The abnormal expression of tumor-associated proteases and lowered extracellular pH are important signatures strongly associated with cancer invasion, progression, and metastasis. However, their malignant effects were mainly identified using cell and tissue studies.
      To non-invasively visualize the heterogeneous distribution of these abnormal indicators in vivo and further disclose their collective behaviors, a protease-triggered dual-ratiometric fluorescent probe has successfully been constructed based on a FRET system comprised of Fe₃O₄ nanoparticles, pH-sensitive ratiometric fluorescent dye, NIR dye, MMP-9 specific peptide substrate linker, and folic acid as a tumor active targeting moiety. In addition to MRI compatibility enabled by Fe₃O₄ portion of the nanoprobe, this rational design allows these intravenously delivered nanoparticles to simultaneously map the protease activity of MMP-9 and microenvironmental pH of tumors. Systematic studies further demonstrate that the dual-ratiometric systems provide not only instant but also quantifiable information on the local protease activity of MMP-9 and pH in tumors. The current study also demonstrated that the overexpression of MMP-9 is well-correlated, in both time and location, with abnormal pH in vivo. The synergistic effects of these two characteristics largely govern the heterogeneous invasion of malignant tumors.
      Therefore, the key novelty of the current study lies in the quantitative and real-time detection of multiple tumor microenvironmental factors through non-invasive multimodality imaging. Thus, we believe that the general concept of the probe design demonstrated herein may represent a powerful tool for studying and characterizing abnormal tumor signatures in vivo.
                                                                                                                                                                                                          Tiancong Ma et al.