Extracellular vesicle-mediated approaches for the diagnosis and therapy of MASLD: current advances and future prospective

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an asymptomatic, multifaceted condition often associated with various risk factors, including fatigue, obesity, insulin resistance, metabolic syndrome, and sleep apnea. The increasing burden of MASLD underscores the critical need for early diagnosis and effective therapies. Owing to the lack of efficient therapies for MASLD, early diagnosis is crucial. Consequently, noninvasive biomarkers and imaging techniques are essential for analyzing disease risk and play a pivotal role in the global diagnostic process. The use of extracellular vesicles has emerged as promising for early diagnosis and therapy of various liver ailments. Herein, a comprehensive summary of the current diagnostic modalities for MASLD is presented, highlighting their advantages and limitations while exploring the potential of extracellular vesicles (EVs) as innovative diagnostic and therapeutic tools for MASLD. With this aim, this review emphasizes an in-depth understanding of the origin of EVs and the pathophysiological alterations of these ectosomes and exosomes in various liver diseases. This review also explores the therapeutic potential of EVs as key components in the future management of liver disease. The dual role of EVs as biomarkers and their therapeutic utility in MASLD essentially highlights their clinical integration to improve MASLD diagnosis and treatment. While EV-based therapies are still in their early stages of development and require substantial research to increase their therapeutic value before they can be used clinically, the diagnostic application of EVs has been extensively explored. Moving forward, developing diagnostic devices leveraging EVs will be crucial in advancing MASLD diagnosis. Thus, the literature summarized provides suitable grounds for clinicians and researchers to explore EVs for devising diagnostic and treatment strategies for MASLD.

Introduction

Lifestyle is known to determine an individual’s quality of life by influencing various factors, including physical and mental well-being. Metabolic dysfunction-associated steatotic liver disease (MASLD) is a hepatic pathology in developed countries that affects approximately one-fourth of the population at the global level. In the rapidly growing era of the urban lifestyle, the adoption of a sedentary lifestyle accompanied by unhealthy dietary patterns has significantly contributed to health-related diseases, especially noncommunicable diseases [1, 2], such as obesity [3], diabetes mellitus [4], hypertension [5], dyslipidemia [6], cardiovascular diseases [7], metabolic syndrome [8], and musculoskeletal diseases [9]. Furthermore, it has also been shown to affect quality of sleep [10], life expectancy [11], productivity [12], and social interaction [13].

The liver, which is the metabolic center of the human body, is strongly affected by the combined effects of an inappropriate diet and a state of physical inactivity. It has been reported that overnutrition causes an energy imbalance in metabolic processes and the accumulation of fatty acids in the liver. In addition, reduced fatty oxidation in the liver as a result of physical inactivity exacerbates liver health [14, 15]. MASLD is one such outcome of hepatic pathology [16] and was previously known as nonalcoholic fatty liver disease.

MASLD is a liver pathology characterized by the sequential progression from simple benign to more severe forms and mainly consists of simple steatosis or metabolic dysfunction associated with the steatotic liver and metabolic dysfunction associated with steatohepatitis. MASLD is associated with additional complications, such as fibrosis, cirrhosis, and end-stage hepatocellular carcinoma (HCC) [17, 18]. It is a condition of hepatic steatosis that is primarily associated with cardiometabolic risk factors such as obesity, insulin resistance, dyslipidemia, and hypertension, with the exclusion of other identifiable causes [19].

According to a systematic review conducted in 2022, the worldwide prevalence of MASLD (NAFLD) is 32.4%, with an increase from 25.5% between 1990 and 2022. Although its prevalence has increased in women, it is lower than that in men [20]. In terms of mortality, there is a 1.93-fold greater risk of death in the MASLD population than in the general population, according to a study conducted in 2020 [21].

The pathology of this disease during the initial stage manifests as a silent killer with no potential symptoms but later results in severe health complications in the absence of an early diagnosis [22]. In such circumstances, histopathological analysis represents the sole diagnostic method for most hepatic diseases, and MASLD follows suit without exception. The conventional diagnostic method for MASLD involves a cell-penetrating method of reaping the tissue sample, which is often associated with the risk of death [23]. Even though biochemical parameters can elucidate some of the metabolic variations associated with hepato-pathophysiology, understanding the hepatic ailment by employing these parameters alone would be misleading, resulting in misinterpretations. Despite these noninvasive techniques, there is no single biomarker that accurately assists in diagnosis and staging, whereas imaging techniques lack sensitivity in detecting disease progression [24, 25]. Early diagnosis remains the only effective strategy for addressing the disease before it progresses too far.

Liquid biopsy is an emerging convenient alternative way of diagnosing and monitoring molecular changes associated with various diseases, including cancer [26, 27]. This minimally invasive technology simplifies the task of sampling and reduces the risk associated with diagnosis. Presently, the technique is widely utilized in cancer screening, particularly for screening genetic aberrations originating from any combination of components such as extracellular vesicles (EVs) [28,29,30], circulating tumor cells [31], and cell-free DNA [32]. These circulating components with greater accessibility can be utilized in real-time monitoring of disease progression, enabling the selection of appropriate personalized therapy [33].

EVs present in circulating blood can provide valuable information about the physiological status of parent cells [34, 35]. EVs are tiny, heterogeneously sized entities delimited by a lipid bilayer. These tiny vesicles, irrespective of their size, carry a variety of molecular cargos. They are incapable of replicating themselves, compelling these particles to obtain information about the parent cells [36]. EVs are primarily engaged in cellular communications. Additionally, they can influence cell signaling cascades, leading to the activation of multiple pathways, thereby participating in both normal physiological and pathophysiological functions [37]. EVs are a heterogeneous class of particles encompassing ectosomes produced by outward budding, whereas plasma membrane fusion of parent cells and exosomes originate in the endosomal network, which is released upon fusion of the multivesicular body to the plasma membrane, and apoptotic bodies, which are released as blebs of cells undergoing apoptosis [36, 38, 39].

Many molecular cargos, such as proteins, nucleic acids, and lipids, are enclosed within them, which can attain signature confirmation upon reaching certain physiological states or disease stages [40,41,42,43,44,45,46,47]. Even highly fragile molecules such as RNA can remain intact and protected from degradation by RNases when they are sorted and packed carefully within EVs [48]. Multiple studies have experimentally curated EVs as a source of biomarkers for liver-related diseases. EVs have been extensively explored in the past and continue to be investigated for the treatment of a variety of liver diseases, including MASLD [49], alcoholic fatty liver disease (AFLD) [50], drug-induced liver injury (DILI) [51], autoimmune hepatitis (AIH) [52], HCC [53], and viral hepatitis [54].

Review aim

The aim of this study was to explore and summarize recent advancements in the diagnosis and treatment of MASLD, with a particular focus on the role of EVs in clinical applications. This review expounds on commonly employed diagnostic biomarkers in clinical settings for MASLD diagnosis. Additionally, this study highlights recent discoveries of blood-based biomarkers with promising diagnostic potential for MASLD. With respect to EV-centered approaches, this review aims to understand the effective utilization of these cellular vehicles in MASLD diagnosis and therapy, covering current advances and prospects.

In line with this aim, the relevant literature was selected primarily from the PubMed database, with a focus on clinically significant data. The publications from 1999 to November 2024, with a particular emphasis on research from the past decade, were included. This approach aligns with trends shown in Fig 2., which demonstrate a substantial rise in studies related to MASLD in the last ten years, reflecting the growing public health focus on its diagnosis and treatment. Similarly, research on EVs has expanded rapidly over the same period, especially in the fields of diagnostics and therapy. The overlap in these research timelines provided a strong rationale for concentrating literature selection within this timeframe.

Extracellular vesicles

EVs, which were previously disregarded as cellular debris [55], are tiny, heterogeneous classes of naturally occurring nanoparticles delimited by the plasma membrane. These tiny particles lack the ability to self-replicate and are produced by parental cells to the extracellular spaces to perform a plethora of physiological functions [56]. The classification of EVs is still debatable; however, on the basis of the biogenetic pathway, EVs can be either ectosomes or exosomes [57]. Ectosomes are formed by the outward membrane blebbing of cells, whereas exosomes are formed by the inward blebbing of the endosomal membrane followed by the formation of multivesicular bodies, which release exosomes by exocytosis upon fusion with the plasma membrane [36, 58]. The biogenetic mechanisms of ectosomes and exosomes are intricate complex cellular mechanisms involving sorting mechanisms that specifically load specific molecular cargo, such as RNA, lipids, and proteins, into vesicles, making them distinct from parental cells [59]. Interestingly, exosome biogenesis is eukaryote specific, as it requires endosomes, whereas ectosomes are produced by both prokaryotic and eukaryotic cells [60, 61].

Biogenesis of circulating EVs

Biogenesis of ectosomes

Ectosomes were first described as subcellular particles derived from platelets in normal serum and plasma, and they were often termed “platelet dust” [55]. Later, ectocytosis was described using stimulated neutrophils. Several studies on ectocytosis termed them shedding bodies or shedding particles and oncosomes to determine their functions and roles in cellular communication. They are produced by a biogenetic process that involves the vertical transfer of molecular cargo to the plasma membrane, which is subsequently packed in lipid bilayer particles via a distinct pathway. Parent cells utilize a distinct contractile machinery that enables cells to pinch off these vesicles at the cell surface [62].

The complexity of this biogenetic pathway is intermediate and is neither as complicated as the biogenesis of exosomes nor as simple as the production of apoptotic bodies due to indiscriminate plasma membrane blebbing [63].

Ectosomes originate from membrane blebbing, which is usually associated with specific changes in the lipid and protein components at specific sites of the plasma membrane altering its properties, such as its rigidity and curvature [64]. The formation of ectosomes is achieved through the dynamic interplay of phospholipid redistribution and the contraction of cytoskeletal proteins [60].

A diverse range of eukaryotic cells produce ectosomes under normal physiological conditions as well as during disease conditions. Under disease conditions, the highly regulated biogenetic pathway can undergo abrupt changes, leading to the aberrant shedding of ectosomes [64]. The biogenetic pathway and the factors influencing the biogenesis of ectosomes under normal physiological conditions and under altered physiological conditions are summarized below.

