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Langmuir 2002, 18, 1024-1029
Interaction of Octaethylene Glycol n-Dodecyl Monoether with Dioctadecyldimethylammonium Bromide and Chloride Vesicles P. C. A. Barreleiro,† G. Olofsson,† W. Brown,‡ K. Edwards,‡ N. M. Bonassi,§ and E. Feitosa*,§ Center for Chemistry and Chemical Engineering, Physical Chemistry 1, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden, Department of Physical Chemistry, University of Uppsala, P.O. Box 532, S-751 21, Uppsala, Sweden, and Physics Department, IBILCE/UNESP, 15054-000, Sa˜ o Jose´ do Rio Preto, SP, Brazil Received June 11, 2001. In Final Form: November 1, 2001 Several different methods were used to investigate the vesicle-to-micelle transition induced by the addition of the nonionic surfactant octaethylene glycol n-dodecyl monoether (C12E8) to spontaneously formed vesicle dispersions of dioctadecyldimethylammonium bromide and chloride (DODAX, X ) Cl- and Br-). Dynamic light scattering reveals that fast mode micelles are formed upon addition of C12E8. The micellar mode becomes progressively dominant as the C12E8/DODAX molar ratio (R) is increased until the vesicle-to-micelle transition is complete. Turbidity, calorimetry, fluorescence quantum yield, and anisotropy measurements indicate two critical compositions: the first, Rsat, when the vesicle bilayer is saturated with C12E8 and the second, Rsol, which corresponds to the complete vesicle-to-micelle transition. Below Rsat the vesicles swell due to incorporation of the surfactant into the vesicle bilayer, and above Rsat mixed micelles and bilayer structures coexist. The determined Rsat and Rsol range from 0 to 1 and 4 to 6, respectively, depending on the surfactant counterion and the experimental method used. Cryo-transmission electron microscopy micrographs show that when R ≈ 4, micelles coexist with extended bilayer fragments. In pure DODAX (1.0 mM) dispersions, unilamellar vesicles are observed. According to the DSC results, C12E8 lowers the gel-to-liquid crystalline transition temperature, Tm, of DODAX and broadens the main transition peak which disappears around R ≈ 5 and 6 for DODAC and DODAB, respectively.
1. Introduction Dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) are long (C18) double chain cationic surfactants that, in excess water, self-assemble above the gel to liquid-crystalline phase transition temperature (Tm) into giant closed-bilayered (vesicle) structures.1-5 The structural organization of these amphiphiles depends on the amphiphile concentration, vesicle preparation method, solvent composition, temperature, and additives.1-7 Probe or bath sonication,2,4 ethanol, chloroform, or dichloromethane injection,4 and extrusion5 have been used to increase the curvature of the giant vesicle structures obtained by simply mixing DODAX (X being either Br- or Cl-) with water above Tm.3,5-7 Octaethylene glycol n-dodecyl monoether, C12E8, is one of the most investigated micelle-forming nonionic poly(ethylene oxide)-derivative surfactants (CmEn, m and n are the number of carbon atoms and polymer monomers, * Corresponding author. E-mail:
[email protected]. † University of Lund. ‡ University of Uppsala. § IBILCE/UNESP. (1) (a) Fendler, J. H. Membrane Mimetic Chemistry, Wiley-Interscience: New York, 1982. (b) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 21, 209. (2) Cuccovia, I. M.; Feitosa, E.; Chaimovich, H.; Sepulveda, L.; Reed, W. J. Phys. Chem. 1990, 94, 3722. (3) Feitosa, E.; Brown, W. Langmuir 1997, 13, 4810. (4) Cuccovia, I. M.; Sesso, A.; Abuin, E.; Okino, P. F.; Tavares, P. G.; Campos, J. F. S.; Florenzano, F. H.; Chaimovich, H. J. Mol. Liquids 1997, 72, 323. (5) Feitosa, E.; Barreleiro, P. C. A.; Olofsson, G. Chem. Phys. Lipids 2000, 105, 201. (6) Feitosa, E.; Karlsson, G.; Edwards, K. Manuscript submitted to publication. (7) Benatti, C. R.; Tiera, M. J.; Feitosa, E.; Olofsson, G. Thermochim. Acta 1999, 328, 137.
