Electron spin resonance study of phosphatidyl choline vesicles using 5-doxyl stearic acid

Colloids and Surfaces B: Biointerfaces 25 (2002) 225– 232 www.elsevier.com/locate/colsurfb

Electron spin resonance study of phosphatidyl choline vesicles using 5-doxyl stearic acid Namita Deo, P. Somasundaran * NSF IUCR Center for Ad6anced Studies in No6el Surfactants, Langmuir Center for Colloid and Interfaces, Columbia Uni6ersity, New York, NY 10027, USA Received 1 December 2000; accepted 13 November 2001

Abstract Harshness and skin irritation are related to surfactant– skin lipid interactions. Delipidization of the stratum corneum is caused by surfactant penetration into extracellualar lipid bilayers; disrupting the lipid microstructure and thereby impairing skin barrier properties. The mechanisms of interactions of sodium dodecyl sulfate (SDS) with lipid as a model membrane are investigated in this work by studying the vesicle to micelle structural transition, which occurs during such interactions of the membrane. The optical density of the phosphatidyl choline liposome was found to increase upon the addition of up to 2 mM SDS and then to decrease gradually on further increase in SDS concentration. Electron spin resonance (ESR) spectrum of 5-doxyl stearic acid (5-DSA) in liposome before and after interaction with SDS solutions at different concentrations showed the rotational correlation time of 5-DSA to increase markedly, possibly due to the penetration of 5-DSA into the lipid bilayers. SDS addition decreased the rotational correlation time of 5-DSA until it attained a constant value above a certain SDS concentration suggesting complete solubilization of liposome and formation of mixed micelles. The ESR spectrum of 5-DSA inside SDS micelle was also seen to be quite different from that obtained after complete solubilization of liposome and, this also indicates the formation of mixed micelles. © 2002 Published by Elsevier Science B.V. Keywords: 5-Doxyl stearic acid; Sodium dodecyl sulfate; Electron spin resonance

1. Introduction Biological membranes composed of regularly packed amphiphilic molecules with a polar head group and a hydrophobic tail are of fundamental importance in the biochemistry of living systems

* Corresponding author. Tel.: + 1-212-854-2926; fax: +1212-854-8362. E-mail address: [email protected] (P. Somasundaran).

as they provide suitable microenvironments for controlled transport of solutes. In an aqueous environment these molecules form bilayers where the packing of the constituent molecules is determined by the lipid head group –water interactions, the intramolecular interactions of the alkyl chains and the shape of the molecules. Due to the complexities of natural membranes phospholipid vesicles have been used as model systems to investigate the effect of different surfactants on the disruption of biomembranes.

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Considerable insight into the behavior of membranes has been obtained from electron spin resonance (ESR) studies. As the membrane is not itself paramagnetic, it is necessary to dope the membrane with a suitable paramagnetic probe molecule. Stable nitroxide radicals have been widely used as spin probes in studies of biological membranes and membrane mimetic systems [1– 3]. Spin probe and spin-labels are extensively used in investigation of micelle chemistry, as well as other organized molecular assemblies [4– 7]. ESR has been used to investigate the microenvironment of nitroxide spin probes in micelles and vesicles by measuring the nitrogen-coupling constant and ESR spectra line widths [8]. The coupling constant is affected by the local polarity of the nitroxide. A more polar environment gives larger values of coupling constant because of the greater electron density in nitrogen. The line widths are controlled by rotational and lateral diffusion of the spin probes, which in turn is affected by viscosity and temperature of the local environment [9,10]. Larger ordering parameters of X-doxyl stearic acids (X-DSAs) at the interfacial regions of micelles or vesicles compared with that of the hydrocarbon region indicates oriented XDSA with its polar head group at the interface. Broader line width results from the slower tumbling rate of the spin probe with a longer relaxation time, which can be controlled by changing the local viscosity around the nitroxide probe. In this paper, vesicle to micellar structural transition was studied in the presence of sodium dodecyl sulfate (SDS) by ESR technique using 5-doxyl stearic acid (5-DSA)as the spin probe molecule and phosphatidyl choline as the model liposome membrane.

2.1. Liposome preparation Unilamellar liposome was prepared by the method followed by Helenius [11]. Lipidic film was formed by rotary evaporation to remove the organic solvent in which lipid was dissolved. The dried lipid film was then dispersed in phosphate buffer and sonicated at 40 °C for 1 h. The resulting liposome was sized through a polycarbonate filter of 0.2 mm size. The phospholipid concentration in liposome suspensions studied was 1.0 mM.

