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.

Development of highly stable and low toxic cationic liposomes for gene therapy.

Cationic liposomes with high stability and low cytotoxicity for gene therapy have been developed. Luciferase plasmid DNA (pLuc) was used as a model gene. The empty liposomes and niosomes were prepared by freeze dried empty liposomes (FDEL) method. The entrapment of pLuc in the liposomes was by reconstitution of the lyophilized dried vesicles with the plasmid solution. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM) and cryo-TEM. Cytotoxicity of the vesicular formulations was investigated on mouse melanoma cell lines (B16F10) by MTT assay. Cationic lposomes and niosomes containing the cationic lipid DDAB were less cytotoxic than other bilayer vesicular formulations. The pLuc entrapped in the cationic DPPC/Chol/DDAB liposomes (at 1:1:1 molar ratio) exhibited higher stability than other vesicular formulations and the pLuc in solution when stored at 4, 30 and 50 degrees C for 8 weeks. The entrapment efficiency determined by gel electrophoresis and gel documentation of the pLuc in this liposomal formulation was 100%. Luciferase gene expression of pLuc-loaded in cationic liposomes (lipoplexes) in HeLa cell lines was evaluated from luciferase activity determined by a luminometer at 24 and 48 h incubation. Percentages of cell proliferation of the lipoplexes on HeLa cell line at 24 and 48 h incubation were evaluated by the WST-1 assay. When the amount of DPPC or cholesterol was increased in the lipoplexes, the higher amount of DDAB was needed to protect pLuc from enzymatic degradation. However, DPPC and cholesterol exceeded 33 and 50% mol, respectively gave no gene expression. The DPPC/Chol/DDAB (at 1:1:1 molar ratio) lipoplex has demonstrated moderately lower luciferase gene expression and low cytoxicity. This lipoplex with the DDAB/pLuc weight ratio of 14:1 was the most desirable formulation for gene therapy because of its high stability, high luciferase gene expression and low cytotoxicity.