On the Formation of Discoidal versus Threadlike Micelles in Dilute

In particular, we have compared the structure of the micelles formed in mixtures of .... sample grew larger after 1 month of storage (compare Figures ...
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Langmuir 2008, 24, 1731-1739

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On the Formation of Discoidal versus Threadlike Micelles in Dilute Aqueous Surfactant/Lipid Systems Emma Johansson,*,† Maria C. Sandstro¨m,† Magnus Bergstro¨m,‡ and Katarina Edwards† Department of Physical and Analytical Chemistry, Physical Chemistry, Uppsala UniVersity, Box 579, 751 23 Uppsala, Sweden, and Department of Pharmacy, Pharmaceutical Physical Chemistry, Uppsala UniVersity, Box 580, 751 23, Uppsala, Sweden ReceiVed August 27, 2007. In Final Form: October 12, 2007 In a recent study, we showed that the surfactant 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2000) induced mixed micelles of either threadlike or discoidal shape when mixed with different types of lipids. In this study, we have exchanged the PEG-lipid for the more conventional surfactants octaethylene glycol monododecyl ether (C12E8), hexadecyltrimethylammonium bromide (CTAB), and sodium dodecyl sulfate (SDS). Cryo-TEM investigations show that also these surfactants are able to induce the formation of long-lived discoidal micelles. Generally, the preference for either discoidal or threadlike micelles can be tuned by the choice of lipids and environmental conditions in much the same way as observed for the lipid/ PEG-lipid system. Our investigation showed, furthermore, that the choice of surfactant may influence the type of mixed micelles formed. It is argued that the formation of discoidal rather than threadlike micelles may be rationalized as an effect of increasing bending rigidity. Our detailed theoretical model calculations show that the bending rigidity becomes significantly raised for aggregates formed by an ionic rather than a nonionic surfactant.

Introduction Polyethylene glycol (PEG)-lipids, which are often used to sterically stabilize liposomes intended for drug delivery,1-3 are micelle-forming surfactants.4 In a previous study, we investigated the effect of a PEG-ylated phospholipid, 1,2-distearoyl-snglycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), on aggregate structure in various lipid mixtures.5 Our results revealed that lipid components that increase the bending rigidity, as well as reduce the spontaneous curvature of the monolayer, tend to promote formation of discoidal rather than threadlike, cylindrical, mixed micelles. Discoidal micelles were furthermore observed at temperatures below the gel-to-liquid crystalline phase transition temperature of the lipid component. In this case not only increased bending rigidity but also reduced lipid/PEG-lipid miscibility help explain why cylindrical micelles do not form, considering the necessity of partial component segregation for the formation and stabilization of the relatively large bilayer discs.5 PEG-lipids are unique surfactants in that they consist of a long flexible polymer attached to a lipid anchor. The large polar head groups make the PEG-lipids well-suited to accumulate at, and form a protective hemispherical cap around, the hydrophobic rim of a bilayer disc. Importantly, the large polymeric head groups also effectively prevent the discs from fusion or self-closure. In this study we wanted to explore how exchanging the PEGlipids for other more conventional surfactants would affect the aggregate structure. The surfactants were chosen to include both * Corresponding author. Tel: (+46) 18 4713630. Fax: (+46) 18 4713654. E-mail: [email protected]. † Department of Physical and Analytical Chemistry, Physical Chemistry. ‡ Department of Pharmacy, Pharmaceutical Physical Chemistry. (1) Blume, G.; Cevc, G. Biochim. Biophys. Acta 1990, 1029, 91-97. (2) Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11460-11464. (3) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171-99. (4) Johnsson, M.; Edwards, K. Biophys. J. 2001, 80, 313-23. (5) Sandstrom, M. C.; Johansson, E.; Edwards, K. Langmuir 2007, 23, 41924198.

