Poster W15 - A Kupffer cell-targeting Nanoparticle Platform to Mitigate Alcohol-associated Liver Disease

Introduction: Alcohol-associated liver disease (ALD) and inflammation is the most prevalent form of liver disease with increased risk for hepatocellular carcinoma development. About 46% of the liver disease deaths in 2022 were attributed to alcohol abuse. Despite its rapidly rising incidence, current therapies for ALD are not consistently effective and safe. We report the development of a targeted poly lactic co glycolic acid (PLGA) and carboxymethyl chitosan (CMC) polymer combination-based nanoparticle platform (PLGA NPs) for anti-inflammatory drug delivery. The NPs were surface-decorated with INT-777 to form PLGA+INT NPs; INT-777 is a highly selective and potent semisynthetic bile acid agonist of G-protein coupled bile acid receptor (Gpbar1), that can attenuate pro-inflammatory cytokine production by Kupffer cells (KCs). The anti-inflammatory drug dexamethasone (DEX) was encapsulated within the CMC layer of the NPs (PLGA+INT+DEX NPs). CMC, a pH-responsive biodegradable polymer, enabled pH-triggered release of DEX in response to the acidic pH in the inflamed liver microenvironment.

Materials and

Methods: PLGA polymer NPs were developed using a standard single emulsion process and then passively coated with CMC. INT-777 conjugation to the NH2 end groups of the NPs was done using carbodiimide chemistry. NP size, surface charge and stability were confirmed using dynamic light scattering (DLS), CMC coating by Fourier transform infrared spectroscopy (FTIR), DEX release by UV-visible spectrometry, and in vitro cytocompatibility and targeting capabilities by MTT cell viability assays and cell uptake studies involving incubation of HepG2 cell line modeling normal hepatocytes and THP-1 monocytes modeling KCs, with different concentrations of NPs for 24h and 2 h respectively. The Bin Gao chronic ethanol feeding mouse model of ALD given a 5%(v/v) ethanol diet daily was used to determine biodistribution of fluorescent dye-tagged NPs at 24 and 72h post administration. NP therapeutic efficacy was determined following intraperitoneal administration of NPs every 3 days for 25 days, following which the mice were euthanized, and several analyses were carried out, including aspartate aminotransferase (AST) and alanine transferase (ALT). Inflammation and lipid droplet formation were analyzed using Hematoxylin and Eosin (H&E) and Oil Red O (ORO) staining.

Results, Conclusions, and Discussions: The PLGA+INT NPs were stable and had spherical morphologies (Fig. 1(a)) with an average diameter and zeta potential of 232.53±33.46 nm and -30.80±1.54 mV respectively. The CMC coating was confirmed using FTIR. The encapsulated DEX was released in a pH-responsive manner with greater release observed at pH 6.0 than at pH 7.4 (Fig. 1(b)). The cytocompatible PLGA+INT NPs showed significantly higher dose-dependent uptake in differentiated THP-1 monocytes than in HepG2 cells. Significant NP fluorescence was observed in the livers of treated mice at 24 and 72 hours after injection, indicating the liver selectivity and KC-targeting capabilities of the NPs. Liver inflammation in the Bin Gao mouse model was confirmed when elevated alanine transferase (ALT) and aspartate aminotransferase (AST) levels was observed on day 11 in the ethanol-fed mice group compared to the control group. Treatment with PLGA+INT+DEX NPs led to led to significant reduction in AST and ALT levels, which were similar to baseline levels observed in control untreated mice given control, non-ethanol diet (Fig. 1(c) and 1(d)). These results were further confirmed by hematoxylin and eosin (H&E) and oil red o (ORO) staining, which revealed small droplet steatosis and periportal inflammation in the ethanol-fed mice groups and relatively lower inflammation in the PLGA+INT+DEX NP group, which was comparable to the control group.

We successfully synthesized PLGA+INT+DEX NPs for KC-targeted drug delivery. Significantly higher macrophage uptake in vitro and liver accumulation in vivo of the NPs was observed. In vivo studies using the Bin Gao chronic ethanol feeding mouse model confirmed that 5% (v/v) ethanol diet caused liver inflammation in C57BL/6N mice after 10 days of liquid diet.

Acknowledgements (Optional): We gratefully acknowledge funding support from NIH grants R21AA029750 and R37CA283937 awarded to JUM.