Precision-targeting and dual silencing osteoclastogenesis and inflammatory pathways for the treatment of radiation-induced bone deterioration.

Ironizing radiation (IR)-induced bone loss remains a significant clinical challenge, largely driven by elevated osteoclastogenesis. Targeting key regulators of osteoclast differentiation and function may offer a novel therapeutic approach for preserving bone integrity under irradiated conditions. In this study, we evaluated the biocompatibility, cellular uptake, and therapeutic potential of a dual microdroplet (MD) system engineered to co-deliver inhibitors targeting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and cathepsin K (CTSK), two key regulators of osteoclast differentiation and function. The biocompatibility and cellular uptake of dual MDs were evaluated in vitro using human bone marrow-derived mesenchymal stem cells (hBMSCs) and RAW264.7 macrophages. Functional assays, including tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring analysis, and cytokine profiling, were performed in vitro. The therapeutic efficacy of dual MDs was further evaluated in vivo using a model of radiation-induced bone loss. Dual MDs demonstrated excellent biocompatibility and robust cellular uptake under both mock-IR and post-IR conditions. Treatment with dual MDs significantly inhibited macrophage fusion, suppressed TRAP activity, and disrupted F-actin ring formation, indicating impaired osteoclast maturation. In addition, dual MDs downregulated key pro-inflammatory cytokines, including G-CSF, IL-6, and TNF receptors. In vivo, dual MDs preserved bone microarchitecture and significantly reduced the expression of CTSK, NFATc1, and TNF-α in irradiated bone tissue. Our findings demonstrated that dual MDs effectively inhibit radiation-induced osteoclastogenesis and inflammation by targeting NFATc1 and CTSK. This dual-targeting approach presents a promising therapeutic strategy for mitigating bone loss in radiation-associated skeletal disorders.