Bioorthogonal and synthetic biology-engineered bacterial outer membrane vesicles for enhanced photo-immunotherapy.

Bacterial outer membrane vesicles (OMVs) have emerged as versatile platforms for cancer immunotherapy, yet their clinical translation is hindered by limited drug-loading capacity, suboptimal tumor accumulation, and rapid clearance, which collectively compromise the induction of robust antitumor immunity. Here, we engineer a multifunctional nanocarrier, OMV-mIL2-ICG-DBCO, that integrates cytokine delivery, photothermal therapy, and bioorthogonal targeting to overcome these obstacles. Using synthetic biology, parental bacteria are modified to autonomously produce interleukin-2 (mIL2)-enriched OMVs, thereby endowing the vesicles with intrinsic immunostimulatory activity. The photosensitizer indocyanine green (ICG) is subsequently encapsulated into OMVs via electroporation, enabling near-infrared light-triggered hyperthermia, immunogenic tumor cell death, and enhanced release of tumor-associated antigens. In parallel, metabolic glycoengineering with azide-functionalized sugar precursors induces N group expression on tumor cell surfaces, while OMVs are functionalized with dibenzocyclooctyne (DBCO), allowing highly specific bioorthogonal conjugation and selective vesicle accumulation within tumors. In vivo, OMV-mIL2-ICG-DBCO exhibits markedly improved tumor homing, potent photothermal ablation, and strong activation of antitumor immune responses, resulting in significant inhibition of primary tumor growth and reduced off-target toxicity compared with non-targeted controls. Collectively, this bioorthogonally targeted, photo-immunotherapeutic OMV platform provides an effective strategy to amplify antitumor immunity and represents a promising candidate for the treatment of breast cancer. STATEMENT OF SIGNIFICANCE: Bacterial outer membrane vesicles (OMVs) are promising bioinspired nanocarriers with inherent immunogenicity, but their therapeutic efficacy is limited by poor tumor targeting and low drug-loading capacity. Here, we develop a dual-engineered OMV platform that combines genetic IL-2 expression with bioorthogonal glycol-metabolic labeling (N3/DBCO) to achieve programmable immune modulation and tumor-specific delivery. The vesicles are further loaded with indocyanine green via electroporation, enabling photothermal-triggered immunogenic cell death that promotes antigen exposure and dendritic cell maturation. This synergistic integration of biological and chemical engineering endows OMVs with precise targeting, potent immune activation, and minimized off-target photothermal effects. The strategy introduces a biorthogonal targetable photo-immunotherapeutic nanoplatform, offering a versatile and generalizable framework for engineering living-derived biomaterials in cancer immunotherapy.