Protective Effects of GalNac-Modified Red Blood Cell-Derived Extracellular Vesicles Against Liver Diseases.

OBJECTIVE: The high morbidity and mortality of liver diseases warrant the development of more effective therapeutic methods. Extracellular vesicles (EVs) are optimal drug delivery vehicles, albeit with insufficient targeting specificity. The aim of this study was to develop modified red blood cell-derived EVs (RBC-EVs) for targeted drug delivery into hepatocytes, and verify their therapeutic efficacy in animal models of acute liver failure (ALF), non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). METHODS: RBC-EVs were extracted and modified with triantennary N-acetyl galactosamine sequences (GalNac-RBC-EVs), then loaded with drugs or oligonucleotides. The GalNac-RBC-EVs uptake by hepatocytes was monitored both in vitro and in vivo. Models of ALF, NAFLD and HCC were established using C57BL/6J mice. RBC-EVs/GalNac-miR-155-ASO was prepared for the treatment of ALF by anchoring GalNac-miR-155-ASO to RBC-EVs. GalNac-RBC-EVs loaded with PJ34 (GalNac-RBC-EVs/PJ34) were used for treating NAFLD. Finally, GalNac-Rab7-siRNA and PJ34 were co-loaded into RBC-EVs (RBC-EVs/GalNac-Rab7-siRNA/PJ34) as a therapeutic agent for HCC. The therapeutic efficacy, biosafety and stability of the modified RBC-EVs were analyzed as per standard protocols. RESULTS: GalNac-RBC-EVs exhibited higher hepatocyte accumulation via asialoglycoprotein receptor (ASGPR)-mediated uptake both in vivo and in vitro. In ALF, RBC-EVs/GalNac-miR-155-ASO reduced inflammation through suppression of pyroptosis (P<0.0001), apoptosis (P<0.0001), and mitigation of necroptosis (P<0.0001). For NAFLD/HCC treatment, GalNac-RBC-EVs/PJ34 decreased hepatic triglyceride (P<0.0001) and attenuated HCC growth (P<0.001) via PARP-1 inhibition (P<0.0001). Co-delivery of GalNac-Rab7-siRNA enhanced PJ34 efficacy (P<0.0001) by prolonging drug retention. No significant toxicity was observed in vital organs (ALT/AST levels, P>0.05; histopathology scores, P>0.05). CONCLUSION: Drug-loaded GalNac-RBC-EVs selectively targeted the hepatocytes, and displayed significant therapeutic efficacy in ALF, NAFLD or HCC without any side effects. Altogether, RBC-EVs modified with GalNac are a promising drug carrier for the effective and precise treatment of various liver diseases.