[⁶⁴Cu]Cu(DDC)₂ NPs: A Novel PET Probe for Noninvasive Visualization of NPL4 Expression in Tumors <i>In Vivo</i>

Nuclear protein localization 4 (NPL4) plays a key role in the ubiquitination pathway and has emerged as a promising target for cancer therapy. The ditiocarb-copper complex, Cu(DDC)₂, an anticancer metabolite derived from the antialcoholism drug disulfiram (DSF), exhibits a high affinity for NPL4. Thus, quantifying NPL4 expression in tumors is crucial for ubiquitination research and for developing NPL4-targeted diagnostic and therapeutic strategies. In this study, we replaced the cold copper ion in Cu(DDC)₂ with the positron-emitting isotope copper-64 and developed three methods for visualizing NPL4 in tumors in vivo using positron emission tomography/computed tomography (PET/CT): (1) an <i>in vivo</i> "synthesis-free" method for preparing [⁶⁴Cu]Cu(DDC)₂, (2) an <i>in vitro</i> synthesis method, and (3) a stabilization method using PEG5000-PLA5000 (PP) to enhance [⁶⁴Cu]Cu(DDC)₂'s hydrophilicity by preparing [⁶⁴Cu]Cu(DDC)₂ NPs. Micro-PET/CT imaging showed minimal uptake of [⁶⁴Cu]Cu(DDC)₂ in NPL4-positive tumors with the <i>in vivo</i> "synthesis-free" method, resulting in poor lesion visualization. However, in vitro synthesized [⁶⁴Cu]Cu(DDC)₂ and [⁶⁴Cu]Cu(DDC)₂ NPs successfully visualized NPL4-positive U87MG tumors. Compared to [⁶⁴Cu]Cu(DDC)₂, [⁶⁴Cu]Cu(DDC)₂NPs demonstrated significantly higher tumor uptake (7.2 ± 0.7% ID/g vs 3.8 ± 0.6% ID/g at 12 h postinjection, <i>P</i> = 0.001) and tumor-to-muscle (T/M) ratio (7.8 ± 1.2 vs. 3.2 ± 0.7, <i>P</i> = 0.001). Tumor uptake of [⁶⁴Cu] Cu (DDC)₂NPs was consistent with NPL4 expression levels and was inhibited by an excess of Cu(DDC)₂. The optimal PP stabilizer concentration was determined to be 0.0005%. This study successfully developed a PET probe, [⁶⁴Cu]Cu(DDC)₂NPs, and established a novel imaging modality for <i>in vivo</i> visualization of NPL4 expression, potentially guiding future NPL4-targeted therapies.