ionic and nonionic species. The three selected surfactants, octaethylene glycol monododecyl ether (C12E8), hexadecyltrimethylammonium bromide (CTAB), and sodium dodecyl sulfate (SDS), are all commercially available and have been used in numerous studies of phase and structural behavior.6-8 The surfactants were mixed in different ratios with lipids in three different states: one lipid in the fluid liquid-crystalline state, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC); one lipid in the more ordered gel-state, 1,2-dipalmitoyl-sn-glycero3-phosphatidylcholine (DPPC); and one lipid mixture in the liquid ordered state, POPC supplemented with 40 mol % cholesterol. Cryo-transmission electron microscopy (cryo-TEM) was used to systematically investigate and compare the aggregate structures in the surfactant/lipid systems. The technique is well-suited for characterization of samples where different aggregate structures coexist and where it is hard, or even impossible, to interpret data from indirect techniques such as light scattering. Rationalizing the Structural Behavior of Aggregates Formed in Surfactant/Lipid Mixtures. The structural behavior of aggregates formed in the process of self-assembling amphiphilic molecules may be rationalized by means of considering the bending properties of a single monolayer. Hence, a range of different properties of amphiphilic aggregates, such as size, shape, polydispersity, and flexibility, may be deduced from the three bending elasticity constants, spontaneous curvature (H0), bending rigidity (kc), and saddle-splay constant (khc).9 The spontaneous curvature represents the sign and magnitude of the preferential curvature of a single surfactant layer. It has recently been demonstrated that H0 is directly related to the transition between micelles and various bilayer structures.10 Accordingly, micelles predominate when H0 > 1/4ξ, where ξ is (6) Gutberlet, T.; Kiselev, M.; Heerklotz, H.; Klose, G. Physica B 2000, 276, 381-383. (7) Rydhag, L.; Gabra´n, T. Chem. Phys. Lipids 1982, 30, 309-324. (8) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113. (9) Porte, G. J. Phys.-Conden. Matter 1992, 4, 8649-8670. (10) Bergstrom, L. M. ChemPhysChem 2007, 8, 462-472.

10.1021/la702637h CCC: $40.75 © 2008 American Chemical Society Published on Web 01/24/2008

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Figure 1. The length-to-width ratio of a tablet-shaped micelle plotted against the bending rigidity according to theoretical calculations as given in ref 10. The spontaneous curvature was set to H0 ) 1/3ξ with ξ ) 12 Å.

the thickness of the hydrocarbon part of the monolayer, whereas bilayers are expected to be present when H0 < 1/4ξ. The bending rigidity quantifies the resistance against deviations from a uniform mean curvature H ) H0 and must be a positive quantity for stable aggregates to exist. As a result, rigid and monodisperse objects with a uniform shape are expected to form at high values of kc,whereas more flexible, polydisperse, and geometrically heterogeneous structures form at low values of kc. For instance, small vesicles with a comparatively large difference in curvature between the outer and inner layers are favored by low values of kc. Likewise, it has recently been demonstrated that the length-to-width ratio of tablet-shaped micelles is strongly influenced by kc, but virtually not by H0 and khc.10 In accordance, it was found that very long rodlike or threadlike micelles may only form at values of kc below or close to kT, whereas more discoidal micelles are expected to form at bending rigidities appreciably larger than kT (Figure 1). The saddle-splay constant has the interesting property of determining the topology of the surfactant layer, i.e., the number of handles or holes present in the film. Moreover, khc may influence the size of amphiphilic aggregates insofar that it increases with increasing khc, and eventually, a transition from discrete to connected structures is expected. This means that khc, for a given topology, plays a role rather similar to H0, and as a consequence, it might be difficult to distinguish from each other effects that are due to the two quantities. In order to rationalize our experimental findings, we have investigated the spontaneous curvature and bending rigidity as calculated from a detailed model. (We have not focused on the saddle-splay constant, since its effect due to electrostatics turns out to be rather small in magnitude, i.e., less than kT, at the comparatively large electrolyte concentrations present in our systems.) In particular, we have compared the structure of the micelles formed in mixtures of lipid in the liquid crystalline state and ionic or nonionic surfactant. Materials and Methods Materials. Egg phosphatidylcholine (EPC) was obtained from Lipid Products (Nutfield, UK). 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Octaethylene glycol monododecyl ether (C12E8; Sigma 98%), hexadecyltrimethylammonium bromide (CTAB; SigmaUltra 99%), sodium dodecyl sulfate (SDS; SigmaUltra 99%), and cholesterol were purchased from Sigma-Aldrich (Stockholm, Sweden). All other salts and reagents were of analytical grade and were used as received.

Johansson et al. Sample Preparation. Lipids and surfactants in desired amounts were codissolved in chloroform and the solvent was thereafter removed in a gentle stream of N2 gas followed by further evaporation in vacuum overnight. The dried lipid films were hydrated in HEPESbuffer (20 mM HEPES, 150 mM NaCl, pH 7.4) at 55 °C for approximately 30 min with intermittent mixing. The samples were extruded 15 times through a polycarbonate filter (pore size 100 nm) in a Mini-Extruder from Avanti Polar Lipids Inc. (Alabaster, AL). For samples with DPPC, the temperature was kept above 55 °C by placing the extruder in a heating block. Lipid concentrations were 3 mM, and 2 mL of the samples was stored for 24 h at room temperature before observation. Turbidity Measurements. Changes in size and structure of the aggregates were followed by measuring the apparent absorbance of the lipid/surfactant mixture. A Hewlett-Packard 8453 UV-visible spectrophotometer was used at a wavelength of 350 nm. After incubation in room temperature for 24 h, the sample was vortexed and the turbidity was measured. Cryo-Transmission Electron Microscopy. Cryo-TEM images were obtained by use of a Zeiss EM 920 A transmission electron microscope (Carl Zeiss Inc., Oberkochen, Germany) operating at 80 kV. A small drop of sample was placed on a copper grid coated with a perforated polymer film. Excess solution was thereafter removed by blotting with filter paper. This procedure was performed in a custom-built environmental chamber under controlled humidity and temperature (25 °C). Immediately after film preparation, the grid was plunged into liquid ethane held at a temperature just above its freezing point. The vitrified sample was then transferred to the microscope for analysis. To prevent sample perturbation and the formation of ice crystals, the specimens were kept cool (below -165 °C) during both the transfer and viewing procedures. A more detailed description of the cryo-TEM procedure can be found elsewhere.11,12

