Construction Of Non-viral Gene Carrier OTMCS-PEI-R18 Modified By RGD-TAT-NLS

Gene therapy is a method designed to express the therapeutic proteins in intracellular environment of target issue. Compared with the traditional therapeutic regimens such as surgery, chemotherapy and radiotherapy, gene therapy has greater potential to eradicate cancer.The key to gene therapy is finding an ideal gene delivery vector. Non-viral gene vectors are now drawing more and more attention due to its controlled structure, high absorption capability, low cytotoxixity and hypoimmunity. Polyethylenimine(PEI) is a kind of typical cationic polymer, which can form a condensed complex with DNA spontaneously through electrostatic interactions with its large amount of positive charge. Due to its highly effective gene transfection, PEI has gained itself remarkable interest in the field of non-viral gene delivery system. However, preclinical studies of PEI vectors revealed several drawbacks: the contradiction between toxicity and transfection efficiency, lack of specific tumor targeting, low transfection capacity in vivo.To solve these problems, we synthesised a new non-viral gene vector. First low molecular weight(LMW) PEI was linked with amphiphilic chitosan OTMCS to obtain degradable PEI derivates, then used tumor-targeting peptide RGD, in conjunction with the cell-penetrating peptide(CPP) TAT(49-57) and nuclear loclization peptide NLS to yield a trifunctional peptide R18, and lastly adopted R18 to modify the PEI derivates. The new gene vector was characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency was validated by in vitro and in vivo studies. Chemical inhibitors specific to various intracellular pathways and confocal laser scanning microscopy have been applied to investigate its cellular transport. PEI 25 KDa, OTMCS-PEI, OTMCS-PEI-R13 were conducted as controls.Part one of this article gave an overview on developing status of PEI as gene carriers. The main reason for limiting its clinical application was discussed andimprovement strategies aiming at these problems were analyzed.Part two focused on synthesis and characterization of OTMCS-PEI-R18. At first the trifunctional peptide R18 was prepared by the solid phase method. The structure and purity of R18 were demonstrated respectively by MS and HPLC. Then amphiphilic chitosan(OTMCS) was crosslinked with PEI 2 KDa to get a high molecular weight PEI derivate OTMCS-PEI. Lastly OTMCS-PEI was modified by R18 to obtain OTMCS-PEI-R18. OTMCS-PEI and OTMCS-PEI-R18 was characterized through1H-NMR method. The results indicated that R18 was synthesized successfully with high purity as 95.56%. OTMCS-PEI and OTMCS-PEI-R18 has been also prepared successfully.Part three was about physical characterization of OTMCS-PEI-R18. Degradation of the polymer was estimated by gel permeation chromatography. Buffering capacities was measured by acid-base titration. The particle size and zeta potential of OTMCS-PEI-R18/DNA complexes were also measured. The DNA condensation capacity, digestion resistance to DNase I, FBS and sodium heparin were determined by agarose gel electrophoresis. The cytotoxicity of these polymers on Hela and B16 cells were measured by MTT assay. The results indicated that OTMCS-PEI-R18 degraded slowly at 37℃ and turned into micromoleculars after 60 h. The particle sizes of these complexes were about 150-450 nm, zeta potential and buffer capacity were suitable for in vivo application. OTMCS-PEI-R18 was able to condense DNA effectively at a w/w ratio of 1.0, and could protect DNA from being digested by DNase I, FBS and sodium heparin at high concentrations. Furthermore,OTMCS-PEI-R18 showed significantly lower cytotoxicity compared with PEI 25 KDa.In part four, plasmid p EGFP-N2 and p GL3-Control were used as reporter genes to measure gene transfection efficiency of the vectors in vitro, and animal tumor model was established to investigate distribution and transfection efficiency in vivo.The transfection efficiency of OTMCS-PEI-R18 was demonstrated to be much higher than the other controls in vivo and in vitro, and with the raw ratio of R18 increased,the transfection efficiengy enhanced accordingly. The distribution of the complexes in different tissures also changed, more DNA complexes accumulated in tumor due to the modification of R18.In part five, intracellular transport mechanism of OTMCS-PEI-R18/DNA was investigated by using chemical inhibitors to disrupt receptor-mediated endocytosisand cytoskeleton structure. Confocal laser scanning microscopy has been applied to visually reveal intracellular fate of FITC-labeled OTMCS-PEI-R18 at different time points. OTMCS-PEI-R18/DNA complexes enter cells by clathrin-mediated endocytosis, caveolin-mediated endocytosis and macropinocytosis fisrtly, then being transport to nucleus with the aid of microfilements and motor protein, and lastly get into nucleus by receptor-mediated manners, not by relying completely on cell division way because of the introduction of NLS. The confocal laser microscopy images also proved that OTMCS-PEI-R18/DNA accumulated in nucleus eventually.We developed a new gene vector by crosslinking LMW PEI with amphiphilic OTMCS, then conjugated with trifuctional piptide R18 with charactersitic of targeting tumor, promoting celular uptake and nucleus transport. The vector system showed proper physicochemistry property, low cytotoxicity and high transfection efficiency in vitro and in vivo. In addition, the mechanism experiment revealed its special receptor-mediated intracelular transport routine, which is suitable for in vivo transfection.