Construction Of High Performance Gene Vectors Based On Polyaspartic Acid

Gene therapy is an effective treatment for cancer and genetic diseases.With the progress of the Human Genome Project,gene therapy is also used to treat more new diseases,such as skin repair and bone defect repair.At present,gene vectors are still the bottleneck restricting the development of gene therapy.Gene vectors mainly include viral gene carriers and non-viral gene vectors.Viral gene vectors have immunogenicity and insertion mutation,which limit their applications.Non-viral gene vectors mainly include cationic polymers,while their obvious toxicity and low transfection efficiency are urgent to be solved.A large number of reports have demonstrated that the introduction of biocompatible and degradable structures in the design of gene vectors can significantly reduce toxicity and increase transfection efficiency.Polyamino acid has good biocompatibility,biodegradability,secondary structure similar to biological proteins,and biological functions.Among them,polyaspartic acid(PAsp)is simple to be synthesized and easy to be modified,providing diversification of avenues for the construction of new high-performance gene vectors.The main contents are as follows:Cellulose nanocrystals(CNCs),a kind of natural polysaccharide nanoparticle with excellent biocompatibility and stable nanoscale size,are good for the construction of nanoscale gene vectors.The cationic carrier(PGEA),which is based on the ring-opening reaction of poly(glycidyl methacrylate)(PGMA)and ethylenediamine(EA),has promising transfection performance.In the second chapter,a novel rodlike supramolecular nanoassemblies(CNC@CB[8]@PGEA)consisting of assembly unit Np modified PAsp derivatives-grafted CNCs(CNC-PAsp-EA/Np)and another assembly unit MV grafting hydroxyl-rich PGEAs(MV-PGEA)were proposed via typical CB[8]-based host-guest interactions.CNC@CB[8]@PGEA was used for the delivery of tumor suppressor gene to treat hepatocellular carcinoma(HCC).CNC@CB[8]@PGEA combined the unique properties of CNC,PGEA,and PAsp.More importantly,PAsp might benefit the disassembly and degradability of assemblies after cellular uptake.Compared with the assembly unit MV-PGEA,CNC@CB[8]@PGEA could effectively condense the expression constructs(plasmid pc3.0-miR-101 and plasmid pc3.0-MEG3)of short(miR-101)and long(MEG3)ncRNAs,and has promising transfection performance in HepG2 cells and excellent anti-tumor efficiency in vitro.Furthermore,compared with the separate delivery of p3.0-MEG3 and p3.0-miR-101,the codelivery of pc3.0-MEG3 and pc3.0-miR-101 mediated by CNC2@CB[8]@PGEA had joint suppression effects and demonstrated remarkable anti-tumor ability in vitro and in vivo.Polyrotaxane,a typical type of supramolecule,is suitable for constructing degradable gene carriers.The disassembly of polyrotaxane could benifit the release of plasmids.In the third chapter,one novel ABA-triblock hydroxyl-rich cationic PR(PP-PGEA)was proposed as one promising gene delivery systems,where CD-PGEA(composed of one CD core and three ethanolamine(EA)-functionalized poly(glycidyl methacrylate)(PGEA)arms)units on PEG backbone as B block was end-capped by degradable poly(aspartic acid)(PAsp)segments as polypeptide A block.Here,the degradability of PAsp segment could facilitate the slip of CD-PGEA,which helped the release of plasmid and improved transfection efficiency.Compared with CD-PGEA,PP-PGEA could more effectively condense plasimid,and has high transfection efficiency in HepG2 and C6 cells.In addition,PP-PGEA/p53 has been proved to possess promising anti-cancer activity in vitro and in vivo.Tobramycin is an aminoglycoside antibiotic,which is mainly used clinically for wound infections and soft tissue infections caused by Gram-negtive bacteria P.aeruginosa and Gram-positive bacteria S.aureus and other sensitive bacteria.In the fourth chapter,novel gene vectors T-PAsp-T(TPT)with biodegradability and antimicrobial ability were prepared via tobramycin initiating the ring-opening polymerization of BLA-NCA to produce PBLA-grafted tobramycin(T-PBLA),and subsequent aminolysis reaction of T-PBLA and tobramycin and ethylenediamine(ED).The degradation of polyaspartic acid can promote the release of plasmid.It is proved that TPT can condense the plamid into nanoparticles suitable for endocytosis and has high transfection performance in mouse embryo fibroblasts(NIH3T3)and mouse fibroblasts(L929)cells,as well as good antibacterial activity in vitro.Furthermore,TPT/pEGF nanocomplexes have been proved to promote the proliferation and migration of epidermal cells,and proved to accelerate skin tissue healing in the rats with skin defects.In summary,two classes of high-performance gene carriers based on degradable PAsp and polysaccharides grafted PGEA were used to treat cancer in this thesis.The high performance of rodlike supramolecular nano-assembly(CNC@CB[8]@PGEA)and ABA-triblock polyrotaxane(PP-PGEA)carriers was proved via a series of in vitro and in vivo assays.Simultaneously,a novel aminoglycoside gene carrier(TPT)based on PAsp was proved to achieve synergistic treatment of antibiosis and wound healing in vitro and in vivo.The construction of the above three classes of high-performance novel gene vectors is of great significance for the application of gene carriers.