Gut microbiome-specific nanoparticle-based therapeutics for liver diseases

Abstract

Alcohol-associated liver disease, metabolic dysfunction-associated steatotic liver disease, and metabolic dysfunction-associated steatohepatitis are chronic liver diseases (CLDs) driven by metabolic dysregulation, immune dysfunction, and gut microbiome alterations. Current treatments are inadequate and provide only symptomatic relief in most cases, underscoring the urgent need for forward-looking approaches. The disturbances in gut and liver communication contribute towards disease progression, making microbiome-based therapeutic strategies an area of growing interest. Nanoparticles have emerged as a powerful tool for drug delivery with high targetability, stability, and targeted release. Further, artificial intelligence offers a transformative approach by accelerating nanoparticle design, optimizing microbial therapy formulations, predicting treatment responses, and personalizing interventions based on patient-specific microbiota compositions. Herein, we give an overview of important liver diseases, key nanocarrier types, and the approaches wherein nanocarriers have been integrated to modulate gut microbiota for the therapy of CLDs. We also describe future directions and the challenges, which need to be overcome for wide scale application and tailored use of gut microbiome-focused nano-drug delivery carriers for the therapy of CLDs. Despite current hurdles, the integration of nanotechnology, microbiome therapeutics, and artificial intelligence-driven precision medicine holds immense promise for reshaping the treatment landscape of CLDs.

Key Words: Nanomedicine; Chronic liver diseases; Gut microbiome; Microbial metabolites; Precision medicine

Core Tip: Chronic liver diseases are driven by metabolic, immune, and microbiome dysregulation. In this review, we detail recent advances in nanomedicine that enable tailored modulation of the gut microbiota, which can improve chronic liver diseases. Nanoparticles increase pharmacological effects and can reduce off-target effects. Further, artificial intelligence and machine learning can aid in production of precision nanomedicine.

INTRODUCTION

Hepatic disease constitutes a worldwide health burden with a steep rise in prevalence during the past decades causing significant morbidity and mortality[1-3]. In terms of pathology, a (chronic) liver insult or injury can progress from inflammation to fibrosis with a risk of ultimately leading to liver cirrhosis and/or hepatocellular carcinoma (HCC)[4]. Liver fibrosis is an important pathological feature, which frequently develops in the setting of persistent liver inflammation, and represents tissue degeneration due to increased synthesis and accumulation of extracellular matrix[5]. Liver fibrosis is a major concern in chronic liver diseases (CLDs), such as alcohol-associated liver disease (ALD), metabolic dysfunction-associated steatotic liver disease (MASLD) with its progressive form metabolic dysfunction-associated steatohepatitis (MASH), and primary sclerosing cholangitis (PSC). MASLD was previously known as non-alcoholic fatty liver disease (NAFLD), whereas MASH used to be known as non-alcoholic steatohepatitis[6]. The terminology was introduced, as the older terms NAFLD/non-alcoholic steatohepatitis were perceived as stigmatizing and scientifically less precise[7]. The global prevalence of MASLD is approximately 38% and the rate is even higher in patients with diabetes and obesity. MASH is the second leading indication for liver transplantation[8]. The prevalence of ALD is about 4.3% in the United States population[9]. To date, there are largely no curative medical treatment options available for liver fibrosis, and standard of care is often supportive only and associated with significant morbidity and mortality[10,11]. An important reason for the failure of therapies is the lower therapeutic efficacy due to poor biodistribution of the pharmacologically active drugs in the liver secondary to the prevailing fibrosis[12]. In addition, the gut microbiome plays a critical role in physiological homeostasis and it is well understood that an aberrant gut microbiome is one of the hallmarks of CLDs[13,14].

Nanotechnology has emerged as an attractive platform for complex unmet medical needs. It opens avenues ranging from drug targeting to molecular diagnostics and provides an array of biotherapeutics and tremendous possibilities for nanomedicine[15,16]. The traditional pharmaceutical formulations lack specific targeting, and are cleared rapidly and therefore require increased or frequent dosing, which may result in serious off-target adverse effects[17]. The recent advances in nanocarrier designs have led to precise control of size, shape, multi-functionality and biocompatibility, and tailored drug release; thus, overcoming some of the major challenges of conventional drugs and dosage forms[18]. A variety of base materials has been used for synthesizing tailored nanocarriers including polymers, dendrimers, liposomes, metal, silicon, and carbon-based materials; and are used for drug delivery, therapeutics, and diagnostics[19-23].

The human gut microbiome is a complex ecosystem of trillions of microbes living in the gastrointestinal tract, and has an important role in maintaining physiological homeostasis[24]. The gut microbiome regulates normal digestion, immunity, and metabolism[25,26]. Imbalance of the physiological microbial community has been implicated in the pathogenesis of ALD, MASLD, and PSC[27]. Advances over the last decade in nanomedicine have opened new opportunities for tailored modulation of specific segments of the gut microbiota, providing novel therapeutic strategies for treating CLDs. Nanomedicine employs the use of nanoscale materials for therapeutics and diagnostics, enabling the targeted delivery of microbial metabolites, probiotics, or genetic material precisely to specific gut areas, hence increasing pharmacological effects while reducing the risk of off-target effects[28-30]. Nanoparticle-based drug carriers have been used to encapsulate drugs, including antibiotics, prebiotics, or anti-inflammatory agents, for their controlled release within the gut[31,32]. Further, the recent integration of artificial intelligence (AI) for the design and development of nanomedicine has drastically increased the pace of identifying effective therapeutics[33]. AI-based approaches, for instance, machine learning (ML) and deep learning, have provided valuable means to optimize the design and synthesis of nanocarriers, forecasting their complex interactions with biological systems, and predicting new drug targets within the gut microbiome[34,35]. Herein, we provide an overview of important CLDs, the complex challenges in their pathology, and the need for identification of novel therapeutic approaches. We provide insights into the role the gut microbiome plays in the pathogenesis of CLDs and how their modulation provides unique avenues. We highlight the role of nanocarriers for the delivery of gut microbiome-based drug candidates to achieve desired therapeutic benefits.

