pH-Targeted Drug Delivery using pH Low Insertion Peptides (pHLIPs) for Liver Fibrosis Treatment

Introduction: : Liver fibrosis is a progressive condition marked by excessive scar tissue in the liver, often resulting from chronic damage due to viral hepatitis, alcohol abuse, or Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). If left untreated, it progresses to cirrhosis. Initially symptomless, it may later cause fatigue, abdominal discomfort, weight loss, jaundice, and swelling, with symptoms varying by cause. Alcohol consumption significantly increases the risk of morbidity and mortality, contributing to numerous diseases. When consumed in excess, alcohol is metabolized into acetaldehyde and excreted by the liver. The buildup of acetaldehyde leads to lipid accumulation, inflammation, and fibrosis, resulting in a decrease in the pH of the microenvironment. Current treatment options for liver fibrosis are limited, with concerns of side effects and the development of drug resistance, especially with Sorafenib, an FDA-approved drug for hepatocellular cancer. This report explores Sorafenib-loaded nanoparticles, conjugated to pH Low Insertion Peptides (pHLIP) that become activated in low pH environments, for the treatment of liver fibrosis.

Materials and

Methods: : A double emulsion method was used to formulate nanoparticles (NPs) of maleimide-functionalized poly (lactic acid-co-glycolic acid) (PLGA) polymer containing Sorafenib, a multikinase inhibitor known for its antifibrotic properties, and conjugated with pHLIP. The NPs were characterised by their particle size, polydispersity index, and zeta potential. Additionally, the cytocompatibility and cellular uptake kinetics of coumarin-6-dye-containing pHLIP-conjugated NPs were assessed in healthy and Lipopolysaccharide (LPS)-treated HepG2 model hepatocytes and LX-2 hepatic stellate cells following 24h and 2h incubation, respectively. For in vivo studies, biodistribution of the fluorescent NPs was assessed in C57BL/6N mice given 4% ethanol diet in combination with carbon tetrachloride injections to induce liver fibrosis. Animals were euthanized 24h following NP administration, and tissues were imaged by EVOS fluorescence microscopy to assess NP accumulation.

Results, Conclusions, and Discussions:: Our results indicate that the Sorafenib-loaded NPs measured 250 nm in size, with a polydispersity index of 0.346, a zeta potential of -23.8 mV, and an encapsulation efficiency of 50.56%. Additionally, the pHLIP-conjugated NPs had a size of 314 nm, a polydispersity index of 0.357, a zeta potential of -31.6 mV and a conjugation efficiency of 88.17%. Cytocompatibility studies of synthesised NPs revealed approximately 80% cell viability at a high concentration of 2000 µg/mL. Significant uptake of pHLIP-conjugated NPs was observed in LPS-treated HEPG-2 and LX-2 cells compared to unconjugated NPs. EVOS images of excised tissues showed NP accumulation in both the spleen and liver, with significantly higher accumulation observed in the liver.

Conclusions: We successfully synthesized pHLIP-conjugated PLGA NPs for drug delivery to treat liver fibrosis. The NPs were cytocompatible and demonstrated significantly higher accumulation in the liver of fibrotic mouse models than in the other organs. Ongoing and future studies will focus on assessing the ability of the NPs to deliver anti-fibrotic therapies effectively to inflamed and fibrotic liver cells both in vitro and in vivo.

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