Caspase-3-Responsive Dual-Enhanced 1H/19F MRI Bimodal Probe for In Vivo Tumor Apoptosis Imaging

Caspase-3 (Cas-3) is a crucial cysteine protease involved in executing cell apoptosis, a process that is a hallmark of tumor cell apoptosis response to chemotherapy. Developing molecular imaging probes that specifically detect apoptotic signals during tumor treatment is considered to be one of the most important and effective strategies for reducing cancer-associated death rates and improving treatment outcomes. However, achieving a dual-enhanced bimodal probe in a single molecule remains a significant challenge. In this study, we developed a ¹H/¹⁹F dual-enhanced magnetic resonance imaging (MRI) probe, CF₃DEVDFFFK(Fmoc)-Gd, responsive to caspase-3 for in vivo imaging of apoptotic cells. Upon interaction with caspase-3, CF₃DEVDFFFK(Fmoc)-Gd efficiently splits into two components CF₃DEVD and FFFK(Fmoc)-C₂-Gd, where FFFK(Fmoc)-C₂-Gd subsequently self-assembles into nanofibers. This process activates both ¹⁹F MRI and ¹H MRI, with longitudinal relaxivity (r₁) increasing from 9.38 ± 0.22 to 23.24 ± 2.33 mM^-1 s^-1 at 0.5 T and turning on the ¹⁹F MRI signal due to the absence of the paramagnetic relaxation enhancement (PRE) effect. In vivo imaging results demonstrated that, after systemic administration, CF₃DEVDFFFK(Fmoc)-Gd effectively accumulated in apoptotic 4T1 tumor tissues, resulting in significantly enhanced ¹H MRI signals for visualization of caspase-3 activity in doxorubicin-treated apoptotic 4T1 tumor tissues, with signal intensity three times greater than that of Gd-DTPA. ¹⁹F MRI further complemented ¹H MRI, with a notable recovery of the ¹⁹F MRI signal after intratumoral injection. These results confirm that CF₃DEVDFFFK(Fmoc)-Gd effectively reports tumor apoptosis through combined ¹H and ¹⁹F MRI, offering a promising approach for the preliminary assessment of antitumor efficacy in vivo.