Metabolic glycoengineered exosomes enable in situ PD-L1-Fc assembly for targeted immunotherapy in ulcerative colitis

Ulcerative colitis is a chronic inflammatory bowel disease driven by aberrant immune activation, disruption of the mucosal barrier, and dysbiosis of the gut microbiota. Although current immunomodulators and biologics improve outcomes in some patients, their efficacy is frequently limited by systemic immunosuppression‑related adverse effects and recurrent disease relapses. Therefore, therapeutic strategies capable of precisely targeting the diseased intestinal mucosa are urgently needed. An exosome‑based delivery system was developed that integrates metabolic glycan engineering with in vivo bioorthogonal click chemistry to enable in situ assembly and localized action of a fusion protein consisting of programmed death ligand‑1 and the Fc fragment of immunoglobulin (PD‑L1‑Fc) within inflamed intestinal tissue. The platform follows a two‑step in vivo targeting scheme: orally administered azide‑modified exosomes (N₃‑Exos) selectively accumulate in inflamed colonic segments; subsequently, intraperitoneal injection of dibenzocyclooctyne (DBCO)‑modified PD‑L1‑Fc allows covalent coupling to the exosomes at the lesion site via a strain‑promoted azide‑alkyne cycloaddition reaction, leading to the direct assembly of PD‑L1‑Fc‑modified exosomes in situ. In a murine model of experimental colitis, orally administered N₃‑Exos showed marked accumulation in inflamed colon and sustained retention. The two‑step combination treatment significantly attenuated body weight loss, corrected colon shortening, reduced disease activity index scores, and alleviated histological damage including inflammatory cell infiltration and crypt destruction. Goblet cell numbers and mucus layer integrity were restored. Immunologically, the treatment expanded regulatory T cells, T helper 1 (Th1) cells, and T follicular helper (Tfh) cells in the colonic lamina propria, while promoting M2 macrophage polarization and reducing the M1/M2 ratio. Colonic levels of interleukin‑10 (IL‑10) were increased, whereas IL‑12, tumor necrosis factor‑α, and IL‑6 were decreased. Furthermore, the gut microbiota was reshaped in community structure, with reduced abundance of pathogenic Enterobacteriaceae (including Escherichia‑Shigella) and enrichment of short‑chain fatty acid‑producing taxa ( Muribaculaceae , Lactobacillaceae ) and the mucus‑protective genus Akkermansia . An innovative approach for the in situ synthesis and precise delivery of PD‑L1‑Fc is presented. This strategy reshapes the mucosal immune microenvironment, restores epithelial barrier integrity, and normalizes gut microbiota homeostasis, offering a new paradigm for precision immunotherapy of ulcerative colitis with significant translational potential. Schematic illustration of a two-step exosome-based strategy for ulcerative colitis. Orally delivered azide-modified exosomes target inflamed colon, then undergo in vivo click reaction with injected DBCO-PD-L1-Fc to generate PD-L1-Fc decorated exosomes in situ, modulating mucosal immunity and restoring barrier and microbiota homeostasis