Milk-derived extracellular vesicles functionalized with anti-tumour necrosis factor-α nanobody and anti-microbial peptide alleviate ulcerative colitis in mice

Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EV_VHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EV_LV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EV_LV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.