Aminopeptidase-Responsive NIR Photosensitizer for Precision Targeting and Eradication of <i>Pseudomonas aeruginosa</i> Biofilms

The emergence of resistance in <i>Pseudomonas aeruginosa</i> represents a significant global health challenge, particularly due to the hurdle of effectively penetrating biofilms with antimicrobials. Moreover, the rise of antibiotic-resistant pathogens has driven the urgent need for developing innovative therapeutic approaches to overcome antibiotic resistance. Antibacterial phototherapy strategies have shown great potential for combating pathogens due to their broad-spectrum antimicrobial activity, spatiotemporal controllability, and relatively low rate of resistance emergence. However, due to the lack of bacterial specificity and penetration, photosensitizers cause considerable damage to mammalian cells and normal tissues and are less effective against bacterial biofilms. Herein, we developed a novel dual-targeting antibacterial strategy to construct a near-infrared photosensitizer, Cy-NEO-Leu. Cy-NEO-Leu showed great bacterial targeting affinity, penetrating and accumulating in biofilms. At the site of infection, it was specifically activated by <i>P. aeruginosa</i> aminopeptidase (PaAP), producing Cy-NEO-NH₂, which demonstrated outstanding photothermal (PTT) and photodynamic (PDT) properties, with a photothermal conversion efficiency of up to 70.34%. Both <i>in vitro</i> and <i>in vivo</i> results demonstrated that Cy-NEO-Leu significantly reduced the biofilm biomass and bacterial viability in <i>P. aeruginosa</i> biofilms. Moreover, phototherapy with Cy-NEO-Leu further activated the immune system, enhancing therapeutic efficacy and promoting wound healing. RNA-seq analysis revealed that the antibacterial mechanism of Cy-NEO-Leu-mediated phototherapy involves disruption of the transcriptional and translational processes of <i>P. aeruginosa</i> under laser irradiation. Overall, our results present a promising therapeutic approach against <i>P. aeruginosa</i> biofilms and inspire the development of next-generation antimicrobials.