Construction Of Novel Apoptosis Molecular Probe And Imaging In Vivo And In Vitro^99mTc-CP3-peptide And Its Biological Distribution

Objective To constructure a 99mTc labeled caspase-3 polypeptide apoptosis molecular probe(99mTc-CP3-peptide)based on aspartic glutamic acid-valine-aspartic acid(DEVD)as the core and investigate the biological distribution in vivo and the apoptosis model of lung adenocarcinoma and its preliminary imaging in vitro.Methods Used "click chemistry" to synthesize99mTc-CP3-peptide with domestic multi-functional.modules,detected radiochemical purity with HPLC at 0,1,2 and 4h time points after labeling.In vitro,99mTc-CP3-peptide was mixed with lung cancer A549 cells after paclitaxel chemotherapy.After stationary 30min,the radioactive counts(Cin)and extracellular radioactivity counts(Cout)were detected with a gamma counter.Then the rate of apoptosis was detected by flow cytometry,and the correlation between Cin/Cout and the rate of apoptosis was analyzed.99mTc-CP3-peptide was injected via tail vein of tumor bearing nude mice(3.7MBq for each mouse),to detect the biological distribution of 5min,15min,30min,60min,120min,240min respectively.Static SPECT imaging was performed,ROI outlined,and the ratio of tumor/contralateral normal muscle tissue was calculated.HE staining was performed to observe the morphological changes of tumor tissues after imaging.The data of one-way analysis of variance,two sample t test and linear correlation analysis was used to analyze the data.Results 99mTc-CP3-peptide of radiochemical purity is(99.33±0.12)%?(99.30±0.38)%?(99.21±0.13)%?(99.33±0.12)%at 0?1?2?4h.There was no statistical difference at each time point(P>0.05).An in vitro investigation showed that the Cin/Cout in the tumor cell chemotherapy group was(10.27 + 2.02)vs(1.09±0.03)in control group 24h after paclitaxel chemotherapy.The chemotherapy group of Cin/Cout was 7.3 times as high as that of the control group.Flow cytometric detection of tumor cells in the same state,the rate of apoptosis in the chemotherapy group was(75.62 + 2.57)%vs(3.42±0.32)%in contorl group.The ratio of Cin/Cout in the chemotherapy group and the control group was positively correlated with the percentage of apoptotic cells detected by flow cytometry(r=0.970,P<0.05).In vivo distribution of tumor bearing mice showed that:99mTc-CP3-peptide is rater fast in blood clearance,and mainly metabolized through kidney.The values of radioactive uptake were low for important organs such as heart,spleen and lung etal,but it is.slightly higher in the liver and slower in metabolism.The value of tumor tissue uptake reached a peak(4.26 + 1.03)%ID/g after injection of 1h.Clear images of the tumor bearing mice can be obtained 1h after injection of drugs.The ROI showed that the ratio of T/NT in the chemotherapy group was(3.83 + 0.11),which was significantly higher than that of the control group(1.57 + 0.09),(t=16.19>P<0.05).After imaging,HE staining of tumor tissue showed that a large number of nuclear retraction,nuclear fragmentation and apoptotic cells were found in the tumor tissue of the chemotherapy group and only a small amount of that were in control group,which was in accordance with the chemotherapy group and the control group of T/NT.Conclusion 99mTc-CP3-peptide,a kind of imaging agent targeting caspase-3 activity,which has good biological distribution,and can be applied to apoptosis imaging of animal models,has a potential clinical value in monitoring tumor cell apoptosis.