W

Article pubs.acs.org/Langmuir

Influence of Diffusive Transport on the Structural Evolution of W/O/ W Emulsions Herzi Sameh,† Essafi Wafa,*,† Bellagha Sihem,‡ and Leal-Calderon Fernando*,§ †

Institut National de Recherche et d’Analyse Physico-Chimique, Pôle Technologique de Sidi Thabet, 2020 Sidi Thabet, Tunisia Institut National Agronomique de Tunisie, 43, Avenue Charles Nicolle 1082-Tunis-Mahrajène, Tunisia § Laboratoire Chimie et Biologie des Membranes et des Nanoobjets, Université Bordeaux 1, CBMN, UMR 5248, Allée Geoffroy St Hilaire, F-33600 Pessac, France ‡

ABSTRACT: Double emulsions of the W/O/W type are compartmented materials suitable for encapsulation and sustained release of hydrophilic compounds. Initially, the inner aqueous droplets contain an encapsulated compound (EC), and the external phase comprises an osmotic regulator (OR). Over time, water and the solutes dissolved in it tend to be transferred from one aqueous compartment to the other across the oil phase. Water transfer being by far the fastest process, osmotic equilibration of two compartments is permanently ensured. Since the transport of the EC and OR generally occurs at dissimilar rates, the osmotic regulation process provokes a continuous flux of water that modifies the inner and outer volumes. We fabricated W/O/W emulsions stabilized by a couple of amphiphilic polymers, and we measured the inward and outward diffusion kinetics of the solutes. The phenomenology was explored by varying the chemical nature of the OR while keeping the same EC or vice versa. Microscope observations revealed different evolution scenarios, depending on the relative rates of transfer of the EC and OR. Structural evolution was mainly determined by the permeation ratio between the EC and the OR, irrespective of their chemical nature. In particular, a regime leading to droplet emptying was identified. In all cases, evolution was due to diffusion/permeation phenomena and coalescence was marginal. Results were discussed within the frame of a simple mean-field model taking into account the diffusive transfer of the solutes.

1. INTRODUCTION Double emulsions of the water-in-oil-in-water (W/O/W) type are compartmented materials comprising small aqueous droplets dispersed in larger oil globules, the latter being dispersed in an external aqueous phase.1−3 These structures are important for technologies utilizing encapsulation of hydrophilic actives such as drugs, nutrients, cosmetics, and reactants.3−7 The actives can be loaded within the innermost droplets, and the high efficiency of their encapsulation makes it possible to implement strategies such as isolation/protection from incompatible environments, taste masking, sustained or stimulus responsive delivery, etc. Double emulsions are generally produced following a two-step sequential emulsification procedure, and they are kinetically stabilized by two emulsifiers of opposite solubility (water soluble and oil soluble). Being metastable, such materials undergo a destabilization progress that provokes structural changes and progressive leakage of the encapsulated actives. The aging of double emulsions may involve both coalescence and diffusion phenomena. Coalescence, i.e., film rupturing, may occur at several levels:8−10 (i) between the inner droplets, (ii) between the oil globules, and (iii) between the globule and the inner droplets dispersed within it. Alternatively, the oil globules are © 2012 American Chemical Society

permeable to hydrophilic species that can migrate from the internal phase to the external one and vice versa without film rupturing. The transportas a passive processalways involves a concentration gradient exerted by the whole set of molecules dissolved in the two aqueous compartments (emulsifiers, low-molecular weight neutral molecules, electrolytes) and leads to equilibration of the concentrations. The transfer may occur through the oil phase or through the thin liquid films that can form between the interfaces. Several possible mechanisms have been proposed to account for the diffusive transport of hydrophilic species: (i) direct solubilization in the oil phase (for neutral molecules), (ii) transport via the hydrophilic surfactant polar headgroup in the case of water,11,12 (iii) transport through the oil phase into reverse micelles,1,3,4,13−16 and (iv) formation of thermally activated transient holes in the thin liquid films separating the internal droplets and the globule surface.8−10 Stability under storage conditions with minimal leakage of the encapsulated species is a necessary prerequisite for W/O/W Received: August 28, 2012 Revised: November 23, 2012 Published: November 24, 2012 17597

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within a simple mean-field model based on Fick’s law. We demonstrate that the structural evolution of double emulsions results from the interplay of solute diffusion and osmotic regulation phenomena. We are thus addressing an original and complex composition ripening instability that is of both scientific and technological relevance. Our findings are generic and may help in rationalizing a large number of observations about the kinetic evolution of W/O/W emulsions.

