Cracking the egg: Isolation and discovery of distinct nonvesicular extracellular particles from Schistosoma mansoni eggs

Extracellular particles (EPs) derived from helminths are emerging as promising immunomodulatory agents, yet their heterogeneity challenges reproducible purification and characterisation. This study focused on purifying and characterising extracellular particles (EPs) from Schistosoma mansoni eggs using tangential flow filtration (TFF) and differential ultracentrifugation (dUC). Contrary to expectations, cryogenic transmission electron microscopy (cryoTEM) revealed that egg-derived EPs are nonvesicular (5-30 nm) nanoparticles (eggNPs) rather than membrane-bound extracellular vesicles. TFF demonstrated superior performance over dUC, yielding higher recovery (76.19% vs. 38.90%), greater purity, and lower endotoxin contamination. Despite limitations in standard protein quantification methods, enriched protein profiles and detection of S. mansoni tetraspanin-2 in TFF-purified eggNPs highlights their distinct molecular signatures. The study also identified contaminants in cryopreservation buffers, underscoring the need for optimised storage conditions. Compared to dUC, TFF preserved particle integrity, with cryoTEM images showing fewer aggregates and distinct EggNP morphology. This work proposes TFF as a scalable and reproducible method for isolating S. mansoni-derived eggNPs, providing a foundation for their exploration as immunomodulatory agents. These findings not only expand the understanding of helminth EPs but also emphasise their therapeutic potential, particularly in inflammatory and immune-dysregulation disorders.