Tc-99m-labelled gold nanoparticles capped with HYNIC-peptide/mannose for sentinel lymph node detection

The aim of this research was to prepare a multifunctional system of technetium-99m-labelled gold nanoparticles conjugated to HYNIC-GGC/mannose and to evaluate its biological behaviour as a potential radiopharmaceutical for sentinel lymph node detection (SLND). Methods: Hydrazinonicotinamide-Gly-Gly-Cys-NH2 (HYNIC-GGC) peptide and a thiol-triazole-mannose derivative were synthesized, characterized and conjugated to gold nanoparticles (AuNP, 20 nm) to prepare a multifunctional system of HYNIC-GGC-AuNP-mannose by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and in the thiol-mannose derivative. The nanoconjugate was characterized by transmission electron microscopy (TEM), IR, UV-Vis, Raman, fluorescence and X-ray photoelectron spectroscopy (XPS). Technetium-99m labelling 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 rat liver homogenized tissue (mannose-receptor positive tissue). Biodistribution studies were accomplished in Wistar rats and images obtained using a micro-SPECT/CT system. Results: TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with HYNIC-GGC-NH2 and thiol-mannose through interactions with thiol groups and the N-terminal amine of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. Tc-99m-EDDA/HYNIC-GGC-AuNP-mannose (Tc-99m-AuNP-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of Tc-99m-AuNP-mannose in rats (footpad), radioactivity levels in the popliteal and inguinal lymph nodes revealed that 99% of the activity was extracted by the first lymph node (popliteal extraction). Biodistribution studies and in vivo micro-SPECT/CT images in Wistar rats showed an evident lymph node uptake (11.58 +/- 1.98 %ID at 1 h) which was retained during 24 h with minimal kidney accumulation (0.98 +/- 0.10 %ID) and negligible uptake in all other tissues. Conclusions: This study demonstrated that Tc-99m-AuNP-mannose remains within the first lymph node during 24 h and therefore might be useful as a target-specific radionanoconjugate for SLND using 1-day or 2-day, conventional protocols. (C) 2011 Elsevier Inc. All rights reserved.