Three-in-One Injectable Hydrogel Orchestrating Host-Bacteria Targeting, Retention, and Autophagy for Methicillin-Resistant Staphylococcus aureus -Infected Mastitis Therapy

Abstract MRSA mastitis is clinically refractory and characterized by progressive inflammation driven by intracellular bacteria that form persistent niches in mammary epithelial cells and macrophages to evade antimicrobial clearance. Conventional antibiotics, particularly β-lactams, are widely used but suffer from poor intracellular accumulation, failing to reach these reservoirs. Even nanocarrier-based strategies struggle to achieve efficient endosomal escape and precise intracellular targeting. Herein, we report a fibronectin-functionalized, fluorinated PLGA nanoparticle encapsulating amoxicillin and sulbactam (Fn-F-PLGA@A/S) engineered with a 2.5% (w/w) fluorinated core for efficient endosomal escape. The fibronectin decoration enables sequential targeting of both host integrins and bacterial fibronectin-binding proteins (FnBPs). Fn-F-PLGA@A/S achieved efficient intracellular delivery, clearing 90% of intracellular MRSA in macrophages and epithelial cells within 4 h in vitro. To prolong mammary retention, Fn-F-PLGA@A/S was loaded into a thermosensitive hydrogel. In a murine MRSA mastitis model, a single intramammary dose of the gel formulation achieved >90% bacterial reduction, attenuated TNF-α and IL-6, and preserved alveolar architecture and tight junctions. Proteomic analysis revealed upregulation of AMPK-mediated autophagy, suggesting that beyond direct antibiotic action, the potent intracellular clearance achieved by Fn-F-PLGA may involve the engagement of host autophagic machinery, potentially facilitating the sequestration of MRSA within degradative compartments. Collectively, by integrating sequential targeting, sustained local retention, and autophagic engagement, this platform provides a robust strategy to address the key limitations of conventional antibiotics against intracellular pathogens in refractory infections.