Engineered Streptococcus salivarius K12-Derived Extracellular Vesicles Functionalized with an Anti-PD-L1 Nanobody to Promote Recurrent Aphthous Ulcer Healing

Background: Recurrent aphthous ulcer (RAU) is a common inflammatory disorder of the oral mucosa characterized by painful ulceration and delayed tissue repair, while rapid salivary clearance and mechanical stimulation limit the efficacy of conventional topical therapies. Streptococcus salivarius K12-derived extracellular vesicles (EVs) are potential cell-free agents for inflammatory oral mucosal disease. We investigated whether surface functionalization with a nanobody selected against human PD-L1 (αPDL1-VHH) could improve their biological activity. Methods: K12-derived EVs were surface-functionalized through a sequential enzymatic ligation strategy, in which endogenous Sortase A-associated machinery was exploited for linker anchoring, followed by OaAEP1-mediated site-specific conjugation of αPDL1-VHH. Proteomic profiling was performed to identify Sortase A and the LPXTG cell wall anchor motif in K12-EVs. The cellular association of EV-αPDL1-VHH was evaluated in PD-L1-expressing cells. Anti-inflammatory activity was assessed in LPS-stimulated RAW264.7 macrophages, and therapeutic effects were evaluated in an acetic acid-induced mouse oral ulcer model. Transcriptomic analysis was performed to investigate the molecular responses to treatment. Results: Proteomic profiling identified Sortase A in K12-EVs and revealed enrichment of the LPXTG cell wall anchor motif, supporting the molecular basis of the endogenous enzymatic ligation strategy. EV-αPDL1-VHH exhibited enhanced association with PD-L1-expressing cells compared with unmodified EVs. In LPS-stimulated RAW264.7 macrophages, EV-αPDL1-VHH markedly suppressed the expression and secretion of pro-inflammatory cytokines and showed greater anti-inflammatory activity than unmodified EVs or free αPDL1-VHH. In an acetic acid-induced mouse oral ulcer model, EV-αPDL1-VHH accelerated wound closure, increased epithelial thickness, and improved histological recovery. Transcriptomic analysis revealed broad transcriptional remodeling and modulation of inflammatory and mTOR-associated pathways, accompanied by increased expression of the stress-responsive regulators DDIT4 and SESN2. Conclusions: PD-L1-targeted engineering enhanced the therapeutic activity of K12-derived EVs and promoted oral mucosal healing, highlighting EV-αPDL1-VHH as a promising targeted probiotic-derived EV platform for inflammatory oral mucosal diseases.