Folic acid-conjugated hydroxyapatite nanoparticles induce 4T1 cell apoptosis via multiple pathways to potentiate breast cancer inhibition: a mechanistic study

An increasing number of nanomaterials are being applied in adjuvant tumor therapy, and prior studies have reported that inorganic nanoparticles can exert tumor-suppressive effects. Among these materials, hydroxyapatite (HA) nanoparticles have garnered substantial attention owing to their strong biocompatibility and inhibitory activity against tumor cell proliferation. However, insufficient cancer cell targeting of HA nanoparticles limits their antitumor efficacy. In this study, we developed folic acid (FA)-conjugated HA nanoparticles (HF nanoparticles) that enhance HA-mediated tumor inhibition by binding to overexpressed FA receptors on cancer cell membranes and exploiting the elevated FA demand in cancer metabolism. In in vivo experiments, HF nanoparticles significantly inhibited tumor growth compared with HA nanoparticles alone, and the former exhibited a synergistic antitumor effect with doxorubicin. Subsequent in vitro mechanistic analyses revealed that tumor-targeted HF nanoparticles induced intracellular calcium overload in 4T1 cells, causing calcium homeostasis imbalance, which disrupted mitochondrial membrane integrity, promoted cytochrome c release, and increased reactive oxygen species accumulation, ultimately activating apoptotic signaling and accelerating apoptosis. Additionally, these exogenous nanoparticles remodeled the immune landscape of the tumor microenvironment: HF nanoparticles promoted M1 macrophage polarization and elevated tumor necrosis factor-α expression, interfering with cancer cell cycle progression and division while activating caspase-related apoptotic genes, further intensifying apoptosis. Overall, this study demonstrated that HF nanoparticles can be engineered as highly targeted antitumor agents and drug delivery carriers, providing nanobiomaterials for more efficient tumor therapy.