Experimental Results Aggregate Structures in DPPC, POPC, and POPC/ Cholesterol Systems. In Figure 2 the aggregate structures found in pure DPPC, POPC, and POPC/cholesterol systems are shown. All preparations contain liposomes, which are slightly aggregated. DPPC liposomes appear polygonal (Figure 2a), whereas POPC liposomes are smooth and spherical (Figure 2b). Liposomes composed of POPC/cholesterol show a tendency for forming somewhat elongated or tubular structures (Figure 2c).13 Aggregate Structures in Surfactant/DPPC Systems. Turbidity. The turbidity profiles obtained from mixtures of DPPC and the three different surfactants are shown in Figure 3. All three surfactant/DPPC mixtures first display a small drop in turbidity and then a peak followed by a large decrease in turbidity. For C12E8, the initial decrease in turbidity is not as pronounced as for the other surfactants. The turbidity could not be measured for all CTAB/DPPC ratios. From ca. 40 to ca. 80 mol % CTAB the lipid mixture was very viscous, and light scattered from small air bubbles trapped in the samples interfered with the measurements. Cryo-TEM. When 11 mol % C12E8 was included in the DPPC mixture (corresponding to the peak in the turbidity curve), large liposomes and open structures were observed with cryo-TEM (Figure 4a). Upon inclusion of more surfactant, the liposomes shrunk in size, and discoidal structures of varying size were formed. At 30 mol % of surfactant only discs were present in the sample and their size was found to be heterogeneous. At C12E8 concentrations corresponding to 39 mol %, the aggregate (11) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; Mcdowall, A. W.; Schultz, P. Q. ReV. Biophys. 1988, 21, 129-228. (12) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf. A 2000, 174, 3-21. (13) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258-66.

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Figure 3. Turbidity profiles of DPPC together with C12E8 (squares), CTAB (triangles), and SDS (circles).

Figure 2. Cryo-TEM images of samples containing (a) DPPC, (b) POPC, and (c) POPC/cholesterol. Scale bars indicate 100 nm.

structure was completely dominated by small discs with a diameter of approximately 20 nm (Figure 4b). Unilamellar and multilamellar liposomes together with a small amount of discs were found in samples containing 19 mol % CTAB. At 38 mol % CTAB, the sample contained mainly discs. This sample appeared a little viscous, and as seen in Figure 4c, some of the discs appeared aggregated. At 49 mol % CTAB, the mixture was very thick and viscous, and cryo-TEM revealed a large number of aggregated discs together with bilayer sheets (Figure 4d). At 90 mol % surfactant, the mixture was no longer viscous and contained mostly globular micelles. Large, and frequently multilamellar, liposomes dominated the structure in samples supplemented with 35 mol % SDS. At 46 mol % SDS, both liposomes and larger discs were present in the sample (Figure 4e). When 53 mol % SDS had been added, discs with a diameter of about 40 nm were the only structures present (Figure 4f). As described above, cryo-TEM revealed that the surfactants induced formation of either small discs or, at high surfactant concentration, globular micelles. Importantly, no cylindrical micelles could be observed in any of the surfactant/DPPC mixtures investigated. A summary of aggregate structures found in the surfactant/DPPC systems can be viewed in Table 1.

