Bioorthogonal Engineering of Bacterial Outer Membrane Vesicles for NIR-II Fluorescence Imaging-Guided Synergistic Enhanced Immunotherapy

The efficacy of immunotherapy in treating triple-negative breast cancer (TNBC) has been restricted due to its low immunogenicity and suppressive immune microenvironment. Bacterial outer membrane vesicles (OMVs) have emerged as innovative immunotherapeutic agents in antitumor therapy by stimulating the innate immune system, but intricate modifications and undesirable multiple dose administration severely hinder their utility. Herein, a two-step bacterial metabolic labeling technique was utilized for the bioorthogonal engineering of OMVs. At first, d-propargylglycine (DPG, an alkyne-containing d-amino acid) was introduced into the incubation process of probiotic <i>Escherichia coli</i> 1917 (Ecn) to produce DPG-functionalized OMVs, which were subsequently conjugated with azide-functionalized new indocyanine green (IR820) to yield OMV-DPG-IR820. The combination of phototherapy and immunostimulation of OMV-DPG-IR820 effectively arouses adaptive immune responses, causing maturation of dendritic cells, infiltration of T cells, repolarization of the M2 macrophage to the M1 macrophage, and upregulation of inflammatory factors. Remarkably, OMV-DPG-IR820 demonstrated tumor-targeting capabilities with guidance provided by near-infrared II (NIR-II) fluorescence imaging, leading to remarkable inhibition on both primary and distant tumors and preventing metastasis without causing noticeable adverse reactions. This study elucidates a sophisticated bioorthogonal engineering strategy for the design and production of functionalized OMVs and provides novel perspectives on the microbiome-mediated reversal of TNBC through a precise and efficient immunotherapy.