W Nanoemulsions Prepared by Phase

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Stability and Tunability of O/W Nanoemulsions Prepared by Phase Inversion Composition Manal Hessien, Nigel Singh, ChonHoon Kim, and Eric Prouzet* Chemistry Department & Institute of Nanotechnology, University of Waterloo, 200 University Av. W, Ontario, N2L 3G1 Canada

bS Supporting Information ABSTRACT: We report on an analysis of the parameters that control both the stability and tunability of O/W nanoemulsions prepared by the phase inversion composition (PIC). These nanoemulsions are prepared with Tween 80 and Span 80, two nonionic surfactants, that can be mixed to adjust the hydrophilic lipophilic balance (HLB). We used a process mixture design method, which combines mixture and process design with phase diagrams, to describe the cross-link between parameters like composition, temperature of preparation, and HLB. Nanoemulsions, stable for several days, are obtained by this method, and they remain unchanged even at high concentration. We have identified the different critical distances of interactions that control the degree of freedom in the motion of the oil droplets. The diameter of these oil droplets could be adjusted between 50 and 300 nm. Different parameters, among them the temperature of preparation, the surfactant over oil ratio (S/O), and the HLB, allow control the final size of the nanoemulsions. As these parameters can exhibit opposite effects on the oil droplet size, the process mixture design method allowed us to illustrate these cross-interactions.

’ INTRODUCTION Porous materials are usually synthesized with the help of organic templates trapped within an inorganic framework, and the porosity being further opened by removing the organic phase. Depending on the size of the organic template, different types of porous materials have been synthesized so far. With single molecules, 0.2-1.5 nm microporous crystallized molecular sieves like zeolites are obtained.1 Shifting from single molecules to surfactant-based micellar assemblies leads to 2.0-10.0 nm mesoporous materials like MCM-41, SBA-15, or MSU silica.2-5 On a larger scale, the use of colloidal particles or latex as templates allows the preparation of reverse opal-like structures with regular pores above 300 nm, which provide specific photonic properties.6,7 Until now, no specific templates have been developed for the synthesis of porous materials that could fit within the intermediate size range (between 50 and 300 nm), even though such materials would find extremely important applications in catalysis, chromatography, purification, or photonics. Oil-in-water nanoemulsions seem the best candidates because they present an oil droplet size that lies within the suitable range,8 and the interest in these systems is demonstrated by the still limited, but increasing literature on this topic.9-13 However, the potential use of nanoemulsions as templates in the synthesis of porous materials requires additional criteria to the simple preparation of such a nanoemulsion. Indeed, these templates must be as versatile as possible to adapt to a suitable structure, they r 2011 American Chemical Society

must be stable enough to stand a high concentration without suffering any phase transition or Ostwald ripening, they have to be rigid enough to stand the mechanical constraints created by the polymerization of the inorganic framework, and they must exhibit a tunable size to allow preparation of a full range of porous materials from a single composition. Moreover, a suitable nanoemulsion that would contain surface molecular groups able to interact with the inorganic precursors would contribute to a favorable interaction between the organic entities and the inorganic precursors. We demonstrated previously that the synthesis of mesoporous silica with polyoxyethylene (PEO) nonionic surfactants can take advantage of the interaction between the PEO chains and silica precursors.14 Liu et al. used such nonionic surfactants for the preparation of paraffin oil-in-water nanoemulsions by the phase inversion composition (PIC) method, a low energy emulsification process. They reported the preparation of stable nanoemulsions with diameters ranging from 100 to 200 nm.15 Surfactants used in this system are a combination of Span 80, a sorbitan monooleate with a low hydrophilic-lipophilic balance (HLB = 4.3), and Tween 80, an ethoxilated sorbitan monooelate ester with a high HLB (HLB = 15). As these two surfactants possess the same Received: November 27, 2010 Revised: January 5, 2011 Published: February 02, 2011 2299

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backbone, they can mix easily, leading to a controlled change in the final HLB. In our way to synthesize nanoemulsion-based porous materials, we present in this report a complete study on the evolution and stability of nanoemulsions made within the (Tween 80:Span 80:paraffin oil:water) system. This study explored the influence of different parameters like temperature, concentration, and HLB, and their cross-interaction. These results, which extend beyond previous analyses,15 demonstrate the high stability of these nanoemulsions and how they evolve as a function of the synergy among different parameters.

