Wang Chuan, Chen Beibei, He Man, Hu Bin
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China.
ACS Nano. 2021 Feb 23;15(2):3108-3122. doi: 10.1021/acsnano.0c09649. Epub 2021 Feb 11.
It has been well established that the early-stage interactions of nanoparticles with cells are governed by the extracellular protein corona. However, after entering into the cells, the evolving protein corona is the key to subsequent processing of nanoparticles by cells. To identify the protein corona around intracellular nanoparticles, it is essential to maintain its original compositions during cell treatment. Herein, we develop a paraformaldehyde (PFA) cross-linking strategy to stabilize corona compositions when extracting protein coronas from cells, providing original information on protein coronas around intercellular gold nanoparticles (AuNPs). The stability of the protein corona after PFA cross-linking was carefully investigated with several characterization methods, and the results demonstrate that PFA cross-linking successfully prevents the dissociation and exchange of corona proteins. Then the recovered intracellular protein corona around AuNPs from living HepG2 cells with a PFA cross-linking strategy was subjected to nanoHPLC-MS/MS for proteomic analysis. It was found that the compositions of intracellular protein coronas are dominated by cell-derived proteins and undergo significant variation of protein species and amounts over time during internalization. Time-resolved analysis provides relevant proteins involved in nanoparticle cellular uptake and transportation, indicating that AuNPs are endocytosed mainly by a clathrin-mediated uptake mechanism and directed into an endolysosomal pathway toward their final destination. Such proteomic-based results are verified by pharmacological inhibition and TEM imaging analysis. This work provides a universal strategy to study compositions of protein corona around intercellular nanoparticles and could be a footstone to link the formation of protein corona around nanoparticles to their biological function in cells.
纳米颗粒与细胞的早期相互作用由细胞外蛋白冠决定，这一点已得到充分证实。然而，进入细胞后，不断演变的蛋白冠是细胞对纳米颗粒后续处理的关键。为了识别细胞内纳米颗粒周围的蛋白冠，在细胞处理过程中保持其原始组成至关重要。在此，我们开发了一种多聚甲醛（PFA）交联策略，在从细胞中提取蛋白冠时稳定其组成，提供细胞内金纳米颗粒（AuNPs）周围蛋白冠的原始信息。通过几种表征方法仔细研究了PFA交联后蛋白冠的稳定性，结果表明PFA交联成功地防止了冠蛋白的解离和交换。然后，采用PFA交联策略从活的HepG2细胞中回收AuNPs周围的细胞内蛋白冠，进行纳米液相色谱-串联质谱（nanoHPLC-MS/MS）蛋白质组学分析。发现细胞内蛋白冠的组成以细胞来源的蛋白质为主，在内化过程中蛋白质种类和数量随时间发生显著变化。时间分辨分析提供了参与纳米颗粒细胞摄取和运输的相关蛋白质，表明AuNPs主要通过网格蛋白介导的摄取机制被内吞，并进入内溶酶体途径到达其最终目的地。基于蛋白质组学的结果通过药理学抑制和透射电镜成像分析得到验证。这项工作提供了一种研究细胞内纳米颗粒周围蛋白冠组成的通用策略，可能成为将纳米颗粒周围蛋白冠的形成与其在细胞中的生物学功能联系起来的基石。
