Hybrid Exosome-Bilosome Oxygen Nanobubbles for Liver Fibrosis Attenuation: Antifibrotic Efficacy Investigation in Multilineage Spheroid Platform

Abstract Liver fibrosis refers to the excessive buildup of scar tissue in the liver, which can lead to cirrhosis and hepatocellular carcinoma. It often presents with a hypoxic hepatic microenvironment and elevated oxidative stress. In this work, we present a hybrid Exosome-Bilosome Oxygen-encapsulating Nanobubble (EBON) with antifibrotic properties that has the potential to protect the liver from localized hypoxia and oxidative stress-induced hepatic damage. Grapefruit-derived exosome-like vesicles utilized here may contribute to the therapeutic potential of the EBON formulation by delivering antioxidant, anti-inflammatory, and antifibrotic cargo, alongside oxygen. EBON has a sub-50 nm size with an oxygen loading capacity of about 50 mg/L and a shelf life of half a year. EBON has demonstrated excellent protection against cellular hypoxia, reactive oxygen species, and superoxide-induced damage in LX-2 and HepG2 cell lines. Additionally, EBON has shown a significant antifibrotic effect in palmitic acid and oleic acid-induced lipotoxicity and oxidative stress-mediated fibrosis model in LX-2/HepG2 multilineage hepatic spheroids. RT-qPCR results confirmed suppression of hypoxia (HIF-1α, VEGF-A), oxidative stress (Nrf2), and pro-fibrotic signaling (α-SMA, TGF-β1, and COL1A1) associated genes in the in vitro hepatic spheroid model. Additionally, the safety profile of EBON has been established in a 14-day murine oral gavage model, validating short-term oral tolerability. We propose that the hybrid Exosome-Bilosome Oxygen encapsulating Nanobubble reported here is a proof-of-concept platform with antifibrotic potential for addressing hypoxia associated with liver fibrosis.