Dual Targeting and Cascaded Aggregation of Luminogen for Washing-Free and Enhanced Fluorescence Imaging of Intracellular Bacteria

The invasion and intracellular survival of <i>Staphylococcus aureus</i> (<i>S. aureus</i>) within phagocytic cells is a major cause of serious antibiotic resistance and recurrent infections in clinical settings, making the detection of intracellular <i>S. aureus</i> highly desirable prior to initiating effective treatment. In this study, we proposed a dual-targeting and cascaded aggregation-induced emission luminogen (AIEgen) probe for washing-free and enhanced fluorescence imaging of intracellular <i>S. aureus</i>. The AIEgen probes consist of an AIE molecule (TPE), cleavable peptide (YVADC(StBu)GKK), targeting peptide (RP)₃, and the disulfide Cys(StBu) and 2-cyanobenzothiazole (CBT) units, YVADC(StBu)GK(TPE)K(RP)₃G-CBT (YTR-CBT). Upon encountering elevated levels of intracellular biomarkers (GSH and Caspase-1) associated with bacterial infection, the YTR-CBT probe undergoes a CBT-Cys click reaction, triggering its self-assembly into AIEgen nanoparticles (TR-CBT-NPs) and consequently activating the AIEgen fluorescence. These nanoparticles then undergo cascaded aggregation on the surface of <i>S. aureus</i>via the (RP)₃ targeting unit, leading to a significantly enhanced fluorescence signal around the intracellular bacteria compared to that of control groups lacking either the targeting unit or bacterial infection. Moreover, the AIEgen probe specifically imaged intracellular bacteria without interference from extracellular bacteria, enabling a washing-free imaging strategy. This capability allowed for rapid monitoring of therapeutic efficacy in a mouse model of septic arthritis involving intracellular bacteria, demonstrating its considerable potential for clinical application.