J. Phys. Chem. B 2006, 110, 10177-10185
10177
Charge-Induced Unilamellar Vesicle Formation and Phase Separation in Solutions of Di-n-Decylmethylamine Oxide Hideya Kawasaki,† Vasil M. Garamus,‡ Mats Almgren,§ and Hiroshi Maeda*,† Department of Chemistry, Faculty of Sciences, Kyushu UniVersity, Fukuoka 812-8581, Japan, GKSS Research Centre, Max-Planck Street, D-21502 Geesthacht, Germany, and Department of Physical Chemistry, Uppsala UniVersity, Box 579, S 751-23 Uppsala, Sweden ReceiVed: March 2, 2006; In Final Form: March 28, 2006
A double-tail amine oxide surfactant, di-n-decylmethylamine oxide (2C10MAO), was prepared, and the effects of protonation on aggregate structure were examined by small-angle neutron scattering (SANS), cryotransmission electron microscopy (cryo-TEM), turbidity, electric conductivity, and solubilization of an oilsoluble dye at various degrees of neutralization, X, defined as the mole ratio of HCl/2C10MAO. The surfactant makes an L2 phase in the nonprotonated state (X ) 0) in water. The L2 phase is in equilibrium with an aqueous L1 phase. On protonation, unilamellar vesicles (ULVs) are formed over a wide range of compositions (0.05 < X< 0.4-0.5 at C ) 10 mM) as observed by cryo-TEM. At X ) 0.2, the ULV is stable over a wide concentration range (3 mM e C < 0.1 M), but an LR phase replaces the vesicle phase at C > 0.1 M. SANS results show that the mean radius of the ULV is about 25 nm and the bilayer thickness is about 2 nm, consistent with the extended configuration of the alkyl chains of the surfactant. An important contribution to the enhanced stability of the bilayer structures over the L2 phase is suggested to be the translational entropy of the counterions. The enhanced stability of the bilayers diminishes as the counterion concentration increases either by an increase of X or by the addition of a salt. When the counterion concentration exceeds a critical value, the ULV solutions transform into the L2 phase (or L2/L1 two-phase system at low surfactant concentrations). The critical composition X* is about 0.4-0.5 in water, but it is below 0.4 in D2O. The critical NaCl concentration is below 5 mM at X ) 0.2. The stability of ULVs against multilamellar vesicles is ascribed partly to undulation forces and partly to the adjustable nature of the spontaneous curvature of amine oxide monolayers. The characteristics of the ULV of the surfactant remain the same within a temperature range 25-50 °C at X ) 0.2. An iridescent lamellar phase and possibly an L3 phase were observed in a very narrow X range (0 < X < 0.02) prior to the vesicle phase.
Introduction Liposomes for uses as pharmaceutical carriers should be robust structures, with little leakage, long circulation times, and long shelf lives.1 Typically, such liposomes are made from phospholipids having a large fraction of long saturated acyl chains, mixed with a large fraction of cholesterol, and stabilized by polyethylene glycol (PEG) lipids. Such liposomes are kinetically trapped structures and described as dispersions of a lamellar phase in an aqueous solution. In other systems vesicles form spontaneously, for instance, from surfactants such as salts of carboxylic acids on a decrease of the pH,2 from various surfactants mixed with alcohols,3 or from mixtures of cationic and anionic surfactants.4 Sometimes it is claimed that a system of spontaneously formed vesicles also represents an equilibrated state. Although vesicles may well be equilibrium structures in some systems, it is difficult to prove it.5 It is, however, often easy to show that they are not equilibrated. The vesicle size distribution must be independent of its history, otherwise equilibrium does not prevail.6 Vesicles in true dynamic equilibrium would disappear and reform * Author to whom correspondence should be addressed. Phone: +81 92 681 8080. E-mail:
[email protected]. † Kyushu University. ‡ GKSS Research Centre. § Uppsala University.
