GalNAc-modified five-component lipid nanoparticles for liver-targeted delivery of p65 siRNA to alleviate cytokine storm in liver injury via NF-κB targeting

Abstract

Acute liver injury (ALI) often triggers a systemic cytokine storm, in which the NF-κB subunit p65 plays a central role in driving inflammatory progression in hepatocytes. Delivering p65 siRNA to hepatocytes can effectively inhibit cellular inflammation and apoptosis. However, siRNA is inherently unstable and lacks tissue specificity. Lipid nanoparticles (LNPs) represent a mature delivery system with inherent liver tropism, yet their targeting efficiency remains suboptimal. To enhance hepatocyte-specific delivery, we developed a galactose-N-acetylgalactosamine (GalNAc)-modified five-component LNP (G-LNP) for liver-targeted delivery of p65 siRNA. GalNAc modification enables active targeting via asialoglycoprotein receptor (ASGPR)-mediated endocytosis, thereby improving hepatic accumulation and intracellular internalization. The optimized G-LNP formulation (1% DSPE–PEG2000–GalNAc) exhibited a particle size of 87 nm, a low PDI, and high encapsulation efficiency. In vitro, G-LNP–siP65 significantly enhanced transfection efficiency in AML-12 hepatocytes in an ASGPR-dependent manner, down-regulated p65 mRNA expression, alleviated CCl₄-induced oxidative stress (increased SOD and decreased MDA), and reduced pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β). In vivo bioluminescence imaging confirmed superior and sustained liver accumulation of G-LNP–Luc compared to unmodified LNP. Furthermore, in a CCl₄-induced ALI mouse model, the G-LNP–siP65 treatment dramatically reduced serum ALT/AST levels, restored redox balance, and suppressed hepatic NF-κB expression, leading to marked amelioration of histopathological damage. Moreover, systemic safety evaluation demonstrated negligible hemolytic activity and a favorable hematological profile, as evidenced by reduced neutrophil percentage and decreased serum CRP levels. Collectively, GalNAc-modified five-component LNPs provide a potent and safe liver-targeted platform for p65 siRNA delivery, effectively mitigating cytokine storm and liver injury via NF-κB silencing. This strategy holds promise for nucleic acid-based therapies against acute and chronic liver diseases.