Targeting Chronic Liver Diseases: Molecular Markers, Drug Delivery Strategies and Future Perspectives

Abstract

Chronic liver inflammation, a pervasive global health issue, results in millions of annual deaths due to its progression from fibrosis to the more severe forms of cirrhosis and hepatocellular carcinoma (HCC). This insidious condition stems from diverse factors such as obesity, genetic conditions, alcohol abuse, viral infections, autoimmune diseases, and toxic accumulation, manifesting as chronic liver diseases (CLDs) such as metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease (ALD), viral hepatitis, drug-induced liver injury, and autoimmune hepatitis. Late detection of CLDs necessitates effective treatments to inhibit and potentially reverse disease progression. However, current therapies exhibit limitations in consistency and safety. A potential breakthrough lies in nanoparticle-based drug delivery strategies, offering targeted delivery to specific liver cell types, such as hepatocytes, Kupffer cells, and hepatic stellate cells. This review explores molecular targets for CLD treatment, ongoing clinical trials, recent advances in nanoparticle-based drug delivery, and the future outlook of this research field. Early intervention is crucial for chronic liver disease. Having a comprehensive understanding of current treatments, molecular biomarkers and novel nanoparticle-based drug delivery strategies can have enormous potential in guiding future strategies for the prevention and treatment of CLDs.

Graphical Abstract

1. Introduction

Chronic and unresolved liver inflammation is a major public health concern and is projected to be the principal cause of terminal liver disease in adults and children in the next few decades (J. Chen et al., 2020; Czaja, 2014). Persistent liver inflammation can progress to fibrosis and cirrhosis, eventually causing liver failure. Fibrosis and cirrhosis are also potent risk factors for hepatocellular carcinoma (HCC), which is the fastest growing cause of cancer-related deaths worldwide (Huang et al., 2023). Chronic liver disease (CLD) is the term used to refer to the different stages of gradual deterioration of the liver over time. Nearly 4.5 million adults in the United States have been diagnosed with CLDs, and over 56,000 deaths are expected due to this condition annually (2 million deaths per year worldwide) (“CDC/Chronic Liver Disease and Cirrhosis,” 2023).

CLDs generally occur as a consequence of alcohol abuse, obesity, viral infection, autoimmune disease or due to the accumulation of certain drugs and toxins in the liver. Liver inflammation is intricately linked to metabolic disorders (“CDC/Chronic Liver Disease and Cirrhosis,” 2023; Koyama and Brenner, 2017a) as it performs several essential metabolic functions include the uptake, metabolism, and secretion of various substances such as dietary amino acids, carbohydrates, lipids and vitamins as well as hemoglobin, bile salts, iron, copper, ammonia, and drugs (Tanwar et al., 2020). The global obesity epidemic has fueled the increase in cases of MASLD (metabolic dysfunction-associated steatotic liver disease), formerly known as NAFLD (non-alcoholic fatty liver disease) - an umbrella term used to describe a range of diseases from isolated hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH), also known as nonalcoholic steatohepatitis (NASH), which is a severe form of MASLD commonly associated with type 2 diabetes, obesity, hyperlipidemia, and insulin resistance (Koyama and Brenner, 2017a). In June 2023, a consensus statement by multiple societies (Delphi) introduced the term metabolic dysfunction-associated steatotic liver disease (MASLD) and phased out the use of the term NAFLD in the context of fatty liver disease nomenclature (Rinella et al., 2023). MASLD affects over 25% percent of the population in the United States, and about 20% of these adults will progress to NASH about 3 years post diagnosis (“NASH Definition & Progression,” 2022; Roth et al., 2019). MASH patients experience progressive, irreversible liver fibrosis and inflammation leading to cirrhosis, hepatocellular carcinoma (HCC) and end-stage liver disease (Mahjoubin-Tehran et al., 2021). MASH is also projected to overtake hepatitis as the primary reason for terminal liver disease and liver transplants in adults and children in the coming years (Li et al., 2020). Despite its prevalence and the urgent medical need for effective medications to prevent, stop or reverse MASH, there are currently no approved drugs available for treatment. Alcohol liver disease (ALD) is another type of CLD that arises from chronic alcohol use and accounts for 30% of the world’s cases of primary HCC and HCC associated deaths (Ganne-Carrié and Nahon, 2019).

Infection due to viral hepatitis has also been implicated in the development of liver inflammation (Koyama and Brenner, 2017a; Liou et al., 2022). The five primary viruses responsible for viral hepatitis, hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), and hepatitis E (HEV), have caused significant morbidity and mortality over many years, primarily due to their widespread prevalence and incidence. Globally, it is estimated that over 250 million individuals are infected with the chronic form of the hepatitis B virus (HBV), and more than 70 million carry the hepatitis C virus (HCV). Additionally, there are over 1.5 million cases of hepatitis A virus (HAV) annually (González Grande et al., 2021). Out of these, HBV, HCV and HDV are the predominant causative agents of viral hepatitis where the inflammation advances to fibrosis, cirrhosis and possibly HCC. HDV is a defective virus that requires HBV to complete its life cycle in human hepatocytes (Mentha et al., 2019).

The liver is also involved in metabolizing or breaking down drugs in the blood. Drug-induced liver injury (DILI) and inflammation occurs when certain chemotherapeutic agents, immunomodulatory agents, herbal products and antibiotics or their metabolites accumulate in the liver (He et al., 2022). DILI has been linked to over 1000 medications and is a prevalent causative factor for acute liver failure in the United States, contributing to 50% of all fulminant cases (Giordano et al., 2014). DILI can be categorized into idiosyncratic or intrinsic liver injury. Idiosyncratic DILI is liver injury that occurs by taking the recommended dose of any conventional regulatory approved drugs. Intrinsic DILI is due to overdose of drugs such as acetaminophen/ paracetamol (APAP) (Teschke, 2023). In most cases, the inflammation and liver damage are observed after several months of taking the medication, when it reaches toxic levels in the liver. About 20% of the DILI patients progress to chronic DILI, which is characterized by inflammation, diminishing bile ducts, cirrhosis and eventually liver failure (Q. Wang et al., 2021). The clinical manifestation of DILI is usually varied and complicated, often resembling the symptoms of other liver diseases such as autoimmune hepatitis and acute viral hepatitis (He et al., 2022), which delays diagnosis of this condition. In some cases, symptoms continue even after cessation of intake of the causative drug, therefore the patients will need lifelong therapy to control the inflammation. Autoimmune hepatitis is an immune-mediated disease where the immune system begins to attack liver cells including hepatocytes and biliary epithelial cells. If left untreated, this can progress to fibrosis, cirrhosis and liver failure. Autoimmune hepatitis has also been associated with increased risk for HCC development (Richardson et al., 2022; Sharma et al., 2022)

Due to the silent nature and variable clinical presentation of CLDs, symptoms develop late, at which point precise and effective treatments are critical to prevent disease progression (Marcellin and Kutala, 2018). A major drawback of current anti-inflammatory, anti-viral and immunosuppressive therapies for liver inflammation is that they are not safe and consistently effective (Czaja, 2014). Several therapies in clinical trials to treat hepatic inflammation have failed due to poor pharmacokinetics or toxicity issues (Bansal et al., 2016). Several MASH Phase II/III clinical trials failed recently due to poor drug pharmacokinetics and lack of efficacy (Bansal et al., 2016; Mullard, 2020). Therefore, there is a critical need for novel drugs and drug delivery strategies that can target the liver to provide sustained, effective treatment to prevent the progression of chronic liver inflammation to end-stage liver disease.

Nanoparticle-based drug delivery strategies can potentially overcome several limitations of traditional drug delivery by shrouding the drug from harsh conditions within the body, evading phagocytosis and rapid clearance, and preventing non-specific accumulation of the drugs in healthy tissues and organs. Depending on the composition, release rates and surface modifications used, these nanoparticles facilitate the targeting of specific cell populations in the liver that are implicated in fibrosis and inflammation and deliver therapies that can mitigate and potentially reverse these diseases. This review provides an overview of potential molecular targets for treating liver inflammation, some of the recent advances in the use of nanoparticle-based drug delivery strategies for the treatment of liver diseases and the future outlook of this critical area of research.

2. Molecular targets and pathways in CLD

The liver and the intestinal barrier together form the gut-liver axis, which is the body’s defense system against gut-derived bacteria, bacterial products like lipopolysaccharide (LPS), food antigens and other toxins (Albillos et al., 2020; Kobayashi et al., 2022). LPS plays a key role in the induction and progression of inflammation, fibrosis and steatosis in many chronic liver conditions. In diseases like MASLD and ALD, the intestinal barrier integrity is compromised, causing LPS and other toxins to be carried by the blood from the gut to the liver (Szabo and Bala, 2010). In cases of acute injury, the healthy liver protects itself via a controlled immune response where there is balance between pro- and anti-fibrosis mechanisms. The wound-healing responses include providing mechanical stability through fibrosis, removal of cellular debris by inflammatory cells, and promotion of liver regeneration through inflammatory signals. However, in CLDs, the wound healing mechanism is impaired leading to an exaggerated or dysfunctional immune response that can contribute to further tissue injury and damage by replacing the liver parenchyma with scar tissue and distorted vasculature. Hepatocellular death serves as the initial trigger, leading to the activation of inflammatory and fibrogenic signaling pathways, which in turn causes activation of hepatic stellate cells (HSCs) and other cell types, over-deposition of extracellular matrix including collagen, and release of fibrogenic factors to cause inflammation and fibrosis (Berumen et al., 2021; Lee and Friedman, 2011)(Fig. 1).

Figure 1.

Schematic representation of the inflammation pathway that is triggered by hepatocytes death, followed by activation of HSCs, and fibrogenesis.

There are several molecular markers on liver cells that are involved in the wound-healing responses and that can be targeted for therapy. The detection of pathogens and their products is done by pattern recognition receptors (PRRs) expressed by several liver cells including hepatocytes, non-parenchymal cells like liver sinusoidal endothelial cells (LSECs) and HSCs, and immune cells such as Kupffer cells (KCs) and dendritic cells (Protzer et al., 2012; Tanwar et al., 2020). PRRs can be classified into five main categories: Toll-like receptors (TLRs), Nucleotide oligomerization domain (NOD) like receptors (NLRs), C-type lectin receptors (CLRs), and retinoic acid-inducible gene-I (RIG-1) like receptors (RLRs) and absent in melanoma-2 (AIM2) like receptors (ALRs) (Emery et al., 2021; Li and Wu, 2021). These receptors recognize conserved molecular structures called pathogen-associated molecular patterns (PAMPs), which are specific to pathogens, and damage-associated molecular patterns (DAMPs) released by stressed, dying hepatocytes. Among the five types of PRRs, TLRs play a critical role as key sensors of the innate immune system in pathogen recognition, including viruses (Kawai and Akira, 2006). TLR3 can detect viral double-stranded RNA, leading to the production of interferon (IFN)-I or IFN-α/β (Alexopoulou et al., 2001). TLR4, one of the most studied TLRs, recognizes multiple endogenous ligands including LPS. It is found in both parenchymal and non-parenchymal cells of the liver, and its signaling is involved in a variety of liver injuries, inflammation and fibrinogenesis. TLRs activate three main pathways implicated in inflammation: the mitogen-activated protein kinase (MAPK) pathway (includes ERK, p38, and downstream c-Jun N-terminal kinase (JNK)), the nuclear factor-kappa B pathway (NF-κB), and the IFN regulatory transcription factor pathway. Stimulation of TLRs can induce the production of both pro-inflammatory and anti-inflammatory cytokines (Bhat et al., 2002; Javaid and Choi, 2020). Other pathways and potential molecular targets that can be considered for nanoparticle-based drug delivery are described below.