Aggregate Structures in Surfactant/POPC Systems. Turbidity. The turbidity profiles for SDS/POPC, CTAB/POPC, and C12E8/POPC are shown in Figure 5. Similar to the turbidity curves for surfactant/DPPC mixtures (Figure 3), the curves display a small drop and then a peak followed by a large decrease in turbidity. The peak generally represents large and sometimes open liposomes as observed by cryo-TEM. Cryo-TEM. The first transition structure was a lacelike structure observed between liposomes and globular micelles in the C12E8/ POPC system. A representative picture of these structures, which occurred at 20 mol % C12E8, is given in Figure 6a. At 40 mol % C12E8, threadlike micelles, some of them heavily entangled and likely branched, were observed (Figure 6b). As more surfactant was added, the threadlike micelles became shorter and disentangled, and finally, globular micelles were formed. Intact and open liposomes together with a few smaller discs were seen in cryo-TEM images of samples containing 49 mol % CTAB. At 70 mol % CTAB, the sample contained many different structures: large liposomes, elongated liposomes, and open lamellar structures with either irregular or circular shape (discs). Many discs were still observed at 80 mol % surfactant (Figure 6c). In the latter sample, cylindrical micelles were also frequently observed (inset Figure 6c). At 90 mol % CTAB, only cylindrical and globular micelles were present (Figure 6d). Liposomes and small discs, with an approximate diameter of 15-30 nm, were observed when 70 mol % SDS was mixed with POPC (Figure 6e). At 80 mol % SDS, only short threadlike and globular micelles were detected (Figure 6f). As reported above, threadlike micelles form at high surfactant to lipid ratio in all three surfactant/POPC mixtures. The type of structures formed at lower concentration of surfactant depends, on the other hand, on whether C12E8, CTAB, or SDS is included in the mixture. The summary of aggregate structures found in the surfactant/POPC systems can be viewed in Table 1. Aggregate Structures in Surfactant/POPC/Cholesterol Systems. Turbidity. The turbidity profiles for mixtures of POPC and cholesterol together with CTAB or SDS were similar, whereas the turbidity measurement for C12E8/POPC/cholesterol resulted in a quite different profile (Figure 7). The most striking difference was that whereas a small amount of a charged surfactant lowered the turbidity, C12E8 instead induced a large rise in turbidity. Cryo-TEM. Only two structures, globular micelles and liposomes, were present over the entire concentration range in the C12E8/POPC/cholesterol system. The latter were often found

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Figure 4. Cryo-TEM images of samples containing DPPC and (a) 11 mol % C12E8, (b) 39 mol % C12E8, (c) 38 mol % CTAB, (d) 49 mol % CTAB, (e) 46 mol % SDS, and (f) 53 mol % SDS. The arrows indicate discs positioned face-on, and the arrowheads indicate discs positioned edge-on. Scale bars indicate 100 nm. Table 1. Summary over the Aggregate Structures Found in Surfactant/Lipid Systems DPPC

POPC

POPC/cholesterol

C12E8

discs

threadlike micelles

CTAB

discs

SDS

discs

discs f threadlike micelles discs f threadlike micelles

liposomes and globular micelles discs discs

to be large and multilamellar (Figure 8a). The number of liposomes decreased, and the amount of globular micelles increased, with increasing surfactant to lipid ratio. Large liposomes and bilayer fragments were found in samples containing POPC/cholesterol together with 50 mol % CTAB. As more surfactant was included, discs became the most frequent structure observed, and some of them appeared slightly aggregated. In samples containing 67 mol % CTAB, almost only small discs, with a diameter of around 30 nm, were observed (Figure 8b). The structures formed in the SDS/POPC/cholesterol system were similar to those found in the CTAB/POPC/cholesterol

Figure 5. Turbidity profiles of POPC together with C12E8 (squares), CTAB (triangles), and SDS (circles).

system. With increasing amount of SDS, first small discs and then globular micelles were formed.

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Figure 6. Cryo-TEM images of samples containing POPC and (a) 20 mol % C12E8, (b) 40 mol % C12E8, (c) 80 mol % CTAB, (d) 90 mol % CTAB, (e) 70 mol % SDS, and (f) 80 mol % SDS. The arrow indicates a threadlike micelle. Scale bars indicate 100 nm.

Figure 7. Turbidity profiles of POPC/cholesterol together with C12E8 (squares), CTAB (triangles), and SDS (circles).

In the CTAB and SDS systems, discs were formed with increasing amounts of the surfactants, while no such aggregate structure were found in the C12E8 samples. A summary of the

aggregate structures found in the surfactant/POPC/cholesterol systems can be viewed in Table 1. Effect of Storage on the Aggregate Structures. To elucidate if the sample structure was stable over time, one sample containing a homogeneous population of mixed micelles was selected from each system and stored at 4 °C for 1 month and then reinvestigated with cryo-TEM. The discs formed in the C12E8/DPPC (39 mol % surfactant) sample grew larger after 1 month of storage (compare Figures 4b and 9a). The CTAB/DPPC (38 mol % surfactant) sample was still slightly viscous after 1 month of storage. Cryo-TEM confirmed that there were still some liposomes present and showed that the discs were slightly more aggregated. The structure did not change appreciably in the SDS/DPPC (53 mol % surfactant) samples and still only small discs were present after 1 month of storage. The stored C12E8/POPC (60 mol % surfactant) mixture displayed threadlike micelles both before and after storage, but after 1 month, some liposomes were also present in the sample. For CTAB/POPC (90 mol % surfactant), the amount of cylindrical micelles seemed to have decreased and more globular micelles appeared to be present. For SDS/POPC (80 mol % surfactant),

