Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression

In order to identify the important factors involved in cationic liposome-me diated gene transfer, in vitro transfection efficiencies by plasmid DNA com plexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios we re evaluated using four types of cultured cells with respect to their physi cochemical properties. Significant changes were observed in the particle si ze and zeta potential of the complexes as well as in their structures, asse ssed by atomic force microscopy, which depended on the mixing ratio. In tra nsfection experiments, except for RAW 264.7 cells (mouse macrophages), effi cient gene expression was obtained in MET-2 cells (mouse bladder tumor), NL H3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [P-32]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected b y the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxi city induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition o f negatively charged liposomes, was restricted as the mixing ratio increase d. Furthermore, confocal microscopic studies using HUVEC showed that the 1: 5 complexes exhibited a dispersed distribution in the cytoplasm whereas a p unctuate intracellular distribution was observed for the 1:10 complexes. Th is suggests that there was a significant difference in intracellular traffi cking, probably release from the endosomes or lysosomes, of the plasmid DNA /cationic liposome complexes between these mixing ratios. Taken together, t hese findings suggest that the DNA/liposome mixing ratio significantly affe cts the intracellular trafficking of plasmid DNA complexed with the cationi c liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection. (C) 2000 Elsevier Science B.V. All rights reserved.