半乳糖化壳聚糖-低分子聚乙烯亚胺/DNA复合物的肝靶向性研究(英文)

Hepatocyte-targeted gene transfection of galactosylated chitosan-graft low molecular polyethyleneimine/DNA complexes

目的：研究半乳糖化壳聚糖-低分子聚乙烯亚胺（galactosylated chitosan-graft-polyethyleneimine,GC-PEI）/DNA复合物在体内外的肝靶向性。方法：GC-PEI与增强型绿色荧光蛋白（enhanced green fluorescentprotein,EGFP）质粒（pEGFP-Cl）在0.01 mol/L PBS,150 mmol/L NaCl,5%葡萄糖溶液中自组装成3种不同溶媒介导的GC-PEI/DNA复合物,检测复合物粒径大小与形态,Zeta电位以及结合和保护DNA的能力;并进一步测定GC-PEI聚合物的毒性,研究复合物的肝靶向转染效率。结果：在GC-PEI与DNA质量比为1∶1-2.5∶1时,GC-PEI聚合物能有效地结合和保护所携带的DNA免受核酸酶和血清的降解。复合物粒子呈规则的球形,有明显的核壳结构。GC-PEI聚合物在检测细胞中未显示出明显毒性;动物体内急性毒性实验显示：通过尾静脉注射50-300μg的GC-PEI聚合物入小鼠后,实验小鼠2周内无急性毒性反应和死亡发生。荧光显微镜和流式细胞仪检测证实GC-PEI/DNA复合物在肝细胞系（QSG7701/core,L02）中的绿色荧光蛋白（green fluorescent protein,GFP）表达明显高于非肝细胞系（SGC-7901,HBE）细胞。体内实验表明转染48 h后,小鼠肝组织在荧光显微镜下可以检测到明显的绿色荧光,而其他主要脏器未见明显荧光。结论：GC-PEI聚合物能够在体内外特异性将外源基因或DNA导入肝细胞,具有良好的肝靶向性。

Objective To investigate the hepatocyte targeted specific property of galactosyla-ted chitosan-graft-polyethyleneimine（GC-PEI）/DNA complexes in vitro and in vivo.Methods With the plasmid expressing enhanced green fluorescent protein（pEGFP-C1） as the reporter gene,the formation of GC-PEI/DNA complexes was induced to self-assemble in 0.01 mol/L phosphate buffered saline（PBS）,150 mmol/L NaCl,or 5% glucose solution（GS）.The complexes were cha-racterized by the particle size,Zeta potential,DNA binding and protection capacity,and further tested for cytotoxicity and hepatocyte targeted transfection activity.Results With the GC-PEI/DNA mass ratio from 1∶1 to 2.5∶1,the GC-PEI/DNA complexes effectively bound and protected the DNA from degradation of DNaseⅠand the serum,which presented as a well-formed sphere or compacted nucleocapsid structure at a diameter of 50-200 nm.The GC-PEI copolymer showed no obvious toxicity in the tested cell lines.Acute toxicity assay revealed that the mice grew well in 2 weeks with GC-PEI dosage from 50 to 300 μg.The assay by flow cytometry and fluorescent microscope showed that the transfection efficiency in hepatocyte lines（L02,QSG7701/core） was higher than that in non-hepatocyte lines（SGC7901,HBE） in vitro.In vivo,the GFP was obviously expressed in the liver tissue and not expressed in other organs 48 h after the transfection.Conclusion GC-PEI copolymer may carry the exogenous gene specifically to hepatocytes in vitro and in vivo,which has very good liver targeted specific property.