¹³¹I/MTX-loaded engineered outer membrane vesicles for sequentially coordinated radio-metabolic-immunotherapy of...

The efficacy of radiotherapy for brain tumors is limited by tumor cell radio-resistance and the immunosuppressive microenvironment, which often counteracts the immunogenic effects of radiation-induced antitumor responses. To address this, we developed statherin-enriched engineered outer membrane vesicles (OMVs), co-labeled with the radionuclide ¹³¹I and loaded with the metabolic inhibitor methotrexate (MTX), termed ¹³¹I-O/M-P, to enable integrated radiotoxicity, metabolic intervention, and immune activation. Upon delivery of ¹³¹I-O/M-P to the tumor site, ¹³¹I promptly emits β-rays that induce DNA double-strand breaks, triggering immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs), thereby initiating antitumor immune responses. Subsequently, the released MTX is internalized by tumor cells, where it inhibits dihydrofolate reductase (DHFR), blocks nucleotide synthesis, impairs DNA repair capacity, and exacerbates DNA damage accumulation, thereby further promoting apoptosis and immune activation. Meanwhile, the intrinsic adjuvant properties of OMVs synergize with the ICD response to enhance T cell activation and infiltration. In both subcutaneous and orthotopic brain tumor mouse models, ¹³¹I-O/M-P significantly enhanced CD8⁺ T cell infiltration, reduced T cell exhaustion phenotypes, and suppressed tumor growth. In summary, we present a temporally coordinated radio-metabolic-immunotherapy strategy, offering a novel therapeutic approach for advancing radio-immunotherapy in brain tumors.