emulsions to be of practical use. The impact of these structures designed as delivery systems would be of significant importance provided that the stability and release mechanisms are more clearly understood and monitored. In practice, a large set of parameters may influence the kinetic evolution of double emulsions: the emulsifiers nature (monomeric or polymeric) and their concentration,4,8−10,14,17,18 the osmotic pressure mismatch between the aqueous compartments,11,12,19,20 the average diameter and volume fractions of the inner droplets and oil globules,1,8−10,21 the oil chemical nature,10,22 etc. Among them, the nature of the surface-active species plays a major role because most of the aging mechanisms involve the interfaces. Pioneering studies on double emulsions were performed in the presence of short surfactants.1,8−10 However, the intrinsic instability (fast coalescence and diffusion) of such materials did not allow viable technological developments to be envisioned. Recent studies in the presence of amphiphilic polymers, proteins, and solid colloidal particles2−4,21−25 reveal that coalescence can be inhibited and that diffusive transport may be extremely slow. The storage stability of these materials based on macromolecular or particulate stabilizers paves the way to new developments of drug or nutrient delivery systems based on double-emulsion technology. In general, the inner droplets in W/O/W emulsions contain an encapsulated compound that determines the osmotic pressure of the internal aqueous phase. In order to avoid water transfer and maintain the internal droplet volume, a species called “osmotic regulator” is dissolved in the external aqueous compartment to match the internal osmotic pressure. Once the double structure is fabricated, both the encapsulated compound and the osmotic regulator undergo a diffusive transfer from one aqueous compartment to the other, in opposite directions. The different hydrophilic species, including water and solutes, cross the oil phase boundary at different rates. Water migration typically occurs within several minutes or hours whatever the oil type,12,19 whereas migration of hydrophilic solutes such as ions or small neutral molecules like lactose is much slower and may occur within a time scale of several weeks or months.10,13,14,22 The comparatively fast exchange rate of water ensures almost “instantaneous” osmotic equilibration. In fact, matching the osmotic pressures of the 2 aqueous compartments in the initial conditions does not guarantee the absence of water transfer over time since the osmotic regulator and the encapsulated compound are generally transported at different rates. Thus far, the impact of such differential diffusivity has been disregarded despite its crucial impact on the kinetic evolution of double emulsions. It is within the scope of the present study to evaluate how the relative exchange rates of the species involved in the osmotic regulation process influence the structure of double emulsions and delivery of the encapsulated compound. For that purpose, we fabricated several W/O/W double emulsions with similar features (same chemical nature and concentrations of the surface-active species, same droplet and globule diameters and fractions) but with various encapsulated compounds and osmotic regulators. Following the preliminary work of Bonnet et al.,22 we could obtain systems where release was mainly due to diffusion/permeation phenomena. By measuring the kinetics of transfer of the solutes and inspecting the double-emulsion structure by optical microscopy, we were able to identify the whole set of evolution scenarios, depending on the differential migration rate of the osmotic regulator and encapsulated species. Experimental results were interpreted

2. MATERIALS AND METHODS 2.1. Materials. The oil phase used for formulation of W/O/W emulsions was either Miglyol (mixture of triglycerides with hydrophobic chains C8/C10 in the mass ratio 55/45, density at 20 °C = 0.95 g·cm−3) from Stéarinerie Dubois Fils (France) or sunflower oil (extra virgin, density = 0.92 g·cm−3) from Lesieur. The lipophilic surface-active species, polyglycerol polyricinoleate (PGPR) (esters of polyglycerol and polyricinoleate fatty acids, Mw ≈ 1800 g·mol−1), was purchased from Palsgraad (France), and the hydrophilic one, sodium caseinate (SC) (Mw ≈ 20 000 g·mol−1, containing 1.2 wt of Na+ ions), was obtained from Lactoprot (Germany). The salts NaCl, CsCl, MgCl2 (hexahydrate 99%), and CaCl2 were from Acros Organics (Belgium), and sodium azide (SA, NaN3) was from Merck (Germany). Glucose, lactose, glycerol, sorbitan monooleate (Span 80), and tetrahydrofuran (THF) were purchased from Sigma-Aldrich (Germany). All species were used as received. Water used in the experiments was deionized with a resistivity close to 15 MΩ·cm at 20 °C. 2.2. W/O/W Emulsion Formulation and Preparation. W/O/W emulsions were prepared at room temperature using a two-step emulsification procedure, as described in ref 22. Unless otherwise specified, the internal aqueous phase contains a chloride salt XCln (Xn+ being the cation and n the salt stoichiometry, Xn+ = Cs+, Na+, Mg2+, Ca2+) at an initial concentration C01,Xn+. The solution was manually dispersed at 80 wt % into the oil phase (Miglyol or sunflower oil) containing PGPR (30 wt %). The obtained crude W/O emulsions was then submitted to a strong shear by means of a Couette’s cell (concentric cylinders geometry, Ademtech SA, France). The final droplet size depends on the applied shear rate, on the interfacial tension, and on the viscosity ratio between the dispersed and the continuous phases.26 Since the oils used in this study exhibited different properties, the shearing conditions were adapted to obtain similar average sizes for the water droplets. Emulsification was performed with a gap of 200 μm, and the applied shear rates were 1600 s−1 for sunflower oil and 2600 s−1 for Miglyol. Under such condition, the average droplet size was always close to 1 μm. Once fragmented, the resulting W/O emulsions were diluted with oil to set the final droplet fraction to 40 wt % and the PGPR concentration to 5 wt % with respect to the oil phase. In the second step, the W/O emulsions were incorporated into an aqueous phase, up to 70 wt %. The external aqueous phase contained 12 wt % SC, 0.08 wt % SA (bactericide agent), and the osmotic regulator (glucose or lactose) at a concentration C02,OR. This latter was fixed to match the osmotic pressure of the chloride salt dissolved in the inner droplets. We adopted the van’t Hoff approximation stating that at constant temperature the osmotic pressure is proportional to the sum over all solute concentrations, leading to C02,OR = (n + 1)C01,Xn+. The W/O/W emulsions were fragmented in the Couette cell with a gap of 100 μm, and the applied shear rate was 6300 s−1 for Miglyol and 7000 s−1 for sunflower oil. The concentrated multiple emulsions were diluted with water containing the osmotic regulator (concentration C02,OR) and SC to set the final globule concentrations at 10 wt % and the SC concentration at 3.1 wt %. The maximum surface concentration of SC has been measured at various water/triglyceride interfaces27,28 and is on the order of 3.5 × 10−3 g·m−2. Thus, the total amount of protein was always large enough to fully cover the globule interfaces. Considering the average globule size (∼10 μm, see below) in our emulsions, the presence of only 0.25 wt % SC in the external aqueous phase was sufficient to achieve saturation. 17598

