Spontaneous Formation of Nanovesicles in Mixtures of Nonionic and

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Spontaneous Formation of Nanovesicles in Mixtures of Nonionic and Dialkyl Chain Cationic Surfactants Studied by Surface Tension and SANS† I. Grillo,*,‡ J. Penfold,§,| I. Tucker,⊥ and F. Cousin# Institut Laue LangeVin, 6 rue Jules Horowitz, B.P. 156, 38042 Grenoble Cedex 9, France, ISIS Facility, STFC, Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, U.K., Physical and Theoretical Chemistry Laboratory, Oxford UniVersity, South Parks Road, Oxford, U.K., UnileVer Research and DeVelopment Laboratory, Port Sunlight Laboratory, Quarry Road East, Bebington, Wirral, U.K., and CEA Saclay, Laboratoire Le´on Brillouin, F-91191 Gif-sur-YVette, France ReceiVed July 29, 2008. ReVised Manuscript ReceiVed September 29, 2008 Surface tension and small-angle neutron scattering have been used to characterize the surface properties and the structure of the aggregates formed in the dilute part of the ternary system didodecyldimethyl ammonium bromide, DDAB/tetraethylene glycol monododecyl ether, C12E4/D2O. For surfactant molar ratios, Rn, between 0.3 and 1 (pure DDAB), the surface tension measurements show the existence of two break points at concentrations of around 10-5 and 10-3 mol/L, respectively. The SANS measurements have shown that the first break point corresponds to a critical micellar concentration (cmc) and the second one corresponds to a critical vesicle concentration (cvc). In the intermediate composition range, Rn ) 0.3-0.8, very small unilamellar vesicles (nanovesicles) are formed with the inner radius varying between 28 and 85 Å and a bilayer thickness of ∼23 Å. At Rn ) 0.8, we observed a transition from small vesicles (V) to large bilamellar or multilamellar vesicles (BLV, MLV) with a relatively large lamellar periodicity of around 1000 Å. In the nonionic-rich region below Rn ) 0.3, more classical surface tension behavior was observed, with only one break point corresponding to the onset of formation of small globular micelles.

Introduction Surfactant mixtures are commonplace in a wide range of industrial, technological, and domestic applications, where the synergism between different surfactants can provide an enhancement of properties such as detergency or tolerance to water hardness. As a result, the behavior of mixed surfactants in solution and at interfaces has been extensively studied both experimentally and theoretically.1,2 The dialkyl chain cationic surfactants, in combination with the polyoxyethyene glycol nonionic surfactants, are an important class of mixed surfactants because they are the major components of formulations such as hair and clothing conditioners. Although the phase behavior of the dialkyl chain cationic surfactants is well established,3-10 there is comparatively little on the phase behavior of the dialkyl chain cationic/nonionic † Part of the Neutron Reflectivity special issue. * Corresponding author. E-mail: [email protected]. ‡ Institut Laue Langevin. § Rutherford Appleton Laboratory. | Oxford University. ⊥ Port Sunlight Laboratory. # CEA Saclay.

(1) Scamehorn, J. F., Abe, M., Eds.; Mixed Surfactant Systems, 2nd ed.; Marcel Dekker: New York, 2004. (2) Holland, P. M.; Rubingh, D. N. In Cationic Surfactants; Holland,P. H., Rubingh, D. N., Eds.; Surfactant Science Series; Marcel Dekker: New York, 1990; Vol. 37. (3) Fontell, K.; Ceglie, B.; Lindman, B.; Ninham, W. Acta Chem. Scand. 1986, A86, 247–256. (4) Dubois, M.; Zemb, Th. Langmuir 1991, 7, 1352–1360. (5) Zemb, Th.; Gazeau, D.; Dubois, M.; Gulik-Krzywicki, T. Europhys. Lett. 1993, 21, 759–766. (6) Dubois, M.; Zemb, Th.; Fuller, N.; Rand, R. P.; Parsegian, V. A. J. Chem. Phys. 1998, 18, 7855–7869. (7) Hass, S.; Hoffmann, H.; Thunig, C.; Hoinkis, E. Colloid Polym Sci 1999, 277, 856–867. (8) Brady, J. E.; Evans, D. F.; Warr, G. C.; Greiser, F.; Ninham, B. W. J. Phys. Chem. 1986, 90, 1853–1859. (9) Caboi, F.; Monduzzi, M. Langmuir 1996, 12, 3548–3558. (10) Tucker, I.; Penfold, J.; Thomas, R. K.; Grillo, I.; Barker, J. G.; Mildner, D. F. R. Langmuir 2008, 24, 6509–6520.

