A General Approach to Design Dual Ratiometric Fluorescent and Photoacoustic Probes for Quantitatively Visualizing Tumor Hypoxia Levels In Vivo

Herein, we describe an energy balance strategy between fluorescence and photoacoustic effects by sulfur substitution to transform existing hemicyanine dyes (Cy) into optimized NIRF/PA dual ratiometric scaffolds. Based on this optimized scaffold, we reported the first dual-ratio response of nitroreductase probe AS-Cy-NO₂, which allows quantitative visualization of tumor hypoxia in vivo. AS-Cy-NO₂, composed of a new NIRF/PA scaffold thioxanthene-hemicyanine (AS-Cy-1) and a 4-nitrobenzene moiety, showed a 10-fold ratiometric NIRF enhancement (I₇₇₃/I₇₃₃) and 2.4-fold ratiometric PA enhancement (PA₇₃₀/PA₆₇₀) upon activation by a biomarker (nitroreductase, NTR) associated with tumor hypoxia. Moreover, the dual ratiometric NIRF/PA imaging accurately quantified the hypoxia extent with high sensitivity and high imaging depth in xenograft breast cancer models. More importantly, the 3D maximal intensity projection (MIP) PA images of the probe can precisely differentiate the highly heterogeneous oxygen distribution in solid tumor. Thus, this study provides a promising NIRF/PA scaffold that may be generalized for the dual ratiometric imaging of other disease-relevant biomarkers.