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Table 1. Initial Compositions of the W/O/W Emulsions Formulated within This Studya oil globules (10 wt %)b internal aqueous phase

oil phase

emulsion no.

ϕ0i , wt %

1

4b

glycerol (0.3 M)c

6b

1b

lactose (0.3 M)c

9b

4b

CsCl (0.075 M)c

6b

2 3 4 5 6 7 8 9 10 11 12

composition

CsCl (0.075 M)c + lactose (0.15 M)c NaCl (0.075 M)c CaCl2 (0.075 M)c MgCl2 (0.075 M)c MgCl2 (0.1 M)c

ϕoil, wt %

composition miglyol oil (95 wt %)c + PGPR (5 wt %)c

external aqueous phase (90 wt %)b OR glycerol (0.3 M)c

other solutes SC (3.1 wt %) b + SA (0.08 wt %)c

lactose (0.3 M)c lactose (0.3 M)c glycerol (0.3 M)c CsCl (0.075 M)c lactose (0.15 M)c glucose (0.15 M)c lactose (0.15 M)c lactose (0.15 M)c lactose (0.225 M)c sunflower oil (95 wt %)c + PGPR (5 wt %)c

lactose (0.3 M)c

13 a

OR = osmotic regulator; SC = sodium caseinate; SA = sodium azide; of the double emulsion. cWith respect to the corresponding phase.

glucose (0.3 M)c

ϕ0i

= inner droplet fraction; ϕoil = oil fraction. bWith respect to the total mass nm) spectroscopy (Perkin-Elmer AAnalyst 100) as previously described.22 Glucose was titrated using the 3,5-dinitrosalycilic acid assay according to Miller.29 The concentration of chloride ions (Cl−) was measured using an Ag/AgCl-specific electrode (Radiometer, France) which is sensitive to the chemical activity of chloride ions. All analyses were conducted in triplicate. To carry on titrations, we had to separate the aqueous phases from the whole emulsion volume. We now describe the two separation methods adopted to collect the inner and outer aqueous phases. Outer Phase. A small amount of sample was collected from the stock volume of the double emulsion. The sample was then centrifuged at 1100g (g being the earth gravity constant) for 30 min (Jouan CR 1000) in order to separate the globules from the external aqueous phase. We checked that the applied centrifugation did not lead to coalescence phenomena that would produce release of the inner solutes. In a separate control experiment, the cream was dispersed again after centrifugation and the globules were observed under the microscope. Both the internal droplet size and the droplet concentration within the globules remained apparently invariant. Moreover, droplet sizing experiments based on static light scattering (see above) were carried out, and we checked that the average globule diameter before and after centrifugation was the same. Inner Phase. A small amount of double emulsion was submitted to two consecutive centrifugation steps at 1100g for 30 min. After each centrifugation step, the subnatant was removed and replaced by a solution containing either NaCl (when glucose was to be analyzed) or lactose (when Xn+ or Cl− were to be analyzed) at a concentration matching the initial osmotic pressure of the external phase. Each time, the cream layer was manually redispersed. These two washing steps aimed at eliminating most of the solute present in the external phase. Then, the double emulsion was centrifuged again at 7000 rpm for 15 min in order to concentrate the globules within a dense cream. The fraction of external water in the cream was negligible (lower than 5% of the total volume) and devoid of solute owing to the previous washing steps. The cream was collected, weighted, and submitted to a 5-fold dilution with THF. The initially transparent mixture was stored at rest in a vial open to the atmosphere (within an extractor hood) to allow complete THF evaporation for 5 h. The amount of water evaporated was reintroduced after weighting the residual mass of sample. In the final state, the system was composed of two macroscopically separated phases, one being the inner aqueous phase (bottom) and the other being the oil phase (top). The reliability of the process was demonstrated by measuring the solute