continuously and thus have a limited lifetime as individual entities. Their usefulness as drug carriers would then, probably, be rather limited. However, vesicles that form and disintegrate as a response to changes in some controllable factor, such as pH or temperature, would be highly desirable. Different ways to make drug carrier liposomes pH- or temperature-sensitive are actively investigated as methods to make the carriers deliver their load in a controlled way. Of particular interest are pHsensitive vesicles that allow encapsulation and targeted release of other substances. A number of different amphiphiles carrying pH-sensitive groups such as gemini surfactants,7 fatty acids,8 double-chain sodium bis-(2-ethylhexyl) phosphate,9 and mixtures of histidine and sodium dodecyl benzenesulfonate10 can form vesicles in different pH regions. Alkylamine oxide surfactants can be protonated to various degrees. In oleydimethylamine oxide solutions a reversible change from threadlike micelles to a mixture of vesicles and bilayers occurs at the half-protonated state (R ) 0.5).11 Vesicles (unilamellar and multilamellar) also form spontaneously by the addition of sodium 2-naphthalenesulfonate to micellar solutions of half-protonated tetradecyldimethylamine oxide.12 In the present study, we synthesized a new double-tail alkylamine oxide surfactant, di-n-decylmethylamine oxide (2C10MAO), which exists as either a nonionic or cationic
10.1021/jp061335r CCC: $33.50 © 2006 American Chemical Society Published on Web 05/04/2006
10178 J. Phys. Chem. B, Vol. 110, No. 20, 2006 (protonated form) species depending on the pH of the aqueous solutions. We have investigated the vesicle formation of 2C10MAO as a function of the degree of protonation and the surfactant concentration by means of small-angle neutron scattering (SANS), cryo-transmission electron microscopy (TEM), dynamic light scattering (DLS), conductivity, and turbidity measurements. Effects of temperature and salt addition were also examined. Here we report the formation of small unilamellar vesicles (〈R〉 ≈ 25 nm) in the ternary system 2C10MAO/ HCl/water, which requires only gentle manual shaking. The vesicles are stable over a wide concentration range (3-75 mM) and a temperature range 25-50 °C for a few months and show moderate polydispersity (σR ) 0.25-0.40). The vesicles are disrupted by further addition of HCl followed by phase separation into two solution phases. Recently, a study on another double-tail amine oxide, dodecyloctylmethylamine oxide, was reported from the Hoffmann group.13 Experimental Section Sample Preparation. 2C10MAO surfactants were synthesized from di-n-decylmethylamine (Kanto Chemical Co.) through the oxidation in ethanol by hydrogen peroxide following, for the most part, the similar procedure as for other alkyldimethylamine oxides, and they were purified by the extraction of the unreacted amine with hexane and recrystallized two times from hot acetone. The purity of 2C10MAO samples was confrimed by 1H NMR (>98% in purity). Samples of different degrees of protonation (R) were prepared as follows: The prescribed amounts of HCl in H2O were added to the nonionic 2C10MAO solution in H2O. In the present study, the degree of neutralization X defined as the mole ratio n(HCl)/n(2C10MAO)is used in place of R. Turbidity. Turbidity measurements were performed at 25 °C with a Jasco Ubest-50 UV-vis spectrophotometer, equipped with a thermostat cell holder and a magnetic stirring device, using quartz cells of 1 cm path length. Turbidity was measured at 400 nm and expressed in transmittance. Small-Angle Neutron Scattering. Small-angle neutron scattering experiments were made with the SANS-1 instrument at the FRG1 research reactor at the GKSS Research Centre, Geesthacht, Germany.14 Four sample-to-detector distances (from 0.7 to 9.7 m) were employed to cover the range of scattering vectors q from 0.005 to 0.25 Å-1. The neutron wavelength λ was 8.1 Å with a wavelength resolution of 10% (full width at full maximum). The solutions were prepared in D2O (99% purity). The samples were kept in quartz cells (Hellma) with a path length of 1, 2, and 5 mm depending on the concentration of 2C10MAO. The samples were placed in a temperature-controlled holder, for isothermal conditions T ) 25.0 ( 0.5 or 50.0 ( 0.5 °C. The raw spectra were corrected for the background from the solvent, sample cell, and other sources by conventional procedures.15 The two-dimensional isotropic scattering spectra were azimuthally averaged, converted to an absolute scale, and corrected for detector efficiency by dividing by the incoherent scattering spectra of pure water that was measured with a 1-mmpath-length quartz cell. The average excess scattering length density per unit mass ∆Fm of the surfactant in deuterated water was determined from the known chemical composition (∆Fm ) -5.14 × 1010 cm/g). The smearing induced by the different instrumental settings is included in the data analysis. For each instrumental setting the ideal model cross section was smeared by the appropriate resolution function when the model
Kawasaki et al.
Figure 1. Turbidity (expressed as transmittance, T, at 400 nm) of the 2C10MAO aqueous solutions at 25 °C as a function of the degree of neutralization X. The surfactant concentration is fixed to 10 mM. Vertical dotted lines a and b indicate approximate positions of the lower bound of the ULV region and of the phase separation, respectively.