Mechanism of ectosomes biogenesis under normal physiological conditions

Changes in lipid composition

The structural properties and shapes of lipids depend upon their hydrophilic head groups, hydrophobic acyl chain length, and saturation. The compositions of the inner and outer leaflets of the plasma membrane are distinct from each other; the inner leaflet predominantly harbors amino phospholipids such as phosphatidyl serine (PS) and the external leaflet is enriched with sphingomyelin and phosphatidylcholine. In general, vesicle formation is associated with a change in lipid composition assisted by PS and the local recruitment of lipid-modifying enzymes such as aminophospholipid translocases, flippases, floppases, gelsolin, scramblases, and calpains [61, 65]. The lipid composition is strongly influenced by these enzymes, in which flippases translocate specifically PS into the inner leaflet and floppases translocate lipids outward. However, the enzyme scramblase promotes the unspecific bidirectional distribution of lipids across the plasma membrane [65,66,67,68].

Membrane asymmetry collapses during ectosome biogenesis, with an increase in the cytosolic Ca²⁺ concentration activating floppases and scramblases while simultaneously inhibiting flippases. The biodistribution of PS induces signals that release ectosomes. The induction of the budding/vesicle formation signal occurs due to surface exposure of phosphatidylserine, wherein the translocation of PS occurs from the inner leaflet to the outer leaflet of the plasma membrane [69].

Activation of the contractile machinery

The formation of ectosomes is a well-orchestrated cellular event wherein phospholipid redistribution coincides with the contractile machinery, which is primarily governed by cytoskeletal proteins. The cytoskeleton contractile machinery relies on a set of enzymes such as ADP-ribosylation factor 6 (ARF6) and myosin light chain kinase (MLCK). ADP-ribosylation factor 6 (ARF6) is a small GTPase protein that activates Phospholipase D and activated phospholipase D recruits extracellular signal-regulated kinase (ERK). ERK recruited at the plasma membrane activates MLCK via phosphorylation [70]. The biogenesis of ectosomes is completed through cytoskeletal contractions regulated by enzymes that govern the interaction between actin and myosin [60]. The phosphorylation of MLCK at Thr18/Ser19 induces the actin-myosin-based cytoskeletal contraction by generating the necessary force required for ectosome budding/shedding [70]. This enhances the activity of myosin II and the enhanced activity of Myosin II enables it to engage in highly efficient interactions with actin filaments increasing the cellular contraction [71]. A study on the regulation of the Rho/MLC pathway by ADP-ribosylation factor 1 (ARF1) for controlling breast cancer cell invasion demonstrated that ARF1 also functions like ARF6 and plays a crucial role in the contractile machinery of the cytoskeleton [72].

Ectosome biogenesis in disease and altered physiological conditions

Ectosomes biogenesis can be abruptly altered under pathological and altered physiological conditions. Under altered physiological conditions, biogenesis can be affected by several factors. Some of the factors affecting the biogenesis of ectosomes and the biogenetic mechanism involved are discussed below.

ARRDC1-mediated ectosome biogenesis

Ectosome biogenesis invariably exploits the tumor-suppressing gene 101 (TSG101) protein, and the endosomal sorting complex required for transport (ESCRT) machinery to produce ectosomes. A study on arrestin domain-containing protein 1-mediated ectosomes (ARMMs) demonstrated that Arrestin Domain Containing 1 (ARRDC1) recruits TSG101 to the surface of cells to produce ectosomes. The ectosomes produced are distinct from exosomes, as they are devoid of late endosomal markers such as CD63 and lysosomal associated membrane protein 1 (LAMP1), indicating that these vesicles are released by direct plasma membrane budding [73].

Hypoxic ectosome biogenesis

Investigation of the role of hypoxia-inducible factors (HIFs) in breast cancer invasion and metastasis revealed that hypoxia in breast cancer cells induces an increase in the expression of the Ras-related protein Rab-22A (RAB22A) which colocalizes with increased expression of ectosomes formation. Moreover, RAB22A had a limited influence on ectosomes formation under nonhypoxic conditions. The study suggested selective recruitment of RAB proteins under hypoxia conditions for the shedding of ectosomes [74]. Hypoxia can exacerbate liver inflammation and fibrosis through the activation of hypoxia-inducible factors in MASLD.

Hyaluronan production and ectosome biogenesis

Hyaluronan synthesis coincides with various physiological events involving rapid tissue remodeling phases, such as embryonic development, inflammation, wound healing, and malignant tumor formation. Rilla et al. (2013) revealed that hyaluronan synthesis enhances the secretion of ectosomes. It is hypothesized that the ectosomes are shed either from tips of hyaluronan synthase (HAS)-induced microvilli or through budding of the plasma membrane. It is believed that cells that synthesize high quantities of hyaluronan generally harbor microvilli, which can serve as platforms for the formation of ectosomes. HAS activity is also influenced by cholesterol, and cellular cholesterol influences the secretion of microvesicles; thus, this study hypothesizes that microvesicle secretion occurs at the plasma membrane because of confirmational changes caused in lipid rafts due to HAS-induced hyaluronan synthesis [75].

RhoA-mediated ectosome formation

Ras-related C3 botulinum toxin substrate 1 (RAC1) and the Ras homolog gene family, member A (RhoA) signaling are important for promoting invadopodia or ectosomes in tumor cells. The Rho family proteins Rac 1 and Rho A act against each other, and the action of these proteins determines the switching of the tumor cell phenotype between ameboid and mesenchymal phenotypes, which are distinct from each other; the former is involved in the shedding of ectosomes and later in the utilization of invadopodia. Tumor cell-derived ectosome formation is driven primarily by the Rho-ROCK pathway, which involves ARF6 activation downstream [76]. RhoA-mediated ectosome formation may be actively involved in the production of cancer ectosomes in HCC.

Biogenesis of exosomes

Exosomes originate from the endosomes on exocytosis of multivesicular bodies. The biogenesis of exosomes is the most complex and well-coordinated cellular event. The complex cellular events of exosome biogenesis include several key events, such as endocytosis, early endosome formation, formation of multivesicular bodies (MVB), intraluminal vesicle (ILV) formation with molecular cargo sorting, multivesicular body maturation, and exosome release. Early endosome formation is the first step of exosome biogenesis and begins with the endocytosis, which can be clathrin-mediated, caveolin-mediated or clathrin-or-caveolin independent endocytosis [59].

Endocytosis and early endosome formation

Clathrin-mediated endocytosis

Cellular uptake was first visualized via glutaraldehyde fixation via electron microscopy in 1960, which led to the discovery of vesicles coated with proteinaceous substances. Clathrin was then identified as a major protein of proteinaceous coating around the vesicles being taken up. Clathrin-mediated endocytosis has been explained in detail in previous studies. It involves a clathrin-coated vesicle cycle with five stages: nucleation, cargo selection, clathrin coat assembly, vesicle scission, vesicle formation, and budding. Briefly, nucleation begins with membrane invagination driven by F-BAR (Fes/CIP4 Homology-Bin/Amphiphysin/Rvs) domain-containing proteins (FCHO proteins), epidermal growth factor receptor pathway substrate 15 (EPS15) and intersectins. The nucleation model then recruits clathrin for budding and adaptor protein complex 2 (AP2) for cargo selection. Clathrin then stabilizes the vesicle, while Dynamin enables scission. Heat shock cognate 70 (HSC70) disassembles the coat, allowing clathrin recycling [77].

Clathrin-independent endocytosis

Clathrin-independent endocytosis poses challenges due to membrane flexibility and restrictions in capturing molecular cargo in small areas. Caveolae-mediated endocytosis is one of the major types of clathrin-independent endocytosis machinery. Small pits on the plasma membrane characterized by proteins such as caveolin and cavins called caveolae can dynamically detach from the membrane to form endocytic carriers [78].

In addition to caveolae-mediated endocytosis, several clathrin-independent endocytosis pathways, including the clathrin-independent carrier/GPI-AP-enriched early endosomal compartment (CLIC/GEEC) pathway and the ARF6-associated pathway, are involved in endocytosis. The detailed mechanisms of these pathways are not well understood; however, reorganization of the actin cytoskeleton is a common key factor in all of these pathways [78, 79].

Early endosome formation

Endocytosis results in the formation of pleomorphic structures known as early endosomes. They play a central role in regulating the recycling and breakdown of membrane elements. Few components of early endosomes are recycled, whereas others are transported into trans-Golgi networks. The molecular cargo predetermined for late endosomes or EVs is sorted into intraluminal vesicles (ILVs), which results in the formation of multi vesicular endosomes (MVEs) [80]. Multivesicular bodies were initially considered important components of the endosomal lysosomal degradation pathway [81]. These multivesicular bodies have multiple fates; they can be sorted toward late endosomes, followed by delivery to lysosomes or the plasma membrane. The molecular cargo destined for degradation follows the former path, whereas the cargo involved in cellular communication through exosomes follows the latter path.

Cargo sorting, multivesicular body formation, and maturation

Although exosomes are tiny, they carry a wide variety of molecular cargo, including proteins, lipids, metabolites, and various forms of RNA, such as messenger RNA (mRNA), microRNA (miRNA), long noncoding RNA (lncRNA), circular RNA (circRNA), and PIWI-interacting RNA (piRNA) [59, 79, 82, 83]. The physiological state of parent cells from which exosomes are produced greatly influences the molecular profile of their cargo. Interestingly, exosomes attain definitive cellular functions on the basis of the molecular cargo they carry [84]. Hence, precise sorting of these cargoes is crucial for exosome biogenesis and function.

Protein cargo sorting

Ubiquitylation and farnesylation are two important posttranslational protein modifications that play prominent roles in the segregation of certain proteins into ILVs [85]. The sorting of molecular cargo occurs through ESCRT-dependent and ESCRT-independent pathways. ESCRT plays a key role in the formation of ILVs by incorporating specific protein cargo. The key components of the pathway include Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HRS/ESCRT0), ESCRT (I, II, III), ALG-2-Interacting Protein X (ALIX), and Syntennin-1 [86]. They play critical roles in membrane scission during ILV formation, cargo selection, incorporate syndecans and other cargos into ILVs, and help in the formation or secretion of exosomes.

