Bioorthogonal engineering of an antioxidant-armored probiotic biohybrid potentiates therapy for ulcerative colitis

Abstract Oral administration of probiotics represents a promising therapeutic strategy for ulcerative colitis (UC), offering the potential to modulate gut immunity and restore mucosal homeostasis. However, the clinical efficacy of conventional probiotics is fundamentally limited by their susceptibility to the pathological oxidative stress within the inflamed intestine, which severely compromises their viability and function. To overcome this critical barrier, we developed a bioorthogonal engineering strategy to construct a robust probiotic-based biohybrid, Lr-DRA. This was achieved by metabolically labeling Lactobacillus reuteri (Lr) with azido groups, followed by site-specific, covalent conjugation of a potent phenolic antioxidant, DBCO-modified rosmarinic acid (DRA). This design ensures the stable integration of therapeutic components without impairing probiotic vitality. In a murine model of dextran sulfate sodium (DSS)-induced colitis, orally administered Lr-DRA demonstrated superior therapeutic efficacy. The surface-anchored DRA exerted protective effects by neutralizing oxidative stress. This protective action significantly enhanced the survival and colonization of the probiotic carrier Lr. Consequently, Lr-DRA treatment markedly alleviated clinical symptoms, suppressed pro-inflammatory cytokines, restored intestinal barrier integrity (as evidenced by upregulated tight junction proteins Zonula Occludens-1 [ZO-1] and Occludin), and promoted mucosal healing. This work establishes a versatile and modular platform for fabricating advanced living therapeutics. By simply varying the probiotic chassis and the conjugated bioactive payload, this strategy can be readily adapted to develop targeted treatments for a broad spectrum of gastrointestinal and metabolic disorders.