scattering intensity was compared to the measured one by means of least-squares methods.1 The parameters in the models were optimized by conventional least-squares analysis, and the errors of the parameters were calculated by conventional methods.17 Cryo-Transmission Electron Microscopy. The electron microscopy investigations were performed with a Zeiss 902 A instrument, operating at 80 kV. Specimens were prepared by a blotting procedure, performed in a chamber with controlled temperature and humidity. A drop of the sample solution was placed onto an electron microscopy (EM) grid coated with a perforated polymer film. Excess solution was then removed with a filter paper, leaving a thin film of the solution on the EM grid. Vitrification of the thin film was achieved by rapid plunging of the grid into liquid ethane held at its freezing point. The virtrified specimen was then transferred in a cold stage to the microscope and investigated at 108 K.18 Dynamic Light Scattering. The average size of the vesicles was measured by an ELS-8000 (Ootsuka Electronics Co.) with a 633 nm wavelength He-Ne laser as the light source using a square cell for the dynamic light-scattering mode. Sample solutions were introduced into the cell after being filtered through a Millipore membrane with pore size of 0.45 µm. The time-dependent autocorrelation function of the scattered light intensity was measured at a scattering angle of 90° after the filtration of the solutions. The average vesicle diameters were obtained with the method of Marquadt. The DLS measurements were usually repeated at least three times, and the average values are reported. Results Phase Behavior. Figure 1 shows the turbidity of the 2C10MAO aqueous solutions at 25 °C (a fixed surfactant concentration, C )10 mM) as a function of the degree of neutralization X defined as the mole ratio n(HCl)/n(2C10MAO), which is close to the degree of ionization or protonation R except at low concentrations or at X values close to or greater than unity. For curved bilayers, the degree of ionization generally differs for outer and inner monolayers at a given X. Nonionic 2C10MAO surfactants (X ) 0) are insoluble in water, and a liquid-liquid phase separation occurs. The same behavior was observed at 4 °C, and hence the Krafft temperature is supposed to be below 4 °C. The top layer is slightly turbid and can solubilize an oil-soluble dye Sudan III, while the bottom layer is a colorless transparent solution (Figure 2a). The two phases are both isotropic as examined with a polarizing optical
ULV Formation and Phase Separation in 2C10MAO
J. Phys. Chem. B, Vol. 110, No. 20, 2006 10179
Figure 2. Pictures of 2C10MAO aqueous solutions at 25 °C in the presence of Sudan III: (a) X ) 0; (b) X ) 0.2.
microscope. Thus, at X ) 0, two phases of L2 and L1 are likely to coexist. A slight increase of X induces a change from the two-phase state to single phase with iridescent colors, which is likely to be a lamellar phase with the spacings between the lamellar layers of several hundred nanometers. The region is observed only in a very narrow range of X, X < 0.02. Around X ) 0.02, an unresolved multiple phase region was observed in which the L3 phase seems to be present as suggested by TEM results to be discussed below. On a further increase of X, the turbidity decreases dramatically. Results of DLS in the region show the presence of large aggregates (C ) 10 mM, X ) 0.2). A crude estimate of the diameter of the particles without correction for electric interaction was about 200 nm. The scattering intensity correlation curves did not change significantly over 62 days (Supporting Information, Figure S1), suggesting a stable aggregate size distribution. In the region, the whole solution is uniformly colored when Sudan III is added (Figure 2b). Under a polarizing optical microscope, no objects were observed. In the region of X larger than 0.4-0.5, however, a phase separation occurs despite an increase of the ionization of the aggregates. The critical composition for the phase separation is denoted as X*. The two solution phases appear isotropic on examination with a polarizing microscope. The separation of phases occurred very slowly and was accelerated either by
Figure 3. Cryo-transmission electron micrographs of 2C10MAO aqueous solutions (X ) 0.2) at different surfactant concentrations, C: (a) C ) 10 mM; (b) C ) 50 mM; (c) C ) 100 mM. The bar in the figure corresponds to 100 nm.
warming or by adding salt. After the two solutions separated, only the top solution phase (likely to be an L2) became colored when Sudan III was added (not shown), but the bottom solution remained clear and colorless, suggesting that few aggregates such as micelles or vesicles were present. It is rather unusual that phase separations are induced with increasing charge, and the two phases remain separated even at X ) 1. There is a possibility that the degree of ionization of the L2 phase is significantly lower than unity even at X ) 1. The hydrogen ion dissociation property of amine oxides in the L2 phase is not known. Cryo-TEM Pictures. Figure 3 shows cryo-TEM micrographs for the 2C10MAO solutions at different surfactant concentrations
10180 J. Phys. Chem. B, Vol. 110, No. 20, 2006
Figure 4. Cryo-transmission electron micrographs of 2C10MAO aqueous solutions of C ) 100 mM at X ) 0.2. The bar in the figure corresponds to 100 nm.