surfactant mixtures.11-14 The mixed surfactant systems exhibit complex phase diagrams that depend on the nature of the surfactants, temperature, concentration, and ionic strength. The shape of mixed aggregates is linked to the interplay between geometric, steric, and electrostatic constraints and the fine balance between attractive and repulsive molecular forces of the different components. In the purely micellar region, the molecular-thermodynamic theory developed by Eriksson et al.15 and Naragajan et al.16 for single surfactants and later extended to binary surfactant systems17,18 provides some predictability in the shapes and sizes of micellar aggregates. The packing parameter pp introduced by Israelachvili et al.19 (defined as Vc/a0lc, where Vc is the alkyl chain molecular volume, lc is the extended chain length, and a0 is the area per molecule at the aggregate surface) is very effective at predicting the evolution in the shape of surfactant aggregates. Double-chain surfactants with a packing parameter of between 1 /2 and 1 are known to organize into bilayers or vesicles. The single surfactants with packing parameters of less than 1/3 tend to form small globular micelles. The micelle morphology is more elongated for 1/3 < pp < 1/2. Mixtures of single- and doublechain surfactants can be used to fine tune the preferred curvature (11) Penfold, J.; Staples, E.; Ugazio, S.; Tucker, I.; Soubiran, L.; Hubbard, J.; Noro, M.; O’Malley, B.; Ferrante, A.; Ford, G.; Buron, H. J. Phys. Chem. B 2005, 109, 18107–18116. (12) Penfold, J.; Staples, E.; Tucker, I.; Thomas, R. K. Langmuir 2004, 20, 1269–1283. (13) Tucker, I.; Penfold, J.; Thomas, R. K.; Grillo, I.; Barker, J. G.; Mildner, D. F. R. Langmuir 2008, 24, 7674–7687. (14) Tucker, I.; Penfold, J.; Thomas, R. K.; Grillo, I.; Barker, J. G.; Mildner, D. F. R. Langmuir 2008, 24, 10089–10098. (15) Eriksson, J. C.; Ljunggren, S.; Henricksson, U. J. Chem. Soc., Faraday Trans. 2 1985, 81, 833–868. (16) Nagarajan, R.; Ruckenstein, E. Langmuir 1991, 7, 2934–2963. (17) Shiloach, A.; Blankstein, D. Langmuir 1998, 14, 1618–1636. (18) Shiloach, A.; Blankstein, D. Langmuir 1998, 14, 7166–7182. (19) Israelachvili, J.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525–1568.

10.1021/la802435h CCC: $40.75  2009 American Chemical Society Published on Web 11/24/2008

Spontaneous Formation of NanoVesicles

or the shape of the mixed aggregates and can often result in a curvature that is favorable to the formation of vesicles. In recent years, the spontaneous formation and growth of vesicles has been studied extensively. Vesicles have been considered to be models for biological membranes and have been used in applications such as microreactors and for drug encapsulation and drug delivery. One major challenge in such formulations is to generate thermodynamically stable aggregates with a well-defined size. It has been shown that mixing singleand double-chain surfactants favors the spontaneous formation of vesicles, and much of that progress is summarized in a number of comprehensive reviews on the different systems forming vesicles.20,21 Because of the large variety of different surfactant molecules and their possible combinations, this is a field of research that is still evolving and developing. In this study, we have investigated the phase behavior of the mixed surfactants of the cationic double-chain surfactant didodecyldimethylammonium bromide (DDAB) with a nonionic surfactant, monododecyl ether (C12E4). We have concentrated on the dilute part of the phase diagram for a total surfactant concentration lower than 5 × 10-2 mol/L (Φ < 2 vol %). The aim of the study is to link the surface properties as determined by surface tension to the self-assembly of the surfactant molecules in the bulk solution and to establish the potential of such mixtures to form well-defined vesicular structures. Surface tension is a very sensitive technique for identifying the onset of surfactant aggregation and for determining critical micellar concentrations (cmc) or critical aggregation concentrations (cac). In contrast, scattering methods (light, X-ray, and neutron) are powerful techniques for investigating the microstructure of aggregates with typical sizes from a few nanometres to a few tens of micrometers. Small-angle neutron scattering (SANS) offers the unique advantage of D/H isotopic labeling and the use of D2O instead of H2O as the solvent considerably increases the contrast between the solvent and the surfactant. Hence, in combination with the very high flux of the new generation of diffractometers such as D22 at the ILL, it becomes possible to study the structure of surfactant aggregates at very low concentrations close to the cmc and thus to follow the initial stages of the self-assembly or micellization process.

Experimental Details Materials. Tetraethylene glycol monododecyl ether (C12E4, Fluka) and dododecyldimethylammonium bromide (DDAB, Aldrich) with a purity of >98% were used without further purification, and the associated molecular data for the surfactants are given in Table 1. C12E4 is a nonionic surfactant, and the binary phase diagram of C12E4 in water has been established by Mitchell et al.22 as a function of temperature and surfactant volume fraction Φ. At 25 °C, between the cmc (5.63 × 10-5 mol/L - Φ ) 0.002%) and 25%, a two-phase domain is observed where wormlike micelles are in solution. A lamellar phase LR is formed between Φ ) 25 and 80%. The bilayer thickness e has been measured by SAXS and SANS.23 Using the linear swelling relation (e ) d*φ, with φ being the volume fraction and d* being the periodicity), it was found that eC12E4) 33.5 Å in water. DDAB is a cationic dialkyl chain surfactant that forms lamellar phases in a large range of concentrations and temperatures. It has been extensively studied as a function of concentration, temperature, and electrolyte.3-5 At 25 °C, below 0.15%, bluish isotropic solutions are formed (Results and Discussion). Between 0.15 and 3%, a two(20) Khan, A.; Marques, E. F. Curr. Opin. Colloid Interface Sci. 2000, 4, 402–410. (21) Segota, S.; Tezak, D. AdV. Colloids Interface Sci. 2006, 121, 51–75. (22) Mitchell, D. J.; Tiddy, G. J. T.; Waring, L.; Bostock, T.; McDonald, M. P. J. Chem. Soc., Faraday Trans. 1 1983, 79, 975–1000. (23) Grillo, I. Insertion de Particules Anisotropes dans des Phases Lamellaires TensioactiVes. The`se de l’Universite´ Paris XI, 1998.

