Lys3-Bombesin Conjugated to 99mTc-Labelled Gold Nanoparticles for In Vivo Gastrin Releasing Peptide-Receptor Imaging

The gastrin releasing peptide-receptor (GRP-r) is over-expressed in breast and prostate cancer and lymph node metastases. Lys(3)-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 Lys(3)-bombesin/HYNIC-GGC and to evaluate its biological behaviour as a potential radiopharmaceutical for in vivo GRP-r imaging. Methods: Lys(3)-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-Lys(3)-bombesin by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and the amine of Lys(3)-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 Lys(3)-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%. Tc-99m-EDDA/HYNIC-GGC-AuNP-Lys(3)-bombesin (Tc-99m-AuNP-Lys(3)-bombesin) showed specific recognition for GRP-r over-expressed in PC-3 cells. After administration of Tc-99m-AuNP-Lys(3)-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 Tc-99m-AuNP-Lys(3)-bombesin multifunctional system shows specific recognition for GRP-r and suitable properties to be used as a molecular imaging agent.