respectively). C12E8 self-assembles into small, rather monodisperse micelles, with a mean radius of around 33 Å.8a The size of the C12E8 micelles does not, under the experimental conditions used in this study, depend on surfactant and electrolyte concentration, but it increases as the temperature approaches the cloud point of the surfactant (ca. 75 °C8b). When added to vesicle dispersions, C12E8 modifies the permeability of the vesicle bilayer and the vesicle structure and organization and reduces the lipids Tm.9 The amount of surfactant solubilized in the vesicle bilayer determines the structure and morphology of the aggregates. At high enough surfactant concentration the vesicle structure is destabilized and mixed micelles are formed. Nonionic micelle-forming surfactants are especially apt to yield vesicle-to-micelle transition of cationic unilamellar vesicles. Anionic (oppositely charged) micelleforming surfactants tend to yield phase separation, owing to charge neutralization, as reported for the system didodecyldimethylammonium bromide/sodium dodecyl sulfate/water.9 In this study we report on some properties of the ternary DODAX/C12E8/water systems which reveal a possible mechanism for the vesicle-to-micelle transition based on the three-stage Lichtenberg model.10 A variety of techniques have been used in this investigation, including dynamic light scattering, cryo-transmission electron mi(8) (a) Nilsson, P. G.; Lindman, B. J. Phys. Chem. 1983, 87, 4756. (b) Feitosa, E.; Brown, W.; Swanson-Vethamuthu, M. Langmuir 1996, 12, 5985. (9) Inoue, T. In Vesicles; Surfactant Science Series No. 62; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; p 151. (10) (a) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (b) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470.
10.1021/la010876z CCC: $22.00 © 2002 American Chemical Society Published on Web 01/22/2002
Mechanism for Vesicle-to-Micelle Transition
Langmuir, Vol. 18, No. 4, 2002 1025 prises amplifier/discriminator system. For details of the experimental arrangement see ref 13. The algorithm REPES14 was used to perform the inverse Laplace transform analyses of the measured intensity time correlation function, g2(t), to obtain the relaxation time distribution, τA(τ), from the Laplace relation15
g1(t) )
Figure 1. Molecular structures of the fluorescence transdiphenylpolyene probes DPB and DPH and their derivatives B4A and 4H4A used in this investigation.
croscopy (cryo-TEM), differential scanning calorimetry (DSC), spectrophotometry, and fluorescence. The results support our previous findings on the same systems based on titration calorimetric investigations.11 2. Experimental Section 2.1. Materials. High-purity dioctadecyldimethylammonium bromide (DODAB) with purity better than 99.9%, as determined by high-performance liquid chromatography by the manufacturer Avanti Polar Lipids (Alabaster, AL) was used as received. Dioctadecyldimethylammonium chloride (DODAC) was obtained by counterion exchange from DODAB (Eastman Kodak) and recrystallized as described elsewhere.4 Octaethylene glycol dodecyl monoether, C12E8 (Nikko Chemicals), was also used as received. The fluorescence probes trans,trans-1,4-diphenyl-pcarboxy-1-propyl-1,3-butadiene, B4A, and trans,trans,trans-1,6diphenyl-p-carboxipropyl-p′-n-butyl-propyl-1,3,5-hexatriene, 4H4A, were synthesized from trans,trans-1,4-diphenylbutadiene, DPB, and trans,trans,trans-1,6-diphenylhexatriene, DPH, respectively, and kindly supplied by Dr. Laerte Miola.12 DPB and DPH were supplied by Aldrich. Figure 1 shows schematically the molecular structures of DPB, DPH, B4A, and 4H4A. Ultrapure water of Milli-Q-Plus quality was used in the sample preparation. 2.2. Vesicle Preparation. Giant unilamellar vesicles were prepared by simply mixing DODAB or DODAC and water at the concentration of 1.0 mM at 60 °C (above Tm ≈ 45 or 48 °C) to obtain a homogeneous solution.3,5-7 The vesicle dispersions were cooled to room temperature and stored for at least 24 h before the measurements. The probe-containing vesicle dispersions for the fluorescence measurements were prepared using the same procedure. The probes were diluted in CHCl3 in a glass flask, and the solvent was evaporated using a N2 flux to give a thin film of the probes. The vesicle dispersions were than placed in the flask, and the solutions were vortexed. 2.3. Dynamic Light Scattering, DLS. DLS measurements were carried out using a 633 nm He/Ne laser (60 mW) as light source and a detector system consisting of an ITT FW 130 photomultiplier connected to an ALV-Langen Co. multibit, multitau autocorrelator through a digitalized Nuclear Enter(11) Barreleiro, P. C. A.; Olofsson, G.; Feitosa, E. Prog. Colloid Polym. Sci. 2000, 116, 33. (12) (a) Allen, M. T.; Miola, L.; Whitten, D. G. J. Am. Chem. Soc. 1988, 110, 3198. (b) Allen, M. T.; Miola, L.; Shin, D. M.; Suddaby, B. R.; Whitten, D. G. J. Membr. Sci., 1987, 33, 201.
