Experimental Study Of Magnetic Resonance Contrast Agent Gd-PEG-VEGF_125-136 Targeting VEGFR-2 High Expression Tumor Cells

Objective:The magnetic resonance contrast agents currently used in clinical practice were mostly non-targeting,it was difficult to adapt to the development needs of personalized precision medical treatment.The aim of this study was to construct a tumor cell with high expression of VEGFR-2 by constructing a magnetic resonance targeting contrast agent targeting tumor cell vascular endothelial growth factor receptor-2（VEGFR-2）.In vitro targeted imaging was conducted to explore the feasibility,targeting ability and enhancement effect of targeted contrast agents in tumor cell-specific molecular imaging,in order to lay the foundation for in vivo imaging and targeted diagnosis of tumors.Methods:（1）Screening of tumor cell lines with high expression of VEGFR-2:cultured human hepatoma cells（HepG2）,human non-small cell lung cancer cells（NCI-H1299）,human cervical cancer cells（HeLa）,human lung adenocarcinoma cells（A549）,using cellular immunofluorescence and Western Blot assay was used to detect the expression of VEGFR-2 in tumor cell lines,and the tumor cells with the highest receptor expression were screened.（2）Preparation of Gd-PEG-VEGF_125-136:Using peptide VEGF125-136 as a targeting peptide and Gd³⁺-DOTA as a developer,a magnetic resonance targeting contrast agent was constructed by amide bond synthesis method,and then separated and purified by HPLC.The MS performs the test.（3）Magnetic resonance signal detection of Gd-PEG-VEGF_125-136:A gradient concentration of the targeted contrast agent solution was prepared,and the T1 signal was detected by 0.5T Meso MR.（4）Magnetic resonance imaging of VEGFR-2 high expression tumor cell lines in vitro:divided into experimental group,control group and blank group.The VEGFR-high expression tumor cell line（HepG2）was added to Gd-PEG-VEGF_125-136was incubated as an experimental group,and sputum-labeled random peptide NTSP was added as a control group,and no contrast agent was added as a blank group.In vitro cell imaging was performed using 3.0T Siemens magnetic resonance,and the T1WI sequence was used to acquire images and measure the T1SNR（signal to noise ratio）values??of each group.（5）Statistical analysis of data:SNR is the measurement data.The experimental group,the control group and the blank group were compared using the SNK（Student-Newman-Keuls）q test to compare the T1 SNR values??of the same concentration or the different concentrations of T1 SNR in the same group.Differences in values??were considered statistically significant at P<0.05.Results:（1）Immunofluorescence experiments showed that the expression of VEGFR-2 was detected in Hep G2,NCI-H1299,He La and A549 cells.The results of Western Blot showed that the expression of VEGFR-2 in Hep G2 cells was the highest.（2）HPLC showed that the targeted contrast agent Gd-PEG-VEGF_125-136was successfully separated from the reaction product,and mass spectrometry showed that Gd-PEG-VEGF_125-136was successfully coupled with a purity of over 95%.（3）0.5T Meso MR scan showed that as the concentration of Gd-PEG-VEGF_125-136increased,the corresponding T1WI image signal was enhanced.（4）3.0T Siemens MRI scan showed that when the concentration of contrast agent exceeded 0.125m M,the difference of SNR between the experimental group and the control group and the blank group was statistically significant（P<0.05）,and the difference of SNR between the experimental groups was different.Statistically significant（P<0.05）;and concentration（X）was related to T1 SNR（Y）,correlation coefficient r=0.9365,regression equation Y=257.6*X+103.9.Conclusion:In this experiment,a magnetic resonance targeting contrast agent Gd-PEG-VEGF_125-136targeting VEGFR-2 was successfully constructed,which can specifically bind to the high-expression VEGFR-2 cell line Hep G2 in vitro,and showed a magnetic resonance at 3.0T.The T1WI signal enhancement effect lays a foundation for in vivo imaging experiments and is expected to be used for magnetic resonance tumor targeted imaging and early diagnosis.