Application of spin markers for study of liposome prepared by the modified reverse-phase evaporation method

Abstract

The EPR spectroscopy was used to determine the structure and physicochemical properties of liposomes prepared from L-α-phosphatidylcholine dipalmitoyl (DPPC) by the modified reverse-phase evaporation method (mREV). EPR study was carried out in the temperature range from 297 K to 340 K i.e. below and above the phase transition temperatureTCof DPPC. On the basis of EPR spectra of spin marker 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) incorporated into the liposome, the parameterfwas determined. TEMPO dissolves easily in water and in the fluid lamellar smectic liquid-crystaline phase of lipid bilayer but is largely excluded from the solid, gel-phase. Thus TEMPO can be used to observe the change in the partition between aqueous and fluid lipid regions. The change in the relative value offas a function of temperature for DPPC shows that, in the presence of water excess, this phospholipids undergoes a transition from a “gel phase” to a lamellar smectic liquid crystalline phase. On the basis of EPR spectra of spin marker 2-(3carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinloxyl (5-DOXYL) incorporated into liposome, the parametera'Nwas determined. The isotropic14N-hyperfine coupling constanta'Nof nitroxide spin label depends on the local environmental polarity. The increase ofa'Nvalue reflects the rise of polarity of spin label environment. Temperature, cholesterol and pH dependent structural changes were also described.