Kit for preparation of multimeric receptor-specific ⁹⁹mTc-radiopharmaceuticals based on gold nanoparticles

Multivalency is a design principle by which organized arrays amplify the strength of a binding process, such as the binding of multimeric peptides to specific receptors located on cell surfaces. The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition.

The aim of this research was to develop a kit for technetium-99m (⁹⁹mTc) labelling of AuNPs that are conjugated to Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or thiol-mannose to produce receptor-specific multimeric systems.

A freeze-dried kit formulation for the instant preparation of ⁹⁹mTc-ethylenediamine-N,N'-diacetic acid (EDDA)/hydrazinonicotinyl (HYNIC)-Tyr³-octreotide (⁹⁹mTc-EDDA/HYNIC-TOC) (vial 1) and a second vial containing 1.5 ml of AuNP solution (1 nM, 20 nm diameter, surface area=1260 nm², 37,000 surface Au atoms, 1.05 × 10 particles) plus 10 µl of Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or mannose (50 µM, approximately 285 molecules per AuNP) (vial 2) were prepared. Multimeric radiopharmaceuticals were prepared by adding 1 ml of 0.2 mol/l phosphate buffer, pH 7.0, and 1 ml of ⁹⁹mTcO4⁻ (4 GBq) to vial 1, and the mixture was incubated at 92°C for 20 min in a dry block heater. A total of 100 µl (200 MBq) of ⁹⁹mTc-EDDA/HYNIC-TOC solution (122 HYNIC-TOC molecules per AuNP) was added to vial 2. No further purification was carried out. Radiochemical purity was determined by instant thin-layer chromatography-silica gel/2-butanone (Rf values for the radiolabelled AuNP and ⁹⁹mTcO4⁻ were 0.0 and 1.0, respectively), ultrafiltration, size-exclusion high-pressure liquid chromatography and a PD-10 column. The conjugates were characterized by ultraviolet-visible, far-infrared and X-ray photoelectron spectroscopy. In-vitro binding studies were carried out in ανβ3 receptor-positive C6 glioma cancer cells, gastrin-releasing peptide receptor-positive PC3 cancer cells or mannose receptor-positive rat liver cells. Biodistribution studies were carried out in athymic mice with induced tumours (PC-3 or C6 cancer cells) or in Wistar rats (⁹⁹mTc-AuNP-mannose for sentinel lymph node detection). Images were obtained using a micro-single-photon emission computed tomography/computed tomography system.

Radiochemical purity was 96 ± 2% for all of the multimeric radiopharmaceuticals. Far-infrared showed a characteristic band at 279 ± 1 cm⁻¹, which was assigned to the Au-S bond. ultraviolet-visible and X-ray photoelectron spectroscopy also indicated that the AuNPs were functionalized with peptides or mannose. Radiopharmaceuticals showed specific recognition for receptors expressed in cancer cells or rat liver cells. Micro-single-photon emission computed tomography/computed tomography images showed clear tumour uptake and lymph node accumulation. The kit (i.e. vial 1 and vial 2) demonstrated excellent stability during storage at 4°C for 6 months.

Multimeric systems of ⁹⁹mTc-AuNP-peptide/mannose prepared from kits exhibited properties suitable for use as target-specific agents for molecular imaging of tumours and sentinel lymph node detection.