Max Planck Institute for Polymer Research , Ackermannweg 10, 55128 Mainz, Germany.
ACS Nano. 2014 Oct 28;8(10):10077-88. doi: 10.1021/nn502754c. Epub 2014 Sep 29.
Rational design of nanocarriers for drug delivery approaches requires an unbiased knowledge of uptake mechanisms and intracellular trafficking pathways. Here we dissected these processes using a quantitative proteomics approach. We isolated intracellular vesicles containing superparamagnetic iron oxide polystyrene nanoparticles and analyzed their protein composition by label-free quantitative mass spectrometry. The proteomic snapshot of organelle marker proteins revealed that an atypical macropinocytic-like mechanism mediated the entry of nanoparticles. We show that the entry mechanism is controlled by actin reorganization, atypical macropinocytic signaling, and ADP-ribosylation factor 1. Additionally, our proteomics data demonstrated a central role for multivesicular bodies and multilamellar lysosomes in trafficking and final nanoparticle storage. This was confirmed by confocal microscopy and cryo-TEM measurements. By quantitatively analyzing the protein composition of nanoparticle-containing vesicles, our study clearly defines the routes of nanoparticle entry, intracellular trafficking, and the proteomic milieu of a nanoparticle-containing vesicle.
为了实现药物输送方法的合理设计，需要对摄取机制和细胞内运输途径有一个公正的了解。在这里，我们使用定量蛋白质组学方法来剖析这些过程。我们分离了含有超顺磁性氧化铁聚苯乙烯纳米颗粒的细胞内囊泡，并通过无标记定量质谱分析了它们的蛋白质组成。细胞器标记蛋白的蛋白质组快照显示，一种非典型的巨胞饮样机制介导了纳米颗粒的进入。我们表明，进入机制受肌动蛋白重组、非典型巨胞饮信号和 ADP-核糖基化因子 1 控制。此外，我们的蛋白质组学数据表明多泡体和多层溶酶体在运输和最终纳米颗粒储存中起核心作用。这一点通过共聚焦显微镜和冷冻 TEM 测量得到了证实。通过定量分析含纳米颗粒囊泡的蛋白质组成，我们的研究清楚地定义了纳米颗粒进入、细胞内运输以及含纳米颗粒囊泡的蛋白质组环境的途径。
