Self-Organization of the Ternary Didecyldimethylammonium Bromide

Jun 25, 2005 - Elena Junquera,† Patricia del Burgo,† Jasminka Boskovic,‡ and Emilio ... de Madrid, 28040-Madrid, Spain, and Centro de Investigac...
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Langmuir 2005, 21, 7143-7152

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Self-Organization of the Ternary Didecyldimethylammonium Bromide/ Octyl-β-D-glucopyranoside/Water System Elena Junquera,† Patricia del Burgo,† Jasminka Boskovic,‡ and Emilio Aicart*,† Departamento de Quı´mica Fı´sica I, Facultad de Ciencias Quı´micas, Universidad Complutense de Madrid, 28040-Madrid, Spain, and Centro de Investigaciones Biolo´ gicas, CSIC, Ramiro de Maeztu 9, 28040-Madrid, Spain Received March 16, 2005. In Final Form: June 2, 2005 The spontaneous and thermodynamically stable mixed vesicles constituted by a double-chain cationic surfactant with 10 carbon atoms hydrophobic tail, didecyldimethylammonium bromide (di-C10DMAB), and a nonionic single-chain surfactant, octyl-β-D-glucopyranoside (OBG), have been characterized in aqueous media by means of a series of experimental techniques, as well as a theoretical approach. Conductivity data allow for the determination of the concentrations at which the monomer-to-vesicle (CVC*) and/or vesicle-to-micelle (CMC*) transitions occur. Electrophoretic mobilities, obtained from laser-dopplerelectrophoresis experiments, permit the determination of ζ-potentials and, from them, the surface charge density of the vesicle aggregates. Cryogenic transmission electron microscopy (cryo-TEM) provides pictures of the vesicles, their size and shape being, thus, determined. Finally, the sensitivity of the emission spectra of some fluorescent probes, such as the cationic TNS and the nonionic PRODAN, to the polarity of the environment, allow for a complete study of different pre- and post-vesicle microdomains, of variable rigidity and micropolarity. This, in turn, yield interesting information about the vesicle surface and bilayer, as well as, about the existence of clusters and/or nanoaggregates prior to the formation of vesicles, as was proposed by us in a previous paper.

I. Introduction The mixture of a single-chain surfactant and a doublechain surfactant, within the appropriate concentration range, is well-known to form spontaneous and thermodynamically stable mixed vesicles.1-6 This kind of vesicle offers advantages with respect to phospholipid vesicles, since their preparation and their conservation over long periods of time are easier. In addition, it is worth mentioning their potential practical applications, as drug delivery systems, active substances in cosmetics and/or food industries, etc., and their no less interesting uses as simplified models to analyze the behavior of biological membranes.1,7-10 Also important is the fact that the interaction of alkyl surfactants with vesicles may drive, at certain concentrations, the disruption of vesicle structures, which points to these mixed aggregated systems as suitable models to mimic the solubilization and reconstitution processes of lipidic bilayers.11-22 * To whom correspondence should be addressed. Fax: 34913944135. E-mail: [email protected]. Web site: http:// www.ucm.es/info/coloidal/index.html. † Universidad Complutense de Madrid. ‡ CSIC. (1) Rosoff, M. Vesicles; Marcel Dekker: New York, 1996. (2) Barreleiro, P. C. A.; Olofsson, G.; Brown, W.; Edwards, K.; Bonassi, N. M.; Feitosa, E. Langmuir 2002, 18, 1024. (3) Viseu, M. I.; Edwards, K.; Campos, C. S.; Costa, S. M. B. Langmuir 2000, 16, 2105. (4) Viseu, M. I.; Velazquez, M. M.; Campos, C. S.; Garcia-Mateos, I.; Costa, S. M. B. Langmuir 2000, 16, 4882. (5) Junquera, E.; Arranz, R.; Aicart, E. Langmuir 2004, 20, 6619. (6) Junquera, E.; del Burgo, P.; Arranz, R.; Llorca, O.; Aicart, E. Langmuir 2005, 21, 1795. (7) Fendler, J. H. Membrane Mimetic Chemistry; John Wiley & Sons: New York, 1982. (8) Christian, S. D.; Scamehorn, J. F. Solubilization in Surfactant Aggregates; Marcel Dekker: New York, 1995; Vol. 55. (9) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6, 66. (10) Somasundaran, P.; Hubbard, A. Encyclopedia of Surface and Colloid Science.; Marcel Dekker: Santa Barbara, CA, 2002.

Didecyldimethylammonium bromide (di-C10DMAB; see Chart 1) is a commercial double-chain cationic surfactant of the quaternary alkylammonium salt series also used in a wide variety of applications, such as fungicidal and bactericidal, wood preservative, disinfectant, and so on.23,24 The phase behavior of this surfactant in water was characterized many years ago.25 Funasaki et al. studied the formation of inclusion complexes of this double-chain surfactant with cyclodextrins, from emf measurements with ion-selective electrodes.26 However, non studies have been reported on the formation of di-C10DMAB vesicles in the absence and/or presence of a single-chain surfactant. Recently, we have investigated the behavior of mixed ternary cationic-cationic and cationic-nonionic systems in aqueous solution, in the diluted region.5,6 These works (11) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470. (12) Dennis, E. A. Adv. Colloid Interface Sci. 1986, 26, 155. (13) Engberts, J. B. F. N.; Kevelam, J. Curr. Opin. Colloid Interface Sci. 1996, 1, 779. (14) Ribosa, I.; Sanchez-Leal, J.; Comelles, F.; Garcia, M. T. J. Colloid Interface Sci. 1997, 187, 443. (15) Lopez, O.; Cocera, M.; Coderch, L.; Parra, J. L.; Barsukov, L.; de la Maza, A. J. Phys. Chem. B 2001, 105, 9879. (16) Ollivon, M.; Lesieur, S.; Grabielle-Madelmont, C.; Paternostre, M. Biochim. Biophys. Acta 2000, 1508, 34. (17) Deo, N.; Somasundaran, P. Colloids Surf. A-Physicochem. Eng. Aspects 2001, 186, 33. (18) Deo, N.; Somasundaran, P. Langmuir 2003, 19, 7271. (19) Fontana, A.; De Maria, P.; Siani, G.; Robinson, B. H. Colloids Surf. B-Biointerfaces 2003, 32, 365. (20) Majhi, P. R.; Blume, A. J. Phys. Chem. B 2002, 106, 10753. (21) Kawasaki, H.; Imahayashi, R.; Tanaka, S.; Almgren, M.; Karlsson, G.; Maeda, H. J. Phys. Chem. B 2003, 107, 8661. (22) Tan, A. M.; Ziegler, A.; Steinbauer, B.; Seelig, J. Biophys. J. 2002, 83, 1547. (23) Argy, G.; Bricout, F.; d’Hermies, F.; Cheymol, A. C. R. Acad. Sci., Ser. III 1999, 322, 863. (24) Budavari, S. The Merck Index; Merck & Co., Inc.: Whitehouse Station, 1996. (25) Warr, G. G.; Sen, R.; Fennell-Evans, D.; Trend, J. E. J. Phys. Chem. 1988, 92, 774. (26) Funasaki, N.; Neya, S. Langmuir 2000, 16, 5343.

