| Objective:Human epidermal growth factor receptor type 2?HER2?is the important treatment target of breast cancer.Overexpressed HER2 is closely related to the growth,metastasis,invasiveness and prognosis of breast cancer.At present methods for determining the expression of HER2 are based on invasive biopsy techniques.However,not all lesions are easily biopsied.Radionuclide-labeled HER2 targeting molecular imaging probes and HER2targeting-mediated anti-tumor drugs can play an important role in the diagnosis and treatment of HER2-positive breast cancer.This experiment explored the vitro binding properties and the vitro anti-proliferative ability of99Tcm-HER2:V2-pemetrexed molecular probe on human breast cancer cells.Method:The HER2-targed binding protein HER2:V2 was synthesized by solid-phase synthesis of Fmoc/tBu polypeptide.The N-termina was linked anti-tumor drug pemetrexed.The C-terminal was joined with 4 amino acids Gly-Gly-Gly-Cys-,forming a strong chelating group similar to the N3S structure,which is 99Tcm-labeled by ligand exchange method.The radiochemical purity and labeling rate of 99Tcm-HER2:V2-pemetrexed were determined by reversed-phase high performance liquid chromatography and paper chromatography.The molecular probe were separately incubated in human fresh serum and physiological saline at different times,derterming the radiochemical purity of the molecular probes at different time points of the two media to understand the in vitro stability.The MDA-MB-453 cells at log phase were plated into two 24-well plates that the experimental group and the blocking group were set.The experimental group was added with the corresponding molecular probes and the blocking group was added with the corresponding 99Tcm-ZHER2:V2-pemetrexed and excess blocking peptide at the same time.The cell sediment was collected and counted using a gamma counter.The radioactivity count of the experimental group subtracted from the radioactivity count of the block group to obtain a specific radioactivity count.The equilibrium dissociation constant?Kd?was determined.The human breast cancer MDA-MB-453 cells and MDA-MB-231 cells in log phase were respectively inoculated into 6-well plates and subjected to cell uptake,retention,internalization,and blocking experiments.The anti-proliferation of HER2:V2-pemetrexed against HER2 high expression human breast cancer MDA-MB-453 cells and HER2-negative human breast cancer MDA-MB-231cells was determined by MTT assay.Results:The retention time of 99Tcm-HER2:V2-pemetrexed molecular probe was about 12.608 min.After labeling for 20 min,the labeling rate was?97.45±0.83?%?n=3?.The radiochemical purity was high.The radiation purity of the molecular probe in human fresh serum and physiological saline under the condition of 37? water within 8h was higher than 92% and the intro stability was good.The equilibrium dissociation constant Kd value of MDA-MB-453 cells against 99Tcm-HER2:V2-pemetrexed was 14.17 nM.The uptake rate of HER2 high-expressing human breast cancer MDA-MB-453cells gradually increased with time gone,and was higher than that of HER2low-expressing MDA-MB-231 cells?t or t'=9.163-18.784,P=0.000-0.011?.The retention rate of MDA-MB-453 cells decreased slowly from 1h?82.62±1.72?% to 8h?63.43±1.08?%.The internalization rate of HER2-expressing human breast cancer MDA-MB-453 cells increased gradually except 6h,and increased to?41.29±2.54?%at 8h.After MDA-MB-453 cells blocking experiments was added with excess unlabeled ZHER2:V2-pemetrexed,the uptake rate of MDA-MB-453 cells were significantly decreased,from the original?13.90±2.23?%to 500 times?1.92±0.13?%,1000 time?1.89±0.12?%?t=9.282,P=0.001,t=9.307,P=0.001?.MTT antiproliferation experiment assay showed that the inhibition rate of HER2 positive MDA-MB-453 cells was higher than that of HER2 low MDA-MB-231 cells with different concentrations?t=2.922-8.836,P<0.05?.Conclusion:99Tcm-HER2:V2-pemetrexed has good characters of simple preparation method,high labeling rate and good stability in vitro.Tt's showed that the HER2-targeted molecular probe has higher uptake rate,longer residence time and higher inhibitity effect on HER2 high-expression cells than HER2 low-expression cells.It's potential to become the HER2-targeted molecular imaging probe with both diagnosis and treatment. |
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