Systematic Engineering of Human CD9 for Extracellular Vesicle Surface Display of Nanobodies Using Single-Vesicle Imaging

Abstract Extracellular vesicles (EVs) are emerging nanoscale platforms for targeted drug delivery, yet rational design of their surface composition is limited by a lack of systematic, high-throughput approaches for identifying productive protein-display constructs. Here, we engineer the human tetraspanin CD9 as a modular scaffold for displaying nanobodies (VHHs) on recombinant EV surfaces, using a high-throughput single-EV imaging platform to screen candidate constructs at the individual vesicle level. Guided by conservation analysis and structural prediction, we mapped seven insertion sites across CD9’s extracellular loops and termini and identified positions that support functional transgene display. Nonconserved regions within the short extracellular loop (SEL) yielded the highest counts of functional recombinant EVs – an empirical result that runs counter to the a priori expectation that the larger long extracellular loop (LEL) would be more permissive. We demonstrate the platform’s modularity by interchangeably incorporating fluorescent proteins and VHHs against GFP, HER2, EGFR, and PD-L1 at optimized SEL positions, where the displayed VHHs bound their cognate antigens with high specificity (∼70–90% co-localization and minimal off-target binding). Co-transfection of host cells with dual constructs yielded EVs co-displaying two independent functional moieties on ∼80% of vesicles. This work establishes single-EV imaging as a fast and quantitative screening tool for identifying productive surface-display constructs and provides a generalizable workflow for producing multifunctional, transgene-displaying EVs with potential downstream bioanalytical, diagnostic, and therapeutic applications.