The initial compositions of all emulsions formulated within this study are indicated in Table 1. The pH of both internal and external aqueous phases was always close to 6.5. Emulsions were stored at 25 °C. Globules tended to cream after a few hours of settling. To maintain homogeneity, instead of applying continuous mechanical stirring that would accelerate the release process because of convective effects, the samples were turned upside down at regular time intervals (10 h). 2.3. W/O/W Emulsion Characterization. The structural evolution of the double emulsions was followed by means of an Olympus BX51 microscope equipped with a phase-contrast device, an oil immersion objective (×100/1.3, Zeiss, Germany), and a video camera. In parallel, droplet sizing was carried out on both the primary W/O and multiple W/O/W emulsions. The water droplet size distribution of the primary W/O emulsions was measured by static light scattering using a Coulter LS 230 apparatus. To avoid multiple scattering, the W/O emulsion samples were diluted with a dodecane solution containing 0.5 wt % sorbitan monooleate (Span 80). This oilsoluble surfactant was used in order to avoid destabilization of the droplets due to the strong dilution with oil. The measuring cell was filled with the dodecane solution, and a small volume of the sample was introduced under stirring. The volume-weighted average diameter dd[4,3] was obtained from Mie’s theory. The size distribution of oil globules in W/O/W emulsions was also measured by means of static light scattering. To dilute the double emulsions, we used NaCl aqueous solutions with the same osmotic pressure as that of the external phase in order to avoid water transfer phenomena at the time scale of the measurements (5 min). The mean diameter, dg[4,3] was obtained using the Fraunhofer model which in principle applies to optically homogeneous spheres with diameters larger than about 5 μm. To check the accuracy of this model, images of a control system were recorded by means of the video camera of the microscope and the dimensions of about 100 globules were measured to evaluate their average size. Results were within 6% precision, in accord with the mean diameter provided by light scattering using the Fraunhofer model. Three samples of the W/O and W/O/W emulsions were analyzed right after preparation and after 1 month storage at 25 °C. 2.4. Measurement of Solute Concentrations in Aqueous Phases. Concentrations of solutes (Xn+, glucose, Cl−) dissolved in the continuous phase and in the inner droplets were measured right after preparation and at regular time intervals during 1 month storage. Cations Xn+ were titrated by flame atomic absorption (Ca2+ at 422.7 nm; Mg2+ at 285.2 nm) and emission (Cs+ at 852.1 nm, Na+ at 589 17599

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number of inner droplets after osmotic equilibration, dg0 being the initial globule diameter. We also carried out calculations assuming that A is equal to the total surface area of the inner droplets, and the results were qualitatively similar. The permeability coefficient can be considered as a spaceaveraged phenomenological constant reflecting the influence of all physicochemical details involved in the transfer. Of course, it is expected that they depend on the chemical nature and concentration of the emulsifiers, the oil nature, and the temperature. Our strategy aims at modeling the exchange kinetics using a minimum amount of physical constants. One permeation coefficient, namely, POR, is required to calculate the flux of the osmotic regulator. Ionic species of opposite charge are necessarily diffusing in an interdependent way because of the electroneutrality condition. The cationic flux due to Xn+ and Na+ ions will require two coefficients, PXn+ and PNa+, while the anionic flux resulting from the exchange of Cl− and N3− ions will be simply deduced from the electroneutrality constraint (see below). Within this approach, we are thus not introducing any physical constant to address the exchange of anionic species. As stated by Mezzenga et al.,19 Laplace pressure is also contributing to the chemical potential of the encapsulated species and as such may influence their delivery rate. However, in our case, the contribution of Laplace pressure will be neglected as a first approximation because of the low interfacial tensions and the relatively large droplet and globule diameters.31 We estimated that Laplace pressure may have a significant contribution only when the droplet diameter becomes smaller than about 0.3 μm. Within this study, such small size will be attained in a limited set of systems and only in the late stages of the exchange process. Since water molecules can diffuse through the oil phase at a much faster rate than electrolytes and nonionic solutes,12,19,32 osmotic equilibration by water transfer is supposed to occur “instantaneously”. The volumes of the two aqueous compartments Vi (i = 1, 2) are deduced from the osmotic pressure balance, assuming ideal behavior of the solutes

concentrations in the inner droplets right after fabrication. Indeed, at short times, the extent of diffusional transfer is negligible and the concentrations measured are reflecting the amount initially dissolved in the inner phase. The measured concentrations were always in agreement with the expected values, the differences always being lower than 7%.

