Proteomic dissection of nanoparticle-containing vesicles reveals a major role for a macropinocytic-like uptake mechanism and multivesicular bodies during intracellular nanoparticle trafficking

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.

用于药物递送策略的纳米载体合理设计，需要对纳米颗粒的摄取机制与细胞内转运通路具备无偏倚的认知。本研究采用定量蛋白质组学方法解析了上述过程：我们分离得到携带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法分析其蛋白质组成。针对细胞器标志物蛋白的蛋白质组学快照分析显示，一种非典型巨胞饮样介导机制促成了纳米颗粒的细胞内进入。研究表明，该进入过程受肌动蛋白重组、非典型巨胞饮信号通路以及ADP核糖基化因子1 (ADP-ribosylation factor 1) 调控。此外，本研究的蛋白质组学数据证实，多泡体与多层溶酶体在纳米颗粒的细胞内转运及最终储存环节中发挥核心作用，该结论通过共聚焦显微镜与冷冻透射电子显微镜 (cryo-TEM) 实验得到了验证。通过定量分析负载纳米颗粒的囊泡蛋白质组成，本研究明确界定了纳米颗粒的进入途径、细胞内转运过程，以及负载纳米颗粒囊泡的蛋白质组学微环境。