Elsevier

Biophysical Chemistry

Volume 114, Issue 1, 1 April 2005, Pages 53-61
Biophysical Chemistry

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

https://doi.org/10.1016/j.bpc.2004.11.006Get rights and content

Abstract

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (τc) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-α phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 Å were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 °C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-α phosphatidylcholine liposomes above the phase-transition temperature (23.9 °C) did not change microviscosity. At 10 °C, an increase of propofol concentration from 0 to 1.0×10−2 M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10−2 M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 Å) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10−2 M was identified as the saturation limit of propofol in dimyristoyl-l-α phosphatidylcholine liposomes.

Introduction

Many physiological functions associated with biological membranes are known to be profoundly affected by the dynamic properties of the phospholipid matrix. It is believed that the changes in such physical properties of bilayers as phase transitions temperature, lateral phase separations, local dielectric properties, and rotational diffusion modulate the biological activity of many membrane-associated receptors, transport proteins, and enzymes. One of the most widely techniques available to study these properties is fluorescence spectroscopy. It has been shown that environmental fluorescent probes of varying structural complexity report on most of these dynamic phenomena, both in natural as well as in model membrane and micellar systems [1]. One important goal of the work has consisted in determining the apparent or effective membrane microviscosity [2]. However, many membrane microviscosity values measured with different fluorophores depend often on the molecular properties of the probe [3]. Generally, when the fluorophore requires a large volume for motion, whether it be for excited-state formation or nonradiative deexcitation, a wide range of microviscosity values is obtained. It has been suggested that the large probe may cause membrane bilayer distortion [2].

Fluorescent probes report, not only on bilayer microviscosities and phase behaviour, but they also provide information on the local dielectric properties of their environment [4], [5]. Usually, the fluorescence emission characteristics can be related to the dielectric constant of a solvent in a standard solvent scale.

The bilayer microviscosity can be studied by use of luminescence techniques such as steady-state or dynamic depolarization and diffusion-dependent inter- and intramolecular excimer formation[1], [5]. Unfortunately, these techniques have limitations. For example, the concentrations required for effective intermolecular pyrene excimer formation in lipid bilayer often lead to probe aggregation because of nonuniform probe distribution [5]. Microviscosities obtained from intramolecular excimer formation are more dependent on the structural characteristics of the probe rather than on the environment itself [3]. To calculate the microviscosities from steady-state depolarization, we must assume that probe motion in the bilayer is isotropic. This assumption may not be valid for most nonpolar fluorophores [6]. Electron spin resonance (ESR) technique is also currently used to investigate the microenvironment of nitroxide spin probes in membrane liposome by measuring the nitrogen-coupling constant and ESR spectra line width [7]. The line widths are governed by rotational and lateral diffusion of the spin probes, which in turn is affected by viscosity (η) and temperature of the local environment [7]. Thus, the relative anisotropy observed in an ESR spectrum is directly related to the rotational mobility of the probe, a term that can be correlated with the probe's microviscosity [8]. This correlation allows measuring the local microviscosity at different depths inside a liposome membrane. Chandar et al. have determined the microviscosity within sodium dodecyl sulphate (SDS) micelles by comparing the ESR nitroxide spectra in SDS micelles to the spectra of the probe in ethanol–glycerol mixture at known viscosity. They suggest that the microviscosity can effectively be defined as the homogenous solution viscosity, which results in the same spectrum as that in the microenvironment [8].

In this work, we have established standard curves of effective microviscosities by calibration of the ESR spectra of three n-doxyl stearic acids (n-DSA; n=5, 12, 16) probes in glycerol–ethanol mixtures of known viscosities. These curves allow us to quantify the effective microviscosity at different depths inside liposomes by measuring the order parameter (S) and the correlation time (τc) on n-DSA ESR spectra. Local microviscosity of the probe environment in the SDS and 2-propanol were calculated using our method. Our results proved to be consistent with values obtained by other techniques and reported in previous studies. Hence, we have applied our method to samples of biological interest such as 2,6-diisopropylphenol (propofol; PPF) solubilized in liposome solutions.

Several drugs can be solubilized inside the membrane bilayer. Some of them induce a decrease in membrane microviscosity and order. Propofol (PPF) (Fig. 1) is a nonbarbiturate anaesthetic agent commonly used for induction and maintenance of general anaesthesia in clinical practice [9]. It is characterized by a unique phenolic structure not present in any other conventional anaesthetic. Due to its lipophilic property, PPF is presumed to penetrate into and interact with membrane lipids, inducing changes in membrane fluidity [10]. Therefore, quantifying the change in viscosity induced by PPF could be important to better understand the molecular mechanism of its anaesthetic action. It has been previously shown by fluorescence and ESR that PPF promotes the formation of fluid phase domains in membranes [11] and decreases the gel-to-fluid state transition temperature [12]. In this study, we have quantified the membrane microviscosity change induced by PPF incorporation into dimyristoyl-l-α phosphatidylcholine (DMPC) by measuring ESR S parameter and correlation time of n-DSA. A fluidity effect of PPF in DMPC liposomes below the phase transition temperature has been evidenced. The limit of PPF incorporation in DMPC has also been determined. The PPF fluidity effect has been qualitatively confirmed by using Merocyanine 540 (MC540) as another lipid packing probe.

Section snippets

Chemicals

Absolute ethanol and chloroform from Merck (Germany), glycerol, dipalmitoyl phosphatidyl choline (DPPC) and dimyristoyl phosphatidyl choline (DMPC) from Sigma (USA), stearic spin probes; 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy (5-DSA), 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxy (12-DSA) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy (16-DSA) from Aldrich (USA) were with analytical grade and used without further purification.

Liposome preparation

Spin-labeled

Standard viscosity plots

Spin labels were used as viscosity probes because their ESR spectra are very sensitive to polarity and mobility [21]. Fig. 3 shows the spectrum of 16-DSA in ethanol (Fig. 3a), in DMPC liposomes (Fig. 3b), and in glycerol–ethanol mixture (Fig. 3c) at 25 °C. Spin-labeled stearic acids n-DSA were solubilized in glycerol–ethanol mixture of increasing viscosity. In each case, S and τc were calculated from the measured ESR spectra. For strongly immobilized probes corresponding to η above 70, 80, and

Discussion

The ESR spectra of 16-DSA in ethanol, with and without glycerol, and in DMPC liposome are shown in Fig. 3. The spectrum obtained in the presence of DMPC liposome is quite different from that of ethanol alone. When the nitroxide molecule can rotate rapidly and randomly, then all the anisotropy averages out and a sharp three-line spectrum is obtained, as in the case of ethanol (Fig. 3a). In the presence of the DMPC liposomes, the nitroxide apparently cannot rotate freely, suggesting its

Acknowledgment

We would like to thank Doctor Michel Tricot for his kind help in operating viscosimeter.

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