药学学报, 2013, 48(12): 1829-1835
引用本文:
闻真, 李刚, 林东海, 王俊腾, 秦利芳, 郭桂萍. 聚乳酸羟基乙酸共聚物纳米粒在Caco-2/HT29-MTX共培养细胞模型的转运研究[J]. 药学学报, 2013, 48(12): 1829-1835.
WEN Zhen, LI Gang, LIN Dong-hai, WANG Jun-teng, QIN Li-fang, GUO Gui-ping. Transport of PLGA nanoparticles across Caco-2/HT29-MTX co-cultured cells[J]. Acta Pharmaceutica Sinica, 2013, 48(12): 1829-1835.

聚乳酸羟基乙酸共聚物纳米粒在Caco-2/HT29-MTX共培养细胞模型的转运研究
闻真1, 李刚2, 林东海1, 王俊腾1, 秦利芳1, 郭桂萍1
1. 烟台大学 药学院, 山东 烟台 264005;
2. 烟台大学 生命科学学院, 山东 烟台 264005
摘要:
建立Caco-2/HT29-MTX共培养细胞模型,研究不同表面化学性质的PLGA纳米粒在覆盖黏液层的Caco-2/HT29-MTX共培养细胞模型的转运能力。以聚乳酸羟基乙酸共聚物(PLGA)为载体材料,通过单甲氧基聚乙二醇(mPEG)及壳聚糖(chitosan)对其进行表面修饰,采用纳米沉淀法制备PLGA-NPs、mPEG-PLGA-NPs和壳聚糖包裹的PLGA-NPs,并测定其平均粒径及zeta电位。以香豆素-6(coumarin 6)为荧光标记物,通过激光共聚焦显微镜观察纳米粒的转运情况;以呋喃二烯(FDE)为模型药物,HPLC测定纳米粒的转运量。通过加入内吞阻断剂秋水仙素及诺可唑研究纳米粒的转运机制。采用免疫荧光法考察不同表面化学性质的纳米粒对细胞紧密连接蛋白ZO-1的影响。结果表明,纳米粒分散均匀,PLGA-NPs表面荷负电,经mPEG修饰后zeta电位近电中性,壳聚糖包裹后表面荷正电。呋喃二烯的包封率均>75%。mPEG-PLGA-NPs在Caco-2/HT29-MTX共培养细胞的转运能力高于PLGA-NPs和CS-PLGA-NPs,在秋水仙素及诺可唑作用下转运量明显降低,并影响ZO-1蛋白的分布。说明PLGA-NPs和mPEG-PLGA-NPs可能通过胞吞作用和细胞旁路途径进行转运,CS-PLGA-NPs主要以胞吞作用进行转运;mPEG-PLGA-NPs因其表面的亲水性及电荷近中性,具有较好的抗黏性能,能够快速穿过黏液层到达细胞并转运。
关键词:    PLGA纳米粒      呋喃二烯      Caco-2/HT29-MTX共培养细胞模型      黏液      转运     
Transport of PLGA nanoparticles across Caco-2/HT29-MTX co-cultured cells
WEN Zhen1, LI Gang2, LIN Dong-hai1, WANG Jun-teng1, QIN Li-fang1, GUO Gui-ping1
1. Pharmacy School, Yantai University, Yantai 264005, China;
2. Life School, Yantai University, Yantai 264005, China
Abstract:
The present study is to establish Caco-2/HT29-MTX co-cultured cells and investigate the transport capability of PLGA nanoparticles with different surface chemical properties across Caco-2/HT29-MTX co-cultured cells. PLGA-NPs, mPEG-PLGA-NPs and chitosan coated PLGA-NPs were prepared by nanoprecipitation method using poly(lactic-co-glycolic acid) as carrier material with surface modified by methoxy poly(ethylene glycol) and chitosan. The particle size and zeta potential of nanoparticles were measured by dynamic light scattering. Coumarin 6 was used as a fluorescent marker in the transport of nanoparticles investigated by confocal laser scanning microscopy. The transport of furanodiene (FDE) loaded nanoparticles was quantitively determined by high performance liquid chromatography. Colchicine and nocodazole were used in the transport study to explore the involved endocytosis mechanisms of nanoparticles. Distribution of the tight junction proteins ZO-1 was also analyzed by immunofluorescence staining. The results showed that the nanoparticles dispersed uniformly. The zeta potential of PLGA-NPs was negative, the mPEG-PLGA-NPs was close to neutral and the CS-PLGA-NPs was positive. The entrapment efficiency of FDE in all nanoparticles was higher than 75%. The transport capability of mPEG-PLGA-NPs across Caco-2/HT29-MTX co-cultured cells was higher than that of PLGA-NPs and CS-PLGA-NPs. Colchicine and nocodazole could significantly decrease the transport amount of nanoparticles. mPEG-PLGA-NPs could obviously reduce the distribution of ZO-1 protein than PLGA-NPs and CS-PLGA-NPs. The transport mechanism of PLGA-NPs and mPEG-PLGA-NPs were indicated to be a combination of endocytosis and paracellular way, while CS-PLGA-NPs mainly relied on the endocytosis way. PEG coating could shield the surface charge and enhance the hydrophilicity of PLGA nanoparticles, which leads mPEG-PLGA-NPs to possess higher anti-adhesion activity. As a result, mPEG-PLGA-NPs could penetrate the mucus layer rapidly and transport across Caco-2/HT29-MTX co-cultured cells.
Key words:    PLGA nanoparticle    furanodiene    Caco-2/HT29-MTX co-cultured cell    mucus    transport   
收稿日期: 2013-06-24
基金项目: 国家自然科学基金资助项目(30973949);山东省自然科学基金资助项目(ZR2009CM012);山东省优秀中青年科学家科研奖励基金资助项目(BS2012YY039).
通讯作者: 林东海
Email: lindonghai_dds@163.com
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