Dual Ratiometric Single-Molecule Theranostic Probes for Photothermal Therapy and Real-Time Quantitative Evaluation of Therapeutic Efficacy In Vivo

Tracking the quantitative evaluation of therapeutic efficiency in tumors is essential for the precision management of cancer patients. Theranostic probes, which integrate diagnostic molecular imaging and therapeutic capabilities into a single entity, can be used to monitor the treatment process and reflect the therapeutic effect. However, current theranostic probes lack precise quantitative evaluations in vivo due to the use of single-wavelength imaging during tumor therapy. Here we present a portal library of dual ratiometric single-molecule theranostic probes for precise tumor therapy through photothermal therapy (PTT) and quantitative evaluation of tumor cell death in vivo via ratiometric near-infrared fluorescence (NIRF) and ratiometric photoacoustic (PA) imaging. One optimal single-molecule dye is further modified into an activatable probe (AF-1F-NO 2 ), whose dual ratiometric NIRF/PA signal and photothermal activity are only activated in the presence of a tumor biomarker (nitroreductase). By eliciting a dual ratiometric response (NIRF/PA: NIRF 850 /NIRF 750; PA 770 /PA 670 ) to tumor hypoxia, the new probe acts as a hypoxia-activated PTT theranostic agent, enabling real-time quantitative evaluation in vivo during PTT. Thus, this study not only presents the first dual ratiometric single-molecule theranostic probe for PTT and real-time quantitative evaluation of therapeutic efficacy in vivo, but also opens up a promising paradigm for engineering other single-molecule dual ratiometric theranostic probes in combination with more therapeutic modalities for precision medicine.