Modular and Nondisturbing Chimeric Adaptor Protein for Surface Chemistry of Small Extracellular Vesicles

Current chemical strategies for modifying the surface of extracellular vesicles (sEVs) often struggle to balance efficient functionalization with preserving structural integrity. Here, we present a modular approach for the surface modification of sEVs using a chimeric adaptor protein (CAP). The CAP was designed with three key features: a SNAP-tag for stable and modular binding, long and rigid linker to enhance spatial accessibility and conjugation efficiency, and the N-terminal sorting domain derived from syntenin to improve CAP expression on the sEV. We established a postsynthetic method to introduce diverse functional molecules onto sEVs, creating a versatile system termed “sEV-X” (where X represents an organic molecule, protein, or nanoparticle). Quantitative analyses at the single-molecule level revealed a linear relationship between CAP expression and the number of conjugated functional molecules, underscoring the importance of steric hindrance mitigation in sEV surface engineering. Moreover, antibody-conjugated sEVs as drug carriers, demonstrated significant tumor-specific delivery and therapeutic efficacy in a tumor-bearing mouse model, underscoring the potential of CAP-expressing sEVs as a customizable therapeutic vesicle. Overall, the CAP technology may serve as a universal platform for advancing the development of sEV-based therapeutics.