ESCRT associated pathways can also be involved in the formation of ILVs. The syndecan-synthenin and Alix pathways and His-domain protein tyrosine phosphatase pathways also allocate ESCRT III to form ILVs. Syndecan-synthenin and Alix can sort proteins such as CD63, CD81, CD82, CD9, and fibroblast growth factor receptor (FGFR) [87, 88]. The ESCRT- independent pathway for the formation of ILVs involves components of lipid rafts such as ceramides. Ceramides actively participate in ILV formation by playing an important role in membrane budding and curvature. Tetraspanins such as CD63 and TSPN6 can also contribute to ILV formation independent of ESCRT. Chaperones such as heat shock protein 70 (HSP70), HSC70, and GPI-anchored proteins can co-sort cytosolic proteins into ILVs and facilitate the incorporation of lipid domains into ILVs.

Nucleic acid cargo sorting

RNA cargo sorting: RNA cargo can be sorted through multiple pathways, wherein RNAs can be directly incorporated into exosomes due to the presence of particular sequence motifs [89] or can be incorporated with the assistance of RNA-binding proteins such as RNA-induced silencing complex (RISC) and Argonaute 2 (AGO2) [90], ESCRT-assisted RNA sorting, or with the help of RNA binding proteins sequestered within tetraspanins enriched microdomains, or with the help of other proteins such as major vault protein and Y-box-binding protein 1 (YBX1) [91].

DNA cargo sorting: Although protein cargo sorting and RNA cargo sorting have been extensively studied, knowledge of sorting of DNA cargo into EVs is limited. Few recent studies have provided some insights into the potential mechanisms that might be involved in DNA cargo sorting. Yokoi et al. (2019) reported that in ovarian cancer cells, genomic DNA is sorted into exosomes through tetraspanins into multivesicular bodies where micronuclei formed during cancer collapse releasing genomic DNA, which is then shuttled to Multivesicular bodies. Similarly, mitochondria also serve as a precursor for DNA cargo for exosomes. The PTEN-induced putative kinase 1 (PINK1) protein released during mitochondrial damage facilitates the interaction of mitochondria and multivesicular bodies leading to the sorting of the mitochondrial cargo into MVBs [92]. Knowledge about the involvement of the ESCRT mechanism in DNA cargo sorting is lacking. However, some contradictory findings suggest that the extracellular secretion of DNA is histone-mediated and is exosome-independent in nature [93]. Future studies in this domain are essential for enhancing the understanding of the mechanisms involved in the sorting of DNA cargo into EVs.

Exosome release

Multivesicular bodies can attain a secretory or degradative fate, and MVBs destined to reach the secretory face translocate toward the plasma membrane and fuse with the plasma membrane marking the end of exosome biogenesis. Exosome release involves soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins which mediate membrane fusion events. The Fas/Fap-1/caveolin-1 cascade and long non-coding RNA HOX transcript antisense intergenic RNA (lncRNA HOTAIR) regulate SNARE formation in stem cells and hepatocellular carcinoma cells respectively [94, 95]. The fate of MVBs is also strongly influenced by cytoskeletal elements, such as actin and microtubules, which play crucial roles in transport, docking, and membrane fusion. Proteins such as Rab27a, Rab 7, and Rab 31 play essential roles in stabilizing docking sites, promoting exosome secretion, and interacting with motor cytoskeletal proteins. Additionally, divalent cations such as calcium ions play important roles in the regulation of Rab11-mediated exosome secretion pathways [59, 79, 84]. The biogenesis of ectosomes and exosomes is summarized in Fig. 1.

Fig. 1

Extracellular vesicle biogenesis. A Ectosome Biogenesis: Ectosomes are released upon membrane blebbing from the cells, which typically involves specific changes in the lipid and protein components at certain plasma membrane sites. During ectosome formation, horizontal cargo sorting is followed by ectocytosis. B Exosome Biogenesis: Exosomes originate from endosomes via exocytosis of multivesicular bodies. Exosome biogenesis involves several key events, such as endocytosis, early endosome formation, MVB formation, ILV formation with molecular cargo sorting, multivesicular body maturation, and exosome release. Endocytosis can be clathrin-mediated or caveolin-mediated or clathrin or caveolin-independent endocytosis

Clathrin-mediated endocytosis involves nucleation, cargo selection, clathrin coat assembly, vesicle scission, vesicle formation, and budding.

Caveolae-mediated endocytosis begins with invagination of the plasma membrane, which is rich in proteins such as caveolins and cavins. The caveolae then pinches off from the membrane to form vesicles that transport cargo into the cell.

Cargo Sorting, MVB Formation, and Exosome release: The molecular cargo within early endosomes is selectively sorted into intraluminal vesicles that form MVBs, which eventually fuse with the plasma membrane to release their contents as exosomes into the extracellular space.

Exosomes: Exosomes harbor diverse molecular cargo including nucleic acids, proteins and lipids.

EV biogenetic pathways: bridging MASLD pathogenesis

Careful observation of EV biogenesis pathways indicates that various molecular signatures overlap MASLD. For example, CD53, a tetraspanin membrane protein involved in EV biogenesis and immune function, has been upregulated in hepatocytes following a high-fat diet and inflammatory triggers. The inhibition of CD53 was found to prevent diet-induced fat accumulation and liver inflammation, highlighting its role in integrating metabolic and inflammatory signals in hepatocytes and its potential as a therapeutic target for conditions such as MASLD and type 2 diabetes [96, 97]. Similarly, other components of EV biogenesis are implicated in disease pathogenesis and are summarized in Table 1.

Table 1 Role of EV biogenesis components in Liver diseases

MASLD—a historical preview:

NAFLD-MASLD

Obesity, a major physical and physiological change, has been recognized as an important physiological event since prehistoric times. The prehistoric recognition of obesity is evident from the “Venus figurines” from the upper Paleolithic era, such as the Venus of Willendorf. While ancient civilizations, including those in Egypt and China, viewed obesity as a symbol of prosperity and fertility, the medical recognition of obesity began with the Indian physician Sushruta in the sixth century BC, who linked it to overindulgence and inactivity. European physicians and philosophers Hippocrates and Galen’s views on obesity were highly influential in medieval and renaissance Europe. They emphasized diet and exercise as primary ways to manage obesity, which has been practiced in medicine for centuries. Fatty liver disease was not identified as a distinct condition until the early nineteenth century [112]. From 1975–2018, global obesity rates tripled, coinciding with the introduction of food rich in high-fructose corn syrup [113]. Initially, the effects of these diet forms were directly linked with obesity; however, it took a long time to confirm the role of such dietary regimens in metabolic syndrome-related diseases such as MASLD. Although there is historical evidence for fatty liver disease, recent findings define the impact of obesity on fatty liver disease, providing a crucial connection. MASLD is a recently identified condition that is significantly driven by obesity. While the ancient people recognized and recorded obesity, related conditions such as MASLD reveal the long-standing consequences of obesity, which can be traced back to historical observations and practices concerning weight and health. Obesity has been prevalent since prehistoric ages; however, MASLD as a disease has been overlooked by the medical community.

Historical records suggest that the autopsy studies carried out during the nineteenth century revealed that hepatic steatosis was a common ailment affecting one-third of French and German populations, predominantly women and tuberculosis patients [112]. The earliest use of the term “fatty liver” dates to 1825, in Louis’s textbook of anatomy and pathology. It was then Thomas Addison in 1836 who introduced the term “fatty liver,” relating it to the presence of tuberculosis and alcohol consumption through histological differences [114]. Much more emphasis has been placed on understanding the mechanism of cirrhosis, as the initial liver manifestations leading to cirrhosis were not known at the time. Most diagnoses occurred at this advanced stage by 19th-century researchers, which ultimately led to the discovery that fatty infiltration in the liver due to metabolic disorders or alcoholism causes cirrhosis.

In the 1960s, "fatty liver hepatitis" emerged in the German literature, where the histopathological description of the liver with necroinflammation in obese individuals distinguished it from alcoholic steatohepatitis. In 1980, Ludwig et al. used the term nonalcoholic steatohepatitis (NASH) for the first time after inspecting liver biopsies of 20 patients who presented similar traits such as alcoholic steatohepatitis, including significant fat accumulation in the liver with signs of lobular hepatitis, focal necrosis, mixed inflammation, and often Mallory bodies, mostly in obese women with mild liver functional abnormalities and common fibrosis [115]. The term NAFLD was introduced to hepatology by Fenton Schaffner in 1986, and NASH progression to fibrosis and cirrhosis was reported by Randall Lee (American pathologist) in 1989 [116, 117]. Recently, the term NAFLD was changed to MASLD and NASH, now replaced with the term MASH) in early 2020s to better reflect the root cause for the disease, including cardiometabolic risk factors, and to reduce stigmatizing language associated with the words “nonalcoholic" and "fatty.". This change from NAFLD to MASLD was driven by global collaborative efforts led by the American Association for the Study of Liver Diseases (AASLD), the European Association for the Study of the Liver (EASL), and Asociación Latinoamericana para el Estudio del Hígado (Latin American Association for the Study of the Liver) (ALEH) with the Delphi proceedings to achieve consensus among experts from various fields [118, 119].

MASLD diagnosis

The understanding and diagnosis of MASLD have significantly evolved over the past 5 decades. The earliest milestone in diagnosing this condition dates to the post-World War II era when it was observed that nonalcoholic individuals exhibit symptoms similar to those caused by alcohol. The drastic shift in research focus toward understanding MASLD progression and establishing diagnostic criteria occurred in the 1990s leading to the development of histological grading and staging systems for MASH and the assessment of steatosis, ballooning, inflammation, and fibrosis [120]. MASLD scoring systems were introduced in 1999 by the NASH Clinical Research Network with standardized methods and protocols to quantify disease activity, disease stage, and fibrosis which essentially guided clinical trials and research [121].