’ EXPERIMENTAL SECTION Materials. The oily phase was paraffin oil (d = 0.86; Fluka). The emulsion was prepared with deionized water and stabilized by a mixture of two surfactants: Sorbitan monooleate (Span 80, d = 1.08, Mw = 428.6 g; Across) and polyoxyethylene (20) sorbitan monooleate (Tween 80, d = 0.994, Mw =1,310 g; Sigma; see Scheme SI.1) All reagents were used as received without further purification. Methods. Nanoemulsions were prepared by first dissolving the surfactants (Tween 80 and Span 80) into the paraffin oil, under gentle magnetic stirring for 15 min. As the formation of these nanoemulsion is based on a W/O to O/W phase inversion, any trials that proceeded in a different addition order, failed to reach stable nanoemulsions (Figure SI.1). The required amount of water was added drop-by-drop (2 mL min-1) to the mixture of oil and surfactant under slow magnetic stirring (60 rpm). Both agitation speed rate and addition rate were kept constant for all samples. For samples prepared at temperatures between 5 and 80 °C, the temperature of both the oil-surfactant mixture and water were first adapted separately to the required temperature in a heating bath before being mixed according to the previous process. The variation in the (Span 80:Tween 80) ratio allowed us to reach different HLBs, depending on the amount of each surfactant, according to the relationship: HLBmix ¼ HLBT  T%þHLBS  S% ¼ 15  T%þ4:3  S% ð1Þ where HLBT is the HLB for Tween 80 (15), T% is the mass percentage of Tween 80, HLBS is the HLB for Span 80 (4.3), and S% is the weight percent of Span 80. The size of the nanoemulsion droplets was studied by dynamic light scattering, using a DLS 135 (Cordouan Technology). This system allowed us to determine the diffusion coefficient of emulsion droplets, even in very concentrated systems, from which the hydrodynamic diameter was deduced by the Stokes-Einstein equation D ¼ ðkB TÞ=ð3πηDh Þ

ð2Þ

where D is the diffusion coefficient, η is the viscosity of the emulsion, T is the temperature, kB is the Boltzmann constant, and Dh is the hydrodynamic diameter. A 60 mW monomode red laser (λ = 658 nm) with variable intensity was used, and measurements were carried out at different temperatures (from 25 to 70 °C) with a detector set at 135°. The correlator parameters were adapted for each sample (sampling time x number of channels) to collect the whole autocorrelation curve. Accuracy and reproducibility of the results were checked with several measurements run with various recording times. Most of of the autocorrelation curves were fitted with a single size cumulant model, the lack of polydispersity being checked in parallel with Pade-Laplace fits.16,17 When the cumulant fit did not converge, we used the Pade-Laplace fit, which still led to single size results. In that case, the value of the diffusion coefficient cannot be provided. Samples for the Analysis in Temperature and Dilution. A nanoemulsion (HLB = 11.6) was prepared with the following mass composition: Span 80: 2.25 g; Tween 80: 4.77 g; paraffin oil: 20.0 g;

water: 73.0 g. The measurements were carried out with temperature steps of 5 °C, between 25 and 70 °C, with the nanoemulsion being diluted (in mass) with a (nanoemulsion:dilution water) ratio varying from 1:1 to 1:500. Additional samples with a lower water content were also prepared to extend the study range (Table SI.I).

Samples for the Analysis of the Surfactant/Oil Weight Ratio (S/O). A first series of analysis was carried out with nanoemulsions prepared with the same HLB = 11.6, and a S/O varying from 0.2 to 1.0 (Table SI.II). All samples were diluted before measurement with a nanoemulsion:water dilution ratio of 1:1,000 to avoid any interdroplet interference. A second series of analysis was ran with samples prepared with different HLBs (Table SI.III). The relationship between HLB and S/O was based on previous results reporting that nanoemulsions prepared at different temperatures (30, 40, and 50 °C, respectively) exhibit a stability domain that depends on the HLB (11.3, 10.7, and 10.2, respectively), and leads to a different oil droplet diameter (240, 200, and 150 nm, respectively).15 We extrapolated from these results, a possible relationship between the HLB and the nanoemulsion size HLBmix ¼ 8:4 þ 0:012Dh

ð3Þ

Nanoemulsions were prepared with different S/O and HLBmix according to eq3. The value of HLBmix was calculated from an evaluation of the diameter Di, based on the volume (or mass of oil Qi) and surface Si of oil droplets, which depends on S/O (see the Appendix) S1 =S2 ¼ Q 1 =Q 2 ¼ ðn1 =n2 ÞðD1 =D2 Þ2 ¼ D2 =D1

ð4Þ

As our goal was to evaluate if a steady change in Dh could result from a change in HLB, we assumed as a first approximation that variations of the Tween 80:Span 80 ratio has no effect on the surface coverage. This initial assumption was no fully confirmed by the following analyses, but it does not modify drastically the conclusions of this one. Taking as a starting point previous experimental values,15 with D1 = 240 nm, Q1 = 5 wt %, oil = 20 wt %, we estimated the value of a droplet diameter D2 if the amount of surfactant Q2 is modified (i.e., S/O), according to eq 4, as well as the value of the HLB via the Tween 80/Span 80 balance, according to eq 3.

