title: Design and biological evaluation of ⁹⁹mTc-N₂S₂-Tat(49-57)-c(RGDyK): a hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins.
abstract: 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 (99m)Tc-N₂S₂-Tat(49-57)-c(RGDyK) ((99m)Tc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional (99m)Tc-RGD peptide ((99m)Tc-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 Cys(12) side chain of the Tat(49-57)-spacer-N₂S₂ peptide. (99m)Tc-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
  (99m)Tc-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 (99m)Tc-Tat-RGD and 5.6% for (99m)Tc-RGD. The IC50 values were 6.7 nM ((99m)Tc-Tat-RGD) and 4.6 nM ((99m)Tc-RGD). Internalization in C6 cells was higher in (99m)Tc-Tat-RGD (37.5%) than in (99m)Tc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for (99m)Tc-Tat-RGD (6.98% ± 1.34% ID/g at 3h) than that of (99m)Tc-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 (99m)Tc-N₂S₂-Tat(49-57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.
authors.affiliation: Departamento de Materiales Radiactivos, Instituto Nacional de Investigaciones Nucleares, Estado de México, Mexico.
authors.forename: Blanca E
authors.initials: BE
authors.lastname: Ocampo-García
authors.forename: Clara L
authors.initials: CL
authors.lastname: Santos-Cuevas
authors.forename: Luis M
authors.initials: LM
authors.lastname: De León-Rodríguez
authors.forename: Rocío
authors.initials: R
authors.lastname: García-Becerra
authors.forename: David
authors.initials: D
authors.lastname: Ordaz-Rosado
authors.forename: Myrna A
authors.initials: MA
authors.lastname: Luna-Guitiérrez
authors.forename: Nallely P
authors.initials: NP
authors.lastname: Jiménez-Mancilla
authors.forename: Mario E
authors.initials: ME
authors.lastname: Romero-Piña
authors.forename: Guillermina
authors.initials: G
authors.lastname: Ferro-Flores
mesh_terms: D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D015195:Drug Design; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000000981:diagnostic imaging / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008297:Male; D051379:Mice; D064847:Multimodal Imaging; D009842:Oligopeptides / Q000737:chemistry*; D015609:Organotechnetium Compounds / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D010446:Peptide Fragments / Q000737:chemistry*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry*
chemical_list: D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D015609:Organotechnetium Compounds; D010446:Peptide Fragments; D019275:Radiopharmaceuticals; D054322:tat Gene Products, Human Immunodeficiency Virus; C490722:tat peptide (49-57), Human immunodeficiency virus 1; C062178:arginine-glycine-aspartate-O-methyltyrosine amide