3. MODELING 3.1. Basic Principles. In this section, we develop a theoretical mean-field approach to account for the diffusive flux of encapsulated ions (Xn+) from the inner to the outer aqueous phase and that of the osmotic regulator (OR) in the reverse direction. It is assumed that the systems evolve under the effect of diffusion only and that coalescence does not take place. Hereafter, subscripts “1” and “2” will refer to the internal and external phase, respectively, while the exponent “0” will refer to the initial conditions. The initial concentrations of ions Xn+ and of the osmotic regulator in the two phases are C01,Xn+ and C0i,OR (i = 1, 2). Unless otherwise specified, C02,Xn+ = 0 and C01,OR = 0 in the initial state (the two main solutes are initially separated). The species cross the oil membrane, and their concentrations at any time t are Ci,Xn+ and Ci,OR (i = 1, 2). In addition, the external phase contains SC and SA. Due to their large size and hydrophilicity, diffusion of casein molecules across the oil barrier can be ruled out. However, transfer of smaller ionic species like sodium (Na+ from SC and SA) and azide (N3− from SA) does occur. To assess the dynamics of the exchange process and its consequences on the structural evolution, all solutes prone to cross the oil barrier must be taken into account, i.e., Xn+, Na+, Cl−, N3−, and the osmotic regulator. For a solute “s”, the difference in chemical potential between the two compartments is responsible for the molecular exchange with a flux Js proportional to the concentration difference according to the well-known Fick law Js =

dN1, s dt

= PA s (C 2,s − C1,s)

(1)

where A is the surface area involved in the permeation process and Ps is the permeation coefficient. The flux Js is expressed in mol·s−1, the concentration in mol·m−3 and the surface in m2, and consequently, the permeability Ps has the dimension of a speed (m·s−1). In double emulsions, the parameter A is not easy to define since the diffusing species do actually cross two interfaces (droplets and globules) with different surface areas. Moreover, some inner droplets are in direct contact with the globule surface while others are not, and there is a body of evidence that the exchange process depends on the location of the droplets.30 For simplicity, we will assume that A is equal to the total globule surface area and a unique “averaged” permeation coefficient will be considered. Within this hypothesis A=

(V1 + Voil)

⎛ V + V ⎞2/3 oil ⎟ = A ⎜ 10 ⎝ V1 + Voil ⎠

∑ N1,s = s

RT V2

∑ N2,s s

V1 + V2 = V10 + V 20 = VT

(3)

(4)

Because of the electroneutrality requirement it is assumed that

6dg (V10 6dg0

RT V1

where R is the ideal gas constant and T is the absolute temperature. Summations are performed over the number of moles Ni,s (i = 1, 2) of the different solutes “s”, including caseins. The total aqueous volume, VT, is preserved over time, and consequently

Vg 2/3

=

π1 = π2 =

1/3

+ Voil)

N1,Cl− + N1,N−3 = nN1,Xn+ + N1,Na+

(5)

N2,Cl− + N2,N−3 + ZN2,Cas = nN2,Xn+ + N2,Na+

(6)

In eq 6, Z is the electric charge carried on by each casein macromolecule. Considering that our commercial sample contains 1.2 wt % sodium and assuming full dissociation in the aqueous phase, we obtain Z = 10.4. Finally, for each solute “s”, the molar amounts in the two compartments are linked via a conservation equation

0

(2)

where Vg is the volume of the oil globules (Vg = V1 + Voil). The right-side term of eq 2 accounts for conservation of the total

0 0 N1,s + N2,s = N1,s + N2,s

17600

(7)

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In fact, osmotic equilibration (eq 5) induces a permanent flux of water such that dV1 V = T (∑ Js ) dt NT s

regulator, POR, is varied. Figure 1 shows the kinetic evolution of the percentage of Xn+ ions released for three different ratios:

(8)

with the total number of moles in the system given by NT =

∑ (N1,s0 + N2,s0)

(9)

s

From eqs 5 and 6, it follows that the net charge flux across the oil phase is equal to zero (cationic and anionic fluxes offset each other): −(JCl− + JN3−) + nJXn+ + JNa+ = 0. The whole set of equations from 1 to 8 is solved numerically, allowing a straightforward calculation of the concentrations of the cationic species individually Ci,Xn+(t) and Ci,Na+(t) as well as that of the osmotic regulator, Ci,OR(t) (i = 1, 2). For the anionic solutes Cl− and N3−, only the sum of the two concentrations can be obtained: Ci,Cl−(t) + Ci,N3−(t). In the asymptotic regime (t → ∞), we define a “reference state” such that the solutes are homogeneously dissolved over the whole accessible volume VT Cs∞ =

Figure 1. Theoretical curves for the kinetics of release of Xn+ in W/O/ W emulsions at different permeation ratios: PXn+/POR = 0.1 (diamonds), 1 (circles), and 10 (triangles). For the initial conditions, the following set of parameters was adopted: V10 = 0.04 L, V20 = 0.90 L, C01,XCl = 0.075 M, C02,XCl = 0; C01,OR = 0; C02,OR = 0.15 M; C02,NaN3 = 0; C02,SC = 0; d0g = 10 μm; PXn+ = 5.0 × 10−11 m·s−1. Inserts show the evolution of the normalized volume of the inner droplets (V1/V10) and surface area of the globules (A/A0).

