An Enzyme‐Responsive Nucleic Acid‐Based Nanoassembly for Coordinated Co‐Delivery of microRNA and Puerarin in Osteoporotic Bone Regeneration

ABSTRACT The repair of osteoporotic bone defects presents a formidable challenge due to the disrupted osteogenic‐adipogenic equilibrium within the bone marrow microenvironment. To address the limitations of monotherapies in regulating this pathological condition, this study engineers an enzyme‐responsive delivery platform, the puerarin‐reinforced nucleic acid‐based nanoassembly (Pue@NB), via structural reprogramming of tetrahedral framework nucleic acid (tFNA). This system enables precise co‐encapsulation and intracellular co‐delivery of osteogenic microRNA (miR‐335‐5p) and puerarin (Pue). While preserving the biocompatibility and cellular entry efficiency of tFNA, Pue@NB incorporates an embedded DNA‐RNA hybrid bio‐switch that responds to endogenous RNase H, enabling structural transformation and coordinated release of both therapeutic agents. In vitro, beyond promoting the migration of bone marrow mesenchymal stem cells (BMSCs), Pue@NB also exhibits a bidirectional regulatory capacity for BMSCs differentiation: its pro‐osteogenic and anti‐adipogenic effects are accompanied by increased hedgehog‐related signaling markers, upregulated TGF‐β1, and suppressed PPARγ/FABP4 expression. In an ovariectomy‐induced osteoporotic bone defect model, Pue@NB substantially accelerated bone regeneration, restored bone mineral density, and improved trabecular microstructure. This work elucidates a synergistic therapeutic strategy that integrates genetic and small‐molecule drugs via nucleic acid nanostructure reengineering, providing foundations for developing next‐generation dynamic nanomaterials for recalcitrant tissue defects.