Small extracellular vesicles as liquid biopsy targets and precision therapeutics for metabolic dysfunction-associated steatotic liver disease

Table 1 Candidate small extracellular vesicle biomarkers investigated in metabolic dysfunction-associated steatotic liver disease

Cargo	Biomarker application	Study population/evidence	EV isolation/characterization	Key finding/diagnostic performance	Ref.
Nucleic acids
miR-122, miR-192, miR-128-3p	MASLD discrimination and progression	Human: Controls (n = 14), non-MASH MASLD (n = 8), biopsy-proven MASH (n = 6)	Plasma EVs; size-exclusion chromatography and ASGR1 immunocapture; TEM, NTA and EV protein markers	Increased with disease severity; study-cohort AUCs of 0.830, 0.895 and 0.803, respectively	[56]
miR-122-5p, miR-27a, miR-335-5p	Pediatric MASLD biomarker candidates	Human: MASLD (n = 5) vs simple obesity (n = 5)	Serum exosomes; TEM, NTA and western blotting	Upregulated; identified by high-throughput sequencing in an exploratory cohort	[63]
Proteins/EV features
PLIN3/CD63 ratio	Risk stratification, particularly lean MASLD	Human: Controls (n = 57), obese MASLD (n = 83), lean MASLD (n = 87)	Serum EVs; iodixanol density-gradient ultracentrifugation; NTA and western blotting	Decreased in lean MASLD; external validation is still required	[60]
CD63+ and ASGR1+ hepatocyte-derived EVs	MASH progression and liver-specific EV detection	Human: Patients with steatosis and MASH	Circulating EVs; NTA, TEM and flow cytometry; CD63 capture with ASGR1 detection	Increased in MASH and associated with disease severity	[61]
FZD-7	MASLD severity and potential treatment-response marker	Human: Healthy controls (n = 20) and MASLD (n = 95)	Plasma exosomes; differential ultracentrifugation; DLS, TEM, zeta potential and exosomal protein markers	Higher in MASLD and reduced after lifestyle intervention	[67]
Lipids
C16:0 ceramide and S1P	MASH-associated EV lipid candidates	Human: Obese controls (n = 11), simple steatosis (n = 16), early MASH (n = 16); parallel mouse experiments	Plasma EVs; sphingolipid profiling by tandem mass spectrometry	Enriched in MASH; supported by parallel experimental models	[71]
FFA (18:0), LPC (22:6/0:0), FFA (18:1), PI (16:0/18:1)	MASH discrimination (exploratory)	Human: Non-MASH MASLD (n = 43) and MASH (n = 40)	Urinary EVs; EXODUS isolation and LC-MS/MS lipidomics	Four-lipid panel distinguished MASH with a study-cohort AUC of 0.923 (exploratory)	[72]

Evidence categories are reported according to the study population and experimental setting, including human, animal, and combined human/experimental evidence where applicable. Reported area under the receiver operating characteristic curves represent performance estimates from the respective study cohorts unless otherwise stated and should not be interpreted as independently externally validated performance. Confidence intervals and comparisons with established non-invasive tests are reported only when available in the original studies. EV: Extracellular vesicle; MASLD: Metabolic dysfunction-associated steatotic liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; ASGR1: Asialoglycoprotein receptor 1; TEM: Transmission electron microscopy; NTA: Nanoparticle tracking analysis; AUC: Area under the receiver operating characteristic curve; PLIN3: Perilipin 3; FZD-7: Frizzled class receptor 7; DLS: Dynamic light scattering; S1P: Sphingosine 1-phosphate; FFA: Free fatty acid; LPC: Lysophosphatidylcholine; PI: Phosphatidylinositol; EXODUS: Extracellular vesicle isolation by dodecyl sulfate-based enrichment; LC-MS/MS: Liquid chromatography-tandem mass spectrometry.

Table 2 Evidence-classified therapeutic roles and strategies of small extracellular vesicles relevant to metabolic dysfunction-associated steatotic liver disease

Source	Cargo	Experimental model/evidence directness	Mechanism/biological effect	Ref.
Native therapeutic sEVs in MASLD-related models
hUC-MSCs	miR-24-3p	In vitro and animal evidence: NAFLD models	Targets Keap-1 and reduces lipid accumulation, oxidative stress, and inflammation	[82]
hUC-MSCs	miR-627-5p	In vitro and animal evidence: NAFLD models	Represses FTO expression and improves hepatic glucose and lipid metabolism	[85]
hUC-MSCs	Not specifically identified	In vitro and animal evidence: Experimental NASH models	Regulates the Nrf2/NQO-1 pathway and reduces inflammation and oxidative stress	[87]
BM-MSCs	miR-96-5p	Animal evidence: Experimental NASH rat model	Downregulates caspase-2, enhances fatty acid oxidation, and reduces steatosis	[88]
Myeloid cells	miR-223	In vitro and animal mechanistic evidence: NAFLD-associated fibrosis	Myeloid IL-6 signaling promotes miR-223-enriched sEV release and attenuates liver fibrosis	[83]
Kupffer cells	miR-690	Cell-based and animal evidence: NASH models	Targets NADK and suppresses fibrogenesis, inflammation, and de novo lipogenesis	[90]
Engineered MASLD-specific interventions
GalNAc-modified RBC-EVs	PJ34; GalNAc surface modification	Animal evidence: NAFLD mouse model	Enhances hepatocyte-targeted delivery and reduces hepatic triglyceride accumulation through PARP-1 inhibition	[96]
RBC-sEVs	siNUP85	In vitro and animal evidence: MASLD models	Silences NUP85 and attenuates steatosis, inflammation, and oxidative stress, partly through SLC27A1/PPAR-γ signaling	[103]
cRGD-modified ADSC-EVs	Angiotensin-(1-7); cRGD targeting	In vitro and animal evidence: NAFLD-associated fibrosis	Activates Mas receptor signaling, enhances Akt/Foxo1-associated autophagy and metabolic reprogramming, and reduces lipid accumulation and fibrosis	[113]
Pathogenic sEVs as therapeutic targets
Adipocytes	miR-122	Animal and in vitro evidence: NAFLD models	Targets Sirt1 and promotes disease progression	[54]
Hepatocytes	miR-192-5p	Human, animal, and cell-based evidence: NAFLD/MASH	Activates proinflammatory macrophages through Rictor/Akt/FoxO1 signaling	[57]
Hepatocytes	RBP4	Human association, cell-based and animal evidence: NAFLD	Promotes Kupffer-cell M1 polarization, hepatic lipid accumulation, and inflammation	[68]
Transferable proof-of-concept approaches
hESC-derived sEVs	miR-6766-3p	Indirect non-MASLD evidence: In vitro and animal liver fibrosis models	Targets TGF-βRII/SMAD signaling and suppresses hepatic stellate-cell activation and fibrotic progression	[89]
5HT1D/CD47-engineered exosomes	miR-29b; 5HT1D antibody and CD47 surface modification	Indirect non-MASLD evidence: In vitro and animal liver fibrosis models	Enhances targeting of activated hepatic stellate cells and suppresses fibrotic signaling through the TGF-β/SMAD pathway	[114]