10.1021/la050701f CCC: $30.25 © 2005 American Chemical Society Published on Web 06/25/2005

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Chart 1. (a) Di-C10DMAB, (b) OBG, (c) TNS, and (d) PRODAN Molecules

were mainly aimed at studying the effect of a second hydrophobic chain and the ionic or nonionic character of its polar head on the structure and properties of the monomeric and aggregated phases, as well as to analyze the ionic-nonionic interactions in the vesicle surface. With this background, we face in the present work a physical chemistry study of the aqueous solutions of cationicnonionic mixed vesicles constituted by a cationic doublechain surfactant (di-C10DMAB) and a nonionic single-chain surfactant, widely used in the crystallization and solubilization of membrane proteins,27-31 the n-octyl-β-Dglucopyranoside (OBG; see Chart 1). Both surfactants have hydrophobic chains of comparable length but differ on the charge of their polar heads. The parameters that determine and characterize the formation of mixed di-C10DMAB/OBG vesicles in diluted aqueous solutions, such as, the concentration domain, the packing features, the shape, the size, and the charge of the resulting vesicles have been evaluated by means of a variety of highly accurate experimental techniques, as well as, theoretical calculations. The critical vesicle concentrations of the pure, CVC, and mixed, CVC*, vesicles defined as the concentration corresponding to the monomer-to-vesicles transition, have been determined from conductivity experiments. This method has been also used to determine the critical micellar concentration, CMC*, or concentration at which mixed micelles start to form (vesicles-to-micelles transition). The solubilization process of the vesicles is generally described by using a “three stage model”11,32 which defines (a) stage I, where the surfactant monomers partition between the bulk and the bilayer, (b) stage II, where mixed vesicles and micelles coexist, and (c) stage III, where all double-chain surfactants are present in solution as mixed micelles. The structure and morphology of these aggregates have been analyzed by using cryogenic transmission electron microscopy (cryo-TEM), a technique (27) Garavito, R. M.; Rosenbush, J. P. J. Cell. Biol. 1980, 86, 327. (28) Michel, H.; Oesterhelt, D. Proc. Nat. Acad. Sci. U.S.A. 1980, 77, 1283. (29) Baron, C.; Thompson, T. E. Biochim. Biophys. Acta 1975, 382, 276. (30) Stubbs, G. W.; Gilbert, S. H.; Litman, B. J. Biochim. Biophys. Acta 1976, 425, 45. (31) Korsaric, N. Biosurfactants: Production. Properties. Aplications; Marcel Dekker: New York, 1993; Vol. 48. (32) Lichtenberg, D.; Robson; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285.

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which has been revealed as a powerful tool to analyze vesicles of small and medium size, in conjunction with some theoretical calculations based on the effective packing parameter of the mixed system.33 The vesicle/ solution interface has been also studied by means of electrophoretic mobility data. This method permits the zeta potential of the aggregate surface from the experimental electrophoretic mobility to be obtained, by using the Henry equation if the particle size is known. We use in this work the equation proposed by Ohshima34 to calculate the Henry function, f(κDa), as a function of κDa instead of the normally used expressions,35 once demonstrated that the validity limits of these equations are totally wrong for κDa falling within the range 0.5 < κDa < 25, which is the usual experimental range of most reported studies, the present one included. Additionally, steady-state fluorescence spectroscopy experiments have been carried out with the aim of further characterizing the mixed aggregates herein studied. The interaction between fluorophore molecules and auto-aggregated surfactant systems to analyze questions of the structure, interactions, and dynamics of the system is well documented in the literature.36-42 The basis for these studies is the characteristic sensitivity of the photophysical properties of many fluorophores to their immediate microenvironment; the interaction between the fluorophore molecules and the solvent or the auto-aggregated system has a direct effect on the energy difference between the ground and excited states of the fluorophore, affecting the maximum emission wavelength and the fluorescence quantum yield. The anionic probe 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS), and the nonionic probe 6-propionyl-2-dimethylaminonaphthalene (PRODAN) belong to this type of compounds with an electron donoracceptor moiety linked by a single bond to an aromatic ring (see Chart 1). This arrangement confers the molecules characteristic properties, which have been widely explained in the literature.36-46 In fact, the use of TNS and/ or PRODAN is quite advantageous in analyzing the surface and interior of mixed aggregates constituted by cationic and nonionic components. We do expect that these electrochemical, microscopic, and spectroscopic results will shed light to a better understanding of the parameters that govern the spontaneous self-organization processes of mixed aggregates. (33) Israelachvili, J. Intermolecular and Surfaces Forces with Application to Colloidal and Biological Systems; Academic Press: London, 1985. (34) Ohshima, H.; Furusawa, K. Electrical Phenomena at Interfaces. Fundamentals, Measurements, and Applications; Marcel Dekker: New York, 1998. (35) Henry, D. C. Proc. R. Soc. London, Ser. A 1931, 133, 106. (36) Lakowicz, J. R. Principle of Flourescence Spectroscopy; Kluwer Acad./Plenum Pubs.: New York, 1999. (37) Niu, S.; Gopidas, K. R.; Turro, N. J.; Gabor, G. Langmuir 1992, 8, 1271. (38) Krishnamoorthy, G.; Dogra, S. K. J. Colloid Interface Sci. 2000, 228, 335. (39) Zhong, D.; Kumar-Pal, S.; Zewail, A. H. ChemPhysChem 2001, 2, 219. (40) Karukstis, K. K. Encapsulation of fluorophores in multiple microenvironments in surfactant-based supramolecular assemblies; Academic Press: London, 2001; Vol. 3: Nanostructured Materials, Micelles and Colloids. (41) Karukstis, K. K.; Zieleniuk, C. A.; Fox, M. J. Langmuir 2003, 19, 10054. (42) Karukstis, K. K.; McCormack, S. A.; McQueen, T. M.; Goto, K. F. Langmuir 2004, 20, 64. (43) Kosower, E. M. Acc. Chem. Res. 1982, 15, 259. (44) Wang, Y.; Eisenthal, K. B. J. Chem. Phys. 1982, 77, 6076. (45) Parusel, A. B.; Nowak, W.; Grimme, S.; Ko¨hler, G. J. Phys. Chem. 1998, 102, 7149. (46) Lobo, B. C.; Abelt, C. J. J. Phys. Chem. 2003, 107, 10938.

