Chemo‐Enzymatic Functionalization of Bovine Milk Exosomes with an EGFR Nanobody for Target‐specific Drug Delivery

Bovine milk exosomes (BmExo) have been identified as versatile nanovesicles for anti-cancer drugs delivery due to their natural availability and biocompatibility. However, tumor-specific delivery based on BmExo often requires post-isolation modifications of the membrane surface with active-targeting ligands. In this study, we report an alternative approach to functionalize BmExo with nanobody combining facile chemical modification and Sortase A-mediated site-specific ligation, as demonstrated by the development of an epidermal growth factor receptor (EGFR)-targeted drug delivery system. The BmExo membrane was first coated with a diglycine-containing amphiphile molecule, NH₂-GG-PEG2000-DSPE, by hydrophobic insertion. The diglycine as nucleophiles displayed on the membrane enabled the subsequent ligation of the EGFR nanobody (7D12) by Sortase A (SrtA)-mediated site-specific transpeptidation. The successful construction of BmExo-7D12 was confirmed by Western blotting analysis, electron microscopy, and dynamic light scattering (DLS). As a demonstration model, BmExo-7D12 loaded with the chemotherapeutic drug doxorubicin (Dox) was shown to be able to deliver Dox to cancer cells in response to the expression of EGFR as manifested by immunocytochemistry and flow cytometry analysis. Finally, the cytotoxicity assay showed that BmExo-7D12-Dox was more effective in killing tumor cells with high EGFR expression while significantly reduced the non-specific toxicity to EGFR negative cells. In conclusion, these results demonstrate that 7D12-functionalized BmExo can serve as a target-specific delivery system for Dox to selectively kill EGFR-expressing tumor cells. This approach should prove to be versatile and efficient for the generation of protein-ligands modified BmExo.