2.1. Hepatocytes

Hepatocytes make up about 85% of the liver mass and are therefore the primary cells to undergo injury and death upon exposure to hepatotoxic agents. Although hepatocytes have traditionally been viewed as passive targets of injury and death in liver disease, recent studies have highlighted their active role in driving liver inflammation and fibrosis via intercellular communication. Sterile hepatocyte injury and death leads to the release of DAMPs, which are danger signals/ molecules recognized by the innate immune system including KCs via PRRs leading to exacerbated inflammatory response in the liver. Stressed hepatocytes actively undergo phenotypic changes in response to the injury, adapt to the changed microenvironment, and engage in crosstalk with surrounding cell populations such as HSCs and immune cells, resulting in the release of pro-inflammatory signals and cytokines, reactive oxygen species (ROS) production and activation of repair pathways (Tu et al., 2015). Persistent exposure to stress factors leads to impairment of normal repair response and initiation of a dysfunctional fibrotic response resulting in fibrosis and eventually cirrhosis and cell death.

Hepatocyte cell death can occur via intrinsic or extrinsic pathways. Intrinsic pathways can be triggered by cellular stress or by dysfunction of intracellular organelles including mitochondria, endoplasmic reticulum and lysosomes in response to injury (Alkhouri et al., 2011). Extrinsic pathways of hepatocyte death are activated by the family of tumor necrosis factor (TNF) death receptor ligands including TNF-α, FASL, and TNF-related apoptosis-inducing ligand (TRAIL) (Ignat et al., 2020), which are produced in large quantities on exposure to bacteria or bacterial products like LPS. These ligands interacts with their cognate death receptor on the hepatocyte cell surface leading to ROS production, caspase 8 activation and cell death (Alkhouri et al., 2011). Apoptosis-signal regulating kinase 1 (ASK1) in hepatocytes is a member of the MAPK family known to regulate apoptosis and inflammation particularly in MASH by activating the JNK/ p38 MAPK signaling pathway. Lipotoxicity and protein misfolding, which is frequently observed in MNASH hepatocytes, causes endoplasmic reticulum (ER) stress and unfolded protein response (UPR), which, if prolonged, can also result in apoptosis mediated by the Bcl-2 of family proteins (Alkhouri et al., 2011). These and other recent findings confirm that hepatocyte apoptosis drives the pathogenesis of many CLDs. Inhibition of hepatocyte apoptosis is therefore of interest to mitigate inflammation and fibrosis in the liver. Fas, TNF-α and caspases have all been considered as molecular targets in preclinic and clinical studies for the treatment of chronic liver inflammation (Alkhouri et al., 2011). ASK1 inhibition has also been found to reduce liver injury and hepatocyte death in NOD-like receptor protein 3 (NLRP3) mutant mice livers; the NLRP3 inflammasome is elevated in MASH livers and plays a key role in MASH progression (Schuster-Gaul et al., n.d.). Farnesoid-X receptor (FXR) is another possible molecular target. It is usually highly expressed in hepatocytes and is an intrinsic inhibitor of hepatocyte apoptosis. However, it is present in reduced levels in fibrotic patients; therefore, forced FXR upregulation in cells can potentially protect the liver against injury and apoptosis (Wang et al., 2018). Peroxisome proliferator-activated receptors (PPARs) are another group of nuclear receptor proteins of which PPARα expressed in hepatocytes regulates lipid metabolism in the liver. PPARα activation can suppress inflammation via inhibition of NF-κB activation (Tillman et al., 2016); therefore, PPARα is another possible target for treatment. In addition to the inflammation-associated molecular targets mentioned above, hepatocytes are frequently targeted via hepatic transferrin and the asialoglycoprotein receptor (ASGPR) (Hu et al., 2014). The ASGPR is uniquely expressed on hepatocyte cell surfaces and can be targeted using galactose, lactose and glucose sugars (Porterfield et al., 2023) Transporters on the hepatocyte cell surface such as the Organic Anion Transporting Polypeptides (OATP) and Solute Carrier Family 39 Member 14 (SLC39A14) are also of interest for hepatocyte targeting and drug delivery (Zhang et al., 2023).

2.2. Kupffer cells (KCs)

KCs are resident macrophages within the liver sinusoids that play a central role in liver inflammation. Previously thought to be derived from bone marrow-derived monocytes, recent evidence suggests that KCs either self-renew or arise from local progenitors (Porterfield et al., 2023). The role of KCs in the liver are many, ranging from maintaining liver integrity and repairing liver tissues following injury, to initiating and resolving the innate and adaptive immune response (Abdullah and Knolle, 2017; van der Heide et al., 2019). Upon hepatocyte injury, KCs become activated and release proinflammatory cytokines and signaling molecules. Depending on the environmental signals, these activated KCs can exhibit markers of proinflammatory M1-like or anti-inflammatory M2-like macrophages; the balance between these two types of KCs is crucial for the regulation of liver inflammation (J. Chen et al., 2020; Slevin et al., 2020). Therapies that can limit M1 KC polarization and promote M2 polarization are therefore of interest to treat CLDs. The G-protein coupled bile acid receptor (also known as Gpbar1 or TGR5) expressed by KCs is another target of interest as its activation promotes an anti-inflammatory phenotype by suppressing pro-inflammatory cytokine release via cyclic adenosine monophosphate (cAMP) signaling and NF-κB inhibition (Bertolini et al., 2022). The mannose receptor C type 1 is another receptor that has been widely studied for targeted drug delivery to KCs. The mannosyl units available in albumin, for example, can be used for binding to the mannose receptor and uptake by KCs (Maeda et al., 2022). The sensing of pathogens and pathogen-derived products in the liver are done by KCs via TLRs and NLRs, which leads to the release of inflammatory chemokines and cytokines such as TNF-α, IL-8 and CCL2, interleukin (IL)-6 and IL-1β and ROS to recruit circulating immune cells such as monocytes to the region. Through cytokine release and immune cell recruitment, KCs promote the recruitment of HSCs to the region (Abbas et al., 2020). The interactions between KCs and HSCs are crucial for driving liver repair. However aberrant HSC activation can initiate chronic liver fibrosis, as described below.

2.3. Hepatic Stellate Cells

HSCs, which are fat- and vitamin A-storing cells present in the perisinusoidal space between hepatocytes and sinusoidal endothelial cells in the liver, are key players of fibrogenesis. HSC activation is promoted by signals from injured hepatocytes and other cells in the disrupted hepatic microenvironment as well as the infiltration of immune cells into the region in response to injury (Abbas et al., 2020). The activated HSCs lose their intracellular lipid droplets and vitamin A storage capacity and differentiate into myofibroblasts, which drive fibrinogenesis by secreting collagen, extracellular matrix (ECM) proteins and matrix metalloproteinases leading to distortion of both the parenchyma and vascular architecture of the liver (Senoo et al., 2010). These cells upon activation also express profibrotic markers including α-smooth muscle actin (α-SMA) and type 1 collagen. After initial activation, HSCs require signals to perpetuate their activated state and ensure their survival, which are likely mediated by cytokines from either HSCs themselves or inflammatory cells. Following liver damage, transforming growth factor (TGF)-β released by injured hepatocytes, KCs and other liver cells forms a TGFβ type 1 receptor/ TGFβ type 2 receptor (TGFβRI/TGFβRII) heteromeric complex at the HSC cell surface, leading to activation of SMAD and non-SMAD signaling, causing profibrotic gene expression (Higashi et al., 2017). The PAMPs and DAMPs released by hepatocytes are also recognized by TLRs in HSCs. TLR4 signaling in activated HSCs leads to activation of inflammatory signaling pathways such as NF-κB and activator protein 1 (AP-1) and production of chemokines and cytokines (Seki et al., 2007; Seki and Schwabe, 2015). There have been efforts to target all these pathways to suppress fibrinogenesis in the liver. HSCs may produce chemotactic signals that regulate their interaction with inflammatory cell types, promoting recruitment of cells that activate HSCs during fibrogenesis and cells that degrade extracellular matrix and eliminate HSCs during fibrosis regression. The C-X-C motif chemokine ligands (CXCLs) such as CX3CL1 and C-C motif chemokine ligand 2 (CCL2) released by activated HSCs can chemoattract macrophages to the region. An array of different interleukins including IL-1β, 4, 5, 6, 13, 15, 17, 22 and 23 released by different injured or activated liver cells are involved in promoting the activation of quiescent HSCs. The IL-10 superfamily of cytokines has antifibrotic properties and are therefore also considered as targets to inhibit HSC activation and promote apoptosis of activated HSCs (Baghaei et al., 2022; Garbuzenko, 2022; Tsuchida and Friedman, 2017). Other potential targets that prevent HSC activation and have anti-fibrotic effects such as vascular endothelial growth factor (VEGF) inhibition, platelet derived growth factor (PDGF) inhibition, PPAR-γ activation, and FXR inhibition are also being actively explored (Baghaei et al., 2022; Zhang et al., 2006). In addition, mannose receptors have been considered for HSC targeting. However, mannose receptors are also present on KCs and endothelial cells, therefore cell-specific targeting may be limited when targeting these receptors (Xiong et al., 2023). Upregulation of CD44 is seen in activated HSCs in liver fibrosis, therefore CD44 can also be targeted for the treatment of CLDs (Osawa et al., 2021; Tan et al., 2024).

