普鲁兰基肿瘤靶向纳米粒的体外抑瘤效应和细胞摄取途径研究  

Cellular uptake of Tumor-targeted nanoparticles derived from pullulan acetate conjugate

作　　者：郏亚静 陈红丽[1] 唐红波[2] 周志敏[3] 张明明[3] 邢雪琨[1] 丰慧根[1] 

机构地区：[1]新乡医学院生命科学技术学院,新乡医学硕士研究生453003 [2]首都医科大学附属北京妇产医院药剂科,北京100026 [3]中国医学科学院/北京协和医学院生物医学工程研究所,天津300192

出　　处：《医学研究生学报》2015年第2期127-130,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81401519;U1304819);北京市自然科学基金(7132064);河南省科技发展计划(122102210148)

摘　　要：目的叶酸受体在某些肿瘤细胞表面的高表达给靶向治疗提供了理论依据。文中制备肿瘤靶向性自组装纳米药物载体,探讨其体外细胞抑瘤效应及其细胞摄取途径,评价作为靶向纳米药物载体的可行性。方法透析法制备包载表柔比星的乙酰普鲁兰叶酸偶合体(fola te con juga ted PA,FPA)纳米粒(FPA/EPI NP),MTS法考察该抗肿瘤药物载体对于肝癌细胞(Hep G2,叶酸受体阴性细胞株)和宫颈癌细胞(Hela,叶酸受体高表达细胞株)的抑瘤效应。细胞实验分为纳米粒对照组、氯丙嗪组、氯喹组、阿米洛利组和叶酸组。对照组不经各种抑制剂预处理,直接加入FPA/EPI NP悬液,37℃孵育2 h,用流式细胞分析仪检测荧光强度。结果纳米粒呈球形,FPA NP粒径为(204.2±10.9)nm,FPA/EPI NP粒径(273.4±11.0)nm,FPA/EPI NP载药量和包封率分别为(6.45±1.04)%和(72.45±11.50)%。当Hep G2和Hela细胞分别与5、40、200、400和1000μg/m L的FPA NP孵育24 h时,细胞存活率均>95%;孵育72 h时细胞存活率高达90.0%。FPA/EPI NP作用Hep G2细胞24 h时,存活率分别是(92.3±5.2)%、(70.4±4.6)%、(54.0±4.0)%、(41.1±4.1)%和(27.0±3.6)%。与纳米粒对照组相比,用氯丙嗪、阿米洛利、叶酸分别预处理Hela细胞,纳米粒的摄取量均减少(P<0.05);用氯丙嗪、阿米洛利分别预处理Hep G2细胞,纳米粒的摄取量均下降(P<0.05)。FPA/EPI NP作用Hep G2和Hela细胞72 h时半数抑瘤浓度分别为168μg/m L和105μg/m L。结论对于Hep G2细胞,FPA/EPI NP主要通过网格蛋白介导的内吞以及巨胞饮途径进入细胞,对于Hela细胞,主要通过网格蛋白介导的内吞以及叶酸受体介导的途径进入细胞。FPA NP有望成为一种新型的肿瘤靶向药物载体。Objective Folate receptors（ FRs）,overexpressed on the surface of a variety of tumor cells,are potential targets for tumor targeting therapy. This study aimed to prepare an FR-mediated drug nanocarrier with folate conjugated pullulan acetate（ FPA） to target chemotherapeutic agents to FR-overexpressed tumor cells and investigate its tumor-suppressing effect in vitro.Methods The cytotoxicity of epirubicin-loaded FPA nanoparticles（ FPA/EPI NP） against Hep G2 and Hela cells was evaluated by MTS assay. The Hep G2 and Hela cells were divided into five groups to be treated with NPs（ NP control）,chlorpromazine,chloroquine,amiloride,and folate,respectively,followed by detection of the fluorescence intensity by flow cytometry. Results FPA/EPI NP was successfully formulated into NPs,with the mean particle size of（ 268. 5 ± 12. 0） nm,by dialysis with an almost spherical shape.The drug-loading rate and entrapment rate of FPA/EPI NP were（ 6. 45 ± 1. 04） and（ 72. 45 ± 11. 50） %,respectively. The survival rates of the Hep G2 and Hela cells were both 95% after 24 hours of incubation with FPA NP at 5,40,200,400 and 1000 μg/m L and90. 0% after 72 hours. The survival rates of the Hep G2 cells treated with 5,40,200,400 and 1000 μg/m L FPA/EPI NP for 24 hours were（ 92. 3 ± 5. 2）,（ 70. 4 ± 4. 6）,（ 50. 0 ± 4. 0）,（ 41. 1 ± 4. 1）and（ 27. 0 ± 3. 6） %,respectively. Compared with the NP control group,the Hela cells of the chlorpromazine,amiloride and folate groups showed a significantly lower rate of NP uptake（ P〈0. 05）,and so did the Hep G2 cells pretreated with chlorpromazine or amiloride（ P〈0. 05）. At 72 hours,the half maximal inhibitory concentrations（ IC50） of FPA/EPI NP against Hep G2 and Hela cells were168 and 105 μg/m L,respectively. Conclusion Clathrin-mediated endocytosis and macropinocytosis are involved in the internalization of FPA/EPI NP in Hep G2 cells,while clathrin-and FR-mediated endocytosis in that of Hela cells. FPA NP may serve as a new drug carrie

关 键 词：普鲁兰多糖 叶酸受体 纳米粒 靶向抗肿瘤 细胞摄取 

分 类 号：R318.08[医药卫生—生物医学工程]