In Vivo Detection of Phospholipase C by Enzyme-Activated Near-Infrared Probes

• Published in: Bioconjugate chemistry Vol. 22; no. 12; pp. 2434 - 2443
• Main Authors: Mawn, Theresa M, Popov, Anatoliy V, Beardsley, Nancy J, Stefflova, Klara, Milkevitch, Matthew, Zheng, Gang, Delikatny, E. James
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.12.2011
• Subjects: Animals; Cancer; Cell Line, Tumor; Enzyme Activation; Enzymes; Fluorescent Dyes - chemistry; Humans; Male; Medical imaging; Mice; Mice, Nude; Microscopy, Confocal; Phospholipids - chemistry; Prostatic Neoplasms - enzymology; Rodents; Signal transduction; Tumors; Type C Phospholipases - analysis; Type C Phospholipases - metabolism
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc200242v

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC50 of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.