Fabrication Of Multifunctional Gene Vectors With Low Molecular Weight For Gene Delivery

Gene-based therapy has been considered as a powerful approach for treating a wide spectrum of diseases,such as congenital and malignant diseases.However,the delivery efficiency of the conventional vectors is undesirable until now.Vectors based on natural viruses have the advantage of efficient delivery but are hampered by some shortcomings including immunogenicity and carcinogenesis.In contrast,nonviral vectors,such as liposome and polycations are faced with severe challenges of poor transfection capability despite the fact that they are safer and easier to synthesize.Therefore,the development of gene vectors with high transfection efficiency as well as low cytotoxicity remains one of the greastest challenges for gene-based therapy.In this thesis,faced with the difficulties in nucleic acid delivery,we developed a series multifounctional gene vectors with low molecular weight,and tested their gene delivery capabilities both in vitro and in vivo.Firstly,inspired by viral structures that are optimized for gene delivery,we designed a small-molecule gene vector(TR4)with aggregation-induced emission properties by capping a peptide containing four arginine residues with tetraphenylethene(TPE)and a lipophilic tail.This novel vector can self-assemble with pDNA to form nanofibers in solution with efficient pDNA condensation,high stability,and high transfection efficiency in a variety of different cell lines,including stem cells.In the transfection of HeLa cells,the transfection efficiency of TR4 was nearly 70% at a N/P ratio of 4,whereas the transfection efficiency of PEI was just 15%.The self-assembly process induces bright fluorescence from TPE,which makes the nanofibers visible by confocal laser scanning microscopy(CLSM).This allows us for the tracking of the gene delivery process.Subsequently,a transferrin-dressed virus-like ternary nanoparticles with aggregation-induced emission(AIE)was developed for targeted delivery of siRNA.TR4 imitates the viral capsid and endows the vector with AIE properties as well as efficient siRNA compaction.The outer glycoprotein transferrin(Tf)mimics the viral envelope protein and endows the vector with reduced cytotoxicity as well as enhanced targeting capability.In addition,the Tf coating can accelerate the intracellular release of siRNA into the cytosol.Confocal laser scanning microscopy(CLSM)results showed that the TR4@Tf complex adhered to the cell membrane,and the siRNA was released into the cytoplasm after 12 h transfection.This might be due to the specific binding between Tf and transferrin receptor(TfR)in the cell membrane,which will weaken the interaction between TR4@Tf and siRNA,and accelerate the release of siRNA.Therefore,we constructed a virus-mimicking vector with virus-like architecture and infection properties.Finally,to realize in vivo gene silencing,we developed a kind of fluorinated oligoethylenimines(fOEIs)-based nano-assemblies(NAs)with good serum resistant property for siRNA delivery and investigated the mechanism underlying the serum resistance.The as-synthesized fOEIs are amphiphilic and readily form nano-assemblies(NAs).The fOEI NAs achieved very favorable gene silencing ability as well as good serum resistance in the transfection of cells.However,the gene silencing efficiency of aOEI-C12 NAs with similar CMCs and zeta potentials as fOEI NAs in the presence of serum was significantly decreased.The mechanism underlying the serum resistance of the fOEIs NAs is mainly derived from their serum protein adsorption resistance properties.Furthermore,the fOEI NAs showed superior gene silencing efficiency when compared to non-fluorinated NAs in silencing ApoB proteins in livers,suggesting potential in vivo applications as powerful gene vectors.This work will offer a promising solution for the construction of efficient gene vectors with good serum resistance.