(C) for X ) 0.2. Similar large unilamellar vesicles (ULVs) are observed at C ) 10 mM and 50 mM, while deformed and even tubular vesicles are seen at C ) 0.1 M. This is probably mainly due to the crowding of the vesicles at the higher concentration. The wide concentration range in which ULVs or a “vesicle phase” exists is undoubtedly one of the characteristics of 2C10MAO. At the highest concentration examined, 0.1 M, bilamellar vesicles with connections between the two bilayers, interlamellar attachments (ILA) or passages, are found as shown in Figure 4. Figure 5 shows cryo-TEM pictures of 2C10MAO solutions (C ) 10 mM) at different X values. At X ) 0.02, sometimes very large structures with a dense interior, sometimes only ULVs, are seen. The inverted structures look similar to the L3
Kawasaki et al. phase particles reported from sodium cholate/glycerol monooleate mixtures.19 These large structures may be more prevalent than it appears from the cryo-TEM study, since they are so large that they may often escape the holes in the polymer film. The exact phase behavior around X ) 0.02 is not well resolved at present. At X ) 0.2, only large ULVs are observed. At X ) 0.4, ULVs are observed in most cases, but filled particles that are radiationsensitive are observed occasionally. These particles will be related to the L2 phase, and hence X ) 0.4 is expected to be close to X*. At X ) 1, radiation-sensitive filled particles are seen in cryo-TEM pictures of the top solutions. It is surprising that dense particles are formed in this case, nominally the most highly charged state. Cryo-TEM observations indicate that the L2 phase is dispersed in the L1 phase as globular particles. To reduce the interfacial tension between the L1 and the L2 phases, a fraction of surfactant (mostly cationic species) may adsorb onto the interface and act as an emulsifier. The “emulsion” state is stable partly because the density difference between the aqueous medium and the particles is small due to the water pool inside the particles and partly because the particles are charged. This suggestion would explain why the separation of the two phases is slow. In summary, cryo-TEM observations support the suggested phase sequence, provide evidence of ULVs, and indicate the dispersed nature of the L2 phase in the L1 phase in the two-phase region at X > 0.5. SANS Measurements. We have examined the aggregate structures of 2C10MAO in the low turbidity region in Figure 1 by SANS. Figures 6 and 7 show SANS profiles of 2C10MAO solutions (X ) 0.2) at different concentrations from 3 to 300 mM. According to cryo-TEM and DLS at X ) 0.2, one should observe ULVs. Concentration dependence can be separated into two regions: ULVs (2-75 mM) and the lamellar phase (0.10.3 M). For all concentrations in the ULV region the typical slope of q-2 is observed, and scattering intensities normalized to the concentration of 2C10MAO practically overlap at the intermediate and large q regions (q > 0.02 Å-1). This observa-
Figure 5. Cryo-transmission electron micrographs of the 2C10MAO aqueous solutions of C ) 10 mM at different x values: (a) X ) 0.02; (b) X ) 0.2; (c) X ) 0.4; (d) X ) 1. The bar in the figure corresponds to 100 nm.
ULV Formation and Phase Separation in 2C10MAO
Figure 6. SANS profiles of 2C10MAO solutions (X ) 0.2) in D2O at different concentrations, C: C ) 3, 10, 20, 50, 75 mM.
Figure 7. SANS profiles of 2C10MAO solutions (X ) 0.2) in D2O at different concentrations, C: C )100, 200, 300 mM.
tion indicates constant characteristics of bilayers of ULVs over a concentration range: The thickness and the mass density of the bilayer and the size of the vesicles do not change with concentration. This finding of the constant nature of the bilayers is important, since the vesicles appear to change their shapes significantly with the concentration according to the cryo-TEM micrographs (Figure 3). The reason for the irregular shapes observed in the cryo-TEM is probably the repulsive interactions between the vesicles squeezed into the thin aqueous film. Deviations of curves at the lowest q region can be attributed to interactions among vesicles. Samples with C > 0.1 M exhibit Bragg maxima (Figure 7), which is typical of the lamellar phase (LR). The lamellar repeat distance d varies linearly with C-1, from 176 to 727 Å with decreasing concentration from C ) 0.3 M to C ) 0.1 M. In contrast, the scattering profiles for C < 75 mM do not exhibit sharp Braggs peaks, with the scattering intensity decaying as q-2. At 0.1 M, however, TEM pictures (Figures 3 and 4) show that vesicles and lamellar structures are not well observed. This discrepancy is probably caused by different actions of D2O and H2O. At C ) 10 mM, the scattering profiles at different X (0.05, 0.1, 0.2, and 0.3) values are rather similar (Supporting Information, Figure S2). At X ) 0.4, phase separation occurs, and the
J. Phys. Chem. B, Vol. 110, No. 20, 2006 10181
Figure 8. SANS profiles of 2C10MAO solutions (X ) 0.2) in D2O at different temperatures, T: T ) 25, 50 °C.
scattering intensity decays as q-4 in the range q j 0.03 Å when measured on a dispersion prepared by mixing the two separated solutions. Thus, in D2O X* is smaller than 0.4 but larger than 0.4 in H2O. Effects of Salt and Temperature. Vesicle solutions at X ) 0.2 undergo phase separation when NaCl is added to a concentration of 5-10 mM. SANS measurements were carried out on the dispersions prepared by mixing the two separated solutions (Supporting Information, Figure S3). In the low-q range (q < 0.02 Å-1), the scattering intensity decays as q-4, while there is a correlation peak near q ≈ 0.1 Å-1 that is absent for the two-phase solutions without NaCl. A slope of -4 at low-q values means that the dispersed particles are large R > 1000 Å and that the interface between the particles and the solvent is smooth (nonfractal) and sharp. Effects of temperature were examined on solutions of X ) 0.2 (Figure 8). At 10 mM, the scattering curves are almost identical at 25 and 50 °C, indicating that the ULVs of 2C10MAO are stable. At 0.1 M, on the contrary, the lamellar phase found at 25 °C transforms into ULVs at 50 °C. Reduction of Bilayer Structural Parameters by Data Analyses. First of all we have analyzed the parameters of vesicle bilayers. The interval of q from 0.02 to 0.3 Å-1 was analyzed by the indirect Fourier transformation (IFT) method developed by Glatter with the version of Pederesen.20,21 Here we assumed that effects of intervesicle interactions are negligible in this q interval. The scattering intensities for infinitely large disklike aggregates are written in the form of the thickness pair distance distribution function pT(r)
(dΣ(q)/dΩ)/C ) (2π /q2)π
∫0∞ pT(r) cos(qr) dr
(1)
The function is given by20
∫
pT(r) ) (πMS)-1 ∆F(r')∆F(r + r') dr'
(2)
with r the coordinate in the direction perpendicular to a bilayer and ∆F(r) the contrast (difference between the scattering length density of aggregates at the point r and the solvent). MS is corresponding to the mass per unit area of disklike (vesicle) aggregates.
