功能化硅壳荧光纳米载体在A549细胞中的细胞标记  

Cell labeling of functionalized silica-coated fluorescent nanocarriers in A549 cells

作　　者：赵焕英[1,2] 隋雷鸣[2] 李峰[1] 李睿[1] 蔡青[2] 邓君[2] 高尔静[2] 姬曼[2] 杨慧[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京市神经再生修复研究重点实验室,北京市100069 [2]首都医科大学医学实验与测试中心,北京市100069

出　　处：《中国组织工程研究与临床康复》2009年第8期1486-1490,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家重点基础研究发展计划("九七三")(2006CB500706);国家自然科学基金项目(30670655);北京市属高等学校人才强教深化计划项目~~

摘　　要：背景:硅壳荧光磁性纳米载体表面功能化后不仅仍具备荧光性、磁性功能,而且还具有携药和携基因潜能,并有良好的生物相容性,可实现对细胞的标记功能。目的:制备Fe3O4@SiO2(FITC)-3-异氰基丙基三乙氧基硅烷-聚酰胺-胺树状大分子(G2.0)纳米颗粒型多功能纳米载体,并评估纳米载体标记A549细胞的能力,在细胞内的分布及与细胞的生物相容性。设计、时间及地点:观察性实验,于2006-06/2007-06在首都医科大学化学生物学与药学院合成了纳米载体,2007-07/2008-07在首都医科大学神经科学研究所完成了复合纳米载体的生物评估。材料和方法:15μL3-异氰基丙基三乙氧基硅烷和195.12mg聚酰胺-胺树状大分子(G2)在无水甲醇环境中反应24h,得到目的产物3-异氰基丙基三乙氧基硅烷-聚酰胺-胺树状大分子;然后将合成好的Fe3O4@SiO2(FITC)与3-异氰基丙基三乙氧基硅烷-聚酰胺-胺树状大分子在甲醇溶液环境下,避光搅拌48h,用永磁铁分离固体,并经无水乙醇洗后真空干燥,得到最终目标载体。主要观察指标:透射电镜观察纳米载体的大小和细胞内分布,Zeta电位测定其生理情况下电荷情况,激光共聚焦显微镜下观察FITC、DAPI和LysotrackerBlue细胞染色情况以评估载体在细胞内定位,CCK-8评估其对细胞毒性作用,流式细胞计数法评价其标记细胞的效率。结果:透射电镜分析表明,修饰的硅壳纳米载体大小约为80nm,pH=7.4,纳米载体zeta电位为+23.93mV。透射电镜和激光共聚焦显微镜结果表明纳米粒主要存在细胞浆中,且能被溶酶体吞噬。CCK-8结果显示纳米载体的浓度高达1g/L时仍无明显的毒性作用。流式细胞计数结果表明,细胞摄取纳米粒呈浓度和时间依赖性。结论:成功制备了硅壳结构的荧光磁性纳米载体,主要分布在细胞浆中,且细胞相容性好。BACKGROUND： Functionalized silica-coated fluorescent nanocarriers are characterized by not only fluorescence and magnetism but also drug-carrying and gene-carrying potency; in addition, they also have excellent biocompatibility so as to label cells. OBJECTIVE： To prepare Fe304@SiO2（FITC）-3-isocyanatopropyltriethoxysilane （ICP）-polyamidoamine （PAMAM） （G2.0） Nanocarriers as a multifunctional carrier and assess their ability of cell labeling, localization in cell and cellular biocompatibility. DESIGN, TIME AND SETTING： An observational study, of which nanocarriers was synthesized at the School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University between June 2006 and June 2007, and of which biological evaluation was performed at Beijing Institute of Neuroscience, Capital Medical University between July 2007 and July 2008. MATERIALS AND METHODS： 15 μL 3-1CP and 195.12 mg PAMAM（G2.0） were inter-acted in anhydrous methanol for 24 hours to obtain 3-1CP-PAMAM（G2.0） nanocarriers; and then, the synthesized Fe304@SiO2（FITC）-3-1CP-PAMAM（G2.0） nanocarriers were stirred in methanol away from light for 48 hours. The solid product was separated by using permanent magnet and vacuum-dried with anhydrous methanol so as to obtain the final target carrier. MAIN OUTCOME MEASURES： The size and intracellular localization of the carrier were observed by transmission electron microscope; the charge was measured by Zeta potential; fluorescein isothiocyanate （FITC）, 4＇,6-diamidino-2-phenylindole （DAPI） and LysoTracker Blue fluorescent molecular markers were observed under confocal laser microscope in order to evaluate intracellular localization of the carrier; cytotoxicity was evaluated by cell counting kit-8 （CCK-8） assay; the efficiency of cell labeling was evaluated by flow cytometry analysis. RESULTS： Transmission electron microscope revealed that the average size of the nanocarrier was about 80 nm （pH=7.4）, and Zeta potential of Nanocarrier was ＋23.

关 键 词：SiO_2纳米粒子 生物材料 细胞相容性 细胞标记 

分 类 号：R318[医药卫生—生物医学工程]