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-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 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 [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by 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 cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection.
Sakurai
F
F
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Inoue
R
R
Nishino
Y
Y
Okuda
A
A
Matsumoto
O
O
Taga
T
T
Yamashita
F
F
Takakura
Y
Y
Hashida
M
M
D000818:Animals; D002412:Cations; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA; D004996:Ethidium; D005456:Fluorescent Dyes; D015870:Gene Expression; D008081:Liposomes; D051379:Mice; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010957:Plasmids / Q000235:genetics*; D000644:Quaternary Ammonium Compounds; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D002412:Cations; D005456:Fluorescent Dyes; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D000644:Quaternary Ammonium Compounds; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; C094877:dioleoyl phosphatidylethanolamine; D004247:DNA; D004996:Ethidium