Study On Construction And In Vitro Properties Of Polyethylenimine Gene Delivery System Based On Polyglutamic Acid

Objective:Construct a highly efficient and low toxicity gene vector is the key to successful gene therapy.The study was designed a biodegradable copolymer G0-b-PLG-g-PEI(PGLP)composed of zero-generation polyamidoamine dendrimer(PAMAM G0)as inner core and polyglutamic acid grafted low molecules of PEI as surrounding for gene delivery carrier.The physicochemical properties and organisms performance of the polymer are evaluated as gene carrier.Methods:In this paper,L-glutamic acid-?-benzyl ester-N-carboxy anhydride(BLG-NCA)was prepared by triphosgene method,and then ring-opening polymerization of BLG-NCA was initiated by PAMAM G0 as initiator to obtain polyamide-amine-b-polyglutamic acid benzyl ester(G0-b-PBLG),and then aminolysis of G0-b-PBLG with PEI 423 as an ammoniating agent to obtain a triblock copolymer G0-b-PLG-g-PEI(PGLP),the molecular structures of PGLP was confirmed by~1H NMR(Proton nuclear magnetic resonance spectroscopy).PGLP and DNA self-assemble into PGLP/DNA complex by electrostatic binding,the ability of DNA combination and protection were investigated by agarose gel electrophoresis.The particle size and potential of PGLP/DNA were determined by dynamic light scattering(DLS).The safty of PGLP and PGLP/DNA complexes were evaluated by CCK-8 reagent and the hemolysis experiment.The results of in vitro transfection were determined by flow cytometry.Confocal microscopy observetd the uptake of gene by cells and the distribution of DNA in cells.Result:~1H NMR spectrum confirmed the successful synthesis of PGLP.Agarose gel electrophoresis experiments showed that PGLP can effectively compress the encapsulated DNA at w/w?0.3.PGLP can protect DNA from serum enzyme degradation at w/w?3.The dynamic light scattering particle size analyzer detected that the particle size range of the PGLP/DNA complex nanoparticles was 105-200 nm,and the potential was between+10 and+28 mV.Cytotoxicity studies showed that the cytotoxicity of PGLP was significantly lower than that of PEI 25K and PGLP/DNA complexes is safty.Hemolysis tests have shown that PGLP/DNA complexes have good biocompatibility with red blood cells.In vitro cell transfection experiments showed that the optimal transfection ratio of PGLP/DNA complex was w/w=8,the transfection efficiency of PGLP/DNA complexes in various cells(HEK 293T,Hela,Bel7402 and primary rat aortic smooth muscle cells RASMC)was significantly higher than optimal transfection efficiency of PEI 25K or Lipofectamin 2000 in serum and serum-free conditions.PGLP/DNA complexes are more strong anti-serum capacity at optimal transfection ratios.Conclusion:PGLP has the potential as a gene carrier and may become a safe,effective,anti-serum gene vector.