2.4. Liver sinusoidal endothelial cells (LSECs)

LSECs are specialized endothelial cells that line the hepatic sinusoid and form about half of the non-parenchymal cells in the liver. They play a crucial role in maintaining liver homeostasis and are considered as gatekeeper cells of the liver (Du and Wang, 2022). LSECs have fenestrations and are constantly exposed to antigens from the gastrointestinal tract. They have important functions, such as removing macromolecules and particles from the blood, participating in liver regeneration, influencing hepatic fibrosis, and interacting with tumor metastasis. These cells also possess immunological roles and exhibit high endocytic activity (DeLeve and Maretti-Mira, 2017; Khanam et al., 2021). In CLD, impaired LSEC function has been observed, leading to dysregulated responses and chronic inflammation, which further contribute to hepatic fibrosis (Xu et al., 2003). LSECs closely interact with leukocytes during their migration from blood vessels into the liver tissue, forming functional units with pericytes (Seki and Schwabe, 2015). HSCs, as liver-specific pericytes, closely interact with LSECs in both normal and injured liver. In the normal liver, fenestrated LSECs actively suppress HSC activation. However, capillarization of endothelia after injury impairs the ability of LSECs to suppress HSC activity, creating a permissive state for HSC activation (Deleve et al., 2008; Xie et al., 2012). This induces phenotypic changes in HSCs and act as one of the initial triggers for HSC activation, leading to alterations in the ECM and increased tissue stiffness. Recent studies suggest that LSECs, through the expression of specific receptors such as CXCR7, CXCR4, and FGFR1, have a pivotal role in deciding between liver regeneration and HSC activation/fibrosis. The precise mechanisms by which CXCR4 and FGFR1 on LSECs promote HSC activation are yet to be determined (Ding et al., 2014). LSECs themselves are responsive to TLR ligands and can produce various inflammatory mediators (Seki and Schwabe, 2015). Peptides with RXR or RXXR motifs have been previously explored for targeting LSECs (Akhter et al., 2013). Adhesion molecules such as ICAM-1 (Intercellular adhesion molecule-1), ICAM-2 (Intercellular adhesion molecule-2), which are overexpressed by LSECs in inflamed livers are also potential molecular targets for drug delivery applications (Pandey et al., 2020).

Several other cell types are present in the liver, such as dendritic cells (DCs), lymphocytes, and cholangiocytes, which also play a significant role in inflammation or liver injury. DCs are a type of myeloid lineage cells closely related to macrophages. In mice, DCs do not contribute to fibrosis development but have been found to promote the resolution of liver fibrosis through MMP-9-dependent mechanisms (Jiao et al., 2012; Pradere et al., 2013). DCs are professional antigen-presenting cells. The liver also contains various types of lymphocytes, including unconventional T cells, natural killer T (NKT) cells, hepatic innate lymphoid cells (ILCs), γδ T cells, and memory CD8 T cells (Khanam et al., 2021; Wang and Zhang, 2019). These liver-resident lymphocytes have important functions in immunosurveillance and maintaining liver homeostasis. However, under pathological conditions, they can also contribute to hepatitis, fibrosis, and cirrhosis. Cholestatic liver disease is characterized by inflammation and fibrosis, and cholangiocytes play a significant role in these processes by producing inflammatory cytokines and chemokines and interacting with various inflammatory cell types, particularly T lymphocytes. Studies have shown that hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells in cholestatic liver fibrosis, rather than portal fibroblasts (Syal et al., 2012).

3. Current treatments for CLDs

Inflammation can be potentially reversed if diagnosed and treated on time in the early stages of liver disease. However, if left untreated, inflammation can progress to irreversible fibrosis and cirrhosis. The majority of the drug development for treating CLDs has focused almost exclusively on MASLD and MASH. Changes in lifestyle and diet are the most successful ways to prevent liver inflammation without the use of medications. Some common treatments for the various stages of CLDs are represented in Fig. 2.

Fig. 2.

Schematic representation of various stages of CLDs and current treatment options, adapted from Way et al., 2022 and used under aCreative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

MASH is the outcome of several factors working together, such as genetic variations, abnormal lipid metabolism, oxidative stress, changes in immune response, and imbalances in the gut microbiota. The concept of “multiple hits” suggests that liver inflammation, rather than steatosis, is the main driver of MASH advancing to fibrosis, suggesting that multiple mechanisms act in synergy to promote disease progression (Xu et al., 2022). Further, several MASLD/MASH models revealed that the liver is overwhelmed in dealing with primary metabolite substrates, carbohydrates, and fatty acids (FAs), leading to the buildup of harmful lipid species. These substances can cause stress, injury, and death of hepatocytes, ultimately leading to fibrosis and genetic instability, increasing the risk of cirrhosis and HCC in patients. The non-availability of accurate diagnostic and therapeutic biomarkers is a major barrier to screening and providing effective therapies against MASH. Metabolic profiling, transcriptomic profiling gene expression patterns, and measurement of circulating proteins are all methods that researchers use to find new biomarkers for MASH therapy development.

Lifestyle interventions, such as exercise, dietary changes, and weight loss are the major treatment options for MASLD patients without fibrosis development (Vachliotis et al., 2022). A recent clinical trial in low energy total diet replacement showed favorable safety profile and improved severity in patient with MASH (Koutoukidis et al., 2023). However, implementing lifestyle changes can be challenging for patients, particularly in terms of physical activity, and the effectiveness of these modifications is still limited. Furthermore, advanced symptoms and diseases such as hepatic fibrosis, cirrhosis, and HCC can persistently affect the quality of life of patients. Therefore, there should be a special focus on pharmacological treatment in addition to lifestyle-related interventions. MASH patients with obesity and type 2 diabetes are sometimes prescribed drugs such as glucagon-like peptide 1 (GLP-1) agonists and pioglitazone, which do not directly act on the liver but have shown the most promise among all the medications that have been studied to alleviate this condition (Baghaei et al., 2022; Dufour et al., 2022). In 2020, Zydus Cadila’s Saroglitazar was approved for the treatment of non-cirrhotic MASH in India following positive results at the end of a phase III liver biopsy trial. Saroglitazar Magnesium (Lipaglyn) is already approved in India for the treatment of type 2 diabetes (T2D) and dyslipidemia. It acts as a gamma agonist and PPAR agonist, and improved alanine transaminase (ALT), liver fat content, insulin resistance and atherogenic dyslipidaemia in the MASH/ MASLD patients (Fraile et al., 2021; Padole et al., 2022).

A few MASH drugs have entered Phase III clinical trials. One of these is the pan-PPAR agonist lanifibranor, which successfully completed 24 weeks of Phase IIb study with 247 participants (Dufour et al., 2022). The Steatosis, Activity, and Fibrosis (SAF) score had a decrease of at least two points in most of the patients that took 1200 mg of lanifibranor (55%), while the percentage of patients who had a decrease of 2 points with 800 mg of lanifibranor and the placebo were 48% and 33% respectively. MASH was resolved without worsening fibrosis at week 24 of the study in 49% of the patients who received 1200 mg, 39% of the patients who received 800 mg, and in 22% of the patients that received the placebo (Francque et al., 2021). At the time of this review, lanifibranor is being evaluated as part of a phase III study in adults with MASH and liver fibrosis (stage 2 or 3). A completed phase II trial with the GLP1-receptor agonist semaglutide demonstrated that 59% of patients treated with semaglutide had a decrease in MASH symptoms compared to a 20% decrease in patients given the placebo. A GLP-1/glucagon receptor co-agonist efinopegdutide has also demosntrated greater promise than semaglutide during initial phase IIa studies. However, in the past few years, a number of drugs have failed during MASH phase IIb and phase III clinical trials. Obeticholic acid (OCA) has been widely studied for its FXR agonism in MASH research, however, it was recently rejected by the FDA due to its concerning risk-benefit profile (Albhaisi and Sanyal, 2021). From the PPAR agonist group, studies on elafibrinor and seladelpar were terminated. Elafibrinor failed to achieve a significant reduction of MASH following 72 weeks, when compared to the placebo group. At 12 weeks, seladelpar did not result in any significant reduction of liver fat and the study was terminated based on atypical histological findings. Pioglitazone, an FDA-approved drug for type 2 diabetes, drew particular attention as a PPAR agonist, followed by a phase IV clinical trial to check the efficacy in MASH patients. Although the phase IV clinical trial showed a reduction in liver fibrosis, patients with type 2 diabetes showed increased adipose tissue insulin sensitivity due to PPARγ-driven side effects (Bril et al., 2018). Recently, a pioglitazone-derived drug, PXL065 (deuterium-stabilized (R)-pioglitazone), has been effectively used in MASH patients with reduced potential for PPARγ-driven side effects. This phase II clinical trial showed the previously proven efficacy of pioglitazone and PXL065, which requires further clinical testing in pivotal NASH trials (Harrison et al., 2023b). From the fibroblast growth factor (FBF) analogon group, pegbelfermin (FBF21 analog) after 24 weeks and 48 weeks of treatment did not show significant improvement in MASH (Loomba et al., 2024). Another FBF analog, aldafermin (FBF19), showed no reduction in MASH at 24 week treatment (Harrison et al., 2022). However, recent 48 week studied showed that aldafermin 3 mg resulted in significant reduction in enhanced liver fibrosis in patients with compensated MASH cirrhosis (Rinella et al., 2024). As shown in Figure 3, various form of cell deaths, such as apoptosis, necroptosis, pyroptosis and ferroptosis, as well as autophagy, are linked to the progression of MASLD/MASH. So, targeting and inhibiting these cell death pathways have shown promising results. However, From this inhibitor group, cenicriviroc (CCR2/5 antagonist), selonsertib (ASK1 inhibitor), and emricasan (caspase inhibitor) were all terminated; no significant improvement in liver fibrosis was observed over 48 weeks (Wiering and Tacke, 2023).

Fig. 3.

Potential targeted therapies in ALD; the green boxes indicates the potential therapies; (1) Alcohol abstinence will reverse the initial fatty liver stage, beyond fibrosis stage requires clinical intervention; (2) Anti-inflammatory molecules- these molecules inhibit the inflammatory cytokines production and alleviate the inflammations; (3) Antifibrotic drugs-reduce the fibrosis; (4) Growth factors-promote liver regeneration; (5) Antioxidant- protect from oxidative stress or ROS; (6) anti-caspase molecules- inhibit the cell death; (7) TLR agonists prevent the KCs activation and thereby alleviate the inflammation/fibrosis; (8) Probiotic, prebiotic- decrease bacterial overgrowth and regulate gut inflammation, antibiotic- decrease bacterial overgrowth and bacterial translocation and further prevents development of infections; (9) Zinc- decrease gut inflammation. Modified with permission (Louvet and Mathurin, 2015). Copyright 2015, Springer Nature.

