Clusterzymes-driven therapy: ultrasmall Cu4 nanoclusters achieve dual-pronged synergistic effects on antioxidant defense and ferroptosis Inhibition for inflammatory osteolysis

Abstract Inflammatory osteolysis represents a critical complication following orthopedic interventions such as total joint replacement, primarily triggered by persistent inflammatory responses induced by prosthetic wear debris or bacterial components like lipopolysaccharides (LPS). Inflammatory osteolysis, a severe complication of orthopedic interventions like total joint replacement, is driven by prosthetic wear debris or lipopolysaccharides (LPS)-induced persistent inflammation and osteoclast activation. Current therapeutic strategies are limited by significant side effects and their inability to simultaneously halt the synergistic pathological processes of inflammation and osteoclast activation, highlighting an urgent need for novel therapeutic approaches. In this study, we synthesized ultrasmall Cu₄ nanoclusters with potent superoxide dismutase (SOD)-and catalase (CAT)-mimetic activities, enabling efficient reactive oxygen species (ROS) scavenging (80.43% • O 2 − and 93.17% H₂O₂ clearance at 200 µg/mL). we successfully synthesized ultrasmall Cu₄ clusters, which exhibit remarkable enzyme-mimetic activities (superoxide dismutase and catalase-like) and potent reactive oxygen species (ROS)-scavenging capabilities. These clusters specifically target mitochondria, effectively scavenging excessive ROS to mitigate oxidative stress. Furthermore, Cu₄ clusters activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, inhibit the activation of key inflammatory pathways such as nuclear factor-κB (NF-κB), regulate iron homeostasis (Ferro-orange staining showed that the positive cell ratio in the lps group was as high as 54.1%, while it dropped to 9.96% in the 20 µg/mL Cu₄ clusters group) and lipid peroxidation to block ferroptosis, and reduce osteoclast formation. In LPS-induced calvarial osteolysis mice, Cu₄ clusters significantly alleviated bone resorption, restoring bone volume/tissue volume (BV/TV) by 57.6% (91.7% of control group) and reducing osteoclast number to 36.4% of the LPS group.Collectively, these actions result in significant alleviation of inflammation and bone resorption. This study highlights Cu₄ clusters as a promising therapeutic agent for inflammatory osteolysis, with substantial potential for clinical translation. Graphical Abstract