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II. Experimental Section A. Materials. di-C10DMAB, with purities of 99% or greater, and OBG, with purity of 98% or greater, were purchased from Aldrich Co. The potassium salt of 2-(p-toluidino)naphthalene6-sulfonic acid (TNS) is from Sigma, and 6-propionyl-2-dimethylaminonaphthalene (PRODAN) is from Molecular Probes. All were used without further purification. OBG was stored in a dark place at 278 K. Double-distilled water was deionized using a Super Q Millipore system (with a conductivity lower than 18 µS cm-1) and finally was also degassed with a vacuum pump prior to the preparation of the solutions. B. Preparation of the Samples. Appropriate amounts of both components of the ternary system, di-C10DMAB and OBG, were weighted and dissolved simultaneously in water to prepare a solution of a given total surfactant concentration at the desired molar fraction. These initial solutions, after sonicated for 2 h in an ultrasonic bath, were homogeneous and totally clear in visiblescale. Their long term thermodynamic stability was checked by confirming that conductivity, κ, and zeta potential, ζ, results remained unchanged four months later, within the experimental uncertainty. C. Conductometric Measurements. Conductivity data were collected at 298.15 K ((1 mK) with a Hewlett-Packard 4263A LCR Meter. The whole equipment, the preparation of mixtures and the fully computerized procedure was widely described previously.47 The accuracy on the specific conductivity κ, obtained as an average of 2400 measurements for each concentration, is better than 0.03%. The conductivity measurements were made as a function of total surfactant concentration, [S]tot () [di-C10DMAB] + [OBG]), at several constant values of the molar fraction, R1. D. ζ-potential Measurements. A laser Doppler electrophoresis (LDE) technique (Zetamaster 2000, Malvern Instruments Ltd.), previously described,5 was used to measure electrophoretic mobilities at 298.15 ( 0.01 K. The cell used is a Zetasizer 2000 Standard Quartz rectangular capillary electrophoresis cell of 5 × 2 × 50 mm, which is calibrated with a zeta potential transfer standard of ζ ) (-50 ( 5) mV. ζ-Potentials were calculated from the electrophoretic mobility measurements made at the molar fractions studied on conductivity experiments. Each electrophoretic mobility data is taken as an average over 10 independent measurements. E. cryo-TEM Measurements. Samples for cryogenic transmission electron microscopy were vitrified according to the method devised by Dubochet et al.48 and also described in Llorca et al.49 Briefly, 5 µL aliquots of the different samples were applied to glow-discharged carbon coated grids for 1 min, blotted for 5 s, and frozen rapidly in liquid ethane at -180 °C and kept at this temperature throughout the whole procedure. Observations were conducted at a JEOL 1230 microscope operated at 100 kV. Micrographs were recorded on Kodak 4489 film at zero degrees tilt and a nominal magnification of 30 000×. Selected micrographs were digitalized in a Dimage Scan Multi Pro scanner (Minolta) with a sampling window of 7 Å/pixel at the specimen. F. Fluorescence Measurements. Steady-state fluorescence experiments were carried out with a Perkin-Elmer LS-50B Luminescence Spectrometer,50 connected to a PC computer. A 10 mm stoppered rectangular silica cell was placed in a stirred cuvette holder whose temperature was kept constant at 298.15 ( 0.01 K with a recirculating water circuit. The aqueous surfactant solutions previously prepared for conductivity and ζ-potential experiments were also used on fluorescence measurements. An aqueous stock solution of TNS at a concentration of 10.08 µM and an ethanol stock solution of PRODAN at a concentration of 97.22 µM were prepared. An aliquot of 10 µL of TNS stock solution was mixed with 3.85 mL of the surfactant solution, while an aliquot of 155 µL of the PRODAN stock solution was mixed with 3.00 mL of the surfactant solution, the ethanol (47) Junquera, E.; Aicart, E. Rev. Sci. Instrum. 1994, 65, 2672. (48) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129. (49) Llorca, O.; McCormack, E.; Hynes, G.; Grantham, J.; Cordell, J.; Carrascosa, J. L.; Willison, K. R.; Ferna´ndez, J. J.; Valpuesta, J. M. Nature 1999, 402, 693. (50) Junquera, E.; Pen˜a, L.; Aicart, E. Langmuir 1997, 13, 219.

Figure 1. Specific conductivity, κ, as a function of total surfactant concentration, [S]tot, in the very diluted range, at 298.15 K, at constant molar fraction, R1, for the mixed system di-C10DMAB (1) + OBG (2). The inset at the top shows the third derivative of conductivity with respect to concentration for R1) 0. 502. being previously evaporated with nitrogen in this case. As a result, probes concentrations are kept constant at [TNS] ) 2.07 µM and/or [PRODAN] ) 5.02 µM, and surfactant concentrations range from pre- to post-vesicle values, at each molar fraction of the ternary system. All surfactant/probe solutions thus prepared were first stirred for 2-3 h, and subsequently allowed to equilibrate more than 60 h, prior to running the spectra. Excitation wavelengths of 330 and 340 nm were chosen, and emission wavelengths were varied from 340 to 600 nm and from 350 to 650 nm, to record the fluorescent emission of TNS and PRODAN, respectively. In all of the cases, excitation and emission band slits were fixed at 2.5 nm and the scan rate selected at 240 nm/min.

III. Results and Discussion Vesicle Concentration Domain. Conductivity experiments were run to determine the concentration at which mixed di-C10DMAB (1)/OBG (2) vesicles are formed in aqueous solutions, the so-called mixed critical vesicle concentration, CVC*. Figure 1 shows the experimental specific conductivities, κ, as a function of total surfactant concentration, [S]tot, at several constant values of molar fraction, R1, through the whole composition range. The third derivative method51 (∂κ3/∂c3 ) 0) was used to calculate CVC* at each molar fraction. The inset at the top of Figure 1 shows a third derivative plot for R1 ) 0.502, as an example. The CVC* thus determined are plotted as a function of the molar fraction R1 in Figure 2. As can be observed, CVC* decreases with the molar fraction R1, which means that vesicles tend to form at lower concentrations as long as the total content on double-chain surfactant increases. It is worth noting that CVC* values are of the same order of magnitude than those previously reported for di-C12DMAB + OBG, whereas the CVC for the pure di-C10DMAB vesicles ()1.325 mM) is almost 1 order of magnitude higher than that of di-C12DMAB ()0.165 mM).5 It seems that the qualitative rule that states that the CMC is divided per 2 when the chain is enlarged (51) Phillips, J. N. Trans. Faraday Soc. 1955, 51, 561.

