Effect of\nHydroxyapatite Nanoparticle Crystallinity\nand Colloidal Stability on Cytotoxicity

Hydroxyapatite nanoparticles (nHA) have gained attention\nas potential\nintracellular drug delivery vehicles due to their high binding affinity\nfor various biomolecules and pH-dependent solubility. Yet, the dependence\nof nHA cytocompatibility on their physicochemical properties remains\nunclear since numerous studies have revealed starkly contrasting results.\nThese discrepancies may be attributed to differences in size, shape,\ncrystallinity, and aggregation state of nHA, which complicates fundamental\nunderstanding of the factors driving nHA cytotoxicity. Here, we hypothesize\nthat nHA cytotoxicity is primarily driven by intracellular calcium\nlevels following the internalization of nHA nanoparticles. By investigating\nthe cytotoxicity of spherical nHA with different crystallinity and\ndispersity, we find that both lower crystallinity and increased agglomeration\nof nHA raise cytotoxicity, with nanoparticle agglomeration being the\nmore dominant factor. We show that the internalization of nHA enhances\nintracellular calcium levels and increases the production of reactive\noxygen species (ROS). However, only subtle changes in intracellular\ncalcium are observed, and their physiological relevance remains to\nbe confirmed. In conclusion, we show that nHA agglomeration enhances\nROS production and the associated cytotoxicity. These findings provide\nimportant guidelines for the future design of nHA-containing formulations\nfor biomedical applications, implying that nHA crystallinity and especially\nagglomeration should be carefully controlled to optimize biocompatibility\nand therapeutic efficacy.