Rapid extracellular vesicle surface decoration with targeting moieties based on a fluorescein binding single chain variable fragment snorkel

Extracellular vesicles (EVs) are cell-derived nanovesicles with promising potential for drug delivery due to their low toxicity and immunogenicity. However, their clinical application is limited by poor targeting to sites of interest. Existing strategies to engineer targeted EVs often require genetic donor cell modification for each specific target, making the process time-consuming and costly. To overcome this, we developed a versatile targeting platform using the fluorescein-specific single-chain variable fragment (scFv) 4 M5.3, integrated into a CD81-based Snorkel-tag construct for surface display on EVs. A C-terminal HA-tag, separated by a PreScission protease (PS) site, allows selective purification of targeted EVs and removal of unbound targeting moieties. This design enables functionalization of EVs with any fluorescein-conjugated targeting molecule. We tested various construct modifications (cMyc, FLAG, PS-HA), which showed differing expression levels and FITC-antibody binding by HEK293 cells and their EVs. As proof of concept, we generated EVs targeting human HER2 and mouse CCR2 by capturing FITC-labeled antibodies, which bound specifically to HER2+ NCI-N87 and CCR2+ RAW264.7 cells. The technology was also successfully applied to transmembrane protein CD9 and WJ-MSC/TERT273-derived EVs. In summary, we present a robust, adaptable method for generating EVs with customizable targeting, enabling high-throughput target screening and accelerating the development of EV-based therapeutics. • The scFv-Snorkel-tag technology enables the display of FITC-conjugated antibodies on the EV surface. • CD81-scFv-Snorkel-tag-expressing EVs demonstrate targeted binding to HER2- and CCR2-expressing cells in vitro . • CD81-scFv-Snorkel-tag EVs can be selectively purified by anti-HA affinity purification and subsequent PreScission Protease cleavage. • The scFv-Snorkel-tag technology will prevent repeated genetic engineering steps for every selected target, enabling rapid EV target screening and identification. • This study shows the specific purification of targeted EVs, preventing possible off-target effects of heterogeneous EV preparations.