Study On Radionuclide Molecular Imaging Of Acute Cardiac Rejection

Part 1.Radionuclide Molecular Imaging of Acute Cardiac Rejection using 99 mTc Labeled anti-CD4 Monoclonal AntibodyObjective: A novel radiolabeled probe,^99mTc-HYNIC-m Ab CD4,was developed to noninvasively visualize CD4+ T cell-infiltration and realize the early diagnosis of acute rejection（AR）of heart transplantation.Methods:（1）The target probe ^99mTc-HYNIC-m AbCD4 and its control probe ^99mTc-HYNIC-Ig G were prepared,and the radiolabeling rate,radiochemical purity and stability in vitro were determined.（2）CD4+ T lymphocytes were extracted from rat lymph nodes as positive cells and rat alveolar macrophages NR8383 were used as negative cells.The expression of CD4 molecules of these two types of cells were verified by Western Blot.The binding affinity of anti-CD4 antibody to CD4+ T lymphocytes was determined by flow cytometry and immunofluorescence staining.（3）The specificity and affinity of the probe binding to CD4+ T lymphocytes in vitro were verified by cell binding assay.（4）Five days after establishing the rat heterotopic heart transplantation models,rats were intravenously injected with ^99mTc-HYNIC-m Ab CD4（11.1³7 MBq,n = 5）through the tail vein.Single-photon emission computed tomography（SPECT）imaging and biodistribution were performed to evaluate the in vivo AR targeting ability of the probe.（5）Lymphocytes infiltration and CD4 expression in each group were detected by histology.Results:（1）^99mTc-HYNIC-m AbCD4 and ^99mTc-HYNIC-Ig G were successfully synthesized with labeling rate of 73.31 ± 0.95% and 63.10 ± 1.89%,respectively.The specific activity were 8.14 ± 0.11 MBq/μg and 7.01 ± 0.21 MBq/μg（n = 5）,respectively.After purified by PD-10 column,their radiochemical purity were both more than 95%.The radiochemical purity was maintained at more than 85% for 12 h in phosphate buffered saline（PBS）and fetal bovine serum（FBS）,which indicated good stability in vitro.（2）Western Blot showed that the CD4 expression of CD4+ T lymphocytes was significantly higher than that of macrophages.Flow cytometry and immunofluorescence results showed that the fluorescence intensity of the experimental group was significantly higher than that of the control group,and could be successfully blocked.（3）Cell-binding experiments showed that the cell uptake rate of the experimental group increased gradually with the extension of incubation time,and reached the highest at 6 h.Binding of ^99mTc-HYNIC-m Ab CD4 to CD4+ T lymphocytes was higher than that of the macrophages and Ig G probe groups,and m Ab CD4 was effective in blockade of the binding reaction.（4）Further,the rat heart transplantation models were successfully constructed.SPECT imaging and biodistribution results showed significantly higher radioactive uptake of graft heart in the allograft group than that of allograft treatment,isograft and ^99mTc-HYNIC-Ig G groups,indicating that the probe has good specificity for in vivo targeting.（5）Histological examination revealed significant CD4+ T lymphocytes infiltration in graft heart of the allograft group,accompanied by myocardial cells destruction.Conclusion: We have successfully developed a novel radionuclide molecular imaging agent 99 mTc labeled anti-CD4 antibody,which has a high labeling rate and radiochemical purity,and shows good stability in vitro.The targeted probe achieved high affinity and specificity of binding to CD4+ T lymphocytes and accumulation in the transplanted heart where rejection occurred.The results showed that radionuclide molecular imaging with ^99mTc-HYNIC-m Ab CD4 is expected to be a novel non-invasive method for the diagnosis of acute cardiac rejection.Part 2.Radionuclide Molecular Imaging of Acute Cardiac Rejection using 68 Ga Labeled LLP2 A peptideObjective: Very late antigen-4（VLA-4）plays an important role in lymphocyte differentiation and transport,making it an attractive target for developing molecular diagnostic agents.In this study,a VLA-4 targeting molecular probe ⁶⁸Ga-NODAGA-LLP2 A was constructed to realize the early diagnosis of acute cardiac rejection.Methods:（1）The peptidomimetic ligand LLP2 A was modifed with chelator NODAGA-NHS,and then radiolabeled with 68 Ga to prepare a radionuclide molecular probe ⁶⁸Ga-NODAGA-LLP2 A.The radiolabeling yield,radiochemical purity and stability of the product were determined by analytical radio-high performance liquid chromatography（radio-HPLC）.（2）Mononuclear cells were extracted from rat spleen as positive cells and cardiomyocytes H9C2 were used as negative cells.The expression of VLA-4 molecules of these two types of cells were verified by Western Blot.The specificity of labeled peptide targeting VLA-4 was investigated by cell binding assay.（3）Rat models of heterotopic heart transplantation were established,including allograft,allograft treatment and isograft groups.Five days after transplantation,rats were intravenously injected with ⁶⁸Ga-NODAGA-LLP2A（5.5⁷.4 MBq/rat,n = 5）through the tail vein.Positron emission tomography（PET）imaging,ex vivo autoradiography and biodistribution studies were performed 1 hour after injection to assay the in vivo targeting efficiency.Results:（1）⁶⁸Ga-NODAGA-LLP2 A was successfully prepared with a greater than 95% radiolabeling yield and the molar activity was 37-74 MBq/nmol.The tracer exhibited good stability and less than 5% disassociation of 68 Ga was observed both in vitro and in vivo.（2） Western Blot showed that the VLA-4 expression of mononuclear cells was significantly higher than that of cardiomyocytes.The cell uptake rate of mononuclear cells was significantly higher than that of cardiomyocytes in cell binding assay.（3）The tracer exhibited high accumulation with good contrast in the transplanted heart of allograft in static PET/CT images,autoradiography and biodistribution studies,and lower accumulation of radioactivity in the allograft blocking and allograft treatment groups,while no uptake was seen in the isograft and normal groups.Conclusion: ⁶⁸Ga-NODAGA-LLP2 A exhibited high VLA-4 targeting efficacy in vivo and could accumulated specifically in the transplanted heart,rendering it a promising candidate for noninvasive imaging of acute cardiac rejection.