Targeted delivery of berberine <i>via</i> ROS-sensitive polymersomes enhances its hepatoprotective activity in CCl ₄ -intoxicated mice

Carbon tetrachloride (CCl₄) metabolism results in the production of highly reactive free radicals and consequent liver tissue damage, making CCl₄-induced liver injury an ideal model for studying drug delivery systems that respond to reactive oxygen species (ROS). Previously, we demonstrated the hepatoprotective activity of isoquinoline alkaloid berberine (BER) against CCl₄-induced hepatotoxicity in mice. In this study, we aimed to investigate the targeted delivery of BER to ROS-rich injury site. For this purpose, ROS-responsive polymersomes (PS), built as amphiphilic block copolymers bearing a boronic ester-based ROS sensor connected to the hydrophobic polymer backbone with embedded BER, were synthesized in our laboratory. PS exhibited a suitable particle size of 117.8 nm, zeta potential of -12.5 mV, and good physical stability. Mice were administered berberine (BER) and polymersome nanoencapsulated berberine (BER-PS) 6 mg kg^-1 intraperitoneally, 1 h before CCl₄ (10% v/v in olive oil, 2 mL kg^-1) and sacrificed 48 h later. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were markedly decreased and histopathological changes were significantly reduced by BER-PS compared to BER. The expression of oxidative stress markers (4-hydroxynonenal (4-HNE), hem oxygenase-1 (HO-1), 8-hydroxy-2'-deoxyguanosine (8-OHdG)), apoptosis (caspase-3, caspase-9, TUNEL), autophagy (microtubule-associated protein 1 light chain 3 beta (LC3B)-I/II, p62), and inflammation (tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB)) was also more effectively ameliorated by BER-PS. Mechanistically, both BER and BER-PS decreased the expression of phosphorylated extracellular signal-regulated kinase (ERK)1/2 and phosphorylated AMP-activated protein kinase (AMPK). BER-PS also decreased nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), phosphorylated c-Jun N-terminal kinase (JNK)1/2 and phosphorylated protein kinase B (Akt). These results suggest that BER-PS is more successful than BER in ameliorating ROS-mediated CCl₄-induced hepatic injury, which could be related to the specifically targeted delivery of the drug to the site of injury under oxidative stress conditions.