(600a) Molecular Delivery to Cells Facilitated by Corona Ions

Corona discharge is a non-thermal plasma obtained by passing high voltage through a sharp or curved conductive object which results in the localized breakdown of air to create charged ionic species. The charge of these species depends on the polarity of the applied voltage. Corona discharge is common in many processes such as scrubbing impurities from air, photocopying, Kirlian photography, and laser printing. This study focused on determining if corona ions could be applied to biological cells to enhance the delivery of exogenous molecules.

Murine B16-F10 (ATCC CRL-6475) cells were grown in McCoy's 5A medium supplemented with 5 percent (v/v) and 50 µg/ml gentamicin sulfate in a humidified 37°C environment. Cells were plated in 18mm petri dishes 18 hours before each experiment. After aspirating the growth media, cells were exposed to corona ions and either calcein, bleomycin (tagged with Alexa-fluor) or SYTOX-green (neuclic acid probe). These molecules are non-permeant in cells with intact membranes. Corona ions were generated with custom made devices that utilized either a single wire or 9 needles to generate ions. An instrument system composed of a data acquisition card, high voltage power supply and LabVIEW program was used to control ion generation. Cells were exposed to either positive or negative ions for different times to facilitate delivery. Treated samples were incubated for 10 minutes and then spectrafluorametrically analyzed.

Experiments designed to deliver calcein using the single wire corona generator exposed the cells to ions for either 6 or 10 minutes. The resulting spectrafluorametric data indicate that B16 cells exposed to calein and corona ions had increased fluorescence relative to cells exposed to the dye alone. The 6 minute exposure time samples were 15.2 fold higher than the control (exposure to dye alone), and the 10 minute exposure samples were 30.2 fold higher than the controls. The single wire corona generator was also used to deliver AlexaFluor-bleomycin. Cells were exposed to 500 µM concentrations of this conjugate and treated with for either 6 or 10 minutes with corona ions. Samples that were treated with the fluorescing drug and corona charge had increased fluorescence relative to the samples that were exposed to the fluorescing drug alone. The 6 and 10 minute exposure times yielded 4.9 and 14.8 fold increases, respectively, relative to the appropriate controls. The 9 needle generator was used to deliver SYTOX-green nucleic acid stain. There was a significant increase in the amount of SYTOX (P = 0.003) delivered to the cells exposed to corona ions relative to those expose to SYTOX alone.

The results of this study demonstrate that corona discharge can be used as a means to facilitate the delivery of non-permeant molecules into biological cells. Future studies will focus on exploring this novel delivery method for drugs and genes to cells in-vitro and animal models.