Langmuir, Vol. 25, No. 7, 2009 3933 Table 1. Molecular Parameters and Scattering Length Densities M (mol/L) density Vmonomer(Å3)a ehead(Å) etail(Å) Fhead(cm-2) Ftail (cm-2) lc(Å)b Vtail(Å3)b σ (Å2) p

DDAB

C12E4

462 0.946 767 1.3 10.3 8.3 × 109 -3.9 × 109 16.7 700 64.1 0.65

362 1 635 6.5 10.3 6.5 × 109 -3.9 × 109 16.7 350 48c 0.44

a Dehydrated molecular volume is deduced from the density and molar mass according to Vm ) M/d Na, where Na is Avogadro’s number. b Length and volume of the fully extended chain estimated from Tanford.24 c Surface per headgroup taken from the surface tension measurements presented below.

phase region is present, where large spherulites of the lamellar phase LR at its swelling maximum are in equilibrium with the isotropic solution. In the range of 2.5-30%, a swollen lamellar phase LR is formed for the surfactant. Between 30 and 75%, the lamellar phase LR of 78 Å periodicity that is dominated by repulsive electrostatic forces is in equilibrium with a collapsed lamellar phase with L′R ) 31 Å periodicity and stabilized by the hydration force. Above 73%, a single L′R phase is formed. The bilayer thickness of the LR phase is 24 Å, which decreases to 22.6 Å in the L′R phase.6 The composition of the ternary system studied here is described by three parameters: c, the total concentration of surfactant; Rn, the molar ratio of DDAB to total surfactant concentration; and Φ, the total volume fraction of surfactant in the sample.

Rn ) c(mol/L) )

nDDAB nDDAB + nC12E4

nDDAB + nC12E4 VDDAB + VC12E4 + VD2O

Φ)

VDDAB + VC12E4 VDDAB + VC12E4 + VD2O

Experimentally, Rn was varied between 0 and 1: Rn ) 0 describes a sample containing only C12E4, and Rn ) 1 describes a sample with only DDAB. Sample Preparation. The samples were prepared by weighing the appropriate amounts of surfactant and water. H2O was use for the surface tension measurements. For the SANS experiments (and the phase diagram determination), samples were prepared in D2O in order to minimize the incoherent background and to increase the scattering cross section (larger contrast between surfactant and solution). The solutions were prepared in glass tubes that were sealed just after sample preparation and gently shaken and then were kept at rest at room temperature. The most dilute samples ( 0.3, there are three linear domains and two transitions points. The transition at the highest concentration, referred to here as the cac, is ∼100 times higher than the cmc. The cmc of the mixed system is reduced compared to the cmc’s of the pure surfactants in water. In contrast, the addition of a nonionic surfactant to the cationic system shifts the value of cac toward higher concentrations. The behavior of many mixed surfactant systems both in selfassembly in solution and in adsorption at the interface can be understood in the context of the departure from ideal mixing using regular solution theory (RST) as developed by Rubingh and others.2,38 The magnitude of nonideality between the two surfactants is expressed in terms of a single interaction parameter β. From this approach, the mixed cmc c* can be expressed as a function of the total surfactant concentration and cmc of the surfactants as

1 - xDDAB xDDAB 1 + ) c* fDDABcmcDDAB fC12cmc12E4

(11)

where xDDAB is the total molar fraction of DDAB in the system. Activity coefficients fDDAB and fC12E4 are given by fDDAB ) exp[β(1- xDDABm)2] and fC12E4 ) exp[ β(xDDABm)2] with xDDABm being the molar fraction of DDAB in the micelles. The variation in the cmc with composition is shown in Figure 2, and within the experimental error, eq 11 with an average interaction parameter β of -2.66 is consistent with the data. From the application of the Gibbs equation to the surface tension data below the first break point, it is possible to calculate the area per headgroup for C12E4 and DDAB and for the mixtures. The values for C12E4 and DDAB, respectively, are 48 and 64 Å2. These compare with an area per headgroup of 65 Å2 for DDAB, measured by the pendant drop method.39 It can also be compared to the value obtained with DHDAB, a surfactant with the same headgroup as DDAB but with two C16 alkyl chains of 62 Å2.10,40 The area per headgroup for C12E4 is higher than the value of 42 Å2 previously obtained for this surfactant by surface tension measurement41 inferred from measurements of the lamellar phase42 and measured directly by neutron reflectivity.43 The area/molecule values for DDAB and C12E4 and their associated mixtures (summarized in Table 2) are calculated from the Gibbs equation {Γ ) (1/mRT) dγ/d ln c} assuming m ) 1, as would be expected for nonionic surfactants. Calculating the area/molecule for DDAB and assuming m ) 2, as for ionic surfactants, gives an unacceptably large value of ∼128 Å2. Thomas et al.44 have recently discussed the significance of the Gibbs prefactor in the context of the adsorption of a range of different Gemini surfactants adsorbed at the air-water interface. Neutron reflectivity measurements provide a direct measure of the area/molecule and in combination with surface tension provide a numerical value for the relationship of the Gibbs equation with the adsorbed amount. Variations in the Gibbs prefactor were (38) Rubingh, D. N. In Solution Chemistry of Surfactants; Mittal, K. L., Ed.; Plenum Press: New York, 1979; Vol. 1. (39) Ricoul, F.; Dubois, M.; Zemb, Th.; Plusquellec, D. Eur. Phys. J. B 1998, 4, 333–340. (40) Penfold, J.; Sivia, D. S.; Staples, E.; Tucker, I.; Thomas, R. K. Langmuir 2004, 20, 2265–2269. (41) Kjellin, U. R. M.; Claesson, P. M.; Linse, P. Langmuir 2002, 18, 6745– 6753. (42) Acharya, D. P.; Kunieda, H. J. Phys. Chem. B 2003, 107, 10168–10175. (43) Lu, J. R.; Li, Z. X.; Su, T. J.; Thomas, R. K.; Penfold, J. Langmuir 1993, 9, 2408–2416. (44) Thomas, R. K. et al Langmuir 2008, in preparation.