The genomic components of MASLD began to be elucidated in the early 2000s, with significant advancements in 2008, with the identification of the PNPLA3 gene as a key factor in increased hepatic fat content [122, 123]. This discovery highlighted genetic predisposition to NAFLD along with the subsequent discovery of influential genes such as TM6SF and GCKR [124,125,126]. The increasing prevalence of MASLD has prompted the scientific community to focus on its diagnosis, highlighting several noninvasive diagnostic methods between 2007 and 2015. Noninvasive diagnostic methods such as the NAFLD Fibrosis Score (NFS), Fibrosis-4 (FIB-4) index, and vibration-controlled transient elastography were developed and became popular during this period [24, 127, 128]. These noninvasive tools have increased the ability to accurately diagnose advanced fibrosis without the need for liver biopsy. By 2015, fibrosis was identified as a crucial prognostic and diagnostic indicator in MASLD and was utilized for predicting overall and liver-specific mortality. Following 2020, the focus remained on refining and sensitizing the noninvasive diagnostic tools and developing advanced technologies aided with machine learning algorithms and accurate histological assessments. Several research consortia such as Liver Investigation Testing Marker Utility in Steatohepatitis (LITMUS) and Non-Invasive Biomarkers of Metabolic Liver Disease (NIMBLE) are aimed at identifying new biomarkers and validating these biomarkers for the diagnosis of early MASLD stages such as MASH, to reduce the reliance of the whole diagnostic sector for MASLD on invasive liver biopsy procedures. However, despite these recent developments in MASLD diagnostics research, liver biopsy continues to be the gold standard for diagnosing MASH and early-stage fibrosis which emphasizes the need for further advancements in diagnostic methodologies. A historic preview of MASLD from prehistoric times to the present day is presented in Fig. 2.

Fig. 2

Historic preview of MASLD- The understanding of MASLD pathology and the development of therapeutic modalities for MASLD have evolved from prehistoric times to the present day. The historical overview of MASLD highlights the key milestones in understanding of the disease

Guidelines for the diagnosis of MASLD

The diagnosis of MASLD is guided by protocols established by several prominent liver disease associations. These guidelines are produced by the EASL, the Asia–Pacific Working Party on NAFLD (APWP-NAFLD), the American Association for the Study of Liver Diseases (AASLD), the National Institute for Health and Care Excellence (NICE), and the Italian Association for the Study of the Liver (AISF). Each of these organizations provides comprehensive criteria and diagnostic tools, which are summarized in Fig. 3, offering a consolidated reference for clinicians to accurately diagnose MASLD.

Fig. 3

a Guidelines for MASLD diagnosis produced by the European Association for the Study of the Liver (EASL), b Asia–Pacific Working Party on NAFLD (APWP-NAFLD), c American Association for the Study of Liver Diseases (AASLD), d National Institute for Health and Care Excellence (NICE), and e.) Italian Association for the Study of the Liver (AISF)

MASLD diagnosis and challenges

MASLD is a silent nonsymptomatic disease generally diagnosed through unintentional clinical or imaging tests. Although it is a slowly progressive disease with no symptoms during the early stage, fatigue, abdominal discomfort, and jaundice are the initial common indicators for suspecting the presence of disease [129]. In addition, some risk factors such as dyslipidemia, obesity, insulin resistance, type 2 diabetes, metabolic syndrome, improper diet, physical inactivity, and sleep apnea make individuals more prone to the disease than the general healthy population [130].

Existing diagnostic tools

Current diagnostic approaches for MASLD include various invasive and noninvasive techniques.

Noninvasive diagnostic approach-MASLD scoring systems using blood-based biomarkers

The diagnosis of MASLD usually starts with elevated liver enzyme levels, typically elevated alanine aminotransferase (ALT) levels compared with aspartate aminotransferase (AST) levels. The use of these enzyme levels as markers, along with other markers, such as gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP), provides a comprehensive understanding of the liver condition. Although ALT is liver specific, it is also altered in non-MASLD conditions. Furthermore, owing to its low specificity, MASLD cannot be solely dependent on liver enzymes [131, 132]. Even though ALT is frequently used as a biomarker due to its affordability and availability, its results depend upon overall liver function, and it may not exclusively indicate MASLD. For example, ALT has also been utilized in determination of metabolic syndrome [133]. Similarly, several stage-specific and disease-specific molecular signatures for MASLD have been identified in the past decade. The noninvasive blood-based molecular signatures for MASLD with sensitivity and specificity and their expression patterns in disease patients and relevant studies are summarized in Fig. 4 and Supplementary Table S1 [134,135,136,137,138,139,140,141,142,143,144,145,146,147].

Fig. 4

a Biomarkers for MASLD: Sensitivity and specificity with the cutoff value for noninvasive blood-based biomarkers of MASLD-Fibrosis Adult population. b Biomarkers for MASLD: Sensitivity and specificity with the cutoff value for noninvasive blood-based biomarkers of the MASLD—Fibrosis Pediatric Population. c Biomarkers for MASLD: Sensitivity and specificity with the cutoff value for noninvasive blood-based biomarkers of the MASLD spectrum specific adult population. d Biomarkers for MASLD: Sensitivity and specificity with the cutoff value for noninvasive blood-based biomarkers of the MASLD- MASH- Adult population. e Biomarkers for MASLD: Sensitivity and specificity with the cutoff value for noninvasive blood-based biomarkers of the MASLD MASH- Pediatric population

However, the limitations associated with these biomarkers diminish their reliability when used alone for disease identification. Therefore, diagnostic scores or indices are usually used to diagnose the risk and degree of the disease, which are calculated by estimating the synergistic outcomes of clinical parameters and hematological parameters [148]. The diagnostic indices are generally calculated on the basis of the combination of specific panels pertaining to the organ of interest. A panel can be defined as a group of medical diagnostic tests generally recommended by physicians that can provide comprehensive information about a particular organ system, disease state, or function. The appropriate index for a disease state or condition is the one that considers a comprehensive range of diagnostic factors along with clinical parameters to assign a score. This approach ensures a more accurate and holistic assessment of the disease, considering various aspects of the patient's health and specific characteristics of the condition being evaluated. The indices and panels used to evaluate the MASLD risk are listed in Table 2.

Table 2 Noninvasive scores/indices and panels used to evaluate the risk of MASLD

Despite the availability of noninvasive scoring systems to evaluate the risk of MASLD, including the various common panels and indices listed in Table 2, many such statistically derived scoring systems have several limitations that prevent their reliance solely on them for the assessment of MASLD. A major limitation associated with the scoring system is population heterogeneity, as these indices are generally calculated on the basis of small, clustered populations in a hospital setting, and actual disease prediction and risk evaluation become challenging. The lack of validation studies in different populations and the ability to specifically differentiate the risk of MASLD from other liver diseases pose significant hurdles in this domain. Specificity could be achieved by the addition of MASLD-specific biomarkers to the scoring systems or panels, which again limits the widespread utility of such panels or scoring systems.

Noninvasive diagnostic approach—imaging techniques

The evaluation of liver health extends beyond the basic assessment of liver enzymes. In the absence of hepatitis B, C, or other causes of chronic liver disease, elevated liver enzymes drive the clinician’s attention toward identifying underlying conditions such as hepatic steatosis. However, diagnosis of such conditions requires more than just clinical suspicion, as it demands an accurate and efficient way of evaluating the disease. In such a scenario, the evaluation is carried out with the help of imaging techniques or histological techniques. The imaging techniques primarily employed include ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance elastography (MRE), and transient elastography (fibro scan). The commonly employed imaging techniques for MASLD diagnosis are summarized in Table 3. The widespread availability of these noninvasive tools makes them go-to choices for initial screening and diagnosis.

Table 3 Imaging biomarkers/devices/techniques used for MASLD diagnosis

However, despite their advantages, these methods often have drawbacks. Notable drawbacks of imaging techniques include operator dependency, noninterpretable results due to obesity, ascites, availability, operational cost, and sampling variability. Perhaps most crucially, they lack sufficient sensitivity to accurately identify MASH, which is characterized by inflammation and associated with risk factors such as fibrosis cirrhosis [191]. Therefore, for a comprehensive analysis of liver health, combining the morphological and functional status of the liver with the appropriate clinical history is important.

Histological techniques for MASLD

Histopathological studies involve invasive techniques such as liver biopsy which involves the excision of a piece of liver or liver tissue followed by microscopic examination by a pathologist for signs of MASLD. Liver biopsy, an essential diagnostic tool for MASLD and MASH reveals distinct histological characteristics. In MASLD, steatosis is characterized by the accumulation of fat droplets in the hepatocytic cytoplasm, which can be macro or microvesicular. MASLD is also defined by the presence of steatosis in at least 5% of hepatocytes. In addition to steatosis, MASH syndrome is also characterized by hepatocellular ballooning and lobular inflammation. Ballooned hepatocytes, which indicate hepatocellular injury, lack caspase 9 and are linked to the activation of the hedgehog signaling pathway. Furthermore, Mallory-Denk bodies (MDBs), which are cytoplasmic aggregates of keratins, ubiquitin, and p62, are not unique to MASLD and can also be found in other liver diseases. Lobular necroinflammation, which is primarily composed of mononuclear cells, is prominent in Zone 3 and tends to decrease in cirrhosis. Other histological findings include enlarged mitochondria (megamitochondria), glycogenotic nuclei, and occasionally portal inflammation. Fibrosis typically begins in Zone 3 and progresses to bridging fibrosis and cirrhosis, with pediatric patients often showing periportal fibrosis initially. These histological features are crucial for diagnosing and staging MASLD and MASH, providing valuable insights into disease progression and guiding treatment strategies [192]. It is considered the “gold standard” despite its demerits such as invasiveness, sampling error, patient discomfort and pain, and limited monitoring frequency [193]. Addressing the loop limitation, early detection of MASLD significantly contributes to effective management, supported by the accuracy of findings.

