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Doi:10.1016/j.jcis.2007.09.008

M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 where n is the solution refractive index.
The normalized second order correlation functions (g(2)(t)) were analyzed using both GENDIST and CONTIN inverse Laplace algorithms, giving both similar relaxation timedistributions. From the average relaxation times, τ , the appar-ent diffusion coefficients, Dapp, were obtained and using theStokes–Einstein relation, the apparent hydrodynamic radii werecalculated 1/τ = Dappq2 = app q 2. 2.5. Cryogenic transmission electron microscopy, Cryo-TEM Cryo-transmission electron microscopy is a particular tech- nique for the direct visualization that has been used to obtain Fig. 1. Fluorescence spectra of nabumetone solubilized in vesicles of AOT with direct high resolution images of surfactant aggregates such as 0.6% PEG 17k containing different surfactant concentration. Dotted line is the micelles, vesicles and different structures formed in polymer– fluorescence spectrum of nabumetone dissolved in aqueous polymer solution.
surfactant systems The sample (0.1 µl) was depositedon a carbon-coated holey polymer film in a controlled envi-ronment vitrification system, (CEVS) Gatan cryoplunge, wheretemperature and relative humidity are controlled. The excessliquid is blotted away with filter paper and quickly vitrified inliquid ethane. The sample is then transferred to a JEOL JEM2010 FEG electron microscope by means of a Gatan CT 3500Cryo-transfer system. The observations were made to an ac-celeration voltage of 200 kV. The working temperature was−170 ◦C and the images were recorded on a digital cameraGatan MSC794. The pictures were taken at a certain defocusto obtain high-quality images.
3. Results and discussion
3.1. Steady-state fluorescence measurements Fig. 2. Effect of the AOT concentration on the fluorescence quantum yield ofnabumetone solubilized on AOT/PEG 6k aggregates: triangles [PEG 6k] = 1%and circles [PEG 6k] = 10%. The quantum yield is normalized respect to that The emission spectra of nabumetone dissolved in Aerosol of nabumetone dissolved in polymer solutions, see text.
OT vesicles and in AOT–polyethylene glycol vesicles were ob-tained. Some of these spectra are represented in As can cvc, see The cvc values found using this method ac- be seen in the figure the nabumetone emission spectrum con- ceptably agree with those obtained from conductivity or surface sists in a broad band centered at 350 nm and the emission in- tension measurements Differences between the cvc values tensity increases as the surfactant concentration also increases.
obtained with distinct techniques were analyzed elsewhere To quantify differences between emission intensity, the fluores- and shown that the quantum yield of nabumetone can be used cence quantum yields, Φf, is preferred to fluorescence intensity; to estimate the cvc of vesicles As can be seen in the fig- then the variation of Φf with surfactant concentration is repre- ure, the nabumetone quantum yield increases more sharply in sented in Results represented in the figure correspond aggregates dissolved in dilute polymer solutions.
to mixed vesicles of AOT and PEG 6k, similar behavior is The photochemical behavior of this probe has been stud- observed in all mixtures studied. For the sake of clarity, the ied and the results indicated that nabumetone exists in fluorescence quantum yield of nabumetone dissolved in mixed two conformations, the folded conformation, low Φf, respon- polymer–surfactant aggregates was normalized respect to the sible for the fluorescence quenching of the naphthalene ring quantum yield of nabumetone dissolved in aqueous polymer so- by the butanone side chain, and the extended conformation lution (Φ0) with polymer concentration match those in the AOT with high Φf. The folded conformation predominates in aque- ous solutions (≈91%). According to values found, in the Results presented in show that the quantum yield of most dilute surfactant solutions the folded conformation pre- nabumetone increases with AOT concentration until it reaches dominates, while at high surfactant concentrations, the probe a plateau at a particular surfactant concentration, cvc, corre- is transferred to a more hydrophobic microenvironment, and sponding to the vesicle formation. The cvc is obtained from the quantum yield increases. Finally, when the AOT concen- the interception of quantum yield values above and below the tration reaches the cvc the probe nabumetone is incorporated M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 Table 1Critical vesicle concentration values found for Aerosol OT vesicles hope that the anchored polymer molecules destabilize the vesi-cle and lead to the formation of different kind of aggregates.
