Fluorescence Small Peptide Targeting Molecular Probes VEGF127-133＿Cy5.5 Experimental Study On In Vivo Imaging Of Tumor-bearing Mice

Objective:The molecular probe VEGF127-133＿Cy5.5 was prepared by using Cy5.5labeled VEGF127-133 small peptide for targeting tumor VEGFR-2.Investigating the efficiency targeted transplanted tumor VEGFR-2 of transplanted HEP G2 tumor bearing nude mice in vivo,and minimal targeted transplanted tumor size will be confirmed by fluorescence imaging.Methods:（1）Molecular probe preparation:The targeted molecular probe VEGF127-133＿Cy5.5 and the non-targeted molecular probe NTSP＿Cy5.5 were prepared by labeled with CY5.5 in the experimental group VEGF127-133 and the control group NTSP small peptide.and identified their physical and chemical properties by chemical mass spectrometry and fluorescence spectroscopy.（2）Tissue HE and immunohistochemical staining:To identificate of tissue and cell morphology in all ROI of tumor-bearing mice and analyze the expression of VEGFR-2 intensity of semi-quantitative analysis.（3）Targeted Fluorescence Imaging in Tumor Bearing Mice:16 tumor-bearing mice which have 8mm～10mm tumor diameter were randomly divided into two groups,8 in each group.The test group used VEGF127-133＿Cy5.5 and the control group used NTSP＿Cy5.5 by tail vein,the distribution in mice was observed at multiple time points,the fluorescence intensity values of each ROI were measured and the time-fluorescence intensity curve was made.（4）Statistical analysis of data:The fluorescence intensity values were the repeated measurements values,and the ROI fluorescence intensity values were compared by repeated measurement of variance analysis.The fluorescence intensity values of the same time point of different ROI comparison using independent sample t test,P<0.05 for the difference was statistically significant.The correlation between the fluorescence intensity and the expression of VEGFR-2 in the tumor area and non-tumor area was compared by using the bivariate correlation statistic method.（5）Determine the minimum tumor diameter imaging experiment:2mm,3mm,4mm tumor-bearing mice were injected with VEGF127-133＿Cy5.5 probe by the tail vein,and the minimum tumor diameter of target fluorescence imaging was observed by fluorescence imaging of multiple time points in vivo,and the tissue fluorescence imaging out vivo.Results:（1）The target molecular probe and non-targeted molecular probe were successfully prepared and purified by HPLC.The coupling rate of the experimental group was more than 97%and the control group was more than 90%.（2）HE staining and VEGFR-2 immunohistochemistry showed no metastatic tumor in liver,spleen,kidney and muscle.The expression of VEGFR-2 was different in different tissues,transplanted tumor（12 points）>liver（4 points）>kidney（3 points）>muscle（2 points）>spleen（0 points）.（3）The first imaging peak was reached at average 40 min after the injection of the molecular probe in the experimental group,and the second imaging peak appeared at average 80 min.The fluorescence signal was slowly restored,it was still obvious at 300 min.In the control group,the tumor peak was reached at average40 min,and then recovered more rapidly.After 300 min,the intensity was close to that before injection.The difference between the experimental group and the control group was statistically significant(F_MP=38.371,P<0.001).In the experimental group,the peak of the probe was reached after average 40 min of the non-tumor injection group,and then recovered rapidly.Compared with the experimental group,the difference was statistically significant(F_MP=63.769,P<0.001).In the control group,the imaging peak was reached at average 20 min after injection of the molecular probe,and then recovered rapidly.There was no significant difference between the two groups in the non-tumor group(F_MP=3.761,P=0.081).There was a positive correlation between the fluorescence intensity and the expression of VEGFR-2 in the tumor area and non-tumor area,and consistent with the results of in vitro tissue fluorescence imaging.（4）Targeted imaging the smallest tumor diameter test showed:2mm tumor diameter tumor area without fluorescence imaging,3mm and 4mm tumor diameter tumor visible fluorescence imaging.Conclusion:VEGF127-133＿Cy5.5 targeted molecular probe was coupled successfully,which can be tumor-bearing mice living tumor VEGFR-2 target specific targeted fluorescence imaging,and the target imaging peak time was 80 min.The tumor size of the transplanted tumor was 3 mm by the minimum targeted imaging of the small animal live imager.