Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity

Abstract

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).

Supported lipid bilayers with encapsulated quantum dots (QDs) via liposome fusion: effect of QD size on bilayer formation and structure

“…Moreover, the membrane appeared darker than the liposome cavity, which confirms that the QDs were embedded within the lipid bilayers instead of the cavity. This is also in agreement with the study by Kethineedi et al 59 , who observed darker rims of liposomes than the liposome interior upon incorporation of hydrophobic CdSe/ZnS QDs. The fluorescence emission spectra were collected to characterize the fluorescent properties of the QD-embedded POPC/POPE liposomes.…”

Activity-based targeting of secretory phospholipase A2 enzymes: A fatty-acid-binding-protein assisted approach

“…Phospholipase A 2 activity has previously been assayed using titrimetric, colorimetric, fluorometric, and radiometric techniques (Reynolds et al, 1991; Kethineedi et al, 2013). These techniques are time consuming, suffer from limited sensitivity, and present significant safety concerns (Kethineedi et al, 2013).…”

“…These techniques are time consuming, suffer from limited sensitivity, and present significant safety concerns (Kethineedi et al, 2013). For example, early efforts that focused on development of chromogenic PLA 2 assays using sn -2-thioester phospholipids led to PLA 2 -catalyzed formation of sn -2-thiolate to be trapped by reaction with DTNB (5,5’-dithio-bis-2-nitrobenzoic acid).…”

“…The photophysical stability of the fluorophores (Kethineedi et al, 2013) and the effect of the size of the reporter groups on lipid membranes are recognized as important issues in the design of fluorogenic probes and assays (Skaug et al, 2011). We have been engaged in developing new fluorogenic probes and assays for the detection and kinetic characterization of secretory phospholipase A 2 enzymatic activity (Wang, et al, 2013).…”

Quantum dot cluster (QDC)-loaded phospholipid micelles as a FRET probe for phospholipase A2 detection

“…QD-encapsulated liposome as FRET probe has also been developed for monitoring the enzymatic activity of PLA 2 . 26 …”