From fluorescence results, the polymer concentrations cor- responding to vesicle–micelle transition do not present amonotonic behavior with the polymer molecular weight. Thus,the vesicle–micelle transition seems to be favored by the addi-tion of PEG molecules of intermediate weight, PEG 17k. Thisfact can be due to the preferential adsorption of this polymerat the vesicle surface. The polymer coverage of the vesicle sur-face may be controlled by varying polymer concentration orvarying polymer size. The anchoring free energy of a poly-mer molecule consists in two main components the energygained by inserting the polymer into the surfactant monolayerand the entropy lost by constraining the chain by the surface.
Fig. 3. Effect of polymer concentration on the fluorescence quantum yield of These components have an opposite dependence on the poly- nabumetone solubilized in AOT/PEG aggregates: (triangles) PEG 6k, (squares) mer mass; consequently, the polymer adsorption goes through PEG 17k and (circles) PEG 35k. The AOT concentration is 0.03 M. The linesare guide to the eyes.
a maximum at a certain polymer size We also analyze the effect of the polymer molecular weight to the aggregates and the quantum yield remains constant. In on the quantum yield of nabumetone. As can be seen in a previous work, nabumetone was successfully used to the lowest quantum yield values correspond to mixtures withPEG 17k, while no very significant differences are observed determine the critical concentration of pure vesicles of AOT on the quantum yield values of aggregates with PEG 6k and and mixed vesicles formed by AOT and a water-soluble poly- PEG 35k. This fact can be explained if one takes into account mer, PSS or PEG 17k. In the work, the polymer solutions are that nabumetone is strongly quenched by water and that always in dilute regime. The cvc is evaluated from the nabume- nabumetone is located at the vesicle interface Thus, the tone quantum yield vs surfactant concentration curves and the low the quantum yield values in systems with PEG 17k can be values are collected in Taking into account that the due to an increase of the hydration of the surfactant head group cvc of pure AOT vesicles is 7.3 mM the cvc values at the interface. Baran et al. showed that different hydration show two different trends, for dilute polymer solutions the cvc levels can be induced by the adsorption of PEG and correspond decreases as compared with pure AOT vesicles. This classical with two PEG structural configurations: the PEG molecules ad- behavior in polymer–surfactant mixtures indicates the existence sorbed in form of random coil, low hydration level, and the of polymer–surfactant interactions responsible of the stabiliza- strongly adsorbed polymer molecules giving a brush structure, tion of the aggregates This fact is consistent with results highly hydrated surface. Thus in our systems, the fluorescence obtained elsewhere for AOT/PEG 17k in dilute polymer solu- results seem to indicate that PEG 17k induces higher hydration tions Conversely, when polymer concentration increases on the vesicle surface than PEG 6k or PEG 35k. This fact is up to a certain value the cvc is higher than that for pure AOT probably due to the stronger adsorption of this polymer at the vesicles. In addition, the nabumetone quantum yield increases more sharply in aggregates dissolved in dilute polymer solu- All these results clearly show that the properties of vesicle tions. These facts seem to indicate the existence of two different interface plays and important role on the stability of aggregates.
This is consistent with theoretical predictions and experi- presents the variation of nabumetone quantum yield mental observations To obtain more information about theinterface of the different aggregates the electrophoretic mobil- with polymer concentration. The AOT concentration is kept ity was obtained.
constant at 0.03 M. In the figure, we represent the ratio be-tween the fluorescence quantum yield of nabumetone dissolved 3.2. Electrophoretic mobility results in AOT/PEG mixtures and dissolved in pure vesicles of AOT,referred as Φvesicle. The quantum yield of nabumetone increases The effect of the addition of PEG polymers on the elec- as the polymer concentration increases and it reaches a constant trophoretic mobility of AOT vesicles was investigated and the value at a certain polymer concentration: 2.7% for PEG 6k, results are presented in The electrophoretic mobility 1.5% for PEG 17k and 2.5% for PEG 35k. All these facts seem curves decreases as the polymer concentration increases and to indicate the existence of different aggregation processes de- the effect is more pronounced in PEG 17k. The diminution of pending on polymer concentration. In particular, one could the electrophoretic mobility by the addition of polymers was M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 The inverse decay time shows the expected q2 depen-dence in the case of the polymer, while for AOT shows a nonq2 only above a scattering angle of 100 ◦C.