2.2. Determination of particle size distribution and stability of liposome preparation Mean size and polydispersity of liposome were determined using a photon correlator spectrophotometer at 25 °C at a lecture angle of 90°. The polidispersity was less than 0.1 indicating the distribution to be homogenous. The particle size distribution after equilibration with phosphate buffer showed very little change with only slight increase in polydispersity index. The liposome preparation thus appeared to be stable in the absence of surfactants.

2.3. Turbidity measurements Turbidity measurements of 1.0 mM vesicle suspensions before and after interaction with SDS concentration were made at 25 °C with Shimadzu UV-240 spectrophotometer (u= 350 nm, cell length= 5 cm). After surfactant addition the absorbance measurements were taken after subsequent vigorous agitation for different intervals of time. The optical density values were corrected for dilution due to the addition of SDS solution.

2. Experimental 3. ESR measurement method SDS was obtained from Fluka and used as received. Phosphatidyl choline from egg lecithin was purchased from Sigma Chemical Corp. Anionic 5-doxyl-stearic acid (2-(3-carboxypropyl)4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy, free radical) was purchased from Aldrich chemical company. The buffer used was phosphate buffer at pH 7.0.

A Microw-Now instrument, Model-8300A, operating at a microwave frequency of 9.5 GHz was used for ESR measurements. The concentration of the probe used in all the studies was 5× 10 − 6 M. For ESR measurements, 5× 10 − 6 M of 5DSA in chloroform was added to 1 mM of phosphatidyl choline in chloroform and evaporated

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overnight. A lipidic film with the ESR probe was dispersed in the phosphate buffer and the ESR spectrum was measured after interaction with SDS for different intervals of time. In order to get a strong and reproducible signal, 100 mm micropipettes were used as the ESR cell. The ESR spectra of 5-DSA in phosphatidyl choline, in phosphate buffer and in water prior to interaction with SDS were also taken. For comparison purposes, ESR spectra of 5-DSA in the buffer were also taken after interaction with SDS solutions for different intervals. 4. Results and discussion Solubilization of 1 mM phosphatidyl choline liposome by SDS for different intervals is illustrated in Fig. 1. Interaction with 12 mM SDS for 15 min was found to cause a maximum solubilization of 35%. The solubilization increased to about 70% with 1 h of interaction, solubilization of PC liposome thus depending on both the surfactant concentration and the interaction time. Interestingly, at 1 mM of SDS, the optical density of liposome increased from 1.4 to 1.5 after 15 min of interaction. We attribute this to the increase in the size of the vesicle due to adsorption of SDS molecules.

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The ESR spectra of 5-DSA in phosphate buffer, with and without phosphatidyl choline, are shown in Fig. 2. The spectrum obtained in the presence of PC liposome is quite different from that of phosphate buffer alone, without the former anisotropy. 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 case of phosphate buffer. In the presence of the phosphatidyl choline molecule, the nitroxide apparently cannot rotate freely, suggesting its immobilization inside the phosphatidyl choline bilayer. The ESR spectra of 5-DSA in 1 mM of liposome before and after interaction with SDS for 15 min are given in Fig. 3. At 2 mM of SDS, the third peak becomes more an-isotropic indicating adsorption of SDS further hindered the rotation of probe molecule. However, at higher SDS concentrations the spectrum becomes more isotropic, indicating the disruption of bilayers. The degree of mobility as monitored by the spectral anisotropy is calculated in terms of rotational correlation time. The rotational correlation time of 5-DSA in the presence and the absence of 1 mM of liposome and after interaction with SDS were calculated using the following equation:

Fig. 1. Optical density decrease illustrating solubilization of PC liposome by SDS after conditioning for 15 and 60 min (n = 3).

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Fig. 2. ESR spectra of 5-DSA in buffer solution and 1 mM PC liposome.

Fig. 3. The ESR spectra of 5-DSA in 1 mM PC liposome before and on interaction with SDS for 15 min.

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~corr =6.73×10 − 10DH(0)

  I0

I−1

1/2

−1

n

where, DH(0) is the width in gauss of the central field spectrum line, and I0 and I − 1 represent peak-to-peak amplitudes of the central field and high field line, respectively. This equation is valid for the correlation time ~corr B3 × 10 − 9 s [7]. Fig. 4 illustrates the change in rotational correlation time of 5-DSA after interaction with SDS for 15 min. The rotational correlation time increased with SDS concentrations and reached plateau at around 4 mM of SDS. As indicated earlier, apparently in 2 mM of SDS, probe molecules begin to interact with SDS to form association complexes. Above 4 mM of SDS, the probe is proposed to be in a micellar environment hindering free rotation of the probe with the rotational correlation time reaching a high value. On further increase in SDS concentration the rotational correlation time remained more or less constant suggesting reaching micellar region. The change in rotational correlation time of 5-DSA in 1 mM of phosphatidyl choline liposome upon interaction with SDS for different intervals of time is shown in Fig. 5. In 2 mM of SDS solution, the rotational correlation time increased in 15 min from 6.5× 10 − 10 to 8×10 − 10 s, as mentioned earlier and this decrease in mobility is due to the adsorption of SDS on the liposome

