反义寡核苷酸聚甲基丙烯酸酯纳米粒的入胞行为研讨.pdfVIP

反义寡核苷酸聚甲基丙烯酸酯纳米粒的入胞行为研究 1 *2 1 2 3 王文喜 梁文权 钱俊青 陈海靓 翟建峰 1.浙江工业大学药学院 浙江 杭州3100142.浙江大学药学院 浙江 杭州 310031 3.浙江万马药业有限公司,浙江 杭州 310011 摘要 目的 研究反义寡核苷酸 ODNs 聚甲基丙烯酸酯纳米粒的入胞行为,考察在胞内的分布 探讨其入胞机制 方法 采用流式细胞仪测定荧光标记的寡核苷酸的入胞量 考察载体的浓度对 ODNss入胞率的影响 分别采用在37和4 条件下培养 考察ODNs的入胞行为是否具有能量依赖 性 以判定ODNs是通过内吞途径还是通过融合途径进入细胞 将其与氯喹共同孵育 考察氯喹对 细胞内荧光强度的影响 以判定ODNs是否位于溶酶体内 结果 使用聚甲基丙烯酸酯载体后 细 胞内的ODNs的量急剧增加 当载体的浓度达20g/mL时 细胞内的荧光总量增加683倍 当细胞 在4 条件下培养时 仅有少量的ODNs进入细胞 而在37条件下培养细胞内的ODNs大大增加 氯喹对细胞内的荧光强度几乎无影响 结论 ODNs 聚甲基丙烯酸酯纳米粒主要是通过吞噬途经进 入细胞 进入细胞后能有效地逃离溶酶体 关键词 聚甲基丙烯酸酯 纳米粒 反义寡核苷酸 入胞行为 nanoparticles 1 2 1 2 3 WANG Wen-Xi, LIANG Wen-Quan, QIAN Jun-Qing,CHEN Hai-Liang, ZHAI Jian-Feng (1.College of Pharmacy 3. ABSTRACT:OBJECTIVETo study the uptake behavior and to find the mechanism of uptake. METHODFluorescence labeled o were was investigated. Cells and ODNs-NP was incubated for8h under 4 or 37 ODNs-NP entrance cells by endocytosis orconfluence. Intracellular fluorescence intensity was determined after incubated for 2h with chloroquine, to find ODNs localized in lysosome or not. RESULTS Cellular uptake of oligonucleotides was significantly increased when they were absorbed onto nanopaticles, when the vehicle concentration was 20g/mL,intracellular total fluorescence intensity was added 683 folds. ODNs- ,while 37 they weresignificantly increased. But chloroquine fluorescence when incubated under 4 was no affect on intracellular fluorescence intensity. CONCLUTION With the help of polyacrylate nanoparticles, oligonucleotides entranced the cells by the way of endocytosis and can successfully escape the degradation in lysosome. KEY WORDS: polymethacrylate; nanoparticles;