Shimoda, A. | Search bioRxiv

bioRxiv2022

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

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 heterogenous 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 spongy-like 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 m) of SPM. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling.

biochemistry↗

bioRxiv2020

New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes

We have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift). O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/931295v1_fig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@e978aeorg.highwire.dtl.DTLVardef@1f208cdorg.highwire.dtl.DTLVardef@11fdb1dorg.highwire.dtl.DTLVardef@6a7373_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Chemical structure of PKH26. C_FIG

biochemistry↗