Functionalized DNA Nanomaterials Targeting Toll‐Like Receptor 4 Prevent Bisphosphonate‐Related Osteonecrosis of the Jaw via Regulating Mitochondrial Homeostasis in Macrophages

Abstract Imbalance of macrophage polarization characterized by an increase in the percentage of pro‐inflammatory M1 macrophages and a decrease in anti‐inflammatory M2 macrophages is considered a critical pathogenic mechanism of bisphosphonate‐related osteonecrosis of the jaws (BRONJ). Because high levels of Toll‐like receptor 4 (TLR4) mediates mitochondrial dyshomeostasis in Zoledronic Acid (ZA)‐treated M1 macrophages, tetrahedral DNA nanomaterial (TDN)‐modified with TLR4‐siRNA on each vertex (TDN‐TLR4‐4siR) with excellent biocompatibility is synthesized. This novel TDN‐TLR4‐4siR nanomaterial reverses the polarization phenotype imbalance decreasing the percentage of M1 RAW264.7 macrophages. Mitochondrial dynamics analysis shows a shift from short rod‐like ultrastructure to elongated shapes with more mitochondrial network continuity in ZA‐primed M1 macrophages after treatment with TDN‐TLR4‐4siR, along with elevated expression of Mfn1 and Mfn2 . TDN‐TLR4‐4siR further reduces intracellular ROS production and restored mitochondrial membrane potential. Furthermore, decreased sequestra formation and accelerated healing of the extraction wound are observed in the TDN‐TLR4‐4siR group, resulting in decreased incidence of rat BRONJ via reprogramming polarized macrophages. Consequently, this study establishes a novel strategy using TDN‐TLR4‐4siR nanomaterial to regulate mitochondrial homeostasis of polarized macrophages to prevent BRONJ.