∫
+∞
-∞
τA(τ)e-t/τ dτ
(1)
where τ is the relaxation time and g1(t) is the first-order electric field time correlation function, g2(t) - 1 ) β|g1(t)|2, and β is a factor which accounts for deviation from the ideal correlation.15 The relaxation time distribution is usually presented as a τA(τ) versus log τ profile, which provides an equal area representation.15 For each mode of the relaxation time distribution the diffusion coefficient is obtained as D ) Γ/q2, where q ) (4πno/λ) sin θ is the modulus of the scattering vector, Γ ) τ-1 is the relaxation rate, λ is the incident wavelength, no is the solvent refraction index, and θ is the scattering angle. 2.4. Cryo-TEM. The technique uses thin (10-500 nm thick) sample films prepared under controlled temperature and humidity conditions within an environmental chamber. The film is vitrified from the desired temperature by quick freezing in liquid ethane and transferred to a Zeiss EM 902 transmission electron microscope for examination. The specimens were kept cold (below 108 K) during the transfer and viewing procedures. The observations were made in a zero loss bright-field mode and at an electron accelerating voltage of 80 kV. For further details on the experimental setup, see ref 16. 2.5. Differential Scanning Calorimetry, DSC. The DSC measurements were made using a MicroCal MC-2 (MicroCal, Northampton, MA) differential scanning calorimeter equipped with 1.2 mL twin cells for the reference and sample solutions. To avoid air bubble formation, the sample and reference solutions were degassed (Nueva II stirrer, Termolyne) before being transferred to the cells using a Hamilton syringe. The DSC thermograms record the differential power required to maintain the reference and sample at the same temperature while scanning the temperature of the cells at a constant rate (about 53 °C/h in this work). The reference thermogram was recorded under the same conditions by filling the cells with the same solvent. The gel to liquid-crystalline phase transition temperature (Tm) is determined as the temperature at the peak maximum, whereas the area under the peak gives the enthalpy change of the corresponding transition (∆Hm). The shape of the curve gives information about the transition, and the width at half-height (∆T1/2) of the transition peak is related to the cooperative nature of the transition. 2.6. Turbidity Spectra. Turbidity of the solutions was measured using a Hitachi spectrophotometer model U-2001 and a spectrofluorometer Hitachi model F4500 at varying λ. All measurements were made at 25 °C, and the temperature of the spectrophotometer cells was controlled by a thermostated bath. The temperature in the cell was measured using a thermocouple. Reproducibility was evaluated from at least three runs. 2.7. Steady-State Fluorescence. The fluorescence emission and absorption spectra of the fluorophores solubilized in the surfactant solutions were obtained, respectively, using a F4500 Hitachi spectrofluorometer and a U-2001 Hitachi spectrophotometer, at 25 °C. The concentration of the probes was 7.0 µM for B4A and 5.0 µM for 4A4H. The fluorescence quantum yield (Φf) was determined by measuring the fluorescence intensity of the probes incorporated in the aggregates excited at the same experimental conditions and wavelength of the spectrum maximum absorbance (λmax ) 340 nm) and incident light intensity (Io). Under these conditions Φf was derived by equating the fluorescence intensity of the fluorophores in the sample and (13) Feitosa, E.; Brown, W.; Hansson, P. Macromolecules 1996, 29, 2169. (14) Johnsen, R. M.; Brown, W. Laser Light Scattering in Biochemistry; Harding, S. E., Sattele, D. B., Bloomfield, V. A., Eds.; Royal Society of Chemistry: London, 1992; p 77. (15) Brown, W., Ed. Dynamic Light Scattering. The Method and Some Applications; Clarendon Press: Oxford, 1993. (16) (a) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. 1988, 10, 87. (b) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3.