Artificial intelligence in MASLD diagnosis (prediction, diagnosis)

Recently, artificial intelligence (AI) has emerged as an effective tool for predicting and interpreting disease risk, the presence of disease, and patient prognosis. AI is a broad field of computer science consisting of various technologies aimed at performing tasks that require human intervention and intelligence. These technologies can be categorized into machine learning, deep learning, natural learning processing (NLP), robotics, and computer vision. Machine learning, a subset of AI enables the computer to make decisions based on the identification and data of the patterns rather than using technologies. This newly developed technique is widely used in radiological imaging, clinical diagnosis, medicine, risk stratification, etc.

AI can be directly or indirectly employed for the prediction or diagnosis of MASLD. AI plays a crucial role in developing machine learning models, followed by the utilization of such machine learning models to predict disease risk which facilitates the designing of appropriate interventions for overcoming the disease.

Machine learning models for the analysis of reports and results

AI can also be utilized to develop tools for improving interpretations, transparency, and generalizability to increase the efficiency of clinical decision-making. For example, deep learning algorithms can enhance the automated interpretation of elastography, MRI, and CT scan results. An elaborate review on the utilization of machine learning approaches as new tools for the histopathological diagnosis of MASH and MASLD is provided elsewhere with an emphasis on the algorithms and machine learning methods utilized for analysis of histopathological results and images [194].

Machine learning models for MASLD risk assessment and disease prediction

Machine learning models ease statistical analysis when trained well. Thus, machine learning models can accurately predict MASLD risk, providing preliminary insights towards detailed targeted liver examinations.

An investigation by Ma et al. involving 10508 patients, explored 11 machine learning algorithms to develop a diagnostic model for MASLD. They reported that Logistic Regression (LR) achieved 83.41% accuracy, whereas support vector machine (SVM) outperformed other methods in terms of specificity (0.946) and precision (0.725), and the AODE model exhibited the highest sensitivity (0.680). By utilizing the F-measure for analysis, the Bayesian Network (BN) model demonstrated the best performance, outperforming the Fatty Liver Index (FLI) by 9.17% in F-measure score, highlighting its potential for accurate MASLD diagnosis [195].

Docherty et al. (2021) attempted to develop a novel machine learning model to predict MASLD via data from the NIDDK and Optum databases which consist of training an extreme gradient boosting model (XGBoost). This model resulted in a sensitivity of 81%, and a precision of 81% in predicting MASH with high accuracy [196]. A recent similar study used an AI machine learning trained XGBoost model to predict high-risk MASH via NHANES 2017- March 2020 data which achieved high sensitivity (0.82), specificity (0.91), accuracy (0.90), and AUC (0.95), outperforming traditional biomarkers such as FIB-4, APRI, BARD, and MASLD fibrosis scores [197]. A recent approach to the identification of MASLD in patients with diabetes mellitus through machine learning approaches demonstrated high performance, with success rates of correctly identifying 82.24% (815/991) and 75.00%(586/744) of MASLD ( +) and MASLD (-) patients respectively [198].

Similarly, Hassoun et al. recently developed NAIF (NAFLD-AI-Fibrosis), a novel AI-based tool for accurate diagnosis of advanced liver fibrosis in the general adult population, which demonstrated superior sensitivity compared with traditional scoring methods such as the APRI and Fib4 (stage F3/F4). NAIF achieved 72% precision, 61% sensitivity, and 77% specificity using data from the NHANES database [199]. Machine learning models can be trained via XGBoost to detect the risk even in the absence of a few data sets. Specifically, applying explainable AI techniques in the medical field, such as sharply additive explanations, can improve the interpretability, transparency, and generalizability of machine learning models. Such models have enormous applications in medicine, as they facilitate clinical decision-making by converting clinical data into real-world applications. While hurdles and challenges remain, the use of AI in diagnosis is promising and warrants further exploration in the future for its potential application in the medical field. Given the lack of efficient therapies for MASLD, early and accurate diagnosis is essential. Therefore, the availability of various noninvasive biomarkers and imaging techniques plays a significant role in the diagnostic process worldwide.

Results of the Phase 3 Trial: Spotlighting the urgent need for surrogate Biomarkers in MASLD

Resmetirom liver-targeted thyroid hormone receptor-β selective drug was recently approved by the FDA as a new drug for MASH stage of noncirrhotic patients. The drug has shown its efficacy in reducing hepatic fat content, improving fibrosis and MASH resolution, and reducing liver damage [200]. The drug has shown acceptable safety, with mild to moderate common gastrointestinal adverse events. The long-term monitoring of the effect of drug in the MASLD population is essential to determine the effect of the drug on bone, thyroid and gonadal pathology. However, the clinical evaluation of the effect of a drug relies heavily on noninvasive liver fibrosis assessments, yet current imaging techniques have several limitations, making it challenging for clinicians to make informed decisions, and highlighting the urgent need for improved diagnostic markers [201]. Hence, establishing universally accepted parameters will help maintain consistency among healthcare workers and researchers, thereby improving the quality of patient care.

Extracellular vesicles in MASLD

Role of EVs in MASLD

The liver, the largest highly vascularized organ of the human body, is exposed to large amounts of circulating antigens and serves as frontline immune tissue [202]. It is the primary organ responsible for removing circulating EVs, which are mainly eliminated by liver macrophages called Kupffer cells [203]. The liver comprises a heterogeneous population of cell types, including hepatocytes, cholangiocytes, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), Kupffer cells, and a range of other immune cell populations, all of which secrete EVs [83, 204,205,206,207]. The number of EVs and molecular cargo that the individual EVs carry greatly depends upon the physiological status of the cells and is altered under disease conditions [44,45,46,47,48].

Recent studies have shown that liver macrophages [206, 208], hepatocytes [209, 210], and HSCs [211] are involved in the disease pathology of MASLD. The pathophysiology of MASLD is a complex process that initiates with the primary manifestation of hepatocyte cell death, followed by a substantial accumulation of inflammatory cells in the affected area. MASLD is strongly associated with obesity, elevated triglyceride levels, elevated ROS generation, oxidative DNA damage, and impaired hepatic catalase activity reflecting the failure of the antioxidant mechanism ultimately leading to Lipoapoptosis [212], Necroptosis [213] or Pyroptosis [214] of hepatocytes. Oxidative stress in MASLD is often associated with elevated activation of inflammatory pathways such as the c-Jun-N-terminal kinase (JNK)/NFκB pathway [215].

Liver cells are known to release EVs both in healthy individuals and in liver patients. The pathophysiological cascade initiated by liver damage significantly alters the nature, composition, and functional properties of the EVs produced by these cells. Since EVs are involved in intercellular communication, structural damage to parent cells alters EV communication. The EVs released from the damaged liver communicate with surrounding cells and influence the microenvironment within the liver. The inflammatory cells that accumulate at the affected pathological site also cause a significant change in the EV pool by producing many inflammatory EVs in the affected area.

The earliest evidence of EVs as disease indicators came from the study of an animal model fed a high-fat diet showing a significant increase in circulating EVs. This study further provided primary evidence for the severity of steatohepatitis in mice with increased circulating EV concentrations [216]. Furthermore, apoptotic bodies released by hepatocytes increase the expression of death receptor ligands in Kupffer cells inducing apoptosis of hepatocytes leading to inflammation and fibrosis [217]. Similarly, an in vitro study revealed that the application of membrane-bound microparticles derived from murine or human hepatocytes exposed to lipotoxic stress on endothelial cells demonstrated that these particles were proangiogenic, increasing the severity of the steatohepatitis [218].

Although the role of macrophages in exacerbating disease is well known, the precise role of EVs in disease remained unclear until a study by Kakazu et al. elevated this connection. They elucidated the role of EVs in bridging these gaps. The accumulation of saturated fatty acids such as palmitate, a precursor of ceramide, a lipotoxic lipid, leads to ER stress, a commonly observed condition in diseases such as MAFLD. These EVs are enriched in C16:0 ceramide and stimulate macrophage chemotaxis via sphingosine-1-phosphate (S1P) generation. Increased levels of C16:0 ceramide-enriched circulating EVs are observed in both mice and human NASH patents, suggesting their potential as bioactive biomarkers [219]. Several such studies have shown the role of lipotoxic hepatocyte-derived EVs in aggravating inflammation. For example, a study on hypoxia in a fat-laden hepatic cell in which hypoxia-inducible factor 1-alpha (HIF-1α), was stabilized revealed a significant increase in the number of EVs released. Hypoxia-induced promoted inflammatory signals and contributed to increased EV secretion. In addition, when EVs obtained from hypoxic fat-laden tissues were used to treat Kupffer cells, there were phenotypic occurrences of hypoxic conditions in Kupffer cells suggesting the impact of EVs on disease development through crosstalk [220]. The analysis of serum extracellular vesicles by Sakane et al., 2024 revealed that the presence of proteomic signature Fibulin 3 correlated with liver-related events including MASLD and fibrosis [49].

Furthermore, MASLD is associated with elevated levels of inflammatory cytokines including IL-6 and TNF-α [221]. Elevated inflammatory cytokines influence the release and composition of hepatocyte-derived EVs [222]. As mentioned previously, these EVs can exacerbate liver inflammation and promote apoptosis in surrounding cells. The dysregulation of autophagy followed by apoptosis increases lipotoxicity. The role of EVs in autophagy in MASH is elaborately explained elsewhere [223].

Muscles and the liver play crucial regulatory roles in metabolism, working together to perform key metabolic functions such as maintaining the energy balance and regulating of glucose and lipid levels. Dysfunction in any of these genes can aggravate the other, leading to a vicious cycle of muscle and liver deterioration. MASLD is also intricately interlinked with muscle pathology, particularly sarcopenia. MASLD and sarcopenia can coexist in both obese and nonobese individuals [224, 225]. Sarcopenia and obesity-associated MASLD share common pathological manifestations including muscle loss, metabolic dysregulation, inflammation, and insulin resistance. Lean MASLD patients experience muscle wasting due to altered inflammatory and metabolic signaling. Evidence suggests that EVs contribute significantly to the deterioration of skeletal muscles in sarcopenic conditions. The molecular payloads of such EVs can exacerbate key pathological events including inflammation, protein degradation, and mitochondrial functions leading to muscle atrophy and impaired muscle regeneration [226, 227]. Compared with obesity-associated MASLD, EVs are likely to contribute to muscle wasting in non-obese MASLD through different mechanisms. EVs in obesity-associated MASLD contribute to inflammation, insulin resistance, and lipid accumulation in muscles, whereas EVs in nonobese MASLD contribute to oxidative stress, impaired metabolism, and muscle regeneration. Similar to MASLD, a significant limitation associated with sarcopenia is the lack of diagnostic markers to support clinical investigations [228]. Recognition of these profound differences in the role in disease pathology could aid in understanding disease pathology and identification of new molecular signatures for this disease.