10182 J. Phys. Chem. B, Vol. 110, No. 20, 2006
Kawasaki et al.
TABLE 1: Parameters of the Vesicle Bilayer Obtained by the IFT Method: Radius Gyration of Thickness. RT,g, and Thickness in Homogeneous Approximation, T, Mass Per Surface, MS, and Number of DDAO Molecules Per Surface Area, M concentration Dmax (mM) (Å) 3 10 20 50 75
22 22 22 22 22
RT,g (Å)
T (Å)
MS M (10-7 g cm-2) (10-10 mol cm-2)
6.5 ( 0.2 6.1 ( 0.2 5.9 ( 0.2 5.7 ( 0.2 5.6 ( 0.2
22.5 ( 0.7 21.1 ( 0.7 20.4 ( 0.7 19.7 ( 0.7 19.4 ( 0.7
1.5 ( 0.1 1.4 ( 0.1 1.4 ( 0.1 1.3 ( 0.1 1.3 ( 0.1
4.4 4.2 4.2 4.0 4.0
The pair distance distribution function is expressed as a sum of N b-splines evenly distributed in the interval [0,Dmax]. The values of the coefficients are calculated numerically by a leastsquares fitting of the IFT model curve to the experimental data. In the present study, the values of Dmax were carefully chosen to give both good fits to the experimental data and smooth pT(r) functions. At the large q range (q > 0.02 Å-1) the experimental data and the fitted curves coincide very well (data not shown). The Gaussian shape of the pair distribution function is characteristic for almost homogeneous locally planar aggregates formed by 2C10MAO. Here we obtained the first estimation of the crosssection thickness of approximately 22 Å, which is obtained from the maximum distance of pT(r). After determination of the pair distance distribution function, the mass per unit area of the aggregate MS and the radius of gyration of the cross section of the bilayer RT,g will be calculated. The radius of gyration can be written as
∫
RT,g ) [
∞ 2 r pT(r) 0
dr /(2
∫0
∞
pT(r) dr )]1/2
(3)
and
MS ) π
∫0∞ pT(r) dr/∆F2
(4)
Obtained values of RT,g and MS are presented in Table 1. The obtained parameter of the radius of gyration is connected with the thickness of the bilayer in the homogeneous approximation, T, as T ) (12R2T,g)1/2. The bilayer thickness is consistent with the extended configuration of the alkyl chain of the molecule. We obtained a small decrease of T with increasing concentration, which could be due to limitations of the method connected with interactions between vesicles. Further analysis concerned modeling of the intensities scattered by ULVs. Here a few assumptions were made. (i) The vesicles are large and thin; i.e., the radius of vesicles R . T. With this assumption we may use the decoupling approximation of the form factor, as was done for other vesicle solutions.22-24 (ii) The vesicles are polydisperse in radius only. (iii) The interaction between vesicles is due to the effective excluded volume of the vesicles. In this case the scattering intensities of an ensemble of n vesicles are written as a function of scattering from a single vesicle P(q) and the interaction among the vesicles S(q)
dΣ(q)/dΩ ) n[〈|P(q)|〉2S(q) + (〈|P(q)|2〉 - 〈|P(q)|〉2)] (5) The decoupling approximation25,26 relies on the assumption that there is no correlation between interparticle separation and particle size. It was used to calculate the second term of eq 5. Brackets mean averaging over the size distribution.
Figure 9. Scattering intensities of 2C10MAO solutions in D2O (10 mM, X ) 0.2) and the model fit (solid line).
