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Electron Paramagnetic Resonance Study of the Structure of Lipid Bilayers in the Presence of Sodium Dodecyl Sulfate
Journal of Colloid and Interface Science 256, 100–105 (2002) doi:10.1006/jcis.2002.8470
Electron Paramagnetic Resonance Study of the Structure of Lipid Bilayers in the Presence of Sodium Dodecyl Sulfate Namita Deo,∗ P. Somasundaran,∗,1 K. Subramanyan,† and K. P. Ananthapadmanabhan† ∗ NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces, Columbia University, New York, New York 10027; and †Unilever Research USA, 45 River Road, Edgewater, New Jersey 07020 Received August 7, 2001; accepted May 9, 2002
the constituent molecules is determined by the lipid head group– water interactions, the inter- and intramolecular interactions of the alkyl chains, and the shape of the molecules. In human skin, lipid bilayers are known to provide the water barrier function of the outermost layers of skin. Surfactants are commonly used as solubilizing agents in the isolation, purification, and reconstitution or crystallization of membrane proteins. For the efficient use of surfactants, it is important to have an accurate knowledge of how they interact with integral membrane proteins and membrane lipid, under both solubilizing and nonsolubilizing conditions. Harshness (skin irritation) of personal cleansing products is related to surfactant interactions with proteins and lipids in the upper layers of skin (stratum corneum). Cleanser surfactants can damage stratum corneum lipids either by their solubilzation in surfactant micelles or by fluidization of the lipid bilayers by surfactant penetration. The sublytic action of surfactants on the phospholipid bilayers leads to the incorporation of surfactant molecules into the bilayer structures as governed by the equilibrium between bilayers and the aqueous phase (1). This incorporation involves complex perturbations in the physical properties of vesicle membranes, which depend on the type and amount of the surfactants partitioned (2–4). Several spectroscopic tools have been used in the past to investigate the microstructure of biological membranes (5–7). Electron paramagnetic resonance (EPR) has been used to investigate the microenvironment in micelles and vesicles by measuring the nitrogen-coupling constant and EPR spectral linewidths (8) of nitroxide spin probes. The coupling constant is affected by the local polarity in which the nitroxide moiety resides. A more polar environment gives larger coupling constants because of the greater electron density on the nitrogen. The linewidths are controlled by rotational and lateral diffusion of the spin probes, which in turn is affected by the viscosity and temperature of the local environment (9, 10). Larger ordering parameters of doxyl stearic acids at the interfacial regions of micelles or vesicles compared with that of the hydrocarbon region, for example, indicates oriented DSA with its polar head group at the interface. Broader linewidth results from the slower tumbling of the spin probe with a longer relaxation time, which can be controlled by changing the local viscosity around the nitroxide probe.
Harshness (skin irritation) of personal cleansing products is related to surfactant interactions with proteins and lipids in the upper layers of skin (stratum corneum). Cleanser surfactants can damage stratum corneum lipids either by their solubilzation in surfactant micelles or by fluidization of the lipid bilayers by surfactant penetration. The mechanism of interaction of sodium dodecyl sulfate (SDS) with a model phospholipid membrane is investigated in this work by studying the vesicle-to-micelle structural transition, which occurs due to the interaction of a phospholipid bilayer membrane with SDS. It was observed that the optical density as well as the hydrodynamic diameter increased upon the addition of SDS up to 2 mM due to surfactant adsorption on the liposomes and then decreased gradually upon further addition of SDS due to transition of the vesicle to a micelle. Two inflection points were observed on both the surface tension as well as SDS monomer activity plotted vs solubilization, corresponding to the onset and complete solubilization of the liposome, respectively. The electron paramagnetic resonance (EPR) spectrum of 5-doxyl stearic acid (5-DSA), a lipid probe molecule, indicates immobilization of the probe molecule in the lipid bilayer in SDS-free solution. The mobility of 5-doxyl molecules in the liposome changes slightly with SDS concentration up to 2 mM, supporting the hypothesis that SDS molecules adsorb on the liposome without any structural disruption. Upon further addition of SDS, the mobility of the lipid probe increases sharply, indicating the disruption of the bilayer, ultimately resulting in complete solubilization of the liposome into a mixed micelle with SDS. The hyperfine coupling constant value of 5-doxyl molecules in a mixed micelle is observed to be higher than that in a pure SDS micelle, suggesting the core of the mixed micelle is more hydrophobic than that of the SDS micelle. C 2002 Elsevier Science (USA)
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 as they provide suitable barriers and microenvironments for controlled transport of solutes. In aqueous environments these molecules form bilayers where the packing of
1
To whom correspondence should be addressed.
