Multivalent bifunctional chelator scaffolds for gallium-68 based positron emission tomography imaging probe design: Signal amplification via multivalency

The role of the multivalent effect has been well recognized in the design of molecular imaging probes toward the desired imaging signal amplification. Recently, we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds (^tBu₃-1-COOH, ^tBu₃-2-(COOH) ₂, and ^tBu₃-3-(COOH)₃) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for ⁶⁸Ga-based PET probe design and signal amplification via the multivalent effect. For proof of principle, a known integrin α_vβ₃ specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H₃1, H₃2, and H₃3), which present 1-3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin α_vβ₃ binding affinities (IC₅₀ values), enhanced specific binding was observed for multivalent conjugates (H₃2: 43.9 ± 16.1 nM; H₃3: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H₃1: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with ⁶⁸Ga³⁺ within 30 min at room temperature in high radiochemical yields (>95%). The in vivo evaluation of the labeled conjugates, ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin α_vβ₃ positive PC-3 tumor xenografts (n = 3). All ⁶⁸Ga-labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h postinjection (p.i.). The PET signal amplification in PC-3 tumor by the multivalent effect was clearly displayed by the tumor uptake of the ⁶⁸Ga-labeled conjugates ( ⁶⁸Ga-3: 2.55 ± 0.50%ID/g; ⁶⁸Ga-2: 1.90 ± 0.10%ID/g; ⁶⁸Ga-1: 1.66 ± 0.15%ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications as diagnostic gallium radiopharmaceuticals.