Nanoparticle-based systems for liver therapy: Overcoming fibrosis and enhancing drug efficacy

Abstract

Liver diseases are among the most insidious and life-threatening conditions due to their progressive nature and late symptom onset. Cirrhosis and hepatocellular carcinoma account for most liver-related deaths, often following the progression from fibrosis. Fibrosis creates a hostile microenvironment, characterized by portal hypertension, vascular capillarization, intrahepatic vasoconstriction, and extracellular matrix deposition, which severely limits drug efficacy. Advances in pharmaceutical science have prompted efforts to develop liver-targeted drug delivery systems to prevent or reduce the progression of fibrosis, a central feature of many liver diseases. Fibrosis often reduces the in vivo efficacy of both approved and experimental drugs, underscoring the need for improved delivery strategies focused on stability, controlled release, and precise targeting. Nanoparticle (NP)-based systems show promise, either by delivering therapeutic agents, or in some cases, by contributing directly to the therapeutic effects. This review summarizes the main types of NPs explored for liver disease treatment, especially those aiming to reverse fibrosis or prevent its progression, a critical therapeutic target in chronic liver diseases. Additionally, it examines gene delivery and ultrasound-guided microbubble strategies, which can be combined with NPs to improve cell-specific targeting and boost therapeutic effects. Together, these approaches have the potential to address current therapeutic challenges and accelerate the development of liver-targeted treatments for clinical application.

Key Words: Liver-specific therapies; Liver fibrosis; Liver cirrhosis; Hepatocellular carcinoma; Nanoparticles; Nanocarriers; Ultrasound-guided delivery; Gene delivery

Core Tip: Liver diseases, including fibrosis and hepatocellular carcinoma, represent major global health challenges with limited treatment options, partly due to drug delivery barriers. Therapeutic efficacy is hindered by factors such as portal hypertension, vascular alterations, and particularly the fibrotic microenvironment. This review summarizes the latest advances in nanoparticle (NP)-based delivery strategies targeting liver fibrosis, focusing on lipid-based, polymeric, and inorganic NPs. It also explores emerging combinatorial technologies including gene delivery platforms and ultrasound-assisted microbubble systems. Finally, it briefly integrates toxicity issues, translational considerations, and artificial intelligence-guided NPs to provide a comprehensive yet focused overview of promising therapeutic avenues.

INTRODUCTION

The human body is equipped with highly specialized, high-performing, and tightly interconnected systems to maintain homeostasis and optimal conditions for life. Among these, the liver plays a central role, carrying out more than 500 essential functions, including its involvement in metabolism and digestion; contribution to the body’s coagulation and defense mechanisms; and a key role in processing toxins, exogenous substances, and waste products. Due to these functions, the liver is constantly exposed to varying concentrations of drugs, chemicals, and other xenobiotics, all of which can potentially cause liver injury. Hundreds of etiologies can lead to hepatic disease, of which the most significant are microbial infections (e.g., hepatitis viruses, cytomegalovirus, Epstein-Barr virus [EBV], and yellow fever virus), metabolic syndrome (e.g., fatty liver disease caused by obesity, hemochromatosis, and Wilson’s disease), exposure to xenobiotics (e.g., alcohol, drugs, and chemicals), hereditary hepatic diseases, autoimmune disorders, and liver malignancies[1].

Liver diseases currently represent one of the most pressing global health concerns, with their burden continuously rising in parallel with the increasing prevalence of metabolic disorders, alcohol misuse, and viral hepatitis[2]. Despite the importance of liver health, a significant portion of the global population tends to underestimate it until inflammation or damage has already occurred. This tendency contributes to alarming statistics regarding liver disease, which represents a major global health concern. Causing more than 2 million deaths annually, liver disease has emerged as a leading contributor to morbidity and mortality worldwide, accounting for approximately 4% of all deaths. Most liver-related deaths result from cirrhosis and its complications or hepatocellular carcinoma (HCC), while acute hepatitis accounts for only a small proportion. Liver diseases currently rank as the eleventh leading cause of death globally, although this number may be underestimated[3]. Recent global projections indicate that deaths from chronic liver disease may exceed 2 million annually, with an increasing trend expected over the next decade[4].

