胆固醇基修饰的普鲁兰纳米粒的摄取机制及亚细胞分布研究

目的 研究胆固醇基修饰的普鲁兰（CHSP）纳米粒的体外HepG2细胞的摄取机制及亚细胞分布.方法 采用异硫氰酸荧光素（FTTC）标记CHSP,透析法制备FITC标记的CHSP（FITC-CHSP）自组装纳米粒并进行表征.采用MTT法考察CHSP纳米粒对HepG2细胞的毒性.选用选择性的内吞途径抑制剂氯丙嗪、菲律宾菌素和阿米洛利来研究HepG2细胞摄取CHSP纳米粒的机制.最后,采用细胞免疫荧光法对内质网、高尔基体和溶酶体进行染色,使用激光扫描共聚焦显微镜（CLSM）观察不同的孵育时间CHSP纳米粒的亚细胞分布.结果 成功合成了FITC-CHSP,且FITC-CHSP能在水介质中自组装成纳米粒,该纳米粒呈规则球形,平均粒径为（63.0 ±1.9） nm.MTT结果表明CHSP纳米粒对HepG2细胞无明显的细胞毒性.内吞抑制实验表明网格蛋白介导的内吞途径以及巨胞饮途径共同参与了CHSP纳米粒的入胞过程.纳米粒的亚细胞分布实验表明：在研究的孵育时间（4 h）内,并未发现CHSP纳米粒进入高尔基体和内质网;当纳米粒与HepG2细胞孵育30 min时,没有纳米粒定位于溶酶体中,随着孵育时间的延长,大量纳米粒分布于溶酶体中.结论 CHSP纳米粒有望成为细胞内递送治疗剂的一种通用载体.

Study on cellular uptake mechanism and subcellular distribution of cholesterol-modified pullulan nanoparticles

Objective To investigate the cellular uptake mechanism and subcellular distribution of cholesterol-modified pullulan （CHSP） nanoparticles by human hepatocellular carcinoma （HepG2） cells.Methods Fluorescein isothiocyanate （FITC） was conjugated to CHSP,and FITC-labeled CHSP （FITC-CHSP） nanoparticles were prepared by dialysis.The cytotoxicity of CHSP nanoparticles against HepG2 cells was evaluated by MTT assay.Meanwhile,several selective endocytosis inhibitors （chlorpromazine,filipin and amiloride） were selected to study the potential endocytosis mechanism.To study the subcellular distribution of CHSP nanoparticles at different incubation times,immunofluorescence staining was used to identify the Golgi apparatus,endoplasmic reticula （ER） and lysosomes,followed by confocal laser scanning microscopy（CLSM） observation.Results Covalent conjugation with FITC yielded stably labeled CHSP,which was successfully formulated into nanoparticles （mean particle size （63.0±1.9） nm） by dialysis with an almost spherical shape.MTT assay clearly indicated that the CHSP nanoparticles did not show significant toxicity in HepG2 cells.In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were both involved in the internalization of CHSP nanoparticles.The subcellular distribution study demonstrated that CHSP nanoparticles were entrapped in the lysosomes at 1 h after incubation,while colocalization of nanoparticles with either the Golgiapparatus or the ER was not observed during the entire course of the study.Conclusions CHSP nanoparticles may serve as a versatile carrier for intracellular delivery of therapeutic agents.