0 0 N1,s + N2,s

VT

(10)

For species initially dissolved in the inner droplets (X , Cl− ions), the fraction released from the internal to the external phase at time t will be given by n+

R s(t ) =

C2,s(t ) Cs∞

(11)

PXn+/POR = 0.1, 1, and 10. We also plot the evolution of the normalized volume of the inner droplets V1/V10 and of the normalized surface area A/A0. For PXn+/POR = 1, the encapsulated species and osmotic regulator diffuse at the same rate but in opposite directions. Since the total net flux, JT, of the different solutes across the oil membrane is permanently balanced (JT = JXn+ + JCl− + JOR = 0), the inner volume V1 remains constant over time. In such conditions, eq 1 can be solved analytically and the fraction of Xn+ ions released is given by10

Alternatively, for species initially dissolved in the external phase (OR, Na+), we define the extent of incorporation from the external to the internal phase as

Is(t ) =

C1,s(t ) Cs∞

(12)

3.2. Theoretical Predictions. We now discuss some theoretical results about the impact of the permeation ratio PXn+/POR on the structural evolution of double emulsions. The volumes and concentrations adopted to carry out calculations match the experimental conditions. However, at this stage, we will consider systems that only contain the electrolyte XCln and the osmotic regulator. Although not fully reflecting the real composition of the systems, this configuration was adopted because of its simplicity and relevance, as most of the important physics of the relaxation process rely on the exchange of the encapsulated salt and osmotic regulator. The other solutes (Na+, N3−, casein) influence the osmotic pressure and the electric charge balance, but they do not have a noticeable impact on the evolution scenario, especially in the early stages, because of their relatively small concentration. Obviously, these solutes will be considered in forthcoming sections when we get to model experimental results. The following initial conditions are considered over the first set of calculations: V10 = 0.04 L, V20 = 0.90 L, C01,Xn+ = 0.075 M, C02,Xn+ = 0, C01,OR = 0, C02,OR = 0.15 M, C02,Na+ = 0, n = 1, and d0g = 10 μm. Within the van’t Hoff approximation, the osmotic pressures of the two aqueous compartments are perfectly matched at t = 0. One and the same value of the permeation coefficient of the encapsulated ions, PXn+ = 5.0 × 10−11 m·s−1, is adopted, while the permeation coefficient of the osmotic

R Xn+(t ) = 1 −

⎛ V 20 ⎡ V10 V ⎞⎤ ⎢ 0 + exp⎜ −P Xn+A0 0T 0 t ⎟⎥ VT ⎢⎣ V 2 V1 V 2 ⎠⎥⎦ ⎝

(13)

For PXn+/POR = 0.1, the inward permeation of the osmotic regulator is faster than the outward permeation of the encapsulated species. The total net flux of solutes within the droplets being positive, osmotic equilibration tends to increase the inner droplet volume (insert) and dilute the encapsulated species. Compared to the case PXn+/POR = 1, delivery of Xn+ is slowed down because the inner concentration C1,Xn+ is considerably reduced (as well as the concentration gradient) under the effect of swelling. Finally, for PXn+/POR = 10, the permeation of Xn+ is faster than that of the osmotic regulator and a continuous deflation of the inner droplets occurs, as revealed by the significant decrease of the normalized droplet volume (insert of Figure 1). Because of the large initial volume ratio, V20/V10 ≈ 22, the external compartment acts as a reservoir that fixes the overall osmotic pressure. Throughout almost all the process, Xn+ and Cl− remain the most abundant species within the droplets because of the slow inward diffusion of the osmotic regulator. To maintain osmotic equilibrium, the concentration of the 17601

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Table 2. Average Globule Diameters Measured at t = 0 and 30 days for Double Emulsions Encapsulating XCln Electrolytesa

a

emulsion no.