TABLE 2: Results of Fitting: Mean Radius of Vesicle, Standard Deviation, and Thickness of Vesicle concentration (mM)
〈R〉 (Å)
σR
T (Å)
3 10 20 50 75
280 ( 10 240 ( 10 240 ( 10 250 ( 10 270 ( 10
0.40 ( 0.04 0.25 ( 0.03 0.30 ( 0.03 0.25 ( 0.03 0.35 ( 0.03
20.2 ( 0.7 19.5 ( 0.5 20.0 ( 0.5 19.9 ( 0.7 19.8 ( 0.7
The form factor of the vesicle is written as
P(q) ) 4π∆FR2{sin(qR)/(qR)}{sin(qT)/q}
(6)
where R and T are the vesicle radius and thickness, respectively, and ∆F is the scattering length density of 2C10MAO in D2O. The polydispersity in vesicle radius can be accounted for by means of the Schultz distribution f(R)
f(R) ) {(p + 1)/〈R〉}p+1[Rp/{Γ(p + 1)}] exp{-(p + 1)R/〈R〉} (7) 〈R〉 is the average vesicle radius, and p is the polydispersity index. The latter is related to the spread of the radius distribution as
σR ) ∆R/〈R〉 ) (p + 1)-1/2
(8)
The structure factor S(q) can be calculated with the PercusYevick approximation for the closure relation27
S(q) ) qRHS/{1 + 24ηHSG(qRHS)}
(9)
where ηHS is the vesicle volume fraction and RHS is the hardsphere radius. In the present study RHS was set equal to 〈R〉. The detailed expression of the function G(qRHS) can be found in the literature.28 The model of polydisperse vesicles can satisfactory describe the scattering data (Figure 9). The quality of fit becomes worse at high concentration (75 mM) due to limitations of modeling; i.e., one can suppose that vesicles start to deform and interact differently than by hard-sphere-excluded volume interactions. The obtained parameters of vesicle structure are presented in Table 2. We did not observe any significant change of vesicle structure in the concentration range between 3 and 75 mM. Increasing the concentration from 3 to 10 mM seems to decrease the polydispersity of the vesicles. The size of the vesicles
ULV Formation and Phase Separation in 2C10MAO
J. Phys. Chem. B, Vol. 110, No. 20, 2006 10183 solutions, it has been established that dH0m/dR < 0 for R < 0.5 and dH0m/dR > 0 for R > 0.5.29 In water, however, different behaviors have been observed depending on the alkyl chain length. The critical micelle concentration (cmc) increases monotonically with R for dodecyldimethylamine oxide, suggesting dH0m/dR > 0 over the whole range of R.30 For oleyldimethylamine oxide in water, long threadlike micelles at R ) 0 (H0m0 > 0) are transformed into vesicles at R ) 0.5, suggesting dH0m/dR < 0 for R < 0.5.11 Monolayers with negative curvatures are unstable in aqueous media, and hence micelles cannot be a major aggregate species for 2C10MAO. Monolayers with negative curvatures form an L2 phase or bilayer aggregates. Phase Sequence at C ) 10 mM and 25 °C in Water. On the basis of the observation by eye and under a polarizing microscope combined with cryo-TEM pictures and SANS results, the phase sequence of 2C10MAO at C ) 10 mM and 25 °C in water will be summarized as follows
Figure 10. Electric conductivity of 2C10MAO solutions (C ) 30 mM) as a function of X at 25 °C. Filled and open symbols refer to the solutions of the same volume with and without 2C10MAO, respectively. For open symbols, the abscissa X simply denotes the HCl concentration, which is equal to 30X mM.
obtained from SANS is smaller than values obtained by DLS and cryo-TEM. Electric Conductivity. The electric conductivity of 2C10MAO solutions (C ) 30 mM) was measured as a function of X as shown in Figure 10. In the range X < 0.5 the specific conductivity κ of the solutions is much smaller than that of HCl solutions without the surfactant (open circles). In the ULV region, only positive charges on the outer leaflet of bilayers and their counterions in the outer aqueous phase contribute to the conductivity. The counterions are not free but strongly attracted to the positively charged vesicle surfaces. Hence, low conductivities in the ULV region are reasonable. In the range X > 0.5 (two-phase region), measurements were made under vigorous stirring. Conductivity is considered to be contributed mostly from the L1 phase and increases with X at a larger rate than in the ULV region. But the increasing rate dκ/dX is significantly smaller than that for a pure HCl solution, indicating a considerable partition of counterions to water pools in the L2 phase. In the range X > 1, the rate is close to but smaller than that of the HCl solution due to the partition to the L2 phase. Discussion Effects of Protonation on the Spontaneous Curvature of the Surfactant Monolayer. In the discussion below we interpret the results in terms of the spontaneous curvature of the surfactant monolayer H0m. The spontaneous curvature of nonionic 2C10MAO (X ) 0), H0m0, is safely assumed to be negative since the L2 phase is found to be stable, H0m0 < 0. At finite X values, charges are introduced, and this makes H0m less negative or positive on one hand, but on the other hand, H0m becomes more negative as a result of the hydrogen bond between two headgroups. As a result of these two opposing effects coupled with protonation, the change of H0m with the degree of ionization R depends on various factors such as the ionic strength of the medium or the degree of counterion binding. As far as the hydrogen bond contribution surpasses the electric repulsion, we have dH0m/dR < 0 and vice versa. For single-tail amine oxides (H0m0 > 0) in 0.1 or 0.2 M NaCl