EVs as potential biomarkers

The accumulated evidence in the past indicates that EVs are instrumental in the pathogenesis of MASLD, underscoring their importance as biomarkers for disease evaluation. Several studies have identified EVs as promising biomarkers for MASLD.

TRAIL-enriched EVs

A study on how lipid-induced signaling aggravates the inflammatory response through EVs enriched with tumor necrosis factor related apoptosis-inducing ligand (TRAIL) released from hepatocytes demonstrated that lipotoxic stress induced by lipids such as palmitate and lysophosphatidylcholine (LPC) enhances EV secretion which in turn activates inflammation by inducing macrophage activation. The study also demonstrated that the inhibition of EV release ameliorates NASH in murine models. This study highlights the importance of TRAIL-enriched EVs as potential biomarkers for identifying novel therapeutic targets (inhibition of ROCK1-dependent release of EVs by hepatocytes) for NASH [229].

ITGβ1-enriched EVs

Monocyte-derived macrophages infiltrate the liver contributing to the inflammatory response in NASH (MASH). In 2019, Guo et al. reported that EVs enriched with Integrin Beta-1 (ITGβ1), which are released from LPC-treated hepatocytes, mediate monocyte adhesion and promote liver inflammation in a murine model of NASH. They used hepatocytes treated with either vehicle control or LPC for EV isolation and proteomic analysis. The diet-induced NASH murine model was then treated with an anti-integrin β1 (ITGβ1) neutralizing antibody (ITGβ1Ab) or a control IgG isotype. These findings suggest the presence of a new biomarker for NASH. This study also revealed that EVs derived from hepatocytes enriched with ITGβ1, regulate NASH inflammation and that antibodies against ITGβ1 ameliorate NASH in diet-induced murine models of NASH, suggesting a potential anti-inflammatory therapeutic strategy for NASH [210].

S1P-enriched EVs

Hepatocyte lipotoxicity leads to inflammatory macrophage effector responses during NASH. A study on EVs released from palmitic acid-treated hepatocytes revealed that they are enriched with sphingosine-1-phosphate (S1P) and are involved in recruiting macrophages to the liver. The study also demonstrated that EV S1P enrichment is largely influenced by the activity of enzymes upon sphingosine kinases 1 and 2 and that the pharmacological inhibition of these enzymes alleviated EV cargo enrichment and concomitant macrophage recruitment inferring that the sphingosine-1-phosphate (S1P) enriched EVs could be potential biomarkers and therapeutic targets for NASH [230].

Hepatic stellate cell (HSC)-derived EVs

Hepatic fibrosis involves excessive accumulation of extracellular matrix leading to scar formation in the liver mediated by activated HSCs under lipotoxic stress. Lipotoxic stress increases the secretion of exosomes carrying microRNAs by hepatocytes, which upon internalization can activate the proliferation and migration of HSCs. The internalization of EVs not only increases the proliferation and migration of HSCs but also affects the expression of profibrotic factors transforming growth factor-beta (TGF-β), cellular communication network 2 (CCN2), collagen type 1, and alpha-smooth muscle actin (α-SMA) [231, 232]. The isolation and characterization of such exosomes provide a new diagnostic opportunity for monitoring the progression toward fibrosis in MASLD. Similarly, recent studies on EVs secreted by healthy individuals have revealed that these EVs can inhibit progression toward fibrosis largely by alleviating the activation of HSCs or by suppressing the inflammatory pathway [233].

Liver sinusoidal endothelial cell (LSEC)-derived EVs

Chronic liver diseases such as nonalcoholic steatohepatitis cause fenestrated linings of liver arteries, and veins lose their discontinuity due to dedifferentiation of LSECs. LSECs form fenestrated linings in the arteries and veins of the liver. A study involving transcriptomic analysis of LSECs demonstrated that the EVs secreted by the LSECs were potent angiocrine effectors and had a deactivating effect on HSCs. The study also revealed several stage-specific proteomic signatures of EVs in chronic liver diseases revealing new therapeutic targets and potential biomarkers [234].

The accumulating literature clearly identifies tiny EVs as promising vehicles that carry enormous amounts of cellular information that could be exploited as biomarkers for MASLD. The unique characteristics of EVs and their potential advantages over existing biomarkers highlight their importance in diagnostic methods. The diagnostic potential of EVs has been clearly illustrated by a recent investigation by Jiang et al. on plasma exosomal metabolites derived from MASLD patients with impaired fasting glucose. An investigation revealed that exosomes derived from patients presented elevated levels of fatty acids, including linoleic acid, palmitate, ceramide, and oleamide, in their exosomes and reduced phosphatidylethanolamine (PE) levels. The detailed pathway analysis revealed altered linoleic acid metabolism as a characteristic feature of MASLD with impaired fasting glucose. These findings suggest the alteration in specific lipid components of EVs, clearly reflects the early metabolic dysfunction, providing valuable biomarkers for diagnosing disease progression [235].

EVs offer many advantages over existing biomarkers as they can increase the sensitivity and specificity of noninvasive diagnostics, stability, and long-term storage of EVs, enabling the development of standardized protocols and procedures for analysis. The diversity of EV molecular cargo offers a new benefit as it can potentially be utilized for multiparametric diagnostic analysis. The recent development of EV research involving the standardization of EV analysis protocols provides hope for the development of new diagnostic strategies for MASLD using EVs. Despite limitations such as lack of technical advancements for thorough analysis of EV molecular cargo, rapid progress promises a promising future for diagnostic of EVs. The swift advancements of multi-omic approaches for analyzing EVs and continuous research outputs in this domain suggest that EVs could play an essential role in next-generation diagnostic techniques offering more precise early detection of MASLD.

Challenges in utilizing EVs for MASLD diagnostics:

State of EV-based diagnostics for MASLD

Studies on EV-based diagnostics are largely in the proof-of-concept phase, with most studies identifying molecular signatures through omics approaches [236]. While these studies suggest certain molecular species are expressed differently in MASLD, translating this knowledge into clinically relevant methods remains challenging.

Selection and isolation of EV subtypes

A key challenge in EV diagnostics is the selection and isolation of EV subtypes. EVs display heterogeneity in size, composition and function. Isolating specific subtypes of EVs is crucial, especially for the evaluation of disease specific molecular markers. For example, isolating liver-specific EVs from biological fluids is essential for accurate disease profiling in MASLD. While liver-specific markers such as Asialoglycoprotein Receptor (ASGR) protein can assist in isolating liver-specific EV populations [50], incorporating this step would add further complexity to the overall EV isolation process. EV isolation techniques such as size exclusion chromatography (SEC) and ultracentrifugation (UC) are widely used, but they have notable drawbacks [237]. SEC separates EVs into different size fractions, so choosing the right fraction is crucial. Focusing on one fraction risks missing important molecular signatures in the others. Similarly, UC can cause EV rupture, leading to the loss of molecular cargo. Even though bulk precipitation methods are cost effective, they can often introduce several other contaminants further complicating their purity and accuracy.

Challenges in EV data and population studies

Existing databases such as ExoCarta and Vesiclepedia provide some basic information about EVs; however, extensive population-specific information, as seen in genetic databases, is lacking. Similarly, global data on EV cargo across different geographic and ethnic populations are lacking, necessitating extensive validation for EV-based markers.

Knowledge gaps in EV biodistribution and circulation

Factors such as the physiological state of patients, and time and day are poorly understood. Understanding these dynamics is essential for reliable diagnosis.

Need for clinical validation in large cohorts

Despite of the large amount of evidence from in vitro studies, there is a need for validation of these findings in patient-derived samples to understand complex human physiology across various diverse populations before moving into clinical settings.

Exploration of EVs for MASLD therapy

The existing therapeutic modalities for MASLD is are discussed elaborately elsewhere [238], and all the developing therapeutic modalities that are being tested in clinical trials are summarized in Table 4. The clinical trial data clearly suggest that the drugs being tested are specific for a few stages of MASLD. While most small molecules (drugs) have been developed to reduce the inflammation, fat accumulation, and scarring caused by fibrosis other interventions developed have focused on lifestyle modifications, such as diet, exercise, and probiotics. There is a need for precise stage-specific therapeutic modalities for increasing the life expectancy of individuals with MASLD. These synthetic drugs and suggested lifestyle modifications can improve the patient’s condition by slowing inflammation and preventing further accumulation of fat in the liver. In contrast, synthetic drugs do pose challenges in attaining the target that affects non-targeted cells, tissues, or organs causing unwanted side effects. Several siRNA-based therapeutic modalities have been developed for MASLD, even though they show increased efficacy, and fail to translate into the clinic. This is in part due to the failure of the delivery of therapeutic cargo to a suitable site. Similarly, the re-establishment of cellular physiology in the affected liver is possible only when the cellular components of the damaged tissue microenvironment are regenerated. Although the liver is the organ with the highest regeneration capacity, the functional retardation of the cellular components of the liver during MASLD reduces regeneration. These key challenges highlight the importance of MASLD therapeutics.

