ABSTRACT Hepatocellular carcinoma (HCC), the fastest rising cause of cancer-related deaths worldwide with a 5-year survival of <20%, affects more than 41,000 individuals in the United States every year. Heavy alcohol consumption leading to fatty liver, hepatitis and cirrhosis has been identified as a key risk factor in HCC development. Current therapies against alcohol liver disease (ALD) and associated fibrosis are non-specific and ineffective. Alcohol abstinence remains the gold standard for ALD treatment to prevent progression to HCC, however this is often hampered by poor compliance. The goal of this proposal is to develop a novel multifunctional nanoparticle (NP) platform (lipid-PLGA NPs) for treatment of alcohol-associated fibrosis via targeted activation of G-protein-coupled bile acid receptor (Gpbar1) and anti-fibrotic drug delivery. The NPs will also release collagenase to facilitate greater NP penetration into the fibrotic liver tissue. We hypothesize that (i) NP-mediated targeted activation of Gpbar1 – a membrane protein expressed in Kupffer cells (KCs) and not hepatocytes, will suppress NF-kβ and STAT3 signaling responsible for HCC development, and (ii) targeted Gpbar1 activation and concurrent anti-fibrotic drug release will synergistically inhibit profibrotic biomarker expressions and cytokine signaling, leading to attenuation of fibrosis. Preliminary investigations by our collaborative research team confirmed that the NPs can selectively accumulate in the KCs in in vivo mouse models. Our proposed aims are: (1) Characterization and in vitro evaluation of liver tissue penetration properties of the collagenase-containing lipid-PLGA NPs. Physicochemical characterization will be done to ensure that the lipid-PLGA NPs will have optimal properties for accumulation in the liver. A 3D multicellular spheroid model of alcohol-induced liver fibrosis will be used to evaluate cytocompatibility, optimum uptake concentrations, and tissue penetration by the NPs in vitro. (2) Gpbar1 agonist incorporation and in vivo elucidation of safety, biodistribution and Gpbar1 targeting capabilities of the NPs. Gpbar1 targeting, safety and anti-fibrotic effects of the lipid-PLGA NPs will be investigated using a widely studied and reported carbon tetrachloride-plus-alcohol induced mouse models of liver fibrosis. (3) In vitro and in vivo evaluation of synergistic effects of Gpbar1-targeting lipid-PLGA NPs given in combination with anti-fibrotic therapies. In this aim, the synergistic effects of Gpbar1 activation and interleukin-17A signaling inhibition on fibrosis attenuation will be determined following encapsulation of anti-fibrotic therapies within the lipid-PLGA NPs. NP efficacy will be evaluated using histology, biomarker analysis and collagen assays. As a first step towards assessing the translational potential of the formulation, we will then investigate the therapeutic effects of the NPs using novel ALD liver fibrosis-on-a-chip developed using primary murine cells. This innovative project will lead to a paradigm shift in the development and testing of new therapeutic strategies against chronic liver diseases to prevent their progression to HCC.

NARRATIVE Chronic alcohol consumption is a major risk factor for the development of hepatocellular carcinoma (HCC) – the fastest growing type of cancer worldwide. Current therapies to treat and prevent alcohol liver disease progression are non-specific and toxic. The main objective of this proposal is to develop a novel multifunctional nanoparticle formulation that can penetrate deep into the fibrotic liver tissue to provide combination therapy to prevent alcohol- induced liver tumorigenesis.