The Study On The Functional ?-helical Cationic Polypeptides For Anti-inflammatory Gene Delivery

Gene therapy is regarded as a promising strategy in the treatment of various diseases.The usual gene non-viral vector includes cationic polymers,dendrimers,lipids,and polypeptides,which afford desired disadvantages toward gene transfection,which mainly attributed to the biological membranes,including cell membranes,endosomal/lysosomal membranes,and nucleus membranes,which impede nanoparticle to the desired cell organelle.In order to overcome the aforementioned drawbacks,it is necessary to develop gene carriers with potent membrane activities.We recently developed a library of a-helical cationic polypeptides,which possess strong membrane penetration capacities and could mediate“pore formation”on cell membranes.In comparison to endocytosis of the traditional non-viral vector,the helacal polypeptides could efficiently condense DNA and punch a pore on the cell membranes,endosomal/lysosomal membranes,and nucleus membranes,which promote gene cargos enter the cell and avoid the gene experience the endosomal/lysosomal entrapment against effective gene transfection.However,the“pore formation”mechanism could lead to irreversible damage to cell membranes.Furthermore,the strong electrostatic interaction between the cationic polypeptide and negative nucleic acid could hinder the gene release in the cytoplasm,which also led to the poor transfection efficiency of helical polypeptides.Additionally,helical polypeptides with stiff,rod-like structure suffer from low siRNA binding affinity.In order to resolve these points,this paepr do the following work.We incorporate various aromatic groups into the side chains of guanidinated,?-helical polypeptides,attempting to potentiate their non-endocytic as well as“non-pore formation”membrane activities to allow effective gene transfection with reduced cytotoxicity.The other is that we design reducible and biogradeable brush polypeptide,which can be degraded by the GSH in cytoplasm to small linear pollypeptides to promote the gene cargos release,ultimately achieveing the effective gene transfection and reduced cytotoxicity.The detailed work is as follows:Part 1.We first explored the effect of the side chain on the gene transfection efficiency of polypeptides,and found that P2 is optimal polypeptide,based on which,we incorporated various aromatic domains(benzyl,naphthyl,biphenyl,anthryl,and pyrenyl)onto the side-chain terminals of guanidine-rich,helical polypeptides to explore the effect of aromatic types on the transfection efficiency of polypeptide.Meanwhile,with naph-modified polypeptide as an example,we futher explored the effect of the aromatic content on the gene transfection efficiency,the content of naph groups is about 2%,5%,10%,and 15%.We found benzyl(Bn)-and naphthyl(Naph)-modified polypeptides demonstrated the highest DNA uptake level that outperformed the un-modified polypeptide P2 by?4 fold.More importantly,compared with P2,Bn-and Naph-modified polypeptides allowed more DNA cargoes to be internalized via the non-endocytic pathway,which greatly bypassed the endosomal entrapment and accordingly enhanced the transfection efficiency by up to 42 fold,outperforming commercial reagent 25k PEI by 3-4 orders of magnitude.We also found that the aromatic groups can improve the binding capacity of polypeptides to siRNA and gene knockdown efficiency.Part 2.We first synthesize the POB-L-Glu-NCA monomer,then the main chain polymer containing disulfide bond was synthesized via polycondensation reaction of two carboxyl and amino groups.The amino groups of this polymer trigger the NCA monomer to polymerizate to obtain brush polymer.Then we introduce the guanidinium groups to the side chain of brush polymer via the azide-alkyne Huisgen cycloaddition reaction to form the reducible and biodegrable brush polupeptides.Agarose gel and heparin replacement assay showed that RBP/DNA polyplexes could rapidly release DNA after GSH treatment.Through intracellular kinetics investigation,it was found that total cellular uptake of RBP/DNA polyplexes was extremely high and a significant amount of free DNA was disassembled from the polyplexes in the GSH-generating HeLa cells.In addition,RBP/DNA polyplexes exhibited excellent gene transfection efficiency and smaller cytotoxicity in HeLa cells.Part 3.The occurrence and development of acute lung injury(ALI)are closely relative to the excessive expression of inflammatory cytokines(such as tumor necrosis factor a,TNF-?).It is effective in the treatment of ALI by inhibiting the inflammatory factor expression.Using the advantages of RBP in gene delivery,we use it to condense TNF-? siRNA for the treatment of acute lung injury.In vivo transfection showed that RBP/TNF-? siRNA polyplexes markedly attenuated the TNF-? mRNA and protein level compared to the NRBP and LP;Moreover,the MPO activity,the inflammatory factor TNF-? and IL-1?,total protein and total cell count of BALF were dramatically reduced,suggesting that TNF-? gene silencing could effectively modulate inflammatory responses.From HE pathological,we also found that RBP/TNF-?siRNA complexes could reduce lung inflammatory response.We have developed“self-activated”,membrane-penetrating ?-helical polypeptides and reducible and biodegrable brush polypeptide,which affored high gene transfection efficiency and reduced cytotoxicity.Meanwhile,RBP/TNF-? siRNA complexes could efficiently treat the ALI of mouse.