A nanobody against CD63 for non-destructive exosome labeling.

Exosomes, also known as small extracellular vesicles, play crucial roles in cancer progression, inflammation, and tissue regeneration, serving as emerging platforms for diagnostics and therapeutic delivery. However, specific and high-affinity binders for non-destructive exosome labeling remain essential due to their complex and heterogeneous nature. Here, we report a nanobody (2E-Nb) identified by phage display that enables precise targeting of CD63, a tetraspanin highly enriched on exosomal membranes, allowing rapid and stable exosome surface modification. Six unique nanobody sequences were isolated, among which 2E-Nb exhibited superior solubility and binding activity. Structural modeling revealed that aromatic and basic residues in the 2E-Nb CDR3 loop form complementary electrostatic and π-π interactions with the CD63 extracellular domain (CD63-ECD). Bio-layer interferometry (BLI) confirmed high-affinity binding with a dissociation constant (KD) of 4.81 × 10− 8 M, characterized by fast association and moderate dissociation kinetics. Confocal microscopy verified the co-localization of 2E-Nb with CD63 in wild-type but not CD63-knockout H293T cells, confirming target specificity. Furthermore, NanoFCM analysis demonstrated that 2E-Nb effectively recognized native exosomal CD63 isolated from H293T cells and mediated efficient fluorescent labeling. Collectively, our findings establish 2E-Nb as a high-affinity, non-destructive tool for exosome labeling and engineering. This nanobody-based strategy provides a convenient and scalable platform for targeted exosome modification, advancing both fundamental exosome research and translational applications.