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Figure 2. CVC* as a function of the molar fraction, R1, for the mixed system di-C10DMAB (1) + OBG (2).

by a -CH2- group for single -Cn- chain surfactants52 can be also applied to double chain surfactants; thus, it can be observed how CVC divides by 8 on going from diC10DMAB to di-C12DMAB (a total of 4 -CH2- groups have been added). Vesicle concentration domain is limited by CVC*, on the monomers-to-mixed vesicle transition, and by the concentration at which the mixed vesicle are disrupted and solubilized by mixed micelles, CMC/tot. According with the three stages model,11,32 accepted by the groups working on this field, we do believe that prior to this total mixed vesicle-to-mixed micelle transition there exists a concentration at which mixed micelles start to form, the so-called CMC*, and coexist with mixed vesicles up to CMC/tot. The conductivity experiments were repeated in a more concentrated range (0 < [S]tot < around 25 mM), to estimate the CMC*. The change on specific conductivity at this CMC* (vesicles-to-micelles) is expected to be small, and, consequently, more difficult to detect than CVC* (monomers-to-vesicles). Figure 3 shows the results at R1 ) 0.501, as an example, together with those obtained in the highly diluted region, at R1 ) 0.502. The extremely good agreement between the experiments done in both ranges is remarkable, confirming the reproducibility of the technique. Although very small, a break on κ was found (following the third derivative method previously explained) at CMC* ) 12.3 mM, in agreement with CMC* found in the literature for similar ternary systems.4,5 It seems that the presence of the double-chain surfactant, irrespective of its length, halves the CMC* with respect to the value previously obtained for pure OBG (CMC* ) 25.24 mM), by using different experimental techniques.53-55 This fact means that the solubilization of the cationicnonionic vesicles, studied herein and previously,6 due to mixed micelles formation is clearly favored, reinforcing one of the well-known applications of OBG on the solubilization and reconstitution processes of membranes. (52) Attwood, D.; Florence, A. T. Surfactant Systems: Their Chemistry, Pharmacy and Biology; Chapman and Hall: London, 1983. (53) Pastor, O.; Junquera, E.; Aicart, E. Langmuir 1998, 14, 2950. (54) del Burgo, P.; Junquera, E.; Aicart, E. Langmuir 2004, 20, 1587. (55) Lainez, A.; del Burgo, P.; Junquera, E.; Aicart, E. Langmuir 2004, 20, 5745.

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Figure 3. Specific conductivity, κ, as a function of total surfactant concentration, [S]tot, in both the very diluted range (solid symbols, R1 ) 0.502), and the moderately diluted range (crossed symbols, R1 ) 0.501), at 298.15 K, for the mixed system di-C10DMAB (1) + OBG (2).

It is also worth noting that the specific conductivity is clearly higher for either mixed vesicles or mixed micelles of di-C10DMAB/OBG than for those of di-C12DMAB/OBG,6 at a given R1. This feature could be pointing to the fact that di-C10DMAB/OBG mixed aggregates show higher mobilities, although other factors, such as the differences on size and dissociation of the aggregates can affect, as will be discussed later. Vesicle Size and Shape. Cryo-TEM experiments were run on a sample with pure di-C10DMAB vesicles and on samples with mixed di-C10DMAB/OBG vesicles within a concentration range where only mixed vesicles are present, i.e., CVC* < [S]tot < CMC*. Figures 4-6 show, as an example, some of the micrographs obtained for the vitrified samples mentioned above. As can be observed, all of the micrographs show the presence of vesicles. Pure di-C10DMAB vesicles (Figure 4) are spherical and unilamellar, with an average diameter of about 36 nm, quite similar to pure di-C12DMAB vesicles previously reported.5 It seems that two C atoms per hydrocarbon surfactant chain do not affect the overall size and shape of the aggregates. Mixed di-C10DMAB/OBG vesicles (Figures 5 and 6) appear to be also spherical and mainly unilamellar, although it is noticeable a certain contribution of vesicles with onionskin structures. The averaged diameters are around 90 nm at R1 ) 0.501 and [S]tot ) 8.980 mM, and around 103 nm at R1 ) 0.848, [S]tot ) 2.992 mM, clearly higher than those of vesicles consisting of pure double-chain surfactant. However, a clear effect of R1 on the size of mixed vesicles is not apparent. It is worth noticing that at a given molar fraction the sizes of di-C10DMAB/OBG mixed vesicles are comparable in magnitude with those of di-C12DMAB/OBG mixed vesicles, but clearly smaller than those of di-C12DMAB/C12EDMAB.5,6 Given that the size and shape of pure vesicles consisting of double-chain surfactants of the dialkyldimethylammonium salt series seems to be unaffected by the length of the surfactant, these differences on the size of mixed vesicles can only be attributed to the cationic (C12EDMAB) or nonionic (OBG) character of the counterpart. Thus, a nonionic single-chain

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Figure 4. cryo-TEM photographies of a di-C10DMAB solution containing pure vesicles (R1 ) 1) at [S]tot ) 2.935 mM.

Figure 5. cryo-TEM photographies of a di-C10DMAB/OBG solution containing mixed vesicles at R1 ) 0.501 and [S]tot ) 8.980 mM.

Figure 6. cryo-TEM photographies of a di-C10DMAB/OBG solution containing mixed vesicles at R1 ) 0.848 and [S]tot ) 2.992 mM.

surfactant, as OBG, decreases the electrostatic repulsion between the cationic polar heads of a cationic doublechain surfactant than does a cationic single-chain surfactant, as C12EDMAB. With respect to the shape of the aggregates, both the contribution of the repulsive electrostatic interactions between the positive charged quaternary ammonium heads of di-C10DMAB, shielded by nonionic OBG head, and the molecular packing properties, coming from the hydrocarbon tails, must be considered. The packing parameter, P ()v/aolc), proposed by Israelachvili,33 relates the molecular characteristics of a surfactant molecule. The volume, ν, and the critical chain length, lc, of the hydrophobic tail, obtained according with the Tanford’s model,56-58 the optimal headgroup area, ao, estimated from the aggregation number of the single-chain surfactant assuming spherical micelles,50 and P are 593.4 Å3, 14.15 Å, 75 Å2, and 0.56, respectively, for di-C10DMAB. The corresponding parameters for OBG have been previously reported.6 Comparing this P with that of di-C12DMAB ()0.56 in both cases),5 it can be concluded that the decrease on the length of the tail of the double-chain surfactant (2 carbon atoms per chain, on going from di-C12DMAB to di-C10DMAB) does not affect to the packing parameter, that is compatible with a vesicle, as the optimal aggregate. On the other hand, the globular shape of pure OBG micelles6 is consistent with a P ) 0.35 for OBG. These theoretical results are in agreement with the cryo-TEM experiments reported herein and with fluorescence data reported in a previous paper.53 An effective packing (56) Tanford, C. J. Phys. Chem. 1972, 76, 3020. (57) Tanford, C. J. Phys. Chem. 1974, 78, 2469. (58) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley & Sons: New York, 1980.

parameter can be defined as follows, in the case of mixed aggregates:

Peffective )

( ) v

aolc

∑i viX agg i

) effective

∑i

ao,iX agg i

∑i

(1)

lc,iX agg i

where X agg is the molar fraction in the aggregate. i Effective packing parameters, calculated with eq 1, show a gradual decrease from 0.56 of the pure di-C10DMAB vesicles to 0.35 of the pure OBG globular micelles, as would be expected. The presence of only mixed vesicles, or only mixed micelles, or coexistence of both, depends very much on both R1 and [S]tot. For example, at R1 ) 0.5, Peffective results 0.54 at [S]tot ) 15 mM, 0.51 at [S]tot ) 26 mM, 0.49 at [S]tot ) 36 mM, and 0.48 at [S]tot ) 46 mM. Considering that CVC* ) 2.426 mM and CMC* ) 12.3 mM at R1 ) 0.5, and keeping in mind the characteristic values of P for globular micelles (0.33 < P < 0.50) and/or vesicles (0.50 < P < 1), the resulting Peffective indicate that there exists a concentration range, above CMC*, within mixed vesicles and mixed micelles coexist prior to the total disruption and solubilization of the vesicles at higher concentrations, as proposed in a former section (see Figure 3). In addition, it is worth noting the gradual change of Peffective, as [S]tot increases, justifying the gradual change on the specific conductivity found in this region of coexistence of both types of aggregates (see Figure 3). However, at higher R1, for example R1 ) 0.85, the resulting Peffective are all higher than 0.50, independently of [S]tot and even at high [S]tot concentrations (for example, Peffective ) 0.55 at [S]tot ) 40 mM and even Peffective ) 0.54 at [S]tot ) 100 mM), which