0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
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100
STRUCTURE OF LIPID BILAYERS IN THE PRESENCE OF SDS
In the present study, the mechanisms of interaction of surfactants with bilayer lipids have been investigated using sodium dodecyl sulfate as the model surfactant and a phospholipid liposome as a model membrane. Specifically, the vesicle-to-micelle transition that occurs due to interaction of SDS with the membrane has been investigated.
EXPERIMENTAL
Materials Sodium dodecyl sulfate was obtained from Fluka and used as received. Phophatidic acid from egg yolk lecithin and phophatidyl choline also from egg lecithin were purchased from Sigma. The anionic lipid probe, 5-doxyl stearic acid (2(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy, free radical) was purchased from Aldrich. The buffer used was phosphate buffer at pH 7.0. 5-Doxyl stearic acid (DSA) molecule was used as the EPR spin probe and phosphatidic acid (PA) and phosphatidyl choline (PC) were used as ingredients for the preparation of the model membrane. Liposome Preparation Unilamellar liposomes of desired size (about 120 nm) were prepared by the method described in our previous paper (11). A lipidic mixture film (PC : PA composition of 1 : 1 molar ratio in chloroform) was formed by removing the organic solvent by rotatory evaporation. The lipid film was dispersed in phosphate buffer and sonicated in a bath sonication unit at 40◦ C for 1 h. The resulting liposome was sized through a polycarbonate filter of 0.2-µm size. A 1 mM of mixed liposome was used throughout this study. Determination of Particle Size Distribution and Stability of Liposome Preparation Mean size and polydispersity of liposome were determined by photon correlation spectroscopy. Samples were adjusted to the required concentration range with phosphate buffer, and the measurements were taken at 25◦ C at a lecture angle. The particle size distribution of liposome suspensions after 24 h of preparation varied a little and the polydispersity is less than 0.1, indicating that the distribution is homogenous. Likewise, the particle size distribution of the liposomes after the addition of the phosphate buffer and equilibration for 24 h at 25◦ C showed in all those cases a slight increase in the polydispersity index, from 0.1 to 0.15, and was stable in the absence of surfactants. The particle size was also determined after 1 h of interaction with SDS at different concentrations. Absorbance Measurements The absorbance measurements were made at 25◦ C using a Shimadzu UV-240 spectrophotometer (λ = 350 nm, cell length = 4 cm). The absorbance change of each liposome sus-
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pension was monitored before and after 1 h of interaction with SDS at different concentrations (1–12 mM). Surface Tension Measurements Surface tension of SDS in phosphate buffer was measured by the drop volume method (12) before and after 1 h of interaction with liposomes (1.0 mM) at 25◦ C and the critical micelle concentration, CMC, of the SDS in the absence and in the presence of liposomes were determined from the plot of surface tension versus concentration. SDS Concentration in the Bilayer The SDS concentration in the lipid bilayer was determined after interaction of the bilayer with SDS solutions at different concentrations. The mixtures were filtered through 3000 molecular weight cutoff sizes filters to separate the SDS monomer from vesicles and micelles in the bulk. The monomer concentration of the filtrate was analyzed by the two-phase titration method (13). The SDS concentration in the bilayer was estimated by subtracting the monomer concentration in the filtrate and the total initial concentration. EPR Measurements A Microw-Now instrument, Model-8300A, was used for EPR measurements at a microwave frequency of 9.5 GHz. The concentration of the lipid probe used in all the studies was 5 × 10−6 M. The 5 × 10−6 M 5-doxyl stearic acid in chloroform was added to 1 mM 1 : 1 mixtures of phosphatidic acid and phosphatidyl choline in chloroform and the liposome was prepared by the method described above. Then 5-ml samples of 1 mM liposome were taken in a 10-ml vial, and SDS at desired concentrations was added. After the mixtures interacted for different intervals of time, the EPR spectrum was recorded. EPR spectra of 5-doxyl stearic acid in liposomes and in phosphate buffer without SDS were also taken. For comparison purposes, EPR spectra of 5doxyl stearic acid in buffer were also taken after interaction with SDS for different intervals of time. The rotational correlation times and hyperfine splitting constants were analyzed from the EPR spectrum of 5-DSA under various conditions. RESULTS AND DISCUSSION
Figure 1 shows the change in effective hydrodynamic diameter as well as the optical density of mixed liposome after 1 h of interaction with SDS concentrations. With 1 mM SDS, both the optical density and the hydrodynamic diameter of the liposome increased marginally, which we attribute to the adsorption of SDS on the surface of the liposome. As the SDS concentration increased, the optical density and effective hydrodynamic diameter decreased measurably from 1.2 and 120 nm to 0.05 and 23 nm, respectively, at 10 mM of SDS, indicating the disruption of the vesicles. On further addition of SDS, no change in
