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.