Development of new spongy monolith for selective separation of extracellular vesicles

Koji OTSUKA, Department of Material Chemistry, Kyoto University, Kyoto, Japan (otsuka@anchem.mc.kyoto-u.ac.jp)
Seiya Kato, Department Of Material Chemistry, Kyoto University, Kyoto, Japan
Toyohiro Naito, Department Of Material Chemistry, Kyoto University, Kyoto, Japan
Takuya Kubo, Department Of Material Chemistry, Kyoto University, Kyoto, Japan
Kazunari Akiyoshi, Department Of Polymer Chemistry, Kyoto University, Kyoto, Japan

An exosome is one of the extracellular vesicles containing specific proteins, nucleic acids, and sugars. The components of exosomes are not fully identified because the effective isolation method for exosomes based on their chemical characteristics has not been established. To facilitate the exosome studies, we focus on the specific separation of exosomes by the recognition of sugar chains on the surface of exosomes. For typical separations based on sugar chains, lectin affinity chromatography (LAC) is usually employed, which is currently used to purify glycoproteins, but the pore size of the LAC separation media is too small to separate exosomes.

To achieve effective exosome separations, a spongy monolith (SPM), which  consists of poly(ethylene-co-glycidyl methacrylate) (PEGM), as a separation medium for LAC. The SPM contains large through pores of >10 μm in diameter, thus exosomes are assumed to be passed through easily. In addition, we previously achieved the affinity separation of biomolecules under an ultra-high flow rate using the SPM.

In this study, to separate exosomes by the recognition of the surface glycans, we prepared two kinds of LAC columns made of the SPM with immobilized sambucus sieboldiana agglutinin (SSA) or concanavalin A (ConA). Additionally, insulin was employed to block the hydrophobic surface of the SPMs. After preparing the columns, chromatographic analyses were carried out with bovine serum albumin (BSA) to evaluate the hydrophilicity and permeability of the SPMs. Since a sufficient amount of BSA was passed, it suggested that the SPMs were substantially hydrophilized. The interaction between the lectins immobilized on the SPMs and sugar chains was then evaluated. As results, glycoproteins (transferrin and glucose oxidase) were effectively interacted with their respective lectins. Furthermore, the desorption procedures were optimized by changing the elution conditions. And then, we evaluated the interaction with liposomes, which consist of lipid bilayer and have the sugars on the surface, as well as exosomes. Along with glycoproteins, mannose-integrated liposomes were also interacted and rapidly desorbed by using the ConA-immobilized SPM. These results suggest that the lectin-immobilized SPMs will be useful for the separation of exosomes based on recognition of the surface sugar chains.

[1] Kubota, K.; Kubo, T.; Tanigawa, T.; Naito, T.; Otsuka, K. Sci. Rep. 2017, 7: 178.

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Abstract Reference & Short Personal Biography of Presenting Author

Koji Otsuka is a professor of Analytical Chemistry of Materials, Department of Material Chemistry, Graduate School of Engineering, Kyoto University, since 2002.  He received his Ph. D. from Kyoto University in 1986 under the supervision of Prof. Teiichi Ando and Dr. Shigeru Terabe.  After receiving the JSPS Fellowship for Young Scientists (1986–1988), he joined Osaka Prefectural College of Technology as a senior lecturer (1988–1990) and an associate professor (1990–1995), then moved to Himeji Institute of Technology as an associate professor of the Prof. Terabe’s Lab (1995–2002), followed by moving to Kyoto University.

His research interests include the development of micro/nano scale high performance separation techniques using electrophoretic and chromatographic methods.  He has published approximately 180 papers and book chapters.

He received the Award of the Society for Chromatographic Sciences (SCS), Japan (2006), the Award for Creative Work of the Chemical Society of Japan (2009), the Award of the Japan Society for Analytical Chemistry (JSAC) (2014), and the Terabe Shigeru Prize (2014).  He is a member of the Permanent Scientific Committees of the HPLC and ITP Symposium Series and an editor of the Journal of Separation Science (Wiley-VCH). Currently he is serving as the president of the SCS as well as the chair of HPLC2019 Kyoto.