Table 4 Clinical trials on existing therapeutic modalities

The use of EVs as a therapeutic modality is one of the most promising strategies that is gaining importance as an answer to conquering diseases by providing an effective way of specifically treating disease alone. These membrane-bound, naturally produced lipid nanoparticles can protect the molecular cargo from degradation in the biological environment. The inherent property of EVs as cargo carriers is that they can be utilized for delivering therapeutic cargo to the site of action. The diverse range of molecular payloads, including proteins, lipids, microRNAs, and nucleic acids, is selectively, actively, or passively encapsulated in EVs, influencing their functionality. Moreover, EVs derived from stem cells inherently possess the potential to activate cellular regeneration. EVs harbor unique protein barcodes on their surface and can acquire definite bimolecular coronas depending on their surroundings which enables them to interact with the specific organ of interest or receptor of interest. Nucleic acids, chemotherapeutic drugs, small molecules, and even viruses can be essentially packed inside EVs and delivered to a targeted site. EVs are becoming therapeutic vehicles of great importance because of their biocompatibility, reduced immunogenicity, ability to cross biological barriers, versatility in cargo loading, etc. Next-generation EV therapeutics aim to utilize EVs fortified with therapeutic cargo or drugs for delivery to the target of interest to eliminate off-target effects. The inherent characteristics of EVs, including their cargo-specific therapeutic effects and site-specific actions due to protein barcoding, make them suitable candidates for developing therapeutic modalities for any disease.

EVs as therapeutic modalities can be utilized in two different ways: as delivery vehicles and as therapeutic agents. On the basis of these functions, EVs can be briefly classified into two different classes namely naturally occurring EVs and engineered EVs. Naturally occurring EVs carry endogenously packed cargoes derived from parents such as immune cells, and mesenchymal stem cells, with inherent therapeutic potential due to their origin. Whereas artificial/ engineered EVs are EVs that are altered through surface modifications through biological or chemical methods and are subjected to physical or biological treatments for loading the cargo of interest. Although there are multiple ways to load materials onto EVs, selecting a method is crucial for ensuring efficient loading. It depends on the physicochemical properties of cargoes, the source of cargo, and the EV subtype. The effectiveness of EVs as “delivery vehicles” and “therapeutic agents” is diversely supported by studies of different diseases.

EVs naturally produced by cells harbor enormous amounts of cellular components that increase their therapeutic value. The majority of MSC-derived EVs are being therapeutically employed for their potential immunomodulation or immunoregulation and regeneration. As of 2024, there are a dozen clinical trials ongoing in which EVs derived from MSCs have been employed for immune regulation and regenerative medicine. The therapeutic utility of MSC-derived EVs has been reviewed in detail elsewhere with relevant ongoing clinical trials [239]. The role of MSC-derived EVs as therapeutic agents in treating liver diseases is being thoroughly investigated, and the clinical trials registered to employ EVs for liver-related disorders are listed in Table 5.

Table 5 The role of MSC-derived EVs as therapeutic agents in treating liver diseases

EVs as potential therapeutic agents for MASLD

Although there is limited evidence for EVs as therapeutic modalities for treating MASLD, the accumulating evidence on the utilization of EVs for therapeutics and delivery systems in several liver diseases and in vitro models resembling MASLD suggests the scope of EV therapy for MASLD.

EVs in Hepatic steatosis and Inflammation therapy

MSC-derived EVs for Hepatic steatosis and Inflammation

Mesenchymal stem cell-derived EVs from various stem cell sources have shown anti-inflammatory and regenerative effects can be utilized as a therapeutic strategy for treating hepatic steatosis and inflammation. Hepatic steatosis and inflammation are the key pathological events occurring in the initial stages of MASLD, progressing toward severe stages such as fibrosis. Several studies have been conducted to assess the ability of MSC-derived EVs to ameliorate hepatic steatosis and inflammation.

As described in the previous sections, EVs derive their molecular cargo and functionality from parent cells. A study exploring the therapeutic utility of microRNA-136-5p in EVs derived from mice bone marrow-derived mesenchymal cells demonstrated that the inhibition of GNAS/STAT3 signaling pathway and lipopolysaccharide (LPS)-induced inflammation resulting in reduced liver inflammation and enhanced M2 macrophage polarization through the GNAS-mediated PI3K/ERK/STAT3 axis in an animal model of chronic liver damage induced by carbon tetrachloride [240].

A similar study on exosomes derived from human umbilical cord-mesenchymal stem cells (hUC-MSCs) demonstrated their efficacy in mitigating this disease. This study emphasized the influence of MSC-derived exosomal miR-24-3p on reducing lipid accumulation, oxidative stress, and inflammation, leading to improved hepatic function and decreased steatosis in both palmitate-treated mouse hepatocytes in vitro and a high-fat diet-induced NAFLD mouse model invivo. miR-24-3p exerts these protective effects by targeting Kelch-like ECH-associated protein 1 (KEAP-1) signaling, thereby attenuating hepatic lipid metabolism disturbances, inflammation, and oxidative stress [241].

A study on the direct use of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes for their therapeutic potential in nonalcoholic steatohepatitis (NASH) using an MCD-induced mouse model revealed that the intravenous transplantation of hUC-MSC exosomes improved body weight loss and liver damage induced by MCD in mice. Furthermore, it also reduced inflammatory cytokines in liver tissue and induced anti-inflammatory phenotypes in macrophages. Macrophage polarization was evident in both in vitro and in vivo experimental models. Exosomes were also capable of reversing the downregulation of PPARα protein expression in ox-LDL-treated hepatocytes in vitro and in vivo in NASH mouse livers [242].

The potential application of human placenta-derived MSC extracellular vesicles (hPMSC-EVs) in liver regeneration following hepatectomy was investigated in 2022 by Li et al. Intravenously administered hPMSC-EVs before partial hepatectomy could potentially improve liver regeneration in vivo and hepatocyte proliferation in vitro. These findings suggest that hPMSCs-EVs have the potential to prevent hepatic dysfunction and improve liver regeneration, possibly through circ-RBM23 delivery [243]. Similarly, a study on the delivery of inherent RNF31 through EVs derived from mesenchymal stem cells revealed that RNF31 delivery significantly improved liver function by alleviating hepatic steatosis in high-fat diet-fed mice [244].

Non-MSC EVs for hepatic steatosis and inflammation

Literature evidence suggests that breastfeeding reduces the risk of MASLD/NAFLD. A study on the effects of EVs derived from mothers’ milk on NAFLD model mice and primary hepatocytes treated with free fatty acid showed that the breast milk-derived EVs alleviated hepatic steatosis and insulin resistance in NAFLD mice by inhibiting lipogenesis and promoting lipolysis. These effects are likely due to EV cargo (proteins and miRNAs) related to lipid metabolism, suggesting a new therapeutic strategy for NAFLD treatment [245]. Exosomes derived from stem cells of the apical papilla (SCAPs) have also shown significant therapeutic potential for treating nonalcoholic steatohepatitis (NASH) in a methionine-choline deficient (MCD) diet-induced mouse model. A study on these exosomes demonstrated that the administration of SCAP-derived exosomes led to reduced liver damage and hepatic fat accumulation and improved lipid metabolism through the upregulation of p-AMPK and mitochondrial biogenesis factors [246].

Immune cells play a key regulatory role in exhibiting the immune cascade. Macrophage polarization is important in inflammatory and anti-inflammatory reactions. M2 macrophage-derived exosomes loaded with siRNA targeting RIPK3 significantly reduced pro-inflammatory cytokines, improved liver pathology, and balanced Th17/Treg cell ratios in a mouse model of immune hepatitis. These findings suggest that EVs can effectively deliver therapeutic agents to liver cells, suggesting a potential strategy for treating NAFLD [247].

EVs in fibrosis therapy

MSC derived EVs for fibrosis

EVs, a paracrine effector of MSCs, can be employed to regenerate the hepatic cell population for recovery from fibrosis. MSCs have already been explored as a therapeutic modality for various fibrotic conditions, including pulmonary fibrosis, spinal cord injury, scarring, and organ transplantation. Recent studies on the use of MSC-derived EVs in treatment of liver fibrosis have revealed that MSC-derived EVs as are potential therapeutic agents for liver fibrosis. A study on the efficacy of human AMSCs amniotic mesenchymal stem cell-derived extracellular vesicles (AMSC-EVs) in treating hepatic fibrosis revealed that AMSC-EVs delivered miR-200a into hepatocytes suppressing ZEB1/PIK3R3 axis. Suppression of the ZEB1/PIK3R3 axis reduces hepatic fibrosis by inhibiting its antifibrotic- effect [248].

A similar study explored the utilization of Wharton's jelly mesenchymal stem cell (hWJMSC-Exo)—derived exosomes for improving liver function and regeneration during liver fibrosis. This study demonstrated that delivering miR-124 via exosomes from human Wharton's jelly mesenchymal stem cells (hWJMSC-Exos) improved liver fibrosis. Exosomes enriched with miR-124 could significantly reduce inflammation and collagen accumulation. The levels of the fibrotic inflammatory markers IL-6, IL-17, TGF-β, STAT3, α-SMA, and COL in a CCl4-induced mouse model, were significantly decreased the administration of miR-124-enriched exosomes (ExomiR-124). The study also demonstrated that ExomiR-124 also promoted the shift of splenic monocytes from inflammatory to restorative phenotypes, confirming that ExomiR-124 is a promising antiinflammatory and antifibrotic therapeutic option for liver fibrosis [249].

A study investigating the therapeutic efficacy of MSC-derived exosome and medical ozone in a CCL₄-induced liver fibrosis rat model revealed superior efficacy of EVs over ozone in reducing liver enzyme levels, oxidative stress markers, and histological liver damage. The evidence given by the study highlights the therapeutic potential of MSC-MVs and the authors also conclude that future research is needed to optimize dosages and explore combined therapies for enhanced effectiveness [250].

The antifibrotic effect of human-derived EVs was supported by another study in which EV-derived human liver stem cells (HLSCs) were evaluated for their ability to treat NASH, a stage in the MASLD spectrum in immunocompromised mice. EV treatment significantly reduces signs of liver fibrosis and inflammation and downregulates 28 out of 29 fibrosis-associated genes upregulated in the NASH liver [251]. The use of human placental mesenchymal stem cell-derived exosomes (ExoMSC) employed for treating liver fibrosis showed an effective reduction of fibrosis and improved the liver microenvironment in a PSC mouse model and patient-derived organoids autoimmune diseases by inhibiting Th17 differentiation and reducing ER stress. ExoMSCs can downregulate IκBζ expression and PERK/CHOP signaling, suggesting potential therapeutic applications of ExoMSC for PSC and Th17-related liver diseases [252].

