| Objective:The gastrin releasing peptide receptors?GRPRs?overexpress in various tumors,which provide the opportunity for GRPR targeted tumor diagnosis and therapy with the aid of radiolabeled peptide probes.In recent reports,the GPPR antagonists have superiority in specific targeting affinity over the agonists,which have been utilized in tumor radio imaging and therapy.However,antagonists suffer from many shortcomings regarding their binding affinity and biodistribution properties,limiting their applications.In this study,the oligomers?dimer/trimer?of antagonists were designed to improve the properties of the antagonist to improve tumor targeting and stability.Monomer/dimer/trimer of RM26?a GPPR antagonist peptide?were synthesized and functionalized with FITC-or 18F-NOTA-.The binding affinities and cytotoxicity on prostate cancer cells?PC-3?of FITC-labeled[RM26],3P-[RM26]2 and 4P-[RM26]3 were systemically investigated and compared.The tumor uptake and targeting properties of[18F]AlF-NOTA-labeled[RM26],3P-[RM26]2 and 4P-[RM26]3 were observed in PC-3 tumor-bearing mice.We explored the value of GPPR antagonist RM26 homogeneous multivalent vector for targeting prostate tumors radiotherapy and diagnosis.Methods:All animal experiments involved are in compliance with relevant regulations and ethics.?1?Liquid phase synthesis of the corresponding monomer/dimer/trimer:FITC/NOTA-[RM26],FITC/NOTA-3P-[RM26]2,FITC/NOTA-4P-[RM26]3,and then confirmed by UPLC-MS and the purity of the products were characterized by HPLC.?2?Confocal fluorescence images and Flow cytometry analysis cellular uptake on PC-3;The cell viability was determined using MTS assay on PC-3 treated with the tracers.Compared with the monomer FITC-[RM26],explore the uptake,targeting properties,and cytotoxicity of FITC-3P-[RM26]2,FITC-4P-[RM26]3 in the PC-3 cell.?3?The NOTA-conjugated[RM26],3P-[RM26]2,4P-[RM26]3 was labeled with 18F using[Al18F]aluminum-fluoride connecting.We explored the optimal conditions for 18F[AlF]labeling,using C18 column separated and purified the radioactive tracers.The chemical purity and stability in vitro were tested.?4?SPF BALB/cn male nude mice were used to establish models of PC-3cell-bearing mice.We divided into experimental group,control group,and closed group?n=3?.The PC-3 cell-bearing mices were injected with 70100?Ci of the 18F labeled radiotracers?100?L?via the tail vein.The control group were injected with the same volume and radiation dose of 18F-FDG.For the determination of nonspecific uptake in the tumor or receptor-positive organs,the tumor-bearing nude mices were preinjected with excess unlabeled BBN peptide for 5 min.Compared with radiolabeled monomers Al18F-NOTA-[RM26]and 18F-FDG,micro-PET/CT imaging of PC-3 tumor-bearing mice were explored tumor targeted uptake and biodistribution in vivo after injection 5 min,30 min,60 min,120 min.Preliminary evaluation of Al18F-NOTA-3P-[RM26]2,Al18F-NOTA-4P-[RM26]3 for the application of tumor imaging diagnosis and treatment.Results:?1?UPLC/MS analysis of the products of FITC/NOTA-[RM26],FITC/NOTA-3P-[RM26]2,FITC/NOTA-4P-[RM26]3 were correct.The purity of all the ptoducts were over 97%determined by HPLC/UV after separation and purification.?2?The binding to PC-3 was rapid for all three compounds,where the fluorescence was clearly observed within the cells 15 min after the addition of conjugates and increased significantly at 45 min.The results of Fluorometric assay showed the uptake levels were FITC-[RM26]<FITC-3P-[RM26]2<FITC-4P-[RM26]3 by fluorescence-activated cell sorting?FACS?.There was no significant toxicity after treatment with each tracer in MTS experiments.Blocking experiments showed monomer/dimer/trimer of RM26had a specific targeting for PC-3 cells.?3?The Al18F successfully labeled NOTA-[RM26],NOTA-3P-[RM26]2,NOTA-4P-[RM26]3 and the reaction time was 30 min,the radiochemical purities were>95%with HPLC analysis.The tracers bivalency/trivalency was stable in PBS and serum at room temperature for 2h with greater than 90%radiochemical purity.?4?Micro-PET/CT imaging in the PC-3 tumor model showed that Al18F-NOTA-4P-[RM26]3 had the best effect targeting properties,which was consistent with the results of cell experiments.The pre-injected excess unlabeled targeting GRPR BBN showed significantly reduced the uptake to tumor which demonstrated the excellent specific targeting ablility in vivo.Compared with 18F-FDG,the image of 18F[AlF]-NOTA-monomer/dimer/trimer in vivo indicated that the relative uptake of heart,abdominal organs were relatively low,reducing the irradiation of important normal organs.The excretion of radiotracers mainly through the bladder.Conclusion:The results showed dimer and trimer of RM26 derivatives both appeared multivalent effect,increased the specific targeting ability in vitro and in vivo compared to the respective monomer.Therefore they may have practical potential as new imaging and radiotherapy agents with high targeting capabilities in GRPR positive tumors. |
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