Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Abstract

Exosomes are membrane vesicles secreted by cells and distributed widely in all biofluids. Exosomes can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of exosomes is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for exosome labelling. One concern with the use of lipophilic dyes is an increase in the exosome size due to membrane fusion or intercalation. This size shift alone may undermine the validity of exosomes tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled exosomes was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled exosomes were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled exosomes was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for exosomes tracking.