Radiosynthesis And Bio-evaluation Of ¹³¹I/¹⁸F-labeled Riboflavin And Analogues For RFVTs-targeted SPECT/PET Imaging In Brain And Heart Diseases

Aim:The reprogramming of mitochondrial energy metabolism is implicated in the early occurrence and later development of many diseases such as cardiovascular and cerebrovascular diseases and cancer.Riboflavin（RF）is the predecessor of key cofactors flavin mononucleotide（FMN）and flavin adenine dinucleotide（FAD）in the mitochondrial oxidative respiratory chain,which is critical to ATP generation.Riboflavin transporter protein RFVT3 is a key protein responsible for riboflavin absorption and transport.Researches have proved that the expression of RFVT3 in intracranial glioma is positively correlated with malignancy.Clinical trials have also shown that riboflavin can remarkably enhance the prognosis of stroke patients.Consequently,RFVT3 is chosen as a novel target for molecular imaging research in nuclear medicine.Methods:¹³¹I-RF probe was radiolabeled through the iodogen method,and the hydrophilicity and in vitro stability were investigated.Micro-SPECT/CT imaging was performed in a middle artery cerebral occlusion（MACO）model.In addition,the radiation distribution of the probe in stroke regions and normal brain regions was investigated by ex vivo autoradiography,and then the expression of RFVTs in normal brain areas and stroke areas was investigated by pathological staining.In addition,¹⁸F-RF was synthesized based on a click reaction and its distribution coefficient,affinity,and metabolic stability were in vitro and in vivo.Intracellular localization was further investigated by cellular uptake experiments.Preclinical micro-PET imaging and biodistribution were determined in U-87 cell line-bearing nude mice and myocardial ischemical reperfusion injury model in rats.Finally,immunohistochemical staining was chosen for validating the RFVT3 overexpression in glioma and myocardium.Results:¹³¹I-RF showed high radiochemical purity and good stability in vitro.SPECT/CT imaging and ex vivo autoradiography demonstrated that the ¹³¹I-RF uptake in the ischemic site is higher than in normal brain areas.Finally,pathological staining showed the expression of RFVT3 is higher in stroke areas than that in normal brain areas.¹⁸F-RF showed high radiochemical purity,high specific activity,and good metabolic stability in vivo.Micro-PET scans with ¹⁸F-RF in glioma-bearing mice and myocardial infarction rat model revealed high tracer uptake in tumor and infarcted myocardium.In addition,FAD can also be applied for fluorescence imaging of U-87 MG glioma.Finally,RFVT3 overexpression in tumor tissues and infarcted myocardium was shown by immunohistochemical staining.Conclusion:¹³¹I-RF can quickly and clearly diagnose stroke in living animals by SPECT/CT.What’s more,RFVT3-targeted probe ¹⁸F-RF not only demonstrated good stability in vitro and in vivo,but also can clearly display high uptake in glioma and infarcted myocardium by PET imaging,which prove ¹⁸F-RF a promising tracer for imaging RFVT3-positive glioma and myocardial infarction.Nevertheless,new probes with better selectivity,higher affinity and more optimized physicochem-properties were highly needed to further developed for better imaging effects and clinical transformation.