New Lipophilic Fluorescent Dyes for Labeling Extracellular Vesicles: Characterization and Monitoring of Cellular Uptake

Abstract

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.

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis

“…Their fl-NTA data showed also the detection of fl-NPs at a size range of approximately 15 to 30 nm that were not detected when scatter mode detection was employed [29]. Although in another investigation, Shimomura et al studied exosomes stained with Mem and PKH dyes where the size of the Mem-labeled exosomes remained constant [38]. This observation was similar to our observation when the lowest concentration of CMG was used for EV labeling and detection of fl-NPs.…”

“…Thus, such differences in net surface charge were observed for the ZP of fl-NPs at the above-mentioned concentrations of dye. There are some similarities between the ZP values of fl-NPs originated from JAr cells and those described by Shimomura et al, who also reported similar ZP values of Mem dye-stained exosomes [38].…”

A method to study extracellular vesicles secretedin vitroby cultured cells with minimum sample processing and extracellular vesicle loss

“…Methods to label EVs in vitro have used many compounds including amine reactive dyes (carboxyfluorescein succinimidyl ester CFSE) (Morales-Kastresana et al, 2017; Pospichalova et al, 2015), lipophilic dyes (PKH dyes, DiI, DiD, DiR, etc.) (Grange et al, 2014; Morales-Kastresana et al, 2017; Nicola et al, 2009; Pospichalova et al, 2015; Puzar Dominkus et al, 2018; Tamura et al, 2016; van der Vlist et al, 2012; Wiklander et al, 2015) and Mem dyes (Shimomura et al, 2021) among others (Panagopoulou et al, 2020). Labeling of EVs in vitro suffers from the limitations inherent to the method employed for EV separation prior to labeling (discussed below) in addition to specific complications that arise during the labeling procedure, depending on the fluorophore employed.…”

“…Labeling in vitro with CFSE prevents artifacts due to dye aggregation and does not affect the size of EVs (Dehghani et al, 2020), but although some studies suggested that removal of the unbound CFSE is not required (Pospichalova et al, 2015), others detected increased fluorescence in the background noise, possibly as a result of spontaneous hydrolysis of free dye (Morales-Kastresana et al, 2017). Similarly, no large aggregation and no significant change of the apparent size of EVs were observed with the Mem dyes, although excess dye needs to be eliminated after labeling (Shimomura et al, 2021).…”

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements

“…In addition, PKH dyes can increase EV size by enhancing clustering and aggregation [ 103 ]. These limitations can be, at least partially, overcome with the use of recently introduced Mem lipophilic dyes that did not aggregate or change the size of EVs [ 104 ]. In conclusion, although simple and straightforward, direct EV labelling with lipophilic dyes has important limitations and therefore requires careful interpretation to avoid misleading results.…”