Intracellular fate of lipoplex plasmid DNA

A marker for intracellular plasmid distribution is hugely excluded from the nucleus following cationic lipoplex transfection. Cultured HeLa cells and corresponding nuclei isolated using nonionic detergent P-40 (NP-40) were analyzed for a fluorescent plasmid marker using flow cytometry. Nearly all the transfected HeLa cells were positive for the plasmid marker, but only approximately 15% of the corresponding nuclei contained the marker 48 hours after transfection. A transcription-competent, rhodamine (Rh) labeled plasmid encoding for a CMV promoter driven green fluorescent protein (GFP) was used to monitor plasmid delivery and transgene expression, respectively, by flow cytometry. Transfection was conducted with lipoplex formed from 2 μg plasmid and 12 μg cationic liposomes (DOTAP:DOPE, 1:1 mol) per 2×10⁵ HeLa cells. Extracellular plasmid DNA was removed using CellScrub washing buffer prior to analysis. A subpopulation of cultured HeLa cells is better at both uptaking liposome-delivered plasmid DNA and expressing transgene. After transfection, the same fraction of cells was positive for both Rh plasmid marker and GFP transgene expression at two, eight, and 24 hours post-transfection. The intensity of the GFP fluorescence in individual cells was correlated with the amount of plasmid in the cell