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The vesicle-to-micelle transition, also referred to as membrane solubilization has been studied by spectrophotometry, steady-state fluorescence, dynamic light scattering, cryo-TEM, and differential scanning calorimetry. In accordance with our previous report on the DODAX/C12E8 systems, based on isothermal titration calorimetry,11 the results are analyzed in terms of the “three-stage” model proposed by Lichtenberg et al.10 3.1. Turbidity. The turbidity of the ternary C12E8/ DODAX/water systems were monitored by adding aliquots of a concentrated C12E8 micellar solution to a 1 mM DODAB or DODAC dispersion. Figure 2 shows the turbidity as a function of the C12E8/DODAX molar ratio. Measurements were made 6 min after each addition of surfactant. After this time no significant changes were observed on the turbidity curves for at least 48 h. The turbidity curves indicate two critical points, Rsat and Rsol, corresponding to saturation of the vesicle bilayer by C12E8 and complete solubilization of DODAX in mixed micelles
(as discussed below). The critical points Rsat ≈ 0.3 and Rsol ≈ 5 for DODAC and Rsat ≈ 0.5 and Rsol ≈ 6 for DODAB indicate that DODAC is solubilized more easily than DODAB by C12E8. In addition, DODAC dispersions display an overall lower turbidity compared to DODAB dispersions. This fact indicates, in accordance with previous cryoTEM results,6 that smaller aggregates are being formed in the DODAC/C12E8 mixture. The turbidity curves in Figure 2 for C12E8/DODAX/water mixtures resemble the curves for phospholipid vesicles mixed with nonionic surfactants, except that a third critical point below Rsat is sometimes observed for the phospholipid systems. Below this point the turbidity is not affected by the addition of the surfactant (nonswelling region).18 Turbidity curves recorded for the interaction between C12E8 and large unilamellar egg phosphatidyl choline (EPC) vesicles display, however, the same general appearance as that obtained for C12E8 and DODAX.18a On the basis of scattering measurements at different lipid concentrations, Rsat and Rsol were determined to be around 0.5 and 2.0, respectively, in the former system.18a It thus appears that considerably more C12E8 is needed for complete solubilization of DODAB or DODAC compared to EPC. Due to structural changes in the dispersion, the turbidity decreases progressively between Rsat and Rsol. In this intermediate range of C12E8 concentration, mixed micelles and probably bilayer aggregates coexist and the fraction of the latter decreases to zero at Rsol. The actual aggregate structures in this region where bilayer saturation but not complete solubilization is attained has been subject to speculation. Cryo-TEM investigations of various phospholipid/surfactant systems, including EPC/C12E8, suggest that a common sequence of structural changes includes a decrease in vesicle size followed by the formation of open liposomes and bilayer disks.18 Moreover, the gradual breakdown of the vesicles is often accompanied by the formation of cylindrical mixed micelles, which decrease in size with increasing surfactant concentration. The scattering/turbidity curves shown in Figure 2 suggest that the C12E8/DODAX/water systems behaves in a similar way. 3.2. Dynamic Light Scattering, DLS. Figure 3 shows the intensity correlation function for the