Samples for the Analysis of the Influence of the Preparation Temperature. Three series of nanoemulsions (HLB = 11.6) with surfactant:oil ratios equal to 0.25, 0.5, and 1.0, respectively, were prepared at temperatures ranging between 5 and 80 °C (Table SI.IV and V). All DLS analyses were carried out at 25 °C, with the samples being diluted with a dilution ratio of 1:2000, after the existence of a nanoemulsion had been checked on the pristine preparation. Experimental Design. We used an experimental design to study the parallel influence of all parameters. The samples compositions were defined within a (water:oil:surfactant) ternary diagram. The lowest and highest level weight concentrations were chosen to fit within the expected domain of existence of nanoemulsions: 0.45 < water < 0.85; 0.10 < oil < 0.40; 0.05 < surfactant < 0.15 (Figure SI.2). The DesignExpert program V.8.0.4 (Stat-Ease Inc.) was used to define the optimum number of experiments according to the limits selected for this study. Significance of the regression model was tested by the “Prob(F)” statistics test, which tests the full model against a model with no variables, and with the estimate of the dependent variable being the mean of the values of the dependent variable. The value of Prob(F) is the probability that the null hypothesis for the full model is true (i.e., that all of the regression coefficients are zero). For example, if Prob(F) has a value of 0.01, then there is 1 chance in 100 that all of the regression parameters are zero. This low value implies that at least some of the regression parameters are nonzero and that the regression equation does have some validity in fitting the data (i.e., the independent variables are not purely random with respect to the dependent variable). In general, a term that has a probability value less than 0.05 will be considered as significant for the regression model, and a probability value greater than 0.10 is generally 2300

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Figure 1. Evolution at different temperatures of analysis of the apparent hydrodynamic diameter Dh of the nanoemulsion as a function of the dilution ratio. regarded as not significant. Twelve samples out of 9 compositions were prepared, the central composition being replicated four times (Figure SI.2). The effect of HLB and preparation temperature were studied by defining five sets of samples (Table SI.VI). All samples of the same set were prepared on the same day with a random order defined by the program. Samples were left to rest overnight, and on the second day of preparation, (1:2000) diluted samples were prepared for DLS measurement. The results were entered into the Design Expert program that used the DLS values to form a model from which equations linking the different parameters to the final nanoemulsion size could be calculated.

’ RESULTS Influence of Dilution. DLS provides information on the diffusion coefficient D of the scattering objects from which the size is deduced via the Stokes-Einstein formula. Therefore, any factor that could modify the diffusion coefficient could be translated into an apparent size variation, and any interaction that could hinder the free motion of the scattering objects will have such a strong effect on D and then Dh. Our initial aim was to explore the limit of stability of the nanoemulsion structure as the concentration is increased, because such a property is extremely important for further uses of these nanoemulsions as templates. The evolution of the hydrodynamic diameter Dh (resp. the diffusion coefficient D) as a function of the dilution ratio is reported in Figure 1 (resp. Figure SI.3). Dh (resp. D) decreases (resp. increases) drastically as the dilution ratio increases, and a constant value of Dh = 250 nm is finally reached for a dilution ratio greater than 50, as a result of the stable values of the diffusion coefficients beyond this dilution ratio (Figure SI.3). We conclude that the actual hydrodynamic diameter of the nanoemulsion prepared for this study is 250 nm and that a dilution ratio g100 is required for correct determination. In the following, all measurements were carried out with dilutions equal to 1:1000 or 1:2000 to be sure that no interaction between oil droplets could disturb the final value of Dh. This analysis demonstrates also that the nanoemulsion remains very stable even at high concentration since neither multisize distribution, nor transition toward a bicontinuous phase was observed, as demonstrated with the correct single-size fit of all autocorrelation curves (Figures SI.4 to SI.6). We investigated further the stability of the nanoemulsion by preparing additional concentrated samples (Table SI.I). We report these values as a function of

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Figure 2. Evolution at 25 °C of the apparent hydrodynamic diameter Dh (b) and the diffusion coefficient (O) as a function of ln(1 - X) where X is the water content of nanoemulsions after dilution (see Table SI.VII). The dashed lines are provided for visual help.

