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

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Abstract

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 (Lβ') via ripple gel phase (Pβ') 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.

Introduction

Liposomes are artificial spherical lipid bilayer structures. According to many described methods phospholipides, glycolipides and cholesterol are mainly used for their preparation [1]. The properties and further application depend on their chemical structure and the preparation method, e.g. hand-shaking, ethanol injection, ether injection, reverse-phase evaporation, freeze-thawing or dehydratation–rehydratation [1]. Small liposomes with diameter of the order of 100 nm are frequently used as a carrier of drugs due to their better distribution in the organisms. Continuous search for new preparation methods of liposomes is connected with their ability to close and transport solutes in living organisms. This protects solutes from undesirable side reactions of organisms to their fast degradation. Recognition of liposomes by the target tissue, cells or organelles depends on the choice of lipids for liposome membrane preparation. l-α-Phosphatidylcholine dipalmitoyl (DPPC) is the most frequently used among synthetic phospholipids.

Using DPPC for liposomes preparation is more advantageous than of phosphatidylcholine (PC) because the phase transition temperature (Tc) is higher for liposomes obtained from DPPC (∼41 °C) than for ones obtained from PC (∼20 °C). Hence, liposomes obtained from DPPC exhibit higher stability and leaktightness in a wider temperature range. A choice of a suitable lipid ought to prevent liposome bilayers from enlargement during storage. Liposome suspension is destabilized after intravenous injection due to adipose (fatty) exchange of phospholipids under plasma lipoprotein influence [2]. The addition of cholesterol to liposome bilayers prevents a lipid exchange and has an additional stabilizing effect [3]. The maximum cholesterol incorporation in liposome membrane obtained from PC by use of the ultrasonic method was 66 mol% while by the use of other methods it amounted to about 50 mol% [4]. The liposomes characterized by a long time of drug release into the blood circulation are of great interest. Liposomes of prolonged time of circulation in blood can be obtained by covering the liposome surface of hydrophilic polymers. Owing to the small resistance of liposomes to gastric juice (pH 1.9), enzymes of the alimentary canal and bile acids in the intestine (pH 8) their application per os is useless.

It was confirmed that a large quantity of drugs may be incorporated into liposomes using the pH gradient which facilities their transport through liposome bilayers [5]. By selecting a suitable pH buffer inside and outside of the liposome bilayers, it is possible to incorporate weak acids and bases [5].

EPR, 2H NMR spectroscopy and depolarization of fluorescence methods are techniques more often used to study liposome fluidity. To study the interaction between liposome vesicles many different techniques are used, e.g. NMR, EPR, differential scanning calorimetry DSC, electron microscopy, gel filtration, fluorescence spectroscopy.

Taking the above into consideration, the aim of this work was to find with the use of the modified reverse-phase evaporation methods (mREV) optimal conditions to obtain liposomes to be used as drug carrier. For this purpose, the effect of temperature and pH on their properties was studied. The EPR spin marker of properties of amphiphilic drugs was used. To confirm the structure of the obtained liposome bilayers, the conformation and the interactions of the phospholipids in a membrane, the NMR spectroscopy and electron microscopy were applied.

Section snippets

Materials

l-α-Phosphatidylcholine dipalmitoyl (1,2-dihexadecanoyl-sn-glycerol-3-phosphocholine) 99% (DPPC), the spin marker 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl free radical (5-DOXYL) were purchased from Sigma Chem. Co., α,α,α-Tris-(hydroxymethyl)-methylamin 99.9% (TRIS) was purchased from Fluka, cholesterol (5-cholesten-3β-ol) 99+%, chloroform, dichloromethane and hydrochloric acid were purchased from POCH, Gliwice, Poland. D2O 99%, chloroform-d 99%, stab. with Ag, phosphoric

Transmission electron microscopy study

Transmission electron micrographs showed that the liposome vesicle, obtained by using the modified mREV method, were spherical in shape (Fig. 2) and in majority they were less than 100 nm in diameter before filtration. The tendency of the liposomes to aggregate was also observed. The method applied allowed us to obtain liposomes of diameter ≤100 nm and of regular spherical structure.

Electron paramagnetic resonance study

EPR is a useful technique for determining fluidity and the structural changes of the lipid bilayers of liposomes.

Conclusions

Spherical bilayer liposomes of diameter ≤100 nm were prepared with the application of the modified reverse-phase evaporation method. The use of more than 0.5 mol of cholesterol per 4 mol of DPPC for the preparation of liposomes does not significantly affect the fluidity of the obtained liposome membranes. The changes of parameters 2Amax, S and τ determined from EPR spectra were analyzed. It was found that liposome membranes prepared in acid environment were stiffer below transition temperature,

Acknowledgements

This work was supported by grant BW/ICh/TCh/03/04 from the University of Silesia. Authors thank Dr S. Duber from the Interdepartmental Laboratory of Structural Research, Faculty of Earth Science, University of Silesia, for micrographs.

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