Imaging And Biodistribution Of⁹⁹Tc^m Labeled VEGF_125-136 Peptide Fragments In Nude Mice Bearing Human Osteosarcoma

Objective: To synthesize99Tcmlabeled vascular endothelial growthfactor （VEGF125-136） and evaluate its biodistribution and imaging in the nudemice bearing human osteosarcoma，in order to find a drugs that reflect theangiogenesis active, to improve the early diagnosis and targeted therapy oftumors.Methods:1Synthesis and label of⁹⁹Tc^m-VEGF_125-136-MAG₃Adopt the automatic peptide synthesizer, target peptideQKRKRKKSRYKS was synthesized by conventional solid phase synthesismethod, and in the synthetic process directly complete the coupling withMAG3, form VEGF_125-136-MAG₃.Then labeled it with⁹⁹Tc^m. The qualityassurance of radioactive labeling⁹⁹Tc^m-VEGF_125-136-MAG₃was measured byhigh performance liquid chromatography （HPLC）. The pue of radioactivelabeling was determined by Xinhua one as stationary phase and0.9%physiological saline as mobile phase when the ITLG-SG was stationary phaseand ACD was mobile phase respectively on one hour, two hour, six hour andtwelve hour in room temperature, testing the stability in vitro of markers.2Modeling tumor-burdened nude mouse animal modeRecovery bone sarcoma MG63cell line, cultivation by the attachedmethod, DMEM nutrient solution （including10%of fire fetal calf serum）.Harvest logarithmic phase cells and make the single cell suspension with cellconcentration of5×10⁷a/ml. And then inoculation cell suspension with0.2ml/one to the left armpit back subcutaneous of nude mouse. These nude micefeeding in specifc pathogen free environment and waiting the diameter oftumor to0.8cm¹.0cm with no significant difference in weight or the size werechosen to experiment. 3The biodistribution of⁹⁹Tc^m-VEGF_125-136-MAG₃in nude mice bearingosteosarcomaThe mice were randomly divided into four groups by0.5h,1h,2h,4h,and every group respectively contained five. And then inject18.5MBq⁹⁹Tc^m-VEGF_125-136-MAG₃to each mouse. The mice were executed afterimaging and striped the tumor, stomach, heart, lung, liver, kidney, spleen,bone, small intestine and the muscle of posterior limb which were swashed bynormal saline and dried by filter paper. The tissues were measured andweighed to obtain the percentage of injected dose per gram of tissue（%ID/g）.Calculated the%ID/g ratio of tumor tissue and side muscle tissue（tumor tomuscle ratios, T/M）4⁹⁹Tc^m-VEGF_125-136-MAG₃tumor imaging studies in nude mice bearingosteosarcomaTake five tumor-burdened nude mouse to18.5MBq99Tcm-VEGF125-136-MAG3, take other five tumor-burdened nude mouse as a competitive inhibitiongroup (first inject VEGF_125-136-MAG₃200μg before0.5h), and then the micewere anesthetized, fastened and placed to radionuclide scintigraphy imagingrespectively. Delineated the tumor and the normal tissue, the latter wasconsidered the background, and calculated the ratio of T/NT using theinterested region technology （ROI）.Results:1The preparation and evaluation of⁹⁹Tc^m-VEGF_125-136-MAG₃In the help of Beijing zhongke honed company, VEGF_125-136-MAG₃synthesis was finished directly by the automatic peptide synthesizer andconventional solid phase method. In the conditions of reaction volume125μl,peptide content20μg, when99Tcm3.7×10⁷, potassium sodium tartrate dosage10μl（500μg）, stannous chloride dosage5μg, radioactive labeling rate wasbetter. Analyzed by HPLC method under the condition,99Tcm-VEGF125-136-MAG3was unimodal, retention time was6.7min and radioactive labeling rategreater than96%. By paper chromatography, markers of immediateradioactive labeling rate is（97.80±1.46）%（n=5）, mark rate is still greater than85%after12h in room temperature.⁹⁹Tc^m-VEGF_125-136-MAG₃had highradioactive labeling rate, without purification and good stability.2observed and evaluation the modelThe tumors were all located the left fore alar back about one or twoweeks which were hard, nodular. Stripped observation, the tumor was locatedin the skin and muscle which were pale pink, smooth.3Biodistribution of⁹⁹Tc^m-VEGF_125-136-MAG₃in nude mice bearingosteosarcomaThe distribution of⁹⁹Tc^m-VEGF_125-136-MAG₃in kidney was highest,second in distribution and liver, in other parts of the content low. The%ID/gof tumors was higher than the muscle tissue in every group. The T/M were1.60±0.05，3.04±0.07，2.33±0.05，1.70±0.04.4Imaging result of⁹⁹Tc^m-VEGF_125-136-MAG₃in nude mice bearingosteosarcoma⁹⁹Tc^m-VEGF_125-136-MAG₃performed abnormal radioactive strongtogether at tumor location, the concentration degree increased gradually, thetumor imaging was most clear at1h. The T/NT at0.5h,1h,2h,4hrespectively were1.16±0.14、3.10±0.19、2.63±0.14、1.90±0.09. Tumorsite did not see obvious development in competitive inhibition group. TheT/NT were0.99±0.05、1.06±0.06、0.98±0.03、0.97±0.03.Conclusion:⁹⁹Tc^m-VEGF_125-136-MAG₃’s immediate radioactive labelingrate was high, good vitro stability, which could be used for animal model vivoimaging study and be absorbed by bone sarcoma cells that highly expressionof VEGF. Tumor imaging results achieved the desired purpose,⁹⁹Tc^m-VEGF_125-136-MAG₃could provides experiment basis for the earlydiagnosis of tumor as a kind of drugs that be reflected bone sarcomaangiogenesis active, also had laid a solid foundation further for tumor receptorimaging study.