Dual-Targeting PET Tracers Enable Enzyme-Mediated Self-Assembly for the PET Imaging of Legumain Activity

Legumain, a lysosomal cysteine protease overexpressed in a variety of tumors, has been considered a promising biomarker for various cancers. Precise detection of legumain activity in the lysosome represents an important strategy for early diagnosis and prognosis of tumors. Small-molecule probes with the property of target-enabled self-assembly hold great potential for molecular imaging. In this study, we reported two dual-targeting radiotracers ([¹⁸F]SF-AAN-M and [¹⁸F]SF-AAN-HEM) with a property of legumain-mediated self-assembly for positron emission tomography (PET) imaging. Both the radiotracers were synthesized with high labeling yield (>50%) and the radiochemical purity was over 99% via one-step straightforward ¹⁸F-labeling. Both tracers were efficiently activated by the reducing agent and legumain to self-assemble into aggregates and showed enhanced retention in legumain-overexpressed MDA-MB-468 cells and tumors, indicating that the introduction of lysosome-targeting morpholine increased the tumor uptake and extended the retention of radiotracers in legumain-overexpressed tumors. In addition, [¹⁸F]SF-AAN-HEM with a hydrophilic (histidine-glutamate)₃ tag displayed significantly reduced liver uptake with no conspicuous reduction in tumor uptake, affording high signal-to-noise ratios (tumor/liver and tumor/muscle). All of these results suggest that dual-targeting tracer [¹⁸F]SF-AAN-HEM could provide a promising tool for in vivo monitoring legumain activity in tumors.