Figure 2. Mixed cmc as a function of surfactant composition Rn. The solid line is an RST calculation for an interaction parameter β of -2.66.

rationalized in terms of factors due to dimer formation and finite ion binding. It is assumed that the DDAB surface tension data can be explained in such a way. Calculating the Isrealachvili et al.19 packing parameter, pp, for C12E4 and for DDAB using area/molecule values of 48 and 64 Å2 produces pp values of 0.44 and 0.65, respectively. Hence, this would imply that the preferred aggregate morphologies are highly elongated micellar structures for C12E4 and planar structures for DDAB. SANS Measurements. The existence of two distinct break points in the surface tension behavior suggests the formation of different kinds of aggregates in the bulk phase. The aim of the SANS measurements is to quantify the variation in the microstructure with surfactant composition and concentration and to relate the variations in the surface tension data above the cmc to variations in the microstructure of the aggregates in solution. The molar ratios of Rn ) 0.06, 0.13, 0.28, 0.35, 0.47, 0.58, 0.78, 0.84, 0.92, and 1 and four concentrations above the cac (2.5 × 10-2, 10-2, 5 × 10-3, 2 × 10-3 mol/L) and one concentration between the cmc and cac (5 × 10-4 mol/L) were measured in order to evaluate the phase behavior. As discussed earlier, the samples were prepared in D2O in order to improve the difference in scattering length densities between surfactant molecules and solvent (∆F ≈ 5 × 1010 cm-2) and to reduce the incoherent scattering coming from water. The samples with the higher surfactant concentrations (2.5 × 10-2 and 10-2 mol/L) were measured at the LLB. The higher flux of the D22 diffractometer at the ILL was required for the lower concentrations measured. A summary of all of the scattering data obtained is presented in the Supporting Information. Typical scattering curves are presented in Figures 3-8 for Rn ) 0.13, 0.78, 0.92, and 1. The variation in the form of the scattering data illustrates that the nature of the aggregates is strongly dependent on the surfactant composition and concentration. As a function of Rn, three different regions are distinguished where micelles, small vesicles, and large multilamellar vesicles (MLV) exist. In both regions where micelles or small vesicles are formed, the experimental form factor is modified by the structure factor at low q ( 3 × 10-2 Å-1. Inset: 2D plot of the raw data.

Figure 7. Multilamellar vesicle region: scattering intensities for Rn ) 0.84, T ) 25 °C; (black 3) c ) 50 mM, (gray +) c ) 25 mM, (black 4) c ) 10 mM, (blue 0) c ) 5 mM, (green O) c ) 2 mM, (red O) c ) 0.5 mM. The solid lines are the fitted curves, and the parameters are in Tables 3 and 5.

1300 Å. For Rn ) 1, the Caille´ parameter obtained from the Nallet-type analysis varies between 0.075 to 0.22 by increasing the concentration from 5 × 10-3 to 5 × 10-2 mol/L. The data at c ) 2 × 10-3 mol/L should be considered separately since they are at the lower limit of concentration of the MLV phase and the Bragg peak amplitudes are extremely low. In ref 34, η is found at 0.16 for a solution with Φ ) 4% DDAB in D2O. In a recent paper,37 the authors have investigated in a systematic way the elastic constants of the DDAB lamellar phase as a function of concentration and for different counterions, and they obtained values between 0.08 and 0.12. The magnitude of η is in broad agreement with our results. However, in detail there are some differences because the other systems relate to a true lamellar phase where the number of staked bilayers N is much higher than 100. The addition of the C12E4 nonionic molecules (up to 15 mol %) in the charged DDAB bilayers does not strongly modify η, which remains between 0.16 and 0.175. These relatively low values of η are characteristic of rigid bilayers, a result generally found for electrostatically stabilized lamellar phases. For comparison, in lamellar phases stabilized by thermal fluctuations

Figure 8. Scattering intensity of the sample Rn ) 0.84, c ) 25 mM displayed in Iq2 vs q to provide evidence of the structure factor of the lamellar phase. The red line is the fit obtained with the Nallet model.34 N ) 2, η ) 0.16, Table 5.

η is >0.5, in agreement with the predictions of Helfrich.50-52 A similar range of values was reported for DHDAB10 and for other dialkyl chain cationic/nonionic surfactant mixtures.11 It is possible that the MLV structure varies slightly with the sample preparation, agitation, or shearing and with time. Nevertheless, for the very similar system DHDAB/D2O,10 no aging was observed. It is expected that the addition of a nonionic cosurfactant would make such membranes less rigid and would eventually result in fluctuation-stabilized membranes, as discussed and demonstrated by Safinya et al.48,49 The addition of nonionic cosurfactants C12E6 and C12E12 to DHDAB13 resulted in an increase in the Caille´ (47) Feitosa, E.; Jansson, J.; Lindman, B. Chem. Phys. Lipids 2006, 142, 128– 132. (48) Safinya, C. R.; Sirota, E. B.; Roux, D.; Smith, G. S. Phys. ReV. Lett. 1989, 62, 1134–1137. (49) Safinya, C. R.; Roux, D.; Smith, G. S.; Sinha, S. K.; Dimon, P.; Bellocq, A. M. Phys. ReV. Lett. 1986, 57, 2718–2721. (50) Helfrich, W. Z. Naturforsch. 1978, 33a, 305–315. (51) Roux, D.; Safinya, C. R. J. Phys. France 1988, 49, 307–318.

