Study On The Effect Of Monomer Sequence On Gene Transfection In Fluoropolymer Gene Carrier

In the last three decades,gene therapy has shown great advantages over other therapeutic methods in the clinical treatment of many diseases,especially in the genetic and acquired gene phenotype-related treatment.However,the lack of efficient and safe gene delivery vectors greatly limited the development potential of gene therapy.Nowadays,cationic polymer gene carriers have gradually become one of the most popular non-viral carriers.Not only because of their chemical structure and composition diversity,but also because they are easily chemically modified and achieved selective delivery in specific tissues or cells.However,cationic polymers are not efficient in gene delivery.The main reason is that cationic polymers usually bind nucleic acids via ionic interactions,resulting in poor stability of the transfection complexes and easily be dissociated under the salt ions.In order to stabilize the complex,other interactions need to be introduced.Traditionally,conjugating hydrophobic ligands on the surface of cationic polymers was recognized as one of the classic methods.These hydrophobic ligands can significantly facilitate the ability of transmembrane and endosomal escape of the carriers while stabilizing the complex.But aliphatic molecules can easily bind with serum proteins,cationic polymers modified with hydrophobic moieties tend to exhibit poor serum stability.The birth of the fluoropolymer gene carrier has solved this problem and shown very broad application prospects.Due to the strong chemical and biological inertness of fluoropolymer,such gene vectors can maintain the stability of the transfection complex to a large extent both in vitro and in vivo.The current fluoropolymers developed for gene delivery are synthesized by grafting fluorous ligands onto cationic polymers.This strategy has certain limitations,such as the single type of polymer,a low grafting ratio,and poor water solubility.In addition,when the fluorous ligands are randomly distributed on the polymer using the “grafting onto” strategy,it is not available to investigate the effect of fluorous monomer sequence on the efficiency of fluoropolymers in gene delivery,which is an essential parameter in the design of polymer materials.So new strategies of synthesizing fluoropolymers are required.In this study,to expand the family of fluoropolymers for gene delivery and explore the effect of monomer sequence of fluoropolymer on transfection efficiency,we synthesized both statistical and block copolymers of 2-dimethylaminoethyl methacrylate(DMAEMA)and heptafluorobutyl methacrylate(HFMA)via reversible addition-fragmentation chain transfer(RAFT)polymerization.And the fluoropolymers with these two sequences were systematically compared for their assembly behavior,nucleic acid binding ability,gene delivery efficiency,level of cellular uptake,and cytotoxicity.Research suggests that both the block and statistical fluorocopolymers can selfassemble into nanoparticles with size of 200 nm,and their critical micelle concentrations are similar.In terms of nucleic acid binding ability,the block polymer is slightly stronger than the statistical copolymer because of the more concentrated distribution of cationic monomers in the block polymer.It is worth noting that the statistical copolymer showed dramatically higher gene delivery efficiency than block copolymer without significant differences in assembly behavior and nucleic acid binding ability.Through the study of their cellular uptake and endocytic pathways,it was found that the main cause of this phenomenon is that the statistical copolymer can be continuously taken up by the cells in 48 hours,while the cellular uptake of block copolymer is almost saturated at 12 h.Moreover,the statistical copolymer of DMAEMA and HFMA showed a fluorine effect in gene delivery and was much more efficient than non-fluorinated control polymers.This study revealed the structureactivity relationships of fluoropolymers consisted of DMAEMA and HFMA,and provided a new insight into the applications of fluoropolymers in gene delivery.