The influence of temperature, cholesterol content and pH on liposome stability

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-α-phosphatidylcholine dipalmitoyl (DPPC) and 5-cholesten-3β-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. 31P 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 2Amax parameter and observation of liposome structural changes. The transition of a membrane structure from tilted gel (Lβʹ) via ripple gel phase (Pβʹ) to a more fluid liquid-crystalline phase (Lα) 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 1H NMR resonances allows us to propose the model of orientation of phospholipide in the membrane.