ternary DODAC/ C12E8/water system for selected C12E8/DODAC molar ratios R, from 0 to 30, and for the initial concentration of DODAC of 1.0 mM. The inset shows the corresponding relaxation time distributions. The scattering measurements were made about 2 min after the addition of the surfactant to the vesicle dispersion and manual shaking of the sample cuvette. Owing to the short waiting time after mixing of the surfactant components, equilibrium was not attained during the scattering measurements (a more detailed scattering investigation is underway). The nonequilibrium condition yields considerably larger values for Rsat and Rsol relative to those obtained by turbidity and differential scanning calorimetry (shown below), but they confirm the three-stage model for the vesicle-to-micelle transition in the C12E8/DODAC/water system. The pure DODAC vesicle dispersion exhibits a typical close-to-single exponential intensity correlation function, since the fast and slow modes of the distribution are very close together (Figure 3, inset), i.e., the dispersion consists of two populations of large unilamellar vesicles, as already reported.3,6 As R increases to 1.5, the distribution becomes clearly bimodal with well-separated modes, with the fast mode having a lower relative amplitude, AF, than the slow mode,
(17) Lakowicz, J. Principles of fluorescence Spectroscopy; Plenum Press: New York, 1983.
(18) (a) Johnsson, M.; Edwards, K. Langmuir 2000, 16, 8632. (b) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146.
Figure 2. Turbidity at 240 nm (25 °C) as a function of the C12E8/DODAX molar ratio, R, measured 6 min after mixing the solutions of (b) DODAC and (9) DODAB. reference solutions, through eq 2
Φf ) [(If Φfo)/Ifo] [(1 - 10-Afo)/(1 - 10-Af)]
(2)
where Φf, If, Φfo, and Ifo are the quantum yield and fluorescence intensity of the fluorophores in the vesicle and reference solutions, respectively. Af and Afo are the absorbance of the fluorophore in the vesicle and reference solutions measured at λmax. In this work we have used DPB/cyclohexane as reference solution, for which Φfo ≈ 0.44 at 25 °C.12 Fluorescence anisotropy was obtained by measuring the vertically and horizontally polarized excitation and emission radiation of the probes. For the vertically polarized excitation light, the vertically (I|) and horizontally (I⊥) polarized emission radiation were measured; for the horizontally polarized excitation radiation, i⊥ and i| are the measured vertically and horizontally polarized emission radiation. The fluorescence anisotropy, 〈r〉, is given by eq 3
〈r〉 ) (I| - GI⊥)/(I| + 2GI⊥)
(3)
where G ) i⊥/i| is a correction factor which accounts for the “L-type” spectrofluorometer with a single emission channel.17
3. Results and Discussion
Mechanism for Vesicle-to-Micelle Transition
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Figure 4. Cryo-TEM micrographs of a pure 1.0 mM DODAB aqueous dispersion. There we can see large unilamellar vesicle structures with diameters up to 330 nm. Sample vitrified from the temperature of 25 °C. Bars equal 100 nm.
Figure 3. Intensity correlation function and the corresponding relaxation time distribution (inset) at increasing molar C12E8/ DODAB ratio, as shown. T ) 25 °C and θ ) 90°. The initial concentration of DODAC was 1.0 mM (the filtering through a 0.4 µm Millipore membrane may reduce this concentration by 20%).
