Fluorescence labelling of extracellular vesicles using a novel thiol-based strategy for quantitative analysis of cellular delivery and intracellular traffic

Abstract

Extracellular vesicles, including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration. A significant challenge within these studies is the ability to label EVs directly or indirectly with fluorescent probes to allow visualization without compromising functionality. Here, we present a thiol-based fluorescence labelling method allowing comprehensive analysis of the cellular uptake of prostate cancer derived EVs in live cells using confocal microscopy. Labelling of the EVs in this way did not influence their size and had no effect on their ability to induce differentiation of lung fibroblasts to myofibroblasts. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that fluid-phase endocytosis and/or macropinocytosis was involved in EV internalisation. Over a period of six hours EVs were observed to increasingly co-localise with lysosomes, indicating a possible termination point following internalisation. Overall this method provides new opportunities for analysing the cellular dynamics of EVs as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors.

Cell‑to‑cell communication via extracellular vesicles among human pancreatic cancer cells derived from the same patient

“…Dynasore is a small, non-selective inhibitor of dynamin GTPase activity (54). Previously, dynasore has been reported to inhibit the uptake of EVs (55,56). The findings of studies mentioned above are consistent with this study.…”

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

“…Bodipy is another popular dye which some groups prefer over PKH as there is reduced background and limited overlap with signals from EVs. Alternatives to lipophilic dyes include CFSE and maleimide‐thiol coupling of dyes to EVs [144]. Interestingly, some labelling protocols recommend BSA to quench dye labelling of EVs.…”

Exploiting the Natural Properties of Extracellular Vesicles in Targeted Delivery towards Specific Cells and Tissues

“…As an alternative to the commonly used fluorescence probes, Zhang et al 2018 proposed indirectly labelling EVs by covalently conjugating membrane phospholipids with fluorescent markers, which permits reducing the non-specific extracellular labelling [129]. By taking advantage of the sulfhydryl groups on the EV surface, maleimide-conjugated Alexa-fluor dyes were covalently added to the EV surface by simple incubation [130]. Protein-based labelling of EVs has also been achieved by using luciferase reporters on cells to produce bioluminescent proteins that are later transferred to the EVs which permit stable real time monitoring [131,132].…”