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the above parameters was observed, suggesting complete solubilization of liposome. Two different sizes of liposome particles were observed above 1 mM of SDS concentration. As the SDS concentration increased, the larger size fraction decreased and the smaller size fraction increased along the solubilization curve. The hydrodynamic diameter is the average size of both the small and the large size fraction of liposomes. To understand the adsorption behavior of SDS on liposomes, SDS monomer and surface tension measurements were carried out after 1 h of interaction with 1 mM liposome and the results are given in Fig. 2. In the absence of liposome, the critical micelle concentration, CMC, of SDS in the phosphate buffer was determined to be 2 mM. The presence of liposome shifted the CMC of SDS toward higher concentration, as is evident from the surface tension curve. Two inflection points were observed on the surface tension curve for SDS in the presence of 1 mM liposome. The first inflection point is proposed to correspond to the saturation of the bilayer liposome and the onset of the solubilization process. The second inflection point indicates the completion of the solubilization process. After the second in-
flection point the surface tension remains constant, suggesting that the CMC of the mixed system has been reached. The higher CMC value for SDS in the presence of liposome supports the hypothesis that the surfactant is adsorbing onto the liposome surface so that the actual monomer concentration available in the solution for micellization is less than the added amount. This hypothesis is in agreement with the results obtained from the optical density and light-scattering studies (Fig. 1). EPR spectroscopy provides the following structural information related to liposome solubilization: solubilization efficiency, polarity of the probe environment, microviscosity of the probe environment, critical concentration of transition from the vesicle to the mixed micelles, and average aggregate size. The amphiphilic paramagnetic molecule 5-doxyl stearic acid, which comprises a long fatty acid chain and two polar groups (carboxyl and nitroxyl) near the molecular ends, was used as the spin probe to monitor the structural changes of liposome during interaction with SDS. The EPR spectrum of the 5-doxyl nitroxide probe in solution is represented by a sharp three peak signal; the spectral lineshape reports the micropolarity and microviscosity of the probe environment. The EPR spectra of 5-doxyl stearic acid in phosphate buffer in the presence and absence of liposome are shown in Fig. 3. It can be seen that all the peaks are almost completely suppressed in the presence of the liposome. When nitroxide molecules rotate rapidly and randomly, then all the anisotropy averages out and a sharp three-line spectrum is obtained, as in the phosphate buffer. In the presence of liposome, the nitroxide molecule cannot rotate freely because of its localization inside the phospholipid bilayer. The EPR spectra of 5-doxyl stearic acid in 1 mM liposome before and after interaction with SDS at different concentrations for 1 h are illustrated in Fig. 4. At 2 mM SDS no change in the spectrum was observed (figure is not shown). However, at higher SDS concentrations the spectrum becomes progressively more isotropic, and finally at 10 mM SDS a sharp three-line isotropic spectrum was observed. This spectrum is similar to
FIG. 2. The change in surface tension as well as monomer concentration of SDS after one-hour interactions with 1 mM of liposome.
FIG. 3. ESR spectra of 5-doxyl stearic acid in phosphate buffer and in presence of 1 mM of liposome.
FIG. 1. Change in optical density as well as effective hydrodynamic diameter of 1 mM of liposome after one-hour interaction with SDS.
STRUCTURE OF LIPID BILAYERS IN THE PRESENCE OF SDS
FIG. 4.
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EPR spectra of 5-doxyl stearic acid in 1 mM of liposome before and upon interaction with SDS for 1 h.
that of the probe molecules in pure SDS micelles (10 mM SDS), indicating complete disruption of the vesicles and formation of smaller mixed micelles. The rotational correlation time of 5-doxyl stearic acid in the presence and in the absence of 1 mM liposome and after interaction with SDS at various concentrations were calculated from the spectra using the following equation: τcorr = (6.73 × 10−10 )H(0) (I0 /I−1 )1/2 − 1 , where H(0) is the width in gauss of the central field spectrum line and I0 and I−1 represent the peak-to-peak amplitudes of the central field and high field line, respectively. This equation is valid provided the correlation time τcorr < 3 × 10−9 s. Figure 5 illustrates the change in rotational correlation time of 5-doxyl stearic acid in phosphate buffer and 1 mM liposome due
FIG. 5. Change in rotational correlation time of 5-doxyl stearic acid before and after 1 h interactions with SDS.