L2/L1 (X ≈ 0) f LR (X < 0.02) f unresolved region containing L3 phase (X ≈ 0.02) f ULVs (0.05 e X < 0.5) f two phases (L2/L1) (X > 0.5) Contrary to the expectation based on the protonation-induced hydrogen bonding, the actual phase behavior proceeds in the direction of increasing curvature with increasing X in the range X < 0.4-0.5: a normal charge effect. For finite X values, one of the factors that makes bilayers (LR or ULVs) more stable than L2 is the translational entropy of the counterions SC. Counterions are confined within narrow water pools in L2, and hence SC is smaller for L2 than for bilayers. With increasing counterion concentrations Cg, by increasing either X, the salt concentration Cs, or the surfactant concentration at finite X, the difference in SC between L2 and bilayers becomes smaller, and at a critical value X* (or Cs*) a transition from bilayers to L2 (or L2/L1 at low concentrations C) is expected to occur. As far as the SC contribution is important, bilayers are more stable than L2, as observed in the range of 0 < X < X*. X* is about 0.5 in water, and X* < 0.2 in 5 mM NaCl. For vesicles, the charge density of the outer monolayer is supposed to be larger than that of the inner one. With the asymmetric compositions, the overall bilayer curvature Hb is close to -H0m of the inner monolayer (Rin). For the outer monolayer (Rout), we expect that H0m ≈ Hb due to significant charge repulsion that interferes with the hydrogen bonding between adjacent headgroups. However, hydrogen bonding between headgroups belonging to different particles may still occur (although in competition with hydrogen bonding to water), because this does not affect the spontaneous curvatures of either monolayer. Intervesicle hydrogen bonds may lead to aggregation of ULVs. In the ranges X ) 0 and X > X*, a single L2 phase is expected to appear at high concentrations, but actually L2/L1 two phases are observed at low C. In the L1 phases at both X ) 0 and X > X*, the population of micelles is suggested to be very low. As to the relative stability of vesicles against lamella, ULVs are more favored over LR for larger X, lower C, and higher T, due to the contribution of SC and the translational entropy of vesicles. Synergism between Nonionic and Cationic Amine Oxides. Synergism between nonionic and cationic amine oxides has been observed in the case of single-tail amine oxides.29 The synergism arises from the hydrogen bond between the nonionic and the
10184 J. Phys. Chem. B, Vol. 110, No. 20, 2006 cationic headgroups31 and hence develops as X increases until electric repulsive interaction exceeds the stabilizing effects of the hydrogen bond. Accordingly, the synergism is observed in the range of small X, while normal charge effects are dominant in the large X region. In the present study on double-tail amine oxide, on the contrary, normal charge effects are observed in the range of small X, while the hydrogen-bond-assisted L2 phase is favored in the range X > 0.5. Accordingly, the charge-induced phase separation is regarded as a result of the synergism. It is not certain whether hydrogen bonds are formed between two cationic species in the L2 phase, though the L2 phase is observed also at X g 1. Also, it is not known whether the degree of ionization R is close to unity in the range X g 1. Stable Unilamellar Vesicles over a Wide Range of Concentrations and Compositions. It is suggested in the present study that ULVs exist in water as the most stable aggregate structure over a wide range of concentrations (3 mM e C < 0.1 M at X ) 0.2) and compositions (0.05 < X < 0.4-0.5 at C ) 10 mM). This is in sharp contrast with the case of didodecyldimethylammonium bromide (2C12DMABr) where ULVs are observed in a very narrow concentration range in water at 25 °C, between 0.02% (∼0.5 mM) and 0.15% (∼3.2 mM).32 The bending constant of bilayers kb is larger for 2C12DMA than that for 2C10MAO because of the difference in alkyl chain length, and the repulsive undulation force is expected to be larger for 2C10MAO. However, electrostatic repulsion is certainly much larger for 2C12DMA. Strong electric repulsion between bilayers hinders the stacking of bilayers on one hand; however, on the other hand, it makes the effective hard-sphere volumes of the vesicles large, so that the ULV dispersion is no longer stable. The latter situation regarding electric interaction and the undulation repulsion are likely to lead to different behaviors of the two double-tail surfactants. However, vesicles (ULVs) of sodium oleate/octanol mixtures are shown to be stable up to concentrations high enough to form a densely packed gel state without transforming into LR.33 Intervesicle repulsion in this case will be much weaker than that for 2C10MAO vesicles. In