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Figure 7. Intensities vs electrophoretic mobility for a vesicle solution of the mixed system di-C10DMAB (1) + OBG (2), at R1 ) 0.878 and [S]tot ) 9.772 mM

means that for high contents of the double-chain surfactants, mixed vesicles are always present. This feature reveals that, up to moderate concentrations, the existence of the four concentration domains: i) mixture of monomers ([S]tot < CVC*), ii) mixed vesicles (CVC* < [S]tot < CMC*), iii) mixed vesicles + mixed micelles (CMC* < [S]tot < CMC/tot), and iv) mixed micelles ([S]tot > CMC/tot), is very much dependent on R1. These results are in agreement with the mixed aggregation behavior experimentally found in this work in the vesicle domain. Furthermore, a cryo-TEM experiment was run in the expected micelles domain, for a solution of di-C10DMAB/OBG at R1 ) 0.502 and [S]tot ) 38 mM, well above the CMC*. Mixed vesicles could not be visualized in any of the micrographs, confirming that at such concentration all of the vesicles have been solubilized by the mixed micelles, in concordance with the theoretical predictions (Peffective ) 0.49). Vesicle Surface. The charged outer surface of pure and mixed vesicles has been studied via electrophoretic mobility measurements. Figure 7 shows, as an example, the electrophoretic spectrum for a vesicle solution at R1 ) 0.848 and [S]tot ) 9.772 mM (CVC* ) 1.511 mM); the experimental peaks, with an optimum and uniform width, lead an average electrophoretic mobility of (5.42 ( 0.05) × 10-8 m2 s-1 V-1. ζ-Potentials can be obtained from these experimental electrophoretic mobilities, µE, by using the Henry equation.35,59 The Henry function, f(κDa), which appears in this equation, is usually calculated for spherical particles by using the approximations given by34,60

f(κDa) ) 1 +

f(κDa) )

(κDa)3 (κDa)4 (κDa)2 -5 16 48 96 (for spheres with κDa < 1) (2)

9 3 330 75 + 2 2(κDa) 2(κ a)2 (κ a)3 D D (for spheres with κDa > 1) (3)

where κD and a are the Debye length reciprocal and the particle radius, respectively.

Junquera et al.

Figure 8. Henry function f(κDa) vs κDa: 0, tabulated values; b, data from eq 2; 2, data from eq 3; and dashed line according to eq 4.

Figure 8 shows a plot of f(κDa) values61 vs (κDa), together with those calculated with eqs 2 and 3 for (κDa < 1) and (8 < κDa < 30), respectively. It is worth noticing that (i) the abovementioned validity limits of eqs 2 and 3 are incorrect; eq 2 can be applied with a reasonably accuracy only for κDa < 0.5 and eq 3 would be valid only for κDa > 25; and (ii) neither eq 2 nor eq 3 can be used for spherical particles for which κDa falls within the range 0.5 < κDa < 25. However, in most published studies, as is also the case of the vesicle aqueous solutions studied herein, this is, in fact, the experimental κDa range. This feature was already pointed out by Henry,34 who recommended to use an interpolation formula, given that there is a hiatus in the course of calculations of f(κDa). At this respect, we have used in this work the expression derived by Ohshima34 for the Henry function, f(κDa), (relative errors less than 1%) to improve the calculations of zeta potentials from electrophoretic mobilities (dashed line in Figure 8)

f(κa) )

[

2 1+ 3

(

2 1+

1 2.5 κa(1 + 2e-κa)

)] 3

(4)

The Ohshima fit gives very good results in the full κDa range, and specially, as can be observed in the figure, for 0 < κDa < 25, which is the range of ionic strength and size of our vesicle solutions. Table 1 reports κDa, f(κDa) and ζ-potential calculated at several molar fractions, R1, for solutions that contain mixed di-C10DMAB/OBG vesicles. In these calculations, a values were taken from cryo-TEM data. Notice that ζ-potential is almost constant with R1, around (80 ( 5) mV, within the experimental error, although a slight decrease could be pointed as long as the content of the ionic double-chain surfactant, di-C10DMAB, increases. (59) Delgado, A. V. Interfacial Electrokinetics and Electrophoresis; Marcel Dekker: New York, 2002; Vol. 106. (60) Hunter, R. J. Zeta Potential in Colloids Science. Principles and Applications; Academic Presss: London, 1981. (61) Abramson, H. A.; Moyer, L. S.; Gorin, M. H. Electrophoresis of Proteins; Reinhold: New York, 1942.

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Table 1. Values of CVC*, KDa, f(KDa) and ζ at Several Molar Fractions, r1, and Total Surfactant Concentration, [S]tot, of the Mixed System di-C10DMAB (1) + OBG (2) R1

[S]tot (mM)

CVC* (mM)

[S]1 (mM)

κDa

f(κDa)

ζa (mV)

0.401 0.649 0.749 0.749 0.848 1.000

6.076 7.890 9.788 6.116 9.772 2.427

2.807 1.885 1.735 1.735 1.511 1.325

2.434 5.124 7.331 4.581 8.284 2.427

7.0 11.2 13.9 11.0 15.4 2.9

1.19 1.26 1.30 1.26 1.31 1.09

86 80 83 81 76 73

a

Error is estimated to be 6%.

Figure 9. Surface charge density, σζ, as a function of molar fraction, R1, for various [S]tot concentrations of the mixed systems: open squares, di-C10DMAB (1) + OBG (2), and solid squares, di-C12DMAB (1) + OBG (2).