One key endpoint in clinical trials for chronic diseases, including MASLD and MASH, is all-cause mortality. Liver related mortality is typically related to the complications of cirrhosis. 15–20% of NASH patients experience a slow development of cirrhosis. To evaluate the efficacy of a drug on mortality rates, a MASH patient would need to be followed over 10 to 15 years. Since development of MASH occurs over a long duration, design and development of clinical trials for early-stage MASH becomes difficult. It is necessary that achievable endpoints are identified for a reasonable time period in order to see any clinical outcome (Sanyal et al., 2015). Another challenge faced during drug development is the difficulty in identifying one key process of pathogenesis during MASLD progression (Thiagarajan and Aithal, 2019). There are a variety of pathogenic pathways contributing to MASH. One potential reason for the failure of clinical trials is that numerous pathways become activated during the course of the disease, and targeting one single pathway is not sufficient to observe significant efficacy. (Wiering and Tacke, 2023). Therefore, there are efforts now to develop combination therapies that can target different mechanisms of action simultaneously for MASH treatment (Ratziu and Charlton, 2023). In the treatment of ALD, complete alcohol abstinence remains the first line of treatment to prevent liver damage. Nutritional assessment is crucial, considering factors like sarcopenia and frailty commonly seen in ALD (Bhanji et al., 2019; Bunchorntavakul and Reddy, 2020). The degree of disease compensation should be evaluated, with an active search for complications. Severe acute forms of ALD, such as alcohol-associated hepatitis, have high mortality and morbidity. Current treatments focus on reducing immune activation and inflammation pathways (Ayares et al., 2022). Corticosteroids are commonly used in the treatment of ALD, which downregulate IL-10 production and reduce the short-term mortality and incidence of encephalopathy (Ohashi et al., 2018). However, their effectiveness is limited and do not improve long-term survival and may cause side effects. Therefore, alternative therapies, including N-acetylcysteine (NAC), pentoxifylline and anti-tumor necrosis factor TNF antibodies, are being explored. All these therapies alone did not provide any significant improvements but when combined these with corticosteroids, NAC and anti TNFα showed improvement in 28 days survival rate though there was no improvement in long term survival rate (Naveau et al., 2004; Phillips et al., 2006; Spahr et al., 2002). Pentoxifylline is no longer considered as viable option for treatment of acute alcohol-associated hepatitis (Ohashi et al., 2018; Thursz et al., 2015). Treatments also incorporate the use of antioxidants (Ferramosca et al., 2017), while emerging therapies such as metadoxine, IL-22 analogs, and IL-1β antagonists are being investigated (Ohashi et al., 2018). Promising results have been observed with granulocyte colony-stimulating factor, microbiota transplantation, and modulation of the gut-liver axis (Ohashi et al., 2018). In advanced ALD, palliative care is deemed significant (Ayares et al., 2022). Ongoing clinical trials are evaluating the efficacy of new agents like DUR-928 and granulocyte colony-stimulating factors to treat alcohol-associated hepatitis (Pimienta et al., 2022). Figure 3 shows the simplified ALD pathways and current treatment options.

In the case of DILI, once idiosyncratic DILI has been detected, cessation of all non-essential drugs as soon as possible is mandatory, and this alone has a positive impact on liver injury. DILI can be presented as cholestatic, hepatocellular, and mixed; a predominant elevation of ALP defines cholestatic liver injury, while hepatocellular liver injury is defined as a predominant elevation of aspartate aminotransferase (AST) and ALT. A large number of conventional drugs and phytochemicals derived from herbal medicine and other chemicals such as glucocorticoids (Ye et al., 2022), NAC (Ntamo et al., 2021), and polyene phosphatidycholine (Fan et al., 2022), have been discussed as potential treatments for DILI (Teschke, 2023). In a study conducted in 2017, bicyclol (4,4’-dimethoxy-5,6,5’,6’-bis(methylenedioxy)-2-hydroxymethyl-2’-methoxycarbonyl biphenyl) was used to treat DILI caused by cardiovascular drug statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors). This study included 168 stain-induced liver injury patients who were equally randomized into two four-week treatment groups, namely bicyclol 25 mg oral dose three times per day and polyene phosphatidylcholine 456 mg oral dose three times per day as control. Polyene phosphatidylcholine is a commonly used drug to treat DILI in China, and efficacy and safety were evaluated in several clinical trials (Lei et al., 2021) After two and four weeks, serum ALT levels were tested as primary endpoints and serum ALT normalization rates were tested as secondary endpoints. Though there was no statistically significant difference in serum ALT level after four weeks, a reduction of ALT level of bicyclol-treated groups was reported (30.36 ± 17.46 vs 50.71 ± 27.13 U/L) (Naiqiong et al., 2017). A recent phase II clinical trial was developed to evaluate the efficacy of bicyclol in treating idiosyncratic acute DILI. A total of 241 patients were divided into three groups in the final study, with 81, 82, and 78 patients in the low dose bicyclol (25 mg times a day (TID)), high dose bicyclol (50 mg TID), and control (polyene phosphatidylcholine; 456 mg TID) groups respectively. Similar to the previous study, primary endpoint was serum ALT levels, and the secondary endpoints were the normalization of ALT and AST levels. ALT levels were decreased after four weeks by −249.2 ± 151.1, −273.6 ± 203.1, and −180 ± 218.2 U/L in the low-dose bicyclol, high-dose bicyclol, and control groups, respectively. Both bicyclol 25 mg and 50 mg TID showed promising efficacy and a manageable safety profile for treatment of idiosyncratic DILI, while high-dose bicyclol achieved a higher efficacy. A phase III trial with a larger sample size and extended time is required to conclude the results (Tang et al., 2022).

APAP overdose is the most common cause of acute liver failure in North America and Europe. The current gold standard for APAP induced liver injury is NAC, which was developed in the 1970s. Numerous clinical trials have been carried out during the last four to five decades to evaluate the efficacy and safety of NAC, and it has been proven to be a safe and effective drug to treat APAP-induced liver injury (Licata et al., 2022). However, NAC is associated with adverse drug reactions, medication errors, and prolonged durations [46]. Due to these reasons, calmangafodipir, a drug that prevents mitochondrial injury during the oxidative phase of toxicity by acting as a superoxide dismutase (SOD), has been studied recently in phase I clinical trials(Morrison et al., 2019). Calmangafodipir has already been studied in the phase II safety and efficacy study of chemotherapy induced peripheral neuropathy in patients with advanced metastatic colorectal cancer, which is also caused by oxidative stress (Glimelius et al., 2018). A recent phase I study by a Scottish group concluded that a combination of NAC with different doses of calmangafodipir (2, 5, 10 μmol/kg) is safe and tolerated by patients with APAP overdose. Further, they reported that the combination of NAC and calmangafodipir may reduce liver injury biomarkers more than NAC alone (Morrison et al., 2019). Another study was also carried out to check the efficacy of combination of calmangafodipir with NAC for APAP overdose, but no results has been provided so far (Buchan et al., 2019). Fomepizole (4-methylpyrazole); an FDA approved alcohol dehydrogenase inhibitor, known to be a potent and specific cytochrome P450 2E1 (CYP2E1) inhibitor (Benić et al., 2022), can be used to treat APAP overdose. After therapeutic dosing, 5% of APAP undergoes metabolism to N-acetyl-p-benzoquinone imine (NAPQI), a highly hepatotoxic metabolite, by CYP2E1. Therefore, APAP overdose may overwhelm the normal detoxication pathway of the liver (Rampon et al., 2020). The inhibition of CYP2E1 prevents excessive formation of NAPQI and thereby reduces the liver damage causing APAP. The efficacy and safety of the fomepizole as a CYP2E1 inhibitor to reduce the formation of NAPQI has been well studied using animal models and human hepatocytes, although there are limited clinical trials (Akakpo et al., 2018, 2019) (Rampon et al., 2020; Shah and Beuhler, 2020).

In recent years, there have been notable advancements in the treatment and management of viral hepatitis, particularly in cases of HBV and HCV. These developments have been led by new discoveries in viral pathogenesis, which have enabled the development of new drugs and safer, more effective oral treatments. (González Grande et al., 2021). For example, the discovery of the fist nucleotide/nucleoside analogs like lamivudine and adefovir dipivoxil in early 2000s enabled the rapid development in treatments. Though the development of resistance for those drugs limited the progression, that was overcome later with entecavir and tenofovir, which have high genetic barrier to the resistance (Fuentes Olmo and Uribarrena Amézaga, 2011; Koumbi, 2015). However, these advancements have not been as pronounced for HAV and HEV. The most noteworthy progress in these two has been understanding the disease’s epidemiology and pathogenesis rather than developing therapeutic interventions. Research has revealed new modes of transmission, particularly pertinent in specific population segments like individuals with a history of liver disease or those who are immunosuppressed, where the diseases can significantly affect morbidity and mortality (González Grande et al., 2021).

As of now, there are no specific medications designed to combat HAV infection, so the primary approach to treatment involves providing supportive care. The prevention of HAV infection encompasses various measures such as vaccination, immune globulin administration, and adherence to hygienic practices such as handwashing. It also involves precautions like avoiding tap water and raw foods in areas with inadequate sanitation, as well as ensuring the proper heating of foods (Almeida et al., 2021; Nelson et al., 2020; Shin and Jeong, 2018).

In HBV, the persistence of covalently closed circular DNA (cccDNA) in the nucleus poses the primary therapeutic challenge in caring for patients. Two forms of IFN (conventional and pegylated) and five nucleos(t)ide analogs (telbivudine, entecavir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), and besifovir dipivoxil) are antiviral agents employed in the treatment of chronic hepatitis B. Although these drugs effectively suppress HBV replication, lower the risk of cirrhosis, and impede further disease progression, they do not provide a cure and do not demonstrate a proven positive impact on the existing viral hepatocyte reservoir. Several preclinical, phase I, phase II, and phase III studies have been conducted to target different ligands using novel therapeutic drugs (Cornberg et al., 2020; Lobaina and Michel, 2017). For example, nitazoxanide (NTZ), a FDA approved thiazolide anti-infective agent for protozoan enteritis, was used to inhibit the HBV regulatory protein X(HBX)-DDB1 protein interaction and thereby significantly restored the Smc5 protein level and suppressed the viral transcription. HBX-DDB1 promotes transcription of cccDNA with degradation of Smc5/6 (Sekiba et al., 2019). Some of the selected recent clinical trial studies are summarized in Table 1.

Table 1.

Recent and ongoing clinical trials for CLDs, and their liver specific molecular targets, status of the trials. Latest publications for the specific clinical trials are listed. The status of the trials is available in clinicaltrials.gov and hepatitis B foundation (hepg.org).

