Preparation And Gene Transfection Efficiency Of L-(+)-tartaric Acid-based Cationic Lipids

Objective:Gene therapy requires safe and efficient vectors to deliver the gene into target cells or nucleus. The vectors for gene delivery are generally divided into viral vector and nonviral vector. Nonviral vector has the advantages of high loading, excellent stability, high security and easily manufactured in a large scale and so on. Cationic lipid is the most widely studied nonviral vector, which is potential to be applied in gene therapy. In this thesis, L-(+)- tartaric acid was developed firstly as the backbone to design and synthesize cationic lipids for highly effective gene delivery, in which 6-aminocaproic acid was used as the headgroup, and different saturated hydrocarbon chains were introduced as the hydrophobic domain. Method:Cationic lipids TS1-TS3 and TD1-TD3 were designed and synthesized using L-(+)- tartaric acid, 6-aminocaproic acid and saturated hydrocarbon chains as starting materials. 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 mol ratio of lipid and DOPE, and characterized by Delsa? Nano C Particle Analyzer. The toxicity of different blank cationic liposomes was also tested by MTT method on 293 T cells. The gel electrophoresis was carried out to investigate the binding and protecting capacity of different cationic liposome to plasmid DNA.The p EGFP-N1 plasmid was used as the model gene and the transfection efficiency of each cationic liposome was evaluated on 293 T cells by flow cytometry and fluorescence microscopy. The cationic liposomes which performed good transfection activity in the preliminary screening experiments were further optimized and evaluated on He La cells. To elucidate the endocytic uptake mechanisms of TD2/DNA complexes, specific inhibitors were applied to investigate the effect on cellular internalization. Results:In this thesis, we synthesized six L-(+)- tartaric acid-based cationic lipids and the structure of all synthesized lipids was confirmed by MS, 1H NMR and IR.The results of size and zeta potential indicated that the cationic liposomes based on the above-mentioned cationic lipids have acceptable size and zeta potential for the gene transfection. The results of cytotoxicity showed that except for TD1, the cationic liposomes based on the above-mentioned cationic lipids have low cytotoxicity and good biocompatibility as gene vectors.All these derivatives can completely retard DNA over the N/P molar ratio of 3, which showed the strong DNA binding and protecting ability. Compared with naked DNA, TS1-TS3 based cationic liposomes had little transfection efficiency and TD1 based cationic liposomes showed weak transfection efficiency while TD2 and TD3 based cationic liposomes showed promising transfection efficiency among other lipids on 293 T cells. Then we optimized the formulation of TD2 and TD3 based cationic liposomes on 293 T cells. The results indicated that the optimized molar ratio of lipids/DOPE for TD2 and TD3 based cationic liposomes was 1:0.5 and 1:2. The MFI of TD2(MFI 153.3 ± 14.7) and TD3(MFI 190.5 ± 7.8) was comparable to the commercially available transfecting reagent DOTAP(MFI 171.1 ± 14.5), but the percent of positive transfected cells of TD2(47.7% ± 7.0%) and TD3(32.9% ± 2.0%) was higher than DOTAP(24.7% ± 1.8%; P < 0.01). The results of transfection efficiency for TD2 and TD3 at their optimum formulation showed that while the MFI of TD2(MFI 53.8 ± 6.2) and TD3(MFI 54.7 ± 2.0) was lower than DOTAP(MFI 98.0 ± 1.8, P < 0.01), the percent of positive transfected cells of TD3(19.6% ± 0.9%) was comparable to DOTAP(18.3% ± 0.9%) and the percent of positive transfected cells of TD2(25.6% ± 0.4%) was still higher than DOTAP. The effects of specific inhibitors on transfection efficiency indicated that TD2/p DNA was uptaken into cells mainly through caveolae-mediated endocytic pathway. Above all, we can conclude that TD2 based cationic liposomes behaved efficient transfection performance, which can offer an excellent prospect as the nonviral vectors for gene delivery. Conclusion:In this thesis, we constructed six cationic lipids with the new backbone of L-(+)-tartaric acid, in which 6-aminocaproic acid was used as the headgroup, and saturated hydrocarbon chains were used as hydrophobic domain. The results showed that, except for TD1, the cationic liposomes based on the above-synthesized cationic lipids have acceptable size, low cytotoxicity and strong carrying capacity as gene vectors. The TD2 based cationic liposome at its optimum formulation had higher transfection efficiency both in 293 T and He La cell lines than that of DOTAP. The TD2/DNA complex was uptaken mainly through caveolae-mediated endocytosis, which could avoid lysosomal degradation and was beneficial for gene transfection. The results from this study should further facilitate the rational design of nonviral gene delivery systems for clinical applications.