Study On Hepatocyte-targeted Gene Transfection Of Galactosylated Chitosan-graft-low Molecular Polyethyleneimine/DNA Complexes

Objective:To construct the asialoglycoprotein receptors(ASGP-R) -mediated non-viral gene vectors which based on galactosylated chitosan (GC) grafted low molecular polyethyleneimine(PEI),and to investigate the hepatocyte -targeted gene transfer efficiency in vitro and in vivo.Methods:In the first part,the GC-PEI copolymers were prepared by first the chitosan was coupled with lactobionic acid as the specific ligand to ASGR,followed the galactosylated chitosan was cross-linked with low molecular polyethyleneimine by diafiltration method.Taking the plasmid expressing enhanced green fluorescent protein(pEGFP-C1) as the reporter gene,the formation of copolymer/DNA complexes were induced to self-assemble in 0.01M phosphate buffered saline(PBS),150mmol/L NaCl or 5%glucose solution(GS).The binding ability of GC-PEI/DNA complexes to DNA was evaluated by gel retardation and binding assay; The protection effect of copolymer to DNA was studied by DNaseⅠand serum treatment.To determin the optimal GC-PEI/DNA complex and its weight ratio,particle size and morphology of the complexes were measured by the transmission electron microscopy(TEM) and the laser grain analyzer,the zeta potential was also detected by the laser grain analyzer.In the second part,the GC-PEI copolymer's cytotoxicity were determined in five cell lines(QSG7701,QSG7701/core,L02,SGC-7901, HBE) by MTT assay in vitro.The acute toxicity in vivo was determined by injecting the copolymer into mice via tail-vein.In the third part,hepatocyte-targeted transfection efficiency medi -ated by GC-PEI copolymer was evaluated in vitro and in vivo.The GC-PEI/pEGFP complexes were transfected respectively into hepatocyte lines(LO2,QSG7701,QSG7701/core) and non-hepatocyte lines(SGC7 -901,HBE) in vitro,and the lipofectamine ^TM2000 was used as a positive control while naked DNA as a negative one.The expression of green fluorescence protein(GFP) in cells was observed under fluorescent microscope and transfection efficiency was measured by FACS.In addition,serum and flee galactose were added to the cell cultures to study their effects on the transfection efficiency of copolymer.In vivo,two GC-PEI/DNA complexes in two different solution media were transferred into the mice's body through tail-vein injection.The major organs(liver, lung,kidney,heart and spleen) were harvested and made into frozen sections two days later.The GFP expressions were observed under the fluorescent microscope,and the naked plasmid DNA and lipofectamine ^TM2000 were used as controls.Results:①The gel retardation and binding assay showed that the optimal proportion of GC-PEI and DNA was 2.5:1(w/w),though other diluted GC-PEI/DNA complexes could also effectively bind DNA.The binding efficiency of DNA was about 93%at the ratio of 2.5:1.The protection and release assay showed that the binding of GC-PEI copolymer with DNA could protect the DNA from degradation of DNaseⅠand serum;the GC-PEI/DNA complexes presented as a well-formed spherical shape or compacted nucleocapsid structure with a diameter of 50～200nm.The particle size of GC-PEI/DNA complex diluted in 150 mM NaCl(GC-PEI/NaCl/DNA)was largest,the second was GC-PEI/DNA complex diluted in PBS(GC-PEI/PBS /DNA),and the smallest was that in 5%GS(GC-PEI/GS5%/DNA).With the GC-PEI/ DNA mass ratio rising from 0.25:1 to 2.5:1,zeta potential and binding ability of the complexes were increased,while the sizes of the complex were decreased.Severe aggregation occurred in GC-PEI/NaCl/DNA complexes at low mass ratio.②The results of MTT showed that the GC-PEI copolymer had little toxicity in five different cell lines,and the relative cell viability is above 80%.The cells had no visible changes at a high concentration of GC-PEI(50μg/mL)compared with negative control group.In vivo,acute toxicity assay revealed that the mices grew well in two weeks with GC-PEI dosage from 50μg to 300μg.The pathologic changes were not observed in major organs of mices by HE staining.③The assay by FACS and fluorescent microscope revealed that the transfection efficiency into hepatocyte lines(L02,QSG7701,QSG7701 /core) was higher than those in non-hepatocyte lines(SGC7901,HBE) in vitro.The transfection efficiency of GC-PEI/DNA/PBS complexes was higher than that of GC-PEI/DNA/GS5%complexes.The galactose could competively inhibit transfection activity of GC-PEI/DNA in expressing HCV-core-QSG7701 cells.The transfection efficiency of the GC-PEI/DNA complex was moderately decreased in serum compared with that in serum free medium.In vivo,the GFP was obviously expressed in liver tissue 48h post-transfection,and one week later the expression began to decrease.No expression was detected in other major organs in both GC-PEI groups(GCPEI/PBS/DNA,GC-PEI/5%GS/DNA).The green fluorescence was not detected in liver tissues in two control groupes.Conclusions:(1) GC-PEI copolymer could carry the exogenous gene specifically to hepatocytes in vitro and in vivo,which have good liver targeted specific property;(2) GC-PEI/DNA complexes could effectively bind and protect DNA;(3) GC-PEI copolymer has no obvious cytotoxicity to tissues and cells in the extent of detection;(4) How to improve the transfection efficiency in vivo is to be further studied.