Evidence categories are assigned according to the experimental setting and disease model reported in the original studies. Studies conducted in liver fibrosis or other non-metabolic dysfunction-associated steatotic liver disease settings are explicitly classified as indirect non-metabolic dysfunction-associated steatotic liver disease evidence. Table 2 is intended to classify therapeutic evidence according to experimental setting and disease-model relevance; administration route, dose, and extracellular vesicle isolation protocols vary across studies and are not used here as evidence-ranking criteria. sEVs: Small extracellular vesicles; MASLD: Metabolic dysfunction-associated steatotic liver disease; hUC-MSCs: Human umbilical cord mesenchymal stem cells; Keap-1: Kelch-like ECH-associated protein 1; NAFLD: Nonalcoholic fatty liver disease; FTO: Fat mass and obesity-associated protein; NASH: Non-alcoholic steatohepatitis; NQO-1: NAD(P)H quinone oxidoreductase 1; Nrf2: Nuclear factor erythroid 2-related factor 2; IL-6: Interleukin-6; GalNAc: N-acetylgalactosamine; NADK: NAD kinase; RBC-EVs: Red blood cell-derived extracellular vesicles; PARP-1: Poly(ADP-ribose) polymerase 1; cRGD: Cyclic arginine-glycine-aspartic acid; ADSC-EVs: Adipose-derived mesenchymal stem cell-derived extracellular vesicles; RBP4: Retinol-binding protein 4; hESCs: Human embryonic stem cells; BM-MSCs: Bone marrow mesenchymal stem cells; TGF‑βRII: Transforming growth factor-beta receptor II; SMAD: Mothers against decapentaplegic homolog; 5HT1D: 5-hydroxytryptamine receptor 1D; CD47: Cluster of differentiation 47; TGF-β: Transforming growth factor-β.

Table 3 Comparison of commonly used small extracellular vesicles cargo loading and surface engineering strategies for therapeutic delivery

Strategy	Principle	Suitable cargos/ modification	Effect on vesicles	Key advantages and limitations
Passive incubation	Passive diffusion or interaction across the vesicle membrane	Small molecules; hydrophobic drugs	Minimal disruption; cargo integrity generally preserved	Simple and mild, but loading efficiency is low to moderate and nucleic acid loading is limited
Electroporation	Electrical pulses generate transient membrane pores	miRNA; siRNA; mRNA	It may cause membrane disruption, aggregation, or RNA precipitation	Useful for nucleic acids with moderate to high loading, but reproducibility is variable and optimization is required
Sonication	Mechanical energy transiently increases membrane permeability	Drugs; proteins; nucleic acids	It may alter vesicle morphology and membrane components	Generally improves loading efficiency, but excessive treatment can damage cargo and compromise native vesicle properties
Extrusion	Mechanical deformation promotes cargo incorporation	Drugs; nucleic acids	It may alter vesicle size distribution and biological activity	It can yield relatively uniform vesicles, but membrane remodeling may affect native functions
Freeze-thaw cycles	Repeated membrane disruption and resealing	Proteins; small molecules	Possible structural alteration and membrane fusion	Easy to perform, but loading efficiency is usually low to moderate and cargo preservation can vary
Saponin-assisted loading	Mild detergent transiently permeabilizes the vesicle membrane	Small molecules; hydrophilic drugs	It may increase membrane permeability and damage membranes at excessive concentrations	It can improve loading efficiency, but saponin concentration and removal of residual detergent require careful control
Endogenous loading (donor-cell engineering)	Donor-cell modification promotes incorporation of selected cargos during vesicle biogenesis	miRNAs; proteins; therapeutic molecules	Usually preserves physiological vesicle structure and biological activity	Good biological compatibility and cargo enrichment, but manufacturing and quality control are more complex
Chemical surface conjugation	Targeting ligands are covalently attached to molecules on the vesicle membrane	Peptides; antibodies; glycans; other targeting ligands	It may alter membrane proteins or surface properties under harsh reaction conditions	It improves targeting specificity, but reaction chemistry and ligand density must be optimized to preserve vesicle integrity

Chemical surface conjugation represents a surface engineering strategy rather than internal cargo loading. miRNA: MicroRNA; siRNA: Small interfering RNA; mRNA: Messenger RNA.