PATHOLOGY OF CLDs, UNIQUE CHALLENGES, AND THE NEED FOR FORWARD-LOOKING APPROACHES

CLDs, including ALD, MASLD/MASH, and PSC, constitute a major global health burden[36]. Despite advances in unraveling details of their pathologies, treatments are primarily focused on supportive care, highlighting the need for breakthrough therapeutic approaches. ALD constitutes a range of liver pathologies caused by chronic excessive alcohol use, starting with steatosis, which may progress towards alcohol-associated hepatitis, fibrosis, cirrhosis, and HCC. The pathogenesis of ALD involves a complex cascade of events encompassing oxidative stress, inflammation, and gut-liver axis dysfunction[37]. Kupffer cells (KCs) are activated during ALD, through various mechanisms, in particular toll-like receptor 4 signaling but also C-type lectin-like receptor signaling among others[38,39]. KC activation is triggered by microbe-associated molecular patterns, such as bacterial endotoxin [or lipopolysaccharide (LPS)], peptidoglycan, and fungal β-glucan, which translocate from the gut into the portal circulation by breach of the gut barrier caused by ethanol and its metabolites including acetaldehyde, and other microbial products[40]. This pathological phenomenon, widely known as “leaky gut”, plays a crucial role in the pathogenesis of ALD. Microbe-associated molecular pattern receptor activation leads to secretion of pro-inflammatory cytokines, e.g., tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, which orchestrate the liver inflammation and cell death of hepatocytes[41,42]. Moreover, ethanol enhances the migration of immune cells including neutrophils to the liver, further aggravating the inflammatory cascade. In addition, alcohol reduces the number of beneficial microbes, such as Lactobacillus and Bifidobacterium, and increases the content of potentially pathogenic microbes, such as Enterobacteriaceae[43]. This phenomenon is termed “dysbiosis”[44]. The current treatments for ALD are symptomatic and can include corticosteroids and pentoxifylline, with limited therapeutic benefits and significant side-effects. The clinical course of ALD widely varies among patients, invoking inquiry into personalized approaches.

MASLD, earlier known as NAFLD, pathologically shows hepatic steatosis without marked alcohol ingestion. MASLD is linked to metabolic syndrome, obesity and type 2 diabetes[6]. The progression from simple steatosis to MASH, fibrosis, and cirrhosis requires intertwined roles of multiple pathways (Figure 1). Pro-inflammatory cytokines and reactive oxygen species (ROS) contribute to hepatocyte injury and the transition from steatosis to MASH. The nucleotide-binding domain, leucine-rich–containing family, pyrin domain-containing-3 (NLRP3) inflammasome activates caspase-1 and interleukin-1 beta and has a key role in this process[45]. The activation of NLRP3 is stimulated by danger-associated molecular patterns, such as extracellular ATP and cholesterol crystals[46]. NLRP3 inflammasome activation of hepatic stellate cells leads to excessive synthesis of extracellular matrix proteins, or fibrosis[47]. Key mediators of fibrosis include connective tissue growth factor, platelet-derived growth factor, and transforming growth factor beta[48]. The multifactorial pathology of MASLD is a major challenge for developing effective treatments and the disease progression widely differs among patients, highlighting the important role of personalized approaches.

Figure 1 Spectrum of pathology of metabolic dysfunction-associated steatotic liver disease. Regular intake of high fat loaded food can trigger the development of metabolic dysfunction-associated steatotic liver in 15%-30% of healthy population, which is characterized by steatosis, implying fat in > 5% hepatocytes and is reversible in up to 21% of patients. In 12%-40% of patients metabolic dysfunction-associated steatotic liver may progress towards the more aggressive form of the disease metabolic dysfunction-associated steatohepatitis (MASH), which is characterized by steatosis, inflammation, ballooning of hepatocytes and fibrosis. MASH is reversible in up to 11% patients. Among 15%-25% MASH patients there is high risk of the development of liver cirrhosis characterized by severely compromised liver function and 7% of cirrhotic patients may develop liver cancer. The only therapeutic option for liver cirrhosis and liver cancer is liver transplantation. At the cellular level, hepatocytes develop steatosis under the influence of enzymes such as fatty acid synthase, and sterol regulatory element binding protein 1. Persistent steatosis induces hepatic inflammation involving immune cells including Kupffer cells. Long-standing inflammation will activate quiescent hepatic stellate cells to cause fibrosis up to cirrhosis by excessive synthesis of extracellular matrix proteins. Cirrhosis may give rise to hepatocellular carcinoma. The interplay of different hepatic cells culminates in the pathological features of MASH. MASL: Metabolic dysfunction-associated steatotic liver; MASH: Metabolic dysfunction-associated steatohepatitis; MASLD: Metabolic dysfunction-associated steatotic liver disease; ECM: Extracellular matrix; HSC: Hepatic stellate cell; KC: Kupffer cells; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; FASN: Fatty acid synthase; SREBP1: Sterol regulatory element binding protein 1; TGF-β: Transforming growth factor-beta; PDGF: Platelet-derived growth factor. Created with a license from BioRender.com.

Due to complex pathologies, patient heterogeneity, gut-liver axis dysfunction, progressive fibrosis, and limited and largely ineffective treatment options, it becomes crucial to identify novel therapeutic approaches for the treatment of these CLDs.

NANOCARRIERS: WHAT DO THEY HAVE TO OFFER? UNIQUE PROPOSITIONS LEARNT FROM CANCER DRUG DELIVERY

Nanocarriers (1 to 1000 nanometers) have gained wide attention as a transformative technology in drug delivery, providing unprecedented targeting, efficiency, and versatility[49]. These nanocarriers are engineered to encapsulate, protect, and deliver drugs to specific tissues or cells. Their novel features, i.e. high surface area-to-volume ratio, flexible surface chemistry, and ability to cross biological barriers, make them ideal for addressing the complex challenges of CLDs, including ALD, MASLD, and PSC[50].

