目的 制备甘草酸-壳聚糖偶联物并研究HK-2细胞对偶联物的摄取,为肾脏特异性递药系统构建奠定基础。方法 通过甘草酸活性酯与壳聚糖的氨基反应,将壳聚糖与甘草酸共价结合生成偶联物;研究偶联物在木瓜蛋白酶缓冲溶液中的释药行为;采用异硫氰基荧光素标记甘草酸-壳聚糖偶联物,激光共聚焦显微镜观察HK-2细胞对偶联物的摄取情况。结果 经红外光谱、核磁共振波谱法测定,确证了甘草酸-壳聚糖偶联物的化学结构;紫外分光光度法测得偶联物中甘草酸结合率达29.3%;体外释药结果表明,偶联物在木瓜蛋白酶缓冲液中缓慢持续地释放药物;偶联物与HK-2细胞孵育后可较快地为细胞所摄取。结论 本实验成功合成了甘草酸-壳聚糖偶联物,该偶联物可递送甘草酸进入HK-2细胞并有望在肾脏部位缓慢持续释放药物,为肾脏特异性递药系统构建奠定了基础。
Abstract
OBJECTIVE To prepare conjugate of low molecular weight chitosan(LMWC)with glycyrrhizin (GL) and investigate its cellular uptake by proximal tubular epithelial cells (HK-2). METHODS Glycyrrhizin-LMWC (GL-LMWC) conjugate was synthesized by the reaction between the amino group of LMWC and the active ester group of GL. Drug release was studied in phosphate buffer (pH 4.0) containing papain. The uptake of the conjugate by HK-2 cells and its intracellular distribution was studied with laser confocal microscopy. RESULTS The chemical structure of GL-LMWC conjugate was confirmed by IR and 1H-NMR. The GL grafting rate was determined to be 29.3%. The drug liberation from the conjugates was significantly accelerated in the presence of papain, which confirmed the capability of the conjugate to degrade in lysosomes. The conjugate could be internalized by HK-2 cells. CONCLUSION The GL-LMWC conjugate with well-defined structure is successfully synthesized and exhibitts high cellular uptake efficiency. The conjugate has the potential to retaing GL in the kidney for a prolonged duration and to sustain its release locally for better efficacy.
关键词
甘草酸 /
低相对分子质量壳聚糖 /
异硫氰基荧光素 /
细胞摄取
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Key words
glycyrrhizin /
low molecular weight chitosan /
fluorescein isothiocyanate /
cellular uptake
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中图分类号：
R284
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81373982)
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