Non-MSC-derived EVs in fibrosis

Semaglutide, a GLP-1 receptor agonist, shows promise in treating MASLD associated with T2D by modulating the exosome composition. In a study with T2D patients, responders to semaglutide treatment exhibited significant improvements in liver fibrosis markers, as their exosomes reduced stellate cell activation and fibrosis-related protein expression. These findings suggest that semaglutide has therapeutic potential through indirect mechanisms involving exosome-mediated cell signaling, although the exact pathways involved further investigation. Although exosomes or EVs are not directly employed for therapy, the findings underscore the importance of exosome research in the development of MASLD therapies [253].

The therapeutic potential of curcumin is hampered by its hydrophobicity and low bioavailability. Owing to their favorable size, composition, and non-immunogenic properties, small Extracellular Vesicles (sEVs) offer a promising solution for curcumin delivery. This study investigates curcumin-loaded milk sEVs, via passive and active (saponin-assisted) loading methods, which maintain nanoparticle integrity and size. Active loading resulted in significantly increased curcumin encapsulation. In vitro tests revealed greater cytotoxicity in cancer cells versus primary hepatocytes, whereas in vivo studies demonstrated reduced liver damage and fibrosis in a liver fibrosis model, highlighting sEVs as effective curcumin delivery systems [254].

Advances in EV therapy for MASLD

Targeted delivery for MASLD therapy

Surface decoration of EVs alters the EV uptake by cells, which can be utilized for targeting EVs at specific sites. EVs can be surface engineered to achieve specific targeting. A detailed review of how extracellular vesicles target nonparenchymal cells to address liver fibrosis is given elsewhere [255]. EVs decorated with ligands of interest or targeting moieties produced either by direct modification or parent cell modification are utilized for targeting specific receptors, cells, or tissues. Notably, several preclinical evidence experimentally demonstrated the importance of targeting extracellular vesicles for the precise delivery of therapeutic cargo, eliminating off-target effects and enhancing the efficacy of therapy.

A study on the attenuation of hepatic steatosis by Yu et al. employed a unique targeting strategy for delivering pirfenidone-laden EVs to HSCs. This study demonstrated that the HA-modified EVs carrying pirfenidone to HSCs showed superior efficacy in inhibiting HSC activation and reducing collagen synthesis in both the rat HSC-T6 and the BRL cell lines. Furthermore, a previous study also demonstrated that in a murine hepatic fibrosis model, therapeutic strategy could significantly improve hepatic cell morphology and ameliorate hepatic fibrosis [256]. Similarly, the HSTP1 peptide fused with exosomal membrane protein Lamp2b through genetic engineering was employed to target the HSCs for effective reversal of fibrosis via human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes [257].

In a similar study EVs from human adipose-derived stem cells (ADSCs) were exogenously modified to bear vitamin A on their surface and showed enhanced targeting towards HSCs. Compared with bare EVs, exogenously modified EVs can reverse the fibrotic cascade even at tenfold lower doses [258].

The screening of targeting peptides, ligands, or chemical compounds to evaluate their efficacy and specificity provides the ideal candidates for targeting a particular receptor. This ideal targeting moiety can be further employed to target specific hepatic cellular niches. The knowledge about MASLD pathology provides essential grounds for the identification of a suitable cellular population to be targeted for the effective reversal of the disease. Future EV therapeutics rely on the development of essential loading strategies for loading molecular cargo and targeting EVs to the suitable hepatic cellular niche. Advancements in the targeting and loading strategies essentially enhance the therapeutic potential of EVs for overcoming MASLD.

Hybrid EVs for MASLD therapy

The efficacy of EVs can also be enhanced by coupling EVs with other nanocarriers to increase their therapeutic potential. The internalization of EVs is generally driven by stereochemical factors of the cell membrane. Anionic components of cellular membranes often offer electrostatic repulsion toward exosomes, affecting EV internalization. Sato et al. developed a biological nano transporter hybrid via the fusion of liposomes and EVs. The hybrid biological nano transporter aided with PEG surface modifications improved interaction with the target cells facilitating efficient delivery of the therapeutic cargo [259]. Similarly, Piffoux et al. and Mukherjee et al. demonstrated enhanced drug delivery efficiency by mixing MSC-derived EVs with PEGylated liposomes, suggesting a promising strategy for improving drug delivery systems [260, 261]. Evers et al. compared the physicochemical properties and functionality of liposomes with those of hybrid nanoparticles. An effective delivery vehicle was created by fusing engineered EVs with liposomes, which exhibited greater efficacy than liposomes alone [262]. Similar studies on hybrid EVs revealed that the combination of EVs with other nanocarriers can increase the efficacy of carrier systems. However, EVs also pose several hurdles as efficient cargo carriers; stability issues and the natural heterogeneity of EVs, reproducibility, and consistency make accessing this carrier system in the clinic difficult. However, when these limitations are properly addressed, a nanocarrier system coupled with surface-engineered EVs for efficient targeting, when utilized, can effectively enhance therapeutic potential.

Challenges in EV therapeutics

In conclusion, EVs are emerging therapeutic measures for MASLD both as cargo carriers and therapeutic agents. However, emerging EV therapeutics pose considerable challenges for translation into the clinic. The major challenge associated with EV therapeutics is maintaining the consistency of the therapeutic products, as they are biogenic in origin. The heterogeneity of the EV population also creates the need for efficient isolation of clinical-grade EV subpopulations. Producing high-quality EVs with consistent properties remains a significant hurdle, as scalable manufacturing and reproducible manufacturing processes are needed.

The significant strides in improving bioengineering EVs in the past 5 years and efforts to optimize the manufacturing of EVs for clinics are poised to overcome current challenges paving the way for a reliable EV-based therapy for MASLD. The current trend and rapidity in EV research ensure that these challenges will soon be addressed, resulting in the development of effective EV-based therapeutic measures for MASLD. This rapid progress holds promise for significantly improving patient outcomes by providing precise, targeted, and efficient therapeutic strategies for MASLD.

Strengths and limitations

This review consolidates a substantial body of research carried out in the past on EVs and MASLD, making it a literature resource for clinicians and researchers. Key topics regarding EVs and MASLD are simplified with the help of tables and figures to easily grasp the interaction of EVs in MASLD. This study included multiple clinical trials and the concerning issues surrounding MASLD for validating the claims and ensuring their timely relevance. The comprehensive tables aid in the practical application of this knowledge in clinical settings. By presenting both the positive and negative aspects of EVs in diagnostics and therapeutics without bias, this review provides an objective overview of the current state of research, which will help researchers identify existing knowledge gaps and potential areas for future investigations.

This review provides a broad perspective on the disease pathology of MASLD, along with other liver diseases, addressing clinical trials and the role of EVs in disease manifestation. Owing to the limited availability of EV-related studies specific to MASLD in the current literature, other liver diseases with overlapping pathological features were also explored. This approach is based on the rationale that MASLD shares several similar pathological events with other liver diseases, allowing insights into the potential utility of EVs in MASLD by analogy. However, a more focused bioinformatic study would help synthesize precise evidence regarding EV-based diagnostic markers specifically for MASLD.

Conclusion and future prospective

EVs are one such newly explored research field of science with continuous evolution where there has been significant research ongoing in the last 20 years, peaking in 2016 with 20.47% of the total articles from the entire period [263], which is growing rapidly with much wider and more practical applications for systematic problems of the population. The use of EVs as a therapeutic modality has also gained significant attention, as demonstrated by bibliometric data. This is evident from the notable increase in research, particularly the use of exosomes as delivery vehicles, which has grown at an impressive annual rate of 55.8% since 2013 [264]. A noteworthy number of patents were generated, and the number of grants sanctioned almost doubled in 2016 [263]. As the research digs the well of knowledge, the field delves deeper, and a vast area remains to explore. Earlier, the major focus was on EV-related biofluids and cell types. With the growth, the research has focused on the role of these EVs in disease diagnosis and therapy. Current research focuses on the exploration of disease through advanced technologies such as multiomics analysis with the assistance of bioinformatics analysis, thus providing accurate resolutions.

In the future, EV research should focus predominantly on two aspects. First, there is a need for the development of an EV-based rapid disease detection kit that can isolate and characterize organ-specific EVs from circulating biofluids to ascertain the presence of disease at a specific site in the body. Second, there is a need for a device that can accurately characterize the organ-specific EVs through multi-omics approaches, providing detailed insights into the incidence of the disease by analyzing risk factors, pathogenesis, progression of the disease, and severity of the disease along mortality predictions.

Therapeutically, the EV research field is concerned with the development of EVs as efficient therapeutic agents and cargo carriers. Research should focus soon on understanding the mechanism of the components of EVs that are effectively improving the disease, which can be implemented to improve the efficiency of EVs as therapeutic agents more precisely. The large-scale manufacturing of therapeutic EVs involving selectively enriching the effector EV subpopulation would be the immediate aim of EV therapeutics. Furthermore, EV-based research toward standardized platform development can enable accurate delivery of the cargo of therapeutic importance to the targeted site more effectively and efficiently, and improving the disease condition is needed to enhance and extend targeted delivery via EVs. Synergistic approaches to solve or select targeting moieties via omics and bioinformatics and efficient engineering techniques for expressing the targeting moiety along with an EV cargo loading strategy would efficiently pave the way for the use of EVs as therapeutic carriers in the clinic.

The paradigm is shifting toward translational applications in EV research, from identifying the existence of these lipid-layered nanoparticles, and the utilization of EVs as next-generation drug carriers. Among several lifestyle disorders, MASLD is one such silent disease prevailing worldwide due to metabolic syndrome in most of the population. As prominent particles of intercellular communication, through the development of EV-based devices, they can make one of the minimally invasive methods much more efficient in conveying the mechanism of disease progression along with the severity and efficient therapeutic utility. As explore trends in EV research is investigated, they can become “EV” erything for MASLD.