5

6

7

8

9

10

11

12

13

d0g ±0.3 μm dg (t = 30 days) ±0.3 μm

9.2 9.1

10.3 8.8

10.2 12.3

10.7 10.4

11.2 10.2

9.8 10.0

10.3 10.1

10.1 14.9

10.4 10.2

Initial compositions are given in Table 1.

encapsulated electrolyte C1,Xn+ remains close to its initial value and the concentration gradient driving the diffusion process is elevated. Compared to the reference system (PXn+/POR = 1), delivery of Xn+ is thus accelerated. According to eq 2, the exchange surface area A is an increasing function of the internal volume V1. Consequently, the impact of the osmotic regulation on the rate of delivery of Xn+ ions tends to be attenuated by the variation of A. For instance, in the limit PXn+/POR≪ 1, the progressive increase of V1 produces a dilution of Xn+ ions dissolved in the droplets. The resulting slowdown in their delivery is partially compensated by the expansion of the surface area A. In the same vein, the osmotic deflation of the inner droplets in the limit PXn+/POR≫1 which accelerates the delivery is partially counterbalanced by the progressive reduction of A (see inserts of Figure 1).

4. EXPERIMENTAL RESULTS AND DISCUSSION 4.1. Characterization of W/O/W Emulsions. Typical size distributions of the inner droplets and globules can be found in ref 22. The mean diameters of the internal droplets, dd, in the primary W/ O emulsion and of the oil globules, dg, in W/O/W emulsions measured by static light scattering were in the same range for the freshly prepared emulsions, dd = 1.0 ± 0.2 μm and dg = 10.2 ± 1 μm, whatever the encapsulated species (XCln) and the osmotic regulator (lactose or glucose). The primary W/O emulsions were stored for 1 month, and within experimental uncertainty (±0.1 μm), their average droplet size remained invariant. Because the structural parameters of the double emulsions were almost identical at t = 0 irrespectively of the composition of the water compartments, the rates of release and the structural evolutions could be reliably compared. In Table 2, we report the mean globule diameter measured at t = 0 and 30 days for the emulsions encapsulating XCln electrolytes. 4.2. Experimental Evidence for Distinct Evolution Scenarios. This section aims at providing experimental evidence for the scenarios predicted in section 3.2, especially the limits PXn+/POR ≪1 and PXn+/ POR ≫1. We thus carried out proof of concept experiments where we fabricated W/O/W emulsions with almost identical osmotic pressures at t = 0 in the two aqueous compartments and inspected the evolution of the inner droplet fraction under the microscope. For that purpose, we used solutes with very dissimilar permeation coefficients like glycerol and lactose. It is well known that small uncharged molecules, such as glycerol, can diffuse through phospholipid bilayers within seconds.33 Similarly, transfer of glycerol in double emulsions was reported to be a very fast process.34 Instead, lactose has been used as an osmotic regulator for long-term (>1 month) encapsulation of Mg2+ ions in double emulsions based on triglycerides.22,31 The lack of structural changes with no apparent swelling nor deflation of the inner droplets over 1 month31 suggests that the transfer of lactose across the oil phase occurs at a very low rate. In the first set of experiments, glycerol was the encapsulated solute and glycerol or lactose were the osmotic regulators (emulsions 1 and 2, see compositions in Table 1). Double emulsions were fabricated following the protocol described in section 2.2, the only difference being that glycerol was dissolved in the inner aqueous phase instead of an electrolyte. The first system under investigation (emulsion 1) comprises glycerol in the two aqueous compartments at the same concentration of 0.3 M. Figure 2 illustrates the state of the emulsion immediately after fabrication (a) and after 1 h storage at 25 °C (b). The two emulsions are almost identical, with no noticeable change in

Figure 2. Optical microscopy images showing the structural evolution of W/O/W emulsions initially containing glycerol in the inner droplets and glycerol (emulsion 1) or lactose (emulsion 2) in the external phase. the inner droplet fraction or size. This observation is not surprising considering that the initial osmotic pressures in two aqueous compartments are almost identical (only a 20% difference due to dissociation of SC and SA in the external phase) and that there is actually no driving force for the diffusive transfer of glycerol owing to its presence in both compartments at the same concentration. The experiment also confirms that the inner droplets experience neither coalescence nor Ostwald ripening that would potentially lead to a decrease of the inner fraction within the explored time scale (1 h). Emulsion 2 contains glycerol in the inner droplets and lactose in the external phase at the same molar concentration. The microscope images in Figure 2c and 2d reveals a progressive emptying on the globules. Right after fabrication (Figure 2c), we clearly observe that the initial fraction is lower than in Figure 2a, and after 1 h (Figure 2d), the globules contain tiny drops hardly discernible under the microscope. Since coalescence and Ostwald ripening can be ruled out, observations strongly suggest that glycerol diffuses rapidly toward the external phase because of the strong concentration gradient, while during the same time, the transfer of lactose is comparatively negligible. This evolution is thus consistent with our theoretical predictions in the limit PEC/POR ≫1 (EC = encapsulated compound). We now consider emulsions where lactose is the encapsulated species, and lactose or glycerol are the osmotic regulators (emulsions 3 and 4). Compared to the previous systems, we decreased the initial droplet fraction (10% of the globule volume) in order to better discern the swelling induced by rapid transfer of glycerol. In emulsion 3, the two aqueous phases contain lactose at the same concentration, and as expected, no noticeable change is observed after 1 h storage (Figure 3a and 3b). Emulsion 4 contains glycerol in the external phase. By comparing Figure 3a and 3c, it appears that the inner fraction increases significantly within a very short time scale, presumably during the 17602

