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 IC(50) 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.