Abstract
Abstract3b-[N-(N′N′–dimethylaminoethane) carbamoyl] cholesterol (DC-chol) liposomes are the first approved cationic liposomes by FDA for clinical trials, and their safety have been demonstrated in treating cystic fibrosis in vivo. Nowadays, the cationic liposomes composed of DC-Chol and dioleoylphosphatidylethanolamine (DC-Chol/DOPE) have been often used as nonviral gene delivery vectors, while their application is severely hampered by low transfection efficiency and poor serum stability. This protocol describes the preparation of lyophilized PEGylated DC-Chol/DOPE immunoliposomes (LPIL), which are conjugated with the Fab’ of humanized anti-HER2 monoclonal antibody for siRNA delivery through lyophilization/rehydration. Briefly, the cationic liposomes were prepared by a lipid film method, and the Fab’ of humanized anti-HER2 monoclonal antibody was mixed with cationic liposomes containing maleimide-terminated linker. Afterward, the HER2-specific PEGylated immunoliposomes (LPIL) were lyophilized and rehydrated with a certain amount of diluted siRNA solution for encapsulating siRNA. The resultant LPIL had a size of 150 nm and a zeta potential of 40 mv. The results showed that the lyophilization/rehydration method could significantly increase the transfection efficiency and gene silencing of immunoliposomes in HER2-overexpressing cells. In addition, PEGylation degree had evident effects on transfection efficiency and gene silencing activity of the liposomes. Results indicated that 2.5% PEG LPIL showed the best transfection efficiency and gene silencing activity. In view of the serum stability, siRNA in 2.5% PEG LPIL showed the lowest degrading rate, indicating that 2.5% PEG LPIL provided superior protection for siRNA. In conclusion, 2.5% PEG LPIL provided the best transfection efficiency, gene silencing efficiency, and siRNA protection, hence representing a promising approach for gene therapy.KeywordsCationic liposomessiRNADOPEDC-CholLyophilization/RehydrationGene silencing
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