Design, Synthesis And Gene Transfection Efficiency Of Cholesterol-based Cationic Lipids

Objective:Gene therapy must use some vectors to bring the gene to pass through the cell membrane and adjust the expression of the targeted gene effectively. The vectors for gene therapy are divided into viral vector and non-viral vector. Non-viral vector, a new gene transfection system, has lots of advantages, such as low toxicity, low immune response, simple preparation and convenient application. Cationic liposome as a commonly used non-viral vector, has the advantages of easy production, low immunogenicity, protecting gene from nuclease degradation and non-carcinogenic activity, which lead to a broad prospect in the development of novel non-viral vectors for gene transfection. In this thesis, considering the advantages of cholesterol-based hydrophobic group and the development of amino head groups and linkages in gene transfection, we choose cholesterol as starting materials and use a linkage to bridge and introduce different amine groups in its side chain to design and synthesize three series of cholesterol-based cationic lipids. Method:We choose cholesterol, 1, 6-hexanediol, basic amino acid, dimethylamine hydrochloride, diethylamine, triethylamine, 1, 4-dibromoethylene, 1, 4-dibromobutane, 1, 6-dibromohexane and 1, 12-dibromododecane as starting materials to design and synthesize three series of cationic lipid materials,including lipid M with a single amino head group, lipid A with amino acid head group and lipid G as gemini lipids. The structure of all synthesized lipids was confirmed by MS, 1H NMR and IR.Cationic liposomes were prepared by film dispersion method at the equal mole ratio of lipids and DOPE, and characterized by Delsa? Nano C Particle Analyzer.Gel electrophoresis was used to investigate the binding capacity of different cationic liposomes for plasmid DNA. The toxicity of different blank cationic liposomes was also tested by MTT method.Using p EGFP plasmid as a model gene, the transfection efficiency of each cationic liposome was evaluated by flow cytometry and fluorescence microscopy. The cationic lipids which performed good transfection activity in the preliminary screening experiments were further optimized and evaluated in the presence of serum. Results:Three series of cholesterol-based cationic lipid materials were prepared. The structure of synthesized 16 compounds were consistent with that of target ones according to 1H NMR, MS and IR.The particle size and Zeta potential of cationic liposomes prepared by film dispersion method meet the requirements of gene vectors. The results of MTT experiments showed that, IC50 of all kinds of cationic liposomes are greater than the positive control Lipofectamine 2000(IC50 = 33 μM). When compared with positive control DC-Chol(IC50 = 51 μM), the lipids have low toxicity and good biocompatibility, except for cationic liposomes based on lipid A4(IC50 = 48 μM), A5(IC50 = 49 μM), G1(IC50 = 44 μM) and G4(IC50 = 35 μM).The results of transfection experiments showed that cationic liposomes based on lipids M1, M6 and A1-A6 have better gene transfection efficiency than DC-Chol(p < 0.05 or p < 0.01). Among them, the transfection efficiency of cationic liposomes based on M6 and A4 was similar to Lipofectamine 2000(p > 0.05), and A6 showed higher transfection activity than Lipofectamine2000(p < 0.05). After optimization of the lipid-DOPE molar ratio and the N/P charge ratio, M1 and M6 showed the best transfection efficiency in all prepared cationic liposomes, higher than DC-Chol(p < 0.05) and Lipofectamine 2000(p < 0.05) even in the presence of serum. Conclusion:In this thesis, three series of cationic lipids including 16 compounds were constructed through introducing different amino head groups to cholesterol skeleton. The results of cytotoxic activity and gene transfection studies showed that the cationic liposomes based on the above-mentioned cationic lipids have acceptable size, low cytotoxicity and strong carrying capacity as gene vectors. All the cationic liposomes have good transfection activity except for the liposomes based on M3, M5 and G1. In the presence of serum, the transfection efficiency of M1, M6, A2 and A3 was higher than DC-Chol, wherein the M1 and M6 were superior to Lipofectamine2000. Through the further in vivo transfection studies, it is promising to get safe and effective vectors for gene therapy.