Disease	Drug	Target	Status	References
MASLD/MASH (NASH)	Efinopegdutide (MK-6024)	Glucagon-like peptide-1 (GLP-1)/glucagon receptor co-agonist	Approved in 2023 based on Phase IIb	(Romero-Gómez et al., 2023)
	Semaglutide	GLP-1 agonist	Phase II	(Newsome et al., 2021)
	Efruxifermin	FGF21 which activates cell membrane co-receptor complex β-klotho; (anti fibrotic)	Phase IIa completed. Phase III in progress	(Harrison et al., 2021)
	Pegozafermin	FGF21	Phase IIa completed. Phase III pending	(Harrison et al., 2023a)
	Saroglitazar	PPAR- α/γ agonist	Phase II	(Gawrieh et al., 2021)
	Obeticholic acid	FXR agonist	Terminated	(Younossi et al., 2019)
	Resmetirom	Thyroid hormone receptor β (THRβ) agonist	Phase III completed	(Harrison Stephen A. et al., 2024)
	Lenifibranor (IVA 337)	Pan-PPAR agonist	Phase III	(Francque et al., 2021)
	Bempedoic acid	ATP-citrate lyase inhibitor	N/A	NCT06035874 ClinicalTirals.gov
	Cenicriviroc	Chemokine receptor 2 and 5 antagonist	Phase III completed; lack of efficacy	(Anstee et al., 2024)
	Aldafermin	An engineered analog of FGF19, inhibits bile acid synthesis	Phase IIb	(Rinella et al., 2024)
	Vitamin D	Dietary supplement	Explanatory randomized clinical trial	(Ebrahimpour-Koujan et al., 2024)
	PROBILIVER (probiotic supplement)	Dietary supplement	Completed	(Silva-Sperb et al., 2024)
ALD	F-652	Contains IL-22 and immunoglobulin G2	Phase I/II on going	(“Clinical Trials-MayoClinic,” 2024)
HBV	VIZR-228	RNAi gene silencer	Phase II	(“Hepatitis B Foundation: Drug Watch,” 2024)
	Imdurisan (AB-729)	RNAi gene silencer	Phase II
	Bulevirtide (Hepcludex)	Entry inhibitor	Phase III
	REP 2139	Surface antigen inhibitor	Phase II
	Bepirovirsen	Antisense molecules: Binds to the viral mRNA to prevent it from turning to viral protein	Phase III
	Selgantolimod (GS9688)	TLR-8 agonist	Phase II
	Rusotolimod (RG7854)	TLR-7 agonist	Phase II
	VIR- 3434	Monoclonal antibody: neutralize or bind the HBV proteins to reduce infection	Phase II
	Burfiralimab (IgG4)	Monoclonal antibody	Phase II
	ASC22 (KN035 or Envafolimab)	Checkpoint inhibitor PDL1 inhibitor	Phase II
HDV	Hepcludex (Bulevirtide formerly Myrcludex B)	Entry inhibitor	EU approved 2023 Phase III in USA	(Wedemeyer et al., 2023)
	REP 2139	HBsAg inhibitor	Phase II	(Da, 2023)
	VIR-2218+VIR 3434	RNAi gene silencer + Monoclonal antibody	Phase II
	Lonafarnib	Prenylation inhibitor	Phase II	(Yurdaydin et al., 2022)
HCV	Sofosbuvir – velpatasvir		Approved	(Wei et al., 2019)

Over the past few decades, there have been notable strides in the treatment of hepatitis C, prompting the World Health Organization (WHO) in 2017 to establish goals for the eradication of HCV by 2030. For more than two decades, chronic HCV infection has been addressed using IFN, with PEGylation (PEG-IFN) enabling a reduction in the frequency of subcutaneous injections from three times a week to once a week. While the combination of PEG-IFN with ribavirin enhanced treatment effectiveness, it was poorly tolerated and resulted in a cure rate of no more than 50% over 24 to 48 weeks. The introduction of the first polymerase inhibitor (sofosbuvir) and the initial inhibitor of nonstructural protein (NS) 5A (daclatasvir) marked a transformative phase in the history of hepatitis C treatment, characterized by excellent tolerance and a cure rate of around 95%. Direct-acting antivirals emerged as highly effective agents, demonstrating efficacy across genotypes and a high resistance barrier, revolutionizing HCV treatment. Various combinations of direct-acting antivirals with broad efficacy have led to high sustained virologic response (SVR) rates, outstanding safety, and good tolerance, even among patients with advanced fibrosis and cirrhosis. Despite the availability of effective treatments, HCV continues to pose a public health threat. Factors contributing to this include disparities between the effectiveness observed in clinical trials and real-life settings, along with challenges such as low disease awareness, absence of screening programs, gaps in follow-up within health services, and a high rate of reinfection in specific populations.

HDV is an incomplete virus that requires presence of HBV to infect and progress. Despite being incomplete virus, it can cause severe progressive hepatitis (Koh et al., 2019). Compared to HBV infected patients, HDV/HBV coinfected patients have a risk of HCC to 3 times higher and liver decompensation up to 2 times higher (Lampertico et al., 2017). Presently, new therapeutic possibilities that focus on disrupting the life cycle of HDV are undergoing clinical assessment. Medications like bulevirtide, telafarnibe, and REP3702139 are aimed at targeting HDV cell entry, replication, and viral assembly and release, respectively. Among these agents, bulevirtide (previously known as Myrcludex-B) received conditional marketing authorization from the European Medicines Agency in 2020 under the trade name Hepcludex^® (Mentha et al., 2019). There is no prescribed therapy for acute HEV infections, which typically resolve on their own through spontaneous clearance of the virus. However, administering early treatment for acute hepatitis E could potentially abbreviate the illness duration and diminish overall morbidity. In instances of severe acute hepatitis E or acute-on-chronic liver failure, the consideration of ribavirin treatment may be warranted. In isolated cases of acute liver failure, corticosteroids have been employed, resulting in improved liver function parameters (González Grande et al., 2021). Glucocorticoids are also usually prescribed in the treatment of autoimmune hepatitis (Liberal et al., 2013).

4. Nanoparticle-based Hepatic Drug Delivery

Nanoparticle-based drug delivery systems have emerged as a promising approach to overcome the limitations of conventional drug delivery methods. These systems enhance the therapeutic efficacy and reduce the side effects of drugs by improving their solubility, stability, circulation time, and targeting capabilities (Blanco et al., 2015; Parveen et al., 2012). Nanoparticles can be designed to selectively target specific cell types within the liver, allowing for enhanced therapeutic efficacy (Wang et al., 2020). Some of the recently reported work in this area is described below.

4.1. Types of Nanoparticles used for Hepatic Drug Delivery

Several types of nanoparticles have been explored for hepatic drug delivery, including liposomes, polymeric nanoparticles, nanostructured lipid carriers, and inorganic nanoparticles.

Liposomes, which are self-assembled vesicles composed of phospholipid bilayers, have been extensively studied for liver-targeted drug delivery (Gao et al., 2010). Commonly used lipids reported in recent literature for liposomal drug delivery to the liver include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), soy lecithin such as lipoid S100 and dipalmitoyl phosphatidylcholine (DPPC). Modification with targeting moieties or stealth polymers, such as polyethylene glycol (PEG), can improve the specificity and biodistribution of liposomes in the liver (Bartneck et al., 2015; Jagwani et al., 2020; Liu et al., 2020; Wu et al., 2019; Jinhang Zhang et al., 2020). Polymeric nanoparticles, typically composed of biodegradable polymers, offer controlled drug release, enhanced stability, and targeted delivery to specific liver cells (Lu et al., 2008). For instance, a pH sensitive poly (methacrylic acid-co-methyl methacrylate) copolymer-based nanoparticles was used to encapsulate and deliver cyclosporine A (CsA), an antiviral agent, to gastrointestinal tract and liver. The nanoparticles were made using an adaptation of the quasi-emulsion solvent diffusion technique. (Dai et al., 2004). In another study, PEGylated poly (lactic-co-glycolic) acid (PLGA) nanoparticles encapsulating CsA were used as a treatment for HCV. The targeted delivery of CsA to liver was achieved by conjugating liver-targeting peptide. This peptide targeted the liver by binding to heparin sulfate proteoglycans on hepatocyte surfaces using the conserved region I of Plasmodium sporozoites’ circumsporozoite protein. The nanoparticle system showed the advantage of reducing the toxic side effect of free CsA and liver specific CsA nanoparticles showed sustained and prolonged anti-HCV effect (Jyothi et al., 2015). Chitosan extracted from crustacean wastes has also been explored as polymer for nanoparticle synthesis as chitosan has mucoadhesive properties and has demonstrated lipid-lowering effects in the liver (Liang et al., 2018; Sumiyoshi and Kimura, 2006). Polylactic acid (PLA), PEG, and combinations of multiple polymers have also been used (El-Naggar et al., 2019). Further, siRNA loaded dextran-based nanoparticles (Foerster et al., 2016), paclitaxel loaded poly (γ-glutamic acid)-poly (lactide) nanoparticles (Liang et al., 2006), and salvianolic acid B loaded mesoporous silica nanoparticles (He et al., 2010) have been studied as liver-specific drug target therapies. Silymarin coated gold nanoparticles have been studied as a potential candidate to treat liver inflammation and cirrhosis due to its hepatoprotective effect (Abdullah et al., 2021; Clichici et al., 2020; Kabir et al., 2014). Abdullah et al. developed a method of treating hepatic fibrosis using silymarin-coated gold nanoparticles. This nanoparticle system enhances the expression of protective miRNAs, namely miR-22, miR-29c, and miR-219a in the liver, which in turn suppresses the expression of key fibrosis mediators - TGFβR1, COL3A1, and TGFβR2, respectively, resulting in antifibrotic effects (Abdullah et al., 2021). He et al. recently synthesized a novel reduction-sensitive amphiphilic polymer by polymerizing isocyanate and bis(2-hydroxyethyl)-disulfide and then adding hydrophilic PEG. Nanoparticles synthesized using this polymer were responsive to reductive agents like glutathione in the liver tumor environment, for cancer-specific drug release (He et al., 2020). Nanostructured Lipid Carriers (NLCs) are composed of a mixture of solid and liquid lipids, which have shown promising results in improving hepatic drug delivery (Beloqui et al., 2014). NLCs offer advantages such as enhanced drug loading capacity, controlled drug release, and increased stability compared to liposomes. Inorganic nanoparticles, such as mesoporous silica nanoparticles (MSNs), have been investigated for hepatic drug delivery. MSNs can be functionalized with various surface modifications, such as polyethylenimine (PEI) and fusogenic peptides, to enhance their stability, biocompatibility, and cellular uptake. Lipid-polymer hybrid nanoformulations taking advantage of the sustained release properties of polymers and excellent biocompatibility of lipids have also been developed and studied for liver drug delivery (Abdou et al., 2019; Liang et al., 2018). In addition to these formulations, bio-inspired, bioengineered, and biomimetic drug delivery carriers have been developed to address some challenges associated with nanoparticle-based hepatic drug delivery (Yoo et al., 2011). These carriers incorporate biological components to enhance biocompatibility, targeting, and controlled release of drugs in the liver.

Nanoparticle-based hepatic drug delivery systems offer several advantages over conventional liver-targeted therapies. Surface modification of these systems to specifically target different types of liver cells will enhance their efficacy and potentially prevent the progression of chronic liver inflammation to advanced-stage diseases. However, research in the area of nanoparticle-based targeted hepatic drug delivery have been very few compared to other organs and inflammatory diseases. In the sub-sections below, we will summarize recent work on targeting specific liver cell types for drug delivery and potential avenues for future research. Figure 4 represents a typical nanoparticle-based drug delivery system.

Figure 4.

Composition of nanoparticle-based drug delivery vehicles; drugs/therapeutic agents can be delivered to the specific target using surface modifications to the vehicle.

