Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, Berlin 12489, Germany; Humboldt-Universität zu Berlin, School of Analytical Sciences Adlershof, Albert-Einstein-Str. 5-9, Berlin 12489, Germany.
Universidad de Oviedo, C/Julián Clavería 8, Oviedo E-33006, Spain.
J Proteomics. 2020 Feb 10;212:103582. doi: 10.1016/j.jprot.2019.103582. Epub 2019 Nov 12.
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.
纳米粒子的诊断和治疗应用需要全面了解它们与生物分子环境的相互作用。然而，对于活细胞内内化的纳米粒子形成的蛋白质冠的形成，我们还尚未完全理解。在本研究中，我们提出了一种强大的方法，用于电泳和质谱分析在 ~30nm 柠檬酸钠稳定的金纳米粒子上形成的活细胞中的硬蛋白冠组成，即与金纳米粒子表面亲和力最高的蛋白质。金纳米粒子被 MCF-7 细胞内化 24 小时，然后从活细胞培养物中提取硬蛋白冠-金纳米粒子生物缀合物。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，这允许鉴定 108 种硬壳蛋白。用 J774 巨噬细胞重复进行孵育时间为 1.5 小时、3 小时、6 小时和 24 小时的实验，结果表明硬蛋白壳在一段时间内保持不变。因此，所提出的实验方法被证明是一种识别活细胞内化的纳米粒子硬壳蛋白的有价值的工具。