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surface. Upon prolonged contact and at higher SDS concentrations, the rotational correlation time decreases gradually due to the penetration of SDS into the liposome interior structure causing its disruption. The change in rotational correlation time of 5-DSA in 1 mM of phosphatidyl choline upon interactions with SDS is shown in Fig. 6. In the presence of 2 mM of SDS an increase in correlation time was observed only after 15 min of interaction with no significant decrease upon prolonged contact. Thus there is no significant effect by 2 mM of SDS on solubilization of phosphatidyl choline liposome. This observation is in agreement with the results from the solubilization data (Fig. 1). At high SDS concentrations, the probe rotational time is lower due to the penetration of the bilayer by SDS molecules. The hyperfine coupling constant is sensitive to the polarity of the medium in which the radical resides [12–15], i.e. higher the polarity, higher is the (AN), because the high polarity favors the pseudoionic structure II:

Particularly high (AN) values are obtained in protic solvents that are hydrogen-bond donors.

Fig. 4. Rotational correlation time of 5-DSA as a function of SDS concentration (S.D. = 94%).

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Fig. 5. Rotational correlation time of 5-DSA in PC liposome after interaction with SDS for different intervals of time (S.D.= 92 – 5%).

Fig. 6. The rotational correlation time of 5-DSA in PC liposome after interaction with different SDS concentration for different intervals of time (S.D. = 93–5%).

The hyperfine splitting constant (AN) which relates to the polarity around the labeled position, is given by [12]: AN =

(A +2AÞ) 3

where, A is time-averaged electron-nuclear hyperfine tensor (parallel) and 2AÞ is time averaged electron-nuclear hyperfine tensor (perpendicular). The nitrogen hyperfine-coupling constant of 5DSA in PC liposome is illustrated in Fig. 7 and Table 1 as a function of SDS concentration. From

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Fig. 7. Hyperfine coupling constant of 5-DSA in PC liposome as a function of SDS concentration (S.D. = 95%).

Fig. 7 it is obvious that when 5-DSA is surrounded by the phospholipid molecules the AN value is quite low, at about 18.52. The lower AN value was observed because the 5-DSA molecule is surrounded by the hydrophobic phospholipid molecules in the absence of SDS. For 24 h of interaction with different amount of SDS the AN value increased with SDS concentration and remained constant above 6 mM SDS. This suggests complete solubilization of PC liposome and micelle formation. Again this result is consistent with the solubilization result (Fig. 1). In 10 mM SDS, the AN value decreased to 20.09 G, suggesting that the probe molecule is surrounded by SDS micelles. In the presence of PC liposome the AN value is reduced to 18.52 G, due to the probe being in the rigid hydrophobic lipid bilayer structure. It is also obvious from the data that the environment inside the lipid bilayer is more hydrophobic than that of SDS micelle. When the bilayer was treated with 10 mM of SDS for 24 h, the AN value increased from 18.52 to 19.41 G. The above results suggest that water molecule can penetrate into the SDS micelle. The mixed PC/SDS micellar interia is more hydrophobic than SDS micellar interia and the mixed micelle

structure is also apparently more rigid than those of SDS micelle, which hinders water penetration. The location of the probe molecule in the mixed micelle structure further suggests the hypothesis that solubilization of liposome is a micellization process.

5. Conclusions First of all, the EPR method can be used to understand the mechanisms of liposome solubilization by surfactants. The correlation times, the line shapes and the hyperfine coupling constants give the actual position of the nitroxide probe molecule and the polarity of the surrounding environment. Table 1

5-Doxyl stearic acid

AN(G)

Phosphate buffer 10 mM SDS PC liposome (1 mM) PC liposome+10 mM SDS

20.85 20.09 18.52 19.41

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From the shape of the EPR spectra and correlation time, it has been found out that the liposome structure is more rigid than that of mixed micelle. The core of the mixed micelle was found to be more hydrophobic than that of SDS micelle, possibly due to the presence of long chain phosphatidyl choline molecule. This investigation gives further experimental evidence that the liposome solubilization is a micellization processes.

Acknowledgements Authors acknowledge the support of the National Science Foundation (Grant Number 9804618. Industrial/University cooperation research center for adsorption studies in novel surfactants). The financial support of Unilever Research Laboratory, US, is gratefully acknowledged.

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