The surface charge density enclosed by the shear plane, σζ, can be calculated, assuming a Gouy-Chapman double layer, by using the equation62

σζ )

[ ( )

( )]

20rκDkBT 2 zeζ zeζ tanh sinh + ze 2kBT κDa 4kBT

(5)

where e is the elemental charge, z is the valence of the ion, kB is the Boltzmann constant, and T is the absolute temperature. Figure 9 shows σζ, calculated with eq 5, as a function of R1, together with σζ for di-C12DMAB/OBG vesicles,6 whose ζ-potential have been also corrected by eq 4. An average value of (19 ( 4) × 10-3 C m-2 has been obtained, almost constant with R1 within the experimental uncertainty of ζ-potential measurements. A comparable result ((14 ( 4) × 10-3 C m-2) has been found for di-C12DMAB/OBG vesicles. Comparing these σζ with those reported for the cationic-cationic system di-C12DMAB + C12EDMAB,5 also corrected, (σζ ) (9 ( 2) × 10-3 C m-2), it can be concluded that nonionic OBG molecules shield the electrostatic repulsion between the cationic quaternary ammonium heads, thus stabilizing the aggregates, increasing the counterion dissociation degree, and, accordingly, ζ-potential and σζ. This fact would also explain (i) (62) Loeb, A. L.; Overbeek, J. T. G.; Wiersema, P. H. The Electrical Double Layer Around a Spherical Colloid Particle; MIT Press: Cambridge, MA, 1961.

the slight decrease on zeta potential as long as R1 increases, observed in Table 1; and (ii) the higher conductivities observed at a certain R1 for the system studied in this work, compared with those of previous works. Aggregates Microenvironments. The structure and morphology of di-C10DMAB/OBG mixed aggregates has been analyzed by measuring the fluorescent emission of two probes: one anionic, such as TNS, and the other one nonionic, such as PRODAN. Both emit weakly in water but exhibit intense fluorescence upon binding to a macromolecule or membrane surface.36,40 The net charge of the surface assembly is a key factor on the emission features; thus, in the case of positively charged surfaces, as the di-C10DMAB/OBG system of this work, anionic TNS is not repelled and surfactant-TNS interactions allow the probe to be located in a more water-restricted environment, thus enhancing the fluorescence quantum yield. On the other hand, the nonionic character of PRODAN allows for supplying information either of ionic or nonionic aggregates. It is known36 that the ground states of the fluorescent probes used in this work, which are characterized by possessing a naphthalene ring and a phenyl ring (TNS) or a dimethylamino moiety (PRODAN) in their molecules, yield, upon excitation, a locally excited state (LE) (via π f π* electronic transition). However, there exists much controversy in the past decade regarding the existence of other excited states, and the mechanism by which these excited states are formed and deactivated. Some groups propose that an intramolecular charge-transfer excited state is formed, characterized by the coplanar orientation of the rings (ICT state),46,63-68 whereas others point to an intramolecular charge-transfer excited state (TICT state),45,69-71 with a characteristic twist around the NH moiety to achieve a perpendicular configuration between the naphthalene ring and phenyl ring (TNS) or dimethylamino moiety (PRODAN). In either case, the transfer of nitrogen lone pair to the sulfonate (TNS) or acetyl substituents (PRODAN) of the naphthyl ring, is stabilized in polar media.36,40 Recently, an TICT state has been proposed by Zewail et al.,39 from femtosecond studies, for the emission of TNS in water, whereas only the classic LE state is found for TNS immersed in supramolecular assemblies, such as CTAB micelles. This photophysical scheme, however, has been questioned, mainly in supramolecular assemblies.41,42,72-74 In another approach, the emission band of this type of fluorescent probes is believed to consist of different peaks or emission components at different wavelength that can be attributed to a series of microenvironments where the probe may be housed, each microenvironment having (63) Zachariasse, K. A.; van der Haar, T.; Hebecker, A.; Leinhos, U.; Kiihnle, W. Pure Appl. Chem. 1993, 65, 1745. (64) Zachariasse, K. A.; Grobys, M.; van der Haar, T.; Hebecker, A.; Il’ichev, Y. V.; Morawski, O.; Riicker, I.; Kiihnle, W. J. Photochem. Photobiol. A: Chem. 1997, 105, 373. (65) Kawski, A. Z. Naturforsch. 1999, 54, 379. (66) Il’ichev, Y. V.; Kuhnle, W.; Zachariasse, K. A. J. Phys. Chem. A 1998, 102, 5670. (67) Zachariasse, K. A. Chem. Phys. Lett. 2000, 320, 8. (68) Zachariasse, K. A.; Druzhinin, S. I.; Bosch, W.; Machinek, R. J. Am. Chem. Soc. 2004, 126, 1705. (69) Grabowski, Z. R.; Rotkiewicz, K.; Siemiarexuk, A.; Cowley, D. J.; Baumann, W. Nouv. J. Chim. 1979, 3, 443. (70) Rettig, W.; Bliss, B.; Dirnberger, K. Chem. Phys. Lett. 1999, 317, 187. (71) Jo¨dicke, C. J.; Lu¨thi, H. P. J. Am. Chem. Soc. 2002, 125, 252. (72) Asakawa, T.; Mouri, M.; Miyagishi, S.; Nishida, M. Langmuir 1989, 5, 343. (73) Manoj, K. M.; Jayakumar, R.; Rakshit, S. K. Langmuir 1996, 12, 4068. (74) Mandal, D.; Pal, S. K.; Datta, A.; Bhattacharyya, K. Anal. Sci. 1998, 14, 199.

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Figure 10. Emission fluorescence spectra of TNS ([TNS] ) 2.07 µM) immersed on aqueous solutions of di-C10DMAB (1) + OBG (2) at: (i) pre-vesicle concentrations (dash lines, in increasing intensity order), R1) 1, [S]tot ) 0.070 mM; R1) 1, [S]tot ) 0.696 mM; and R1) 0.502, [S]tot ) 0.698 mM; and (ii) mixed vesicle concentrations (solid lines, in increasing intensity order), R1) 0.502, [S]tot ) 5.970 mM; R1) 0.649, [S]tot ) 5.417 mM; R1) 1, [S]tot) 3.113 mM; and R1) 0.502, [S]tot ) 8.132 mM. The inset on the top shows the deconvolution of the emission fluorescence band of TNS immersed on a vesicle solution of di-C10DMAB (R1) 1, [S]tot ) 3.113 mM) on three Gaussian components.

different hydrophobicity, microviscosity, rigidity, and/or solvation characteristics. Accordingly, this approach interprets the overall emission of the probe by deconvoluting the band into the optimum number of reproducible overlapping curves, and assigning them to different probe microenvironments, this assignment being based on their expected and/or known properties. This is, in fact, our approach in the present work. Figures 10 and 11 show the emission spectra of TNS and PRODAN, respectively, for a series of di-C10DMAB/ OBG/probe aqueous solutions at different molar fractions, R1, and total surfactant concentration, [S]tot. These concentrations were chosen to have either monomers or vesicles, according with the CVC* at each molar fraction, obtained from conductivity experiments. Experimental emission spectra were analyzed, after conversion of wavelength to frequency, by deconvolution into overlapping Gaussian curves with a commercial nonlinear leastsquares multi-peaks fitting procedure that uses an iterative Marquardt-Levenberg fitting algorithm. The experimental fluorescence data were thus deconvoluted into the optimum number of reproducible components, based on the photophysical data of probe emission, the center, width, and amplitude of each Gaussian curve being the adjustable parameters. Several features were chosen as control tests to ensure the goodness of the deconvolution: (i) the reproducibility of the final results for the center, width, and amplitude of the peaks, irrespective of the choice of the starting parameters on a series of different fits; (ii) a minimum value in the χ2 parameter; (iii) a random residual plot with no systematic features; and (iv) a maximum value for the square of the multiple correlation coefficient, r2, better than 0.999. The insets on the tops of Figures 10 and 11 show, as examples, the

Junquera et al.

