Metabolically Labeling Near-Infrared-II Excited Fluorophores on Bacterial Outer Membrane Vesicles for Targeted Imaging and Photoimmunotherapy of Gram-Negative Bacterial Peritonitis.

Gram-negative bacterial infections present significant challenges to public health due to their unique outer membrane (OM) structure, which confers reduced sensitivity to microbicides. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria have emerged as versatile platforms for immunotherapy by leveraging their inherent immunogenicity and engineerability. Herein, we developed near-infrared II (NIR-II) excitable fluorophore-labeled OMVs (IR-FCTP-OMVs) for precise imaging and photoimmunotherapy of peripheral and endosomatic Gram-negative infections, such as Escherichia coli-induced peritonitis, by exploiting homologous bacterial targeting and immune activation. d-propargylglycine (DPG)-modified OMVs were generated via the d-amino acid (DAA) labeling method and subsequently conjugated with azide-modified IR-FCTP through copper-catalyzed click chemistry. These IR-FCTP-OMVs selectively targeted Gram-negative bacteria, demonstrating potent bactericidal activity against planktonic and biofilm-forming pathogens under a 1064 nm NIR-II laser irradiation. Notably, in an E. coli-induced peritonitis model, IR-FCTP-OMVs facilitated deep-tissue NIR-II imaging and effective photoimmunotherapy, significantly reducing bacterial loads and inflammation. Furthermore, they promoted dendritic cell maturation and increased CD8+CD3+ T cell populations, indicating the robust activation of both innate and adaptive immune responses. These findings position IR-FCTP-OMVs as a promising tool for the precise diagnosis and targeted treatment of Gram-negative infections, offering a novel approach to combat antimicrobial resistance and enhance clinical outcomes.