a catanionic mixture, hexadecyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzene sulfonate (SDBS), the “vesicle phases” extend up to about 3 wt % (∼67 mM) and over a wide range of compositions.34 These results are rather comparable with those of the present surfactant. In comparison with the two vesicle systems mentioned above, 2C12DMA and CTAT/SDBS, bilayers of 2C10MAO in the present study are much more flexible partly due to short alkyl chains and partly due to versatile spontaneous curvatures that are possible by adjusting the degrees of ionization of both inner and outer monolayers. The moderately polydisperse nature of 2C10MAO vesicles found in the present study, σR ) 0.250.40, is close to the theoretical prediction σR ) 0.283 of Bergstro¨m and Eriksson, which according to them should be valid for all equilibrated vesicle solutions in the dilute limit, independent of the bilayer flexibility.35 It should be noted that the versatile nature of the monolayer spontaneous curvature in bilayers is one of the characteristics of amine oxides. In many surfactant bilayers, flip-flop motions of the surfactants are required to change the compositions of the constituting monolayers. In amine oxide bilayers, however, compositional changes are achieved simply by a transfer of HCl through the bilayer. Since the chemical bond of HCl is partially
Kawasaki et al. of covalent nature, its transport through the nonpolar medium of bilayer will be not unlikely. Conclusions A double-tail amine oxide surfactant, di-n-decylmethylamine oxide (2C10MAO) makes an L2 phase in the nonprotonated state (X ) 0) in water, and the L2 phase is in equilibrium with an aqueous L1 phase. On protonation, unilamellar vesicles (ULVs) are formed over a wide range of compositions (0.05 < X < 0.4-0.5 at C ) 10 mM). At X ) 0.2, the ULVs are stable over a wide concentration range (3 mM e C < 0.1 M), but the LR phase replaces the vesicle phase at C > 0.1 M. The diameter and the bilayer thickness of the ULVs are about 25 and 2 nm, respectively. An important contribution to the enhanced stability of the bilayer structures over the L2 phase is suggested to be the translational entropy of counterions. The enhanced stability of the bilayers diminishes as the counterion concentration increases either by increasing X or by adding a salt consisting of the same counterion species, and eventually bilayer structures transform into an L2 phase when X* is about 0.4-0.5 in water or when it is below 0.4 in D2O. The critical NaCl concentration is below 5 mM at X ) 0.2. The stability of ULVs against multilamellar vesicles is ascribed partly to undulation force and partly to the adjustable nature of the monolayer spontaneous curvature of amine oxides. Acknowledgment. H.M. thanks Dr. Yuji Yamashita and Professor H. Hoffmann for kindly providing him with their data on dodecyloctylmethylamine oxide, which were helpful in interpreting the data in the present study. Mr. Go¨ran Karlsson is thanked for expert help in the cryo-TEM investigations. SANS measurements have been supported by the European Commission under the 6th Framework Program through the Key Action: Strengthening the European Research Area, Research Infrastructures (Contract No. RII3-CT-2003-505925). This work was partially supported by CREST (Japan). Supporting Information Available: Dynamic light-scattering data on ULVs, SANS data at different mixing ratios X, and SANS data in the presence of NaCl. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Torchilin, V. P. Nature ReV. Drug DeliVery 2005, 4, 145. (b) Cevc, G. AdV. Drug Delivery ReV. 2004, 56, 675. (2) Hargreaves W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759. (3) Beck, R.; Gradzielski M.; Horbaschek, K.; Shah, S. S.; Hoffmann, H.; Strunz, P. J. Colloid Interface Sci. 2000, 221, 200. (4) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. Science 1989, 245, 1371. (5) Van Zanten, R.; Zasadzinski, J. A. Curr. Opin. Colloid Interface Sci. 2005, 10, 261. (6) Almgren, M.; Rangelov, S. J. Phys. Chem. B. 2005, 109, 3921. (7) Johnsson, M.; Wagenaar, A.; Engberts, J. J. Am. Chem. Soc. 2003, 125, 757. (8) Edwards, K.; Silvander, M.; Karlsson, G. Langmuir 1995, 11, 2429. (9) Chen, W. J.; Li, G. Z.; Zhou, G. W.; Zhai, L. M.; Li, Z. M. Chem. Phys. Lett. 2003, 374, 482. (10) Gonzalez, Y. I.; Nakanishi, H.; Stjerndahl, M.; Kaler, E. W. J. Phys. Chem. B 2005, 109, 11675. (11) Kawasaki, H.; Souda, M.; Tanaka, S.; Nemoto, N.; Karlsson, G.; Almgren, M.; Maeda, H. J. Phys. Chem. B 2002, 106, 1524. (12) Kawasaki, H.; Imahayashi, R.; Tanaka, S.; Almgren, M.; Karlsson, G.; Maeda, H. J. Phys. Chem. B 2003, 107, 8661. (13) Yamashita, Y. Doctoral Dissertation, Bayreuth University, 2005. (14) Stuhrmann, H. B.; Burkhardt, N.; Dietrich, G.; Ju¨nemann, R.; Meerwinck, W.; Schmitt, M.; Wadzack, J.; Willumeit, R.; Zhao, J.; Nierhaus, K. H. Nucl. Instrum. Methods Phys. Res., Sect. A 1995, 356, 133. (15) Wignall, G. D.; Bates, F. S. J. Appl. Crystallogr. 1986, 20, 28.
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