Versatile functionalization of Bifidobacteria-derived extracellular vesicles using amino acid metabolic labeling and click chemistry for immunotherapy

Extracellular vesicles (EVs) are nanoparticles secreted by various organisms. Methods for modifying EVs functionally have garnered attention for developing EV-based therapeutic systems. However, most technologies used to integrate these functions are limited to mammalian-derived EVs and a promising modification method for bacteria-derived EVs has not yet been developed. In this study, we propose a novel method for the versatile functionalization of immunostimulatory probiotic Bifidobacteria-derived EVs (B-EVs) using amino acid metabolic labeling and azide-alkyne click reaction. Azide D-alanine (ADA), a similar molecule to D-alanine in bacteria cell-wall peptidoglycan, was selected as an azide group-functionalized amino acid. Azide-modified B-EVs were isolated from Bifidobacteria incubated with ADA. The physicochemical and compositional characteristics, as well as adjuvanticity of B-EVs against immune cells were not affected by azide loading, demonstrating that this functionalization approach can retain the endogenous usefulness of B-EVs. By using the fluorescent B-EVs obtained by this method, the intracellular trafficking of B-EVs after uptake by immune cells was successfully observed. Furthermore, this method enabled the formulation of B-EVs for hydrogelation and enhanced adjuvanticity in the host. Our findings will be helpful for further development of EV-based immunotherapy.