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To investigate the cytotoxicity of the homemade peptide cationic liposome CDO14 and its efficacy of RNA interference (RNAi). MTT method was used to determine the cytotoxicity of the liposome to a human lung cancer cell line Luc-A549 that can express luciferase stably. Luciferase siRNA (Luc-siRNA) was transfected into Luc-A549 cells by CDO14. Contents of luciferase in the transfected cells were detected by luminous instrument and contents of total protein in these cells were detected by BCA method. Nude mice were inoculated with Luc-A549 cells in axilla to establish xenograft tumor model. Complexes of Luc-siRNA and the cationic liposomes were injected into the modeling mice via tail vein. Contents of luciferase in the transfected mice were detected by the whole body imaging system. The cytotoxicity of the homemade cationic liposome was similar to that of commercial liposome DOTAP, and lower than that of Lipo2000. The siRNA transfection efficacy mediated by CDO14 was higher than that mediated by DOTAP. The homemade peptide cationic liposome CDO14 is expected to serve as delivery vector in gene therapy because of its low cytotoxicity and high transfection efficiency.

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 μM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 μM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).

In order to study the efficiency of small interfering RNA (siRNA) transfer mediated by cationic liposome, we used luciferase siRNA to evaluate the gene silencing activity in the Hep-2 cells, which were stably transduced with a luciferase gene. The pDNA transfection was studied, and siRNA arrearage assay was conducted to determine the capability of cationic liposome with siRNA. Different concentrations of siRNA was used to silence luciferase gene' activity, and then the result was examined by microplate reader. Cell viability was analyzed after transfection by MTT assay. The results suggested that Lipofectamine 2000 could transfer the pDNA efficiently, and have strong binding capacity with siRNA. The silencing efficiency of luciferase was obtained with low concentration of siRNA. The cell viability was influenced by RNA interference (RNAi) very slightly, but the cell survival rate decreased with the increase of siRNA concentrations. It was well concluded that by optimizing the experimental conditions, cationic liposome can transfer low concentration siRNA to silence target gene's activity efficiently.

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2-3-fold than DOTAP/pDNA control and 70-120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.