Figure 11. Emission fluorescence spectra of PRODAN ([PRODAN] ) 5.02 µM) immersed on aqueous solutions of diC10DMAB (1) + OBG (2) at: (i) pre-vesicle concentrations (dash lines, in increasing intensity order), R1) 0.502, [S]tot ) 0.701 mM; R1) 1, [S]tot ) 0.700 mM; and (ii) mixed vesicle concentrations (solid lines, in increasing intensity order), R1) 0.649, [S]tot ) 5.455 mM; R1) 0.502, [S]tot) 8.134 mM; R1) 0.502, [S]tot) 5.986 mM; and R1) 1, [S]tot ) 3.122 mM. The inset on the top shows the deconvolution of the emission fluorescence band of PRODAN immersed on a vesicle solution of di-C10DMAB (R1) 0.502, [S]tot ) 5.986 mM) on three Gaussian components.

deconvolutions of TNS emission for the solution at R1 ) 1 and [S]tot, ) 3.113 mM, and PRODAN emission for the solution at R1) 0.502 and [S]tot ) 5.986 mM, respectively. Tables 2 and 3 resume the fitting parameters for all the experimental spectra of TNS and PRODAN, respectively. As can be noticed, in all of the cases, the mathematical treatment points to the presence of three microenvironments, in both pre-vesicle and vesicle solutions, which have been assigned as follows. TNS Emission. Two groups of peaks are clearly observed in Figure 10, those corresponding to monomers or prevesicle solutions (dashed lines) and those corresponding to vesicle solutions (solid lines). As can be seen in Table 2, the Gaussian components of the former (first three lines in Table 2) are centered at 396, 414, and 442 nm, whereas the latter (last 4 lines in Table 2) are centered at 424, 447, and 478 nm. Note that λi remains quite constant within each group, irrespective of (i) R1 (from 0.5 to 1); (ii) monomer concentration (from 0.07 to 0.7 mM); and/or (iii) vesicle concentration, [S]v, (from 3.5 to 5.7 mM at R1 ) 0.5). These peaks have been assigned to the following microenvironments: (i) The 442-447 nm emission, the unique peak in common on vesicle and pre-vesicle solutions, has to be assigned to TNS in the bulk solvent. In the presence of vesicles, (ii) the emission at around 424 nm has been attributed to TNS inside the hydrophobic bilayer. This is the more hydrophobic region and, accordingly, the corresponding emission is the most blue shifted one and (iii) the emission at around 478 nm is assumed to come from TNS in the vesicle surface. Notice that TNS experiences a lower energy transition when it is housed in the surface, than that one found in the bulk solvent, indicating that the charged di-C10DMA+ heads yield a higher polar microenvironment than that of the bulk. Furthermore, it is known that TNS hardly fluoresces in

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Table 2. Parameters of the Deconvoluted Gaussian Components of the TNS Fluorescence Emission Spectra at Various Molar Fractions, r1, and Total Surfactant Concentrations, [S]tot of the Mixed System di-C10DMAB (1) + OBG (2): Wavelength λι, Width Wi, and Area Ai in Terms of % Contribution to the Overall Fluorescence Emission Area R1

[S]tot (mM)

CVC* (mM)

0.502 1 1 0.502 0.502 0.649 1

0.698 0.070 0.696 5.970 8.132 5.417 3.113

2.426 1.325 1.325 2.426 2.426 1.885 1.325

[S]v (mM)

λmax (nm)

Imax

λ1 (nm)

A1 %

W1

λ2 (nm)

A2 %

W2

λ3 (nm)

A3 %

W3

r2

3.544 5.706 3.532 1.788

396.0 416.1 401.5 436.0 433.0 435.1 436.5

802.6 11.5 314.1 257.9 352.1 263.1 292.9

395.4 397.1 396.3 424.4 423.8 424.3 424.0

22.4 17.7 17.3 15.3 21.7 24.9 23.6

16 20 17 29 30 31 31

412.3 414.5 415.1 445.5 447.2 448.4 447.4

45.9 49.4 42.5 49.3 46.4 43.2 47.2

28 33 31 45 43 42 43

436.9 445.3 443.9 476.3 478.8 479.2 479.7

31.7 32.8 40.2 35.4 31.8 31.9 29.2

47 59 54 73 67 66 66

0.9993 0.9986 0.9985 0.9998 0.9999 0.9999 0.9999

Table 3. Parameters of the Deconvoluted Gaussian Components of the PRODAN Fluorescence Emission Spectra at Various Molar Fractions, r1, and Total Surfactant Concentrations, [S]tot of the Mixed System di-C10DMAB (1) + OBG (2): Wavelength λι, Width Wi, and Area Ai in Terms of % Contribution to the Overall Fluorescence Emission Area R1

[S]tot (mM)

CVC* (mM)

0.502 1 0.502 0.502 0.649 1

0.701 0.700 5.986 8.134 5.455 3.123

2.426 1.325 2.426 2.426 1.885 1.325

[S]v (mM)

λmax (nm)

Imax

λ1 (nm)

A1 %

W1

λ2 (nm)

A2 %

W2

λ3 (nm)

