Real-time quantitation of intracellular polycation/DNA nanocomplex transport: evidence for directed motion

Multiple particle tracking (MPT) was used to study the mechanism of gene delivery with a non-viral DNA delivery system. Movements of hundreds of individual fluorescent PEI/DNA nanocomplexes were tracked through COS-7 cells and their transport properties were quantified by calculating their mean-square displacements (MSD). Average MSDs of complexes did not change significantly over the first 5 h post-transfection. At a time scale of 10 s, MSD values ranged from 0.21 to 0.57 μm², compared to < 0.32 μm² for 6 kb-sized naked DNA. Velocities calculated for individual particles showed some moved as fast as 0.26 μm/s, while others were almost immobile. Interestingly, 35% of the complexes were transported in a directed fashion 0.5 h after transfection (rather than random diffusion), a phenomenon that could not be detected by other methods such as Fluorescence Recovery After Photobleaching (FRAP). The percentage of cells undergoing directed transport increased to 62 % after 5 h. Studies such as these should allow the systematic optimization of the physicochemical properties of non-viral gene carriers for enhanced gene transfection efficacy.