Nanocarriers are designed to load small molecules, proteins, nucleic acids, and even live bacteria. The number of atoms or molecules that can be loaded into a nanoparticle depends on its size, structure, and the properties of the cargo. For instance, a 100-nm liposomal nanoparticle can encapsulate approximately 10000 to 100000 small molecules, while larger nanoparticles or those with porous structures can carry even more[51]. The loading capacity of a nanocarrier is further modulated by the interaction between the cargo and the nanoparticle matrix, which can be optimized through surface modification or the use of specific polymeric materials (Figure 2).

Figure 2 Classification of different types of nanoparticles with their specific advantages and disadvantages. The figures enlist three major types of nanoparticles: Polymeric, inorganic, and lipid-based. The polymeric nanoparticles include polymersomes, dendrimers, polymer micelles, and nanospheres. Advantages of polymeric nanoparticles are precise control, flexibility, surface modification, and sustained release; while the limitations include risk of toxicity and aggregation. The inorganic nanoparticles comprise silica nanoparticles, quantum dots, iron oxide nanoparticles, and gold nanoparticles. Their advantages are the unique physicochemical properties, tailored design, and versatility. Their solubility and toxicity might pose challenges in delivery. The lipid-nanoparticles can be subdivided into liposomes, lipid nanoparticles, and extracellular vesicles. They offer advantages such as simplicity, high bioavailability, payload flexibility, and high biocompatibility, whereas the low encapsulation remains a limitation. Created with a license from BioRender.com.

There are several advantages that nanocarriers offer. Nanocarriers are functionalized with ligands, such as antibodies or peptides, to actively target desired cell types and/or tissues, minimizing off-target effects[52]. The release of therapeutic agents can be precisely controlled through stimuli-responsive materials, e.g., pH-sensitive or enzyme-sensitive polymers. Nanocarriers protect their cargo from degradation, extending its half-life and improving bioavailability[53]. They can traverse barriers, such as the gut epithelium or the blood-brain barrier, enabling the delivery of drugs to previously inaccessible sites. Further, nanocarriers can be engineered to combine diagnostics and therapeutics, enabling theranostic applications. There are several key types of nanoparticles that have been developed for the treatment of CLDs, each with unique properties and applications.

Lipid-based nanoparticles

Lipid-matrix is used to synthesize liposomes and solid lipid nanoparticles, which are employed due to high biocompatibility and flexibility to load both hydrophilic and hydrophobic drugs[54]. Liposomes, for instance, consist of a phospholipid bilayer surrounding an aqueous core, making them ideal for delivering water-soluble drugs. Liposomal doxorubicin (Doxil) is a successful nanodrug used in cancer therapy, demonstrating reduced cardiotoxicity and improved tumor targeting[55]. Lipid-based nanoparticles, especially liposomes owing to their close similarities to the cellular membrane, can be effectively applied to deliver gut microbiome-modulating agents, such as short-chain fatty acids (SCFAs)[56].

Polymeric nanoparticles

Polymeric nanoparticles are synthesized with biodegradable polymers, such as poly(lactic-co-glycolic acid) and chitosan (CS)[57]. These nanoparticles offer controlled release and can be tailored to respond to specific stimuli, pH or enzymes[58]. Poly(lactic-co-glycolic acid) nanoparticles can deliver anti-inflammatory drugs (e.g. curcumin) to the liver, reducing inflammation and fibrosis in MASLD mouse models[59,60]. CS nanoparticles are known for their mucoadhesive properties, are ideal for oral delivery of gut microbiome-based therapies[61].

Inorganic nanoparticles

Inorganic element-based nanoparticles, such as gold nanoparticles, iron, silver, and silica nanoparticles, offer unique optical and magnetic properties, making them suitable for imaging and targeted therapy[62]. Gold nanoparticles have been used to deliver small interfering RNA (siRNA) to hepatocytes, silencing genes involved in fibrosis and inflammation[63]. Copper sulfide nanoparticles offer unique benefits of semiconductor properties, and photothermal effects making them an attractive tool for liver diseases theranostics[64]. These approaches can be adapted to deliver microbial metabolites or probiotics to the gut-liver axis.

Dendrimers

Dendrimers are highly branched, tree-like structures with a high degree of surface functionality[65]. They can be engineered to carry multiple drug molecules and target specific cells. Polyamidoamine dendrimers have been used to deliver antiviral drugs to hepatocytes, demonstrating enhanced efficacy and reduced toxicity[66]. Similar strategies can be employed for gut microbiome-based therapies[67].

Exosomes

Exosomes are natural nanovesicles released by cells, playing a key role in cell-to-cell communication[68]. They can be engineered to carry therapeutic cargo and target specific tissues and to the gut microbiome as well. Exosomes obtained from mesenchymal stem cells were used to deliver anti-fibrotic microRNAs to the liver, reducing fibrosis in preclinical models[69,70]. This approach can be extended to deliver microbial metabolites or probiotics.

Unique propositions

Nanoparticles provide several unique advantages compared with the conventional drug delivery systems, making them particularly valuable for treating CLDs. These diseases often involve specific cell types, such as hepatocytes, KCs, or hepatic stellate cells. Nanoparticles can be linked with ligands, for instance, galactose or hyaluronic acid, to target these cells, reducing off-target effects and reducing toxicity[71]. The gut-liver axis plays a central role in the pathogenesis of ALD, MASLD, and PSC[72]. Nanoparticles may be tailored to deliver drug directly to the gut epithelium, the liver, or the gut microbiome, addressing the root cause of these diseases. CLDs are multifactorial, requiring simultaneous targeting of multiple pathways. Nanoparticles can be engineered to carry different drugs, enabling synergistic therapy as well as providing avenues to curb drug resistance, which may be of special relevance for targeting fibrosis[73]. CLDs require long-term treatment. Nanoparticles can provide tailored release of therapeutic agents, lowering the frequency of dosing and enhancing patient compliance. Nanoparticles can combine diagnostics and therapeutics, enabling real-time monitoring of pharmacotherapy.

