Development of a specific radiopharmaceutical based on gold nanoparticles functionalized with HYNIC-peptide/mannose for the sentinel lymph node detection in breast cancer

The aim of this research was to prepare a multifunctional system of 99mTc-labelled gold nanoparticles conjugated to HYNIC-G GC/mannose and to evaluate its biological behaviour as a potential radiopharmaceutical for sentinel lymph node detection. Hydrazino nicotinyl-Gly-Gly-Cys-NH2 (HYNIC-G GC) peptide and a thiol-triazole-mannose derivative were synthesized, characterized and conjugated to gold nanoparticles (Au-Np, 20 nm) to prepare a multifunctional system of HYNIC-G GC-Au-Np-mannose by means of spontaneous reaction of the thiol (Cys) present in HYNIC-G GC sequence and in the thiol-mannose derivative. The nano conjugate was characterized by transmission electron microscopy (Tem), IR, UV-Vis, Raman, Fluorescence and X-ray photoelectron spectroscopy (XP S). 99mTc labelling was carried out using EDDA/tricine as co ligands and SnCl2 as reducing agent with further size-exclusion chromatography purification. Radiochemical purity was determined by size-exclusion HPLC and I TLC-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. Tem and the spectroscopic techniques demonstrated that Au-Np were functionalized with HYNIC-G GC and thiol-mannose through interactions with thiol groups of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. 99mTc-EDDA/HYNIC-G GC-Au-Np-mannose (99mTc-Au-Np-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of 99mTc-Au-Np-mannose in rats (foot pad), 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. In order to design a pharmaceutical formulation for the instant preparation of stable m ultimeric systems with target-specific molecular recognition based on gold nanoparticles, a freeze-dried kit formulation of 99mTc-ethylenediamine-N, N-diacetic acid (EDDA)/hydrazino nicotinyl (HYNIC)-Tyr3-octreotide (99mTc-EDDA/HYNIC-TOC, previously approved by the Mexican Ministry of Health) (vial 1) and a second vial containing 1.5 ml of Au-Np solution plus 10 μL of thiol-mannose, Lys3-bombesin, or cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] (c[RGDfK(C)] (approximately 285 molecules per Au-Np) (vial 2) were prepared. M ultimeric radiopharmaceuticals prepared from kit showed a radiochemical purity of 96 ± 2%. The far-infrared spectra showed a characteristic band at 279 ± 1 cm-1, which was assigned to the Au-S bond. UV-Vis and XP S also indicated that the Au-Np were functionalized with peptides or mannose. Radiopharmaceuticals showed specific recognition for receptors expressed in cancer cells or rat liver cells. Micro-SPECT/CT images showed clear tumour uptake and lymph node accumulation. The kit demonstrated excellent stability during storage at 4 C for 6 months. This study demonstrated that 99mTc-Au-Np-mannose remains within the first lymph node during 24 h and therefore might be useful as a target-specific radiopharmaceutical for SLND using 1-day or 2-day conventional protocols. Likewise, m ultimeric systems of 99mTc-Au-Np-mannose, 99mTc-Au-Np-RGD and 99mTc-Au-Np-Lys3-bombesin prepared from kits exhibited properties suitable as target-specific agents for molecular imaging of tumours and sentinel lymph node. (Author)