创建靶向p97–NPL4–UFD1通路的诊疗一体化PET分子探针用以可视化指导双硫仑抗肿瘤精准治疗

Disulfiram (DSF)， an old alcohol-aversion drug, was currently reported to be repurposed for treating the malignant tumors. The active metabolite of this drug, named diethyldithiocarbamate (DTC) , can form a DTC–copper complex (bis (diethyldithiocarbamate)–copper (CuET)) with copper, which will target to p97–NPL4–UFD1 pathway in malignant tumors with an order of magnitude higher comparing to normal tissue and induces an apoptosis-independent cell death. However, there are two problem needed to be resolved before it will be introduced to clinic. Firstly, it needs to determine which patients can obtain the best benefit from this treatment. Secondly, it needs more measures to improve the treatment effect of DSF. Based on the successful establishment of a chelating radiolabeling methodology for positron emitters of 64Cu, in this project, we will innovatively develop a novel PET probe （64Cu-CuET）using 64Cu as a substitute of Cu to participate in CuET synthesis. After that, we will evaluate whether this novel probe can noninvasively visualize the expression of p97–NPL4–UFD1 in tumors by microPET/CT. Furthermore, we will design a high hydrophilic and tumor targeting vector by modifying the dendrimer gold nanoparticle with tumor targeting peptide DA7R and hydrophilic linker PEG, which can encapsulate 64Cu/Cu-CuET complex. This carries cargo of 64Cu/Cu-CuET can be served as theranostics tool, not only for delivering more CuEF to the tumor to enhance the therapeutic effect, but also for visualizing the targeting capacity of the vector in vivo by microPET/CT. This research work will help to guide the antitumor treatment of disulfide and may establish a more effective treatment strategy.

最新研究发现廉价的传统戒酒药双硫仑具有良好的抗肿瘤效能，双硫仑在体内代谢为二硫代氨基甲酸酯后能与金属铜（Cu）相螯合形成双二硫代氨基甲酸铜复合物（bis (diethyldithiocar bamate)-copper，CuET），该复合物能特异性靶向p97–NPL4–UFD1通路，产生明显的抗肿瘤效能。我们在成功建立了正电子核素64Cu螯合标记法的基础上，拟用64Cu取代Cu参与CuET合成，研制一种靶向p97-NPL4-UFD1通路的PET分子探针64Cu-CuET，用microPET/CT显像验证其对高表达p97-NPL4-UFD1的恶性肿瘤的显像能力，创建一种将肿瘤p97-NPL4-UFD1通路可视化的新型PET/CT分子影像技术，用以指导双硫仑抗肿瘤治疗。在此基础上，用肿瘤靶向多肽DA7R和PEG修饰第5代聚酰胺胺树状大分子金纳米颗粒载体，用于负载64Cu/Cu-CuET复合物，建立一种既能将大量CuEF输送到肿瘤靶位以提高治疗效果又能用PET进行可视化研究的诊疗一体化新模式。此研究具有重要的科学意义，将可促进基于p97-NPL4-UFD1通路的显像和治疗进展。

基础研究发现廉价的传统戒酒药双硫仑具有良好的抗肿瘤效能，双硫仑在体内代谢为二硫代氨基甲酸酯后能与金属铜（Cu）相螯合形成双二硫代氨基甲酸铜复合物（bis (diethyldithiocar bamate)-copper，CuET或Cu(DDC)2），该复合物能特异性靶向p97–NPL4–UFD1通路，产生抗肿瘤效能。我们采用正电子核素64Cu与Cu一起参与CuET合成，研制一种靶向p97-NPL4-UFD1通路的PET分子探针64Cu/Cu-CuET，也即64Cu/Cu(DDC)2，通过用microPET/CT显像验证高表达NPL4的恶性肿瘤（脑胶质瘤U87和前列腺癌LnCAP) 能高摄取64Cu/Cu(DDC)2，从而创建了一种将肿瘤p97-NPL4-UFD1通路可视化的新型PET/CT分子影像技术，可用以指导64Cu/Cu(DDC)2抗肿瘤治疗。我们通过U87肿瘤模型的PET/CT显像研究发现二硫代氨基甲酸酯与氯化铜分开给药可能对治疗恶性肿瘤无明显效果。在体外合成64Cu/Cu(DDC)2后再静脉给药，肿瘤部位能较高程度摄取64Cu/Cu（DDC）2。但由于64Cu/Cu(DDC)2为脂溶性化合物，不易溶于水，在浓度大时易形成颗粒样沉淀，无法用于静脉注射，另外在体内脂溶性的64Cu/Cu(DDC)2易于向肝脏聚集，降低肿瘤部位摄取量，影响治疗效果。针对此难题，我们采用了一种水溶性纳米稳定剂来解决此难题，我们将64Cu/Cu(DDC)2与PLA5000-PEG5000相混合，发现64Cu/Cu(DDC)2能溶于PLA-PEG溶液中，并与其形成64Cu/Cu(DDC)2 PLA-PEG纳米（简称64Cu/Cu(DDC)2-NPs），通过胶质瘤U87肿瘤模型的PET/CT显像研究发现，64Cu/Cu(DDC)2-NPs在肿瘤部位的摄取量明显高于64Cu/Cu(DDC)2（约2~5倍），从而创建了一种可溶解64Cu/Cu(DDC)2并便于静脉注射的新方法，同时此新技术有可能能更好地用于治疗恶性肿瘤。我们也用肿瘤靶向分子PSMA修饰PLA5000-PEG5000，将其制备成一种靶向纳米稳定剂PSMA-PLA-PEG，希望能将更多64Cu/Cu(DDC)2输送到肿瘤部位，但PET/CT显像研究发现该载体负载效能并没有优于PLA-PEG。