Success stories in cancer nanodrug delivery, which may be translated to CLDs

Doxil was the first nanodrug approved by the Food and Drug Administration (FDA) demonstrating reduced cardiotoxicity and improved tumor targeting in cancer therapy[55]. Its success highlights the importance of lipid-based nanoparticles for targeted delivery. The pancreatic, lung, and breast cancer therapeutic Abraxane uses albumin nanoparticles to deliver paclitaxel, improving solubility and reducing side-effects; however, the challenge remains the specific delivery for modulation of the gut microbiome[74]. This approach can be adapted for delivering hydrophobic gut microbiome-modulating agents. Onpattro is used for the treatment of adult polyneuropathy caused by hereditary transthyretin-mediated amyloidosis, which works by reducing the production of an abnormal transthyretin protein in the liver. It delivers siRNA to hepatocytes, silencing disease-causing genes[75]. This strategy can be used to deliver microbial metabolites or probiotics. Vyxeos, a liposomal formulation of daunorubicin and cytarabine, demonstrates enhanced efficacy in leukemia[76]. This approach can be applied to deliver combination therapies for CLDs. Genexol-PM uses polymeric micelles to deliver paclitaxel, improving solubility and reducing toxicity[77]. This strategy can be adapted for delivering gut microbiome-based therapies.

NANOCARRIERS FOR DELIVERY OF DRUGS AND MODULATION OF GUT MICROBIOME FOR LIVER DISEASES

Nanocarriers are a tool for the precise and efficient delivery of a wide array of therapeutic agents, including microbes, their metabolites, and nucleic acids, in the treatment of CLDs[28]. These nanoscale delivery systems give multiple advantages, such as enhanced stability, targeted delivery, controlled release, and the ability to overcome biological barriers, making them ideal for modulating the gut-liver axis[78]. By encapsulating and protecting their cargo, nanocarriers ensure the bioavailability and therapeutic efficacy of microbial-derived therapies, which are often hindered by degradation, poor absorption, or off-target effects. For instance, nanoparticles have been successfully employed to deliver SCFAs, probiotics, and bacteriophages to the gut, reducing inflammation, oxidative stress, and fibrosis[79]. Furthermore, nanocarriers enable the delivery of microbial nucleic acids and genetic materials, such as microRNAs and siRNAs, to precisely target disease pathways[80]. The integration of nanotechnology with microbiome science holds immense promise for revolutionizing the treatment of CLDs, offering hope for more effective and personalized therapies (Table 1).

Table 1 Overview of different types of microbiome-inspired nanoparticles trialed in experimental liver disease.

Type of nanoparticle	Animal model used	Details of treatments	Therapeutic outcomes	Microbiome-specific effects/ role in design	Ref.
pH/gut microbiota-responsive NC nanoparticles	High-fat diet-induced steatohepatitis in C57 mice	Nanoparticles containing NC 8 mg/kg/day, by gavage	Reduced weight gain, serum aspartate aminotransferase, alanine aminotransferase and lipid levels, improved liver and intestinal inflammation, and altered diversity of gut microbiota	Abundance of beneficial Bacteroidetes gradually increased, whereas that of Desulfobacterota and Proteobacteria decreased	[84]
Low-molecular weight chitosan-based selenium nanoparticles	High-fat diet-induced liver disease in C57 mice	Nanoparticles were gavaged orally at 200 mg/kg/day	Inhibited hepatic fat accumulation and markedly improved the intestinal barrier by increasing mucus secretion from goblet cells	Increased the abundance of mucus-associated microbiota (Bifido-bacterium,Akkermansia, and unclassified Muribaculaceae) and decreased the abundance of obesity-related bacteria (Anaerotruncus, Lachnoclostridium, and Proteus)	[88]
Macrophages modulating transcription factor XBP1s siRNA-loaded folic acid-modified TPGS nanoparticles	Fat-, fructose- and cholesterol-rich diet model in C57 mice	Injected intravenously (0.05 nmol/L, 100 μL) every 3 days for 4 weeks	Reduced weight gain, dyslipidemia, inflammatory cytokines extrahepatically, and ER stress, fat accumulation, and fibrosis in the liver	Higher relative abundance of Blautia and Bacteroides, and lower abundance of Actinobacteriota, Muribaculaceae and Bifidobacterium	[89]
Prebiotic-like silybin-2-hydroxypropyl-β-cyclodextrin inclusion nano-complex	High fat diet-fed hamsters	100 mg/kg/day silybin equivalent nanoparticles by oral gavage	Decreased hepatic lipid content, oxidative stress, and inflammation	Restored the gut microbiota, increased fecal excretion of bile acids and intestinal integrity	[92]
Hydrogen-based nanocapsule loaded with ammonia borane inside hollow mesoporous silica nanoparticles core	Diet- and genetic (db/db)-induced steatohepatitis in C57 mice	0.8 mg/day/mouse hydrogen in nanoparticles administered through diet	Reduced lipid synthesis and increased fatty acid catabolism	Increased the abundance of Akkermansia muciniphila	[95]
Lactobacillus plantarum ghosts based astaxanthin-loaded nanoparticles	Modified Lieber-DeCarli liquid diet (5% ethanol) in C57 mice	20 mg/kg/day nanoparticles by oral gavage	2.11 times higher plasma bioavailability, reduced oxidative stress	Biomimetic self-guided delivery	[97]
Nanoparticles based on exosome-like nanoparticles released by Lactobacillus rhamnosus GG	Lieber-DeCarli liquid diet (5% ethanol) in C57 mice	On last 3 days by daily gavage of 200 μL of nanoparticles (50 μg protein content)	Protected the intestine from ethanol-induced barrier dysfunction and the liver from steatosis and inflammation	Increase tight junction protein expression in intestinal epithelial cells	[98]
Exosome like nanoparticles derived from Phellinus linteus	Hepa 1-6 liver tumor cells in vitro and in vivo diethylnitrosamine (20 mg/kg) and 40 ppm N-nitrosomorpholine in drinking water for 24 weeks in ICR mice	10 mg/kg/orally protein containing nanoparticles in 200 μL, every 3 days for a total of 5 doses	Strong anti-proliferative, anti-migratory, and anti-invasive effects	Enhanced Lactobacillus, Turicibacter, and Enterorhabdus. Lactobacillus	[104]
Polyethylene glycol (CHO-PEG2000-CHO)-poly (ethyleneimine) (PEI25k)-citraconic anhydride-doxorubicin nanoparticles loaded with plasmid-encoded hsulf-1 enzyme	H22 hepatocarcinoma model	Nanoparticles containing doxorubicin 5 μg, hsulf-1 60 μg, in 200 μL/mouse, subcutaneous injection, for 5 days	Increased cytokine secretion and the intratumoral infiltration of CD4/CD8+ T cells with strong antitumor effects	Non-pathogenic Escherichia coli-based nanoparticles	[107]