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% surfactant) system, the discs grew larger upon storage, and some liposomes were present in the stored sample (compare Figures 8b and 9b). The SDS/POPC/cholesterol (70 mol % surfactant) sample remained stable, and small discs were the dominating structure also after storage. Model Calculations of Bending Elasticity Constants. The three bending elasticity constants may be estimated from detailed model calculations. It turns out that the quantitative outcome depends on the particular constraints imposed on the aggregates during the process of bending. For instance, a self-assembled layer may be bent at constant aggregation number (thermodynamically closed layer) or exchange molecules with a surrounding reservoir of free amphiphile in solution at constant chemical potentials (thermodynamically open layer). It has been demonstrated that the bending rigidity of a thermodynamically closed monolayer must always be larger than the corresponding quantity for a thermodynamically open layer.14-16 As a matter of fact, this may explain the comparatively large bending rigidities that have been found for phospholipids membranes in a crystalline gel state and our observation that discoidal micelles are always found in mixtures containing DPPC. Expressions for H0, kc, and khc have recently been evaluated for pure as well as mixed fluid layers.16,17 It was found that, in the case of layers that are open in the thermodynamic sense, kcH0 may be written as a sum of different contributions, i.e., Figure 8. Cryo-TEM images of samples containing POPC/ cholesterol and (a) 70 mol % C12E8, (b) 67 mol % CTAB. Arrow indicates ice crystal. Scale bars indicate 100 nm.

kcH0 ) (kcH0)hb + (kcH0)el + (kcH0)hg

(1)

The contribution due to the hydrophobic effect may be written as

(kcH0)hb ) -

ξγhb 4

(2)

where γhb is the hydrocarbon-water interfacial tension, which equals about 50 mJ/m2 at room temperature.16,17 The contribution due to electrostatics, as calculated from the Poisson-Boltzmann mean field theory, equals

(

)

xπlBκ-1 (kcH0)el qx 1 ln + (ξ + d) ) kT 2πlB qa 2a

(3)

for a monolayer composed of an ionic surfactant and a nonionic lipid with mole fractions x and 1 - x, respectively, that is open in a thermodynamic sense.17 a denotes the area per aggregated molecule, whereas the surface of charge is located a distance d/2 outside the hydrocarbon-water interface. In other words, d may be considered as the diameter of a spherical head group with the point of charge located at its center. We may note that layer properties, such as thickness, area per molecule, and composition, are curvature dependent and ξ, a, and x in eqs 2, 3, and 7 all refer to the values that minimizes the free energy per aggregated molecule for planar geometry. The parameter

q≡ Figure 9. Cryo-TEM images of stored samples containing (a) C12E8/ DPPC (39 mol % surfactant), (b) CTAB/POPC/cholesterol (67 mol % surfactant). Scale bars indicate 100 nm.

no difference in the aggregate structure could be observed after 1 month of storage. The C12E8/POPC/cholesterol (70 mol % surfactant) sample did not change upon storage and still contained only globular micelles and liposomes. In the CTAB/POPC/cholesterol (67 mol

s

xs

2

(4)

+1+1

is related to the reduced surface charge density s ) 2πlBκ-1/a and assumes values between zero and unity as 0 < s < ∞. The Bjerrum length equals lB ) 7.15 Å and the Debye screening (14) Porte, G.; Ligoure, C. J. Chem. Phys. 1995, 102, 4290-4298. (15) Safran, S. A. AdV. Phys. 1999, 48, 395-448. (16) Bergstrom, L. M. Langmuir 2006, 22, 3678-3691. (17) Bergstrom, L. M. Langmuir 2006, 22, 6796-6813.

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length κ-1 ) 3.04/xcsalt Å for a 1:1 electrolyte with molarity csalt in an aqueous medium at room temperature. The residual head group repulsion contribution (kcH0)hg may be calculated from expressions based on the free energy of mixing head groups with a volume Vhg and water with a molecular volume Vw in the hydrophilic part of the monolayer, i.e.

hg ) °hg +

Vhg 1 - φhg ln(1 - φhg) + ln φhg Vw φhg

(5)

where °hg is the curvature-independent part of the head group free energy and φhg is the volume fraction of head groups in the hydrophilic layer. This particular contribution has been worked out in detail for the various bending elasticity constants and will be published in a forthcoming paper. The bending rigidity may be written as

kc ) kcel + kchg

(6)

Figure 10. The spontaneous curvature as calculated from eqs 3 and 7 for a mixture of a nonionic double-chain lipid and an ionic single chain surfactant. The volume of each chain was set to 350 Å,3 approximately corresponding to an aliphatic C12 chain. The head groups were treated as hard spheres with radius equal to 3 Å. The dash-dotted line represents H0 ) 1/4ξ.

where the electrostatic contribution has been elaborated to give17

[

]