4.2. Hepatocyte-targeted Drug Delivery

Given their central role in many liver diseases, hepatocytes have become a primary target for nanoparticle-based drug delivery systems. However, effectively targeting injured and inflamed hepatocytes with drugs or nanoparticles is challenging due to their swift sequestration by macrophages and KCs, posing difficulties in achieving uptake and retention in hepatocytes (Zhang et al., 2016). In a recent investigation, a hepatocyte-targeting glycodendrimer (GAL-24) was systematically designed and developed, utilizing biocompatible building box and a facile chemical process. GAL-24 was specifically engineered to naturally target the ASGPR on hepatocytes, resulting in notable accumulation in the liver—reaching 20% of the injected dose—just one hour after systemic administration (Sharma et al., 2021). ASGPR is primarily expressed on hepatocytes and minimally on other hepatic cells and exhibits high affinity for carbohydrates, specifically galactose, N-acetylgalactosamine and glucose (D’Souza and Devarajan, 2015). Therefore, it is most frequently used in hepatocyte-targeting drug delivery applications. Several studies focused on ASGPR using different ligands conjugated to PLGA NPs, such as bis(1-O-ethyl-β-D-galactopyranosyl)amine ligand (Raposo et al., 2020) and lactosaminated-human serum albumin peptide (Dhoke et al., 2018). Witzigmann et al reported the development of PEGylated liposomal nanoparticles surface modified with targeting ligands isolated from hepatitis B virus large envelope protein in order to target the sodium-taurocholate cotransporting polypeptide present on hepatocyte cell surface. The particles demonstrated excellent cytocompatibility and greater uptake in liver derived HepG2 cells than in the non-hepatic HeLa cells in vitro. The particles were radiolabeled using Indium-111 and fluorescently tagged, and were observed in the livers of NMRI mice (Witzigmann et al., 2019). In a more recent study, Carmignani et al. reported using polydopamine NPs (PDNPs) to treat MASLD in vitro. The MASLD model was established in an oleic acid treated HepG2 model. The PDNP treatment effectively reduced lipid accumulation, TCs, TCT, and oxidative stress levels in an in vitro model of hepatic steatosis, highlighting the efficacy of polydopamine as an antioxidant and anti-inflammatory compound (Carmignani et al., 2024).

Targeting hepatocytes for treating HCC is another area of interest in recent years. Wei et al. developed oleanolic acid loaded galactosylated chitosan-modified liposomes to target ASGPR (Wei et al., 2023). They reported enhanced anti-tumor efficacy because of the hepatocyte-specific targeting of the liposomes. The efficacy of the ASGPR targeted drug delivery may be hampered under certain conditions, such as liver cirrhosis, where inhibitors in the bloodstream suppress the binding ability of the ASGPR. It has been reported that protein kinase c (PKC) α expressed in liver cancer cells (hepatocytes) has the ability to bind with 18β-Glycyrrhetinic acid (GA), a pentacyclic triterpene acid extracted from traditional Chinese herb licorice (Singh et al., 2018).

4.3. Kupffer cell-targeted Drug Delivery

Due to KCs’ critical role in the initiation and resolution of liver inflammation, targeting them can be useful for treating liver diseases such as MASH and liver fibrosis. Nanoparticles accumulating in the liver tend to be taken up by KCs. Strategies for KC-targeted drug delivery include using nanoparticles functionalized with ligands that bind to KC-specific receptors, such as CD163 or mannose receptors (Yu et al., 2020). Among these, mannose receptors are most commonly used for targeting. Melgert et al. developed a dexamethasone coupled-mannosylated albumin therapeutic system that accumulated almost exclusively within KCs for treatment of inflammation. Although the dexamethasone delivery in this manner unexpectedly resulted in enhanced collagen I and III deposition in the liver of rats, a reduction of reactive oxygen species (ROS) was also reported (Melgert et al., 2001). Inhibition of KC overactivation using benzyl isothiocyanate (H.-W. Chen et al., 2020) and regulating polarization of M1 and M2 KCs have been explored as a potential treatment for MASH and MASLD (J. Chen et al., 2020). In another study, type-I IFN was delivered to mannose receptors on KCs due to its anti-inflammatory and immunomodulatory effects. This was achieved by using an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, attached by combining albumin fusion technology and site-directed mutagenesis. The inhibition of liver injury in Concanavalin A-induced hepatitis mice model showed the efficacy of the treatment while increasing the survival rate (Minayoshi et al., 2018). Similarly, Maeda et al. developed a polythiolated and mannosylated human serum albumin (SH-Man-HSA)-based nano-antioxidant to target mannose receptors on KCs for MASH treatment. Upon co-delivery with a nitric oxide donor which increased hepatic blood flow and mannose receptor expression, the formulation was observed to have a hepatoprotective effect in the methionine/choline-deficient (MCD) model of MASH mice by suppressing ROS production by KCs (Maeda et al., 2022). TLRs and Fc receptors are also used for KC targeting (Colino et al., 2020). For example, the FcγRIIIa (or CD16a) receptor is low affinity receptor present on macrophages like KCs and have been considered for targeting purposes (LI et al., 2016).

In a recent study, Yu and coworkers identified vesicle like NPs (VLNs) as a new component in honey. These honey VLNs (H-VLNs) targets the leucine-rich repeat related family, pyrin domain containing 3 (NLRP3) inflammasome in primary macrophages (KCs). H-VLNs not only have an anti-inflammasome function but also inhibit the transcriptional activities of C-JUN and NF-κB. The microRNAs, miR5119 and miR5108, as well as the phenolic compound luteolin in H-VLNs, are found to suppress both the C-JUN and NF-κB pathways. This investigation mainly focused on analyzing the efficacy of H-VLNs administered through orally in aged (11.5 months) C57BL/6J mice model. These H-VLNs were discovered to shield the livers of aging mice from chronic inflammation, while also hindering the progression of fibrosis and nodule formation. KCs in the liver predominantly uptake H-VLNs, leading to the suppression of the NLRP3 inflammasome, C-JUN, and NF-κB pathways (Liu et al., 2024).

4.4. HSC-targeted Drug Delivery

HSCs are a major non-parenchymal cell type in the liver that play a central role in liver fibrosis. Targeting HSCs can help reduce the production of extracellular matrix proteins and prevent the progression of liver fibrosis (Friedman, 2008). Strategies for HSC-targeted drug delivery include using nanoparticles functionalized with ligands that bind to receptors on the surface of HSCs, such as the PDGF receptor, integrin α_vβ₃ receptor, mannose-6- phosphate/insulin-like growth factor II (M6P/IGF II) receptor(Hu et al., 2019; Zhao et al., 2014), and retinol bunding protein (RBP) receptor. One of the earliest works on HSC targeting was by Beljaars et al., who demonstrated that albumin modified with mannose-6-phosphate (M6P) is taken up by the M6p/IGF II receptor expressed on activated HSC for delivery of antifibrotic agents (Beljaars et al., 1999). Greater molar ratios of M6P to albumin led to greater accumulation in the HSCs of fibrotic rat livers as well as non-parenchymal cells of cirrhotic human liver slices. Azzam et al. pretreated carbon tetrachloride-treated mouse models of liver fibrosis with collagenase-containing nanoparticles to enhance the penetration of therapeutics into fibrotic livers. They then delivered chitosan nanoparticles modified with PDGF receptor-beta (PDGFR-β)-binding peptides via tail vein injection to enhance interactions with PDGFR-β receptors in HSCs. Collagen binding enhanced the retention of the particles in the fibrotic livers of carbon tetrachloride-treated mice. The collagenase nanoparticle treatment led to greater accumulation of peptide-conjugated chitosan nanoparticles in the mice liver (Azzam et al., 2020).

Since HSCs are responsible for storing vitamin A in the body, coating nanoparticles with vitamin A can help direct them to HSC through interactions with the retinol binding protein (RBP) receptor (Hu et al., 2019). Toriyabe et al. developed lipid nanoparticles loaded with siRNA, which were formulated using SS-cleavable proton-activated lipid-like material (ssPalms) containing vitamin A. The siRNA was designed to knock down the gene expression of type I collagen α−1 (Col1a1), a major ECM substance produced by HSCs. Once these nanoparticles enter HSC, they disassemble due to the breakdown of disulfate bonds in ssPalms by intracellular glutathione (GSH), releasing Col1a1 siRNA for gene silencing. In experiments conducted on mice suffering from liver fibrosis induced by carbon tetrachloride (CCl₄), the administration of vitamin A-modified nanoparticles at a dose of 3.0mg siRNA/kg resulted in significantly decreased collagen production compared to control mice (Toriyabe et al., 2017). In another study, researchers developed vitamin A-coupled liposomes named VA-lip-siRNAgp46, using a freeze-dried empty liposome method. The purpose of these liposomes was to deliver siRNA against gp46, which is a rat homolog of human heat shock protein 47 that has a crucial role in liver fibrosis development. The rats that were given VA-lip-siRNAgp46 survived longer than those who were given control liposomes without vitamin A modification (Sato et al., 2008).

Integrin α_vβ₃ is a cell surface receptor that is commonly found in activated HSC. It recognizes cyclic peptide ligands. Xuan et al. developed core-shell nanoparticles using perfluorooctyl bromide (PFOB) liquid as a core, which was stabilized by a PLGA polymer shell. The nanoparticles were modified using cyclic arginine-glycine-aspartic acid (cRGD) octapeptide. These nanoparticles were tested in vitro using rat HSC-T6 cells and showed promising targeting efficiency towards activated HSC. The nanoparticles were also able to detect integrin α_vβ₃ on activated HSC at different stages of liver fibrosis. As a result, they generated high-quality ultrasound images that were able to distinguish liver fibrosis at different stages (Xuan et al., 2017). Studies have indicated that collagen VI is highly produced by activated HSC and is a potential target for liver fibrosis treatment. Du et al. developed sterically stable liposomes using egg phosphatidylcholine, PEG conjugate and cholesterol to encapsulate IFN-α1b. They used cyclic (Arg-Gly-Asp) (RGD) peptide as a ligand for collagen VI. IFN-α1b is known for its anti-fibrotic properties, reducing the production of collagen I and III in the liver (Du et al., 2007). Hepatocyte growth factor (HGF) can reverse the fibrogenic process by inhibiting the production of extracellular matrix while inducing apoptosis of myofibroblasts (Kim et al., 2005; Matsuda et al., 1995). In another study, Li et al. developed RGD peptides that were combined with maleimide-[poly(ethylene glycol)]-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (MAL-PEG-DOPE) and incorporated into sterically stabilized liposomes to HGF to activated HSC. Liposomes labeled with cyclic RGD peptides improved the efficiency of HGF and were more effective in reducing liver cirrhosis in rats induced by thioacetamide (Li et al., 2008). It has been discovered that the IGF II receptor is capable of recognizing M6P (Adrian et al., 2007). In a study conducted by Li et al., they used a desolvation method to create M6P-modified neoglycoprotein-based nanoparticles. They mixed sodium ferulate (SF), a potential treatment for liver fibrosis, with M6P-conjugated bovine serum albumin (BSA) and then added 95% ethanol to form SF-loaded M6P-modified nanoparticles. The study found that when delivered in M6P-modified nanoparticles at an SF dose of 1.0 mg/kg, SF accumulation significantly higher in the liver than in other tissues in mice with BDL-induced liver fibrosis compared to free SF (Li et al., 2009).

