| Background:Positron emission imaging (PET) has become the leading technology in the fieldof nuclear medicine. Commonly used positron emitting nuclide in clinical aredependent on the on site cyclotron, and the most commonly used PET probe18FDG isnot a tumor-specific targeting imaging agent, progresses of anti-tumor targetedtherapy and individualized treatment guidelines has put forward higher requirementsto molecular Imaging. Preparation of targeted PET probes using generator producedpositron emitting nuclide is an important complement to the clinical PET imagingwith18FDG.68Ge/68Ga generator is the most mature generator to produce positronemitting nuclide, half-life and positron abundance of68Ga is particularly suitable for labeling small molecules(such as peptides) and PET imaging. Angiogenesis is a veryattractive target for anticancer treatment and tumor images. APN/CD13played animportant role in tumor angiogenesis. NGR motif-containing peptides can specificallybind to CD13which is overexpression on tumor neovascular.68Ga-labeled NGRmotif-containing peptides are promising tool for tumor angiogenesis imaging andguiding antiangiogenic therapy in CD13-positive tumor.Objective:1. Two new CD13targeting peptides containing cyclic NGR motif monomerand dimer respectively were synthetized and labeled with68Ga, to provide a newapproach for PET imaging of tumor angiogenesis. The lower liver uptake areexpected.2. Optimum labeling conditions for68Ga-labeling will determine in ourlaboratory to lay the foundation for future work and development of automatedlabeling.3. The in vitro pharmaceutical characteristics and in vivo biodistribution of68Ga-NOTA-G3-NGR、68Ga-NOTA-G3-NGR2and68Ga-NOTA-G3-RGD2wereevaluated, and MicroPET iamging in HT1080xenograft bearing mice using the newprobes were performed. The results were compared between68Ga-NOTA-G3-NGRand68Ga-NOTA-G3-NGR2,68Ga-NOTA-G3-NGR2and68Ga-NOTA-G3-RGD2, toprovide the basis for target and probes selection in tumors which co-expressed CD13and integrin αvβ3.Methods:1. Cyclic NGR peptide was synthetized through disulfide bonds;p-SCN-Bz-NOTA was used as bifunctional chelator; p-SCN-Bz binding groups isbeneficial to retain the inherent NOTA ligand binding space and coordination ability;G3modified linker could increase the bond length between the two CNGRC;p-SCN-Bz-NOTA and NGR peptide coupled with a stable thiourea bond.2. The selection segmentation for fractionation method was determinedthrough measurement of leaching efficiency. The optimum conditions of labeling reaction were determined using the fractionation method, including pH value,temperature, reaction time and the amount of peptide.68Ga labeled peptides werepurified using C18cartridge and SCX cartridge to make sure which one was superior.NGR peptide and RGD peptide were labeled with68Ga under optimal reactionconditions, measuring the labeling yield and radiochemical purity to further determinethe optimal reaction conditions.3. Evaluation of pharmaceutical characteristics in vitro of68Ga-labeled peptideprobes by determination of lipid-water partition coefficient (logP), radiochemicalpurity in saline at room temperature and in human serum at37℃at different timepoints, IC50values of binding affinity, cell uptake and efflux of probe.4. MicroPET studies were performed to compare the in vivo uptakecharacteristics of the probes in HT-1080bearing nude mice, and the receptor-specificuptake was also determined using nude mice bearing human HT-1080tumors.Results:1. Successful synthesis of NOTA-G3-cCNGRC monomer and dimer wasconfirmed by mass spectrometry analysis2. The optimal reaction conditions of68Ga labeled NOTA-G3-cCNGRCmonomer and dimer were determined with fractionation method, including pH3.0-3.5,42℃,10min, and15-20μg peptide.68Ga labeled peptides could be purified usingeither C18cartridge or SCX cartridge. Eluted efficiency of SCX cartridge was higherthan C18cartridge, and elution can be used directly. Elution of C18cartridgecontaining ethanol and sometimes need further processing.3.68Ga-NOTA-G3-NGR,68Ga-NOTA-G3-NGR2and68Ga-NOTA-G3-RGD2are all hydrophilic probes, has good stability, specific CD13receptor bindingaffinity in vitro; and all could image CD13positive HT1080tumor clearly with highT/NT values; tumor uptake can be blocked with unlabeled cold peptide. These probesall rapidly excreted through the urinary system, with fast blood clearance, low uptakein liver and other vital organs. All these characteristics are suitable for thetransformation to clinical applications.4. Quantitative analysis of PET images and in vivo biodistribution of these probes revealed there are no significant difference in tumor uptake and in vivodistribution between68Ga-NOTA-G3-NGR and68Ga-NOTA-G3-NGR2,68Ga-NOTA-G3-RGD2and68Ga-NOTA-G3-NGR2.Conclusion:1. NOTA-G3-cCNGRC monomers and dimmers were successfully synthetized.The optimum conditions for the new method that68Ga labeling peptide in mylaboratory is pH3.0-3.5,42℃,10min,15-20μg peptide.2.68Ga-NOTA-G3-NGR and68Ga-NOTA-G3-NGR2are new and promisingimaging probes for CD13-positive tumor angiogenesis imagin, and they are the firstgroup of68Ga labeled NGR cyclic peptide probe in current reports. They have thesimilar effect in HT1080tumor angiogenesis imaging, and the liver uptake was lowerthan previous reports.3.68Ga-NOTA-G3-RGD2and68Ga-NOTA-G3-NGR2are all promising imagingprobes for HT1080tumor which co-expression CD13and αvβ3integrin with equaleffect. This result provide a basis for the preparation of multi-target probe containingboth NGR and RGD motifs and a cocktail kind of anti-angiogenesis drugs used inhuman fibrosarcoma patients. |
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