[ [

] ]

kcel 2(p - q)2 2qa κ-1 px ) 1+ (ξ + d) +ξ+d kT 2πlB pq 2a xπlB p2x(1 - x)(1 - xy)2

qa +ξ+d (2p(1 - x)(1 - xy)2 + 1)a xπlB

2

(7)

for a mixture of an ionic surfactant and a nonionic lipid, where

p≡

s

xs

2

(8)

+1

assumes values in the range 0 < p < 1. The relative difference in tail volume between surfactant and lipid (y) assumes negative values for the case of an ionic surfactant (small tail) and a nonionic lipid (large tail). It turns out that the hydrophobic effect does not contribute explicitly to kc whereas kcel may be considered as a sum of three different contributions. The first term in eq 7 represents kc for an infinitely thin charged surface, and the second term consists of effects arising as a result of the layer having a finite thickness, whereas the last (always negative) term is the result of mixing surfactant and lipid in the aggregates. The spontaneous curvature may be calculated by simply dividing kcH0 in eq 1 with kc in eq 6. The result for a simple case of a nonionic double-chain lipid and an ionic single-chain surfactant with identical chains and hard sphere head groups is shown in Figure 10. It is seen that H0 increases from negative to positive values as an increasing amount of surfactant is added. Hence, we expect smaller and more curved aggregates to form at high surfactant mole fractions, as is clearly observed in the experiments. A transition from bilayer structures (liposomes) to micelles is expected at H0 ) 1/4ξ, the value of which is indicated in the figure. It may be noted that ξ, as obtained by minimizing the molecular free energy of a planar monolayer, is found to slightly decrease and H0 to increase, with increasing x as a result of the enhanced head group repulsion as the fraction of singlechain surfactant is increased. The lipids and surfactants used in the present measurements have different head group sizes and chain lengths. Moreover, the head groups of the lipids DPPC and POPC are zwitterionic, whereas the headgroup is made up of a flexible chain for the nonionic surfactant C12E8. As a result, it is not straightforward to calculate absolute values of kcH0 and kc, needed to determine H0, for real amphiphilic molecules. However, if we want to analyze the different behaviors observed as either an ionic or a

Figure 11. The electrostatic contribution to the bending rigidity (kcel) for a mixture of a single-chain ionic surfactant (SDS) and a double-chain nonionic lipid (POPC) plotted against the mole fraction of ionic surfactant in a planar layer (x) in accordance with eq 7. The three contributions to kc due to the bending of an infinitely thin surface (dashed line), finite thickness effects (dotted line), and mixing effects (dash-dotted line) are also given. The thickness of the hydrocarbon part was set to ξ ) xξ1 + (1 - x)ξ2, where ξ1 ) 12.5 Å and ξ2 ) 16.8 Å corresponding to 75% of the fully stretched tails. The area per aggregated molecule was set to a ) xa1 + (1 - x)a2 with a1 ) 40 Å2 and a2 ) 69.4 Å.2 The relative difference in tail volume is defined as y ) (V1 - V2)/(xV1 + (1 - x)V2), where V1 ) 350 Å3 and V2 ) 936 Å3 are the tail volumes of surfactant and lipid, respectively. The distance between the surface of charge and the hydrocarbon-water interface was set to d/2 ) 3 Å. The hydrophobic interfacial tension was set equal to γhb ) 50.7 mJ/m2 and the Bjerrum length lB ) 7.15 Å. The Debye screening length was set to κ-1 ) 7.7 Å corresponding to [NaCl] ) 150 mM plus sodium counterions from the SDS.

nonionic surfactant is added to an aqueous solution of lipid, it is informative to study the differences in kcH0 and kc, respectively, between a charged and an uncharged system. Expressions for these differences are exactly given by (kcH0)el and kcel in eqs 3 and 7, respectively. The electrostatic contribution to the bending rigidity kcel for a mixture of a nonionic lipid with one palmitoyl and one oleoyl chain (POPC) and an ionic surfactant with a dodecyl chain (SDS) is plotted in Figure 11 together with its three contributions. kcel may be interpreted as the difference between POPC/SDS and POPC/C12E8 aggregates that is due to electrostatic mean-field effects as calculated by the Poisson-Boltzmann theory. In the corresponding calculations for POPC/CTAB aggregates, one has to take into account the different tail length between CTAB and C12E8. However, it turns out that the main features observed in Figure 11 are not changed by this difference. The contribution

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for an infinitely thin surface (first term in eq 7) turns out to be rather small at the comparatively small Debye screening length (κ-1 ) 7.7 Å) in the presence of a substantial amount of added salt ([NaCl] ) 150 mM). The second term in eq 7 due to finite thickness effects is always positive, whereas the mixing term always brings down kc as compared to a one-component surfactant layer. We see that the net effect of charging the aggregates is to raise kc about kT. Moreover, the length-to-width ratio is expected to increase 10 times as kc is decreased from 2 to 1 kT (Figure 1). Hence, the tendency of the ionic surfactants SDS and CTAB to form discoidal micelles together with POPC, rather than threadlike micelles as is observed for C12E8, may be explained as a result of an increasing bending rigidity for charged aggregates. We may note that the bending rigidity has frequently been found to be raised upon increasing the surface charge density, giving rise to increased persistence lengths of polyelectrolytes and wormlike micelles.18,19

