Synthesis Of Phospholipid Fluorescent Probe And Its Application In The Detection Of Phospholipase A 2 Activity

Phospholipases are involved in a wide range of physiological processes including digestion, inflammation response, membrane remodeling, and intercellular signaling. One class of phospholipases that have been intensively studied are the phospholipases A2 (PLA2), which belongs to a group of lipolytic extracellular enzymes and is normally associated with cellular membranes but is also found in soluble form in almost all cell types. PLA2 catalyze the hydrolyzes of sn-2 fatty acyl bonds in phospholipids, liberating free fatty acids and lysophospholipids. The ubiquity of PLA2 in the digestive system, immune system, and central nervous system means that it is involved in many important physiological processes, including phospholipid homeostasis, signal transduction, and inflammation. Dysregulation of PLA2 has been implicated in many common diseases such as atherosclerosis, rheumatoid arthritis, Alzheimer's disease, and cancers. For example, in prostate cancer PLA2 levels have been found to be up to 22-fold higher than at physiological levels. Therefore, the ability to measure and monitor the activity of PLA2 is important in drug discovery and in the clinical diagnostics.Currently, a wide range of analytical techniques have been developed to monitor PLA2 activity. Typical methods for measuring PLA2 activity include discontinuous radiochemical, spectrophotometric, electrochemical, magnetic relaxation, and fluorescent methods. Among these, fluorescent method has attracted increasing attention because of its sensitivity, simplicity, and suitability for in-vivo analysis. PLA2 primarily acts on aggregated phospholipids organized into lipid bilayers, such as the cell membrane and PLA2 activity on free lipid monomers is relatively lower than that on bilayers, monolayers, and micelles. Furthermore, the use of phospholipid monolayer-coated hydrophobic beads as substrates can decrease lag time for enzymatic hydrolysis. Therefore, nanoparticles composed of artificial lipid analogs have been synthesized to assay PLA2 activity.The aim of this paper is to design a simple, rapid, high sensitive and specific fluorescent assays for monitoring the activity and inhibition of PLA2 based on fluorescence method which uses phospholipid monolayer-coated hydrophobic beads as fluorogenic substrates. This thesis consists of three parts.Chapters 1:IntroductionFirstly, we introduce a brief overview of phospholipase A2 and its substrate lecithin. And the detection methods of phospholipase A2 were reviewed. Then we present the formation and application of self-assembly assemblies of phospholipids,such as micelles and vesicles. And some properties of quantum dots and nanographenes 3a were introduced. Finally, the background, purpose and contents of this paper are describe.Chapters 2:Quantum dot cluster (QDC)-loaded phospholipid micelles as a FRET probe for phospholipase A2 detectionThe chapter, we developed a fluorescent assay using quantum dot clusters (QDC)-loaded phospholipid micelle as probe for PLA2 detection. The probe was prepared by encapsulating many small hydrophobic quantum dots (QDs) within the hydrophobic core of micelles that were formed from the coassembly of hydrogenated soy phosphatidylcholine phospholipid (HSPC) and fluorescent lipid (NBD-PC). To act as PLA2-responsive fluorescent probe, small percentage of NBD-PC was also incorporated into micelle shell. The quantum dots clusters (QDCs) were formed and served as a substrate for quenching of the NBD fluorescence. As a result, the probes show almost no fluorescence. The fluorophore NBD was attached to the sn-2 position of the phospholipid, which could be released in in the presence of PLA2 and be away from quencher. As a result, quenching effect disappeared and the probes show an effective fluorescent enhancement. The fluorescence response based on these QDC-loaded phospholipid micelles could be used for the detection of PLA2. Compared with single QD, use of QDC enables improve quenching efficiency. The fluorescent intensity is proportional to the concentration of PLA2 in the range of 5 to 400 U/L with a low detection limit of 3 U/L,which indicates the probe provide high sensitivity for phospholipase A2 detection. In addition to determining phospholipase levels, the probe can be used to identify, study, and screen PLA2 inhibitors.Chapters 3:c-HBC 3a-loaded phospholipid vesicles as a FRET probe for phospholipase A2 detectionThe chapter, we developed a fluorescent assay using c-HBC 3a-loaded phospholipid vesicle as probe for PLA2 detection. The probe was prepared by encapsulating many small hydrophobic c-HBC 3a within hydrophobic cavity of vesicles that were formed from the coassembly of hydrogenated soy phosphatidylcholine phospholipid (HSPC) and fluorescent lipid (NBD-PC). To act as PLA2-responsive fluorescent probe, small percentage of NBD-PC was also incorporated into vesicle shell. c-HBC 3a in the hydrophobic cavity served as a substrate for quenching of the NBD fluorescence. As a result, the probes show almost no fluorescence. The fluorophore NBD was attached to the sn-2 position of the phospholipid, which could be released in in the presence of PLA2 and be away from quencher. As a result, quenching effect disappeared and the probes show an effective fluorescent enhancement. The fluorescence response based on these 3a-loaded phospholipid vesicles could be used for the detection of PLA2. Compared with QDC, use of 3a also enables improve quenching efficiency. The fluorescent intensity is proportional to the concentration of PLA2 in the range of 10 to 400 U/L with a low detection limit of 5 U/L. This work demonstrates that the developed method can provide promising platform for monitoring PLA2 and inhibition because of its sensitivity and simplicity.