Fluorination Strategy

Multi-barriers have limited the gene transfection efficacy of polymeric gene vectors, including formation of gene complexes, cellular uptake, endosome escape and intracellular release of nucleic acids, etc. And cytotoxicity generated by gene vectors also limited their application on clinic. Many modification strategies based on polymeric gene delivery systems have been arised to solve these problems. Dendrimers were modified with various functional ligands such as amino acids, peptide, lipids, nanoparticles and other moieties. Although these strategies improve their gene transfection efficacies to some extent, it still remains many problems in gene delivery and sole modification strategy cannot overcome the multi-barriers simultaneously.In this doctoral dissertation, fluorination strategy was proposed to improve the gene transfection efficacy of traditional polymers. Fluorinated polymers can condense nucleic acids into nanoparticles via self-assembly according to the fluorine effect (F-F interaction) which also help to improve the stability of the carrier/DNA complexes. Fluorinated compounds are both hydrophobic and lipophobic, therefore fluorination can improve the affinity of polymers to cell membrane and across the lipid bilayer of the cell membrane, as well as the endosome/lysosome membrane, facilitating endosomal escape of the polymers. In addition, the fluorinated polymeric gene vectors may have great potential to realize the serum-resistance gene transfection because of the chemical and biological inertness of fluorinated compounds. The main researching methods and results are summerized as follows:Chapter 1:Gene transfection and its multi-barriers were overviewed; The advantages and applications of poly amindoamine (PAMAM) dendrimer as gene vectors were introduced; Commonly used modification strategies to improve the gene transfection efficacy of dendrimers were described; The properties of fluorine and fluorinated compounds were introduced emphatically; Finally, the research strategies and main contents of the thesis were given.Chapter 2: The perfluoroalkyl modified PAMAM dendrimers were designed and synthesized as gene vectors via fluorination strategy. The gene transfection efficacies and gene transfection mechanism of these fluorinated dendrimers were investigated in different cell lines. The results indicate that, G5-F768 can achieve >90% gene transfection efficacy and contain >90% cell viability at low dose which is superior to the commonly used and commercialized reagents. Fluorination can improve the cellular uptake, endosome escape and stability of gene complexes in serum containing medium. Fluorination regulates the condensation and intracellular release of DNA. In addition, fluorination also works well on other cationic polymers in gene delivery and represents a promising tool in the design of high efficient and low cytotoxic gene materials.Chapter 3:Then the influence of different dendrimer generation and grafting degree of perfluoroalkyl groups on gene delivery were investigated. Heptafluorobutyric anhydrides were reacted with PAMAM dendrimer at different ratios. After screening and comparison, the results show that fluorination improves the gene transfection efficiency of dendrimers with different generations. Additionally, different grafting degree of fluorinated groups significantly affect the transfection efficacy of dendrimer. The ratio within 55%～75% will achieve ～80% of gene transfection with fluorinated dendrimer. This research also analyses the structure-function relationship of fluorinated dendrimers.Chapter 4:In this chapter, fluorinated dendrimers were investigated for siRNA delivery. The results show that fluorinated dendrimers can achieve siLuc-mediated gene silencing efficacy. Under low dose of siRNA (0.05 nM), the gene silencing efficacy of fluorinated dendrimers is superior to the commercial Lipofectamine 2000. It works better on higher generation dendrimers with higher fluorination degree such as G5-F782, G6-F7172 and G7-F7327. Fluorination improves the stability and the cellular uptake of dendrimer/siRNA complexes and also regulate the condensation and intracellular release of siRNA. Therefore, Fluorinated dendrimers have a great promising in siRNA delivery which hold enormous value for further study.Chapter 5:Other fluorination methods on PAMAM dendrimer were explored in this chapter. Fluorobenzoic acids (FBAs) were modified on the surface of PAMAM to achieve the fluorinated dendrimers. The transfection efficacies and mechanism of gene delivery of these fluorinated dendrimers were also investigated. The results indicate that FBA modified PAMAM dendrimers can achieve high gene transfection efficacy with low cytotoxicity. Parameters such as the numbers and chemical structure of FBAs modified on dendrimer will impact its gene transfection efficacy significantly. G5-4FBA36 as the representative is superior to several commercial transfection reagents. In addition.fluorobenzoic acids modification improve the cellular uptake and endosome escape of dendrimer/DNA complexes. The aim of this study is to expand the application of fluorination strategy which is significative for further design and development of gene vectors.Chapter 6:Summarized the research content of this dissertation and looking forward to the future of fluorination strategy and its further applications.