Discussion The surfactants used in this study have, similar to DSPEPEG2000, high positive spontaneous (or “intrinsic”) curvature and self-aggregate into small globular micelles in dilute aqueous solution with low or moderate electrolyte concentration.20-22 These micelle-forming surfactants have limited solubility in lipid bilayers and will, if added in high concentration, induce formation of surfactant/lipid mixed micelles. In an earlier study, we showed that two fundamentally different types of mixed micelles, discoidal or threadlike micelles, may form as the concentration of PEG-lipid is increased above the bilayer saturation concentration [and the spontaneous curvature H0 is increased above 1/4ξ (Figure 10)].5 Cryo-TEM investigations revealed that a change from threadlike to discoidal micelles could be induced by cooling the lipid mixture to temperatures below the gel-to-liquid crystalline phase transition temperature, where the bending rigidity is expected to be significantly raised. Discoidal mixed micelles were furthermore promoted by inclusion of components in the lipid mixture that increase the bending rigidity as well as reduce the spontaneous curvature of the lipid mixture. Aggregate Structures in Surfactant/DPPC Systems. All three surfactants (C12E8, CTAB and SDS) were, similar to DSPEPEG2000, found to induce formation of discoidal micelles when mixed with DPPC. The investigations were carried out at 25 °C, i.e., well below TC for DPPC,23 and the bending rigidity for phospholipid membranes is known to be about 10-fold higher in the gel compared to the liquid crystalline phase.24 Further, PEG-lipid/lipid miscibility is presumably reduced in the gel phase, promoting component segregation. These effects help explain why the mixed micelles in all investigated systems adapted a disoidal shape. The extent of the initial drop in turbidity observed when combining less than 20 mol % surfactant with DPPC varies between the ionic and nonionic surfactants. This may be explained by the fact that the charged surfactants, CTAB and SDS, in (18) Jerke, G.; Pedersen, J. S.; Egelhaaf, S. U.; Schurtenberger, P. Phys. ReV. E 1997, 56, 5772-5788. (19) Bergstrom, M.; Pedersen, J. S. Langmuir 1999, 15, 2250-2253. (20) Lasic, D. D. J. Colloid Interface Sci. 1986, 113, 188-193. (21) Rohde, A.; Sackmann, E. J. Colloid Interface Sci. 1978, 70, 494505. (22) Dorshow, R.; Briggs, J.; Bunton, C. A.; Nicoli, D. F. J. Phys. Chem. 1982, 86, 2388-2395. (23) Cevc, G. Phospholipids Handbook; Marcel Dekker Inc.: New York, 1993. (24) Mecke, K. R.; Charitat, T.; Graner, F. Langmuir 2003, 19, 20802087.

Johansson et al.

contrast to the nonionic C12E8, give rise to an electrostatic repulsive force that prevents the aggregation of metastable liposomes. The small peak observed in the turbidity curves appearing at surfactant concentrations just below bilayer solubilization generally represents large and sometimes open liposomes, as shown in Figure 4a. When monitoring the turbidity of such samples, both freshly prepared and, as in the experimental protocol used throughout the study, after 24 h, an increase in turbidity is observed. The turbidity increase is likely due to fusion and growth of the initially formed aggregates explaining the presence of large and open liposomes. Cryo-TEM images of samples containing high concentrations of CTAB display aggregated discs and bilayer sheets, which may contribute to the viscous behavior of these samples. However, we have at present no satisfactory explanation for the observed aggregation behavior. A similar tendency for aggregation has been observed in other systems containing CTAB.25 Aggregate Structures in Surfactant/POPC Systems. TC for POPC corresponds to -3 °C,23 which means that aggregates with the lipids in a fluid state are expected at room temperature. Addition of all three surfactants eventually induced the formation of threadlike micelles. This result was no surprise, since the comparably low bending rigidity of fluid aggregates is expected to reduce the energetic cost of forming geometrically heterogeneous structures. As already discussed, there were some important structural differences between aggregates formed by ionic and nonionic surfactant, respectively. The two charged surfactants induced the formation of bilayer discs at low surfactant/ lipid ratios, because of a higher kc due to electrostatics compared with the nonionic system. Aggregate Structures in Surfactant/POPC/Cholesterol Systems. In a recently published paper,5 we showed that inclusion of cholesterol changed the structure of the mixed micelles formed in the DSPE-PEG2000/EPC system. The long threadlike micelles frequently observed in pure DSPE-PEG2000/EPC mixtures were not detected in samples containing 10 mol % or more cholesterol. The mixed micelles formed in the SDS/POPC and CTAB/POPC systems are similarly affected by complementation with cholesterol. Thus, when POPC/cholesterol was mixed with CTAB or SDS, discs were formed and no threadlike micelles could be observed. A plausible explanation for the observation of discs rather than threadlike micelles may be found if we consider the tendency of cholesterol to induce a liquid ordered state, with high bending rigidity, of the aggregates. This is consistent with the facts that increasing concentrations of cholesterol are known to progressively increase the bending rigidity of phosphocholine membranes26 as well as decrease phospholipid membrane curvature.27 Interestingly, neither threadlike nor discoidal but only globular micelles could be observed in the C12E8/POPC/ cholesterol system. The globular micelles must contain a low amount of POPC in order to be able to maintain their globular shape, and cholesterol is not expected to increase the curvature of the aggregate structures. It is thus likely that the globular micelles are poor in both phospholipid and cholesterol, while these molecules constitute the main components in the large liposomal aggregates. Similar behavior has been observed in egg yolk lecithin/cholesterol/taurocholate systems.28 We have at present no straight forward explanation for the large (25) Horbaschek, K.; Hoffmann, H.; Thunig, C. J. Colloid Interface Sci. 1998, 206, 439-456. (26) Henriksen, J.; Rowat, A. C.; Ipsen, J. H. Eur. Biophys. J. 2004, 33, 73241. (27) Chen, Z.; Rand, R. P. Biophys. J. 1997, 73, 267-76.

