| Gene therapy means the therapeutic delivery of exogenous genetic substances to diseased cells and then repair and compensate genetic defect or abnormality which may cause serious diseases.However,the development of gene therapy remains stagnant because of lack of efficient and safe delivery vectors.At present,gene delivery vectors can be mainly divided into virus and non-virus vectors.Despite virus vectors have high delivery efficiency,serious immune rejection compromises its translation for clinic use.Non-virus vectors(i.e.cationic polymer and liposome)usually exhibit definite chemical structures,designed functions,non-immunogenicity and possible large scale production,which would be the most promising vectors for gene therapy.Successful gene delivery requires accomplishing five tasks:(1)condensing nucleic acids into nanoparticles to avoid degradation;(2)targeting to diseased cells;(3)rupturing endosomes to help endosomal escape of nucleic acids;(4)releasing nucleic acids into cytoplasm;(5)metabolizing into non-toxic substances.To solve this issue,we developed a core-shell delivery system,in which,a polyplex formed by cationic polymers and nucleic acids as the core and a self-assembled tri-block copolymer as the shell.the latter will accomplish the fourth task and the former will solve the rest four tasks.The core is dependent on the biologically responsive cationic polymers.The polymers should be relatively stable during delivery process but degradable in response to tiny intracellular changes,which may decrease the cytotoxicity.However,degradation will lead to the decrease of delivery efficiency.Therefore,the balance between stability and degradation of cationic polymers should be the most important property.Meanwhile,the requirements for delivery of si RNA and DNA are not same,which means vectors need to release them in different time points and positions.Now,the most popular biodegradable linkages are imine linkage,disulfide linkage and ester linkage.We used imine linkage based polymers to deliver si RNA and obtained successful delivery efficiency.But regarding DNA,it can only express when delivered into nucleus.Hence,different biodegradable linkages should be directed against different genetic materials,or even combine them together to improve efficiency.In this project,we chose imine and disulfide linkages as the biodegradable bonds,spermine as the unit,1,4-Phthalaldehyde and dithiodipropionic acid as two linkers to construct two cationic vectors(TPSP and DTDPS)that totally based on different degradable mechanisms.We conducted the physical chemical characterization of polymers and polyplexes formed from them.Also,the degradability and delivery efficiency of si RNA and DNA were tested in vitro and in vivo respectively.The results showed that these two polymers both had good ability to condense nucleic acids.However,when the polymers were mixed with si RNA and DNA respectively,TPSP always showed larger particle size and zeta potential.These two polymers were sensitive to p H changes and GSH.When comparisons were made in vitro and in vivo,TPSP and DTDPS were able to deliver si RNA into 7721 cells,but their ability to deliver DNA was terrible and TPSP showed better silencing effect both in vitro and in vivo.In conclusion,the imine linkage based polymer TPSP is superior to deliver si RNA and DNA. |
