In vivo multiplexed analysis of aminopeptidase activities by hyperpolarized molecular probes for tumor diagnostic applications

Aminopeptidases (APs) in the renin-angiotensin system (RAS) and their activity balance play a crucial role in regulating vascular functions. Multiplexed analysis of RAS-related AP activities is useful for diagnosing diseases, including cancer. Dynamic nuclear polarization coupled magnetic resonance imaging (DNP-MRI) enables simultaneous detection of multiple enzymatic activities in vivo. However, developing practical DNP-MRI probes, especially for multiplexed detection, remains challenging. Here, we report the design of DNP-MRI probes for in vivo multiplexed analysis of AP activities. By integrating quantum mechanical calculations, organic synthesis, and physicochemical/biochemical evaluations, we developed a series of AP-responsive DNP-MRI probes with high enzymatic reactivities and distinguishable chemical shifts. Using these probes, we successfully detected and visualized multiple AP activities in vivo. Furthermore, we performed in vivo multiplex analysis of RAS-related AP activities in tumor-bearing mice, demonstrating the potential of this approach for monitoring the efficacy of anti-angiogenic cancer therapy and for accurate discrimination of tumor types.