Developing Of Polypeptide CNGQGEQc Molecular Probes For Targeting Lung Cancer And Its Fluorescent And PET Imaging Study In Lung Cancer

Backgroud:Molecular imaging based on small molecular polypeptide is the focus of clinical oncology research.cNGQGEQc is a short peptide composed of 8 amino acids.This small molecular peptide that can bind to non-small cell lung cancer was screened out first by Guo linlang et al by means of combinatorial chemical peptide library technology.It was found that cNGQGEQc(a pair of disulfide bonds formed by cysteine at both ends)can bind to non-small cell lung cancer cells through integrin 3 on the cell membrane.cNGQGEQc was successfully radiolabeled with 131I and 99Tcm by our research group previously and was used for SPECT imaging.68Ga is a positron radionuclide,which can more easily obtain from germanium and gallium generator.At the same time the resolution of PET image with 68Ga is better than 131I and 99Tcm in SPECT imaging.So in order to get higher quality images,in this experimental study a molecular probe based on 68Ga-cNGQGEQc was to be synthesized for performing PET imaging studies.Moreover,this study also used fluorescence imaging technique to study the binding characteristics of cNGQGEQc with non-small cell lung cancer.Guo linlang et al have found that cNGQGEQc which d-cysteine at both ends of the peptide molecule forms a pair of disulfide bonds,is a ring polypeptide molecule(hereinafter abbreviated as cNGQGEQc-C).This kind of polypeptide molecule is costly and time-consuming in the synthesis process.In our previous study,we found that the catenulate polypeptide molecule cNGQGEQc(cysteine at both ends does not form a pair of disulfide bonds,abbreviated as cNGQGEQc-L)can also bind to A549 cell,and the synthesis of catenulate peptide molecule is more convenient and economical compared with cyclic peptide.In this study,the catcnulate polypeptide molecule 1.c.cNGQGEQc-L was also studied by fluorescence and PET imaging,and the differences between two different structure polypeptide molecules were compared,so as to find a feasible and more economical polypeptide molecule that can bind to non-small cell lung cancer.Object:1.cNGQGEQc-C,a small molecule polypeptide that can bind to integrin α3 receptor,was selected as the targeting carrier.2.Small molecule polypeptide cNGQGEQc-C was labeled with fluorescein FAM and radionuclide 68Ga.At the cellular and tumor-bearing nude mice level,fluorescence imaging and micro-PET/CT imaging were used to explore the feasibility of polypeptide molecular probe in the diagnosis of non-small cell lung cancer,and to evaluate whether it can be used as a targeted binding molecule with non-small cell lung cancer.3.The catenulate small molecular polypeptide cNGQGEQc-L was synthesized,and the its labeling with fluorescein FAM and radionuclide 68Ga was performed.Fluorescence imaging and micro-PET/CT imaging were used to expolore the feasibility of cNGQGEQc-L as molecular probe for the diagnosis of non-small cell lung cancer.4.The combing differences of cNGQGEQc-L and cNGQGEQc-C with non-small cell lung cancer were compared.Materails and Methods:1.The Fluorescence Inversion Microscope System and Flow Cytometry were used to observe the binding of FAM-cNGQGEQc-C and FAM-cNGQGEQc-L with A549 cells.2.The distribution characteristics of FAM-cNGQGEQc-C and FAM-cNGQGEQ c-L in tumor-bearing nude mice were observed by small animal living imager,and the T/NT ratio of the average fluorescence intensity of tumor to non-tumor organs was calculated.3.The PET probes of’6sGa-DOTA-Ahx-lys-cNGQGEQc-C and 68Ga-DOTA-Ahx-lys-cNGQGEQc-L were synthesized,and the HPLC analysis was performed for quality control.A nude mouse model of non-small cell lung cancer(A549)was established.Two kinds of molecular probes radiolabeled with 68Ga were,respectively,injected into nude mice bearing non-small cell lung caner A549 via tail vein.micro-PET/CT imaging was performed at different phases,and the areas of interest were delineated.The%ID/g value was calculated.4.Software SPPS 22.0 was used for statistical analysis.Results:1.Fluorescence Inversion Microscope System showed that fluorescence probes of both FAM-cNGQGEQc-C and FAM-cNGQGEQc-L polypeptide molecules could bind to A549 cells at the site of cell membrane and enter the cytoplasm.The fluorescence binding intensity of FAM-cNGQGEQc-L with cells was higher than that of FAM-cNGQGEQc-C.2.Flow Cytometry experiments showed that the binding of two kinds of fluorescent polypeptides with A549 cells had the saturation characteristic.3.The fluorescence binding intensity of FAM-cNGQGEQc-C and FAM-cNGQGEQc-L with A549 cells could be significantly inhibited by 4 times the concentration of unmodified polypeptides with cNGQGEQc-C and cNGQGEQc-L.4.Small animal living imager showed that fluorescence probes of both FAM-cNGQGEQc-C and FAM-cNGQGEQc-L could bind to the tumor,which was mainly excreted through the urinary system and biliary tract system.5.The probes of 68Ga-DOTA-Ahx-lys-cNGQGEQc-C and 68Ga-DOTA-Ahx-lys-cNGQGEQc-L were successfully synthesized.The in vitro stability of 68Ga-DOTA-Ahx-lys-cNGQGEQc-L was good,and that of 68Ga-DOTA-Ahx-lys-cNGQGEQc-C was not good enough.6.When the probe of 68Ga-DOTA-Ahx-lys-cNGQGEQc-C was imaging for tumor in nude mice bearing non-small cell lung caner A549,it was observed that there was radioactive concentrated in tumor.While for 68Ga-DOTA-Ahx-lys-cNGQGEQc-L obvious radioactive concentrated in tumor was observed.Conclusions:1.In vitro and in vivo experiments of FAM-cNGQGEQc-C and FAM-cNGQGEQc-L could bind to lung cancer cells and tumors.2.The 68Ga-DOTA-Ahx-lys-cNGQGEQc-C and 68Ga-DOTA-Ahx-lys-cNGQGE Qc-L are successfully synthesized.micro-PET/CT imaging showed that 68Ga-DOTA-Ahx-lys-cNGQGEQc-C and 68Ga-DOTA-Ahx-lys-cNGQGEQc-L can bind to tumor.The radioactive uptake in tumor with 68Ga-DOTA-Ahx-lys-cNGQGEQc-L is better than 68Ga-DOTA-Ahx-lys-cNGQGEQc-C.