Surface Mineralization of Engineered Bacterial Outer Membrane Vesicles to Enhance Tumor Photothermal/Immunotherapy

Gram-negative bacteria can naturally produce nanosized spherical outer membrane vesicles (OMVs) with a lipid bilayer membrane, possessing immunostimulatory capabilities to be potentially applied in tumor therapy. However, the systemic toxicity induced by pathogen-associated molecular patterns (PAMPs) of OMVs is the main obstacle for their clinical translation. Herein, melanin-loaded OMVs were produced with a genetic engineering strategy and further coated with calcium phosphate (CaP) to reduce their toxicity to enhance tumor treatment effects. Wild-type bacterium <i>Escherichia coli</i> Nissle 1917 (EcN) was genetically engineered to highly express tyrosinase to catalyze the intracellular synthesis of melanin, giving melanin-loaded OMVs (OMV^Mel). To reduce the systemic toxicity in tumor therapy, OMV^Mel was coated with CaP by surface mineralization to obtain OMV^Mel@CaP. In comparison with OMV^Mel, OMV^Mel@CaP showed lower systemic inflammatory responses in healthy mice and less damage to the liver, spleen, lung, and kidney, so the administration dose could be increased to enhance the antitumor effect. In the acidic tumor microenvironment, the CaP shell disintegrated to release OMV^Mel to trigger antitumor immune responses. Under costimulation of OMV^Mel acting as immunoadjuvants and the damage-associated molecular patterns (DAMPs) released by the photothermal effect, the efficiency of tumor photothermal/immunotherapy was largely boosted through promoting the infiltration of matured DCs, M1 macrophages, and activated CD8⁺ T cells, decreasing the ratio of MDSCs in tumors.