The following are among the most prominent pathological conditions of the liver: (1) Alcohol-associated liver disease (ALD): Alcohol abuse is highly prevalent in wealthier countries and is likely underdiagnosed and underreported in many poorer regions. It remains one of the leading causes of cirrhosis worldwide[5]. ALD often coexists with viral hepatitis, non-alcoholic fatty liver disease (NAFLD), and other liver conditions. Moreover, affected patients are more likely to develop cirrhosis compared to those with other liver disease etiologies[6]; (2) NAFLD: This liver disease represents a continuum of liver abnormalities, ranging from NAFL to non-alcoholic steatohepatitis (NASH). Its course can vary, and in severe cases, may progress to cirrhosis and liver cancer. NASH is the more advanced stage of NAFLD, a chronic inflammatory liver disease characterized by steatosis, lobular inflammation, and hepatocellular injury, including hepatocyte ballooning, along with varying degrees of fibrosis[7]. In Europe and America, NAFLD is the second-leading cause of end-stage liver disease and liver transplantation; however, it is the leading cause in females[8,9]. Moreover, pediatric NAFLD is emerging as a serious concern, with rising incidence among adolescents and young adults due to obesity and insulin resistance[10]; (3) Viral hepatitis: Most reported viral hepatitis cases are caused by hepatitis viruses. However, non-hepatotropic viruses can also lead to hepatitis, including human cytomegalovirus, human adenovirus, human herpesvirus 6, varicella-zoster virus, herpes simplex virus type 1, and EBV[11,12]. Over the past 24 years, the incidence of hepatitis A virus, hepatitis C virus (HCV), and hepatitis E virus has remained relatively stable, whereas hepatitis B virus (HBV) incidence has decreased due to increased vaccinations[13-17]. However, HCV-related liver disease remains a major cause of liver-related mortality in low-resource regions due to limited access to curative therapies[18]; (4) Primary sclerosing cholangitis (PSC): This rare, chronic cholestatic disease is characterized by inflammation of the bile ducts, primarily driven by autoimmune mechanisms. The condition significantly reduces patient survival and quality of life. PSC is more prevalent in industrialized nations, and inflammatory bowel disease represents the most significant risk factor associated with its development[19,20]. PSC is widely recognized as a significant risk factor for cholangiocarcinoma, gallbladder cancer, and colorectal cancer, contributing notably to premature mortality[21,22]; (5) Primary biliary cholangitis: It is a rare autoimmune liver disease whose incidence has increased in recent years, particularly in Western populations. Women constitute the majority of affected individuals[23,24]; (6) Autoimmune hepatitis: This disease is associated with an elevated 10-year risk of developing cancer, including HCC, colorectal cancer, lymphoma, and nonmelanoma skin cancer, compared with age- and sex-matched controls. Additionally, this cancer risk further increases when autoimmune hepatitis is accompanied by cirrhosis[25,26]; (7) Acute liver failure: It can rapidly progress to multiorgan failure, resulting in severe complications. Although a substantial proportion of cases have unknown causes, recognized etiologies include viral hepatitis (e.g., hepatitis E virus) and drug-induced liver injury[27-29]; (8) Fibrosis and cirrhosis: It is estimated that approximately 40% of patients with chronic liver disease will eventually develop liver fibrosis, which can progress to severe liver impairment and failure. Persistent liver injury typically initiates fibrosis, and sustained fibrosis may advance to cirrhosis. Over time, cirrhosis substantially increases the risk of HCC and ultimately mortality. Additionally, tumorigenic nodules may directly originate within fibrotic liver tissue[30,31]. Liver fibrosis not only represents a major pathological hallmark of chronic liver diseases but also acts as a physical and biological barrier to drug delivery, thereby compromising therapeutic efficacy[32]. Following injury, the liver demonstrates a remarkable ability to regenerate through two distinct repair mechanisms: The generation of new hepatic cells and a wound-healing response characterized by the production of extracellular matrix (ECM) proteins. In cases of chronic liver injury, however, sustained wound healing can result in liver fibrosis, creating an abnormal hepatic microenvironment marked by elevated pro-inflammatory factors and continuous activation of hepatic stellate cells (HSCs). During fibrosis, the ECM progressively accumulates components that are typically absent or scarce in healthy liver tissue, including collagen types I, III, and V, elastin, tenascin, and fibronectin. By contrast, the ECM in a healthy liver primarily consists of collagen types IV and VI, laminins, and proteoglycans within the space of Disse, all of which are gradually replaced as fibrosis advances[33-35]. Liver fibrosis is also influenced by disruptions in intercellular communication networks involving both parenchymal and non-parenchymal cells[36,37]. Individuals suffering from decompensated cirrhosis experience a significantly elevated risk of liver-related complications and death[38]. The primary mechanisms leading to hepatic decompensation are thought to include increased portal pressure, bacterial translocation, systemic inflammation, and hyperdynamic circulation. Clinically, decompensation manifests as ascites, variceal bleeding, jaundice, and hepatic encephalopathy, the latter being the most common presentation[39,40]. Cirrhosis is now recognized as a systemic disease due to its pathological impact on multiple organs beyond the liver; and (9) HCC: It constitutes most primary liver cancer cases, which globally ranks as the sixth most diagnosed cancer and the third-leading cause of cancer-related mortality, following lung and colorectal cancers. Among men, liver cancer is the second-leading cause of cancer death. Chronic inflammatory liver diseases, characterized by progressive and persistent inflammation, represent the primary drivers of HCC. Although HBV and HCV have historically been the most common etiologies, NAFLD is now rapidly emerging as a leading cause of HCC in developed countries, particularly among non-cirrhotic patients. Approximately 76% of HCC cases result from chronic HBV and HCV infections. Other contributing factors include NAFLD, aflatoxin B1 exposure, excessive alcohol intake, obesity, type 2 diabetes, and smoking[41,42]. The pathogenesis of HCC is complex and often resistant to conventional therapies. Chemotherapy, even when combined with radiotherapy and utilizing first-line agents, frequently shows limited efficacy. Additionally, these therapeutic approaches are associated with numerous side effects, negatively impacting patient quality of life and normal tissue metabolism. HCC remains a significant clinical challenge, largely due to the late presentation of symptoms during advanced stages, which contributes substantially to the high mortality rate of the disease[3].

LIVER-TARGETED DRUG DELIVERY

Given the liver's pivotal role in xenobiotic metabolism and its exposure to high concentrations of systemic drugs, it presents both an opportunity and a challenge for therapeutic targeting. Liver complications represent some of the most severe diseases worldwide, often presenting significant physiological barriers that limit effective drug targeting, particularly in conditions such as fibrosis, cirrhosis, and HCC. Given the extensive metabolic activity of the liver and its central role in drug metabolism, developing targeted drug delivery strategies specifically for liver diseases is essential[43,44].

Recent advances have emphasized the need to improve therapeutic selectivity and efficacy while minimizing off-target effects in hepatology. Key pathological features, such as sinusoidal capillarization, excessive ECM deposition, and impaired cell communication, act as major barriers to drug delivery. These structural changes limit the distribution of systemically administered agents, highlighting the need for precisely engineered delivery platforms capable of overcoming them[43].

As previously mentioned, liver fibrosis is a common pathological feature shared by many liver diseases and often progresses to cirrhosis. Fibrotic remodeling disrupts liver structure and hinders drug accessibility and efficacy[45]. Consequently, considerable research has focused on identifying innovative strategies for targeted liver drug delivery aimed at preventing or halting the progression of fibrosis[46]. Although early-stage fibrosis may be reversible, no truly effective treatments exist. Current strategies aim to reduce inflammation, preserve liver function, and regulate ECM turnover by suppressing HSC activity. Therapeutic options include rifaximin (gut-liver axis), albumin (circulatory support), statins (anti-inflammatory), and non-selective beta-blockers (portal pressure control).

Portal hypertension, a hallmark of advanced liver fibrosis and cirrhosis, disrupts hepatic perfusion and sinusoidal pressure. As schematized in Figure 1, this condition can increase intestinal permeability and bacterial translocation, reduce liver perfusion, and promote portosystemic shunting, ultimately compromising drug absorption and metabolism, and access to parenchymal target cells (Figure 1)[47].

Figure 1 Effects of portal hypertension on hepatic and intestinal function relevant to drug absorption and metabolism. Portal hypertension contributes to increased intestinal permeability and bacterial translocation, structural alterations in the liver, elevated vascular resistance, and the formation of portosystemic shunts. These interconnected changes impair hepatic perfusion and detoxification capacity, ultimately compromising oral drug absorption and systemic metabolism.

Sex-specific factors are increasingly recognized as critical modulators of hepatic disease progression and therapeutic outcomes. Emerging preclinical data highlight advanced chronic liver disease, and portal hypertension highlights notable differences between males and females in HSC activation, inflammatory responses, and fibrosis development[48]. These differences can significantly influence drug distribution and the efficacy of liver-targeted therapies. Importantly, estrogen signaling appears to exert protective effects against fibrosis progression, whereas testosterone may promote hepatic inflammation and ECM deposition. These findings underscore the importance of incorporating sex as a biological variable in the design and optimization of novel treatments for liver disease.

