Optimized Biotinylation-Based Loading of Therapeutic Peptides/Proteins into Nanoscale Extracellular Vesicles for Targeted Cancer Therapy

Extracellular vesicles (EVs) are cell-derived, nanoscale membrane vesicles that have attracted attention as promising drug delivery carriers for various drug modalities. Cargo-load efficiency is important for the application of EVs as delivery vehicles. In this study, we developed a nanoscale method to multiply the loading efficiency of peptide and protein cargos into EVs. Fusion proteins were used to load peptide/protein cargoes into EVs, and a biotinylation reaction from the archaeon Sulfolobus tokodaii was used to allow multiple cargoes to be attached to a single molecule of the fusion protein containing the EV-tropic protein. This method significantly increased the loading efficiency of gLuc, a model reporter protein, into large EVs (lEVs). Subsequently, lEVs loaded with therapeutic peptides, protein kinase A (PKA) inhibitor peptides (PKI), or signal transducer and activator of transcription 3 (STAT3) inhibitor peptides (STAT3i), were prepared using an established loading method. PKI-loaded- and STAT3i-loaded lEVs demonstrated inhibitory effects on PKA and STAT3 signaling pathways and regulated M1/M2 marker expression in tumor-associated macrophages (TAMs). Moreover, PKI-loaded EVs enhanced the antitumor effect of the anti-PD-1 antibody in mice with breast cancer by modulating the TAM phenotype in the tumor microenvironment. These results suggest that therapeutic peptides/proteins can be efficiently loaded into EVs using the developed method to elicit the therapeutic potential of cargos in targeted cancer therapy.