Cellular Traffic And In Vitro Transfection Of Functional Peptides Decorated Gelatin-siloxane Nanoparticles As Non-viral Vectors

Gene therapy is a promising method for healing human disease at the level of genome. Conparring to viral gnen vectors, no-viral gene vectors, benefiting much from their good biocompatibility, have lower efficiency of the gene delivery and transfection. Gelatin-siloxane nanoparticles are novel organic-inorganic hybrid biomaterials with good abilities of DNA encapsulation and release. However, the potentials of nanoparticales based on gelatin-siloxane as novel gene vectors have not been systematic studied. Targeting the main barriers of gene delivery in cells, this thesis has operated a series of studies on the potentials of gelatin-siloxane-based nanoparticles as novel gene vectors. The main studis are as follows:(1) Cytocompatibility of gelatin-siloxane nanoparticles (GS NPs) and Tat decorated gelatin-siloxane nanoparticles (TG NPs) were analyzed by MTT assay. The entrances of GS NPs and TG NPs into Hela cells were investigated by both confocal laser scanning microscope (CLSM) and fluorescence activated cell sorter (FACS). Cell membrane-penetrating mechanisms of GS NPs and TG NPs were investigated by inhibited and co-localized experiments.(2) The fate of GS NPs after internalization into cells was investigated by CLSM and transmission electron microscope (TEM). By labeling lysosomes, the enhanced effects of chloroquine, Tat-peptides and Kala-peptides on the escape of GS NPs from lysosomes were discussed. However, chloroquine presents significate cytotoxicity when incubates with cells for long-period time (> 24 h) and Kala-peptides presents an instable effect on the escape of GS NPs. Moreover, the target abilities of Tat-peptides to nucleuses and pALDH leader-peptides to mitochondrias were investigated by labeling nucleuses and mitochondrias with fluorescence probes.(3) By labeling plasmid DNA, TG NPs were confirmed to transport plasmid DNA into Hela cells more effectively than liposomes analysed by FACS. By labeling both TG NPs and DNA, the release and distribution of plasmid DNA in intracellular environment were investigated using CLSM. Usingβ-galatosidase as report gene, the transfection abilities mediated by TG NPs were investigated. The effects of influence factors, such as Tat/GS ratio, TG/DNA ratio, DNA concentration and serum, on the transfection were systematically analysed to obtain the optimal condications for TG NPs medicated transfection.