A Modular Glycoengineered Exosome Platform for Targeted Delivery via Chemoenzymatic Engineering of Surface Sialic Acids

Bovine milk-derived exosomes (mExos) are scalable, biocompatible nanocarriers well-suited for the cross-barrier transport and protection of labile nucleic acid therapeutics. However, achieving precise active targeting for cargo delivery remains challenging, as conventional surface functionalization methods often lack site-specificity, unintentionally altering native physicochemical properties or obscuring essential membrane interactions. Herein, we report a modular glycoengineering strategy that leverages the dense sialic acid coating on mExos to introduce versatile anchoring sites for targeting ligand modification via sequential chemoenzymatic remodeling. Specifically, the native terminal sialic acids on the mExo surface were enzymatically removed and replaced with azide-modified sialic acid analogs, which was achieved efficiently through an integrated multienzyme reaction. In principle, the resulting platform, mExo-N3, enables the glycan-selective conjugation of any dibenzocyclooctyne (DBCO)-modified targeting ligands for targeted delivery, avoiding the limitations of random modifications. To validate this platform, we conjugated mExo-N3 with an anti-EGFR nanobody (7D12) and encapsulated a microRNA-21 inhibitor (miR-21i). The resulting mExo-7D12@miR-21i construct maintained colloidal stability and exhibited specific internalization by EGFR-positive cancer cells. Intracellular delivery of miR-21i restored the expression of tumor suppressors PTEN and PDCD4, inducing apoptosis and suppressing proliferation. Furthermore, in an A431 xenograft model, the targeted system achieved 58.3% tumor growth inhibition in vivo without evident systemic toxicity. By converting native glycans into functional anchoring sites, this approach provides a translatable framework for customizing exosomal delivery vectors into targeted therapeutics.