Targeting ROS-induced osteoblast senescence and RANKL production by Prussian blue nanozyme based gene editing platform to reverse osteoporosis

Blocking the reactive oxygen species (ROS) induced osteoblast senescence and massive RANKL release is the key to alleviate the vicious osteoporotic micro-environment in aged skeleton. In this study, we constructed a composite nanoparticle by confining the growth of Prussian blue nanozyme (PBzyme) on the surface of a sub-50 nm hollow mesoporous silica nanoparticles (HPB), which presented strong ROS scavenging ability. The abundant mesoporous structure made it available to combine receptor activator of nuclear factor kappa-B ligand (RANKL) CRISPR/Cas9 plasmid and skeletal targeting agent alendronate (HPB@RC-ALN) together to modulate the osteoporotic micro-environment. Our results indicate HPB@RC-ALN confined ROS generation and alleviated osteoblast senescence as revealed by dihydroethidium and senescence-associated β-galactosidase staining. The rejuvenated osteoblasts presented robust osteogenic ability as shown by alkaline phosphatase and alizarin red staining in vitro, as well as the micro-computed tomography (μCT) in vivo. Meanwhile, efficient transfection of CRISPR/Cas9 plasmid by HPB@RC-ALN precisely achieved RANKL gene editing target and knocked the RANKL gene out in osteoblasts as confirmed by T7E1 digestion and western blot assays. Hence, the osteoclast formation was significantly suppressed as shutting down the RANKL production by HPB@RC-ALN in the senescent osteoblast. At last, the μCT revealed fully reversed bone volume in ovariectomized mice after HPB@RC-ALN injection into the bone remodeling site. Overall, the constructed nanozyme based gene editing platform achieved bidirectional regulation of osteoblast-osteoclast through rescuing cellular senescence and blocking RANKL production. This strategy provides a new theoretical base for osteoporosis management.