Hacking Extracellular Vesicles: Using Vesicle-Related Tags to Engineer Mesenchymal Stromal Cell-Derived Extracellular Vesicles

Background/Objectives: Extracellular Vesicles (EVs) have shown promise as diagnostic and therapeutic tools as well as pharmacological nanocarriers. Strategies are being explored to develop EVs to enable their monitoring, imaging, loading with pharmacological agents and surface decoration with tissue-specific ligands. EVs derived from mesenchymal stromal cells (MSC-EVs) are of interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this end, canine MSCs were engineered to express the green fluorescent protein (GFP) associated with CD63, syntenin-1, TSG101 and the palmitoylation signal of Lck, which were expected to introduce GFP into EVs. Overexpression of tagged GFP in canine MSCs was detected by Western blotting and examined by confocal and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed high efficiency but exhibited a diffuse localization pattern in the transfected cells. Palmitoylation signal showed low efficiency and low specificity in terms of localization. TSG101 showed a morphological pattern consistent with a specific localization in endosomal structures, but its low expression did not allow further considerations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 was 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 is the most effective tag for canine MSC-EVs engineering. Further studies aimed at better deciphering its contribution and clarifying which part of the molecule is involved in vesicle trafficking could provide insights for EV bioengineering.