title: Lys3-bombesin conjugated to 99mTc-labelled gold nanoparticles for in vivo gastrin releasing peptide-receptor imaging.
abstract: The gastrin releasing peptide-receptor (GRP-r) is over-expressed in breast and prostate cancer and lymph node metastases. Lys3-bombesin is a peptide that binds with high affinity to GRP-r. The aim of this research was to prepare a multifunctional system of technetium-99m labelled gold nanoparticles conjugated to Lys3-bombesin/HYNIC-GGC and to evaluate its biological behaviour as a potential radiopharmaceutical for in vivo GRP-r imaging.
  
  
  METHODS
  Lys3-bombesin and hydrazinonicotinamide-Gly-Gly-Cys-NH2 (HYNIC-GGC) peptides were conjugated to gold nanoparticles (AuNP, 20 nm) to prepare a multifunctional system of HYNIC-GGC-AuNP-Lys3-bombesin by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and the amine of Lys3-bombesin. The nanoconjugate was characterized by transmission electron microscopy (TEM), IR, UV-Vis, Raman, and X-ray photoelectron spectroscopy (XPS). Technetium-99m labelling through the HYNIC-GGC ligand was carried out using EDDA/tricine as coligands and SnCl2 as reducing agent with further size exclusion chromatography purification. Radiochemical purity was determined by size exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in human prostate cancer PC-3 cells (GRP-r positive cells). Biodistribution studies were accomplished in athymic mice with PC-3 induced tumours and images obtained using a micro-SPECT/CT system.
  
  
  RESULTS
  TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with HYNIC-GGC-NH2 and Lys3-bombesin through interactions with thiol groups of Cysteine and the N-terminal and epsilon-amine of Lysine respectively. Radio-chromatograms showed radiochemical purity higher than 95%. 99mTc-EDDA/HYNIC-GGC-AuNP-Lys3-bombesin (99mTc-AuNP-Lys3-bombesin) showed specific recognition for GRP-r over-expressed in PC-3 cells. After administration of 99mTc-AuNP-Lys3-bombesin in mice the pancreas-to-blood ratio was 36 at 1 h demonstrating ability to target in vivo GRP receptor-bearing cells. In vivo micro-SPECT/CT images in mice showed an evident tumour uptake (6.39 +/- 0.83% IA/g at 1 h).
  
  
  CONCLUSIONS
  This study demonstrated that the 99mTc-AuNP-Lys3-bombesin multifunctional system shows specific recognition for GRP-r and suitable properties to be used as a molecular imaging agent.
authors.affiliation: Instituto Nacional de Investigaciones Nucleares, Estado de México, C. P. 52750, México.
authors.forename: Andrei N
authors.initials: AN
authors.lastname: Mendoza-Sánchez
authors.forename: Guillermina
authors.initials: G
authors.lastname: Ferro-Flores
authors.forename: Blanca E
authors.initials: BE
authors.lastname: Ocampo-García
authors.forename: Enrique
authors.initials: E
authors.lastname: Morales-Avila
authors.forename: Flor
authors.initials: F
authors.lastname: de M Ramírez
authors.forename: Luis M
authors.initials: LM
authors.lastname: De León-Rodríguez
authors.forename: Clara L
authors.initials: CL
authors.lastname: Santos-Cuevas
authors.forename: Luis A
authors.initials: LA
authors.lastname: Medina
authors.forename: Eva L
authors.initials: EL
authors.lastname: Rojas-Calderón
authors.forename: Marco A
authors.initials: MA
authors.lastname: Camacho-López
mesh_terms: D000818:Animals; D006046:Gold; D008297:Male; D053768:Metal Nanoparticles; D051379:Mice; D008819:Mice, Nude; D046529:Microscopy, Electron, Transmission; D015394:Molecular Structure; D058726:Nanoconjugates / Q000737:chemistry; D015609:Organotechnetium Compounds; D011471:Prostatic Neoplasms / Q000175:diagnosis; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals; D018004:Receptors, Bombesin / Q000737:chemistry*; D013055:Spectrophotometry, Infrared; D013667:Technetium
chemical_list: D058726:Nanoconjugates; D015609:Organotechnetium Compounds; D019275:Radiopharmaceuticals; D018004:Receptors, Bombesin; D013667:Technetium; D006046:Gold