The diagnostic and therapeutic application of nanoparticles requires comprehensive knowledge of their interaction with the biomolecular surroundings. The formation of the protein corona on nanoparticles that were internalized by living cells is yet to be understood. In this study, we present a robust approach for the electrophoretic and mass spectrometric analysis of the hard protein corona composition formed in living cells on ~30 nm citrate-stabilized gold nanoparticles, i.e., the proteins with the highest affinity towards the gold nanoparticle surface. The gold nanoparticles were internalized by MCF-7 cells for 24 h followed by the extraction of the hard protein corona‑gold nanoparticle bioconjugates from living cell cultures. The extracted proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by ESI-Q-TOF-MS, which allowed to identify 108 hard corona proteins. The experiments were repeated with J774 macrophage cells with incubation times of 1.5 h, 3 h, 6 h, and 24 h, and the results showed that the hard protein corona remained unchanged over time. Therefore, the proposed experimental approach proved to be a valuable tool for identifying hard corona proteins of nanoparticles internalized by living cells.

中文翻译：

---

质谱法分析活细胞中纳米颗粒的硬蛋白电晕。

纳米颗粒的诊断和治疗应用需要全面了解其与生物分子环境的相互作用。在被活细胞内化的纳米颗粒上形成蛋白质电晕尚未被理解。在这项研究中，我们提出了一种在电泳和质谱分析中在约30 nm柠檬酸盐稳定的金纳米颗粒上，即对金纳米颗粒表面具有最高亲和力的蛋白质上，在活细胞中形成的硬蛋白电晕成分的可靠方法。金纳米颗粒由MCF-7细胞内化24小时，然后从活细胞培养物中提取硬蛋白电晕-金纳米颗粒生物结合物。然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离提取的蛋白质，并通过ESI-Q-TOF-MS进行分析，从而鉴定出108种硬质电晕蛋白质。用J774巨噬细胞重复实验，孵育时间为1.5 h，3 h，6 h和24 h，结果表明硬蛋白电晕随时间保持不变。因此，所提出的实验方法被证明是鉴定被活细胞内化的纳米颗粒的硬质电晕蛋白的有价值的工具。