In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH₂ Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

Abstract

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

A High-Affinity, High-Stability Photoacoustic Agent for Imaging Gastrin-Releasing Peptide Receptor in Prostate Cancer

“…Four dyes, IRDyeQC-1, ATTO740, RD-831 and ICG, showed high photoacoustic signal (Figure 1A) but because ATTO740 demonstrated superior photostability (Figure 1B and supplementary information) it was selected as the most suitable signaling moiety for our imaging agent. The dye was conjugated to the targeting peptide through a triple glycine linker that was shown to allow retention of the peptide’s binding affinity (33, 34). The targeting peptide was a D-Phe 6 -bombesin[6–14] with modifications at positions 13 and 14 that impart antagonist properties, high binding and stability toward peptidases (Figure 1C).…”

Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood–Brain Barrier

“…Female SJL/J mice (5-6 week old) were obtained from Harlan (Indianapolis, IN, USA). Mice received AF680-conjugated WT and mut DK17 peptides (200 ng/mouse in a volume of 200μl) via tail-vein injection 61 . The mutant DK17 is only different from wild type due to its replacement of Ile6-Phe8 motif ( Figure 7B ) with Gly residues.…”

Fluorescent Dye Conjugates for Optical Imaging of Cancer

“…For example, an AlexaFluor 680-conjugated BBN analog was shown to specifically target tumor tissue with high selectivity and affinity in vivo. 82 …”