Fluorescent Lipo-Beads for the Sensitive Detection of Phospholipase A₂ and Its Inhibitors

Phospholipase A₂ (PLA₂) is a membrane lytic enzyme that is present in many organisms. Human PLA₂ has emerged as a potential biomarker as well as a therapeutic target for several diseases including cancer, cardiovascular diseases, and some inflammatory diseases. The current study focuses on the development of lipo-beads that are very reactive and highly sensitive to PLA₂. To develop the best supported lipid bilayer formulation, several lipid combinations were investigated using 10 μm porous silica beads. The reactivity of PLA₂ was monitored via the decrease in particle fluorescence because of the release of entrapped fluorescent dye from the particle pores or the disintegration of a fluorescent lipid constituted on the bilayer upon lipid hydrolysis using flow cytometry. The enzyme binding studies indicate that lipo-beads with bulky fluorescent tags in the lipid head group and anionic lipids produce a more pronounced response. The kinetic studies suggest that these lipo-beads are very reactive with PLA₂ and can generate a detectable signal in less than 5 min. The enzyme inhibition studies were also conducted with two known PLA₂ inhibitors, varespladib and quercetin. We find that quercetin can hydrolyze the supported membrane, and thus inhibition of PLA₂ is not observed; however, varespladib has shown significant PLA₂ inhibition on lipo-beads. We have demonstrated that our lipo-bead-based approach can detect annexin-3, a known disease biomarker, as low as 10 nM within 5 min after incubation.