NC: Nitidine chloride; XBP1s: X-box binding proteins 1; siRNA: Small interference RNA; TPGS: Tocopheryl polyethylene glycol succinate; ER: Endoplasmic reticulum; PEG: Polyethylene glycol; PEI: Polyethyleneimine; hsulf-1: Human sulfatase-1; ICR mice: Institute of Cancer Research mice.

Nanocarriers for the treatment of MASLD

The progression from steatosis to MASH has a variety of factors such genetic predisposition, insulin resistance, dysfunctions in lipid metabolism, and mitochondrial anomalies[81]. The gut microbiota has been recognized to be involved in the pathophysiology of metabolic disorders, including MASLD[82,83]. Figure 3 provides the plausible mechanisms by which microbiome-inspired nanocarriers show their therapeutic benefits. Lu et al[84] developed a pH/gut microbiota-responsive system of nitidine chloride (NC)-CS/pectin (PT)-nanoparticles, and studied the effect of colon-targeted delivery system NC-CS/PT-nanoparticles in a high-fat diet (HFD)-induced mouse model of steatohepatitis. The release studies indicated high concentrations of NC in the colon. The in vivo results showed that NC-CS/PT-nanoparticles significantly reduced body weight, reduced aspartate aminotransferase, alanine aminotransferase and lipids. It also improved liver and intestinal inflammation, and altered the gut microbiota in mice. Following treatment with the nanoparticles, the number of beneficial Bacteroidetes enhanced, and Desulfobacterota and Proteobacteria decreased, which were high in the disease group.

Figure 3 Mechanism of action of orally delivered microbiome nanocarriers for the treatment of liver diseases. The enterohepatic circulation plays an important role in the mobility of nanoparticles, wherein the portal vein circulates nanoparticles and their payload to the liver, and the metabolites may then circle back again from the liver to the intestine via the biliary system. In the intestine, microbiome-inspired nanoparticles may potentially affect the microbiome by modifying the microbiome profile, by delivering pharmacologically active microbial metabolites (such as short chain fatty acids) or molecules. Within the liver, liposomes may reduce inflammation by acting on immune cells such as Kupffer cells, may reduce apoptosis of hepatocytes, and/or may reduce the deposition of extracellular matrix proteins including collagen, elastin, and fibronectin. ECM: Extracellular matrix; SCFA: Short chain fatty acids. Created with a license from BioRender.com.

Studies report that selenium polysaccharides possess pharmacological effects in reducing blood lipids, blood glucose, and affect immunity[85]. Further, selenium supplementation increases the growth of useful gut microbiota and associated metabolic pathways[86]. Low-molecular weight CS (LCS) improves intestinal barrier and reduced inflammation in mice on HFD by increasing beneficial gut bacteria Akkermansia and Gammaproteobacteria and reducing inflammatory bacteria Erysipelatoclostridium and Alistipes[87]. Harnessing these properties, Luo et al[88] developed a LCS selenium nanoparticle (LCS-SeNPs) to study if LCS-SeNPs reduce steatohepatitis in mice. LCS-SeNPs decreased liver fat and increased the integrity of intestines. It increased the mucus release from goblet cells. Further, LCS-SeNPs increased the abundance of Bifidobacterium, Akkermansia, and unclassified Muribaculaceae linked with mucosa and reduced obesity-related bacteria (Anaerotruncus, Lachnoclostridium, and Proteus). This led to modulation of several metabolic pathways, including bile acid secretion, purine metabolites, and tryptophan derivation. The glycocholic acid and tauro-beta-muricholic acid levels were lower in the microbiota of LCS-SeNP group indicating that these LCS-SeNPs are promising for the treatment of MASLD. However, being metal-based nanoparticles, the challenge of bioaccumulation and addressing irregular pharmacokinetics will be required, before this formulation may move to the next phase of possible clinical development.

In another work, mice with diet-induced steatohepatitis were treated with macrophages modulating transcription factor X-box binding protein 1 (XBP1) siRNA-loaded folic acid-modified tocopheryl polyethylene glycol succinate-based nanoparticles (FT@XBP1). Mice subjected to the feeding model had high quantity of Firmicutes, Blautia and Bacteroides, and lower relative abundance of Bifidobacterium. FT@XBP1 treatment significantly reduced weight gain, lipids, cytokines extrahepatically, and ER stress, fat accumulation, and fibrosis in the liver. Steatohepatitis in mice led to high Blautia and Bacteroides, and lower levels of Actinobacteriota, Muribaculaceae and Bifidobacterium, and were reversed by FT@XBP1 treatment. FT@XBP1 enhanced the expression of zonula occludens-1 (ZO-1) in the intestine, restored intestinal barrier integrity, and hepatic inflammation indicating promise for the therapy of MASH[89]. Although tocopheryl polyethylene glycol succinate-based nanoparticles are easy to formulate and provide the added advantage of anti-inflammatory effects, their low loading capacity remains a major hurdle for clinical translation of such formulations.