In advanced fibrosis stages characterized by severe complications such as refractory ascites, therapeutic efforts shift toward improving systemic manifestations through anti-mineralocorticoids and diuretics, often combined with furosemide or torasemide. In cases where viral hepatitis underlies fibrosis progression, novel antiviral agents (e.g., entecavir, Epclusa, Vosevi) have demonstrated clinical efficacy. However, their use remains limited by considerable side effects, toxicity, suboptimal pharmacokinetics, and a lack of liver-specific targeting, issues that similarly affect other anti-fibrotic drugs in clinical use or under investigation[49,50].

Biomarkers such as serum N-terminal propeptide of type III collagen (PIIINP) and liver stiffness measurements are being explored to better define treatment windows and improve therapeutic precision[51]. Additionally, artificial intelligence (AI) is increasingly being leveraged to model ligand-receptor interactions, enhance molecular selectivity, and support treatment stratification.

Although various pharmacological strategies are under investigation, their clinical translation is hampered by inadequate drug bioavailability and insufficient accumulation at fibrotic sites. These limitations underscore the urgent need for precision delivery platforms, especially in early or intermediate fibrosis stages where therapeutic interventions may be most beneficial[43,52].

A large number of antifibrotic agents demonstrate robust effects in vitro but fail to replicate these outcomes in vivo, largely due to non-specific distribution. This often necessitates higher dosing, increasing the risk of off-target toxicity and damage to healthy tissues. To overcome these challenges, several advanced drug delivery systems are under development. These include ligand-functionalized nanoparticles (NPs), stimuli-responsive carriers, and computationally designed constructs aimed at enabling localized release within fibrotic tissue. Such targeted strategies, especially those that facilitate receptor-mediated uptake and transport across the dense fibrotic microenvironment, are also highly relevant in the context of HCC, which frequently arises in fibrotic or cirrhotic livers. Addressing these shared delivery barriers is essential for improving the efficacy of both antifibrotic and antitumor nanomedicines.

Hepatic fibrosis results from complex interactions among HSCs, liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs), all central to disease progression and serving as key targets for precision drug delivery. Targeting HSCs is especially promising due to their major role in collagen and ECM production. Yet, challenges remain, including poor solubility, inefficient delivery, and the difficulty of reaching HSCs in vivo[53]. A distinctive feature of HSCs is their ability to store retinyl esters within cytoplasmic lipid droplets. Under healthy conditions, HSCs maintain a non-proliferative, quiescent phenotype. Upon liver injury, they activate into myofibroblasts, which are contractile, inflammatory, and produce the ECM, driving fibrosis[34]. Activation is triggered by oxidative stress, cytokines, and altered cell signaling, prompting HSCs to secrete cytokines and chemokines that fuel inflammation and influence hepatocytes, KCs, LSECs, and immune cells[54]. The primary therapeutic goal in hepatic fibrosis is to specifically target these activated HSCs and interrupt the fibrogenic cascade. However, since HSCs comprise only 5%-8% of liver cells, achieving specific drug delivery remains a key challenge[55].

A promising approach to enhancing targeted delivery involves ligand-receptor interactions, particularly strategies that exploit molecular alterations in activated HSCs. These cells metabolize vitamin A into retinaldehyde and retinoic acid (RA), positioning RA as both a biomarker and a potential therapeutic target[56]. Among the key fibrogenic mediators, platelet-derived growth factor (PDGF) plays a central role in HSC activation by promoting their migration, proliferation, and ECM production[57]. PDGF signals through PDGFR-α and PDGFR-β, with the latter being markedly overexpressed in activated HSCs, making it a promising target for antifibrotic therapies[58]. PDGFR-β also correlates with liver damage and cirrhosis outcomes[59]. Therapeutic compounds like destruxin A5 and dihydroartemisinin inhibit PDGFR-β signaling and show antifibrotic potential[60]. Genetic models confirm that PDGFR-β modulation affects fibrosis severity: Depletion of PDGFR-β is associated with reduced liver fibrosis, whereas enhanced receptor activation accelerates fibrogenesis. In addition, dual-ligand strategies (e.g., combining PDGFR-β- and integrin-targeting peptides) have improved specificity and accumulation in fibrotic areas[61]. These findings collectively emphasize PDGFR-β as a promising therapeutic target for liver fibrosis. However, despite promising preclinical data, clinical translation is lacking. Innovative strategies, like nanobodies (heavy-chain-only antibodies fragments) engineered to bind PDGFR-β, are under development and warrant further study[62].

LSECs also play pivotal roles in hepatic homeostasis and fibrosis progression. In co-culture models, healthy LSECs maintain HSC quiescence and can even reverse activation via nitric oxide (NO) and vascular endothelial growth factor (VEGF) signaling[63-65]. LSECs represent approximately 15%-20% of total liver cells and form the structural lining of hepatic sinusoids, acting as a selectively permeable barrier that controls the exchange of small molecules between the bloodstream and the space of Disse, where HSCs reside. They also contribute, alongside hepatocytes and HSCs, to liver metabolic functions including protein, lipid, and glucose regulation[65-67].

In physiological conditions, NO (primarily induced by hepatocytes) and HSC-derived VEGF preserve LSEC phenotype. In response to injury, LSECs release angiocrine factors to support regeneration. However, chronic injury activates fibroblast growth factor receptor 1 signaling, shifting their phenotype toward a profibrotic state[66]. Fibrosis leads to LSEC defenestration and capillarization, among the earliest pathological changes, thereby impairing their homeostatic and vasodilatory roles. Reduced NO production increases intrahepatic resistance and contributes to fibrosis progression[64]. Activated HSCs further exacerbate this vicious cycle by depositing excessive ECM in the space of Disse, which aggravates the loss of endothelial fenestrations, impairs LSEC function, and hinders drug penetration[55]. The phenotypic alteration of LSECs during fibrosis is closely associated with the activation of signaling pathways regulated by the Hedgehog gene family[68]. Interventions targeting this axis, such as soluble guanylate cyclase activators or liver X receptor alpha agonists, have shown promise in restoring LSEC phenotype and fenestration integrity in preclinical models[69].

KCs, the liver-resident macrophages, also drive fibrosis. Representing approximately 80% of body macrophages, KCs reside in sinusoids and respond to both systemic and local stimuli. Upon activation, they release cytokines (e.g., transforming growth factor beta 1 [TGF-β1], tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin 1 beta) and chemokines (e.g., C-C motif chemokine ligand 3 [CCL3], CCL5), which activate HSCs and fuel fibrosis[70,71]. KCs also promote oxidative stress and lipid peroxidation, facilitating HSC transdifferentiation into myofibroblasts[72]. This process leads to the loss of intracellular vitamin A droplets in HSCs and the acquisition of a contractile, proliferative, and migratory phenotype[34].