ln(1 - X) (0 < X < 1: Water weight amount; Table SI.VII), because the high water content (including the initial water used for preparation and the dilution) makes difficult to follow correctly the evolution of Dh and D at high dilution (Figure 2). Three domains are clearly identified. At higher dilutions (small ln(1 - X)), the value of Dh is constant and corresponds to the actual diameter (Dh = 250 nm). This domain extends from the highest dilution down to 1:50 (X = 0.9947, ln(1 - X) = -5.24). A second region with a steady increase of the apparent hydrodynamic diameter, and the parallel decrease of the diffusion coefficient, is defined for 0.74 < X < 0.9947. Finally, a sharp increase of the hydrodynamic diameter, and a parallel decrease of the diffusion coefficient, is observed for X < 0.74. This three-step evolution of the mobility of the nanoemulsion as a function of the increasing concentration (decreasing water content) demonstrates an increasing interaction between the objets, leading first (0.9947 < X < 0.74) to a limited hampering in mobility, followed by a major restrain in mobility when the weight water content decreases below 0.74. Influence of the Temperature of Analysis. We used the previous samples to test the possible evolution of the measurement temperature on Dh (Figure 3). We report only results corresponding to dilutions high enough to prevent any interaction between the nanoemulsion droplets. This result demonstrates clearly that the size of the nanoemulsion does not depend on the temperature of the nanoemulsion itself, once it has been formed. As for the temperature of preparation, we will see thereinafter that it has a great effect on the nanoemulsion size. Influence of the Surfactant:Oil Weight Ratio (S/O). As the surfactant molecules cover the surface of the oil droplet, it is expected that increasing the surfactant over oil ratio would induce a shrinking of the oil droplet, provided that all other parameters remain contant. A first series of samples was prepared with different S/O and a constant HLB = 11.6 (Table SI.III). DLS measurements were recorded at 25 °C with a 1:1000 dilution ratio. The size of the nanoemulsion decreases as expected, and increasing S/O from 0.25 to 1.0 leads to a variation of Dh (b) from 200 to 135 nm (Figure 4). A second series of samples was prepared with different S/O, but a HLBmix (0) varying from 12 to 9.4 based on eqs 3 and 4. Compared 2301

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Figure 3. Evolution of the hydrodynamic diameter Dh as a function of the temperature of measurement, for the nanoemulsion with dilution ratio high enough to prevent any interdroplet interaction.

Figure 4. Evolution of the hydrodynamic diameter Dh measured at 25 °C, as a function of the surfactant/oil weight ratio: (b) for a constant HLBmix and (0) a HLBmix varying according to the (0) curve.

with the previous one, a variation of Dh (0) toward smaller sizes is observed, Dh being equal to 150 nm instead of 220 nm for S/O = 0.25. This variation is larger as S/O varies from 0.2 to 0.5, decreasing from 170 down to 105 nm, but Dh increases again for S/O values larger than 0.5. This analysis confirms the relevance of S/O to the size control, but the increasing value of Dh above S/O = 0.5 appears to be quite contradictory with the single influence of the parameter alone. Therefore, it seems that both S/O and HLBmix have a combined influence on the variation of Dh. Influence of the Temperature of Preparation. Samples were prepared at temperatures between 5 and 80 °C with S/O equal to 0.25, 0.5, and 1.0. The results of DLS measurements recorded at 25 °C are displayed in Figure 5. As the temperature of preparation increases, we observe that the size of the nanoemulsion drops down, this effect being limited above 60 °C. This trend is more important for the highest S/O values: for S/O = 0.25, Dh decreases from 195 (10 °C) to 170 nm (60 °C) with a 13% shrinkage, for S/O = 0.5, Dh decreases from 160 (10 °C) down to 95 nm (70 °C) with a 40% shrinkage, and for S/O = 1.0, Dh decreases from 117 (10 °C) to 57 (60 °C) with a 51% reduction. These results confirm that the temperature of preparation has a drastic effect on the size of nanoemulsion droplets

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Figure 5. Evolution of the hydrodynamic diameter Dh measured at 25 °C, as a function of the temperature of preparation of nanoemulsions, for different surfactant/oil weight ratio: (0) 0.25, (O) 0.5, and (() 1.0 (HLB = 11.6).

