U46 - In vitro and In vivo Evaluation of Liver-targeting Polymeric Nanoparticle System to Mitigate Alcohol-related Liver Disease (ALD)

Introduction: : Alcohol-related liver disease (ALD) is the most common type of liver disease typically linked to hepatocellular carcinoma. Current treatments for liver inflammation often lack consistent efficacy and safety. We report the development of an innovative nanoparticle (NP)-based drug delivery system using a combination of poly lactic-co-glycolic acid (PLGA) and carboxymethyl chitosan (CMC) for targeted delivery of anti-inflammatory drugs to the liver. To enhance the effectiveness of our NPs, we modified their surface with INT 777, a selective and potent semisynthetic bile acid agonist of Gpbar1, aiming to reduce the production of pro-inflammatory cytokines by Kupffer cells (KCs). CMC, a biodegradable polymer responsive to pH changes, was employed to facilitate the release of the anti-inflammatory drug dexamethasone (DEX) in reaction to the acidic conditions present in the inflamed liver environment.

Materials and

Methods: : NPs were synthesized using amine-terminated PLGA in a single-emulsion process and coated with 1% w/v CMC (PC-1 NPs). INT-777 was then linked to the pre-made NPs through carbodiimide chemistry. In vitro studies using differentiated THP-1 (dTHP1) cells modeling KCs included a cell uptake study, competitive assay, cyclic adenosine monophosphate (cAMP) activity analysis, and pro-inflammatory cytokine release were performed to determine the effectiveness of the NP system. ALD was developed in C57BL/6N mice using an optimized 10-day 5% ethanol liquid diet. The biodistribution study of NPs was conducted by administering coumarin-6-dye-containing NPs through intraperitoneal injection on day 11, followed by organ harvesting and imaging to assess NP accumulation. To evaluate NP efficacy, mice were treated with empty NPs, DEX-loaded NPs, INT-777-conjugated NPs (without DEX) and the final NPs containing both INT-777 and DEX. On day 25, mice were euthanized, and the blood and liver tissues were assessed for inflammatory markers. Lipid droplet formation and cytochrome P450 2E1 (CYP2E1) activity were also carried out using fresh frozen liver tissues fixed in 4% (w/v) paraformaldehyde.

Results, Conclusions, and Discussions:: The NPs were successfully synthesized and characterized for size (218.9±0.749 nm), zeta potential (-30.1±1.181 mV), and stability. The DEX release confirmed the pH responsive drug release, and INT conjugated NPs showed selective uptake in dTHP1s. The dTHP1 cells treated with ethanol and lipopolysaccharide showed elevated interlukin-6 (IL-6), IL-1, and tumor necrotic factor- (TNF-a) expressions; these cytokine levels demonstrated dose-dependent decrease upon treatment with NPs containing INT-777 and DEX. In vivo biodistribution studies confirmed that the majority of the NPs were concentrated in the liver at 24 hr after injection, indicating good hepatic selectivity. Immunohistochemistry analysis of KC marker F4/80 in liver tissue samples confirmed localization of the NPs in KCs. ALD mice treated with NPs containing both INT and DEX demonstrated a reduction in IL-6, IL-1, and TNF- levels comparable to levels in the control diet-fed group. Treatment with NPs containing either DEX or INT did demonstrate similar levels of reduction. These results were further validated by Oil red O staining, which confirmed significantly lower oil droplet formation in the livers of ALD mice given the NPs containing both INT and DEX, when compared to the other treatment groups. The CYP2E1 plays a role in alcohol metabolism, and increased activity of CYP2E1 is associated with higher levels of inflammation due to alcohol consumption. In a comparison between the control group and mice treated with NPs containing both INT and DEX, there was similar CYP2E1 activity. However, the ALD control group and other treatment groups exhibited higher levels of CYP2E1 expression.

Conclusions: We successfully synthesized INT-777 conjugated pH-sensitive DEX-encapsulated PLGA NPs (NPs). The INT-777 conjugated NPs demonstrated selective uptake by Kupffer cells (KCs). In vivo experiments indicated that a 5% (v/v) ethanol diet led to liver inflammation in C57BL/6N mice after 10 days of the diet. Consistent with the in vitro findings, the NPs exhibited hepatic selectivity in vivo. PLGA NPs containing both INT and DEX have the potential to alleviate alcohol-induced liver inflammation in vivo

Acknowledgements and/or References (Optional):: This work was supported by the National Institute of Health grant numbers R21 AA029750 and R37 CA283937 to J.U.M.