Camel milk-derived extracellular vesicles using a two-step PEG precipitation strategy.

Isolation of extracellular vesicles (EVs) from milk is technically challenging due to the presence of high concentrations of casein and other proteinaceous contaminants. In this study, we report an optimized two-step polyethylene glycol (PEG) precipitation protocol designed to improve the recovery and enrichment of camel milk-derived EVs. The performance of this two-step protocol, in which initial precipitation at PEG10% is followed by re-precipitation at Re-PEG5%, was evaluated and compared against several alternative isolation strategies, including single-and two-step PEG10% precipitation, and PEG precipitation combined with ultrafiltration (UF) and size-exclusion chromatography (SEC), with protocols assessed across multiple parameters. A suite of quantitative assessments, including protein-to-lipid and nucleic acid-to-protein ratio measurements and flow cytometry, was performed alongside qualitative characterizations via TEM, SEM, AFM, ATR-FTIR, NMR, DSC, EDS and elemental mapping. Complementary characterizations consistently demonstrated that the two-step Re-PEG5% protocol generated EV preparations with higher purity and reduced co-isolation of non-vesicular contaminants than the two-step PEG10% protocol or protocols involving costlier UF and SEC techniques. This improved purity was accompanied by enhanced cytocompatibility in HEK293 cells, as verified through MTT assays, suggesting that optimization of PEG concentration is a critical determinant of both camel milk EV quality and biological performance. Overall, the physicochemical and biological characterizations performed provide substantial evidence supporting the quality and yield of the EVs isolated from camel milk using a two-step PEG precipitation protocol. This cost-effective and scalable strategy offers a promising, green-chemistry-aligned alternative for EV production in biomedical applications, particularly in resource-limited settings.