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bioRxiv · 10.1101/2025.01.09.632234

Rigorous process for isolation of gut-derived extracellular vesicles and the effect on latent HIV

Anyanwu, N. C. J.·Premadasa, L. S.·Naushad, W.·Okeoma, B. C.·Mohan, M.·Okeoma, C. M.
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Abstract

AimExtracellular particles (EPs) are produced/secreted by cells from all domains of life and are present in all body fluids, brain, and gut. EPs consist of extracellular vesicles (EVs) made up of exosomes, microvesicles, and other membranous vesicles; and extracellular condensates (ECs) that are non-membranous carriers of lipid-protein-nucleic acid aggregates. The purity of EVs|ECs, which ultimately depends on the isolation method used to obtain them is critical, particularly EVs|ECs from the gastrointestinal (GI) tract that is colonized by a huge number of enteric bacteria. Therefore, identifying GI derived EVs|ECs of bacterial and host origin may serve as a window into the pathogenesis of diseases and as a potential therapeutic target. MethodsHere, we describe the use of high-resolution particle purification liquid chromatography (PPLC) gradient-bead-column integrated with polyvinylpolypyrrolidone (PVPP)-mediated extraction of impurities to isolate GI-derived EPs. Results and ConclusionPVPP facilitates isolation of pure and functionally active, non-toxic EVs ColEVs from colonic contents. ColEVs are internalized by cells and they activate HIV LTR promoter. In the absence of PVPP, ColEVs have a direct reductive potential of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) absorbance in a cell-free system. Assessment of the origin of ColEVs reveals that they are composed of both bacteria and host particles. This protocol requires [~]12 hours (5 hours preprocessing, 7 hours isolation) to complete and should be used to purify EVs from sources contaminated with microbial agents to improve rigor. Additionally, this protocol provides a robust tool for researchers and clinicians investigating GI-derived EVs and the translational use of GI-derived EVs for diagnostic and therapeutic use. HighlightO_LIColEVs but not ColECs are present in colonic content (GI tract) and can be isolated with gradient or single bead PPLC column. C_LIO_LIColEVs isolated without PVPP are toxic to cells and they have a direct reductive potential of MTT. Addition of PVPP treatment in the isolation protocol results in clean and non-toxic ColEVs that transactivate the HIV LTR promoter. C_LI

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Anyanwu, N. C. J., Premadasa, L. S., Naushad, W., Okeoma, B. C., Mohan, M., Okeoma, C. M.. 2025-01-09. Rigorous process for isolation of gut-derived extracellular vesicles and the effect on latent HIV. https://doi.org/10.1101/2025.01.09.632234

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