A3 %

W3

r2

3.560 5.708 3.570 1.796

515.9 514.5 494.6 493.4 497.9 495.1

151.6 247.0 538.2 525.6 502.3 664.8

459.8 459.6 433.6 432.0 432.8 440.2

10.2 20.0 4.8 5.1 4.9 6.8

54 66 43 40 42 51

514.9 514.7 498.8 497.8 497.8 498.7

85.0 75.5 93.5 93.3 93.6 91.6

50 49 58 59 59 59

576.6 576.0 577.4 578.6 578.0 578.9

4.8 4.6 1.7 1.5 1.5 1.6

35 36 31 30 30 30

0.9992 0.9993 0.9997 0.9997 0.9997 0.9996

water, but it does in aqueous solutions with small amounts of surfactant (below CVC or CMC).37 It means that in pre-vesicle solutions, part of TNS must be within environments with less polarity than water. Based on that, the other two peaks on the spectra for pre-vesicle solutions have been assigned as follows: (iv) the emission at around 396 nm could be attributed to TNS housed within clusters formed by the di-C10DMA+ and OBG monomers. These clusters should be characterized by a rigid hydrophobic cavity, which would result in an intense blue shifted transition with respect to that found in the bulk.37 (v) We suggest two possibilities for the peak at around 414 nm: the formation of ion-pair complexes, as suggested by some other authors,75 or the presence of certain microdomains in the solution, that must be more loose than the previous commented hydrophobic cavity, since this microenvironment should be more polar than that rigid hydrophobic cavity but less polar than the bulk solvent. Actually, we reported in a previous work the formation of nanoaggregates, prior to the CVC*,6 in a similar mixed surfactant system. Accordingly, other authors37,75 suggest that the association of the fluorescent probe with, for example, monomers or clusters of surfactant in aqueous media could be the responsible for the emission observed at concentrations below the CMC. The previous λi assignment is consistent with the next features also resumed in Table 2: (i) at constant R1, Imax increases with [S]tot in pre-vesicle solutions (R1 ) 1), and with [S]v in vesicles solutions (R1 ) 0.502), indicating that, as surfactant concentration increases, the probe is located in less polar environments; (ii) Imax remains constant at different R1 () 0.502 and 0.649) and constant [S]v () 3.5 mM), confirming that vesicle concentration is the factor that mainly governs the intensity of the peak; (iii) in the absence of vesicles, when the surfactant concentration is extremely low, Imax also results the lowest, but increases dramatically with surfactant concentration. Furthermore, at constant [S]tot ) 0.7 mM (below CVC*), Imax decreases with R1, which clearly indicates that OBG has a participation in the microenvironments by creating, together with di-C10DMA+, a more hydrophobic region than that formed by di-C10DMA+ alone. In conclusion, the micropolarity that TNS experiments within the microdomains below CVC* is lower than that one found within the (75) Karukstis, K. K.; Savin, D. A.; Loftus, C. T.; D’Angelo, N. D. J. Colloid Interface Sci. 1998, 203, 157.

vesicles, this fact producing a blue shift of the spectra with respect to the spectra obtained when vesicles are present. These features, not expected to be found in the pre-vesicle region, in principle, are in agreement with previous results obtained by other authors37 for TNS in surfactant solutions of C16TAB. PRODAN Emission. Accordingly, two groups of peaks can be also observed in Figure 11, where emission spectra of PRODAN in aqueous solutions of the di-C10DMAB/OBG system are shown: those corresponding to pre-vesicle solutions (dashed lines) and those corresponding to vesicle solutions (solid lines). The Gaussian components obtained for pre-vesicle solutions (first 2 lines in Table 3) are centered at 460, 515, and 576 nm, whereas those for vesicle solutions (last 4 lines in Table 3) are centered at 434, 498, and 578 nm. Again, λi remains quite constant within each group of spectra, irrespective of the composition, R1, of the system (from 0.5 to 1) and/or vesicle concentration, [S]v, (from 3.5 to 5.7 mM at R1 ) 0.502). The peaks of PRODAN have been assigned as follows: (i) the emission at around 578 nm, the unique peak in common on vesicle and prevesicle solutions, has been assigned to PRODAN in the bulk solvent. In the presence of vesicles: (ii) the emission at around 434 nm has been attributed to PRODAN inside the hydrophobic bilayer; and (iii) the emission at around 498 nm is assumed to come from PRODAN in the vesicle surface. It is worth noticing that the emission of PRODAN from the vesicle surface is blue shifted with respect to that from the bulk, in contrast with the behavior previously presented for TNS. It can only be due to the characteristics of the probe, since the vesicle surface is the same, irrespective of the probe. In pre-vesicle solutions, the assignment of the other two peaks is similar to that proposed for TNS: (iv) the emission at around 460 nm has been also attributed to PRODAN immersed within clusters formed by the di-C10DMA+ and OBG monomers and (v) the peak at around 515 nm has been attributed to the loose microdomains previously referred for TNS, since the formation of ion-pair complexes is not possible in this case, given the nonionic character of PRODAN. The previous λi assignment is consistent with the next features also resumed in Table 3: (i) at constant R1, Imax remains roughly constant with increasing [S]v in the vesicles domain, which can be explained if the probe is almost totally immersed in the vesicle; (ii) at constant [S]v () 3.6 mM), Imax decreases with R1 varying from 0.502 and 0.649; (iii) in the absence of vesicles, Imax is lower

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than that in its presence, irrespective of R1, whereas at constant [S]tot ) 0.7 mM (below CVC*), Imax increases with R1, which indicates that, in contrast with what we have found for TNS, in the case of PRODAN, the OBG is not the main responsible of the hidrophobicity of the microdomains. In conclusion, the micropolarity that PRODAN experiments in the microdomains below CVC* is slightly higher than that felt within the vesicles, this fact producing a red shift of the spectra compared with those obtained in the presence of vesicles. The global picture of results obtained from the emission spectra of TNS and PRODAN indicates that both probes are predominantly solubilized within the vesicles (notice in Tables 2 and 3 that (A1 + A3) > A2 for TNS and (A1 + A2) > A3 for PRODAN, where Ai are the areas of the deconvoluted bands in terms of % contribution to the overall fluorescence emission area). Nevertheless, the anionic TNS remains within the bulk solvent up to considerable extent (A2 is about 45% of the total fluorescence), whereas the nonionic PRODAN are almost totally housed within the vesicles (A3 is very low). Another interesting aspect is that both probes are solubilized mainly within the vesicle surface (A3 > A1 for TNS and A2 > A1 for PRODAN). In fact, in the case of PRODAN, it seems that almost all of the probe is solubilized in the palisade layer of the vesicle, since A2 is around 93% of the total fluorescence (A2 . A1 + A3). This fact, usually found for both vesicle and micelles aggregates,8,36,76 confirms that the solubilization of fluorescent probes, which are usually globular molecules, within the hydrophobic core of the aggregate may be sterically hindered. Once the Gaussian components of the emission spectra of TNS and PRODAN have been assigned to different

vesicle (hydrophobic core and/or surface) and pre-vesicle (clusters and/or nanoaggregates) microenvironments of different polarity, and using the reported λmax of both probes in a series of pure solvents of known dielectric constant,40,77 several conclusions can be remarked: (i) the results obtained in the case of TNS indicate that the hydrophobic core (λ1 ) 424 nm) presents a polarity similar to that of n-propanol ( ) 21), whereas the vesicle surface (λ1 ) 478 nm) resembles an ethylenglycol environment ( ) 41.4); and (ii) the results obtained from PRODAN spectra reveal an hydrophobic core similar to THF ( ) 7.4) or CHCl3 ( ) 4.8) environments and a palisade with a polarity similar to that of ethanol ( ) 24). Given that the vesicles are the same in both cases, these results point to the fact that PRODAN is located deeper in the hydrophobic core than TNS, and furthermore, it selects zones of vesicle surface richer on nonionic OBG than its anionic counterpart (TNS) does. However, conclusions regarding the effect of the composition of the system, R1, on the polarity of the different microenvironments are speculative, since the spectroscopic parameters within each microenvironment do not change almost with R1. This feature is consistent with the facts that size, zeta potential, and charge density do not seem to be affected by the composition. Acknowledgment. The authors thank the Spanish Ministry of Education, Project No. BQU2002-586. J.B. thanks MEC of Spain (Project No. SAF2002-01715) for supporting cryo-TEM measurements. The authors also thank to O. Llorca for assistance on cryo-TEM experiments.

(76) Zana, R. Surfactant Solutions: New Methods of Investigation; Marcel Dekker Inc.: New York, 1987.

(77) Lide, D. R. CRC Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 2004.

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