and that these systems remain stable even after the system has been cooled down at room temperature. Multiparameter Influence. We combined finally the parameters that have been studied independently in the previous analyses: composition, preparation temperature, and HLB. These analyses were carried out through experimental design with the sample compositions defined according to Table SI.VIII. The response surface maps describe the variation of Dh as a function of the different components (oil, surfactant, and water) at two different HLBs defined by the respective proportions in Tween 80 and Span 80 and three different temperatures of preparation (50, 60, and 70 °C) (Table SI.X). The 2D and 3D response surface maps are displayed in Figures 6 and 7, respectively. The results of sample prepared at 50 °C with an HLB of 9.6 could not be used because the statistic validity of the surface response model deduced from the experimental data was not significant, as illustrated by the discrepancy between the experimental points and the surface (Figure 7) and as proved by the high Prod(F) parameter (Table SI.X). The response surfaces for set nos. 2 (50 °C, HLB = 11.7) and 3 (60 °C, HLB = 10) are quite similar, with Dh exhibiting a minimum along the whole study domain at the center of the response surface, and two maxima, one close to the higher oil concentrations and the other at higher surfactant content. For the set no. 4 (70 °C, HLB = 9.6), the whole response surface is rather flat, fluctuating between 118 and 220 nm, and the minimum has shifted toward the top middle of the study zone. Finally, the variation of Dh for the set no. 5 (70 °C, HLB = 11.7) presents a quasi linear decrease from the highest oil content to the water-surfactant rich domain, with a variation from 250 to 60 nm. These results show that stability and size of these nanoemulsion, depend not only on the composition, but also on additional parameters like HLB, or the temperature of preparation. The origin of the influence of these parameters is discussed in the following.

’ DISCUSSION The PIC mechanism has been described as resulting from a curvature inversion from the initial W/O structure to the final O/ W nanoemulsion, as the amount of water, initially small, becomes progressively larger.18,19 The critical step in the formation of these O/W nanoemulsions is the transition, which explains why 2302

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Figure 6. 2D response surface map for the hydrodynamic diameter Dh deduced from the experimental design, for samples prepared at different temperatures (50, 60, and 70 °C) and different HLB (9.6, 10.5, and 11.7). The response surface map for (T = 50 °C, HLB = 9.6) is statistically not significant.

Figure 7. 3D response surface map superimposed over the phase diagram for the hydrodynamic diameter Dh deduced from the experimental design, for samples prepared at different temperatures (50, 60, and 70 °C) and different HLB (9.6, 10.5, and 11.7). The response surface map for (T = 50 °C, HLB = 9.6) is statistically not significant. 2303

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Langmuir the addition of water to oil leads to nanoemulsions, whereas the addition of oil to water does not.20 This transition from W/O to O/W proceeds via an intermediate bicontinuous phase whose characteristic distance, which depends on S/O, controls the final oil droplet size. Pey et al., using a mixture of nonionic Tween 20 and Span 20 surfactants, established that the oil droplet size decreases as the surfactant/oil ratio increases, and that this size exhibits a dependence on the Tween 20 percentage.20 Fernandez et al. prepared microemulsions with a mixture of nonionic surfactants (Cremophor A6 and Cremophor A25) with a constant HLB and a preparation temperature of 80 °C. They found that increasing the surfactant concentration from 2.5 to 10 wt % resulted in changing the droplet profile from a bimodal at 10 and 0.6 μm to a single modal at 0.3 μm.19 Finally, Liu et al. obtained paraffin oil droplets with diameters as small as 100 nm with a mixture of Span 80 and Tween 80.15 They noticed that stable nanoemulsions with a droplet diameter below 200 nm were formed at 50 °C if both a critical surfactant/oil ratio, and a specific value of the HLB were optimized. However, until now, no study has actually covered the complex and crossed influence of all parameters (surfactant content, Surfactant/Oil ratio, HLB, preparation temperature) and proposed a full description of these systems. Few reports have partially addressed this problem by using methods such as factorial design, but they focused more on process parameters such as the addition rate vs stirring rate, and of the surfactant/oil ratio vs surfactant percentage.20 The present study uses a more sophisticated method based on a process mixture experimental design.21,22 It allowed us to explore the combined influence of different chemical (components proportions, HLB) and physical (temperature preparation) parameters on the final stability and tunability of these nanoemulsions, as a function of the actual phase diagram. Stability of the Nanoemulsions. As stability is a major parameter for the further use of these systems as templates, we explored the limits of stability of these nanoemulsions as a function of the water fraction adjusted either by dilution, or by the preparation of more concentrated nanoemulsion (Tables SI.I and SI.VII). DLS confirms the stability of these nanoemulsions, because the motion of isolated objects is still detected at high concentration. This stability is confirmed by the single size fit for all DLS measurements, which indicates that there is no aggregation or partial merging as the water content is reduced (Figures SI.4 to 6). Nevertheless, the DLS results show that the diffusion coefficient, D, becomes constant only beyond a critical (nanoemulsion:dilution water) ratio of 1:100 (Figure SI.3), and it corresponds to a hydrodynamic diameter Dh of 250 nm (Figure 1). The low values (resp. high values) for D (resp. Dh) at dilution ratios lower than 50 do not correspond to a change in the nanoemulsion size but to an increasing steric cluttering that hinders the free motion of the oil droplets, leading to a decrease of the diffusion coefficient from which Dh is calculated. This observation demonstrates that careful attention must be applied to DLS analyses dealing with these systems, regarding the level of dilution required for correct measurements. The report of D and Dh as a function of the water content X are displayed in Figure 2. The choice of a specific parameter (ln(1 - X)) allowed us to follow the evolution of these parameters even at the highest dilutions. As mentioned, three domains are identified as the water content is modified, which reveals an increasing interaction between the objets initially allowed to move freely (zone I: 0.9947 < X), then partially restrained (zone II: 0.74 < X < 0.9947), and finally rapidly hampered in mobility (zone III: X < 0.74). We can relate these observations to the average water

