The influence of temperature and pH on the structure of liposomes formed from DMPC

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Abstract

The EPR and NMR spectroscopy was used to determine the structure and physicochemical properties of liposomes prepared from dl-α-phosphatidylcholine dimyristoyl (1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine) (DMPC) by the modified reverse-phase evaporation (mREV). EPR study was carried out in the temperature range from 284 to 310 K, i.e. below and above the phase transition temperature Tc of DMPC. On the basis of EPR spectra of spin marker 2-(carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (5-DOXYL) incorporated into liposome, the parameters S (empirical order parameter), 2Amax (line widths) and τ (rotational correlation time) were determined. Temperature and pH dependent structural changes of liposome were also described. 31P NMR study of the liposome in the temperature range from 340 to 350 K allowed to suggest that the single-bilayered liposome were obtained.

Introduction

Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent in their interior. The size of liposome ranges from 20 nm up to several micrometers and they may be composed of one or several concentric membranes, with a thickness of about 4 nm [1]. Liposomes have unique properties due to the amphiphilic character of the lipids, which make them suitable for drug delivery [2], [3], [4], [5]. Liposomes can be classified into three groups: multilamellar vesicles, small unilamellar vesicles and large unilamellar vesicles. When liposomes are prepared by hydration of a dried lipid mixture, they spontaneously form relatively large particles (about 1 μm in size) containing multiple lipid bilayers alternating with aqueous interphases. Such liposomes are called multilamellar vesicles (MLV's). Other procedures such as prolonged exposure to ultrasound or pressure-driven filtration through filters with small pore size, cause MLV's to form small unilamellar vesicles (SUV's, diameter<100 nm). Certain procedures lead to the formation of large unilamellar vesicles (LUV's), which are normally 200–800 nm in diameter and consist of a single phospholipid bilayer with an aqueous phase inside. In some cases, liposomes are determined by the method of preparation [6]. Liposomes interact with the cell surface by two main mechanisms: adsorption and endocytosis. Liposome can be adsorbed to a cell surface directly or by specific interaction with a cell-surface receptor. Liposome systems have become a popular drug delivery platform for several reasons. Liposomes prepared from natural lipids taken from living systems are nontoxic and biodegradable. The so-called controlled drug delivery systems are intended to maintain continuously efficacious drug concentrations over longer time periods. Furthermore, liposomes are unique because they provide an environment that can enclose both hydrophilic and hydrophobic molecules [7].

Hydrophobic molecules are intercalated into the bilayer membrane, and hydrophilic molecules can be entrapped in the internal aqueous region [8], [9]. The structure and dynamics of membrane lipids and their relationship to membrane function are important problems in membrane biophysics.

Our previous studies on DPPC liposome confirm that the modified reverse-phase evaporation method (mREV) allows to obtain small liposomes of diameter of 100 nm [10]. In the present work, we aimed to analyze liposome formed with dl-α-phosphatidylcholine dimyristoyl (1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine) (DMPC). The phase transition temperature (Tc) of DMPC (∼297 K) is lower than that of DPPC (∼314 K) [11]. The choice of the type of phospholipids is important for the application of liposomes as drug carriers [12]. It has been asserted that the maximum penetration of phospholipids membranes takes place around Tc [13]. It is connected with the release of a drug transported by a liposome [14]. Therefore, it is essential to know the physicochemical properties of liposomes obtained by the use of mREV method, formed with phospholipids different than DPPC.

To determine the dynamics of a membrane built of DMPC and to assess the effect of temperature, cholesterol content and pH on these liposomes, the EPR studies with the use of spin marker 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (5-DOXYL), which has the properties of the amphiphilic drug, have been carried out. To confirm the single bilayer structure of obtained liposomes the NMR spectroscopy has been used. The number of the bilayers in the phospholipids vesicles is important since it determines the kinetics of release of the liposome transported drug into the circulatory systems.

Section snippets

Materials

dl-α-Phosphatidylcholine dimyristoyl (1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine) 99% (DMPC), the spin marker 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl free radical (5-DOXYL) from Sigma Chem. Co., praseodimium (III) nitrate hexahydrate 99.9% from Aldrich, α,α,α-Tris-(hydroxymethyl)-methylamine 99.9% (TRIS) from Fluka, cholesterol (5-cholesten-3β-ol) 99+% (Chol), chloroform, dichloromethane and hydrochloric acid from POCH, Gliwice, Poland. D2O 99%, chloroform-d 99%,

Electron paramagnetic resonance study

Electron paramagnetic resonance spectroscopy of spin-labelled lipid molecules is widely used for probing the dynamic structure of lipid bilayers and biological membranes [29], [30]. Since the mobility of spin-labelled molecules in fluid lipid membranes is found to be close to the optimum range of motional sensitivity of nitroxide, the utility of the EPR spectroscopy arises. In principle, from the spin-label spectra the information about the amplitude and rate of motion of the spin-label group

Conclusions

The above study confirms that the modified reverse-phase evaporation method (mREV) allows to obtain small liposome membranes with a single phospholipid bilayer. From the EPR spectra the value of parameters S, 2Amax and τ, characteristic for the structural and dynamic changes of the investigated liposome membranes, were determined. It is ascertained that an addition of cholesterol causes an increase of stiffening of phospholipid acyl chains above Tc while below Tc, in the gel state, cholesterol

Acknowledgements

The authors wish to thank Mr Karl-Heinz Weiss from the Bruker Inc. for his helpful technical advices. This work was supported by the KBN grant 3 T09A 064 28.

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