Acid-Labile Hyperbranched Poly(Ortho Ester Amido Amine) As Gene Vectors:Design,Preparation And Transfection In Vitro

Gene therapy was viewed as an effective approach for treating hereditary diseases,with the continuous research and development,which has performed great prospects and potentials at the treatment of acquired diseases such as cancer.Gene therapy involves the introduction of new genes into cells,to restore or add gene expression,for the purpose of treating disease.Based on what mentioned above,the key of gene therapy was to obtain a suitable gene carrier,which can safely and efficiently introduce exogenous normal genes into target cells.As a kind of gene carrier which can be transfected efficiently,the development and application of virus vector are greatly restricted due to its immunogenicity and other safety problems.Therefore,the non-viral vectors had been fastly developed which due to its good biological safety,easy preparation and low cost,such as the cationic lipids and polymers and so on.The non-viral vectors have a good potential in the system of gene delivery.Though cationic polymers were now well established as a successful gene delivery vector,those reaching to clinical trials were still limited owing to the following mentioned barriers.The major barriers mainly include its toxicity,evading immune system,hemocompatibility,efficient intracellular delivery at a target site,polyplex releases DNA and ultimately the gene expression.The first report on the usage of cationic polymer as gene delivery vector dates back to three decades,with the research and discovery of a large number of researchers,cationic polymers have been used as an indispensable gene carrier for gene therapy.Polycations and negatively charged DNA form polyplexes by positive and negative charge attraction,so that negatively charged DNA can enter the target cells safely and effectively for therapeutic purposes.Cationic polymers as gene carriers could be mainly classified into linear polymers and dendrimers.Compared with the linear cationic polymer,the dendrimers has a good three-dimensional spherical structure,and its surface carries a large number of terminal functional groups,good solubility,low solution viscosity and melt viscosity,and a large number of internal cavity structures.Dendrimers have become a promising class of gene carriers due to these properties.It has been reported that the transfection efficiency of partially hydrolyzed dendrimers was 50 times higher than that of unhydrolyzed dendrimers,this indicates that the imperfection of structure could adjust the transfection performance of dendrimers.In addition,compared with dendrimers,hyperbranched polymers were more practical because of their simplicity of easy operation and low production cost.Therefore,it has become very meaningful to develop a novel degradable hyperbranched cationic gene vector for improving the safety and transfection efficiency.We aim to develop a novel hyperbranched cationic gene vector and study its performance as a gene vector.In this work,linear and hyperbranched cationic polymers were prepared from diethylene glycerol by simply changing the temperature.This paper was mainly carried out through the following parts(1)On the basis of the reported synthesized amino-terminated monomers,the two-terminal double bonded monomers were prepared by reacting them with acrylic anhydride.Polymer POEAMAM was synthesized by Michael addition reaction of acrylic acid ester monomer with N-aminoethyl piperazine.Linear hyperbranched poly(ortho ester amido amine)was synthesized at 20 ? and hyperbranched poly(ortho ester amido amine)(HPOEAMAM)was synthesized at 50 ?.Linear POEAMAM and hyperbranched POEAMAM structures were determined by NMR spectroscopy.The fluorescence properties of hyperbranched POEAMAM and linear POEAMAM were studied by inverted fluorescence microscopy and steady-state fluorescence spectrometer.The proton buffering ability of hyperbranched POEAMAM was verified by acid-base titration experiment.(2)As gene delivery vectors,cationic polymers were required to condense DNA into nano-sized complexes.Firstly,the ability of POEAMAM to compress DNA was validated by agarose electrophoresis,and then the ability of POEAMAM to compress DNA was further validated by heparin sodium replacement test.The par;ticle size of hyperbranched POEAMAM composites was about 100 nm and the potential was about+ 15 mV.At pH 5.5,the release of DNA from HPOEAMAM/DNA complexes increased from 87%to 92%at 48 h,and that from LPOEAMAM/DNA complexes increased from 54%to 72%at 48 h.(3)The cytotoxicity of linear POEAMAM and hyperbranched POEAMAM was detected by MTT assay.The results showed that the cell survival rate was above 95%,indicating that the polymer had excellent biocompatibility.The transfection efficiency of POEAMAM/DNA was investigated by two-dimensional cell model and three-dimensional cell model.The results of inverted fluorescence microscopy and flow cytometry showed that hyperbranched POEAMAM had good transfection efficiency in cells.Laser confocal analysis showed that the transfection efficiency of hyperbranched POEAMAM in 10%serum was better than that of PEI-25K.Here we reported a novel hyperbrached PAMAM(HPAMAM)synthesized by the Michael addition polymerization of acrylic acid ester monomer and 1-(2-aminoethyl)piperazine.We found that the newly synthesized polymers have good biocompatibility and high transfection efficiency.Therefore,hyperbranched POEAMAM have great potential in gene therapy.