4.5. Other Liver Cells Targeted via Nanoparticles

Among other liver cell types, LSECs have been studied the most for targeted drug delivery. LSECs play a crucial role in immunomodulation through their involvement in antigen presentation and innate immunity. Nanoparticles decorated with mannose moieties have proven effective in targeting LSECs. This functionalization enables the nanoparticles to bind to the mannose receptors expressed by LSECs. Studies have shown that linking mannose to lipid nanoparticles (LNPs) causes a redirection of mRNA delivery from hepatocytes to LSECs, offering utility in gene editing applications. By adjusting the molecular composition of nanoparticles, such as changing the pKa of LNPs or modifying the hydrocarbon side chain of cholesterol, the delivery focus can be shifted from hepatocytes to LSECs (Dilliard and Siegwart, 2023). Shifting the pKa from 6.4, which is optimal for hepatocyte delivery, to approximately 7.1 resulted in a change in the distribution of LNPs and gene silencing efficacy from hepatocytes to LSECs. In a recent study, simvastatin loaded functionalized polymeric micelles were developed to target the LSEC by coupling peptides ligands of LSEC membrane receptors CD32b, CD36, and ITGB3. These functionalized micelles with CD32b enhanced the efficacy of simvastatin, reduced portal pressure, and reduced liver fibrosis in a non-decompensate mouse model (Gil et al., 2023). Yu et al. developed 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based lipid nanoparticles containing α-melittin for targeting and activating LSECs in liver metastasis. The activated LSECs modified the cytokine/ chemokine environment in the liver, thereby resisting the formation of metastatic lesions and dramatically improving survival rates in the Actb-EGFP C57BL/6 transgenic mice with enhanced green fluorescent protein (EGFP) fluorescence (Yu et al., 2019). Site-specific targeting of neoplastic hepatocytes without affecting hepatocytes using novel aptamer TLS 9a with phosphorothioate backbone modification (L5) functionalized drug nanocarrier (PTX-NPL5) was investigated to treat HCC. The new PTX-NL5 has shown superiority over the other tested experimental ligands and has shown potential to be used as neoplastic hepatocytes targeted therapy for HCC patients (Chakraborty et al., 2020).

4.6. Non-specific targeting of MASLD/MASH via nanoparticles

There are several recent studies that passively target liver or intestine to treat MASLD effectively. Ursodeoxycholic acid (UDCA), a FDA approved bile acid effective for liver diseases. Jo et al developed a poly(ursodeoxycholic acid-oxalate) (PUOX) NPs that contained hydrogen peroxide (H₂O₂)- scavenging peroxide ester bonds, which released UDCA in a H₂O₂-triggered manner. This NP system showed concurrent antioxidant and anti-inflammatory effects. Two mouse models were created to replicate acute liver injury due to APAP overdose and MASLD model by giving methionine/choline deficient (MCD) diet. In acute liver injury model, a single dose of PUOX NPs was administered intravenously to increase rapid therapeutic action, while MASLD model mice were given multiple oral administration of PUOX NPs for 2 or 4 weeks. In both models, PUOX NPs significantly reduced the ALT levels, the levels of proinflammatory cytokines such as TNF-α and IL-1β, highlighting the potential use of PUOX NPs for ROS-associated liver diseases such as MASLD and liver failure (Jo et al., 2024). MASH involves in oxidative stress caused by overproduction ROS, but small molecule antioxidants have not been approved because of the severe adverse effects that collapse redox homeostasis even in healthy tissues. Therefore, recently, poly(ethylene glycol)-block-poly(cystine) (PEG-block-PCys)-based self-assembling polymer NPs (Nano^cyses) were developed to overcome this disadvantage. Cystine (Cys) released through in vivo degradation by endogenous enzymes to obtain antioxidant effect. In this study, Kaga and Nagasaki further modified poly(L-cysteine) (PCys) chains, where sulfanyl groups were protected by tert-butyl thiol (StBu) and butyryl (Bu) groups to change the reactivity of side chains. These Nano^cys were administered to choline-deficient and L-amino acid-defined high-fat diet (CDAHFD)-induced MASH mouse model via free drinking water. The results showed that Nano^cys significantly suppressed oxidative markers of cell membrane and intracellular protein, such as hepatic malondialdehyde (MDA) and protein carbonyls. Further, release profile of the Cys showed that Cys release is chain length-dependent, where shorter PCys chains released Cys faster than longer ones. Nano^cyses also suppressed intraflammatory cytokines and chemokines such s IL-1β and CCL2 (Koda and Nagasaki, 2024).

The gut microbiota has a significant impact on the development of MASH, although the specific mechanism is not yet fully understood. Studies have shown that the transcription factor XBP1s is involved in regulating inflammation and lipid metabolism, as well as maintaining the integrity of the intestinal barrier. Zhu et al developed XBP1 targeting siRNA loaded folic acid modified TPGS nano-micelles (FT@XBP1). MASH model was developed in mice by giving a fat, fructose, cholesterol (FFC) rich diet. The mice fed with the FFC diet consistently showed higher levels of ER stress-related proteins, including GRP78, PERK, ATF6, IRE1α, and XBP1s. In this study, FT@XBP1 treatment not only significantly inhibited liver lipid accumulation and collagen deposition in the FFC diet-fed mice but also improved high-fat-induced intestinal barrier dysfunction. This was demonstrated by the restoration of the integrity of the intestinal barrier and increased levels of ZO-1 protein expression, which partially restored intestinal flora structure, reduced endotoxemia, and alleviated liver inflammation. This study showed that the inhibition of XBP1s by FT@XBP1 could restore the integrity of the intestinal mucosal barrier and intestinal flora disturbance, inhibit LPS translocation, and subsequently reduce liver inflammation and lipid deposition to prevent MASH progression (Zhu et al., 2024). In another study, nitidine chloride (NC), a potent anti-inflammatory drug, was encapsulated into a chitosan-pectin NP system (NC-CS/PT-NPs) to target gut microbiota. The MASLD model was developed in high fat diet (HFD)-fed mice. The results showed that NPs treated mice showed decreased AST, ALT, and lipid levels (Lu et al., 2024). Recent studies have highlighted the importance of gut microbiota in treating MASLD/MASH. More research needs to be done to understand the role of gut microbiota in the progression of MASLD/MASH. Targeting the intestine and colon to treat MASLD could be another potential approach.

In summary, nanoparticle-based drug delivery systems offer a promising approach for targeted drug delivery in the treatment of chronic liver inflammation. The choice of nanoparticle type and targeting strategy should be carefully considered based on the specific liver disease and cell type being targeted. Continued research in this field holds great potential for the development of more effective and targeted therapies for chronic liver inflammation. Some of the notable nanoparticle-based drug delivery studies in addition to this section are summarized in Table 2, highlighting targeting ligand, applications and major findings.

Table 2.

Summary of additional nanoparticle-based drug delivery systems, their targeting ligands, delivered drug, possible applications, and major finding of the study.

