Impact of Phospholipase A2 hydrolysis on Triplet-Triplet Annihilation Upconversion Liposomes

Abstract

Triplet-triplet annihilation upconverting (TTA-UC) liposomes are of emerging interest because of their potential bioapplications in biosensing/imaging and light driven therapeutic delivery. However, a potential challenge in-vivo is their stability, since liposomes are prone to enzymatic degradation. Here, for the first time, we examine the impact of phospholipase hydrolysis on TTA-UC in liposome. We applied a TTA-UC liposome integrating a BODPY charge transfer sensitizer and perylene emitter pairs within DOPC membrane that shows an intense upconverted blue emission under green excitation. Phospholipase A2 (PLA2) enzyme was applied as the phospholipase as it is ubiquitous in the body and upregulated in a number of conditions. Surprisingly, we observed that PLA2 treatment resulted in only a relatively modest decrease in TTA-UC intensity on exposure of the lipsosome to enzyme. In the presence of imipramine, a competitive inhibitor of PLA2, or absence of Ca2+ on which hydrolysis depends, the enzymatic action is inhibited and TTA-UC intensity is indistinguishable from that in enzyme-free solution. Characterization of Ca2+ dependent enzymatic activity, drug-based inhibition and impact of hydrolytic procucts on membrane packing were assessed at pore-suspended lipid bilayer using confocal-based fluorescence lifetime imaging (FLIM), fluorescence lifetime correlation spectroscopy (FLCS), and electrochemical impedance spectroscopy (EIS). In the absence of the inhibitor, FLIM and FLCS studies show that enzymatic lipid cleavage increases lipid packing and decreases membrane fluidity without significant damage to the bilayer. Thus, we conclude that decreased TTA-UC output is due to the the increased viscosity of the membrane on hydrolysis. Electrochemcial impedance confirm these observations where membrane admittance decreases in response to phospholipid hydrolysis inidicating tighter lipid packing of the membrane with hydrolytic product. Nanoscale imaging using atomic force microscopy (AFM) in liquid mode at a mica-supported lipid bilayer further confirmed that PLA2 caused lipidosis in presence of Ca2+, resulting in nanoscale pore formation, whereas in the absence of either Ca2+ or imipramine treated PLA2 did not induce lipid hydrolysis. Overall, our findings provide a molecular basis for understanding enzymatic action in general at a liposome bilayer model and show, for the first time, the influence of enzyme hydrolysis on TTA-UC integrity and efficiency in liposome.