3D correlative light and electron microscopy reveals the uptake and processing of inorganic-organic hybrid nanoparticles into cancer cells

Inorganic-organic hybrid nanoparticles (IOH-NPs) are a promising drug delivery system in oncology due to their high drug-load capacity. In this study, we established a 3D correlative light and electron microscopy (CLEM) workflow that combines confocal fluorescence microscopy (FM) with focused ion beam scanning electron microscopy (FIBSEM) to unambiguously identify and visualize the (sub)cellular uptake and processing of reference fluorescently labeled and zirconium-based IOH-NPs in murine H8N8 breast cancer cells. The 3D-CLEM workflow was set up without the need to add external fiducial markers since image correlation was achieved using lipid droplets as intrinsic correlative landmarks. We observed that all H8N8 breast cancer cells had taken up IOH-NPs after 4 h, and most IOH-NPs were found in clusters within the H8N8 cells. IOH-NPs were internalized by endocytosis within 2 h with increasing cellular concentrations over time and accumulated in endolysosomal vesicles over 24 h, while the overall endolysosomal volume increased between 2 and 6 h after IOH-NP incubation and returned to its original value thereafter, remaining stable for up to 48 h. The 3D-CLEM workflow also revealed changes in the morphology and density of the IOH-NPs inside endolysosomal vesicles, suggesting the dissolution of IOH-NPs after 2 h. We also observed mitochondrial swelling in IOH-NP exposed cells, suggesting stress responses even without drug load. The 3D-CLEM workflow provides new insights into the cellular tracking and processing of IOH-NPs and supports the development of novel nanomedicine strategies.