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Figure 8. Variation of the water thickness separating two nanoemulsion droplets as a function of the water content. The vertical dashed lines correspond to the breaks in the evolution of the diffusion coefficient measured by DLS.

Scheme 1. Characteristic Distances in the Nanoemulsion (Droplet Diameter =250 nm), as a Function of the Water Content, Deduced from Results Displayed in Figure 8

distance between two oil droplets, as a function of the water content (Figure 8; see the Appendix). The oil droplets can move freely only if the water thickness between two droplets is greater than 1.0 μm (X > 0.9947) (Figure 8, inset), which corresponds to a water shell thickness of 500 nm around each oil droplet. Below this value, there is a progressive hindering of the nanoemulsion motion down to a water content X = 0.74. This value corresponds to a water thickness of 120 nm (Figure 8), that is, a water shell thickness of 60 nm around each droplet (Scheme 1). For X < 0.74, the diffusion coefficient D decreases extremely rapidly as a result of oil droplets coming into close contact. As noticed before, these oil droplets are extremely stable since they do not merge even when the water content becomes so small that the whole nanoemulsion is almost gelled. This stability is also demonstrated by DLS analyses carried out at different temperatures (Figure 3) that do not reveal any change in the structure or size of the oil droplets. This finding confirms that, unlike methods based on mechanical emulsification, low energy emulsion methods like the PIC process lead to a very stable structure because it results from a fine physical-chemical balance. These nanoemulsions are stable for days, especially when they are stored as diluted, whereas nanoemulsions prepared by high energy mechanical mixing are very often stable only within a couple of hours or days.8 2304

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Langmuir Tunability of the Nanoemulsions. As mentioned, the size of the nanoemulsion droplets can be theoretically modified if we find the correct balance between the total surface of oil droplets and their size, the oil volume being constant. Different ways can be followed to achieve this balance, the obvious method being simply to increase S/O. Our analyses (Figures 4 and 5) confirm this evolution, and we can assign it to the higher concentration of surfactant that allows for the formation of a larger surface of oil droplets whereas the overall oil volume remains constant, which can be solved only by creating smaller droplets.10,13,23 However, this effect is limited, and we observe (Figure 4) that it is more important if we reduce in parallel the HLB according to eq 3. Once the nanoemulsions have been prepared, their size remains independent from any further temperature fluctuation, but the initial temperature of preparation has a huge influence on the final droplet size (Figure 5). We assign this phenomenon to the increasing hydrophobicity of nonionic surfactants (here, Tween 80) resulting from the increase of the preparation temperature. Without any specific information on the actual structure of the bicontinuous phase, we assume that Tween 80 becomes more hydrophobic and shifts progressively from the O/W interface into the oil phase, a shift that should modify the structure of the intermediate bicontinuous phase, leading to smaller droplets. However, this single parameter does not allow to fully explain the evolution of Dh as a function of the preparation temperature when S/O varies (Figure 5). We observe that this reduction of size reaches a limit when the temperature increases (60 °C for S/O = 1.0 and 70 °C for S/O = 0.5) and that it can even increases as observed for S/O = 0.25. It is most probable that the full understanding of this evolution will be provided by a close analysis of the intermediate bicontinuous phase, which must be investigated by other analyses, especially by ultrasmall angle X-ray scattering. We further observed also that decreasing HLBmix by increasing the Span 80/Tween 80 ratio leads also to a reduction of the oil droplet size (Figure 4). This result seems somewhat counterintuitive because Span 80 is characterized by its small molecular area (0.46 nm2), compared to Tween 80 (2.48 nm2).11 We would expect that decreasing HLBmix would lead to a smaller equivalent surface, hence a larger droplet size, provided S/O being constant. Nonetheless, HLBmix is defined by weight, not molar proportions (eq 1), and decreasing HLBmix by increasing the proportion in Span 80 has a first consequence of dramatically increase the number of moles of Span 80 because the molar weight of Span 80 is only 0.32 times that of Tween 80, as demonstrated by the calculation of the number of molecules of surfactants required for changing the HLB, and the resulting effect on the total surfactant surface area with the decreasing HLB (Figure 9). This confirms that the actual effect of decreasing HLBmix results mostly in a higher number of Span 80 surfactants molecules being involved, leading finally to an increasing of the total surface area of the oil droplets. Multiparameter Influence. The previous parameters were discussed independently, but the whole stability of these nanoemulsions, and their tunability, results from a combination of all parameters. Therefore, only the process mixture design method, which combines an experimental design and phase diagrams, can describe precisely the evolution of the nanoemulsion as a result of cross-interactions between these parameters. The hydrodynamic diameter Dh was chosen as a response in this study. It was calculated with the equations deduced from the design experimental points (Figure 7 and Table SI.XI), for different HLBs

