Expression of luciferase plasmid (pCMVLuc) entrapped in DPPC/cholesterol/DDAB liposomes in HeLa cell lines.
The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 x 10(6) RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the zeta potential of 7.17 +/- 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 x 10(6) RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.
Manosroi
Aranya
A
Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand. pmpti005@chiangmai.ac.th
Thathang
Korakot
K
Manosroi
Jiradej
J
Werner
Rolf G
RG
Schubert
Rolf
R
Peschka-Süss
Regine
R
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D002412:Cations; D002460:Cell Line; D002784:Cholesterol / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008156:Luciferases / Q000235:genetics; D010316:Particle Size; D010957:Plasmids / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013010:Sonication
D002412:Cations; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; C015831:dimethyldioctadecylammonium; D015060:1,2-Dipalmitoylphosphatidylcholine; C081581:colfosceril palmitate; D002784:Cholesterol; D008156:Luciferases