Yoon et al[90] evaluated the effect of probiotic treatment with Bifidobacterium breve and Bifidobacterium longum and nanoparticles based on these strains in mice with diet-induced steatohepatitis and explored the molecular mechanisms using multi-omics approaches. The metagenomic investigations revealed a significantly higher relative abundance of the Bacteroidetes phylum in the stool of control and probiotic-treated groups mice. Similarly, gut metabolomics evidenced that SCFAs and tryptophan metabolites were repleted to normal levels by probiotics, whereas bile acids were partially normalized. Further, the levels of inflammatory cytokines such as tumor necrosis factor-alpha were significantly decreased, demonstrating promise for treatment of patients with MASLD. In another work, a prebiotic-based atorvastatin nano-amorphous (PANA) formulation was developed to improve the efficacy of atorvastatin against diet-induced steatohepatitis in mice by improving liver and gut health. Oral treatment of PANA increased drug concentration in the liver compared with pure drug. Further, PANA intervention effectively restored gut health, indicated by increased beneficial microbes and decreased deleterious microbes, and improved intestinal immunity, barrier integrity, and inflammation. In comparison with the free drug, PANA reduced body weight, lipids, steatosis, and inflammation. PANA reduced the M1-type monocytes, and M1/M2 ratio, and lowered pro-inflammatory factors indicating that the nanoformulation ameliorated the bioavailability leading to improved efficacy compared with the free drug[91]. However, their stability, shelf-life, risk of immunogenicity, and correct analytical characterization remain regulatory and translational challenges, which should be adequately addressed before such formulations may move to the next stages of development.

Ren et al[92] developed a prebiotic-like silybin-2-hydroxypropyl-β-cyclodextrin inclusion (SHβCD) nano-complex of silybin to increase efficacy. The results showed that silybin formed a 1:1 stoichiometric inclusion complex with high solubility and bioavailability. In HFD-fed hamsters, SHβCD restored the gut microbiota and intestinal integrity. SHβCD was more effective in decreasing hepatic lipid content, oxidative stress, and inflammation than silybin without nano-complex. Hepatic transcriptomics revealed reduced inflammation and metabolism. This work sheds light on the utility of nano-cyclodextrin-based complexes for the therapy of MASLD. β-carotene is a unique phytochemical, which has been shown to reduce several metabolic diseases. However, it has poor oral bioavailability due to hydrophobicity and environmental sensitivity. Dong et al[93] synthesized cationic lipid-assisted nanoparticles for delivery of β-carotene. In a diet-induced liver disease mouse model, β-carotene nanoparticles reduced the development of insulin resistance, inflammatory injury, and hepatic steatosis. β-carotene nanoparticles attenuated the phosphatidylinositol 3-kinase/serine/threonine kinase/mechanistic target of rapamycin pathway and peroxisome proliferator-activated receptor gamma gene expression involved in steatosis development. In addition, the nanoparticles remodeled the composition of gut microbiota in mice with diet-induced steatohepatitis by reducing the Firmicutes/Bacteroidetes ratio and enhancing the abundance of Ligilactobacillus, a beneficial bacterium known to enhance SCFA production. Carotene is prone to oxidation, and has poor water solubility and on the other side cationic lipids may trigger toxic reactions. These hurdles remain a bottleneck for the wide scale application and clinical translation of carotene-based nanoparticles for liver diseases.

Hydrogen-rich water mildly reduces liver fat in patients with MASLD[94]. However, 1 L hydrogen-rich water delivers only a maximum of 1.6 mg hydrogen gas. Jin et al[95] produced a hydrogen-based nanocapsule by loading ammonia borane inside mesoporous silica nanoparticles for high and prolonged hydrogen-release in the gut. The hydrogen-nanocapsule reduced diet- and genetic (db/db)-induced steatohepatitis. It enhanced the abundance of Akkermansia muciniphila. Further, hepatic transcriptomics showed improved liver metabolic profile with lower lipid synthesis and increased fatty acid catabolism indicating potential benefits against MASLD. Poor hydrogen loading, need for functionalization, biocompatibility, and scale-up costs remain major challenges for further development of this formulation.

Nanocarriers for the treatment of ALD

Astaxanthin (AXT), a novel phytochemical, has been shown to reduce ethanol-induced liver disease in mice by lowering ROS, and lipids[96]. However, it has poor oral bioavailability. In an interesting work by Huang et al[97], whey protein isolate and galactose were used to synthesize dual-targeted AXT-loaded nanoparticles. Lactobacillus plantarum ghosts (LPGs) contain bacterial structure as a biomimetic strategy to deliver AXT. The use of LPGs improved the absorption, and enhanced its intestinal adhesion. AXT-loaded nanoparticles/LPGs significantly attenuated the expression of oxidative stress-related markers to improve ethanol-induced liver disease in mice.

During ALD, gut dysbiosis-induced disruption poses a major pathological challenge. Previous research indicates that probiotic Lactobacillus rhamnosus GG has beneficial effects in experimental ethanol-induced liver disease in mice by improving the intestinal barrier function. Gu et al[98] showed that exosome-like nanoparticles (ELNs) derived from Lactobacillus rhamnosus GG increase tight junction proteins and lower LPS-induced inflammation in macrophages. In vivo, oral treatment reduced ethanol-induced gut-barrier dysfunction, liver steatosis, and inflammation. Administration of bacteria producing aryl hydrocarbon receptor agonists improves ethanol-induced liver disease[99]. Nuclear factor erythroid-2-related factor 2 was reported to be involved in the inhibition of bacterial translocation and endotoxin release in ethanol-treated mice. This protective effect was mediated by increased tryptophan metabolites released by bacteria indicating the potential value of this approach for the treatment of human ALD[98]. The heterogeneity, batch-to-batch variation, risk of immunogenicity, lack of mechanistic understanding, and scaling remain challenges for ELNs-based formulations.

Plant-based ELNs contain various bioactive agents. In a recent work, Goji berry (Lycium barbarum L., Wolfberry)-derived ELNs were synthesized and trialed in ethanol-fed mice. Treatment with ELNs increased the expression of hepatic antioxidant-associated genes and gut barrier-related genes. Further, 16S ribosomal RNA sequencing revealed that ELNs altered gut microbiota by increasing the relative abundance of Akkermansia. In addition, metabolomics showed increased L-asparagine, L-serine, D-glucuronic acid, as well as unsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid in the mouse cecum, which affect the biosynthesis of leukotriene, a key metabolite involved in chronic inflammation, indicating the potential mechanism of action of ELNs[100]. These studies demonstrate the role of nanocarriers in modulating the gut microbiome to improve ALD. Further, in silico and AI-driven strategies might provide sharper tools for drug delivery and improved pharmacological effects to manipulate gut microbiota. However, as mentioned earlier overcoming the delivery and safety challenges associated with ELNs remains an area of concern for clinical translation of these nanoparticles.