Interestingly, KC-HSC interactions are bidirectional. In vitro and in vivo studies have shown that antifibrotic compounds such as Schisandrin B act via KCs by inhibiting nuclear factor kappa B and p38 mitogen-activated protein kinase and engaging cannabinoid receptor 2, thus reducing their fibrogenic activity[73]. In NASH and alcoholic steatohepatitis models, KCs are major reactive oxygen species (ROS) producers. A novel nano-antioxidant (SH-Man-HSA), targeting KCs via the mannose receptor C-type 1, attenuates fibrosis, inflammation, and apoptosis while enhancing survival[74].

These findings highlight the need for integrated therapeutic strategies that address the cellular cross-talk among HSCs, LSECs, and KCs. Such therapies would greatly benefit from site- and cell-specific drug delivery systems capable of overcoming the structural barriers of the fibrotic liver microenvironment.

NP-BASED DRUG DELIVERY SYSTEMS FOR LIVER TARGETING

The origins of nanomedicine trace back to the 1970s with the conceptualization of nanoscale carriers for anticancer drugs. Since then, the field has evolved rapidly, especially after the United States Food and Drug Administration (FDA) approval of Doxil^® (PEGylated liposomal doxorubicin) in the 1990s, which represented a milestone in clinical nanotechnology[75].

NPs have transformed targeted drug delivery, offering promising strategies for treating liver diseases. Their small size, modifiable surfaces, and capacity to encapsulate diverse therapeutic agents enable site specific accumulation and controlled release. These features improve biocompatibility, bioavailability, and therapeutic precision while reducing systemic toxicity. NPs can navigate the complex hepatic architecture, characterized by fibrotic ECM and sinusoidal capillarization, making them ideal for liver-targeted therapy[76].

NP physicochemical properties, including size, shape, and surface charge, critically affect pharmacokinetics and hepatic interaction[77]. NPs larger than 400 nm are rapidly cleared by the reticuloendothelial system (RES), while those < 200 nm show prolonged circulation and deeper tissue penetration. Neutral or negatively charged NPs minimize opsonization and benefit from hydrophilic coatings like polyethylene glycol (PEG) for extended circulation. To ensure consistent performance, safety, and clinical translatability of NP systems, standardized characterization protocols are essential. Frameworks such as those developed by the FDA/National Cancer Institute nanotechnology characterization lab provide valuable guidance in this regard[78].

These principles apply particularly in hepatic fibrosis, where effective therapeutic delivery to activated HSCs (aHSCs), KCs, and LSECs remain critical for modulating fibrogenic activity. Moreover, the fibrotic microenvironment, which features ECM deposition, sinusoidal capillarization, immune cell polarization, creates physical and biological barriers to NP penetration[48]. Designing effective liver-targeted therapies therefore requires careful consideration of these cell specific barriers and mechanisms[79].

In addition to tissue-level barriers, intracellular trafficking represents a major hurdle: Most NPs are sequestered within endolysosomal compartments following uptake, limiting the efficacy of cargo delivery unless lysosomal escape is achieved. This challenge is particularly relevant for gene therapies and RNA-based treatments, where cytoplasmic release is required for functional activity[80].

Four principal internalization pathways dominate NP-cell interactions: Clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and phagocytosis. Clathrin-mediated uptake, involving approximately 100 nm vesicles, is common for positively charged particles and receptor-targeted systems. Caveolae-mediated endocytosis occurs via 50-80 nm flask shaped vesicles associated with lipid rafts and often bypasses lysosomal degradation. Macropinocytosis allows non-specific uptake of larger particles (> 200 nm) through actin-driven membrane ruffling. Phagocytosis is classically attributed to professional phagocytes such as macrophages, neutrophils, and dendritic cells, but it can also occur in non-specialized epithelial cells such as hepatocytes. This mechanism facilitates the engulfment of micron-sized particles (> 500 nm) via actin-mediated phagosome formation. The internalization pathway not only determines cellular uptake efficiency but also dictates intracellular trafficking, localization, and the therapeutic efficacy of nanocarriers. For hepatic delivery in particular, designing NPs with optimized size (typically < 200 nm to penetrate sinusoidal fenestrae), tailored surface properties, and effective endosomal escape mechanisms is critical for maximizing delivery outcomes[81,82].

Nanocarrier-mediated liver targeting occurs through passive or active mechanisms. Passive targeting exploits physicochemical properties of NPs and pathological features of diseased tissue (e.g., enhanced permeability and retention effect), but lacks cell-level specificity. By contrast, active targeting leverages ligands on NP surfaces that bind specific receptors on target cells, and can also use physical stimuli (pH, temperature, magnetic fields) to refine delivery (Figure 2)[83].

Figure 2 Major cellular internalization pathways of nanoparticles and their intracellular fate. Nanoparticles can be internalized through four primary mechanisms: Macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and phagocytosis. These pathways determine the intracellular routing of nanoparticles, leading to lysosomal degradation, entry into the endoplasmic reticulum (ER), the Golgi apparatus, or exocytosis. Clathrin-mediated endocytosis and macropinocytosis typically result in fusion with lysosomes, whereas caveolae-mediated uptake may bypass the lysosomal route, enabling alternative trafficking. Phagocytosis, mainly performed by specialized cells, leads to phagolysosome formation, with possible degradation or release of residual content. Created in BioRender.com (Supplementary material).

In addition to uptake route, NPs differ in size, shape, surface charge, and core composition, which influence biodistribution, biodegradability, and intracellular trafficking. Broadly, NPs are categorized into organic and inorganic types, each offering specific advantages and limitations for liver targeting (Tables 1 and 2).

Table 1 Overview of different inorganic and organic nanoparticles used for liver targeting, including their target cells, internalization mechanisms, main benefits, and common limitations.

