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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 study the hepatic targeting of galactosylated chitosan-graft-polyethyleneimine (GC-PEI) / DNA complex in vitro and in vivo. METHODS: GC-PEI and enhanced green fluorescent protein (EGFP) plasmids (pEGFP-Cl) were self-assembled into three different media in 0.01 mol / L PBS, 150 mmol / L NaCl and 5% The GC-PEI / DNA complex was used to detect the particle size and morphology, Zeta potential and the ability to bind and protect DNA. The toxicity of GC-PEI polymer was further studied, and the liver-targeted transfection efficiency . Results: At the mass ratio of GC-PEI to DNA of 1: 1-2.5: 1, the GC-PEI polymer can effectively bind and protect the DNA from nuclease and serum degradation. The composite particles are regularly spherical with obvious core-shell structure. The GC-PEI polymer showed no obvious toxicity in the test cells. The acute toxicity test in animals showed that there was no acute toxicity reaction within 2 weeks after injection of 50 ~ 300μg GC-PEI polymer into the tail vein And death happened. Fluorescence microscopy and flow cytometry demonstrated that the expression of green fluorescent protein (GFP) in GC-PEI / DNA complex was significantly higher in hepatocyte cell line QSG7701 / core (L02) than in non-hepatocyte cell line SGC- 7901, HBE) cells. In vivo experiments showed that 48 h after transfection, the mouse liver tissue under the fluorescence microscope can detect significant green fluorescence, while other major organs showed no significant fluorescence. CONCLUSION: GC-PEI polymers can specifically introduce foreign genes or DNA into hepatocytes in vitro and in vivo with good liver targeting.