3940 Langmuir, Vol. 25, No. 7, 2009

Figure 9. Typical autocorrelation function derived from 0.5 mM DDAB/ C12E4 with Rn ) 0.58 in D2O. The solid red line is a fit to the CONTIN fit from which the particle size distribution is determined. The error bar is smaller than the symbol.

parameter, which is indicative of a more flexible membrane. C12E12 was more effective than C12E6, consistent with a greater disruption of the membrane structure by the larger headgroup of C12E12. In contrast, C12E314 acts in a similar way to C12E4 and has little impact upon the membrane rigidity, which eventually results in phase separation. For Rn ) 0.92 and 0.84, intermediate concentrations (5 × 10-3, 2 × 10-3, and 10-3 mol/L) cannot be fitted with a simple model. The scattering curves shows the form factor of a small vesicle, with a characteristic oscillation and minimum at around 3 × 10-3 Å-1, on the top of which the typical oscillations from the MLV are superimposed at low q. Hence, in this region large MLV and small vesicles are in coexistence. Below the cac (c ) 5 × 10-4 mol/L), the samples are fully transparent, and no phase separation occurs even after several months at rest, and a strong decrease in the scattering intensity, indicative of the formation of much smaller aggregates, occurs. Nevertheless, an upturn at low q is present for Rn ) 1 and 0.92, which is an indication that larger aggregates are still present in solution. The SANS data and its interpretation have clearly provided evidence for the relationship between the surface tension properties and the nature of the aggregates formed in solution. It can be concluded that the second break point in the surface tension corresponds to the transition from micelles to small unilamellar vesicles or large multilamellar vesicles, which is referred to as a critical vesicle concentration (cvc). DLS Experiments. An example of the fit to the data and a typical volume-averaged particle size distribution (in this instance for 0.5 mM, Rn ) 0.58 DDAB C12E4 mixtures) are shown in Figures 9 and 10. The reproducibility of the particle size distributions obtained is excellent. Furthermore, they are in good agreement with the analysis of the SANS data. There are several observations that can be made from the light-scattering data. First, in the clear region the data are consistent with very small particles, and there is no indication of growth when proximate to a phase boundary. Second, in the region where coexistence is proposed between vesicles and bilamellar vesicles two components are consistently present in the particle size distributions. The smaller object is between 20 and 40 nm depending on the ratio of surfactants, Rn. The second and larger component in this two-phase region grows with increasing solution concentration and can be on the order of a micrometer. Although it is difficult to be precise in general, the number density of these larger objects increases with solution concentration whereas the

Grillo et al.

number density of the smaller objects changes less strongly with solution concentration. Overall, the light scattering is entirely consistent with the phase behavior derived from the analysis of the SANS data. Furthermore, it serves to corroborate the presence of small vesicles in these mixtures. Phase Diagram. The dilute part of the phase diagram for total surfactant concentration below 5 × 10-2 mol/L (Figure 11) is obtained by combining the results of visual inspection, surface tension, and SANS. The horizontal axis corresponds to the total concentration of surfactant, and the vertical one corresponds to the molar ratio Rn between the two surfactants. The white part represents the region where the samples are optically fully transparent and not birefringent. The gray parts correspond to samples that are optically turbid. In the upper part, the samples have a homogeneous milky aspect after preparation. A phase separation between a turbid, birefringent upper phase and a transparent bottom phase occurs after a few days at rest. This optically turbid region starts at concentrations just higher than the cvc. In the gray part at the bottom, the samples are turbid but not birefringent, and no phase separation occurs. In the nonionic-rich part, a single phase of globular micelles is present. They are relatively small (R2 ≈ 21-24 Å) and prolate. Their size increases with the concentration and number of C12E4 molecules. Between molar ratios of 0.3 and 0.8 and in a relatively narrow domain of concentrations (10-3 mol/L < c < 5 × 10-2 mol/L), small unilamellar and relatively monodisperse vesicles are formed. The mean radius (distance from the center to the midplane of the bilayer) varies between 40 and 100 Å. However, over much of this range there is lamellar (BLV, MLV)/small-vesicle coexistence. Above Rn ) 0.8, complex behavior is observed, which is characterized by the formation of large aggregates with a radius above 1000 Å. Depending on the concentration and Rn, one finds bilayer vesicles (BLV), multilamellar vesicles at the highest concentrations, and a region where BLV and small micelles are in coexistence. The phase diagram of DDAB/C12E4/D2O has broad similarities with the trends observed in the following ternary systems: DHDAB/C12E6/H2O,12 DHDAB/C12E12/H2O,13,14,53 SDBS/ C12E8/2 mM CaCl2, and SDBS/C12E23/2 mM CaCl2.54 In such systems, as a general tendency, solutions rich in charged dialkyl chain surfactants have lamellar structures (either a true LR phase, MLV, BLV, or ULV), and solutions rich in nonionic molecules are micellar. However, the unusual feature of this phase diagram is the occurrence of the region at low surfactant concentrations and at intermediate compositions (Rn ) 0.3-0.8) where the microstructure consists of very small relatively monodisperse vesicles.

