Internalization and Cellular Fate of Protein Corona‐Coated Nanoparticles by Multimodal Multi‐Scale Microscopy

Abstract Upon exposure to biological environments, nanoparticles are rapidly coated with biomolecules, predominantly proteins, which alter their colloidal stability, biodistribution, and cell interactions. Despite extensive efforts to investigate the nanoparticles' fate, only a few studies use high‐resolution characterization methods that allow in‐depth characterization, and the existing methodologies are unable to differentiate particles internalized at the onset of incubation from those taken up toward the end of an incubation period. In this study, these limitations related to incubation disparities are overcame and precisely monitored the spatiotemporal displacement of colloidally stable protein corona‐coated nanoparticles within cells. An unprecedented application of cryogenic X‐ray nanotomography, combined with high‐resolution, super‐resolution, and correlative microscopy techniques, revealed the migration of nanoparticles to the perinuclear region while monitoring the evolution of cellular organelles in fully hydrated cells under near‐native conditions, without the need for contrasting agents. Notably, this tracking indicates the progressive fusion of vesicles carrying the nanoparticles intracellularly. This strategy demonstrates the potential for uncovering the temporal aspects of nanoparticle behavior within cells and can be adaptable to a wide range of nanoparticles and cell types, offering a versatile and powerful tool to follow nanoparticles in cellular environments.