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to interaction with SDS for 1 h. It is evident that the rotational correlation time of 5-doxyl stearic acid in phosphate buffer increases with the addition of SDS, reaching a plateau around 4 mM SDS. Apparently at 2 mM SDS, the probe molecule begins to interact with SDS to form an association complex. Above 4 mM SDS, the probe molecules are proposed to be inside the micelle and hence the rotation of the probe molecule is hindered, causing an increase in the rotational correlation time. Above 4 mM SDS the rotational correlation time remained constant, suggesting that the CMC of SDS is reached. In contrast to the above, the rotational correlation time of 5-doxyl stearic acid in 1 mM liposome increased first with SDS up to a concentration of 2 mM and then gradually decreased. This decrease in mobility at low concentration is consistent with the earlier observations of increased optical density and hydrodynamic radius in this range, attributed to the adsorption of SDS on the vesicle surface, which apparently further hinders the rotation of the probe molecules. At higher SDS concentration the rotational correlation time decreased gradually, supporting the hypothesis that the liposomes are being gradually solubilized under these conditions. Increase in the SDS concentration above 10 mM caused no further decrease in rotational correlation time, indicating that complete solubilization of liposome and mixed micelle formation have already occurred. It is known that the nitrogen hyperfine-coupling constant of the nitroxide radical is highly sensitive to the polarity of the medium in which the spin system is inserted (14–17). In particular, the higher the polarity, the higher the (AN ) value. The hyperfine splitting constant (AN ), which relates to the polarity around the labeled position, is given by (18) AN = (A + 2A⊥)/3, where A is the time-averaged electron-nuclear hyperfine tensor (parallel) and 2A⊥ is the time-averaged electron-nuclear hyperfine tensor (perpendicular). The change in the hyperfine splitting constant of 5-doxyl stearic acids due to 1-h interactions with SDS is illustrated in
Fig. 6. The hyperfine splitting constant of 5-doxyl molecules in the absence of liposome decreases with increase in SDS concentration, reaching a plateau at 4 mM SDS. The initial decrease in AN value is attributed to the onset of the micellization process. Once the CMC of SDS is reached, AN remains constant, indicating that the probe molecules are solubilized in the surfactant micelle. In contrast to this, in the presence of liposomes, the AN value of 5-doxyl stearic acid molecules is lower in the beginning due to a more nonpolar environment (19, 20) in the vesicle membrane. This value increases with SDS concentration, indicating the transfer of probe molecule from a nonpolar environment to a relatively polar environment as the vesicle is disrupted. This change in polarity of the environment may be due to the difference in packing in a compact liposome vs that in loosely packed mixed micelles. It is also evident that the AN value of the probe molecule is lower in this mixed micelle than in SDS micelle. This is probably because these mixed micelles contain the longchain phosphatidic acid and phosphatidyl choline, which make the core of the micelle relatively more hydrophobic and bigger, which was also suggested by the light-scattering results. All the above evidence clearly suggests that liposome solubilization by surfactants occurs via a micellization process. CONCLUSIONS
Interactions of mixed liposome with sodium dodecyl sulfate were monitored using light scattering, surface tension, and EPR spectroscopy. The above liposome solubilization was clearly shown to be a process involving first adsorption of sodium dodecyl sulfate on liposome and then dissolution of liposome to form mixed micelles. The CMC increase for SDS upon interacting with liposome shows utilization of SDS for the solubilization of liposome. EPR spectroscopy further gave direct evidence for the disruption of the bilayer by SDS. The change in the rotational correlation time of a lipid probe incorporated into the liposome bilayers showed that solubilization of the above liposome is a micellization process. It was also evident from the hyperfine splitting constant value that the mixed micelles formed between the surfactant and the phospholipids have higher hydrophobicity compared to that pure SDS micelle due to the presence of longchain phospholipids in the mixed micelle. The EPR study gives direct evidence that liposome solubilization is a micellization process. ACKNOWLEDGMENTS Authors acknowledge the support of the National Science Foundation (Grant 9804618, Industrial/University cooperation research center for adsorption studies in novel surfactants). The financial support of Unilever Research Laboratory, U. S. is gratefully acknowledged.
REFERENCES FIG. 6. Change in hyperfine splitting constant value of 5-doxyl stearic acid after 1 h interactions with SDS.
1. Small, D. M., Bourg´es, M., and Dervichia, D. G., Nature 211, 816 (1966). 2. Small, D. M., in “The Bile Acids” ( P. P. Nair and D. Kritchevsky, Eds.), Vol. 1, p. 249. Plenum Press, New York, 1971.
STRUCTURE OF LIPID BILAYERS IN THE PRESENCE OF SDS 3. Lichtenberg, D., Robson, R. J., and Dennis, E. A., Biochim. Biophys. Acta 737, 285 (1983). 4. Helenius, A., and Simons, K., Biochim. Biophys. Acta 415, 29 (1975). 5. Ribosa, I., Garcia, M. T., Para, J. L., del la Maza, A., et al., Int. J. Cosmetic Sci. 14, 131 (1992). 6. Schubert, R., Beyer, K., Wolburg, H., and Schmidt, K. H., Biochemistry 25, 5263 (1986). 7. Bayerl, T., Klose, G., Blanck, J., and Ruckpaul, K., Biochim. Biophys. Acta 858, 285 (1986). 8. Bratt, J. P., and Kevan, L. J., Phys. Chem. 977, 7371 (1993). 9. Baglioni, P., Rivera-Minten, E., Dei, L., and Ferroni, E., J. Phys. Chem. 94, 8218 (1990).