Dilute Aqueous Surfactant/Lipid Systems

rise in turbidity when a small amount of surfactant was included in the C12E8/POPC/cholesterol system. It is noteworthy and has also been observed when Triton-X was added to cholesterol-supplemented liposomes.29 Effect of Storage on Aggregate Structures. DPPC, POPC, and POPC/cholesterol samples containing SDS remained stable over time. Aggregate size and structure did, as judged from cryoTEM images, not change significantly upon 30 days of storage at 4 °C. The stable structure and absence of aggregation may in part be explained by the ability of the surfactant to generate an electrostatic repulsion that stabilizes the lipid/surfactant aggregates. It might also be possible that the spontaneous curvature is sufficiently raised by the ionic surfactant (cf. eq 3) so that comparatively small aggregates with high spontaneous curvature are thermodynamically stable. For samples containing C12E8 (except the C12E8/POPC/cholesterol sample) or CTAB, structural changes were noted after 30 days of storage. The relatively large head group of C12E8 can perhaps offer some steric stabilization but apparently not enough to prevent the aggregates from fusion and growth into larger structures (compare Figures 4b and 9a). For CTAB, the changes in aggregate structure were very pronounced (compare Figures 8b and 9b), which may be rationalized as an effect of decreased spontaneous curvature for the surfactant with longer tail (CTAB) as compared to the one with shorter tail (SDS).16 The reason why CTAB as a charged surfactant cannot stabilize the aggregates to the same extent as SDS may also be connected to the comparably more shielded charge on CTAB than on SDS. (28) Kaplun, A.; Konikoff, F. M.; Eitan, A.; Rubin, M.; Vilan, A.; Lichtenberg, D.; Gilat, T.; Talmon, Y. Microsc. Res. Tech. 1997, 39, 85-96. (29) Alonso, A.; Saez, R.; Villena, A.; Goni, F. M. J. Membr. Biol. 1982, 67, 55-62.

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Conclusions Results of this study show that conventional surfactants, similar to PEG-lipids, can induce the formation of either threadlike cylindrical or large discoidal mixed micelles when mixed with phospholipids. The type of micelles formed is intimately coupled to the phase state of the lipids, but can also be influenced by the inherent properties of the surfactant. As an illuminating example, the uncharged surfactant C12E8 does in mixtures with POPC induce a gradual transition from bilayers to threadlike micelles, whereas the charged surfactants CTAB and SDS at low surfactant concentrations promote formation of discoidal aggregates. Our experimental observations do, in combination with theoretical model calculations, suggest that the bending rigidity of the lipid mixture has a decisive influence on the structure of the mixed micelles formed. The PEG-stabilized bilayer discs may find important biotechnical and pharmaceutical applications. This is probably not true for the discs formed in the systems investigated in the present study. First, the amount of surfactant needed to create homogeneous discs is rather high, and second, the discs formed are comparably small. Moreover, the discs formed in samples containing C12E8 or CTAB are not stable over time. The high monomer aqueous solubility, in particular of SDS and CTAB, further reduces the number of potential applications for the surfactant-stabilized discs. Acknowledgment. Financial support from The Swedish Research Council, the Alice and Knut Wallenberg Foundation and O.E. and Edla Johansson Science Foundation are gratefully acknowledged. LA702637H