In the case of vesicles the non q2 behavior has been pre- dicted when Helfrich interfacial elasticity is properly consid-ered in the calculation of the scattering function a moredetailed study on this matter will be presented in a future paper.
All the experiments corresponding to mixtures present mul- tiexponential correlation functions and the ILT treatment givescomplex distribution functions. presents the distribution function, on the apparent hydrodynamic radius scale (R two different concentrations of PEG 6k and illustrates the be-havior found for the rest of polymer molecular weights.
Fig. 4. Effect of the mixture composition on the electrophoretic mobility of At low polymer concentrations the distribution functions vesicles composed of 0.03 M of AOT and variable polymer concentrations: show two components, the one corresponding to the highest ra- PEG 6k (triangles), PEG 17k (circles) and PEG 35k (squares). The arrows point dius, shows similar q2 inverse decay time dependence to the the polymer concentration at which the fluorescence quantum yield reaches a pure AOT vesicles, with a apparent hydrodynamic radius of constant value.
(57.7 ± 0.6 nm). The second component corresponds to thelowest radius and has the expected q2 dependence for all the previously observed for AOT/PEG 17k in dilute polymer con- scattering angles. The average radius (6.6 ± 0.4 nm) found for centrations, and was interpreted using the diffuse double layer this component is compatible with an elongated micellar ag- theory According to it, the PEG adsorption on the inter- gregate. collects the rest of the R values along with face gives a relatively thick layer on the surface, which shifts the amplitudes of the different modes at the scattering angle of the slip boundary toward the bulk solution decreasing the elec- trophoretic mobility At high polymer concentrations, the distribution functions In all these systems, when the polymer concentration in- also show two components, both have linear inverse decay time creases, the electrophoretic mobility decreases in absolute value q2 dependences. The average R values found for 10% PEG and reaches a plateau. The end of the plateau corresponds to 6k ([AOT] = 0.03 M) were 1.5 ± 0.1 nm and 299 ± 4 nm, and in the polymer concentration at which the fluorescence quantum are collected the rest of the values. The size of the low- yield reaches a constant value, arrows in A further poly- est component is compatible with spherical micelles while the mer addition gives smaller absolute values of μe that may be an highest size component may correspond to very large rod like indication of the transition from vesicles to micelles.
micelles or to superstructures of elongated micelles embedded The electrophoretic mobility measurements are consistent in a polymer network.
with fluorescence results. Thus, the aggregates with PEG 17kpresent the smallest electrophoretic mobility in absolute value.
3.4. Cryo-transmission electron microscopy, Cryo-TEM This fact indicates that PEG 17k forms the densest polymerlayer responsible of both the lowest quantum yield values of To reveal the structures formed at different AOT–polymer nabumetone and the lowest electrophoretic mobility in absolute compositions and to help elucidate the light scattering results, the systems were analyzed by means of Cryo-TEM. This tech-nique allows direct examination of microstructures after rapid 3.3. Light-scattering measurements freezing of the hydrated specimens. Cryo-TEM pictures ob-tained from an aqueous solution of Aerosol OT 0.01 M are In order to obtain information on the effect of polymer ad- in We have obtained a large number of pictures from dition on aggregates size, dynamic light-scattering experiments aqueous solutions of 0.01 and 0.03 M of AOT; pictures showed have been performed at 30.0 ◦C with different polymer concen- in were chose for the high quality of images and be- trations, and keeping constant the AOT (0.03 M). For compar- cause are the most representative of the size distribution of AOT ative purposes, the size of pure components, AOT aggregates vesicles. The figure shows that monodisperse small unilamel- and aqueous polymers were determined. In the former the AOT lar vesicles, SUV, with a radius of about 20 nm predominate concentration is also 0.03 M and in the latter, the polymer in this system. also shows the existence of multilamel- concentrations were the highest used in mixed systems, which lar vesicles, MLV, in a small proportion as compared with AOT in all cases is well above the overlapping concentration vesicles. The biggest aggregates were observed by optical mi- (c∗(PEG 35k) = 0.9% w/w, c∗(PEG 17k) = 1.4% w/w, c∗(PEG croscopy shows the distribution of the outer vesicle 6k) = 3% w/w).
