Spatiotemporally Asynchronous Dual-Modality Probe: Real-Time and Accurate Monitoring of Neutrophil Recruitment in Phototherapy Prognosis

The persistent issue of poor prognosis in tumor recurrence and metastasis, even with promising treatments such as photodynamic therapy, underscores the urgent need for noninvasive imaging tools to investigate underlying mechanisms. However, achieving real-time, crosstalk-free monitoring of multiple biomarkers within pathological processes remains challenging, largely due to the absence of effective molecular design strategies. Herein, we propose a “spatiotemporal asynchronous” probe design strategy and develop HD-LN, a sequentially activatable fluorescence/photoacoustic (FL/PA) probe for real-time monitoring of neutrophil recruitment after tumor phototherapy. HD-LN is first activated by tumor-overexpressed leucine aminopeptidase (LAP), leading to fluorescence activation and restoration of photodynamic (PDT) activity. After phototherapy, it is further activated by recruited neutrophil elastase (NE), resulting in fluorescence quenching together with enhanced photoacoustic signaling. Through this sequential activation of FL/PA signals, we successfully demonstrated that poor PDT prognosis correlates with neutrophil recruitment and NETs-associated changes during treatment, while inhibition of NE expression significantly improves therapeutic outcomes and prevents tumor metastasis. Furthermore, by leveraging the sequential signal changes, HD-LN first enabled in vivo screening of NE inhibitors. This work not only provides insights into overcoming poor PDT prognosis but also establishes a novel strategy for real-time, crosstalk-free monitoring of multiple biomarkers during physiological processes.