	NPs type	Target cells	Mechanism of action	Advantages	Limitations
Metal/metal oxide	Gold NPs	Kupffer cells, HSCs	Passive targeting; accumulation in hepatic tissue; modulation of inflammatory pathways in cytoplasm	High stability, ease of synthesis, surface functionalization, enable imaging	Non-biodegradable; liver accumulation risk, potential cytotoxicity
Silica-based	Silica NPs	Kupffer cells	Phagocytosis by Kupffer cells; drug release from NP surface within endolysosomal compartments	High stability, customizable surface enables functionalization	Poor biodegradability, potential long-term retention and chronic toxicity
Carbon- based	Carbon nanotubes	Hepatocytes, HSCs	Membrane penetration or endocytosis; direct cytoplasmic delivery of drug or gene (lysosomal bypass possible)	High drug/gene loading, tunable size and shape, and be surface-functionalized for targeting	Non-biodegradable; accumulation and inflammation risks, potential cytotoxicity and oxidative stress
	Fullerenes (C60)	Hepatocytes, Kupffer cells	Passive uptake, ROS scavenging in cytoplasm	Strong antioxidant activity, high liver cellular uptake and accumulation, potential anti-inflammatory effects	Non-biodegradable, accumulation risk, potential hepatotoxicity and oxidative stress
Lipid-based	Liposomes	HSCs (SPARC/RA-mediated)	Receptor-mediated endocytosis; drug release via lysosomal degradation or cytoplasmic escape (formulation-dependent)	Biodegradable, high biocompatibility, encapsulate both hydrophilic/lipophilic drugs and genetic material, surface modifiable for targeting	Cost, possible drug leakage and instability during storage
	Solid Lipid NPs (solid lipid NPs and NLCs)	HSCs, hepatocytes	Endocytosis; gradual drug release within endolysosomal compartments (no lysosomal escape unless specifically engineered)	Biodegradable, good biocompatibility, solid core improves stability, suitable for controlled drug/gene release	Limited loading for hydrophilic drugs (improved in NLC), possible formulation-dependent instability
Polymeric	Polymeric NPs (e.g., PLGA, chitosan)	HSCs (via HA receptor)	Receptor-mediated endocytosis; pH-sensitive release with lysosomal escape (formulation-dependent)	Biodegradable, controlled release, high specificity via receptor-mediated targeting	Some polymers may trigger immune response, potential accumulation risk for some formulations
	Nanomicelles	HSCs	Endocytosis; cytoplasmic drug release (lysosomal escape possible depending on composition)	Biodegradable, small size (< 50 nm), enhance solubility of poorly water-soluble drugs, good stability in circulation	Possible low loading capacity, potential premature drug release and rapid clearance

HA: Hyaluronic acid; HSCs: Hepatic stellate cells; NLCs: Nanostructured lipid carriers; NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic) acid; RA: Retinoic acid; ROS: Reactive oxygen species; SPARC: Secreted protein acidic and rich in cysteine.

Table 2 Overview of drug delivery nanoparticles, including representative surface ligands, typical drug-payload capacity, and predominant internalization mechanisms.

Nanoparticle	Representative surface ligands	Typical drug-payload capacity	Predominant cellular internalization pathway
Gold nanoparticles	PEG, folic-acid, RGD peptide, anti-HER2 Ab	5-10 wt% (approximately 80-150 doxorubicin molecules per 20 nm gold NP)	Receptor-mediated clathrin-dependent endocytosis
Silica nanoparticles	PEG, folic-acid, iRGD peptide	25-30 wt% (paclitaxel approximately 250 mg/g MSNP)4	Clathrin-mediated endocytosis; caveolae contribution reported
Carbon nanotubes	PEG, transferrin, RGD peptide	approximately 20 wt% (doxorubicin 2.5 mg/mg carbon nanotube)	Energy-dependent endocytosis and direct membrane penetration
Fullerenes (C60 derivatives)	PEG-C60, malonic-acid-C60, amino-C60	5-15 wt% for hydrophobic drugs	Caveolae-mediated endocytosis and passive diffusion
Liposomes	DSPE-PEG, antibody fragments, folic-acid	10-15 wt% (Doxil® approximately 14 wt% doxorubicin)	Endocytosis/membrane fusion; phagocytosis by macrophages
Solid lipid nanoparticles	PEG, polysorbate 80, lactoferrin	5-12 wt% (curcumin 85 mg/g solid lipid NP)	Clathrin- & caveolae-mediated endocytosis
Polymeric Nanoparticles (PLGA, PLA)	PEG, folic-acid, aptamers, anti-EGFR Ab	5-20 wt% (paclitaxel 120 mg/g PLGA NP)	Receptor-mediated endocytosis (clathrin & caveolae)
Nanomicelles	PEG-PLA or PEG-PCL cores with folate or RGD	10-30 wt% hydrophobics (docetaxel 250 mg/g)	Clathrin-mediated endocytosis; macropinocytosis

AB: Antibody; DSPE: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; EGFR: Epidermal growth factor receptor; HER2: Human epidermal growth factor receptor 2; iRGD: Integrin RGD; MSNP: Mesoporous silica nanoparticle; NP: Nanoparticle; PCL: Polycaprolactone; PEG: Polyethylene glycol; PLA: Polylactic acid; RGD; Arginine-glycine-aspartic acid; wt%: Weight percentage.

Inorganic NPs (e.g., silica, titanium dioxide, and gold) offer structural and functional stability but may accumulate in the liver, raising toxicity concerns. These materials generally exhibit low or no biodegradability and are prone to endolysosomal sequestration, which may impair drug release and contribute to cytotoxicity in a formulation-dependent manner. Recent evidence supports both long-term accumulation and variable toxicity profiles. For instance, bovine serum albumin-coated gold NPs persisted in the liver, spleen, and kidneys up to 120 days post-injection and elicited inflammatory and fibrotic responses in renal tissue, despite preserved organ function[84]. However, other studies reported no significant chronic toxicity in mice over 12 months, despite prolonged hepatic and splenic retention. Preclinical rodent studies show that silica NPs (30-50 nm) accumulate in the liver, leading to oxidative stress, inflammation, and metabolic disruption[85], while mesoporous silica formulations demonstrated acceptable tolerability in early phase human trials[86]. Nevertheless, gold NPs have also shown anti-inflammatory and antifibrotic effects in liver models[87].

Organic NPs, especially lipid- and polymer-based systems, are valued for their biodegradability, tunability, and safety profile. Biodegradable variants include lipid-based NPs (LNPs), polymeric NPs (PNPs), and protein-based carriers; non-biodegradable types include carbon nanostructures[88].

Recent in vivo toxicity assessments of PNPs (e.g., poly(lactic-co-glycolic) acid [PLGA] with/without chitosan coating) in rodent models have shown excellent biocompatibility, with chitosan-modified formulations exhibiting even lower inflammatory responses compared to uncoated controls[89]. Additionally, the internalization pathway not only determines cellular uptake efficiency but also influences intracellular trafficking, localization, and ultimately the therapeutic efficacy of nanocarriers (e.g., anisamide-tethered lipidoids). A preclinical liver fibrosis study demonstrated effective RNA delivery to activated liver fibroblasts, achieving approximately 65% gene silencing and reducing collagen deposition[90].

