| Background:Neuroendocrine neoplasms(NENs)are a class of tumors that originate from neuroendocrine cells.The early diagnosis rate is low,and many patients are already at an advanced stage when they are diagnosed..Due to the high expression of somatostatin receptor(SSTR)on the cell surface of most NENs,SSTR imaging is currently considered to be the most sensitive method for detecting NENs.However,the common insulinomas in gastroenteropancreatic NENs express low SSTR but high glucagon-like peptide-1 receptor(GLP-1R).In view of the heterogeneity of tumor itself,in order to improve the detection rate of NENs,this experiment intends to construct a new type of somatostatin receptor and glucagon-like peptide-1 receptor that are commonly highly expressed in NENs cells.The dual-target molecular probe DOTA-exendin-4-TOC can target SSTR and GLP-1R at the same time,and it is radiolabeled with 68Ga to explore 68Ga-DOTA-exendin-4-TOC as a novel NENs targeting drug feasibility.Objects:1.To construct a novel dual-target molecular probe DOTA-exendin-4-TOC that can simultaneously target SSTR and GLP-1R.2.Establish the labeling method of 68Ga-labeled dual-target molecular probe DOTAexendin-4-TOC.3.To explore the possibility of 68Ga-DOTA-exendin-4-TOC as a new molecular probe for NENs tumor imaging.Methods:1.The dual-target molecular probe DOTA-exendin-4-TOC was synthesized,and analyze and identify it.2.68Ga-labeled DOTA-exendin-4-TOC was performed by the bifunctional chelator method and the radiochemical purity of the labeled product was determined by Radio-HPLC analysis.3.In vitro stability of the labeled product was measured by Radio-HPLC.In vivo stability of the probe was performed by gamma counter.Also Octanol-Water partition coefficient of the labeled product was performed by gamma counter.4.In vitro cell uptake assay was utilized to evaluate the binding affinity of 68GaDOTA-exendin-4-TOC to receptors SSTR and GLP-1R.5.We selected the RIN-M5F cell line that expresses both GLP-1R and SSTR,the INS1 cell line that expresses high GLP-1R and the AR42J cell line that expresses high SSTR to establish the tumor-bearing nude mouse model.They were pathologically verified.6.68Ga-DOTA-exendin-4-TOC was injected via tail vein to study its radioactivity distribution and MicroPET/CT imaging in tumor models.Results:1.The purity of the synthesized DOTA-exendin-4-TOC is higher than 95%.The molecular weights are consistent and meet the experimental requirements.2.The labeling method of 68Ga-labeled DOTA-exendin-4-TOC is simple,and the product has high radiochemical purity(higher than 95%),the product peak is single.3.68Ga-DOTA-exendin-4-TOC was incubated in PBS buffer solution for 60 min and 120 min for radiochemical purification The radiochemical purity of concentration is still greater than 95%,and it has good in vitro stability.4.In the Octanol-Water partition experiment,the Octanol-Water partition coefficient logP(n-octanol/PBS)of 68Ga-DOTA-exendin-4-TOC was-2.02 ± 0.05,suggesting that the prepared labeled polypeptide was a hydrophilic substance.5.The results of in vitro cell uptake experiments showed that RIN-M5F cells,INS-1 cells,and AR42J cells had a significant uptake effect on 68Ga-DOTA-exendin-4TOC.The uptake curves of 68Ga-DOTA-exendin-4-TOC by RIN-M5F cells and INS-1 cells were similar,which shows a peak at 60min;while AR42J cells were more sensitive to 68Ga-DOTA-exendin-4-TOC.Its uptake value of 68Ga-DOTAexendin-4-TOC was higher than that of the other two cells.The uptake of 68GaDOTA-exendin-4-TOC by AR42J cells continued to increase from 0 min to 120 min.6.The tumor-bearing nude mouse model of RIN-M5F cells,INS-1 cells and AR42J cells were successfully established,and the models were confirmed by pathology verify.7.MicroPET/CT imaging of tumor-bearing nude mouse model of RIN-M5F cells,INS-1 cells and AR42J cells shows that the three t tumor-bearing nude mouse model had uptake of 68Ga-DOTA-exendin-4-TOC,and the uptake of the three kinds of tumors at 60min and 120min were respectively,RIN-M5F tumor uptake values was 0.87 ± 0.11(60 min)、0.80 ± 0.78(120 min),INS-1 tumor uptake value was 1.57 ± 0.12(60 min)、1.20 ± 0.10(120 min),AR42J tumor uptake value was 1.33±0.12(60 min),1.50 ±0.10(120 min).8.The radioactivity distribution results of RIN-M5F cells,INS-1 cells and AR42J cell bearing-tumor models showed that INS-1 cells and AR42J cell tumors had uptake of 68Ga-DOTA-exendin-4-TOC,while the uptake of 68Ga-DOTA-exendin-4-TOC by RIN-M5F cell tumors was low;68Ga-DOTA-exendin-4-TOC was concentrated in the kidney in nude mice.Conclusions:1.In this study,68Ga-DOTA-exendin-4-TOC is successfully synthesized and radiolabeled.The preparation method by bifunctional chelating agent is simple,the radiochemical purity is high(higher than 95%),and it has good stability in vitro and in vivo.68Ga-DOTA-exendin-4-TOC is a hydrophilic substance that can be excreted through the urinary system.2.In vitro cell uptake experiments show that 68Ga-DOTA-exendin-4-TOC can be specifically inhibited by RIN-M5F cells,INS-1 cells and AR42J cells.3.MicroPET/CT imaging and radioactivity distribution experiments show that RINM5F cells,INS-1 cells,and AR42J cell tumor models can specifically uptake 68Ga-DOTA-exendin-4-TOC.In the follow-up study,our team will continue to improve the receptor-ligand affinity of the dual-target molecular probe. |
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