Screening And Structure-activity Relationship Of Functional Polymers For Gene And Protein Delivery

Cationic polymers are widely used as non-viral biomacromolecule vectors with good solubility,high tissue penetration ability,controllable structure and facile production.However,their applications are limited by moderate delivery efficacy and high cytotoxicity.To address this problem,various ligands have been modified on polymers.Despite improved transfection efficiency and biocompatibility,rational design of efficient biomacromolecule carriers remains challenging.The reason is that ligand-functionalized polymers used in biomacromolecule delivery are reported by independent researchers using different polymer scaffolds and cell lines.It is hard to provide structure-function relationships of these materials based on current knowledge and experiences,which are essential for the design of ideal polymeric carriers for biomacromolecule delivery.In this dissertation,we proposed a strategy of library screening to find out the structure-activity relationships of the modified groups on polymers and aquire efficient carriers for the delivery of biomacromolecules at the same time.Detailed methods and results are summarized as follows:(1)Screening of efficient si RNA carriers in a library of surface-engineered dendrimers.A library of surface-engineered dendrimers was built by conjugating several kinds of ligands including alkyl chains,fluoroalkyl chains,amino acids,heterocyclic compounds,benzene derivatives and other compounds on generation 5(G5)polyamidoamine(PAMAM)dendrimer.Gene silencing results showed ten surface-engineered dendrimers induced more than 75% gene knockdown efficacy to several target genes in different cell lines with low toxicity.Efficacy of the lead material E9-2 is much superior to commercial lipid carriers such as Lipofectamine?2000(Lipo 2000)in hard-to-transfect cells such as stem cells.The structure-activity relationships studies demonstrated that ligands in the library either have appropriate hydrophobicity(fluorous chains or aliphatic rings)or combine hydrophobic ligands(phenyl group)and functional ligands(fluorine atoms,bromine atoms,boronic acid or guanidine groups)can significantly improve the gene silencing efficacy of dendrimers.(2)Screening of efficient protein carriers in a library of fluorinated dendrimers.A library of fluopolymers was synthesized by modifying G5 PAMAM dendrimer with 22 types of fluorous ligands at different grafting ratios.With the increase of chain length,protein delivery efficacy first gradually improved and then reduced.Dendrimer conjugated with an average number of 71 fluoroalkyl chains containing eight fluorine atoms is the most efficient polymer.This lead material efficiently transported proteins including BSA,?-Gal,saporin,as well as peptides into cells,and bioactivities of the proteins or peptides were maintained after transduction.The fluoropolymers were much more efficient than their non-fluorinated analogue with similar conjugation ratios,suggesting the beneficial effect of fluorine atoms in intracellular protein delivery.This phenomenon can be explained by the fluorous effect of fluorinated polymers during endocytosis and endosomal escape.Polymers with longer fluorous chains and higher fluorination degrees showed higher efficacy in protein delivery.However,they are not soluble in aqueous solutions and should be dissolved in dimethyl sulfoxide to complex with the proteins.(3)Screening of efficient protein carriers in a library of guanidine analogue modified dendrimers.After well understanding the beneficial of hydrophobic ligands and functional ligands combination,we combined different hydrophobic ligands with guanidine to form different kind of guanidine anoglues.Branched polyethylenimine with a molecular weight of 25 k D(b PEI 25 k D)was modified with 7 guanidine analogues at different grafting ratios.Protein delivery results showed that the combination of phenyl and guanidyl groups improved the transfection efficacy of b PEI 25 k D most efficiently.The efficiency of b PEI 25 k D modified with a number of 60 4-guanidinobenzoic acid showed more than ten times higher transfection efficacy than the commercial protein transfection reagent PulsinTM.Modification of 4-guanidinobenzoic acid on G5 PAMAM dendrimer,generation 2(G2)PAMAM dendrimer,b PEI with a molecular weight of 1.8 k D(b PEI 1.8 K)all achieved much improved transfection efficiencies.The modified polymers efficiently delivered various proteins and peptides into cells without the need for protein modification,while maintaining high protein activity and excellent biocompatibility.In summary,by comparing si RNA delivery efficacy of the materials in a library of surface-engineered dendrimers we aquired more than ten efficient si RNA carriers,and learn that appropriate hydrophobicity or combining both hydrophobic ligands and functional ligands can significantly improve the gene silencing efficacy of dendrimers.According to this theory,we bult a library of fluorinated dendrimers and a library of guanidine analogue modified polymers respectively.We acquired several protein carriers with robust efficacy and low cytotoxity,and futher validate and complement our theory of structure-activity relationships of cationic polymers in the delivery of biomacromolecules,which is beneficial for the design of ideal polymeric carriers for biomacromolecule delivery.In addition,the efficient si RNA and protein carriers screened from three librarys have shown great potential for transformation and may be developed for scientific research or even clinical treatment in the future.