LNPs such as liposomes, solid lipid NPs (SLNs), and stable nucleic acid lipid particles (SNALPs) are well-established for liver therapy[91-93]. Liposomes encapsulate hydrophilic and hydrophobic agents and are used in antifibrotic strategies such as dexamethasone and naringenin loaded formulations. Albumin-modified liposomes targeting secreted protein acidic and rich in cysteine (SPARC) and galactose-functionalized liposomes targeting asialoglycoprotein receptor (ASGPR) demonstrate enhanced liver uptake and antifibrotic effects[93,94]. Studies on the regulation of ASGPR expression in hepatocytes and its zonal distribution under different physiological conditions provide key insights for optimizing ASGPR-targeted nanocarriers[95,96]. However, efficient endosomal escape remains a critical factor, as lysosomal degradation may limit the efficacy of non-modified liposomal systems unless the formulation facilitates membrane fusion or pH triggered release into the cytoplasm.

SLNs offer higher drug-loading capacity and controlled delivery. Cationic SLNs derived from low-density lipoproteins effectively delivered small interfering RNA (siRNA) (e.g., small interfering connective tissue growth factor [siCTGF]), reduced fibrosis markers, and improved liver pathology in vivo in rats[97]. Nevertheless, lysosomal escape is typically limited for SLNs, and drug release often occurs within the endolysosomal compartments unless specific modifications are introduced. PNPs (e.g., PLGA, PEG-polylactic acid [PLA], hyaluronic acid [HA]-based) also support sustained delivery and targeted uptake. Examples include curcumin-loaded HA-PLA NPs and docetaxel-conjugated carboxymethylcellulose, both of which reduced ECM accumulation and HSC activation[98-100]. Additionally, chitosan-coated PLGA NPs have been developed as a sustained drug release strategy in vitro, showing promising stability and biocompatibility[91]. These systems often exploit receptor mediated endocytosis and may include pH-sensitive or redox-responsive polymers to promote lysosomal escape and cytoplasmic delivery.

Carbon-based non-biodegradable NPs (e.g., redox-responsive fullerenes, carbon nanotubes [CNTs]) exhibit strong antioxidant effects but raise safety concerns due to tissue accumulation and potential hepatotoxicity. Nonetheless, carefully engineered CNTs offer drug-loading potential and cell-specific targeting if toxicity is mitigated[101-104]. Their uptake primarily occurs via passive diffusion or scavenger receptor-mediated pathways, often leading to cytoplasmic ROS scavenging; however, long-term biodistribution remains a critical safety consideration.

LNPs, including liposomes and SNALPs, are primarily internalized through clathrin-mediated endocytosis, particularly when functionalized with targeting ligands such as galactose for hepatocytes or vitamin A for HSCs. After endocytosis, LNPs typically enter endolysosomal compartments, where endosomal escape determines bioavailability, especially in gene delivery. Cationic lipids promote fusion with endosomal membranes, facilitating cytoplasmic release[75].

Polymeric micelles (e.g., silibinin-loaded hyaluronic acid) combine small size with targeted delivery, achieving high selectivity for fibrogenic cells and evading RES clearance. Supersaturated micelle systems have improved oral bioavailability of poorly soluble agents like silibinin while reducing gastrointestinal toxicity[55,105]. Their internalization typically occurs via receptor mediated endocytosis, and depending on composition, controlled cytoplasmic release can be achieved through polymer degradation.

PNPs, composed of PLGA, PEG-PLA, or chitosan, can be internalized via multiple routes, including clathrin and caveolae mediated endocytosis. Endosomal escape remains a key challenge; accordingly, polymeric systems often incorporate pH-sensitive or membrane-disruptive moieties to facilitate cytoplasmic delivery[106].

Recent studies have shown that remodeling the fibrotic niche with NPs, for example, restoring LSEC fenestrations or normalizing ECM stiffness, can improve drug delivery[107]. Liu et al[108] demonstrated that spherical nucleic acid NPs can reverse LSEC capillarization and re-establish endothelial fenestrations, enhancing NP delivery into fibrotic mouse liver tissue. Similarly, HA-coated polymeric micelles have been shown to preferentially accumulate in fibrotic liver, where they release curcumin locally and downregulate pro-fibrotic markers[109]. In another study, porous silicon NPs delivering anti-TGF-β1 siRNA significantly reduced fibrosis in carbon tetrachloride (CCl₄)-induced mice, as evidenced by decreased collagen deposition, alpha smooth muscle actin (α-SMA) levels, and serum alanine aminotransferase/aspartate aminotransferase[110].

Such findings underscore the importance of designing nanocarriers that not only target hepatic cells but also navigate and modulate the altered liver architecture, where anatomical and vascular remodeling severely hinders drug access to fibrogenic cell populations[47].

These delivery advantages are particularly significant when combined with gene therapy or genome-editing platforms such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Among gene-editing approaches, CRISPR/Cas9 systems encapsulated in lipid or polymer-based NPs have shown promising antifibrotic effects by selectively silencing genes involved in fibrogenesis, such as TGF-β1 and collagen type I, alpha 1 (COL1A1). This technology offers unparalleled specificity but is limited by delivery challenges, which NPs are helping to overcome. LNPs delivering Cas9 mRNA and guide RNAs efficiently edit liver genes in vivo, reducing fibrotic markers and improving liver function[111].

Further supporting this strategy, in 2024, Wang et al[112] described multistage delivery NPs responsive to environmental stimuli, which enhanced intracellular delivery of Cas9 systems and reactivated silenced microRNAs in fibrotic liver tissue. Another notable preclinical example comes from Chen et al[113], where lipid-polymer hybrid NPs carrying siRNA against PDGFR-β significantly reduced collagen deposition and α-SMA expression in CCl₄-induced fibrosis models.

Emerging approaches are also exploring biomarker-guided delivery systems to further enhance therapeutic precision. Non-invasive fibrosis biomarkers, such as transient elastography and serum PIIINP levels, may provide valuable information to optimize treatment timing and stratify patients for NP-based therapies. Although still under investigation, these tools may improve clinical translation by aligning therapeutic interventions with disease staging[114,115].

In parallel, combinatorial nanocarriers capable of co-delivering small molecules and siRNA, responsive materials, and image-guided or AI-informed delivery systems are gaining momentum. AI is already being applied to simulate biodistribution, optimize ligand-receptor interactions, and design nanomaterials with enhanced liver specificity[52].

Advancements in nanotechnology have also enabled the development of gene delivery systems, gene therapies, and microbubble-assisted delivery, each designed to meet the increasing demand for highly selective and effective treatment options. These methods facilitate the encapsulation of therapeutic molecules or genetic material with disease-modifying potential, allowing direct and accurate liver delivery while minimizing off-target effects. In this context, NPs significantly enhance the efficiency and specificity of these emerging therapeutic strategies.

