| Objective To prepare the 99mTc-labeled novel cell-penetrating peptide-mediated targeting chemokine receptor 4(CXC Chemokine Receptor 4,CXCR4)messenger RNA(mRNA)targeting small interference RNA(Small interference RNA,siRNA)molecular probe(99mTc-PF 14/siRNA),and to study its value in gene imaging of breast cancer tumor-bearing mice.Methods The CXCR4 mRNA targeted siRNA and control siRNA with irrelevant sequence were designed and synthesized.Using the indirect labeling method,the dual-function chelating agent HYNIC was firstly coupled with siRNA,and then the siRNA with labeled with 99mTc to obtain the monomer targeting probe 99mTc-HYNIC-siRNA for CXCR4 mRNA and the monomer control probe 99mTc-HYNIC-control-siRNA.PD-10 column was used to separate and purify the labeled product,and the radiochemical purity was determined by paper chromatography.The cell penetrating peptide PF14(PepFectl4,PF14)was incubated with the monomer probe at room temperature to obtain the multivalent molecular probe 99mTc-PF14/siRNA and the control probe 99mTc-control-PF14/siRNA.Human breast cancer cell line MDA-MB-231 cells were cultured in vitro,and the cells in the logarithmic growth stage were prepared into PBS cell suspension at a concentration of 1×107 cells/ml.The tumor bearing animal model was constructed by inoculation into the right anterior axilla of BALB/c nude mice at a dose of 0.2 ml/mouse.When the tumor diameter was about 1.0?1.5 cm,36 MDA-MB-231 tumor-bearing nude mice were selected and divided into 3 groups with 12 in each group by random number table method.The tail vein was injected with 99mTc-PF14/siRNA,99mTc-control-PF14/siRNA and 99mTc-HYNIC-siRNA,respectively.At 1 h,2 h,4 h and 6 h after injection,3 nude tumor-bearing mice were sacrificed for dislocation after eyeball blood sampling.The tissues,organs and tumors were weighed and the corresponding radioactivity counts were measured with ?-well counter.The uptake rate(%ID/g)of probes per gram of tissue in various tissues,organs and tumors was calculated.And the distribution of probes in breast cancer animal models of the three groups was compared.Fifteen MDA-MB-231 cell-bearing nude mice were randomly divided into 3 groups of 5 mice and injected with 99mTc-PF14/siRNA,99mTc-control-PF14/siRNA and 99mTc-HYNIC-siRNA.SPECT imaging was performed 1 h,2 h,4 h,and 6 h after administration of the tail vein to observe the radioactive concentration of tumor sites in each group over time.The ratio of the radioactive count at the tumor site at each time point to the same area of the contralateral normal site(Target to nontarget ratio,T/NT)was measured by the region of interest technique,and the difference of imaging between the three groups was compared.All experimental data were statistically analyzed using statistical software SPSS 23.0,and Two-sample t-test was used to compare the differences between the two groups.P<0.05 was considered statistically significant(1 h:10.73±0.26%ID/g vs.3.26±0.13%ID/g,P<0.01;2 h:11.36±0.15%ID/g vs.4.49±0.12%ID/g,P<0.01;4 h:12.45±0.08%ID/g vs.5.21±0.23%ID/g,P<0.01;6h:13.61±0.14%ID/g vs.6.41±0.10%ID/g,P<0.01).Results In this experiment,the targeted probe 99mTc-HYNIC-siRNA and the control probe 99mTc-HYNIC-control-siRNA were successfully prepared,with the marking rate of 57.28±1.54%and 55.09±2.81%,and the radiochemical purity of both of them reached 93%by paper chromatography.At room temperature,99mTc-HYNIC-siRNA and 99mTc-HYNIC-control-siRNA were incubated with PF14 for 30 minutes to obtain multivalent molecular probes 99mTc-PF14/siRNA and 99mTc-control-PF14/siRNA.Biological distribution experimental results showed that the targeted probes 99mTc-PF 14/siRNA and 99mTc-HYNIC-siRNA were mainly distributed in tumor,liver and kidney.The radiation distribution of the two groups of probes in tumor tissues increased gradually with the extension of time,while the radiation distribution in other organs and tissues decreased gradually.At any time point,the%ID/g value of tumor tissues in the 99mTc-PF14/siRNA group was higher than that in the 99mTc-HYNIC-siRNA group,with statistically significant differences.The control probe 99mTc-control-PF14/siRNA was mainly distributed in liver and kidney,and was less distributed in tumor tissues.SPECT imaging results showed that after 1 h of injection of 99mTc-PF14/siRNA and 99mTc-HYNIC-siRNA in tumor bearing nude mice with breast cancer MDA-MB-231 cells,significant concentrations of radioactivity were observed in the tumor sites of both groups.With the extension of time to 6 h,the tumor imaging became clearer,while the radioactivity distribution of other tissues or organs was gradually sparse.At 1 h,2 h,4 h and 6 h after injection,the T/NT ratio of 99mTc-PF14/siRNA group(11.58±0.18,14.72±0.50,22.31±0.98,26.03±0.94)was significantly higher than that of 99mTc-HYNIC-siRNA group(3.58±0.21,6.17±0.54,6.46±0.24,7.64±0.47),with statistically significant differences(t=49.18,19.96,27.03,30.05,all P<0.01).After the injection of 99mTc-control-PF14/siRNA into the control group,the radioactivity mainly concentrated in the abdominal cavity and bladder,and no obvious concentration of radioactivity was observed in the tumor sites of tumor-bearing nude mice within 1?6 h.Conclusion PF 14-mediated CXCR4 mRNA targeting molecular probe 99mTc-PF14/siRNA is simple to prepare,showing better CXCR4 mRNA targeting imaging performance in breast cancer model,and superior to 99mTc-HYNIC-siRNA,with good application potential. |
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