The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A 1 , A 2 (PLA 2 ), C (PLC) and D (PLD) based on their site of action. Here we report the design, synthesis and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA 2 or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore Pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged Black Hole Quencher-3 (BHQ-3) to the phospholipid head group or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C 6 , C 12 ) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids were assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC 6 -PyroC 6 -PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA 2 , Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA 2 was negligible due to steric hindrance at the sn-2 position. In contrast, the C 12 -spacered PyroC 12 -PtdEtn-BHQ demonstrated a remarkable selectivity for PLA 2 and the best relative PLA 2 /PLC sensitivity, significantly outperforming previously known probes. These results open an avenue for future in vivo experiments and for new probes to detect PL activity.Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A 1 , A 2 (PLA 2 ), C (PLC) and D (PLD) based on their site of action (1,2) (Figure 1).Since PLA 2 is elevated in a number of diseases (e.g. prostate and breast cancer, rheumatoid arthritis, etc) (3-5) we have chosen to develop probes that distinguish PLA 2 from other PLs. In addition, since increased PLC is detected in melanoma, ovarian and breast cancers and PLC has been implicated in maintaining the high levels of phosphocholine (PC) and phosphoethanolamine (PE) characteristic of many human tumors (6-10), we are also developing probes to specifically detect the actions of this enzyme. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe primary focus of this work is to develop methods to investigate the expression and activity of PLs in vivo in mammals. The above diseases can be detected, monitored and in some cases treated using soft tissue penetrating near infrared (NIR) light (11,12). As a result we are employing optical imaging using NIR fluorophores (NIRF).Our approach is to design self-quenching reporter probes that release fluorescent moieties only upon c...
In this paper 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. Due to 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 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.
There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce upon separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.
This information can be used to calibrate the transabdominal pulse oximeter as a measurement of fetal arterial saturation. With these results, we can advance the accurate, no-risk, noninvasive transabdominal fetal pulse oximeter for human use. This research may contribute to the more accurate diagnosis of the diseases of the fetus including Hypoxic Ischemic Encephalopathy.
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