ABSTRACT
UNLABELLED: 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-NH(2) (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 SnCl(2) 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-NH(2) and thiol-mannose through interactions with thiol groups and the N-terminal amine of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. (99m)Tc-EDDA/HYNIC-GGC-AuNP-mannose ((99m)Tc-AuNP-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of (99m)Tc-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 (99m)Tc-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.
Subject(s)
Gold/chemistry , Lymph Nodes/metabolism , Mannose/metabolism , Metal Nanoparticles/chemistry , Oligopeptides/chemistry , Oligopeptides/metabolism , Organotechnetium Compounds/chemistry , Animals , Humans , Lectins, C-Type/metabolism , Lymphatic Metastasis , Mannose Receptor , Mannose-Binding Lectins/metabolism , Microscopy, Electron, Transmission , Oligopeptides/pharmacokinetics , Radiochemistry , Rats , Rats, Wistar , Receptors, Cell Surface/metabolism , Sentinel Lymph Node Biopsy , Spectrum Analysis , Sulfhydryl Compounds/chemistryABSTRACT
The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, (99m)Tc-EDDA/HYNIC-Lys(3)-bombesin ((99m)Tc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type (99m)Tc-N(2)S(2)-Tat(49-57)-Lys(3)-bombesin ((99m)Tc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of (99m)Tc-Tat-BN and to compare its cellular internalization with that of (99m)Tc-BN. Structures of N(2)S(2)-Tat-BN and Tc(O)N(2)S(2)-Tat-BN were calculated by an MM procedure. (99m)Tc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N(2)S(2)-Tat-BN and 300 kcal/mol for Tc(O)N(2)S(2)-Tat-BN. (99m)Tc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of (99m)Tc-BN (p<0.05). The tumor-to-muscle radioactivity ratio was 8.5 for (99m)Tc-Tat-BN and 7 for (99m)Tc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.
