The EPR and NMR spectroscopy was used to determine the influence of temperature (297-340 K), cholesterol and pH on liposome membranes obtained by modified reverse-phase method (mREV). For preparation of liposomes, L-a-phosphatidylcholine dipalmitoyl (DPPC) and 5-cholesten-3b-ol (cholesterol), main component of the membrane, were used in the molar ratio DPPC/cholesterol 4, 8 and 16 at pH range 1.9-8.4. The liposomes not containing cholesterol were prepared as reference. 31 P NMR spectra point to the bilayer structure of the membrane. Incorporation of the 4-dimethyl-2-(carboxypropyl)-2-tridecyl-3-oxazolidynyloksyl (5-DOXYL) into the liposome membrane allowed for the estimation of the 2A max parameter and observation of liposome structural changes.
The transition of a membrane structure from tilted gel (L b' ) via ripple gel phase (P b' ) to a more fluid liquid-crystalline phase (L a ) with temperature increase was observed. The greatest changes in properties of the obtained membrane were found for cholesterol concentration 0.5-1.0 mol. The S order parameter, characteristic for liposome structural changes decreases with temperature increase and decreasing pH. For liposome without cholesterol, the values of this parameter are slightly lower while a distinct change is observed at the temperature range 310 -315 K and for pH equal to 1.9 and 5.0. This confirms higher membrane fluidity in these conditions. The values 7.4-8.4 of pH only slightly affect the membrane fluidity as well as the cholesterol content higher than 0.5 mole per 4 moles of DPPC.
The temperature dependence of 1 H NMR resonances allows us to propose the model of orientation of phospholipide in the membrane.