【公開日：2023.07.31】【最終更新日：2023.05.16】
課題データ / Project Data
課題番号 / Project Issue Number
22UT0050
利用課題名 / Title
エクソソームの表面分析
利用した実施機関 / Support Institute
東京大学
機関外・機関内の利用 / External or Internal Use
内部利用（ARIM事業参画者以外）/Internal Use (by non ARIM members)
技術領域 / Technology Area
【横断技術領域 / Cross-Technology Area】（主 / Main）計測・分析/Advanced Characterization（副 / Sub）-
【重要技術領域 / Important Technology Area】（主 / Main）次世代バイオマテリアル/Next-generation biomaterials（副 / Sub）-
キーワード / Keywords
形状・形態観察, 分析,電子顕微鏡/Electron microscopy,赤外・可視・紫外分光/Infrared and UV and visible light spectroscopy,バイオアダプティブ材料/ Bioadaptive materials,DDSマテリアル/ DDS material
利用者と利用形態 / User and Support Type
利用者名（課題申請者）/ User Name (Project Applicant)
WANG CHENYU
所属名 / Affiliation
東京大学
共同利用者氏名 / Names of Collaborators in Other Institutes Than Hub and Spoke Institutes
ARIM実施機関支援担当者 / Names of Collaborators in The Hub and Spoke Institutes
寺西亮佑
利用形態 / Support Type
（主 / Main）機器利用/Equipment Utilization（副 / Sub）,技術補助/Technical Assistance
利用した主な設備 / Equipment Used in This Project
報告書データ / Report
概要（目的・用途・実施内容）/ Abstract (Aim, Use Applications and Contents)
Extracellular vesicles (EVs) play an important role in intercellular communication by carrying molecules between cells, both locally and systemically. Among which, exosome is an important category, of whose size believed to be in the range of 30−150 nm. All of which consist of cell-derived lipid-bilayer membranes and serve as vehicles carrying different types of cellular cargos, such as membrane lipids, cell-specific proteins, mRNA, DNA and microRNA delivering to the neighboring or distant recipient cells. Therefore, Exosomes are recognized as promising biomarkers and potential theranostic nanocarriers. We employed a simple and fast method to control the surface properties of exosomes via polydopamine coating. Furthermore, the polydopamine coating allowed for surface PEGylation of exosomes via secondary reactions, which opens exciting avenues for the application of exosomes. The morphology of exosomes after surface functionalization was characterized by TEM.
実験 / Experimental
The exosomes were derived from cell culture supernatant, and isolated by size exclusive column and ultracentrifuge. The isolated exosomes were resuspended in pH 7.4 Tris buffer solution, which were first coated with PDA by self-polymerization of dopamine (Exo@PDA). Additionally, PDA coatings possess abundant catechol and amine groups capable of subsequent reactions, such as Michael addition or Schiff-base reaction, and we coated the Exo@PDA with PEG-thiol (PEG-SH) via Thiol-Michael addition to form Exo@PDA@PEG. The morphology of Exo@PDA@PEG were observed by transmission electron microscope (TEM, JEM-1400).
結果と考察 / Results and Discussion
The bare exosomes exhibited round shape with smooth surface and had a diameter of around 70–100 nm. The membrane structure of exosomes was clearly observed by cryo-TEM as shown in Figure 1 a,d. As for PDA coated exosomes (Exo@PDA), their outline was similar with the morphology of bare exosomes (Figure 1b). It was most likely because the thickness of PDA coating layer was only several nanometers. There was an ultrathin layer formed on the surface of exosome from the TEM image on Figure 3e, which was the sample of PDA coated exosomes after uranyl acetate staining. After PEGylation, the surface of PEGylated exosomes (Exo@PDA@PEG) became slightly rougher compared to bare exosomes (Figure 1c). In addition, the exosomes were encircled with annular structure upon PEG grafting on exosomes (Figure 1f).
図・表・数式 / Figures, Tables and Equations
Figure 1. Morphology of exosomes and surface-functionalized exosomes. Cryo-TEM images of bare exosomes (a), Exo@PDA (b), and Exo@PDA@PEG (c). TEM images of bare exosomes (d), Exo@PDA after negative staining (e), and Exo@PDA@PEG (f).
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
成果発表・成果利用 / Publication and Patents
論文・プロシーディング（DOIのあるもの） / DOI (Publication and Proceedings)
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
- C. Wang, H. Ejima, “Polydopamine-Assisted Surface Functionalization of Exosome”, The 71st Annual Meeting of the Society of Polymer Science, Japan, Online, May 25–27, 2022 (Poster)
特許 / Patents
特許出願件数 / Number of Patent Applications：0件
特許登録件数 / Number of Registered Patents：0件