General Discussion The existence of a cmc and a cvc in the dilute binary system DDAB/water has not been previously demonstrated as clearly in the literature. Some papers discuss a cac, but in most of cases only one transition point is observed and the reported values are 0.047555 and 0.05 mM,56,57 in good agreement with the value reported here (0.0463 mM). By conductivity measurements, Junquera et al. reported a cvc at 0.165 mM but did not detect a cmc.58 More recently conductivity measurements59,60 reported (52) Grillo, I.; Levitz, P.; Zemb, Th. Eur. Phys. J. E 2001, 5, 377–386. (53) Tucker, I. DPhil Thesis, Oxford, U.K., 2007. (54) Penfold, J.; Thomas, R. K.; Dong, C. C.; Tucker, I.; Metcalfe, K.; Golding, S.; Grillo, I. Langmuir 2007, 23, 10140–10149. (55) Caria, A.; Regev, O.; Khan, A. J. Colloid Interface Sci. 1998, 200, 19–30. (56) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M.; Lindman, B. J. Phys. Chem. B 1999, 103, 8353–8363. (57) Viseu, M. I.; Vela´zquez, M. M.; Campos, C. S. Langmuir 2000, 16, 4882–4889.

Spontaneous Formation of NanoVesicles

Langmuir, Vol. 25, No. 7, 2009 3941

Figure 10. Particle size distribution obtained from an analysis of the experimentally determined autocorrelation function presented in Figure 9, fitted to a Rayleigh Gans Debye model. The different colors are the results of three different experiments.

Figure 11. Phase diagram for the dilute part of the ternary system DDAB/ C12E4/D2O. The gray part delimits the optically turbid samples that separate into two phases a few days after the preparation. The white parts are domains where the samples are optically transparent. The symbol (+) indicates points where SANS measurements have been made. L1 is a micellar phase, V is the domain where small vesicles are formed, BLV is the region of bilayer vesicles, and MLV is the region of multilamellar vesicles. The red squares indicate the cvc.

a first transition at 7.8 × 10-2 mM and a second at 0.6 mM, in relatively good agreement with the values reported here. However they attributed the first point to the cvc and the second one to the growth and formation of giant vesicles. Pulsed field gradient NMR measurements56 have shown a fast and a slow diffusing component above the cac (0.05 mM) and below 3 mM, and this was interpreted as the coexistence of micelles and vesicles. The differences in the results reported in the literature for the same binary system show how the values obtained for the transition can depend on the technique used, and this highlights the need for complementary techniques to characterize the structure of aggregates. Between the cmc and the cvc, the lightscattering and microscopy techniques that are frequently used are not sensitive to the size of the micelles and so are not detected. The SANS measurements using a very high flux spectrometer such as D22 was able to demonstrate the formation of small micelles and thus identify the existence of a true cmc in the DDAB/D2O system as predicted by Marques et al.56 The existence of two transitions in the surface tension is also reported in several other mixed systems containing DDAB, for example, in (58) Junquera, E.; Arranz, R.; Aicart, E. Langmuir 2004, 20, 6619–6625. (59) Segota, S.; Heimer, S.; Tezak, D. Colloids Surf., A 2006, 274, 91–99.

cationic-cationic mixtures: DDAB/DTAC/water,57,61,62 DDAB/ C12EDMAB/water,58 in a catanionic mixture DDAB/SDBS/ water,59 and in cationic-nonionic systems DDAB/OGB/water.63 However, the nature of the different aggregates and their sizes are not always clearly established. The formation of microaggregates (50-200 Å) at the first transition and their growth in size after the second transition (∼2000 Å) are found in refs 58 and 63. In refs 57 and 61, the sequence monomers f vesicles f micelles is reported. The vesicles, characterized by DLS and cryo-TEM, are relatively large from 400 to 6000 Å with a high polydispersity in size. The existence of two break points is hence not a classical behavior, and only a few other examples are found in the literature.64,65 The fact that this feature is frequently obtained in the presence of DDAB suggests that it is strongly linked to the particular nature of the DDAB molecules. There are few examples of small vesicles with a radius below 100 Å reported in the recent literature. They have been observed in a mixture of Lecithin and bile salt,66 in the ternary system DDAB/SDS/water,67 and the in quaternary systems TDMAO/ HCl/hexanol/water68 and triton X-100/octanol/CPCl/water.69 These small vesicles are often not present in a single phase but are in coexistence with other types of aggregates as multilamellar vesicles or micelles. Their region of existence is in general extremely small. In the present study, the dilution of the surface charge induces a high bending of the bilayers, whereas Oberdisse et al.69 reports the formation of small vesicles in the nonionicrich part of their phase diagram. However, the small size of vesicles and their relatively narrow polydispersity would imply, following the discussion of Oberdisse et al.69,73 and Jung et al.,74,75 a relatively high bending elasticity and a finite spontaneous (60) Soltero, J. F. A.; Bautista, E.; Pecina, E.; Puig, J. E.; Manero, O.; Proverbio, Z.; Schulz, P. C. Colloid. Polym. Sci. 2000, 278, 37–47. (61) Viseu, M. I.; Edwards, K.; Campos, C. S.; Costa, S. M. B. Langmuir 2000, 16, 2105–2114. (62) Treiner, C.; Makayssi, A. Langmuir 1992, 8, 794–800. (63) Junquera, E.; del Burgo, P.; Arranz, R.; Llorca, O.; Aicart, E. Langmuir 2005, 21, 1795–1801. (64) Han, F.; He, X.; Huang, J.; Li, Z.; Wang, Y.; Fu, H. J. Phys. Chem. 2004, 108, 5256–5262. (65) Fan, Y.; Cao, M.; Yuan, G.; Wang, Y.; Yan, H.; Han, C. C. J. Colloid Interface Sci. 2006, 299, 928–937. (66) Hjelm, R. P.; Thiyagarajan, P.; Alkan, H. J. Appl. Cryst. 1988, 21, 858– 863. (67) Bergstro¨m, M.; Pedersen, J. S. J. Phys. Chem. B 2000, 104, 4155–4163. (68) Beck, R.; Gradzielski, M.; Horbascheck, K.; Shah, S. S.; Hoffmann, H.; Strunz, P. J Colloid Interface Sci. 2000, 221, 200–209. (69) Oberdisse, J.; Porte, G. Phys. ReV. E 1997, 56, 1965–1975. (70) Agarwal, V.; Singh, M.; McPherson, G.; John, V.; Bose, A. Colloids Surf., A 2006, 281, 246–253. (71) Lee, J.-H.; Agaewal, V.; Bose, A.; Payne, G. F.; Raghavan, S. R. Phys. ReV. Lett. 2006, 96, 048102.