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
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Eirovitch, E., Nayeem, A., and Freed, J. H., J. Phys. Chem. 88, 3454 (1984). Deo, N., and Somasundaran, P., Colloids Surf. A 186, 33 (2001). Dennis, E. A., Arch. Biochem. Biophys. 165, 764 (1974). Li, Z., and Rosen, M. J., Anal. Chem. 53, 1516 (1981). Janzen, E. G., Top. Stereochem. 6, 117 (1971). Stout, G., and Engberts, J. B. F. N., Org. Chem. 39, 3800 (1974). Knauer, B .R., and Napier, J. J., J. Am. Chem. Soc. 98, 4395 (1976). Abe, T., Tero-Kubota, S., and Ikegami, Y., J. Phys. Chem. 86, 1358 (1982). Yamagata, Y., and Senna, M., Langmuir 16, 6136 (2000). Lassmann, G., Ebert, B., Kuzetsov, A. N., and Damerau, W., Biochim. Biophys. Acta 310, 298 (1973). 20. Shirley, S., Polnaszek, C. F., and Smith, I. C. P., Biochim. Biophys. Acta 515, 375 (1978).
Electron Paramagnetic Resonance Study of the Structure of Lipid Bilayers in the Presence of Sodium Dodecyl Sulfate Namita Deo,∗ P. Somasundaran,∗,1 K. Subramanyan,† and K. P. Ananthapadmanabhan† ∗ NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces, Columbia University, New York, New York 10027; and †Unilever Research USA, 45 River Road, Edgewater, New Jersey 07020 Received August 7, 2001; accepted May 9, 2002
the constituent molecules is determined by the lipid head group– water interactions, the inter- and intramolecular interactions of the alkyl chains, and the shape of the molecules. In human skin, lipid bilayers are known to provide the water barrier function of the outermost layers of skin. Surfactants are commonly used as solubilizing agents in the isolation, purification, and reconstitution or crystallization of membrane proteins. For the efficient use of surfactants, it is important to have an accurate knowledge of how they interact with integral membrane proteins and membrane lipid, under both solubilizing and nonsolubilizing conditions. Harshness (skin irritation) of personal cleansing products is related to surfactant interactions with proteins and lipids in the upper layers of skin (stratum corneum). Cleanser surfactants can damage stratum corneum lipids either by their solubilzation in surfactant micelles or by fluidization of the lipid bilayers by surfactant penetration. The sublytic action of surfactants on the phospholipid bilayers leads to the incorporation of surfactant molecules into the bilayer structures as governed by the equilibrium between bilayers and the aqueous phase (1). This incorporation involves complex perturbations in the physical properties of vesicle membranes, which depend on the type and amount of the surfactants partitioned (2–4). Several spectroscopic tools have been used in the past to investigate the microstructure of biological membranes (5–7). Electron paramagnetic resonance (EPR) has been used to investigate the microenvironment in micelles and vesicles by measuring the nitrogen-coupling constant and EPR spectral linewidths (8) of nitroxide spin probes. The coupling constant is affected by the local polarity in which the nitroxide moiety resides. A more polar environment gives larger coupling constants because of the greater electron density on the nitrogen. The linewidths are controlled by rotational and lateral diffusion of the spin probes, which in turn is affected by the viscosity and temperature of the local environment (9, 10). Larger ordering parameters of doxyl stearic acids at the interfacial regions of micelles or vesicles compared with that of the hydrocarbon region, for example, indicates oriented DSA with its polar head group at the interface. Broader linewidth results from the slower tumbling of the spin probe with a longer relaxation time, which can be controlled by changing the local viscosity around the nitroxide probe.
Harshness (skin irritation) of personal cleansing products is related to surfactant interactions with proteins and lipids in the upper layers of skin (stratum corneum). Cleanser surfactants can damage stratum corneum lipids either by their solubilzation in surfactant micelles or by fluidization of the lipid bilayers by surfactant penetration. The mechanism of interaction of sodium dodecyl sulfate (SDS) with a model phospholipid membrane is investigated in this work by studying the vesicle-to-micelle structural transition, which occurs due to the interaction of a phospholipid bilayer membrane with SDS. It was observed that the optical density as well as the hydrodynamic diameter increased upon the addition of SDS up to 2 mM due to surfactant adsorption on the liposomes and then decreased gradually upon further addition of SDS due to transition of the vesicle to a micelle. Two inflection points were observed on both the surface tension as well as SDS monomer activity plotted vs solubilization, corresponding to the onset and complete solubilization of the liposome, respectively. The electron paramagnetic resonance (EPR) spectrum of 5-doxyl stearic acid (5-DSA), a lipid probe molecule, indicates immobilization of the probe molecule in the lipid bilayer in SDS-free solution. The mobility of 5-doxyl molecules in the liposome changes slightly with SDS concentration up to 2 mM, supporting the hypothesis that SDS molecules adsorb on the liposome without any structural disruption. Upon further addition of SDS, the mobility of the lipid probe increases sharply, indicating the disruption of the bilayer, ultimately resulting in complete solubilization of the liposome into a mixed micelle with SDS. The hyperfine coupling constant value of 5-doxyl molecules in a mixed micelle is observed to be higher than that in a pure SDS micelle, suggesting the core of the mixed micelle is more hydrophobic than that of the SDS micelle. C 2002 Elsevier Science (USA)
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 as they provide suitable barriers and microenvironments for controlled transport of solutes. In aqueous environments these molecules form bilayers where the packing of
1
To whom correspondence should be addressed.
