Guangdong Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515 Guangdong, China.
Artemisinin Research Center, and Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
J Colloid Interface Sci. 2023 Oct 15;648:497-510. doi: 10.1016/j.jcis.2023.05.045. Epub 2023 May 10.
Nanoparticles (NPs) have broad application prospects in the field of biomedicine due to their excellent physicochemical properties. When entering biological fluids, NPs inevitably encountered proteins and were subsequently surrounded by them, forming the termed protein corona (PC). As PC has been evidenced to have critical roles in deciding the biological fates of NPs, how to precisely characterize PC is vital to promote the clinical translation of nanomedicine by understanding and harnessing NPs' behaviors. During the centrifugation-based separation techniques for the PC preparation, direct elution has been most widely used to strip proteins from NPs due to its simpleness and robustness, but the roles of multifarious eluents have never been systematically declared. Herein, seven eluents composed of three denaturants, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea (Urea), were applied to detach PC from gold nanoparticles (AuNPs) and silica nanoparticles (SiNPs), and eluted proteins in PC have been carefully characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chromatography coupled tandem mass spectrometry (LC-MS/MS). Our results showed that SDS and DTT were the main contributors to the efficient desorption of PC on SiNPs and AuNPs, respectively. The molecular reactions between NPs and proteins were explored and verified by SDS-PAGE analysis of PC formed in the serums pretreated with protein denaturing or alkylating agents. The proteomic fingerprinting analysis indicated the difference of the eluted proteins brought by the seven eluents was the abundance rather than the species. The enrichment of some opsonins and dysopsonins in a special elution reminds us that the possibility of biased judgments on predicting NPs' biological behaviors under different elution conditions. The synergistic effects or antagonistic effects among denaturants for eluting PC were manifested in a nanoparticle-type dependent way by integrating the properties of the eluted proteins. Collectively, this study not only underlines the urgent need of choosing the appropriate eluents for identifying PC robustly and unbiasedly, but also provides an insight into the understanding of molecular interactions during PC formation.
纳米粒子（NPs）由于其优异的物理化学性质，在生物医学领域具有广泛的应用前景。当进入生物流体时，NPs 不可避免地会遇到蛋白质，并随后被它们包围，形成所谓的蛋白质冠（PC）。由于 PC 已被证明在决定 NPs 的生物学命运方面起着关键作用，因此如何精确地表征 PC 对于通过了解和利用 NPs 的行为来促进纳米医学的临床转化至关重要。在用于 PC 制备的基于离心的分离技术中，由于其简单性和稳健性，直接洗脱已被最广泛地用于从 NPs 上剥离蛋白质，但各种洗脱液的作用从未被系统地声明。在此，七种洗脱液由三种变性剂、十二烷基硫酸钠（SDS）、二硫苏糖醇（DTT）和尿素（Urea）组成，用于从金纳米粒子（AuNPs）和硅纳米粒子（SiNPs）上分离 PC，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和色谱串联质谱（LC-MS/MS）仔细表征 PC 中洗脱的蛋白质。我们的结果表明，SDS 和 DTT 分别是有效去除 SiNPs 和 AuNPs 上 PC 的主要贡献者。通过对用蛋白质变性或烷化剂预处理的血清中形成的 PC 的 SDS-PAGE 分析，探索和验证了 NPs 与蛋白质之间的分子反应。蛋白质组学指纹分析表明，七种洗脱液洗脱的蛋白质的差异在于丰度而不是种类。在特殊洗脱液中某些调理素和失调素的富集提醒我们，在不同洗脱条件下预测 NPs 生物学行为时，存在有偏差判断的可能性。通过整合洗脱 PC 的变性剂的性质，以依赖于纳米粒子类型的方式表现出变性剂之间的协同或拮抗作用。总的来说，这项研究不仅强调了需要选择合适的洗脱液来稳健和无偏地识别 PC，而且还深入了解了 PC 形成过程中的分子相互作用。