diameter for 200 vesicles taken for Cryo-TEM. Results show Both pure AOT and PEG solutions give single exponential an almost monomodal distribution of vesicles. Qualitatively, correlation functions that, when analyzed using reg- the light scattering results agree with Cryo-TEM ones show- ularized inverse Laplace transforms (ILT) (GENDIST, CON- ing that Aerosol OT solutions consists in rich monodisperse TIN), lead to single peak decay time distributions vesicles system. However, the radius measured by light scat- M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 Fig. 5. (A) Normalized second order correlation functions, g(2)(t), and (B) the inverse decay time values vs the square wavevectors for pure AOT 0.03 M and forpure PEG 35k (5% w/w).
tering (RH = 68.5 nm, for AOT 0.01 M), is significantly larger In dilute polymer solutions, below the overlapping one, than the radius measured by Cryo-TEM (diameter = 40 nm).
Cryo-TEM shows no clear images, probably due to vesicle as- This can be partly because scattering yields an intensity aver- sociation in the freezing process and the formation of too big as- age while Cryo-TEM gives a number average and so the former semblies Thus, we use Cryo-etch SEM to confirm this fact.
weights larger aggregates that scatter more light and shifts the The procedure of preparation of samples and some of these im- hydrodynamic radius toward larger values ages are in Supplementary material section. The pictures show Cryo-TEM images for AOT solutions with polymer con- big colloidal assemblies probably due to vesicle association in centrations above the overlapping one show that the system the freezing process. The formation of aggregates of similar is formed by spherical micelles with a micelle radius be- structure was observed in saline surfactant solutions or in tween 2.5 and 3 nm in agreement with light scattering results.
binary colloidal systems The structure of these assemblies In addition, some large aggregates of different size were ob- is independent of the freezing procedure and preserves some vesicles inside the colloidal aggregates.

M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 Fig. 6. Distribution functions on an apparent hydrodynamic radius scale forAOT 0.03 M and two different concentrations of PEG 6k.
Table 2Apparent hydrodynamic radius and amplitude values found using dynamic lightscattering for aggregates formed in Aerosol OT and PEG polymers in aqueoussolutions 67.3 ± 0.4 57.7 ± 0.06 6.6 ± 0.4 1.5 ± 0.1 60.5 ± 0.06 2.4 ± 0.05 1.7 ± 0.02 3.4 ± 0.1 2.4 ± 0.2 Note. Amplitudes of the different modes, Ai , correspond to 90◦ while the RHvalues are obtained from the inverse decay rate vs square wave vector plots.
Cryo-TEM and light scattering results indicate that monodis- perse small unilamellar vesicles (SUV) form pure AOT solu-tions. The addition of small concentrations of PEG 6k, PEG 17k Fig. 7. Cryo-TEM micrographs from an aqueous solution of Aerosol OT 0.01 M and PEG 35k leads to the appearance of elongated micelles sample; (a) monodisperse small vesicles area; (b) multilamellar and small vesi- coexisting with SUV's. With a further increase in PEG con- cles area; (c) histogram of vesicle sizes from Cryo-TEM images.
tent, SUV disappears and the size of the elongated micellesdecreases to a radius compatible with spherical micelles. Forthe highest PEG concentrations all the polymers are well above or to superstructures of elongated micelles embedded in a poly- the overlapping concentration, in these cases coexisting with mer network. According to the results found in this work, the spherical micelles, very large aggregates have been detected.
addition of PEG on AOT water solutions induces the following These aggregates may correspond to very large rod like micelles M.M. Velázquez et al. / Journal of Colloid and Interface Science 316 (2007) 762–770 SUV −→ SUV + elongated micelles assemblies formed in the freezing process in solutions with [dilute polymer regime] Aerosol OT and PEG 6k. The surfactant concentration remains constant at 0.03 M AOT while that PEG 6k concentrations were −→ Spherical micelles + large aggregates 0.03 and 1%, respectively.
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