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: Lixin Ma, Ping Yu, Bhadrasetty Veerendra, Tammy L Rold, Lauren Retzloff, Adam Prasanphanich, Gary Sieckman, Timothy J Hoffman, Wynn A Volkert, Charles J Smith
• Journal: Molecular imaging (Mol Imaging) 2007 May-Jun Vol. 6 Issue 3 Pg. 171-80 ISSN: 1535-3508 [Print] England
• PMID: 17532883 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Fluorescent Dyes
• Peptide Fragments
• Receptors, Bombesin
• bombesin (7-14)
• Bombesin

Topics

• Animals
• Bombesin (analogs & derivatives, analysis, chemical synthesis)
• Breast Neoplasms (diagnosis)
• Cell Line, Tumor
• Female
• Fluorescent Dyes (analysis)
• Humans
• Mice
• Mice, SCID
• Microscopy, Fluorescence
• Peptide Fragments (analysis, chemical synthesis)
• Receptors, Bombesin (analysis)
• Transplantation, Heterologous