UNLABELLED: 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.
UNLABELLED: 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 humanprostate 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: Amir R Jalilian; Blanca Ocampo-García; Wanvimol Pasanphan; Tamer M Sakr; Laura Melendez-Alafort; Mariano Grasselli; Ademar B Lugao; Hassan Yousefnia; Clelia Dispenza; Siti Mohd Janib; Irfan U Khan; Michał Maurin; Piotr Ulański; Say Chye Joachim Loo; Agnes Safrany; Joao A Osso; Adriano Duatti; Kattesh V Katti Journal: Pharmaceutics Date: 2022-05-15 Impact factor: 6.525