AS. These modes are most probably associated with mixed DODAC/C12E8 micelles and mixed DODAC/C12E8 vesicles. Further increase in R yields a progressive increase in AF and decrease in AS, and the latter vanishes around the concentration ratio R ≈ 30 (inset). The apparent hydrodynamic radii of the aggregate species in the mixture were not estimated owing to the nonequilibrium conditions. 3.3. Cryo-TEM. The cryo-TEM measurements were performed after 48 h. At low concentration (e.g., 1 mM) DODAB molecules self-assemble in water, above Tm, as unilamellar vesicles.6 As shown in the cryo-TEM micrograph (Figure 4) the vesicles are rather polydisperse in size and shape. The presence of faceted vesicles is due to the vitrification of the sample at a temperature below the gel-to-liquid crystalline phase transition temperature. A detailed investigation of the structure and size of these “spontaneously formed” vesicles will be reported elsewhere.6 Figure 5 shows cryo-TEM micrographs for 1 mM DODAB dispersion in the presence of 4.0 mM C12E8, i.e., at a C12E8/DODAB molar ratio in the intermediate region. As expected the sample is dominated by globular micelles (Figure 5c). The presence of bilayer structures, in the form
Figure 5. Cryo-TEM micrographs of 1.0 mM DODAB dispersion in the presence of C12E8 4.0 mM (molar ratio C12E8/DODAB ) 4.0). They depict (a) vesicle, (b) extended lamellae, and (c) micellar structures in different scan areas of the same sample. Bars equal 100 nm.
of open vesicles (Figure 5a) and extended bilayer sheets (Figure 5b), indicates, however, that the vesicle-to-micelle transition is not yet complete at R ) 4. 3.4. Differential Scanning Calorimetry, DSC. The effect of addition of C12E8 on the DSC heating curves of DODAC and DODAB vesicles is shown in Figure 6. The first thermograms in Figure 6 (R ) 0) correspond to pure 1.0 mM DODAB and DODAC in water. In good agreement with previous reports,5 well-defined peaks corresponding to the gel to liquid-crystalline phase transition temperatures of 45.5 ( 0.1 and 47.9 ( 0.1 °C for DODAB and DODAC, respectively, were obtained. Upon addition of C12E8, a progressive broadening of the main transition peak and a lowering of Tm is observed, indicating that the incorporation of C12E8 molecules in the DODAX bilayer has a disordering effect. The main peak is well-defined, and only a small decrease in Tm is observed (∆T ≈ 3-4 °C) for molar ratios up to R ≈ 1.0 for DODAB and 0.4 for DODAC. At higher R the peak shifts to lower temperatures and becomes increasingly broader to disappear at about R ) 5.0 and 6.0 for DODAC and DODAB, respectively. These results, together with those from light scattering and turbidity measurements suggest that the vesicle-to-
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Figure 6. DSC upscans for 1 mM DODAX dispersions: (a) X ) Cl- and (b) X ) Br-. The C12E8/DODAX molar ratios are indicated in the figure. Curves have been offset from each other to avoid overlap.
micelle transition is not sharp. The well-defined peak at low R indicates the existence of mixed vesicles with Tm lower than that for the pure DODAX vesicles. At intermediate R, the broad and low-intensity peak indicates that other structures than vesicles dominate the dispersion. As discussed earlier we probably have a mixture of bilayer and micelle structures in this region. Finally, at high R, the thermograms without peak indicate the presence of mixed micelles only. Differential scanning calorimetry measurements were also performed on probed DODAB and DODAC vesicles (Figure 7). The transition temperature is not significantly affected by the presence of the probes B4A and 4H4A. 3.5. Steady-State Fluorescence. Owing to their amphiphilic characteristics (see Figure 1), the probes are most probably anchored in the hydrophobic core of the aggregates with the carboxyl group facing the aggregate interfaces. Figure 8a shows the effect of C12E8 on the emission spectra of B4A in the DODAB dispersion, and Figure 8b shows the effect of the molar ratio R on the corresponding fluorescence quantum yield (φf). The addition of C12E8 does not change considerably the fluorescence spectrum of B4A incorporated in the aggregates but the intensity of the peaks, leaving roughly
Barreleiro et al.
