Non-Steroidal Anti-Inflammatory Drug Induced Membrane Fusion: Concentration and Temperature Effects

Abstract

Membrane fusion is a critical step in many biological events. The fusion process is always induced by different fusogenic agents of which proteins and peptides form the largest group. The mechanistic details of the fusion process vary depending on the nature of the fusogenic agents. However, membrane fusion induced by small drug molecules at physiologically relevant concentration has not been observed. Only recently our group has shown that three painkillers, namely, meloxicam, piroxicam, and tenoxicam, belonging to the oxicam group of non-steroidal anti-inflammatory drugs (NSAIDs) share this property. In this work, we present the effect of drug concentration and temperature on the kinetics of the fusion process. Small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage whereas transmission electron microscopy (TEM) was used to image the fusion process and to calculate the average diameter of the vesicles. The results show that, in this fusion process, lipid mixing and content mixing are two sequential events and can occur even at a very low drug to lipid ratio (D/L) of 0.018. For a D/L ratio greater than 0.045, leakage of the vesicles leading to rupture compete with the fusion thereby inhibiting it. Temperature variation in the presence of drugs gives linear Arrhenius plots and is used to calculate the activation energies for the lipid mixing and content mixing, which are less compared to that seen in SUVs with a smaller diameter of 45 nm. Thermodynamic parameters of the transition state are calculated. The fusogenic property of the drugs has been interpreted in terms of the ability of the drugs to introduce membrane perturbation even at such low D/L ratios as studied here.