Bacterial metabolism-activated turn-on fluorescent liposomal probe for diagnosing bacteria-associated tumors

Tumor-resident bacteria have emerged as key regulators of tumor progression, metastasis, and therapeutic resistance, yet their detection remains challenging because of their low abundance and predominantly intracellular localization. Here, we report a bacterial metabolism-activated fluorescent liposomal probe for the selective detection of intratumoral bacteria. The probe was constructed by covalently conjugating hydrophilic D-alanine, a bacterial metabolizable substrate, to a hydrophobic fluorophore, yielding an amphiphilic cationic metabolic-labeling molecule, 3-HFD. In aqueous media, 3-HFD self-assembled into fluorescence-quenched nanoaggregates through aggregation-caused quenching, thereby minimizing background fluorescence. These nanoaggregates were encapsulated into charge-reversible, membrane-fusogenic DCPA-H₂O liposomes to promote tumor accumulation and intracellular delivery. In the weakly acidic tumor microenvironment, DCPA-H₂O underwent protonation, enhancing cellular internalization and membrane fusion. Following intracellular release, the cationic probe bound with negatively charged bacteria and was metabolically incorporated into the bacterial cell wall during peptidoglycan biosynthesis, resulting in aggregate disassembly and fluorescence recovery. This metabolism-triggered fluorescence turn-on strategy enabled selective visualization of intratumoral bacteria while minimizing false-positive signals from unactivated probes. In both bacteria-associated tumor-bearing mouse models and clinical tumor specimens, the liposomal probe detected all bacteria-positive samples identified by the gold standard, demonstrating its potential for the diagnosis of bacteria-associated tumors.