Polymeric Nanomaterials For Gene Delivery

Gene therapy is a revolutionary high-tech biological therapy,whose principle is to import the normal nucleic acid(DNA)or the sequences with silent effect(siRNA)into target cells to play a role in the treatment.However,even gene therapy possesses undisputed superiority compared with traditional therapy,it still has several obstacles: 1)a number of biomembrane barriers in cell structure(cell membrane,endo/lysosome membrane and nuclear membrane,etc.)prevent the nucleic acid from penetration,leading to the reduced uptake;2)the nucleic acid entering cells via endocytosis is captured by endosome/lysosome and will be further degradated under the condition of lysosomal acidic environment and hydrolytic enzymes,resulting in limited transfer efficiency to cytoplasm;3)in the presence of large amounts of substances such as protein and enzymes in the blood,nucleic acid is easy to be degraded and brings a series of side effects.In order to overcome these barriers,it's necessary for us to choose appropriate gene vectors to ensure safe targeting of nucleic acid molecules efficiently and controllably.As one of cell penetrating peptides' derivatives,?-helical cationic polypeptide is a kind of membrane-penetrating cationic polymer with the ability of effectively overcoming biomembrane barrier,promoting nucleic acid into cells,and endosomal escape.Polymer vesicle,which derives from liposomes,is a kind of gene delivery vectors with hollow structure.The dense hydrophobic membrane and hollow hydrophilic inner of polymer vesicles provide stable load of hydrophilic/hydrophobic drugs,nucleic acid,and protein,as well as preventing the leakage of loaded molecules and ensuring the whole stability in blood circulation.Based on the above understanding,we present a brief literature overview on the gene therapy,the barrier of gene delivery,gene vectors,photodynamics therapy and ulcerative colitis in chapter 1.In chapter 2,a photosensitizer(PS)-embedded,star-shaped helical polypeptide was developed,which combined the merit of multivalency-enhanced intracellular DNA uptake and light-strengthened endosomal escape to enable efficient gene delivery with low toxicity.The amino-terminated 5,10,15,20-Tetrakis-(4-aminophenyl)porphyrin(TAPP)was used as an initiator to initiate ring opening polymerization(ROP)of N-carboxyanhydride(NCA)and connect guanidine groups via efficient “click chemistry” reaction,to ultimately yield star-shaped helical polypeptide.The star polypeptide could condense DNA tightly and form into nanocomplex(~150 nm).Compared with linear polypeptide,the star polypeptide with topological structure could promote DNA release and cell uptake level.The light-induced ROS made almost complete endosomal release of the DNA cargo via photochemical internalization(PCI),and further led to an increment of the transfection efficiency.Compared with the in vitro transfection efficiency of star polypeptide without light,the star polypeptide with light irradiation led to a 6~8 fold increment of the transfection efficiency in He La,B16F10,and RAW 264.7 cells,outperforming commercial reagent 25 k PEI by up to three orders of magnitude.Such multivalency-and PCI-potentiated gene delivery efficiency was also demonstrated in melanoma-bearing mice following intratumoral injection.It afforded notably higher transfection efficiency and lower cytotoxicity than its linear analogue.In chapter 3,a vesicle based on TKPR peptide-functionalized reversibly crosslinked polymersomes(TKPR-RCP/RCP)mediated by TNF-? siRNA and hydrophilic drug(dexamethasone sodium phosphate,DSP)was developed,which combined the advantages of high-efficiency targeted delivery and high biocompatibility with negligible toxicity for the treatment of dextran sulfate sodium(DSS)-induced ulcerative colitis(UC).Obviously,TKPR-RCP with thick hydrophobic membrane could remain stable for a long time.TKPR siRNA and DSP were loaded completely and tightly into the aqueous lumen of the polymersomes,and released quickly from TKPR-RCP in the reducing environment.The TKPR-RCP promoted the distribution of siRNA in ulcerative colon and then efficiently entered macrophage.In LPS-induced RAW 264.7 cells,TKPR-RCP NPs loading with TNF-? siRNA and DSP dislayed superior gene knockdown capacity in vitro.In DSS-induced UC in mice,daily administration of TNF-? siRNA and DSP-loaded TKPR-RCP NPs significantly decreased the level of TNF-? m RNA and protein content in colon.It could also improve body weight loss,colon length shortening,bloody stools,and decrease disease activity index,indicating the efficient anti-inflammation ability.The blood routine indexes and biochemical indexes of the mice after administration were negatively changed,showing excellent biocompatibility of TKPR-RCP.