Figure 7. DSC upscans for 1 mM DODAX dispersions: (a) X ) Br- and (b) X ) Cl- in the absence (i) and presence of 4H4A (ii) and B4A (iii) probes.
unchanged their position. Initially the spectral intensity increases (up to about Rsat ≈ 1) and then decreases, suggesting that initially the probes migrate to the bilayer interior due to a decrease in the surface charge density of the mixed vesicles relative to the pure DODAB vesicles. The initial increase in φf as C12E8 is solubilized in the DODAB bilayer was unexpected since the bilayer becomes less densely packed owing to the decrease in Tm and φf should decrease instead. Above Rsat φf decreases owing to the formation of mixed micelles and rupture of the vesicle membranes and the probes “feel” a less ordered phase owing to the formation of smaller aggregates. Around Rsol ≈ 4, the vesicle-to-micelle transition is complete, and above Rsol, φf is constant indicating that there are no changes in the mixed micellar structure. About the same behavior was observed for the fluorescence anisotropy, 〈r〉, of B4A (Figure 9a) and the more bulky probe 4H4A (Figure 9b). In the latter case the first stage was not detected probably because of the lower sensitivity of this probe to the vesicle swelling and mobility of the surfactants in the mixed bilayer. The initial increase in anisotropy of B4A is, as in the case of the quantum yield, due to the movement of the probes to the bilayer interior. In the intermediary region the decrease in 〈r〉 indicates the formation of more isotropic microenvironments, such as the interior of smaller aggregates; above Rsol the anisotropy signal is roughly
Mechanism for Vesicle-to-Micelle Transition
Figure 8. Fluorescence emission spectra of B4A in the C12E8/ DODAB/water system, at selected ratios R. Letters a through d indicate the concentration ratios R ) 0, 0.9, 4.3 and 9.3, respectively. (b) Effect of C12E8/DODAB ratio on the fluorescence quantum yield of B4A. Arrows indicate Rsat and Rsol. The initial concentration of DODAB was 1.0 mM. Measurements were made at 25 °C.
constant indicating no structural change. The Rsat and Rsol values obtained from the anisotropy curves are about 1.0 and 4.5, respectively, in good agreement with those obtained by the quantum yield of B4A (Figure 8b). 4. Conclusions Surfactant solubilization in the C12E8/DODAX/water system is a process that ultimately yields a vesicle-tomicelle transition. We have used different techniques and methods to show that C12E8 and DODAX molecules mix together to form mixed vesicles and/or micelles in solution. The structures formed in the DODAX/C12E8 mixture are distinguishable by the techniques used and depend on the molar ratio R ) C12E8/DODAX. As C12E8 is added to the vesicle dispersions the gel to liquid-crystalline phase transition temperature characteristic of the bilayer decreases and the main peak broadens. The results from different methods reported here and elsewhere11 suggest the following mechanism based on the three-stage model for the vesicle-to-micelle transition. Up to the first critical point (Rsat) the vesicle swells and
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Figure 9. Effect of C12E8/DODAB ratio on the anisotropy of B4A (a) and 4H4A (b). Arrows indicate Rsat and Rsol. The initial concentration of DODAB was 1.0 mM. Measurements made at 25 °C.
the bilayer becomes saturated with surfactant. Beyond Rsat the addition of surfactant induces formation of mixed micelles which coexist with DODAX-C12E8 mixed vesicles and/or bilayer fragments. As R is further increased the amount of mixed micelles increases at the expense of the bilayer aggregates. The solubilization of DODAX molecules in the micellar structures continues up to the second critical point (Rsol) where the vesicle-to-micelle transition is complete. Thus, a plausible mechanism for the vesicleto-micelle transition is as follows: vesicles f mixed vesicles f mixed vesicles + mixed micelles f mixed micelles. Intermediate structures such as open vesicles and bilayer fragments are likely to form between Rsat and Rsol. Acknowledgment. E.F. (Grant 98/09772-4) and N.M.B. (Scholarship 98/04010-9) wish to thank Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) for financial support. P.C.A.B. acknowledges the PRAXIS XXI, JNICT for financial support, scholarship BD/13788/ 97. K.E. thanks The Swedish Research Council for Engineering Sciences for financial support. We express our gratitude to Go¨ran Karlsson for running the cryoTEM experiments. LA010876Z