0021-9797/02 $35.00
C 2002 Elsevier Science (USA)
All rights reserved.
100
STRUCTURE OF LIPID BILAYERS IN THE PRESENCE OF SDS
In the present study, the mechanisms of interaction of surfactants with bilayer lipids have been investigated using sodium dodecyl sulfate as the model surfactant and a phospholipid liposome as a model membrane. Specifically, the vesicle-to-micelle transition that occurs due to interaction of SDS with the membrane has been investigated.
EXPERIMENTAL
Materials Sodium dodecyl sulfate was obtained from Fluka and used as received. Phophatidic acid from egg yolk lecithin and phophatidyl choline also from egg lecithin were purchased from Sigma. The anionic lipid probe, 5-doxyl stearic acid (2(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy, free radical) was purchased from Aldrich. The buffer used was phosphate buffer at pH 7.0. 5-Doxyl stearic acid (DSA) molecule was used as the EPR spin probe and phosphatidic acid (PA) and phosphatidyl choline (PC) were used as ingredients for the preparation of the model membrane. Liposome Preparation Unilamellar liposomes of desired size (about 120 nm) were prepared by the method described in our previous paper (11). A lipidic mixture film (PC : PA composition of 1 : 1 molar ratio in chloroform) was formed by removing the organic solvent by rotatory evaporation. The lipid film was dispersed in phosphate buffer and sonicated in a bath sonication unit at 40◦ C for 1 h. The resulting liposome was sized through a polycarbonate filter of 0.2-µm size. A 1 mM of mixed liposome was used throughout this study. Determination of Particle Size Distribution and Stability of Liposome Preparation Mean size and polydispersity of liposome were determined by photon correlation spectroscopy. Samples were adjusted to the required concentration range with phosphate buffer, and the measurements were taken at 25◦ C at a lecture angle. The particle size distribution of liposome suspensions after 24 h of preparation varied a little and the polydispersity is less than 0.1, indicating that the distribution is homogenous. Likewise, the particle size distribution of the liposomes after the addition of the phosphate buffer and equilibration for 24 h at 25◦ C showed in all those cases a slight increase in the polydispersity index, from 0.1 to 0.15, and was stable in the absence of surfactants. The particle size was also determined after 1 h of interaction with SDS at different concentrations. Absorbance Measurements The absorbance measurements were made at 25◦ C using a Shimadzu UV-240 spectrophotometer (λ = 350 nm, cell length = 4 cm). The absorbance change of each liposome sus-
101
pension was monitored before and after 1 h of interaction with SDS at different concentrations (1–12 mM). Surface Tension Measurements Surface tension of SDS in phosphate buffer was measured by the drop volume method (12) before and after 1 h of interaction with liposomes (1.0 mM) at 25◦ C and the critical micelle concentration, CMC, of the SDS in the absence and in the presence of liposomes were determined from the plot of surface tension versus concentration. SDS Concentration in the Bilayer The SDS concentration in the lipid bilayer was determined after interaction of the bilayer with SDS solutions at different concentrations. The mixtures were filtered through 3000 molecular weight cutoff sizes filters to separate the SDS monomer from vesicles and micelles in the bulk. The monomer concentration of the filtrate was analyzed by the two-phase titration method (13). The SDS concentration in the bilayer was estimated by subtracting the monomer concentration in the filtrate and the total initial concentration. EPR Measurements A Microw-Now instrument, Model-8300A, was used for EPR measurements at a microwave frequency of 9.5 GHz. The concentration of the lipid probe used in all the studies was 5 × 10−6 M. The 5 × 10−6 M 5-doxyl stearic acid in chloroform was added to 1 mM 1 : 1 mixtures of phosphatidic acid and phosphatidyl choline in chloroform and the liposome was prepared by the method described above. Then 5-ml samples of 1 mM liposome were taken in a 10-ml vial, and SDS at desired concentrations was added. After the mixtures interacted for different intervals of time, the EPR spectrum was recorded. EPR spectra of 5-doxyl stearic acid in liposomes and in phosphate buffer without SDS were also taken. For comparison purposes, EPR spectra of 5doxyl stearic acid in buffer were also taken after interaction with SDS for different intervals of time. The rotational correlation times and hyperfine splitting constants were analyzed from the EPR spectrum of 5-DSA under various conditions. RESULTS AND DISCUSSION
