| BackgroundBombesin and gastrin releasing peptide(GRP)share a homologous 7-amino acid amidated C-terminus,and has a high affinity for GRP receptors in the human body.They can promote the proliferation of tumor cells as autocrine or paracrine growth factor.The study found that most of the tumor cell membrane expression of high GRP receptors,including prostate cancer,breast cancer.By blocking the GRP receptor can cut off the receptor-mediated signal transduction pathway and inhibit the growth of cancer,which suggested that GRPR can be used as an ideal molecular targeting receptor for the diagnosis and treatment of cancer.However,natural bombesin has many shortcomings,so it needs to be cut to make it better for imaging.Objective1.Construction of a target against tumor GRP receptor of the bombesin derivative antagonist peptide DPhe-Gln-Trp-Ala-NMeGly-His-Sta-Leu(host peptide,ATBBN)and the bombesin receptor agonist peptide Gln-Trp-Ala-Val-Gly-His-Leu-Met(control peptide,AGBBN).2.The feasibility of using molecular probes for prostate cancer and hepatocellular carcinoma imaging was evaluated from the cell,tissue and living body level by fluorescence imaging and microPET/CT imaging by using fluorescent and 68Ga markers.If the targeting image was feasible,it also speculated that can be used for targeted therapy of the tumor.Methods1.The fluorescent probe of FAM-ATBBN was synthesized by solid phase peptide synthesis method,separated and purified by high performance liquid chromatography(HPLC)and identified by mass spectrometry.vitro experiments were performed on prostate cancer PC3 cell lines with high expression of GRPR.Cell uptake and saturation experiments were performed using inverted microscope,confocal microscopy and flow cytometry.The targeting of the peptide was verified from the cell level.Prostate cancer xenograft models were established and confirmed by histopathology.FAM-ATBBN was injected into the tail vein of the xenograft models to perform the fluorescence uptake experiment.The area of interest was draw and the ratio of tumor/non-tumor was calculated.2.The PET molecular probes 68Ga-NOTA-ATBBN,68Ga-NOTA-AGBBN were and s68Ga-NOTA-PEG4-ATBBNynthesized by chelating method.The products were purified,separated and controlled by HPLC.68Ga-NOTA-ATBBN and 68Ga-NOTA-AGBBN was injected into the tail vein of the xenograft models to perform the dynamic and static micro PET/CT imaging.The area of interest was drawn and the value was expressed as as a percentage of the injection dose per gram of tissue(%ID/g).The ratio of tumor/non-tumor was calculated.3.Statistical analysisSPSS 21.0 statistical software was used for analysis.Tumor/non-tumor ratio comparison was using two independent samples t test.P<0.05 was considered as statistically significant difference.Results.1.The synthesized FAM-ATBBN was a powdery yellow powder with the main peak molecular weight of 1543.1 and purity of 99.12%.The vitro binding assay showed that FAM-ATBBN was uptake by PC3 cell membrane,and the fluorescence uptake intensity increased with the increase of concentration.Nude mice generally showed high fluorescence uptake at 1 hour,2 hours and 3 hours,and the fluorescence uptake value was higher than 2 hours(t=-3.818,P=0.019;t=-4.597,P=0.010;t=-4.68,P=0.009;t=-2.859,P=0.046;t=-5.628,P=0.032).2.The total reaction time of Syntheting 68Ga-NOTA-ATBBN and 68Ga-NOTA-AGBBN was 25 minutes.Yield was 50%.Radioactivity specific activity was 1.1×1010Bq/mmol and the radioactive purity was more than 92%.The results of micro PET/CT scans showed that PC3 prostate cancer was uptake by two imaging agents,but the tumor/non-tumor ratio was not statistically significant.HepG2 hepatocellular carcinoma was uptake by 68Ga-NOTA-AGBBN,and the tumor/background ratio was significantly higher.3.Radioactive purity of 68Ga labeled PEGylated ATBBN was up to 98%.Tumor/non-tumor ratio slightly increased.In addition to tumor/bone ratio was statistically significant,the remaining tumor/non-tumor ratio was not statistically significant.4.HepG2hepatocellular carcinoma showed significant uptake of 68Ga-NOTA-AGBBN.Tumor/brain and tumor/muscle ratio was higher than 3.0.Immunohistochemistry showed HepG2 hepatocellular carcinoma with GRPR expression.Conclusions1.This study successfully synthesized the fluorescent molecular probe FAM-ATBBN and PET molecular probe 68Ga-NOTA-ATBBN,68Ga-NOTA-AGBBN and 68Ga-NOTA-PEG4-ATBBN;2.In vitro cell fluorescence uptake experiment,in vivo fluorescence uptake experiment and mircoPET/CT imaging confirmed ATBBN targeting prostate cancer GRP receptor.tumor/brain,tumor/muscle ratio is high,which was suitable for prostate cancer imaging.3.There were no significant differences in the micro PET/CT imaging results between the two molecular probes of 68Ga-NOTA-ATBBN and 68Ga-NOTA-AGBBN.4.PEGylated ATBBN improved radioactive purity,but did not improve miro PET imaging results.5..Micro PET/CT imaging preliminary display 68Ga-NOTA-AGBBN can be used as a molecular probe targeting hepatocellular carcinoma,which may have good potential in molecular imaging of hepatocellular carcinoma,but still need more experiments to verify the results. |
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