Impact of phospholipase A₂ hydrolysis on triplet-triplet annihilation upconversion liposomes

Triplet-triplet annihilation upconversion (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 liposomes. We applied a TTA-UC liposome integrating a BODIPY charge transfer sensitizer and perylene emitter pair within a DOPC membrane, that exhibits 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 under 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 liposomes to the enzyme. In the presence of imipramine, a competitive inhibitor of PLA₂, or absence of Ca²⁺ on which phospholipase hydrolysis depends, the enzymatic action is inhibited and TTA-UC intensity is indistinguishable from that in enzyme-free solution. Ca²⁺-dependent enzymatic activity, drug-based inhibition and the impact of hydrolytic products on membrane packing were characterized in pore-suspended lipid bilayers, using confocal-based fluorescence lifetime imaging (FLIM), fluorescence lifetime correlation spectroscopy (FLCS), and electrochemical impedance spectroscopy (EIS). 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 the decreased TTA-UC output is due to the increased viscosity of the membrane upon hydrolysis. Electrochemcial impedance confirms these observations, where membrane admittance decreases in response to phospholipid hydrolysis indicating tighter lipid packing of the membrane with hydrolytic products. Nanoscale imaging, using atomic force microscopy (AFM) in liquid mode at a mica-supported lipid bilayer, further confirmed that PLA₂ causes phospholipid hydrolysis in the presence of Ca²⁺, resulting in nanoscale pore formation, whereas either in the absence of Ca²⁺ or with imipramine-treated PLA₂, it 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 liposomes.