GFP Farnesylation\nas a Suitable Strategy for Selectively\nTagging Exosomes

Exosomes are small extracellular vesicles (EVs) constituting\nfully\nbiological, cell-derived nanovesicles with great potential in cell-to-cell\ncommunication and drug delivery applications. The current gold standard\nfor EV labeling and tracking is represented by fluorescent lipophilic\ndyes which, however, importantly lack selectivity, due to their unconditional\naffinity for lipids. Herein, an alternative EV fluorescent labeling\napproach is in-depth evaluated, by taking advantage of green fluorescent\nprotein (GFP) farnesylation (GFP-f), a post-translational modification\nto directly anchor GFP to the EV membrane. The performance of GFP-f\nis analyzed, in terms of selectivity and efficiency, in several typical\nEV experimental setups such as delivery in recipient cells, surface\nengineering, and cargo loading. First, the capability of GFP and GFP-f\nto label exosomes was compared, showing significantly higher GFP protein\nlevels and fluorescence intensity in GFP-f- than in GFP-labeled exosomes,\nhighlighting the advantage of directly anchoring the GFP to the EV\ncell membrane. Then, the GFP-f tag was further compared to Vybrant\nDiD lipophilic dye labeling in exosome uptake studies, by capturing\nEV intracellular fluorescence in a time- and concentration-dependent\nmanner. The internalization assay revealed a particular ability of\nGFP-f to monitor the uptake of tagged exosomes into recipient cells,\nwith a significant peak of intensity reached 12 h after administration\nby GFP-f but not Vybrant-labeled EVs. Finally, the GFP-f labeling\ncapability was challenged in the presence of a surface modification\nof exosomes and after transfection for siRNA loading. Results showed\nthat both procedures can influence GFP-f performance compared to naïve\nGFP-f exosomes, although fluorescence is importantly maintained in\nboth cases. Overall, these data provide direct insight into the advantages\nand limitations of GFP-f as a tagging protein for selectively and\naccurately tracking the exosome route from isolation to uptake in\nrecipient cells, also in the context of EV bioengineering applications.