Temporal Dynamics and Uptake Mechanisms of Carbonated Hydroxyapatite Nanoparticles in Murine F‑OST Cells

Carbonated hydroxyapatite (CarboHA) is a biomaterial gaining attention for its biocompatibility, bioactivity, and expanding applications in bone regeneration, aesthetic biostimulation, dentistry, and drug delivery systems. However, the impact of synthesis conditions on its physicochemical properties and cellular internalization mechanisms remains poorly understood. In this study, CarboHA nanoparticles were synthesized using a wet-chemistry method at three temperatures: 5 °C, 37 °C, and 90 °C. The particles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and zeta potential analysis. To enable fluorescent tracking during internalization studies in primary murine osteoblast (F-OST) cells, rhodamine was adsorbed onto the nanoparticles. Endocytic pathways were examined using selective inhibitors for clathrin-mediated, caveolin-mediated, lipid raft-mediated endocytosis, macropinocytosis, and phagocytosis and quantified via fluorescence microscopy and image analysis. The synthesis temperature significantly influenced crystallinity and morphology, which in turn dictated cell uptake mechanisms. Single-particle optical tweezers assays revealed increased adhesion times with higher synthesis temperatures. CarboHA synthesized at 5 °C produced smaller, less crystalline particles internalized predominantly through clathrin- and caveolin-mediated pathways, whereas highly crystalline nanoparticles produced at 90 °C favored macropinocytosis and phagocytosis. CarboHA synthesized at 37 °C demonstrated intermediate behavior, engaging multiple internalization routes. Together, these findings establish a clear relationship between synthesis temperature, nanostructural features, and cellular adhesion/internalization mechanisms, highlighting how controlling synthesis conditions enables the design of CarboHA-based materials optimized for specific biomedical applications, including resorbable bone grafts with tunable remodeling profiles and nanocarriers engineered for targeted intracellular delivery.