Mitochondria-Targeted Cassic Acid Base Nanoprodrug for Enhanced Anti-Inflammatory and Osteogenic Effects in Osteoarthritis

CATPCS prodrug-based nanodrug delivery system (PNDDS), a mitochondria-targeted nanoprodrug combining Cassic acid (CA) with a PEG-modified chitosan-tripolyphosphate (TPP) molecule (CATPCS), overcomes the limitations of free Cassic acid in treating osteoarthritis (OA) and bone regeneration. FTIR and DLS confirm that lyophilized CATPCS self-assembles into stable, homogeneous spherical nanoparticles (230.8 ± 45.5 nm, PDI: 0.180, ζ potential: 30.5 ± 9.2 mV). For targeted delivery, HR-TEM scans verified this spherical morphology. <i>In vitro</i> studies show that IL-1β-stimulated chondrocytes internalize and colocalize CATPCS more effectively than free CA and CA NPs owing to the positively charged TPP moiety. CATPCS and CA NPs have in vitro cytotoxicity lower than that of free CA, indicating biocompatibility. In IL-1β-stimulated chondrocytes, CATPCS significantly reduced the level of ROS and RNS, increased cartilage-specific markers (COL2A1, ACAN), and lowered inflammatory factors. Increased levels of alkaline phosphatase (ALP) and alizarin red staining (ARS) protected mitochondrial membrane potential, reduced chondrocyte apoptosis, and increased osteogenesis. Comparable to Diclofenac (DG), CATPCS decreased mechanical allodynia, cold hypersensitivity, and thermal hyperalgesia in MIA-induced OA rats. CATPCS reduced oxidative stress by lowering MDA and restoring SOD and GPx activity according to biochemical studies. Histopathology demonstrated that CATPCS reduced inflammation and preserved the OA animals' articular cartilage thickness. Mitochondrial-targeted CATPCS PNDDS is an excellent osteoarthritis and bone regeneration therapy because of its enhanced delivery, anti-inflammatory, antioxidant, and osteogenic effects.