While these technologies are still evolving, they represent a crucial step toward integrating diagnostics and therapeutics into a unified framework, enabling real-time monitoring, personalized treatment, and improved prediction of therapeutic outcomes.

In conclusion, liver-targeted NP systems hold transformative potential for the treatment of fibrosis and related hepatic disorders. Ongoing innovations in NP engineering, combined with genome editing, AI, and biomarker integration, are steadily advancing the field toward clinically viable, highly selective, and effective liver-targeted therapies.

The following chapters will further explore two critical and emerging strategies that complement NP-based delivery for liver fibrosis: NP-mediated gene delivery systems and ultrasound-targeted microbubble-assisted delivery. Both approaches show promise in overcoming existing limitations in therapeutic targeting and tissue penetration.

NP-BASED GENE DELIVERY FOR LIVER FIBROSIS

Gene therapy targeting the liver has the potential to reduce or block the expression of defective genes by delivering therapeutic genetic material directly to the tissue. Genetic modification could alter myofibroblasts and promote liver regeneration. However, for successful gene therapy, effective and tissue-specific delivery of genetic material to fibrotic livers is crucial[116,117]. In this context, NP-based platforms, particularly lipid and polymeric carriers, have gained traction as efficient and selective gene delivery tools, offering improved targeting to hepatic cells while minimizing systemic exposure. This approach takes advantage of the liver’s distinctive blood vessel structure and its porous endothelium, which remain accessible even in early fibrosis, making the organ especially suited for NP-mediated gene transfer.

Gene delivery systems primarily rely on vectors, which can be viral or non-viral. This section focuses exclusively on non-viral NP-mediated strategies, as viral vectors, although efficient, raise concerns regarding immunogenicity, scalability, and long-term safety[118]. Among non-viral vectors, LNPs and PNPs are the most explored for liver-targeted gene therapy. Cationic LNPs are widely used due to their ability to encapsulate nucleic acids and promote intracellular delivery via electrostatic interactions, shielding the cargo from nuclease degradation during circulation[119]. While their transfection efficiency is generally lower than viral systems, LNPs offer better safety margins, cost-effective manufacturing, and suitability for multiple treatment cycles—features that are especially valuable in chronic liver conditions.

siRNAs are among the most widely used RNA tools in antifibrotic gene therapy. Delivery systems like cationic liposomes, micelles, and PNPs have been optimized to target aHSCs[120]. The reduced perisinusoidal space and impaired fluid exchange in fibrotic liver tissue have even been used as selective delivery cues. Ligands like biotin-linked insulin-like growth factor 2 receptor (IGF2R) peptides, cholesterol, and vitamin A have been used to functionalize neutravidin-based siRNA nanocomplexes targeting poly(rC)-binding protein 2 (PCBP2), a key regulator in fibrosis[121,122]. In the context of liver fibrosis, several successful preclinical applications of NP-based gene therapy have emerged. One example is the use of vitamin A-coupled liposomes carrying siRNA against heat shock protein 47 (HSP47), which effectively reversed fibrosis and promoted liver regeneration in a dimethyl-nitrosamine-induced rat model. Another example includes an siRNA/peptide nucleic acid hybrid nanocomplex targeting PCBP2, which demonstrated potent antifibrotic activity and collagen suppression in both in vitro and CCl₄-induced in vivo models[123]. Ligand-modified NPs offer additional specificity by directing delivery to aHSCs by engaging surface receptors like IGF2R or SPARC. Studies employing galactose, mannose, cholesterol, or vitamin A ligands have shown enhanced delivery efficiency to fibrotic regions[121,122,124].

Size is also a crucial parameter, NPs smaller than 200 nm are typically preferred to navigate liver sinusoids and reach perisinusoidal targets. Among emerging technologies, gold nanorods coated with cationic polymers have been used to deliver CRISPR/Cas9 plasmids in fibrotic mouse livers, offering high protection, nuclear access, and significant reductions in fibrotic markers. Similarly, LNPs delivering mRNA and guide RNAs have shown successful in vivo editing of fibrosis-related genes such as COL1A1 and TGF-β1, resulting in reduced ECM deposition and inflammation[125].

Polymeric platforms, such as PEGylated L-tyrosine polyurethane NPs, have also demonstrated efficient transfection and immune evasion. In comparative studies, transfection efficiency was significantly higher in cancer cell lines than in normal HSCs, highlighting the need for further refinement in fibrosis-specific contexts[108]. Recently, hybrid LNP/PLGA carriers combining PEG surface modification and ROS-responsive linkers showed enhanced siRNA release in oxidative fibrotic environments, leading to marked suppression of α-SMA and TGF-β1 in preclinical models[126]. In a parallel study in 2023, Han et al[90] developed anisamide-functionalized lipidoid NPs (AA-T3A-C12) that delivered siRNA against HSP47 with > 60% silencing efficiency in aHSCs and significantly reversed fibrosis in CCl₄-induced mice. Importantly, the clinical candidate BMS-986263, a retinoid-conjugated LNP delivering siRNA against HSP47, has advanced to Phase II trials, showing promising reduction in fibrotic markers in patients with advanced liver fibrosis[127]. Recently, hybrid LNP/PLGA carriers combining PEG surface modification and ROS-responsive linkers showed enhanced siRNA release in oxidative fibrotic environments, leading to marked suppression of α-SMA and TGF-β1 in preclinical models[126].

One of the most promising NP-based gene delivery platforms currently undergoing clinical evaluation is STP705, a histidine-lysine polymer conjugated with siRNAs targeting both cyclooxygenase-2 and TGF-β1. In mouse models, STP705 induced synergistic apoptosis in fibrotic fibroblasts and suppressed multiple profibrotic markers[128,129]. This platform has progressed to Phase I clinical trials for non-hepatic indications[130], while a related compound, STP707, is now in Phase I evaluation for solid tumors and PSC, a rare liver fibrosis subtype[131].

Despite these promising developments, several limitations persist. These include limited nuclear entry for DNA constructs, transient expression in the case of mRNA, and reduced transfection efficiency in advanced fibrotic livers due to ECM density and altered sinusoidal architecture. Notably, transfection efficiency has been significantly higher in cancer cell lines than in normal HSCs, underscoring the need for further refinement in fibrosis-specific settings[108]. Furthermore, many NP formulations have yet to progress beyond preclinical stages, and few have been tested in large animal models or long-term toxicity studies. Clinical translation will require enhanced delivery precision, durable gene expression, and standardized protocols for safety and efficacy evaluation.

In summary, NP-based gene delivery systems offer a versatile and scalable alternative to viral vectors for treating liver fibrosis. While still largely in experimental phases, platforms like LNPs, polymeric nanocarriers, and peptide-conjugated siRNAs have demonstrated strong potential in reversing fibrotic pathology and restoring liver function. Continued optimization and rigorous clinical validation will be crucial to bring these innovations into therapeutic reality.

