Design And Synthesis Of Dual Ratiometric Fluorescent And Photoacoustic Molecular Probes For Tumor Imaging,Therapy,and Prognostic Analysis

By allowing real-time visualization and real-time diagnostics,near-infrared fluorescence（NIRF）imaging has greatly improved the therapeutic outcome in cancer diagnosis,image-guided surgery,and therapeutic evaluation.However,it is unable to deliver high spatial resolution（NIR?I,<1 mm）.Luckily,a new nonionizing imaging technique,photoacoustic（PA）imaging,which incorporates optical excitation with ultrasound detection,allows for detailed visualization of deep disease-relevant biomarkers with high spatial resolution,although the spatial resolution and sensitivity of PA imaging are inadequate.Thus,the underlying imaging objects can be combined with photoacoustic and fluorescence imaging to gain complementary optical absorption and fluorescence information.It is well known that single NIRF/PA intensity-based probes are easily affected by substantial interferences,such as changes in environment polarity,p H,photobleaching,probe concentration,probe distribution,and laser power.Considering ratiometric NIRF/PA probes with two emission bands,these interferences can be effectively mitigated because of the built-in self-calibration mechanism.Therefore,dual ratiometric NIRF/PA probes can be quantitatively analyzed using quantitative NIRF/PA imaging.Unfortunately,currently commonly used near-infrared dyes are far from being an ideal scaffold for developing high-performance NIRF/PA dual-ratio molecular probes due to the following reasons:（1）The absorption and emission wavelengths of near-infrared dyes whose NIRF/PA properties can be easily regulated,such as hemicyanine,are not long enough.The construction of near-infrared dual-ratio probes cannot be matched with current commercial in vivo fluorescence imaging and photoacoustic imaging instruments.（2）Many near-infrared dyes are highly fluorescent emitting molecules,but their fluorescence generation pathways（radiative processes）and photoacoustic generation pathways（nonradiative processes）are always competing with each other,which will inevitably affect the output of fluorescence and photoacoustic signals,so it is difficult to balance the energy distribution of fluorescence and photoacoustic in a single near-infrared dye molecule.In this work,we describe an energy balance strategy by sulfur substitution to transform existing hemicyanine dyes（Cy）into optimized NIRF/PA dual ratiometric scaffolds.For this purpose,a series of NIRF/PA dual ratiometric scaffolds have been constructed based on the energy balance strategy.Furthermore,these new NIRF/PA dual ratiometric probes have also been successfully applied for quantitatively visualizing tumor hypoxia levels in vivo and tumor diagnosis-therapy-quantitative prediction in vivo.The main contents are as follows:（1）We have described a general energy balance approach by sulfur substitution to transform existing hemicyanine dyes（Cy）into optimized NIRF/PA dual ratiometric scaffolds.The partial fluorescence quenching induced by the sulfur substitution will contribute to achieve an effective balance between the radiative and nonradiative decay pathways,and ultimately benefits the dual-mode imaging of NIRF and PA.The extending of the absorbtion and emission wavelengths,not only matches well the lowest excitation wavelength of the commercial instrument,but also alleviates auto fluorescence as well as facilitates ratiometric calibration.（2）We prepared a novel dual-ratio NIRF/PA response of nitroreductase probe AS?Cy?NO₂which allows quantitative visualization of tumor hypoxia levels in vivo has been rational designed and prepared.The dual ratiometric NIRF/PA imaging accurately quantified hypoxia with high spatial resolution in a breast cancer xenograft?bearing mouse model.More importantly,the 3D maximal intensity projection（MIP）PA images of the probe can precisely differentiate the highly heterogeneous oxygen distribution in solid tumor.（3）We have proposed a synergistic balanced regulation strategy by introducing superabsorption units in a sulfur-substituted hemicyanine dye to constructed dual ratiometric FL/PA single-molecule theranostic scaffolds.The artistical introduction of superabsorption units can not only incerase the molar extinction coefficient of hemicyanine dyes(ε_max=6.7×10⁴ in PBS,up 1.9?fold),but also effectively boosting PCE（63%,up 1.4?fold）.Fortunately,the p H-induced wavelength red-shift in the absorption and emission spectra without influence by superabsorption units introduction,which will provide a favorable precondition for achieving FL/PA dual ratiometric imaging,diagnosis,therapy,and quantitative prediction therapeutic efficacy in vivo.（4）We have designed and synthesized a single molecule（AF?1F?NO₂）that specifically activates its ratiometric fluorescent(FL₈₅₀/F₇₅₀),ratiometric photoacoustic(PA₇₇₀/PA₆₇₀),and photothermal signals upon response to tumor-overexpressed enzyme（nitroreductase,NTR）for turn-on PTT and quantitative evaluation of PTT efficacy in vivo.Using the distinct switch in F₈₅₀/F₇₅₀ and PA₇₇₀/PA₆₇₀ ratios in response to NTR,AF?1F?NO₂ could permit noninvasive and quantitative dual-modality imaging of NTR in vivo,confirming a degraded NTR levels in 4T1tumors following hypoxia-induced PTT by itself.In particular,three-dimensional（3D）images of the probe can precisely verify the smaller heterogeneity oxygen distribution in 4T1 tumor-bearing mice after PTT.The dual ratiometric theranostic probe may be applied for"three-in-one"diagnosis,therapy,and quantitative predictions of therapeutic efficacy in vivo for other therapeutic modalities.