Strategies In The Design Of High Efficient And Safe Dendrimer-based Gene Vectors

As one kind of synthetic cationic polymers, dendrimers with excellent monodispersity, controllable size and surface functionality, good solubility and biocompatibility, are emerging as a new class of non-viral gene vectors. Amine-terminated dendrimers have well-defined numbers of primary amine which can condense nucleic acid into nano-scale size. Tertiary amine groups in the interior pockets have pH buffering ability, which enhances the endosomal escape of the dendrimer/DNA complexes. There have been a lot of research works about dendrimer based gene delivery. Dendrimer-based gene transfection reagents such as SuperFect(?) and PolyFect(?) have already entered the market. However, dendrimer-based gene vectors are associated with the "malignant" correlation between transfection efficacy and cytotoxicity. (1) Dendrimers with high molecular weight have high transfection efficacy but devastating toxicity. (2) Dendrimers with higher nitrogen to phosphorus (N/P) ratios to DNA result in superior transfection efficacy but worse biocompatibility. Therefore, there is an urgent need to break up correlation between transfection efficacy and cytotoxicity.The research in this dissertation focuses on solving the problem about the correlations above. The strategies designed are as follows:(1) A reversible cross-linking strategy was proposed to prepare high efficient and low toxic gene vectors. Disulfide containing linker was used to cross-link low molecular weight PAMAM dendrimers (molecular weight:3256 Da) to form supramolecular structures. The synthesized materials can effectively condense DNA and protect DNA from DNase degradation. Disulfide cross-linked dendrimers showed much improved gene transfection efficacy than G2 and G5 dendrimers. The most efficient material (DCD3) exhibited comparable efficacy to branched PEI with a molecular weight of 25 kD and is more biocompatible than these cationic polymers. We verified that the disulfide bond reduction occuring in the endocytic pathway regulated DNA release through polymer and played a key role for efficient and low toxic gene transfection. In summary, we used the cross-linking strategy to prepare low generation PAMAM dendrimer-based gene vectors with high gene transfection efficacy and low cytotoxicity.(2) A fluorination strategy was proposed to prepare high efficient and low toxic gene vectors. A series of fluorinated dendrimers were synthesized by a facile route. The fluorophilic effect between fluorocarbon chains of the fluorinated dendrimer improves the stability of the carrier and DNA complexs, which makes the fluorinated dendrimers form stable complex and achieve high transfection efficacy with low toxic at low N/P ratios. For example, the fluorinated dendrimer G5-F768 showed 90% EGFP gene expression at N/P ratio of 1.5 in HEK293 cells and N/P ratio of 2:1 in HeLa cells. Even at an extremely low N/P ratio of 0.5:1,50% EGFP gene expressions were observed. Besides in HEK293 and HeLa cells, the fluorinated dendrimer G5-F768 also showed excellent gene transfection efficacy and biocompatibility in several cell lines such as CHO, COS-7, MB231, A549, U2OS, NIH3T3 and MG63 cells and these polymers show superior efficacy compared with several commercial transfection reagents such as Lipofectamine 2000. High efficacy of G5-F768 achieved at low N/P ratio about 2:1 and gene transfection with low N/P ratios leads to low cytotoxicity. In summary, using fluorination strategy, we prapared high efficient and low cytotoxic gene materials with low N/P ratios.(3) Based on the low molecular dendrimers and low N/P ratios, fluorination strategy was used for designing high efficient and low cytotoxic gene materials. Transfection efficacies of PPI dendrimers with relatively low molecular weight are not as high as other representative cationic polymers. Here, fluorinated PPI dendrimers were synthesized by modification G3, G4 and G5 PPI dendrimers with heptafluorobutyric acid. These fluorinated PPI dendrimers achieved high transfection efficacy with low toxicity at low N/P ratios. For example, fluorinated low molecular weight G3 PPI dendrimers (molecular weight:1687 Da) showed 80% EGFP gene expression at N/P ratio of 1.6. These Fluorinated materials showed comparable or superior transfection efficacies to four commercial transfection reagents such as Lipofectamine 2000, jetPEI, SuperFect and PolyFect. The optimized N/P ratios of fluorinated PPI dendrimers were about 2 and the transfected cells maintained high cell viability around 90%. These fluorinated PPI dendrimers showed high EGFP transfection efficacy on 3D multicellular spheroids. Such a fluorination strategy allows us to design efficient and low cytotoxic gene materials based on low molecular weight PPI dendrimers with low N/P ratios.(4) The fluorination strategy was uesd to prepare high efficient and low toxic siRNA vectors. A library of fluorous compounds modified PAMAM dendrimers (>70) was built to discover more vectors with excellent gene transfection efficacy and low toxicity. Gene silencing results showed about ten fluorinated dendrimers induced more than 50% gene knockdown efficacy with low toxicity. And three fluorinated dendrimers achieved 80% gene knockdown efficacy at extremely low N/P ratios of 0.84:1,0.75: 1 and 1.5:1. While G5 PAMAM dendrimer only showed extremely low gene silencing efficacy (< 10%) at N/P ratio of 18:1. Above on, we developed several siRNA vectors with high gene transfection efficacy and low cytotoxicity based on the fluorination strategy.Taken together, based on "malignant" correlation between transfection efficacy and cytotoxicity in dendrimer gene delivey, reversible cross-linking strategy and fluorination strategy were proposed to prepare several high efficient and low cytotoxic dendrimer-based gene vectors.