Development Of ?-glutamyltranspeptidas-based PET Probes For In Vivo Imaging

Cancer has become one of the leading causes of human death in the 21st century.How to improve the efficiency of early cancer diagnosis has become an arduous challenge in modern medicine.And in recent years,the development of tumor-related biomarkers undoubtedly opens a door of hope for early cancer diagnosis.Among them,?-glutamylaminotransferase?GGT?,as a nucleophilic hydrolase that plays an important role in regulating the intracellular redox balance and maintaining cell homeostasis,is involved in tumorigenesis,invasion and drug resistance processes.It has been reported that the overexpression of GGT is often regarded as a sign of diseases and cancers,such as liver and gallbladder diseases,colon cancer,cervical cancer,ovarian cancer and breast cancer etc.Nowadays,GGT has become a potential biomarker in disease diagnosis.Therefore,the development of effective methods for accurately detecting GGT activity in organisms is essential for the diagnosis of early cancer.Molecular imaging technology has been achieved numerous landmark advances in vivo tumor diagnosis.Compared with traditional diagnostic methods?such as H&E staining?,it has higher sensitivity and diagnostic efficiency,and can truly realize the real-time,non-invasive and comprehensive tracking of occurrence and development progression of tumors in vivo.Among them,positron emission tomography?PET?imaging has become the most dynamic imaging method in modern medical imaging due to its high sensitivity and no tissue penetration depth limitation.In view of this,combining the advantages of GGT targeted specific uptake and"one-step"fluorine-18 radiolabeling,we rationally designed and synthesized two GGT-targeted PET imaging probes and used them to detect GGT activity in vivo herein.The work was mainly carried out in the following two aspects:?1?We rationally designed a GGT-based fluorine-18 labeled small-molecule probe,[¹⁸F]?-Glu-Cys?StBu?-PPG?CBT?-AmBF₃(¹⁸F-1-P)by applying a in vivo biocompatible reduction self-assembly motif,?Cys?StBu?-PPG?CBT??and ingeniously decorating with a GGT-recognizable substrate,?-glutamate??-Glu?,a PET imaging group([¹⁸F]AMBF₃)for enhancing PET imaging to detect GGT level of tumors in living nude mice.The probe had exceptional stability under physiological conditions,but could be efficiently cleaved by GGT.Then a reduction-triggered self-assembly triggered by GSH was happened and formed nanoparticles(¹⁸F-1-NPs)progressively that could be directly observed by transmission electron microscopy?TEM,119 nm?.In vitro cell experiments,¹⁸F-1-P showed GGT-targeted uptake contrast of 2.7-fold to that of ¹⁸F-1 for the detection of intracellular GGT activities.Moreover,compared to GGT-deficient L929 normal cells,the higher uptake in GGT overexpressed HCT116 tumor cells??4-fold?demonstrated that¹⁸F-1-P can distinguish some GGT-overexpressed tumor cells from normal cells and have good tumor specific detection ability.In vivo PET imaging revealed enhanced and durable radioactive signal in tumor regions after ¹⁸F-1-P co-injecting with 1-P,thus allowed real-time detecting endogenous GGT level with high sensitivity and non-invasive effect.?2?For further detection of GGT activity in vivo,we modified the probe based on the first one and proposed a design concept of a dual GGT targeting water-soluble PET imaging probe,([¹⁸F]?-Glu-Cys-PPG?CBT?-AmBF₃)₂(¹⁸F-2-P)for the detection of GGT levels in vivo.The probe was introduced two GGT target groups and two radiolabeled groups([¹⁸F]AMBF₃)to improve the targeting and intracellular fluoride density,and replacing hydrophobic tert-butylmercaptocysteine with cystine to improve its water solubility.The results showed that the probe had good radiostability and low cytotoxicity.The results of uptake in negative and positive cells showed that¹⁸F-2-P had good targeting specificity,and the uptake in HCT116 cells reached 2.81±0.04%AD at 2 h,which was increased compared to the probe¹⁸F-1-P?2.59±0.07%AD?.PET imaging in vivo showed that the maximum uptake at the tumor site was obtained at 10 min?4.43±0.56%ID/g?after the probe was injected into the tail vein,which was significantly higher than that of the probe¹⁸F-1-P injection alone?3.20±0.29%ID/g?.These studies showed that the dual GGT targeting design was beneficial to improving the specific uptake of the probe at the tumor site and had some reference significance for the design of the GGT targeting probe.