Figure 1 shows the change in effective hydrodynamic diameter as well as the optical density of mixed liposome after 1 h of interaction with SDS concentrations. With 1 mM SDS, both the optical density and the hydrodynamic diameter of the liposome increased marginally, which we attribute to the adsorption of SDS on the surface of the liposome. As the SDS concentration increased, the optical density and effective hydrodynamic diameter decreased measurably from 1.2 and 120 nm to 0.05 and 23 nm, respectively, at 10 mM of SDS, indicating the disruption of the vesicles. On further addition of SDS, no change in
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the above parameters was observed, suggesting complete solubilization of liposome. Two different sizes of liposome particles were observed above 1 mM of SDS concentration. As the SDS concentration increased, the larger size fraction decreased and the smaller size fraction increased along the solubilization curve. The hydrodynamic diameter is the average size of both the small and the large size fraction of liposomes. To understand the adsorption behavior of SDS on liposomes, SDS monomer and surface tension measurements were carried out after 1 h of interaction with 1 mM liposome and the results are given in Fig. 2. In the absence of liposome, the critical micelle concentration, CMC, of SDS in the phosphate buffer was determined to be 2 mM. The presence of liposome shifted the CMC of SDS toward higher concentration, as is evident from the surface tension curve. Two inflection points were observed on the surface tension curve for SDS in the presence of 1 mM liposome. The first inflection point is proposed to correspond to the saturation of the bilayer liposome and the onset of the solubilization process. The second inflection point indicates the completion of the solubilization process. After the second in-
flection point the surface tension remains constant, suggesting that the CMC of the mixed system has been reached. The higher CMC value for SDS in the presence of liposome supports the hypothesis that the surfactant is adsorbing onto the liposome surface so that the actual monomer concentration available in the solution for micellization is less than the added amount. This hypothesis is in agreement with the results obtained from the optical density and light-scattering studies (Fig. 1). EPR spectroscopy provides the following structural information related to liposome solubilization: solubilization efficiency, polarity of the probe environment, microviscosity of the probe environment, critical concentration of transition from the vesicle to the mixed micelles, and average aggregate size. The amphiphilic paramagnetic molecule 5-doxyl stearic acid, which comprises a long fatty acid chain and two polar groups (carboxyl and nitroxyl) near the molecular ends, was used as the spin probe to monitor the structural changes of liposome during interaction with SDS. The EPR spectrum of the 5-doxyl nitroxide probe in solution is represented by a sharp three peak signal; the spectral lineshape reports the micropolarity and microviscosity of the probe environment. The EPR spectra of 5-doxyl stearic acid in phosphate buffer in the presence and absence of liposome are shown in Fig. 3. It can be seen that all the peaks are almost completely suppressed in the presence of the liposome. When nitroxide molecules rotate rapidly and randomly, then all the anisotropy averages out and a sharp three-line spectrum is obtained, as in the phosphate buffer. In the presence of liposome, the nitroxide molecule cannot rotate freely because of its localization inside the phospholipid bilayer. The EPR spectra of 5-doxyl stearic acid in 1 mM liposome before and after interaction with SDS at different concentrations for 1 h are illustrated in Fig. 4. At 2 mM SDS no change in the spectrum was observed (figure is not shown). However, at higher SDS concentrations the spectrum becomes progressively more isotropic, and finally at 10 mM SDS a sharp three-line isotropic spectrum was observed. This spectrum is similar to
FIG. 2. The change in surface tension as well as monomer concentration of SDS after one-hour interactions with 1 mM of liposome.
FIG. 3. ESR spectra of 5-doxyl stearic acid in phosphate buffer and in presence of 1 mM of liposome.
FIG. 1. Change in optical density as well as effective hydrodynamic diameter of 1 mM of liposome after one-hour interaction with SDS.
STRUCTURE OF LIPID BILAYERS IN THE PRESENCE OF SDS
FIG. 4.
