Abstract

Osteoporosis is a prevalent metabolic bone disorder characterized by an imbalance between bone resorption and formation. Current therapeutic options are constrained by side effects and low bioavailability. Icariin (ICA), a naturally derived osteotropic flavonoid, exhibits osteogenic and anti-osteoclastogenic properties; however, its clinical application is limited due to poor solubility and low oral bioavailability. This study introduces β-cyclodextrin-modified gold nanoclusters (CGNCs) as a nanocarrier for efficient delivery of ICA. The synthesized ICA-loaded CGNCs (ICA-CGNCs) have a hydrodynamic diameter of approximately 2.16 nm, demonstrating excellent dispersity, sustained release kinetics, and storage stability. In vitro, ICA-CGNCs showed good cytocompatibility in both MC3T3-E1 pre-osteoblasts and RAW264.7 macrophages. In MC3T3-E1 cells, ICA-CGNCs promoted osteogenic differentiation, as indicated by increased ALP activity, enhanced mineralization, and upregulated osteogenic genes (ALP, BMP2, RUNX2, and COL1A1); immunofluorescence further showed elevated nuclear β-catenin and RUNX2 signals, supporting the involvement of osteogenic pathway-associated events in this cell model. In RAW264.7 cells, ICA-CGNCs suppressed RANKL-induced osteoclastogenesis by reducing TRAP-positive multinucleated osteoclast formation, disrupting F-actin ring organization, and downregulating osteoclast markers (TRAP, CTSK, MMP9, and NFATc1), which was supported by decreased bone resorption pit formation and attenuated nuclear accumulation of NF-κB p65 and NFATc1·In a glucocorticoid-induced zebrafish osteoporosis model, ICA-CGNCs effectively promoted cranial and vertebral bone mineralization without systemic toxicity, outperforming both free ICA and CGNCs alone. This study establishes the ICA-CGNC platform as a dual-action nanotherapeutic strategy with significant potential for osteoporosis treatment and bone regeneration.