title: 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.
authors.affiliation: Department of Radiology, The Radiopharmaceutical Sciences Institute, Columbia, MO, USA.
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.forename: Bhadrasetty
authors.initials: B
authors.lastname: Veerendra
authors.forename: Tammy L
authors.initials: TL
authors.lastname: Rold
authors.forename: Lauren
authors.initials: L
authors.lastname: Retzloff
authors.forename: Adam
authors.initials: A
authors.lastname: Prasanphanich
authors.forename: Gary
authors.initials: G
authors.lastname: Sieckman
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Hoffman
authors.forename: Wynn A
authors.initials: WA
authors.lastname: Volkert
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Smith
mesh_terms: D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D001943:Breast Neoplasms / Q000175:diagnosis*; D045744:Cell Line, Tumor; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000032:analysis* / Q000138:chemical synthesis; D018004:Receptors, Bombesin / Q000032:analysis*; D014183:Transplantation, Heterologous
chemical_list: D005456:Fluorescent Dyes; D010446:Peptide Fragments; D018004:Receptors, Bombesin; C118536:bombesin (7-14); D001839:Bombesin