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EPR spectra of 5-doxyl stearic acid in 1 mM of liposome before and upon interaction with SDS for 1 h.
that of the probe molecules in pure SDS micelles (10 mM SDS), indicating complete disruption of the vesicles and formation of smaller mixed micelles. The rotational correlation time of 5-doxyl stearic acid in the presence and in the absence of 1 mM liposome and after interaction with SDS at various concentrations were calculated from the spectra using the following equation: τcorr = (6.73 × 10−10 )H(0) (I0 /I−1 )1/2 − 1 , where H(0) is the width in gauss of the central field spectrum line and I0 and I−1 represent the peak-to-peak amplitudes of the central field and high field line, respectively. This equation is valid provided the correlation time τcorr < 3 × 10−9 s. Figure 5 illustrates the change in rotational correlation time of 5-doxyl stearic acid in phosphate buffer and 1 mM liposome due
FIG. 5. Change in rotational correlation time of 5-doxyl stearic acid before and after 1 h interactions with SDS.
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to interaction with SDS for 1 h. It is evident that the rotational correlation time of 5-doxyl stearic acid in phosphate buffer increases with the addition of SDS, reaching a plateau around 4 mM SDS. Apparently at 2 mM SDS, the probe molecule begins to interact with SDS to form an association complex. Above 4 mM SDS, the probe molecules are proposed to be inside the micelle and hence the rotation of the probe molecule is hindered, causing an increase in the rotational correlation time. Above 4 mM SDS the rotational correlation time remained constant, suggesting that the CMC of SDS is reached. In contrast to the above, the rotational correlation time of 5-doxyl stearic acid in 1 mM liposome increased first with SDS up to a concentration of 2 mM and then gradually decreased. This decrease in mobility at low concentration is consistent with the earlier observations of increased optical density and hydrodynamic radius in this range, attributed to the adsorption of SDS on the vesicle surface, which apparently further hinders the rotation of the probe molecules. At higher SDS concentration the rotational correlation time decreased gradually, supporting the hypothesis that the liposomes are being gradually solubilized under these conditions. Increase in the SDS concentration above 10 mM caused no further decrease in rotational correlation time, indicating that complete solubilization of liposome and mixed micelle formation have already occurred. It is known that the nitrogen hyperfine-coupling constant of the nitroxide radical is highly sensitive to the polarity of the medium in which the spin system is inserted (14–17). In particular, the higher the polarity, the higher the (AN ) value. The hyperfine splitting constant (AN ), which relates to the polarity around the labeled position, is given by (18) AN = (A + 2A⊥)/3, where A is the time-averaged electron-nuclear hyperfine tensor (parallel) and 2A⊥ is the time-averaged electron-nuclear hyperfine tensor (perpendicular). The change in the hyperfine splitting constant of 5-doxyl stearic acids due to 1-h interactions with SDS is illustrated in
Fig. 6. The hyperfine splitting constant of 5-doxyl molecules in the absence of liposome decreases with increase in SDS concentration, reaching a plateau at 4 mM SDS. The initial decrease in AN value is attributed to the onset of the micellization process. Once the CMC of SDS is reached, AN remains constant, indicating that the probe molecules are solubilized in the surfactant micelle. In contrast to this, in the presence of liposomes, the AN value of 5-doxyl stearic acid molecules is lower in the beginning due to a more nonpolar environment (19, 20) in the vesicle membrane. This value increases with SDS concentration, indicating the transfer of probe molecule from a nonpolar environment to a relatively polar environment as the vesicle is disrupted. This change in polarity of the environment may be due to the difference in packing in a compact liposome vs that in loosely packed mixed micelles. It is also evident that the AN value of the probe molecule is lower in this mixed micelle than in SDS micelle. This is probably because these mixed micelles contain the longchain phosphatidic acid and phosphatidyl choline, which make the core of the micelle relatively more hydrophobic and bigger, which was also suggested by the light-scattering results. All the above evidence clearly suggests that liposome solubilization by surfactants occurs via a micellization process. CONCLUSIONS
Interactions of mixed liposome with sodium dodecyl sulfate were monitored using light scattering, surface tension, and EPR spectroscopy. The above liposome solubilization was clearly shown to be a process involving first adsorption of sodium dodecyl sulfate on liposome and then dissolution of liposome to form mixed micelles. The CMC increase for SDS upon interacting with liposome shows utilization of SDS for the solubilization of liposome. EPR spectroscopy further gave direct evidence for the disruption of the bilayer by SDS. The change in the rotational correlation time of a lipid probe incorporated into the liposome bilayers showed that solubilization of the above liposome is a micellization process. It was also evident from the hyperfine splitting constant value that the mixed micelles formed between the surfactant and the phospholipids have higher hydrophobicity compared to that pure SDS micelle due to the presence of longchain phospholipids in the mixed micelle. The EPR study gives direct evidence that liposome solubilization is a micellization process. ACKNOWLEDGMENTS Authors acknowledge the support of the National Science Foundation (Grant 9804618, Industrial/University cooperation research center for adsorption studies in novel surfactants). The financial support of Unilever Research Laboratory, U. S. is gratefully acknowledged.
REFERENCES FIG. 6. Change in hyperfine splitting constant value of 5-doxyl stearic acid after 1 h interactions with SDS.
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