Rothen-Rutishauser Barbara, Kuhn Dagmar A, Ali Zulqurnain, Gasser Michael, Amin Faheem, Parak Wolfgang J, Vanhecke Dimitri, Fink Alke, Gehr Peter, Brandenberger Christina
Adolphe Merkle Institute, University of Fribourg, Marly, Switzerland.
Nanomedicine (Lond). 2014 Apr;9(5):607-21. doi: 10.2217/nnm.13.24. Epub 2013 Jun 5.
We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches.
MATERIALS & METHODS: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment.
Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy.
We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology.
我们通过两种定量显微镜方法研究了荧光标记的聚合物包被金纳米颗粒（NPs）在不同生物学条件下的细胞摄取机制。
使用经特异性和细胞毒性测试的内吞抑制剂评估摄取机制。通过激光扫描显微镜或透射电子显微镜分析金纳米颗粒的细胞摄取情况，并使用来自同一实验的细胞通过体视学进行定量。
仅使用氯丙嗪（网格蛋白介导的内吞作用）和甲基-β-环糊精（小窝蛋白介导的内吞作用）可实现最佳抑制剂条件。两种成像技术均显示甲基-β-环糊精介导的细胞内纳米颗粒显著抑制（63 - 69%）以及氯丙嗪介导的增加（43 - 98%），表明主要通过小窝蛋白介导的内吞作用摄取。透射电子显微镜成像显示超过95%的纳米颗粒位于细胞内囊泡中，并且检测到的每个细胞的纳米颗粒事件比激光扫描显微镜多约150倍。
我们强调在受控实验条件下并结合体视学以足够的显微镜分辨率研究纳米颗粒与细胞相互作用的重要性。
