Abstract
Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter Erp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.
Highlights
Extracellular vesicles (EVs) have gained signi cant attention as promising drug carriers for personalized nanomedicine over the recent decade
Five widely available puri cation methods were studied for their ability to separate the labelled EVs from the unbound dye: ultracentrifugation (UC), ultracentrifugation with discontinuous iodixanol density gradient (UCG), ultra ltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC)
In ultracentrifugation with discontinuous density gradient (UCG), because of their buoyant density and bigger size compared to uorescent dye molecules, the EVs oat to the higher interface between the layers with smaller densities while the unbound dye is expected to stay mostly at the bottom of the gradient in the highest density layer. Both UF and SEC are based on the size separation: in UF, the EVs concentrate above the lter membrane as most of the unbound dye is washed to the ltrate, and in SEC, the EVs elute from the column before the unbound dye
Summary
Extracellular vesicles (EVs) have gained signi cant attention as promising drug carriers for personalized nanomedicine over the recent decade. Paper carbocyanine dyes are commonly used in membrane staining.[31] BPC12 is a molecular rotor dye and its viscosity dependent uorescence could be used to study the integrity of the EVs during cell up take and consequent trafficking inside the cell.[32] BP is a neutral, nonpolar lipid stain.[33] Both these BP dyes are very hydrophobic and will intercalate deep into the EV membrane.[34] All the other studied dyes are at least partially at the hydrophilic part of the EV membrane and exposed to the surroundings. As an example of loading EVs with a biologically active molecule, the labelling was studied with a Ptx-OG, a tubulin targeting anti-cancer agent Paclitaxel labelled with uorescent dye OG.[7,36] Being somewhat hydrophobic, Ptx part will intercalate into the EV membrane
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