A Preliminary Study On The Synthesis Of Enhanced CT Contrast Agent And Its Affinity For Human Breast Tumor Cells

Cancer has always been a disease that endangers human health.Early diagnosis is the key to cure the tumor.At present,X-ray-computed tomography(CT)is the most widely used method to examine tumor noninvasively.The contrast agents can effectively improve the contrast and clarity of X-ray imaging and improve the sensitivity of CT.However,the current use of iodinated contrast agents for the diagnosis of early tumors is not sensitive.Therefore,it is of great significance to develop new target-specific CT imaging contrast agents.Sigma-2 receptor was highly expressed in a variety of tumor cell lines and it can be used as a binding site for the tumor cells.We select SV119,sigma-2 receptor ligand,as a target carrier,coupled with triiodo-benzene backbone to obtain new targeted specifically CT contrast agents,and study its affinity with the tumor cells.In this paper,after comparing and analyzing the existing structures of iodinated contrast agents,three novel structures of intermediates of triiodobenzene contrast agents were designed.A new highly efficient synthetic targeted vector SV119 was designed.One-pot reaction was used to design and synthesize 9-benzyl-9-azabicyclo[3.3.1]nonan-3-one.Through a series of reactions,the target product SV119 was successfully obtained and coupled with three intermediates respectively to get three new targeted contrast agents.The target product SV119 was successfully obtained and coupled with three intermediates to obtain three novel targeted contrast agents.The structure of the synthesized compounds was characterized by elemental analysis,infrared and nuclear magnetic resonance techniques.It was proved that the chemical structure of SV119,the three contrast agents and their conjugates were consistent with the designed target structures.Early research in our group has found that sigma-2 receptor proteins are also highly expressed in breast cancer cell lines that express high levels of sigma-1 receptor protein.Then the mammalian expression plasmid containing sigma-1 receptor gene(pcDNA3.1-sigma-1)was transfected into MCF-7 breast cancer cells by liposome transformation to construct MCF-7-sg1 which highly expressed sigma-2 receptor protein.The result of PCR confirmed that the MCF-7-sg1 genome contained sigma-1 gene,the MCF-7-sg1 cells can expressed the target product by Western blot.Through comparative analysis of expression levels,the MCF-7-sg1 engineered cell line with the highest expression level was screened for using in this study.To prove the new targeted contrast agents can bind specifically to tumor cells,we used the characteristics of rhodamine B that can fluoresce to synthesis of RB-SE by the DCC method,then coupled with SV119 to get RB-SV119.The fluorescence emission spectrum was measured and it was found that RB-SV119 had the function of emitting fluorescence under the action of excitation light.Therefore,RB-SV119 can be used as a fluorescent probe to study the contrast agents' affinity for tumor cells.Two ligands(AC927 and BD1047)that specifically bind to the sigma receptors were used as competitive reagents to bind sigma-2 protein on the surface of MCF-7-sg1 cells with the fluorescent probe RB-SV119.The fluorescence emission of cells under excitation light proved that RB-SV119 can specifically bind to tumor cells.In order to study the affinity of the novel targeted contrast agents with tumor cells,a competitive assay of contrast agents and fluorescent probe with MCF-7-sg1 cells was designed.The MCF-7-sg1 cells were first incubated with novel synthetic contrast agents,and then the MCF-7-sg1 cells were incubated with the fluorescent probe RB-SV119 at room temperature.Fluorescent emission under excitation light of tumor cells in each group was observed.In the situation,it was found that the contrast agent II and contrast agent III group cells had no fluorescence,while the contrast agent I cells had slight fluorescence.Experiments have shown that contrast agent I has weaker binding capacity to tumor cells,and contrast agent II and contrast agent III have specific binding force on tumor cells that express sigma-2 receptor.