Preclinical assessment of nanoparticles conjugated with 64Cu-DOTA-PEG-BBN targeting gastrin-releasing peptide receptors

1508 Objectives: Gold andiron oxide based nanoparticleshave emerged as innovative tools for cancer imaging and therapy, as benefit of their size, biocompatibility and pharmacokinetics. Their surface functionalization with high number of recognizable biomolecules (gastrin-releasing peptides, GRP, namely bombesin, BBN, bombesin-like short chain peptide, BBN(7-14), and Neuromedin B, NMB) forms high-specific targeted systems, suitable for multimodal PET-MRI or PET-CT imaging aimed for early detection of tumours and/or therapy monitoring and follow-up. The aim of this study was to investigate preclinically and comparatively evaluate the potential as imaging agents of activated N-Hydroxysuccinimide gold nanoparticles, coated with polyethylene glycol (PEG) and conjugated to 64Cu-DOTA-BBN/BBN(7-14)/NMB and iron oxide nanoparticles coated with PEG and conjugated to 68Ga-DOTA-BBN/BBN(7-14)/NMB, respectively. Methods: The morphology and size distribution of gold/iron oxide nanoparticles were examined by TEM/SEM coupled with energy dispersion spectroscopy (EDS) techniques and, after functionalization, by UV-Vis spectroscopy and DLS. The gold/iron oxide NPs were fconjugated to PEG(4)-BBN(7-14), PEG(4)-BBN, and PEG(4)-NeuromedinB, previously radiolabelled with Cu-64 using DOTA as chelating agent. The radiolabelled nanoconjugates were incubated with neuroendocrine colorectal cancer cells HT-29 and HCT116, and prostate cancer cells DU-145, all over-expressing GRPRs. We investigated the specific binding of Bombesin (BBN), Bombesin-like peptide sequence BBN(7-14) and Neuromedin B (NMB) to GRP receptors in the presence of cold competitor antagonist ligands. In-vivo PET imaging was performed on high-resolution micro-PET-CT system, on tumor bearing nude mice (Nu/J) inoculated with HT-29, HCT116 and DU-145 tumors, respectively. Results: In vitro binding kinetics assessment showed an increased retention of over 30% of radioisotope inside tumor cells in the case of AuNP-PEG(4)-64Cu-DOTA-BBN(7-14), compared to 64Cu-DOTA-PEG(4)-BBN(7-14) alone. Similarly, IONP-PEG(4)-64Cu-DOTA-NMB was better uptaken and internalized, compared to 64Cu-DOTA-PEG(4)-NMB alone. These results are attributed to the increased concentration and multiple binding sites of BBN-like peptides to GRPRs allowed by their exposure on the surface of NPs. Subsequently, higher internalization rate is promoted, by receptor-mediated endocytosis. Biodistribution/imaging studies, and the results show high and stable tumor uptake of the radiolabelled nanoconjugates, fast blood-clearance and allowed for determination of key PKs parameters, path of accumulation and excretion rate. Conclusions: The combination of size-controlled distribution of nanoparticles, functionalization with high-specific targeting peptides and Cu-64 radioisotope labelling resulted in highly promising imaging/therapeutic scaffolds, based on gold/iron oxide NPs coated with PEG(4) and bearing specific targeting 64Cu-DOTA-BBN analogs. Their preliminary in vitro and in vivo evaluation shows good results regarding uptake, retention, stability and PKs parameters. Further investigations are to be performed to determine the detailed dosimetry and therapeutic efficacy to extend the potential use to targeted radionuclide therapy, by use of highly cytotoxic potency of Auger-electrons emitted by Cu-64, inside the tumour, and specifically affecting the tumour cell metabolism. Research Support: This work was supported by a grant of the Romanian Ministry of Research and Innovation, UEFISCDI project number 64PCCDI/2018.