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Figure 9. Evolution of the total surface of surfactant area, and the number of moles of surfactants (calculated from Table SI.3) as a function of the HLB.

and preparation temperatures (Table SI.X). As mentioned, the set no. 1 (50 °C, HLB = 9.7) cannot be used because the model fitting was not statistically significant. The central set no. 3 (Table SI.VI; 60 °C, HLB = 10.5) gives a good indication of the parameters that govern the tunability of these nanoemulsions (Figures 6 and 7). The response for this set is symmetrical along the diagonal of the surface, starting at the water pole. This symmetrical profile means that Dh remains minimum along this axis, whatever the concentration in water, as illustrated by the curves deduced from the response surface equation (Table SI.X; Figure 10). Unlike the previous ones, these curves are plotted as a function of the oil:surfactant weight ratio (O/S), which is more pertinent for the present analysis than S/O. We observe only a small increase of Dh and shift of the minimum Dh toward smaller O/S, as the water content increases from 50 to 80%. We mentioned already that O/W nanoemulsions prepared by the PIC method are obtained through a progressive addition of water, leading from an initial W/O system, to the final formation of the O/W nanoemulsion, via an intermediate bicontinuous mesophase.18-20 The intermediate mesophase corresponds to a critical volume of water required for the phase inversion, this critical volume being dependent on the components (oil and surfactant). The curves displayed in Figure 10 demonstrate that the oil droplets that have been formed, once the critical water amount has been added, remain unchanged whatever the further addition of water, and that the critical parameter is the oil/surfactant ratio (resp. surfactant/oil) that must remain roughly equal to 2.5 (resp. 0.4) for this preparation temperature and HLB. When this ratio varies with either an oil content increase, or a surfactant content increase (Figures 6 and 7), the value of Dh begins to increase, as illustrated by the evolution of Dh for iso-concentrations in surfactant (Figure SI.11.a), that is, lines parallel to the base of the analysis area, and in oil (Figure SI.11.b), that is, lines parallel to the right side of the analysis area. Similar curves, obtained from the other sets, are displayed in Figures SI.7 to SI.9. The value of Dh increases progressively as we increase O/S (Figure SI.11.a), which corresponds also to a parallel increase of the oil/water ratio, since the surfactant percentage is constant. This progressive increasing of Dh finds its origin in the progressive decreasing of the water content. 2305

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increase in Dh. Finally, the set no. 5 (70 °C, HLB = 11.7) combines two factors that must lead to a size reduction of the oil droplets. This trend is confirmed with the lowest sizes obtained (Dh = 65 nm). It seems surprising to observe a totally different shape (flat surface) with a minimum size obtained for the highest volumes of the surfactant content, that is, the smallest O/S ratio (Figure SI.9). However, this can be well understood in the light of the previous results: the expected optimum O/S ratio as observed in set no. 3 is certainly shifted toward lower values, and we observe only the right part of the curve illustrated in sets nos. 3 and 2. These analyses confirm that a full understanding of the complex behavior of these nanoemulsions and benefit from multiparameter analyses allowed by experimental design.

Figure 10. Evolution of the hydrodynamic diameter (Dh) as a function of the oil:surfactant weight ratio, for the (T = 60 °C, HLB = 10.0) samples, at different values of water content.

The study area was obviously selected in an area of stability of the nanoemulsion, but increasing the oil/water ratio will shift the system closer to the critical water amount required to achieve well-defined nanoemulsions. It is still above the critical minimal value required to obtain the W/O to O/W transition, but it results in larger oil droplets. On this other side, reducing O/S below 2.5, that is, increasing the amount of surfactant for a given volume of oil (Figure SI.11.b) will create an excess of surfactant, compared with the optimum value (O/S = 2.5) for the available oil volume. The initial spherical droplets should evolve toward ellipsoidal or rod-like objects as observed for the spherical to rodlike transition of pure surfactant micelles, interpreted by the single angle DLS measurement as an increase of Dh. Compared with set no. 3, set no. 4 (50 °C, HLB = 11.7) corresponds to a higher hydrophilicity (lower temperature of preparation) that is expected to lead to a higher droplet size and a higher total droplet surface (higher HLB) that is expected to lead to a smaller droplet size. As a result of these two opposite trends, we observe the same response surface as for set no. 3, but the region of minimum for Dh is larger and expanded to the left side of the study area (Figure SI.7). Compared with set no. 3, set no. 2 (70 °C, HLB = 9.6) exhibits a higher hydrophobicity that leads to smaller sizes and lower HLB that leads to larger sizes. Here again, the opposite influence of these parameters gives a constant value of Dh, marked by a rather flat response surface (Figure SI.7) with 120 nm < Dh < 200 nm as soon as the oil:surfactant ratio becomes greater than 1. For this set again, it is demonstrated that increasing the surfactant concentration (O/S < 1) leads to an

