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bioRxiv · 10.64898/2026.04.23.720326

Surface functionalization of small extracellular vesicles derived from Caco-2 and HEK293T cells in the neutralization of Shiga toxin 1 subunit B

Mikolajczyk, K.·Bereznicka, A.·Czernek, L.·Gualerzi, A.·Forleo, L.·Bedoni, M.·Lodej, N.·Migdal, P.
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Abstract

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC), which are responsible for severe foodborne infections that can progress to hemolytic-uremic syndrome (HUS). Currently, no specific anti-toxin therapies are available. In this study, we devised a glycoengineering strategy utilizing Functional-Spacer-Lipid (FSL) conjugates to create small extracellular vesicles (sEVs)-based decoy receptors for Shiga toxin type 1 (Stx1). sEVs isolated from human Caco-2 and HEK293T cells were functionalized with Gb3 trisaccharide (Gal1[->]4Gal{beta}1[->]4Glc)-containing FSL conjugates, yielding Gb3-decorated vesicles, displaying the Gal1[->]4Gal epitope. Characterization of FSL-modified sEVs confirmed that FSL incorporation did not adversely affect sEV morphology, size distribution, or surface charge. Western blotting and bead-assisted flow cytometry verified the presence of exosomal markers (CD9 and CD63) and the Gb3 epitope on modified vesicles. Gb3-tagged sEVs from both cell types exhibited high specificity in binding Stx1B, while control vesicles carrying Galili epitope (Gal1[->]3Gal{beta}1[->]4GlcNAc), lacking Stx1B binding, demonstrated negligible binding. Gb3-expressing Caco-2 cell-based assays revealed that Gb3-decorated sEVs markedly reduced Stx1B binding to Caco-2 cells, indicating effective competition with cellular receptors. Furthermore, glycoengineered sEVs did not impair Caco-2 cell viability at concentrations sufficient for Stx1B sequestration. These findings establish FSL-mediated glycoengineering as a rapid and versatile approach for generating sEV-based decoy receptors that effectively bind Stx1B. Gb3-containing human sEVs may serve as an agent for neutralizing Stx1B and potentially other glycan-binding toxins, supporting the development of promising next-generation anti-toxin therapeutics.

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Mikolajczyk, K., Bereznicka, A., Czernek, L., Gualerzi, A., Forleo, L., Bedoni, M., Lodej, N., Migdal, P.. 2026-04-27. Surface functionalization of small extracellular vesicles derived from Caco-2 and HEK293T cells in the neutralization of Shiga toxin 1 subunit B. https://doi.org/10.64898/2026.04.23.720326

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