Shedding light on parasite-derived extracellular vesicles: from isolation to human intestinal models

Helminth extracellular vesicles (EVs) are increasingly recognized as key players in host–parasite interactions, yet their study remains technically challenging. Here, we investigated EVs secreted by Anisakis sp., a zoonotic nematode associated with allergic reactions, gastrointestinal inflammation, and possibly cancer, using an in-vitro model based on human intestinal organoids (HIO). EVs features were assessed through multiple methodologies: isolation strategies included size exclusion chromatography (SEC), SEC combined with Amicon filtration, ultracentrifugation, and a polymer-based precipitation method (Exoquick); characterization was performed through transmission and scanning electron microscopy (TEM and SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA and interferometric-NTA), and tunable resistive pulse sensing (TRPS). Notably, combining SEC and Amicon filters yielded cleaner preparations suitable for advanced imaging assays and functional experiments. Nevertheless, iNTA detected internal contents indicative of complex bioactive cargo, and AFM and DLS suggested the presence of a surfaceassociated biomolecular corona, as shown by size reductions upon proteinase K treatment (e.g., DLS: 243 nm untreated vs. 174 nm treated). Functional studies involved treating 2-D cultures of HIO with Anisakis L3 EVs. Structural changes in epithelial architecture, particularly junctional organization and cell differentiation markers, were investigated through immunofluorescence and western blot analyses. Preliminary results did not reveal significant phenotypic differences between EVtreated and untreated organoids after 24 h. HIO were further analyzed via small RNA sequencing to reveal EV-induced transcriptional effects. No substantial alterations in miRNA expression profiles were observed. Subtle context-dependent effects, insufficient exposure time or interactions limited to specific cell subpopulations may have contributed to the absence of measurable responses. Although functional effects on HIO were not evident under current conditions, this integrated workflow lays the foundation for standardized protocols for nematode EV isolation and functional studies. Ongoing comparative studies are aimed at validating the presence and composition of the corona, a crucial mediator of EV functions, and evaluating the influence of isolation methods on EVs for functional studies, exploring host-parasite interplay.