Plasmid containing pCMV-GFP was propagated in transformed Escherichia coli cells. E coli cells were grown in standard Luria Bertani medium at pH 7.0 to a cell density of ~3 to 4 × 109 cells/mL. Cells were harvested by centrifugation and the plasmid DNA was extracted and purified using a Qiagen Plasmid Maxi Kit (Qiagen, Santa Clarita, CA) as per the manufacturer?s recommended protocols. In brief, the plasmid purification procedure involved alkaline hydrolysis of the cells and isolation of the plasmid by binding to an anion-exchange resin of proprietary composition. The concentration of the purified plasmid preparation was 0.87 µg/µL determined using a UV-visible spectrophotometer (SpectraMax Plus, Molecular Devices, Sunnyvale, CA) using OD260 (optical density at wavelength 260 nm). The OD260/OD280 ratio was 1.964, indicating that the plasmid preparation was sufficiently pure and could be used for transfection purposes. Standard agarose gel electrophoresis on a 0.8% wt/wt agarose gel was conducted to investigate the plasmid structural integrity and revealed 2 major bands. The high-mobility band was attributed to the most compact or supercoiled form of plasmid DNA. The other band with low mobility indicated the overall nonsupercoil content in the plasmid preparation.
Formation of DNA-Anionic Liposome Complexes
For a single transfection experiment, 225 ng of DNA was diluted into a liposome suspension equivalent to 40 µg lipid in a centrifuge tube. Anionic lipoplex formation was achieved by addition of calcium chloride solution. Ca2+ ions were introduced into the formulation using stock solution of a 2 M calcium chloride solution that had been previously filter sterilized through a 0.2-µm filter. The amount of plasmid DNA was identical in all experiments irrespective of the Ca2+ concentrations (~7 mM to 132 mM) introduced into each complexation tube. The final volume of the lipoplex suspension applied to the cells was 300 µL. The complexes were incubated for 25 minutes at room temperature.