Polymer	Drug/molecules	Main targeting ligand/cells	Applications	Major findings	Refer ences
Maleimide modified PLGA	Lamivudine	ASGPR	Can terminate viral DNA synthesis of HBV and HIV-1 infection. reduce the liver damage	Lactosaminated-HAS conjugated NPs were able to target hepatocytes/liver; 2.17-fold increase in cell uptake and 3.84-fold increase in retention in HepG2 cells.	(Dho ke et al., 2018)
Mesoporou s silica NPs coated with chitosan	Lysine	Glycyrrhi zin (GL) receptor	Target hepatocytes cells for liver regeneration and deliver anti-inflammatorydrugs.	Glycyrrhizin-conjugated, chitosan coated, lysine embedded mesoporous silica NPs were successfully established as a novel therapeutic system for liver regeneration	(Hou et al., 2020)
Catalytic NPs containing enzyme in Hyaluronic acid (HA) networks	Superoxide dismutase (SOD)	HA receptor (CD 44)	Target hepatocytes and effectively remove the ROS in the liver due to drug induced toxicity	Ultra-small calcium phosphate NPs were crystalized and decorated on the surface of the NPs for the efficient endosomal escape after cellular uptake. Can use different cationic, anionic, and neutral polymers to target different tissues/organs	(Lee et al., 2018)
Polyethylen e sebacate NPs	Doxorubici n	ASGPR	Target hepatocytes and a treatment for HCC	ASGPR ligand, pullulan (Pul), arabinogalactan (AGn), and Combination of Pul-AGn were attached by adsorption. AGn created slight renal toxicity in mice. Pul NPs successfully reduced the tumor volume, collagen content, and HCC biomarkers in mice.	(Pran athart hihar an et al., 2017)
RNA NPs	Paclitaxel/miR122	ASGPR	Target hepatocytes and cancer cells. Co-delivery of paclitaxel and miR122. Can be used in other liver inflammation treatments.	The RNA NPs contain3 copies of hepatocytes targeting ligands, one copy of miR122, and 24 copies of paclitaxel. MiR122 downregulated the tumor oncogenic factor and the drug efflux transporter. RNA NPs accumulated in HCC tumor and effectively inhibited tumor growth after 22 days of IV administration	(H. Wang et al., 2021)
α-melittin peptide-NPs	Mellitin containing RXR or RXXR and RKR sequences	LSECs	RKR sequence in mellitin targets and modulates LSECs to become antigen presenting cells (APCs). Can further investigate the possibilities of using α-mellitin NPs to break LSEC-mediated immunogenic tolerance.	α-melittin-NPs mainly targeted the LSECs in vivo via mellitin peptide sequence. Intravenous administration of NPs effectively elicited LSEC activation and reversed the immune microenvironment. Inhibited liver metastasis through generating protective T-cell immunity via coordination with NK cells. Showed 80% survival rate in the spontaneous liver metastatic tumor model (mouse).	(Yu et al., 2019)
Small interfering RNA (siRNA) loaded stable nucleic acid Lipid NPs (SNALP)	siRNA, pPB peptide (C*SRNLI DC*), high mobility group box-1 (HMGB1) protein or heat shock protein 47 (HSP47)	Activated HSC	Can be used in cirrhosis treatment due to anti-inflammatory and antifibrotic effect of the NP system	pPB peptide mediation enables targeted delivery of siRNA loaded SNAP to HSCs in vitro and in vivo. HSP47-siRNA@SNAP-pPB produced only antifibrotic activity. HMGB1-siRNA@SNAP-pPB produced better therapeutic effect due to dual antifibrotic and anti-inflammatory activity.	(Jinfa ng Zhan g et al., 2020)
Liposomes	vsmodegib (VIS)	Integrin αvβ3	Target the HSCs effectively and reduce the liver fibrosis. Can be used to deliver different drugs/therapeutic agents to target HSCs.	The cyclic peptide (cRGDyK) used to target αvβ3 in HSCs. VIS loaded cRGDyK NPs effectively inhibit the Hedgehog (Hh) pathway, which plays an active role in activation of HSCs and fibrosis. Alleviates liver fibrosis.	(Li et al., 2019)
Peptide NPs	Riociguat (guanylate cyclase simulator), JQ1 (anti-fibrosis agent)	Activated HSCs, LSECs	Riocigyat reverses the capillarization of LSECs due to liver fibrosis, which prevent substance exchange between blood and Disse space. Potential treatment for chronic liver fibrosis	Riocigyat facilitates JQ1-peptide-NP delivery to activated HSCs and thereby inhibit the proliferation of HSCs and decrease the collagen deposition in the liver. This system exhibited excellent anti-fibrosis efficacy in MASH induced mouse models.	(Li et al., 2023)
Mannan modified PLGA NPs	Simvastatin	Mannose receptor on LSECs	Simvastatin converts activated HSCs to quiescent state by stimulating kruppel-like factor (KLF2)-nitric oxide (NO) pathway and up-regulate the expression of Chemokine (C-X-C motif) ligand 16 (CXCL16) on LSECs to reverse capillarization.	Mannan was selected to target mannose receptor on LSECs. Simvastatin did not show any obvious toxicity. CXCL16 expression on LSECs induced by simvastatin attracted CXC6+ NKT cells in the liver to inhibit tumor growth. Simvastatin notably reduced the tumor growth in fibrotic HCC mouse model (BL57C/6). The lower dose administered through tail vein injection showed higher efficiency than oral administration of free simvastatin.	(Yu et al., 2022)
PEGylated and thiolated hollow mesoporou s silica NPs	Obeticholic acid (OCA), ferroptosis inhibitor liproxsatin-1, NO donor S-nitrosothiol	Farnesoid X receptor (FXR)/hepatocyt es	OCA, an agonist of the FXR, has ability to reduce liver fibrosis and inflammation, though recently failed in phase III trials. Combining OCA with ferroptosis blocker and NO donor, NO provides protection against oxidative stress-associated pathology in MASH, can significantly improve the ability to resolve liver fibrosis and prevent cell death	Ferroptosis is a newly recognized of ion-dependent regulated form of a cell death due to uncontrolled lipid peroxidation. This cell death is considered as a critical cause of hepatocytes death and inflammation in NASH. This multifunctional NP system exhibited potent therapeutic action against MCD diet-fed MASH mouse model. The NP system improved gut microbiota dysbiosis in MASH mice. This study demonstrated that a synergistic approach targeting FXR, ferroptosis, and fibrosis simultaneously could be an effective therapeutic strategy.	(Fu et al., 2024)
Hyaluronic acid-bilirubin NPs (HABNs)	Bilirubin	CD44 receptor on activated HSCs	The NP system can successfully inhibit the ROS/oxidative stress activity in activated HSCs, thereby inhibits the HSC proliferation, ECM production and fibrosis.	Bilirubin has antioxidant and anti-inflammatory properties. Intravenously administered HABNs effectively targeted activated HSCs in liver, particularly in a CDAFD diet-induced MASH murine model. Murine model showed that HABNs were able to inhibit HSC activation, proliferation and collagen production.	(Shin n et al., 2024)
Palmitic acid-modified albumin (PSA) NPs	Sorafenib	CD44 receptor on activated HSCs and SR-A receptor on macropha ges	The NP system can target HSCs and KCs to effectively deliver the drugs. Sorafenib can be used to treat liver cirrhosis effectively.	Chondroitin sulfate(CS), a natural polysaccharide, has affinity for the CD44 receptor on HSCs and scavenger receptor A (SR-A) on activated macrophages (KCs) PSA NPs were modified by surface adsorption of CS. CS-PSA NPs effectively targeted and suppressed HSCs and macrophages, reducing the expression of α-SMA and inhibiting secretion of proinflammatory and pro-fibrotic factors.	(Tan et al., 2024)
Mesoporou s silica NPs	siRNA	ASGPR receptor in Hepatocy tes	The biomimetic siRNA delivery system was used to target proprotein convertase subtilisin kexin type 9 (PCSK9) gene in a high-fat diet induced NAFLD/MASLD mouse model	Hepatocytes targeting was achieved through integrating triantennary N-aceetylgalactosaine (GalNAc)-engineered cell membrane with silica NPs. Exhibited strong hepatocytes targeting and KC escaping. Targeting efficiency can be tailored through GalNAc-engineering strategy, insertion order, and cell membrane sources. Reduced both serum and liver LDL-C, TG, and TC levels.	(He et al., 2024)

5. Conclusions and Future Perspectives

The prevalence of CLDs is on the rise, accounting for about 2 million annual deaths (1 out of every 25 deaths) worldwide (Devarbhavi et al., 2023). Viral hepatitis, alcoholic liver disease and MASLD have been identified as the key disease conditions that progress to cirrhosis; most liver disease-related deaths have been attributed to complications from cirrhosis and HCC. The goal of the WHO’s global hepatitis strategy is to reduce new viral hepatitis-related cases and deaths by 90% and 65% respectively between 2016–2030 (Devarbhavi et al., 2023; “Hepatitis,” 2024). October 2023 was designated as National Liver Awareness Month in the United States to encourage people to determine whether they are at risk of liver disease. These initiatives underline the recognition of CLDs as a major contributor to global morbidity and mortality. Issues relating to non-specificity and toxicity of developed therapeutics against CLDs can be addressed, in part, by encapsulating them within biocompatible and biodegradable nanoparticle formulations that can deliver medications in a targeted manner to specific cells in the liver. Due to the central role of the liver in metabolism and clearance, nanoparticles tend to accumulate in the liver following administration. However, in the absence of a targeting component, the nanoparticles may not accumulate in the cells of interest, which may reduce efficacy. Different types of including liposomes, polymeric nanoparticles, NLCs, and inorganic nanoparticles have been explored for hepatic drug delivery. Liposomes and protein micelles, due to their biocompatibility and low toxicity, are the most extensively researched in this area (Akbarzadeh et al., 2013; He et al., 2019; Mustafai et al., 2023). However, particle stability and optimal drug encapsulation and release kinetics remain barriers to effective drug delivery. Among approaches used to improve drug encapsulation, freeze-thaw cycling where liposomes are permeabilized by freezing with liquid nitrogen followed by thawing at 37°C, has been studied. Cholesterol incorporation imparts stability and improves fluidity in liposomal formulations (Nsairat et al., 2022; Pisani et al., 2023). Another challenge for hepatocyte-targeted liposomal drug delivery is the opsonization and clearance of liposomes arriving in the liver by KCss, LSECs and other members of the reticuloendothelial system. This can be evaded by PEGylation of the liposomes (Warner et al., 2022). Controlled drug delivery can be achieved using sophisticated design strategies such as the use of multi-functional nanoparticles incorporating polymers that can respond to specific stimuli in the target cells (e.g. pH, enzymes) or the development of intelligent drug delivery systems that can sense and adapt to the changes in the body. For sustained drug release, lipid-polymer hybrid formulations have also been developed and studied. These formulations combine the non-immunogenicity and biocompatibility of lipids with the sustained and controlled release properties of polymers, which aids in efficient drug release.

To further enhance the efficacy of nanoparticle-based drug delivery carriers, their surfaces can be modified to incorporate targeting ligands that are highly specific towards biomarkers upregulated by specific populations of liver cells in various disease conditions. Biomarker targeting ensures retention of the particles at the target site and enhances internalization of the nanoparticles by the cells of interest. However, the occurrence of several complex underlying mechanisms in CLDs, many of which are still poorly understood, makes the identification of therapeutic targets and receptors for therapy difficult. Some of the commonly targeted biomarkers on liver parenchymal and non-parenchymal cells for drug delivery are discussed herein. While identifying biomarkers for targeted drug delivery, sex-specific differences must also be considered. Reports show that MASLD prevalence is greater in males than females, however females have greater tendency to develop MASH and advanced fibrosis from MASLD (Nagral et al., 2022; Yuan et al., 2019). Women also tend to present with more severe forms of cirrhosis and alcoholic hepatitis than men following excessive alcohol intake (Saunders et al., 1981). Therefore, identifying unique biomarkers responsible for CLD progression in female patients would be advantageous for delivering targeted therapies.

Targeting to specific cell types can also be improved by developing nanoparticles that can overcome biological barriers known to hinder drug delivery. Particle diameters are a key factor governing deposition of the particles in different parts of the liver. Nanoparticles with a diameter of 150 nm or below can evade uptake by KCs and pass through the sinusoids via the fenestrations along the endothelial barrier, for accumulation in the hepatocytes. Smaller particles (50 nm or below) can accumulate deeper in the tissue in the space of disse, which is the perisinusoidal space (Mishra et al., 2013). The biological barriers to be overcome varies depending on the route of administration. For example, if oral administration is used, the formulations will need to be protected from enzymatic degradation and acid hydrolysis and must be able to pass through the gastrointestinal membrane to arrive at the liver (Moosavian et al., 2021).

Another challenge to the development and testing of drug delivery formulations is the lack of predictive preclinical models that can accurately recapitulate key mechanisms and cell-cell interactions seen in CLDs. Although murine models are widely used to study new therapeutics, the observations from these studies often do not translate to efficacy in human trials. Research carried out recently in liver-chips using primary hepatocyte cultures predicted toxicities and responses to therapies that were not detected using in vivo preclinical models, pointing towards the increasing relevance of these liver-chips in research (Jang et al., 2019). Cost, efficacy and precise control of various parameters (e.g., flow, cell arrangements, mechanical stresses) must be considered while developing tools for preclinical testing of new therapeutics and drug delivery strategies. They must also aid in high-throughput generation of human relevant data. Finally, manufacturing and regulatory challenges must be accounted for, for clinical translation of nanoparticle-based medicines. Scale-up of the formulations with negligible batch-to-batch variations can be technically challenging and costly. Moreover, the regulatory pathways for approval of nanoparticle-based medicines are still evolving, which can delay their introduction to the market. It is recommended to foster close collaborations between researchers, manufacturers, and regulatory agencies. This could help to streamline the production process, ensure the consistent quality of nanoparticles, and facilitate the regulatory approval process. With continued research and innovation, it is anticipated that nanoparticle-based drug delivery systems will play an increasingly important role in the targeted treatment of liver diseases in the future and contribute towards treating and potentially reversing these conditions.