Nanocarriers for the treatment of HCC

HCC is one of the most aggressive cancers, posing a severe threat to health and survival. The heterogeneity of the tumor tissue, intratumoral fibrosis, patient-specific genetics and epigenetic factors make it an extremely challenging clinical condition[101]. A wide variety of novel interventions have been explored for the therapy of HCC such as eradication of resident cancer stem cells, photothermal therapy, actively targeting nanoparticles, and epigenetic therapy among others[102,103]. Unfortunately, current pharmacotherapies for HCC cause severe side-effects and have limited efficacy. There has been growing interest in natural food-derived nanoparticles due to their potential therapeutic benefits, including anticancer and anti-inflammatory activities. In a recent work, Zu et al[104] extracted and purified natural ELNs from Phellinus linteus. ELNs were found to contain different types of functional components, such as lipids and pharmacologically active small molecules. In vitro, ELNs showed high internalization efficiency in Hepa 1-6 liver tumor cells and elicited strong anticancer effects. In addition, in an in vivo HCC model, ELNs increased ROS and improved the gut microbiome. The ELN increased beneficial bacteria, such as Lactobacillus, Turicibacter, and Enterorhabdus. Lactobacillus which are known to increase tumor necrosis factor-related apoptosis-inducing ligand production, causing high cell-mediated cytotoxicity. Turicibacter is responsible for synthesis of the SCFA butyric acid, which has strong immunomodulatory properties. The low loading capacity, risk of immunogenicity, and variations in batch uniformity remain critical regulatory hurdles for clinical translation of ELNs.

Bacterial magnetosomes (BMs) are organelles produced by magnetotactic bacteria for living in response to geomagnetic fields. BMs have two components: Biomineralized inorganic magnetic crystals (either Fe₃O₄ or Fe₃S₄) and an organic magnetosome membrane made of phospholipids and proteins. BM synthesis is regulated by biomineralization providing limited size distribution, uniform morphology, good magnetic properties, and crystal structure. Harnessing this property, doxorubicin- and daunorubicin-loaded BMs were prepared and showed enhanced anticancer efficiency in a mouse model of breast cancer. The formulation showed enhanced growth inhibitory effect in vitro and in vivo without any toxicity to normal tissues. The drugs-loaded BMs induced p53 and Caspase 3 gene expression and downregulated Myc expression, leading to apoptosis[105]. The low yield of BMs, genetic instability, challenges of purification, endotoxin contamination, and susceptibility to oxidation are pertinent formulation challenges for the clinical development of BMs.

Aspartate β-hydroxylase (ASPH) is overexpressed on both murine and human HCC cells. In one study, nanoparticle lambda (λ) phage vaccine constructs were prepared against ASPH-positive liver cancer. The HCC mice were immunized before and after the subcutaneous implantation of a syngeneic C57/Bl mouse-derived BNL 1ME A.7R.1 HCC cell line. Prophylactic and therapeutic immunization inhibited HCC growth and progression. High numbers of ASPH-antigen-specific CD4+ and CD8+ lymphocytes were observed in the spleen. Furthermore, vaccination led to antigen-specific T helper 1 and T helper 2 cytokine secretion by immune cells, indicating the potential of phage-based nanoparticles for the delivery of agents for treatment of HCC[106].

Bioengineered bacterial strains can effectively target and accumulate within tumor tissues, induce an immune response, and be used as novel drug delivery vehicles. However, conventional bacterial therapy has challenges, such as low drug loading capacity and difficulty to release cargo, leading to poor therapeutic outcomes. Yang et al[107] constructed a non-pathogenic Escherichia coli inspired drug/gene delivery vehicle and an in-situ hepatitis B surface antigen producer. Polyethylene glycol (CHO- polyethylene glycol 2000-CHO)-poly (ethyleneimine) (polyethyleneimine 25k)-citraconic anhydride-doxorubicin nanoparticles loaded with plasmid-encoded human sulfatase-1 enzyme were linked on the surface of antigen. In a murine HCC model, the nanoparticles actively targeted and colonized tumor sites. The Ag significantly increased cytokine secretion and the intratumoral penetration of CD4/CD8+ T cells, providing a strong immune response. At the same time, the nanoparticles released human sulfatase-1 and doxorubicin into the tumor with high anticancer effects, presenting a novel approach for the therapeutic intervention of HCC. The cytotoxicity of polyethyleneimine, plasmid stability, off-target effects, and heterogenous release due to varying pH of the tumor tissue remain some of the preclinical hurdles that need to be addressed before this formulation may see the light of the day.

Reduction of the liver inflammatory and immunosuppressive microenvironment inflicted by the gut microbiota-secreted LPS is critical for inhibiting HCC and metastasis. However, synergistically targeting LPS-mediated inflammation and gut microbiota is a challenging feat. Yao et al[108] developed a “Trojan-horse strategy” with an oral dextran-carbenoxolone conjugate. They mixed prebiotic and glycyrrhetinic acid homologs, for targeted delivery of glycyrrhetinic acid to HCC. In an HCC model, a reduction in the numbers of LPS-associated microbiome was seen, especially Helicobacter. A high increase (37-fold) in the abundance of Akkermansia, was detected, which has been shown to increase immune response. In addition, dextran-carbenoxolone increased natural killer T cells (5.7-fold) and CD8+ T cells (3.9-fold) and reduced M2 macrophages (59% reduction). These studies clearly indicate the potential of nanocarrier-based therapeutics for the treatment of liver cancer and their potential for further refinement and tailoring. AI-driven approaches can massively aid in the synthesis of nanosystems that can effectively target the tumor tissue as well as the microbiome at the same time, providing higher accuracy with enhanced therapeutic effects. The cytotoxicity and side-effects of carbenoxolone, limited release, cumbersome complex-design, and its stability are major challenges for the future development of this formulation.

