Nanoclusters of small amphiphilic molecules in a raft-mimicking model membrane

Biological membranes are considered as laterally heterogeneous, containing disordered and ordered regions ranging in size from a few nanometers to micrometers. Pulsed dipolar EPR spectroscopy (PDS) is specifically designed to determine distances between spin-labeled molecules in the range from 1.5 to typically 8 nm. A non-steroidal anti-inflammatory drug ibuprofen, due to its amphiphilic nature, is integrated into the cell membrane. In this study, we investigated spin-labeled ibuprofen (ibuprofen-SL) in a lipid raft-mimicking bilayer formed by a mixture of dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC) and cholesterol (35:35:30 mol%). Two variants of PDS were used: double electron-electron resonance (DEER, also known as PELDOR) and the simple 2-pulse electron spin echo (2p ESE) method. Analysis of PDS data in the time and frequency domains shows that ibuprofen-SL molecules at a threshold concentration of about 1 mol% form 6-membered clusters in the membrane. The clusters apparently have a prismatic structure oriented along the normal to the membrane, with a maximum diagonal of about 4 nm, and are most likely located in cavities formed between nanodomains approximately 30 nm in diameter.