Correlative Synchrotron X-ray Microscopy Reveals Dose- and Division-Dependent Nanoparticle Redistribution in Macrophages

Abstract

Understanding the intracellular fate of nanoparticles is essential for designing safer and more effective nanomedicines, yet most studies rely on static observations and lack high-resolution, near-native volumetric information. Here, we establish a synchrotron-based correlative X-ray microscopy framework to investigate how fluorescent silica nanoparticles (SiNPs) redistribute within macrophages as a function of concentration and successive cell-division cycles. SiNPs were internalized by RAW 264.7 macrophages at different concentrations and analyzed using a synchrotron-based correlative X-ray microscopy workflow integrating cryogenic soft X-ray tomography (cryo-SXT), cryogenic structured illumination microscopy (cryo-SIM), and coherent X-ray ptychography, with confocal fluorescence microscopy used to establish population-level uptake tendencies. Cryo-SXT reveals a concentration-dependent redistribution of nanoparticle-containing vesicles from peripheral endosomes toward the perinuclear region, while correlative cryo-SIM confirms strict vesicular confinement, with no evidence of free nanoparticle diffusion into the nucleoplasm. At higher doses, nanoparticles approach the nuclear region via vesicles extending into nuclear-envelope invaginations, rather than by true nuclear entry. Successive cell divisions redistribute the intracellular nanoparticle load and promote stable perinuclear clustering, identifying a long-term sequestration route in macrophages. Coherent X-ray ptychography further reveals nanoscale deformations of the nuclear envelope associated with dense perinuclear vesicles. Together, these results establish synchrotron-based correlative X-ray microscopy as a mechanistic, multiscale platform for unveiling the dynamic intracellular fate of nanoparticles and providing mechanistic insight into their apparent nuclear localization.