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Grillo et al.

curvature. The finite spontaneous curvature probably arises from nonideal mixing between the two layers of the vesicle bilayer, resulting in anisotropy in packing and in charge. To that extent, the results presented here are consistent with the arguments of Jung et al.74,75 in that the narrow polydispersity and the relatively rigid membrane (high bending elasticity) are the criteria for forming small unilamellar vesicles. Although it is likely that anisotropy in the packing across the bilayer, resulting also in asymmetry in the charge interaction, is responsible for the required spontaneous curvature. However, this has not been measured and will be the subject of future studies. Others76-79 have discussed spontaneous vesicle formation in charged bilayers, lamellar-to-vesicle transitions, and the contribution of the electrostatic interaction to the membrane bending elasticity and spontaneous curvature. In particular, the role of different counterion screening in the inner and outer parts of the bilayer has been discussed,77,78 and this remains an additional factor. Measurements with different counterions and different electrolyte concentrations would help to discriminate between these different factors and are the subject of future work. The existence of large unilamellar vesicles is widely reported in the literature, and in contrast, there are relatively few examples of bilayer vesicles reported. In the dilute region (c < 60 mM) of binary system DHDAB/D2O,10 the existence of BLV has been confirmed by SANS, USANS, and light scattering, with very similar behavior to that of DDAB/D2O. The radius of the particles is on the order of 1400 Å at the maximum swelling, nearly twice the lamellar spacing measured at 850 Å. The addition of C12E12 molecules to DHDAB induces the coexistence of BLV and micelles;13 BLV formation has been evidenced by both scattering techniques and cryofractures in an aqueous solution of CTAB and phenol derivatives70 after the addition of a biopolymer to the CTAT/SDBS vesicle phase71 and in aqueous solutions of SDBS and imidazoline mixtures.72 In these three examples, the BLVs have radii on the order of 1000 to 2000 Å and exhibit relatively high size polydispersity, similar to that of the structures reported here for DDAB. A feature of the vesicle region of the phase diagram is that the Caille´ parameter is consistent with that expected of a relatively rigid membrane in which the charge interaction dominates. It would be expected that the addition of a nonionic cosurfactant (such as the C12E4 added here) would result in a more flexible membrane in which thermal fluctuations would eventually dominate, as reported by Safinya et al.48 This is not observed here and the Caille´ parameter is relatively insensitive to the addition of C12E4. A similar trend was also reported for DHDAB/ C12E3,14 and was attributed to reduced electrostatic screening as a result of the reduced counterion concentration as the cationic is replaced by the nonionic cosurfactant. The shape of aggregates can be qualitatively understood in a rough approximation by the variation of the average packing parameter pav. For mixed aggregates, pav is expressed as (72) Gonza´les, Y. I.; Stjerndahl, M.; Danino, D.; Kaler, E. W. Langmuir 2004, 20, 7053–7063. (73) Oberdisse, J. Eur. Phys. J. B 1998, 3, 463–469. (74) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Lampietro, D. J.; Kaler, E. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1353–1357. (75) Jung, H. T.; Lee, S. Y.; Kaler, E. W.; Coldren, B.; Zasadzinski, J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15318–15322. (76) Mitchell, D. J.; Ninham, B. W. Langmuir 1989, 5, 1121–1123. (77) Lekkerkerker, H. N. W. Physica A 1989, 159, 319–328. (78) Fogden, A.; Hyde, S. T.; Lundberg, G. J. Chem. Soc., Faraday Trans. 1991, 87, 949–955.

pav )

∑ Vixiagg i

∑ i

lixiagg

∑ a0ixiagg

(12)

