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

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.