Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of 99mTc-N2S2-Tat(49–57)-c(RGDyK) (99mTc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional 99mTc-RGD peptide (99mTc-EDDA/HYNIC-E-[c(RGDfK)]2). Methods: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys12 side chain of the Tat(49–57)-spacer-N2S2 peptide. 99mTc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. Results: 99mTc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for 99mTc-Tat-RGD and 5.6% for 99mTc-RGD. The IC50 values were 6.7 nM (99mTc-Tat-RGD) and 4.6 nM (99mTc-RGD). Internalization in C6 cells was higher in 99mTc-Tat-RGD (37.5%) than in 99mTc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for 99mTc-Tat-RGD (6.98%±1.34% ID/g at 3h) than that of 99mTc-RGD (3.72%±0.52% ID/g at 3h) with specific recognition for α(v)β(3) integrins. Conclusions: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid 99mTc-N2S2-Tat(49–57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.