i

agg i

x is the molar fraction of compound i in the aggregate, which can be calculated using RST for a given interaction parameter β. For the DDAB/C12E4/D2O system, pav varies between 0.44 (pure C12E4) and 0.65 (pure DDAB) and is higher than 0.5 for Rn > 0.25. This is in broad qualitative agreement with the phase behavior (Figure 11) where the vesicles and thus the formation of the bilayer are observed for Rn > 0.3. The packing parameter allows one to predict the shape of the aggregates but does not give any information on their sizes. Furthermore, it does not explicity treat the charge and variations in the charge with mixing. However, in a sense this is implicitly included because the variation in the area/molecule reflects the changes in the headgroup interaction between the charged and uncharged extremes. Oberdisse et al. have developed a quantitative electrostatic cell model69 based on a numerical solution of the PoissonBoltzmann equation. The only free parameter is the intrinsic bending elasticity modulus introduced to explain the instability of the flat bilayer in favor of small highly curved vesicles. This model gives good agreement with the experimental data and explains the size increase with the increase in surfactant concentration, as observed in the DDAB/C12E4/D2O system. The mixture of surfactants with packing parameters corresponding to different kind of aggregates results in complex phase diagrams. The mixed aggregates adopt intermediary shapes, consistent with the packing constraints. Several studies done with a charged surfactant having a preferred flat curvature and a nonionic surfactant forming globular micelles exhibit similar phase succession from globular micelles (which are more or less elliptic) to vesicles and lamellae; the extent of the different domains varies with the nature of the surfactants.10,12,14 In the system SDBS/C12En, larger headgroups induce higher curvature, and the micellar phase persists until a richer charged surfactant ratio is reached.54 The coexistence of lamellae (vesicles) and micelles has been demonstrated in a range of different systems. There are striking similarities here with the formation of biomembranes, where the main ingredients are dialkyl chain lipids, and membrane solubilization studies. In their pioneering work, Gershfeld et al.80,81 established the criteria for the spontaneous formation of phospholipids ULV and MLV above a critical solution temperature and their transition from micelles to vesicles. In the context of membrane solubilization studies,82 the solubilization of membranes by a range of ionic and nonionic surfactants has been investigated, resulting in the disruption of the membrane and the formation of mixed micelles and mixed lamellar/micellar coexistence. One of the many examples of this is the work of Ko¨nig et al.,83 where the lamellar/micellar transition upon the addition of C12E5 to DMPC is described. In general, however, detailed structural information is not available. An exception to this is the work of Ricoul et al.84 on DDAB/glycolipid mixtures. The DDAB/C12E4/D2O phase diagram can be closely compared to that work in which DDAB/LS (laurylsaccharose)/ water is established for slightly higher concentrations and where the different aggregates are in osmotic equilibrium with a swollen (79) Radlinska, E. Z.; Zemb, Th. N.; Dalbiez, J.-P.; Ninham, B. Y. W. Langmuir 1993, 9, 2844–2850. (80) Gershfeld, N. L.; Stevens, W. F., Jr.; Nossal, R. J. Faraday Discuss. Chem. Soc. 1986, 81, 19–28. (81) Gershfeld, N. L.; Mudd, C. R.; Tajima, K.; Berger, R. L. Biophys. J. 1993, 65, 1174–1179. (82) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146–163. (83) Ko¨nig, S.; Me´le´ard, P.; Roux, D. NuoVo Cimento D 1994, 16, 1585–1594.

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lamellar phase.84 The large polar volume of LS induced the formation of small ellipsoidal micelles for a molar ratio between surfactants of Rn ) [DDAB]/{[DDAB] + [LS]} < 0.5. For Rn ) 0.58, Φ ) 0.5%, the formation of very small vesicles is also found. The phase diagram for higher Rn was not studied. They described quantitatively the transition from a swollen lamellar phase to micelles, the lamellar/micellar coexistence, and the swelling behavior in terms of the osmotic pressure in the coexisting phases. Given the more complex phase behavior observed here and, in particular, the existence of the very small vesicle region, it is not clear that such an approach would be applicable here. Radlinska et al.85 have also reported the coexistence of vesicles and micelles of DDA salts with hydroxide and carboxylates as counterions. It is clear that the formation of very small unilamellar vesicles is a feature that is unique to the dialkyl C12 chain surfactant/ cosurfactant system presented here.

properties correspond to strong structural changes in the solution behavior. The SANS measurements carried out on high-flux SANS diffractometer D22 have allowed us to study very dilute samples and to identify the first transition as a cmc and the second one as a cvc. For DDAB in D2O, it is the first time to our knowledge that the formation of micelles before the multilamellar phase is clearly demonstrated by scattering techniques. With increasing DDAB concentration and composition for DDAB/C12E4, aggregates are formed in the following sequence of globular prolate micelles f very small vesicles f multilamellar large vesicles. Geometrical constraints associated with changes in the average packing parameter can be used to rationalize the succession of phase sequences. These different types of aggregates are formed spontaneously and are stable over a period of several months. The study of their formation and growth by a real experiment time experiment to follow the early stages of formation will be reported in a forthcoming paper.

Summary

Acknowledgment. The Laboratoire Le´on Brillouin (Saclay, France) and the Institut Laue Langevin (Grenoble, France) are acknowledged for the beam time allocated on PAXY and D22.

The dilute part of the phase diagram of the ternary system DDAB/C12E4/D2O has been characterized using a combination of surface tension and SANS. The first feature of this system is the evidence of two break points in the surface tension for molar ratios higher than 0.3. These transition points in the surface (84) Ricoul, F.; Dubois, M.; Zemb, T.; Plusquellec, D. Eur. Phys. J. B 1998, 4, 333–340. (85) Radlinska, E. Z.; Ninham, B. Y. W.; Dalbiez, J.-P.; Zemb, Th. N. Colloids Surf. 1990, 46, 213–230.

Supporting Information Available: General overview of the experimental SANS profiles for the ternary system DDAB/C12E4/D2O measured at the ILL and LLB, with Rn ) 0.00, 0.13, 0.2, 0.28, 0.37, 0.47, 0.58, 0.78, 0.85, 0.92, and 1. This material is available free of charge via the Internet at http://pubs.acs.org. LA802435H