肝细胞癌靶向多功能纳米载体的应用

Application of hepatocellular carcinoma targeted multifunctional nanocarriers

目的：合成精氨酸－甘氨酸－天冬氨酸（ RGD）修饰的具备MRI显像和基因传输功能的纳米载体，研究载体对肝细胞癌的靶向基因传输功能和MRI示踪能力。方法制备载体RGD－聚乙二醇（ PEG）－聚乙烯亚胺（ PEI）－超顺磁性氧化铁纳米颗粒（ SPION）；利用凝胶阻滞实验分析载体复合小分子干扰RNA（ siRNA）的能力；经电位及粒度测定仪测量载体复合siRNA后的粒径和电位；用免疫细胞化学实验分析Bel－7402细胞表面αvβ3的分布；用流式细胞仪、激光共聚焦显微镜分析载体对Bel－7402细胞的基因传输功能；验证载体对Bel－7402细胞的MRI显像功能。结果氮磷比（ N／P）＝2．8，RGD－PEG－PEI－SPION完全复合siRNA；Bel－7402细胞膜表达大量αvβ3；N／P＝10，载体复合siRNA后粒径趋于稳定，为（85．2±5．6）nm，电位为（＋12．4±1．2）mV，载体对Bel－7402细胞的基因转染率达到71．2％±2．1％，激光共聚焦显微镜下胞质内可见大量表征载体的红色荧光和表征siRNA的绿色荧光；与RGD－PEG－PEI－SPION孵育的Bel－7402细胞于T2＊WI呈现显著的低信号强度。结论本研究成功合成RGD修饰、MRI可视的载体RGD－PEG－PEI－SPION；载体可靶向、高效的传输siRNA至肝细胞癌，并且对肝细胞癌具备敏感的MRI显像能力。

Objective To synthesize the RGD-modified magnetic resonance imaging （ MRI）-visible gene transfer nanocarrier and assess its gene delivery ability and MRI visibility for hepatocellular carcinoma.Methods The multifunctional nanocarrier RGD-PEG-PEI-SPION was constructed.And the degree of binding between nanocarrier and siRNA was determined by agarose gel electrophoresis.The zeta potential and particle size of cationic polymer vectors were measured with a Zeta-Plus instrument.Immunocytochemical assay was performed for detecting the expression of αvβ3 in Bel-7402 cells.The active targeting ability of nanocarrier to Bel-7402 cells was evaluated by flow cytometry and laser confocal microcopy.The MRI visibility of nanocarrier to Bel-7402 cells was assessed.Results RGD-PEG-PEI-SPION could condense siRNA entirely at a N/P ratio of 2.8;the membranes of Bel-7402 cells possessed an enrichment of αvβ3 receptors.At a nitrogen/phosphate ratio of 10,the particle size of RGD-PEG-PEI-SPION/siRNA attained a constant size of 85.2 ±5.6 nm,the zeta potential reached ＋12.4 ±1.2 mV,the gene transfection efficiency of nanocarrier to Bel-7402 cells attained 71.2%±2.1% and the cells showed significantly stronger RGD-PEG-PEI-SPION （ red） and siRNA （ green） fluorescence under laser confocal microcopy.The cells incubated with RGD-PEG-PEI-SPION exhibited significantly lower normalized MR T2＊WI signal intensity.Conclusion A RGD-modified MRI-visible gene delivery nanocarrier RGD-PEG-PEI-SPION has been successfully synthesized.It has a higher transfection efficiency of transferring siRNA into Bel-7402 cells and could sensitively detect hepatocellular carcinoma with MRI.