ULTRASOUND-TARGETED MICROBUBBLE-MEDIATED DELIVERY

Ultrasound microbubbles offer an approach for precise targeted drug delivery with minimal off-target effects. They are gas-filled vesicles (1-10 µm) coated with lipids or proteins, which can carry or be co-administered with therapeutic compounds. Upon ultrasound stimulation (20 kHz to 20 MHz), they oscillate or rupture (sonoporation), enhancing local release and uptake of co-delivered agents like drugs, NPs, or genetic material. Therapeutic agents can be attached via approaches such as biotin–avidin coupling[132]. Microbubble responsiveness depends on the compressibility of their core, which enables controlled oscillation under ultrasound stimulation[133].

Ultrasound microbubbles, traditionally used as contrast agents, are increasingly investigated for therapeutic delivery. In fibrotic liver tissue, characterized by elevated ECM density and interstitial pressure, drug and gene delivery is significantly impaired. Microbubble oscillation transiently disrupts endothelial and ECM barriers, enhancing NP and gene vector uptake. Recent studies have shown that PNPs combined with microbubble-assisted ultrasound significantly increase gene transfection efficiency in fibrotic liver models, surpassing traditional NP- or ultrasound-therapy used alone. These hybrid systems merge the targeting and payload capacity of NPs with the physical penetration boost provided by ultrasound-activated microbubbles. This synergistic enhancement is especially relevant in delivering nucleic acids across dense fibrotic matrices. For example, a study aimed at evaluating the transfection of HSC-T6 cells used a complex consisting of lipid microbubbles and cationic nanoliposomes carrying the hepatocyte growth factor gene. Twenty-four hours after transfection, the treated cells exhibited increased green fluorescence and were induced to die, indicating more efficient transfection compared to groups treated with only microbubbles and plasmid. The results led to the conclusion that combining lipid microbubbles with NPs enhances gene transfection efficiency and overcomes the limitations of carrying a small amount of genetic material and insufficient targeting seen in conventional therapies[134,135].

Ultrasound-guided microbubbles also significantly enhance NP delivery to the liver, improving treatment efficacy for liver diseases like fibrosis and cancer (Figure 3). Although NPs typically enter cells via endocytosis or caveolae-mediated pathways, microbubbles, in combination with ultrasound, facilitate increased cellular uptake and targeted delivery of NPs, thus overcoming barriers to effective drug delivery in the liver. Specifically, cavitation from microbubble collapse can create temporary pores in cell membranes, improving intracellular access for large biomolecules or nanocarriers. The associated shear forces and fluid microstreaming can also promote endosomal escape, an essential step for successful gene delivery.

Figure 3 Schematic representation of therapeutic strategies for liver fibrosis. Nanoparticles, microbubbles paired with ultrasound, or a mix of the two can be used to deliver drug-based and gene-based therapies. Microbubble oscillation transiently disrupts endothelial and extracellular matrix barriers, enhancing nanoparticle uptake. These delivery systems aim to improve tissue penetration and therapeutic efficacy.

An example of ultrasound microbubble therapy effectively combined with NP treatments comes from a study that used ultrasound-targeted destruction of cationic liposome-bearing microbubbles to deliver artificial miRNA targeting CTGF to the livers of rats with hepatic fibrosis[136]. Using a biotin-avidin method, cationic liposomes were coupled with microbubbles, and their administration resulted in decreased fibrosis, demonstrating the potential of this approach as an effective treatment for hepatic fibrosis. In a mouse model of HCC, ultrasound-targeted microbubbles were used to deliver c-Myc antisense oligodeoxynucleotide to malignant cells, leading to the inhibition of tumor development and proliferation. Liver cells express ASGPRs, which specifically recognize and endocytose glycoproteins with terminal galactose groups, also known as galactose-specific receptors. The study employed the polymeric compound galactosylated poly-L-lysine as a targeting ligand for ASGPR, efficiently promoting the expression of the therapeutic construct in liver malignant cells, resulting in decreased c-Myc levels[137]. A limitation of this study is that ASGPR expression is highly abundant in healthy and well differentiated liver cells but may be lower in poorly differentiated hepatocytes[95,96]. As a consequence, targeting ligands may preferentially bind to healthy hepatocytes, leading to non-specific distribution and reduced effectiveness against tumor tissue. In addition, the ASGPR main peptide RHL-1 exhibits zonal expression, with the highest concentration in pericentral hepatocytes and a gradual decrease toward the periportal zone in a healthy male rat liver. This expression gradient is also dynamic and may vary depending on gender and different pathophysiological conditions[96]. Thus, the study presents intriguing opportunities for using NP selective targeting in combination with hepatic zonation of specific molecules.

Recent developments include smart microbubble formulations co-loaded with CRISPR/Cas9 or siRNA constructs, which respond to ultrasound intensity and tissue stiffness, allowing real-time imaging-guided release[138]. By uniting imaging, physical targeting, and gene delivery in one platform, ultrasound-responsive microbubbles open new possibilities for precision liver therapies. By improving delivery specificity, cellular uptake, and tissue penetration, these combined strategies pave the way for next-generation treatments for hepatic fibrosis and other chronic liver diseases.

CONCLUSION

NP-based drug delivery systems have markedly advanced liver therapy by enabling controlled release, protection of labile compounds, and targeted administration of poorly soluble drugs, yet their clinical implementation continues to face significant hurdles. As fibrosis and tumor progression reshape hepatic architecture, characterized by ECM deposition, vascular capillarization, loss of fenestrations, and increased tissue pressure, conventional drugs become less effective, placing hepatic stellate cells at the center of therapeutic strategies. While many antifibrotic compounds appear effective in vitro, their performance in vivo is still hampered by poor tissue penetration, rapid clearance, and off-target effects. Lipid- and polymer-based NPs can help to overcome these barriers by exploiting passive liver accumulation or ligand-mediated recognition. Their performance may be further enhanced when combined with complementary approaches, including ultrasound-assisted microbubbles or gene-based therapies. At the same time, the growing availability of advanced therapeutics, such as peptides, antibodies, and nucleic acids, introduces new requirements for stability, efficient intracellular uptake, and preservation of biological activity. Functionalization with targeting moieties is essential for specificity, but if not carefully optimized it may paradoxically reduce efficacy or even induce toxicity. Taken together, these opportunities and challenges suggest that NPs should be viewed not as definitive solutions but as adaptable platforms whose ultimate success will depend on achieving a delicate balance between precision and safety, and on aligning their versatility with the increasingly complex therapeutic landscape of liver disease.