CLINICAL TRANSLATION AND REGULATORY CHALLENGES

The preclinical research has demonstrated promising benefits of gut microbiome-specific nanoparticle-based therapeutics in mouse models of liver disease; however clinical translation remains in its infancy. Till date, there are no FDA-approved nanoparticle-based formulations that specifically target the gut-liver axis, and clinical trials are scarce. Most existing human studies involving microbiome-based or microbiome-inspired targeted approaches are exploratory and are yet to be studied in the context of MASLD or MASH[109]. There are several key challenges with microbiome-based nanoparticle formulations. One of the critical barriers is the risk of immunogenicity. Nanoparticles, especially those with cationic or synthetic polymeric surfaces, may cause activation of innate immune responses or complement pathways, potentially increasing liver inflammation. Further, batch-to-batch variability in nanoparticle production, especially in formulations involving biological components (e.g., prebiotics, bacterial components), challenges reproducibility and scalability. This is one of the important considerations for regulatory approval, as Good Manufacturing Practice-compliant synthesis of microbiome-based nanoparticles requires stringent control over physicochemical parameters such as size, charge, and ligand density.

The regulatory ambiguity around these formulations also complicates drug development. These formulations occupy a hybrid space between biologics, drugs, and devices, lacking clear regulations from the FDA or European Medical Agency. In addition, the lack of standardized biomarkers for microbiome-liver interactions also limits the design of clinically meaningful endpoints for trials. Overall, bridging the gap between preclinical promise and clinical applicability of nanoparticle-based formulations for CLDs will not only require robust safety and efficacy data in humans but also cutting-edge innovations in nanoparticle design, industrially scalable production, and regulatory alignment.

FUTURE PERSPECTIVES AND THE ROLE OF AI

The complex pathology of CLDs reflects the intricate interplay between genetics, immune dysregulation, metabolic disturbances, and environmental factors, particularly the gut microbiome. The gut-liver axis plays a pivotal role in these conditions, where microbial dysbiosis contributes to disease progression through mechanisms such as increased intestinal gut permeability, endotoxemia, and aberrant immune activation. Understanding these complex interactions has led to the exploration of nanoparticle-mediated therapies as a transformative approach to delivering microbiome-based therapeutics to precisely target gut-liver interactions. The encapsulation of probiotics, engineered bacteria, or drug molecules within nanoparticles has demonstrated potential in restoring microbial equilibrium, reducing hepatic inflammation, and ameliorating fibrosis. Fecal microbiota transplantation, though effective, remains limited by variability and regulatory concerns, but nanoparticle-encapsulated microbiota formulations could overcome these challenges by enabling controlled and targeted microbial delivery.

Clinical investigation for nanoparticle-based therapies is required in the future, yet major hurdles persist. The safety and immunogenicity of nanoparticle formulations, the potential unintended alterations in gut microbial ecology, and the long-term consequences of genetic and microbial interventions remain critical concerns requiring extensive evaluation. Additionally, regulatory complexities associated with the convergence of nanotechnology, microbiology, and gene therapy present significant barriers to widespread clinical adoption. However, the integration of AI holds immense promise in overcoming these challenges by optimizing every stage of therapy development, from designing precision-engineered nanoparticles with ideal physicochemical properties to predicting host-microbiome interactions and identifying optimal microbial consortia for therapeutic interventions.

AI-driven computational models can accelerate drug discovery, simulate nanoparticle interactions at the cellular level, and analyze vast clinical datasets to personalize treatment strategies. ML algorithms can further refine patient selection criteria for clinical trials, enabling a more targeted approach to therapy that accounts for individual genetic, metabolic, and microbiome profiles. As AI continues to evolve, its role in real-time monitoring of treatment efficacy through advanced imaging and biomarker analysis will facilitate adaptive therapeutic strategies, ensuring maximum efficacy with minimal side-effects. Despite the formidable challenges, the convergence of nanotechnology, microbiome science, and AI-driven personalized medicine offers an unprecedented opportunity to revolutionize the treatment landscape for CLDs. With continued advancements in biocompatible nanoparticle systems, refined microbial engineering techniques, and AI-powered predictive analytics, the future holds immense promise for transforming treatment of liver diseases into manageable, and potentially reversible, conditions. The path forward requires a multidisciplinary effort, bringing together hepatology, toxicology, microbiology, materials science, computational biology, and regulatory expertise to translate these groundbreaking innovations into safe, effective, and accessible therapies for patients worldwide.

CONCLUSION

The convergence of nanotechnology, microbiome science, and AI presents an unprecedented opportunity to revolutionize the treatment of CLDs by addressing their complex pathology at multiple levels. These conditions, driven by intricate interactions between metabolism, immune dysfunction, and gut microbiota, have long challenged conventional treatment approaches, necessitating innovative solutions that can precisely target disease mechanisms. Nanoparticles provide an advanced platform for targeted delivery enabling more effective and sustained therapeutic interventions. While clinical trials continue to explore the safety and efficacy of these cutting-edge strategies, challenges such as immunogenicity, scalability, regulatory approval, and interpatient variability must be systematically addressed to enable clinical translation.

AI is poised to play a transformative role in overcoming these barriers by optimizing nanoparticle formulations, predicting therapeutic responses, refining microbial consortia design, and personalizing treatment strategies through predictive analytics and ML models. AI-driven drug discovery, precision medicine, and real-time monitoring of therapeutic outcomes will further accelerate progress in this field, making previously untreatable liver diseases more manageable and potentially reversible. Despite existing challenges, the future of hepatology is poised for a paradigm shift, where engineered nanoparticle-based microbial therapies, guided by AI, will enable a more effective, individualized, and holistic approach to liver disease management. Continued collaboration across scientific disciplines, regulatory advancements, and technological breakthroughs will be essential to realizing this vision, ensuring that these promising therapies transition from experimental innovation to clinical reality.