Shimomura Takashi, Seino Ryo, Umezaki Kaori, Shimoda Asako, Ezoe Takatoshi, Ishiyama Munetaka, Akiyoshi Kazunari
Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202, Japan.
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Bioconjug Chem. 2021 Apr 21;32(4):680-684. doi: 10.1021/acs.bioconjchem.1c00068. Epub 2021 Mar 13.
PKH dyes, which are currently the most widely used fluorescent probes for extracellular vesicle (EV) labeling, have some limitations. For example, these dyes tend to aggregate, leading to formation of EV-like nanoparticles that can be taken up by cells. Moreover, it has been suggested that PKH dyes trigger an enlargement of EVs because of membrane fusion or intercalation. To overcome these limitations, we developed three novel extracellular vesicular-membrane-binding fluorescent probes-Mem dye-Green, Mem dye-Red, and Mem dye-Deep Red-for monitoring EV uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold and amphiphilic moieties on the cyanine. The three dyes have different photophysical characteristics. To investigate the characteristics of the Mem dyes for EV labeling, we performed nanoparticle tracking, zeta potential measurements, and confocal microscopy. The dyes enable highly sensitive fluorescence imaging of EVs. They can also be used to observe EV dynamics in live cells. The Mem dyes show excellent EV labeling with no aggregation and less particle enlargement.
PKH染料是目前用于细胞外囊泡（EV）标记的应用最为广泛的荧光探针，但存在一些局限性。例如，这些染料容易聚集，导致形成可被细胞摄取的类EV纳米颗粒。此外，有人认为PKH染料会因膜融合或嵌入而引发EV增大。为克服这些局限性，我们开发了三种新型细胞外囊泡膜结合荧光探针——Mem染料-绿色、Mem染料-红色和Mem染料-深红色，用于监测细胞对EV的摄取。这些染料含有一个作为荧光支架的花菁基团以及花菁上的两亲性部分。这三种染料具有不同的光物理特性。为研究Mem染料用于EV标记的特性，我们进行了纳米颗粒跟踪、zeta电位测量和共聚焦显微镜检查。这些染料能够对EV进行高灵敏度荧光成像。它们还可用于观察活细胞中的EV动态。Mem染料显示出出色的EV标记效果，无聚集且颗粒增大较少。
