Synthesis,Characterization And Biological Application Of Poly-L-Carnosine

Until today,diseases such as cancer,hereditary diseases,neurodegenerative diseases and cardiovascular diseases are still difficult to cure,seriously threatening human life and health.Gene therapy is a molecular biomedical technology that efficiently import exogenous genes(normal genes or therapeutic genes)into diseased tissue cells to correct or replace defective genes to achieve therapeutic gene diseases by physical,chemical and biological methods.Designing and preparing a safe and efficient gene vector is the key problem in the field of gene therapy.The application of viral gene vectors have been limited because of its issues.Thereby,non-viral gene carriers are becoming increasingly attractive,cationic polymer gene carriers are typical representatives of non-viral gene carriers.In this dissertation,we designed and synthesized a novel cationic homopolymer poly-l-carnosine(PCar)and its crosslinked polymers(X-PCar)based on 1-carnosine,an endogenous dipeptide with multiple excellent physiological properties.The structure of PCar has been characterized in detail.The physicochemical properties of the complexes formed by PCar and X-PCar with plasmid DNA were investigated,the cytotoxicity and in vitro transfection efficiency of PCar and X-PCar were examined.The work was mainly divided into the following two parts:1.Firstly,we synthesized camosine with imidazole amine and primary amine protected by tert-butyloxy carbonyl based on the amino protection strategy.Afterwards,we synthesized monomer methacrylamide containing carnosine through multi-step reaction.Subsequently,based on the traditional free radical polymerization methods,we used azobisisobutyronitrile as an initiator to initiate free radical polymerization of monomers under certain conditions to obtain monodispersed PCar with number average molecular weight of approximately 7.6 kDa and polymer dispersity index of less than 1.2.Acid-base titration assay results showed that PCar has better buffering ability in physiological conditions.Agarose gel electrophoresis test demonstrated that PCar has great condensation ability to plasmid DNA.The results of dynamic light scattering and surface Zeta potential measurements indicated that PCar and plasmid DNA can form nanoparticle with smaller size and higher Zeta potential.In vitro cell viability and transfection were evaluated in HeLa cells.The cytotoxicity of PCar is lower than that of 25 kDa bPEI.Although the gene transfection efficiency of PCar/DNA complexes in HeLa cells is lower than 25 kDa bPEI/DNA complex,it is still expected to be a new type of effective cationic polymer gene carrier.2.Based on the synthesized PCar,we synthesized X-PCar by introducing several crosslinking agents(ethylene glycol diglycidyl ether,L-lysine diisocyanate and dithiodipropionic acid),these crosslinking reactions have higher reaction efficiency.Agarose gel electrophoresis test showed that each of X-PCar has better condensation ability to plasmid DNA.Ethidium bromide competition experiments confirmed that X-PCar have great combination ability with plasmid DNA.The results of dynamic light scattering and surface Zeta potential measurements demonstrated that X-PCar can condense plasmid DNA to form stable complexes.The cytotoxicity of X-PCar were investigated in HeLa cells and QSG cells,the results indicated that X-PCar have lower toxicity to HeLa cells and QSG cells than 25 kDa bPEI.The in vitro transfection experiment of complexes to HeLa cells manifested that X-PCar have higher transfection ability than PCar.The above studies have pioneered the application of carnosine in the field of gene carriers,and established the effective synthetic method of novel functional polymers based on carnosine.