(M1130-11-71) A Kupffer Cell-Targeting Nanoparticle System to Mitigate Alcohol-Related Liver Disease (ALD)

Purpose: Alcohol-related liver disease (ALD) is the most prevalent form of liver disease commonly associated with hepatocellular carcinoma. From 2019 to 2040, an estimated 1,003,400 individuals are expected to die from chronic ALD in the United States, 35% of whom will be under the age of 55. The current anti-inflammatory, anti-viral, and immunosuppressive therapies for liver inflammation are not consistently effective and safe. To meet the urgent need for targeted and effective interventions against ALD, we report the development of novel nanoparticles (NPs) using a poly lactic co glycolic acid (PLGA) and carboxymethyl chitosan (CMC) polymer combination for liver-targeted anti-inflammatory drug delivery. The G-protein coupled bile acid receptor TGR5 (Gpbar1) expressed primarily in Kupffer cells (KCs) mediates proinflammatory cytokine production in the liver. Therefore, we surface-decorated our NPs with INT 777, a highly selective and potent semisynthetic bile acid agonist of Gpbar1 that can attenuate reactive oxygen species (ROS) production and proinflammatory cytokine secretion by KCs. CMC, a pH-responsive biodegradable polymer, was used to trigger the release of the anti-inflammatory drug dexamethasone (DEX) in response to the acidic pH in the inflamed liver microenvironment. Therefore, concurrent NP-mediated Gpbar1 stimulation and pH-responsive DEX release in the acidic microenvironment have an additive effect in mitigating chronic hepatic inflammation.

Methods: PLGA NPs loaded with DEX were prepared by single solvent emulsion evaporation technique in 5% (v/v) polyvinyl alcohol (PVA) solution. These NPs were then directly added to CMC solution with 1% v/v PVA, for passive coating of CMC. Size distribution, polydispersity index (PDI) and zeta potential of the NPs were determined by dynamic light scattering. pH-responsive drug release kinetics were studied under two different pH values: 6 and 7.4. INT 777 was conjugated to NPs using a standard carbodiimide coupling reaction; the conjugation was confirmed using X-ray photo electron spectroscopy (XPS) analysis. Cytocompatibility studies following 24 h NP treatment was assessed with HepG2 human hepatoma and THP-1 human monocyte (KC mimic) cells using colorimetric WST-8 assay. Coumarin-6 dye - loaded NPs were used to analyze the cellular uptake of NPs after treatment of inflammation-inducing lipopolysaccharide (LPS) with and without INT NPs. Cyclic adenosine 3,5-monophosphate (cAMP) activity was determined after LPS treatments to THP-1 cells by cAMP assay kit (Promega). A standard NIAAA mouse model of ALD was developed using 12-week-old C57BL/6 mice and used to assess NP biodistribution 24 h following intraperitoneal injection. The fixed sections of the liver and other organs were also analyzed using hematoxylin and eosin staining. In addition, lipid droplet accumulation in the liver tissue was evaluated using Oil red O staining. The biodistribution of the NPs in all the organs were analyzed using fluorescence microscope (EVOS M5000, Thermo Fisher, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the blood were quantified using ALT and AST assay kits (Abcam) according to the manufacturer’s protocol.

Results: The synthesized INT 777-tagged PLGA-CMC NPs (PLGA-CMC-INT NPs) had a hydrodynamic diameter of 185.47 +/- 1.31 nm, zeta potential of -20.26 +/- 0.55 mV, and PDI value of 0.231 +/- 0.01. The release study showed the pH-responsive release of DEX, with higher release observed at pH 6. XPS data confirmed the surface conjugation of INT 777 to the PLGA-CMC NPs. Synthesized NPs were cytocompatible with HepG2 and THP-1 cells up to a high concentration of 1000 µg/ml. Cell uptake study using THP-1s, representing KCs, demonstrated significantly higher uptake of the NPs after LPS treatment compared to the HepG2, representing hepatocytes, implying the KC-targeted nature of the INT. Dose-dependent uptake of the NPs was observed in both cell types. The NIAAA mouse model of ALD was successfully developed following ten days of ethanol diet. Biodistribution of NPs confirmed that the majority of the PLGA-CMC-INT NPs accumulate in the liver, not in other organs. The Oil red O staining confirmed the presence of oil droplets in the liver histological sections of ethanol-treated mice.

Conclusion: The stable PLGA-CMC-INT NPs were successfully synthesized and characterized. Significantly higher uptake of PLGA-CMC-INT NPs than PLGA-CMC NPs (without INT 777) was observed in THP1 cells, indicating that INT can target the KCs in the liver when there is inflammation. The in vitro cytocompatibility studies showed that the PLGA-CMC NPs and PLGA-CMC-INT NPs are biocompatible. PLGA-CMC-DEX NPs showed pH-responsive release of DEX as expected. In vivo studies confirmed that ethanol diet caused liver inflammation in the mice, and INT-tagged NPs accumulated in the liver during the first 24 hrs.

Acknowledgements:We would like to acknowledge funding support from the National Institute of Health (NIH) grant (1R21AA029750-01) and the Rhode Island IDeA Network of Biomedical Research Excellence (RI-INBRE) grant.

Figure 1:Quantitative uptake of INT+ and INT- nanoparticles on both DTHP-1 (differentiated THP-1 cells, representing Kupffer cells) and HepG2 (representative hepatic cells) cells treated with and without bacterial lipopolysaccharide (LPS) (* represents significance (p < 0.05) with all other three groups in DHP-1, # p < 0.05 in HepG2).

Figure2: NP biodistribution 24 h following intraperitoneal injection; (i) NPs without INT, 20X magnification; (ii) NPs with INT, 20X magnification; (iii) NPs with INT, 40X magnification