’ CONCLUSION Nanoemulsions prepared by low energy emulsification method have began to focus the interest of researchers for both fundamental and applied developments. These systems are complex, and their structure depends on a close crossinteraction of parameters whose actual influence has to be known. Our study allowed us to identify the characteristic parameters that govern both stability and tunability of nanoemulsions prepared with a combination of nonionic surfactants. First, these systems are very stable, even at high concentration and no phase transition is observed within a couple of days. Moreover, unlike nanoemulsions prepared by mechanical processes, the present systems are stable over weeks, especially when they are diluted. It must be also noted that interactions between the oil droplets, occur even at rather high dilution, and that DLS analyses must get rid of these interactions, by providing a sufficient dilution. These nanoemulsions, once prepared, are not modified by temperature within a 25 to 80 °C temperature range. The tunability of these systems is controlled by various parameters. The surfactant/oil ratio is a parameter that can help to reduce the oil droplet size but the size control depends also on other parameters and an optimum must be find within the phase diagram, once the temperature of preparation and the HLB have been defined. The temperature of preparation has a major influence and its origin should be found in the control of the hydrophobic/hydrophilic balance of the Tween 80 during the formation of the intermediate bicontinuous phase. The HLB, which results from a mixture of Tween80 and Span80, has also an influence on the nanoemulsion size, but this influence results mostly from a variation in the coverage surface of the surfactant layer than from a direct effect of the hydrophilic/hydrophobic balance. Finally, the present study illustrates the importance to use a multiparameters analysis such as process mixture experimental design, to span correctly the cross interactions in these complex systems. ’ APPENDIX Relationship between S/O and the Diameter of Oil Droplets. The total volume of oil VT is distributed among all

droplets of the nanoemulsion, with a volume that can be assigned to the oil alone, the contribution of surfactants being neglected V T ¼ ni ðπDi 3 =6Þ with VT the total volume of oil, Di the droplet diameter, and ni the number of droplets. The total surface of droplets ST is ST ¼ ni πDi 2 2306

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For the same volume of oil VT, the diameter of droplets can change from D1 to D2, if the surfactant/oil ratio changes, according to V T ¼ n1 ðπD1 3 =6Þ ¼ n2 ðπD2 3 =6Þ n1 =n2 ¼ ðD2 =D1 Þ3 This change in the droplet size will induce a parallel change in the total droplet surface Si, which has to be adjusted by a modification in the surfactant/oil ratio S1 ¼ n1 πD1 2 leading to :

and

S2 ¼ n2 πD2 2

S1 =S2 ¼ Q 1 =Q 2 ¼ ðn1 =n2 ÞðD1 =D2 Þ2 ¼ D2 =D1

Water Distance between Two Oil Droplets. Example of a calculation for a sample with the following composition: Water = 73 g, oil = 20 g, Tween80 = 4.77 g, Span80 = 2.25g with density equal to 1.0, 0.86, 0.994, and 1.08, respectively. The total volume of this sample is equal to 103.48 cm3 (1.031023 nm3). The hydrodynamic diameter (Dh) of an oil droplet is 250 nm, and its volume is V = πDh3/6 = 8.18106 nm3. As the total oil volume is 23.26 cm3 = 23.261021 nm3, the number of oil droplets in this composition, with 20 g of oil, is equal to 23.261021/8.18106 = 2.841015. The whole volume can be divided among the oil droplets by assigning a cubic cell to each oil droplet. The volume of one cubic cell is equal to 1.031023 /2.841015 = 36.3106 nm3. The length of this cube (l) is equal to 330 nm, and the water thickness is equal to 330-250 = 80 nm. The length l corresponds to the diameter of a sphere made of the oil droplet (Dh = 250 nm) plus a water shell of 40 nm. Hence, in this example, the water distance between two oil droplets is equal to 80 nm.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Additional tables of data as well as figures and schemes. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Fax: þ1 519 746-0435. Tel: þ1 519 888-4567  38172. E-mail: [email protected].

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