approach to deliver plasmid DNA for in vitro and in vivo applications. We investigated plasma assisted delivery in vitro in mouse melanoma cells (B16F10) using a novel surface plasma device, which is operated in air. We evaluated the influence of applied voltage and distance between the surface device and cell monolayer. We found no significant effect on the viability of cells. Highest expression following delivery of a plasmid encoding green fluorescent protein was achieved with an applied voltage of 11.25 kV at a 2 mm distance and 5 s exposure time. To better understand the influence of oxidative damages and stress on cells after plasma delivery, a mRNA expression study was performed. Our results indicated that TNFα mRNA was significantly upregulated. The mRNA response may be attributed to the RONS generated by plasma; however, this mRNA upregulation was not adequate to be reflected in a coordinate protein upregulation. From the results reported here, it is clear that this novel plasma device could be used for plasmid delivery.

Membrane damage and active but nonculturable state in liquid cultures of Escherichia coli treated with an atmospheric pressure plasma jet.

Electrical discharge plasmas can efficiently inactivate various microorganisms. Inactivation mechanisms caused by plasma, however, are not fully understood because of the complexity of both the plasma and biological systems. We investigated plasma-induced inactivation of Escherichia coli in water and mechanisms by which plasma affects bacterial cell membrane integrity. Atmospheric pressure argon plasma jet generated at ambient air in direct contact with bacterial suspension was used as a plasma source. We determined significantly lower counts of E. coli after treatment by plasma when they were assayed using a conventional cultivation technique than using a fluorescence-based LIVE/DEAD staining method, which indicated that bacteria may have entered the viable-but-nonculturable state (VBNC). We did not achieve resuscitation of these non-culturable cells, however, we detected their metabolic activity through the analysis of cellular mRNA, which suggests that cells may have been rather in the active-but-nonculturable state (ABNC). We hypothesize that peroxidation of cell membrane lipids by the reactive species produced by plasma was an important pathway of bacterial inactivation. Amount of malondialdehyde and membrane permeability of E. coli to propidium iodide increased with increasing bacterial inactivation by plasma. Membrane damage was also demonstrated by detection of free DNA in plasma-treated water.