Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media

Abstract

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongylike monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 μm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer

“…However, the amount of NPs collected from the culture supernatant was much less than that of the LNPs we had expected. SEVs have membrane proteins such as CD9 and CD81 on their surface, which may cause nonspecific adsorption to the hydrophobic surface of TiO 2 –SPM. , Additionally, because these proteins may cover the phospholipids on the SEV surface, hybridized TiO 2 might require a longer adsorption time to SEVs than that of LNPs. In previous reports, incubation took 5 min until sufficient binding between SEVs in urine and TiO 2 particles was confirmed .…”

“…Here, we developed a novel method for purification of SEVs using a highly permeable spongy-like monolithic polymer (spongy-monolith; SPM) hybridized with titanium oxide (TiO 2 ) by thermal mixing (Figure ). SPM provides a flow path suitable for low-pressure chromatographic separation of SEVs without clogging due to its μm-sized through-pores that are much larger than the diameter of SEVs . In addition, SPM consists of poly­(ethylene- co -glycidyl methacrylate) (PEGM), a flexible and inexpensive polymer, applied to separation media with various shapes and sizes.…”

“…SPM provides a flow path suitable for low-pressure chromatographic separation of SEVs without clogging due to its μm-sized through-pores that are much larger than the diameter of SEVs. 28 In addition, SPM consists of poly(ethylene-co-glycidyl methacrylate) (PEGM), a flexible and inexpensive polymer, applied to separation media with various shapes and sizes. Metal oxides, such as titanium oxide, selectively capture the abundant phosphate groups on the lipid bilayer surface of SEVs via bidentate binding.…”

Efficient Selective Adsorption of SARS-CoV-2 via the Recognition of Spike Proteins Using an Affinity Spongy Monolith

“…As a previous example, we selectively adsorbed and desorbed IgG toward the SPM immobilized protein A by controlling the pH . Furthermore, we successfully separated the extracellular vesicles, exosome, due to the difference in the surface sugar chains using lectin-immobilized SPMs . The study showed that SPM provided the effective separation for bio-related nanoparticles compared to typical porous particle adsorbents.…”

“…32 Furthermore, we successfully separated the extracellular vesicles, exosome, due to the difference in the surface sugar chains using lectin-immobilized SPMs. 34 The study showed that SPM provided the effective separation for bio-related nanoparticles compared to typical porous particle adsorbents. According to our studies, paying attention to the spike protein of SARS-CoV-2 that protrudes on the surface of the virus, we conceived that immobilizing an anti-spike protein (AS) binding the spike protein RBD to the SPM selectively adsorbs and desorbs SARS-CoV-2.…”