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Figure 3. Optical microscopy images showing the structural evolution of W/O/W emulsions initially containing lactose in the inner droplets and lactose (emulsion 3) or glycerol (emulsion 4) in the external phase. second emulsification step. The swollen state is preserved after 1 h, as revealed by Figure 3d. To interpret this evolution, it can be argued that the inward diffusion of glycerol occurs at a much faster rate than the outward diffusion of lactose. This system is thus illustrating the limit PEC/POR ≪1. 4.3. Generalization. In order to prove the generality of the abovedescribed phenomenology, we carried out a series of experiments with an electrolyte, CsCl, initially dissolved in the inner droplets at 0.075 M. In this case, it was necessary to follow the emulsions for 1 month to assess their behavior because of the slow permeation of the ionic species. An emulsion containing CsCl in both aqueous compartments at the same concentration was first prepared to check the stability of the inner droplets within the globules in the absence of diffusive flux (emulsion 5). By comparing Figure 4a and 4b, it can be concluded that the inner droplet fraction and size does not exhibit any significant change after 1 month storage. The average globule diameter was measured by light scattering and remained close to 10 μm (Table 2), thus confirming the structural stability of this double emulsion. When lactose was used as the osmotic regulator in the external phase (emulsion 6), the inner droplet fraction after 1 month storage was noticeably lower than the initial one, as observed in Figure 4c and 4d. Concomitantly, the average globule diameter measured by light scattering decreased from 10.3 ± 0.3 to 8.8 ± 0.3 μm. Inner droplets were smaller on average after 1 month storage. The smallest ones, whose size is at the resolution limit of the microscope (0.2−0.3 μm), are not distinguishable on the image of Figure 4d because of their intense Brownian motion. Since coalescence of the inner droplets on the globule surface is unlikely, we hypothesize that the evolution of emulsion 6 is mainly due to the diffusive transfer of the inner phase (CsCl and water) toward the external one. This evolution scenario suggests that the permeation coefficient of the encapsulated species is much larger than that of the osmotic regulator, i.e., PXn+/POR ≫1. In emulsion 7, glucose was used as an osmotic regulator instead of lactose (Figure 4e and 4f). Glucose diffuses more rapidly than lactose across the oil phase owing to its smaller molecular size. Emulsions were carefully observed under the microscope, and we noticed that in the initial state the inner droplets were submitted to a slow but yet discernible Brownian motion. One month later, they were tightly

Figure 4. Optical microscopy images showing the structural evolution of W/O/W emulsions initially containing CsCl in the inner droplets and CsCl (emulsion 5), lactose (emulsion 6), or glucose (emulsion 7) in the external phase. packed and Brownian motion was not discernible anymore. Swelling was confirmed by a slight increase in the average globule diameter from 10.2 ± 0.3 μm at t = 0 to 12.3 ± 0.3 μm at t = 30 days. This evolution can be qualitatively interpreted considering that the permeation coefficient of glucose exceeds that of CsCl. The osmotic pressure equilibration process would drive the transfer of water toward the inner droplets, as expected in the limit PXn+/POR ≪1. One of the most direct consequences of swelling is the increase of the average droplet size. However, the increment of the inner droplet diameter was not large enough to be easily detectable under the microscope. In general, tracking any change in the average droplet is cumbersome because of the cubic dependence of the volume on the diameter: a kfold variation in volume only induces a k1/3-fold variation in diameter. 4.4. Kinetics of the Diffusive Phenomena. In the two following sections, we estimate the permeation coefficients, based on the kinetic evolution of the solute concentrations in the two aqueous compartments. We then analyze the evolution scenario in terms of swelling or deflation of the inner droplets, considering the permeation ratio PXn+/ POR. The generality of the approach is checked by following the evolution of the materials under variable initial osmotic conditions (from nearly iso-osmotic to hyperosmotic conditions) and varying the chemical nature of the solutes. We simultaneously measured the concentration of Cs+ in the external phase and that of glucose and Na+ ions in the inner aqueous phase for emulsion 7. Data are reported in Figure 5. The fraction of 17603

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Cs+ released was calculated from eq 11, and the fraction of glucose and Na+ incorporated in the inner droplets was given by eq 12.

same values of PXn+ and PNa+ as in Figure 5 were adopted and POR was used as the only adjustable parameter. The best fit was obtained for POR = 0.6 × 10−11 m·s−1 (PXn+/POR = 8.3). Although the calculations slightly underestimate the measured values at short times, it can be stated that the model reproduces the general trends of the process in the limit PXn+/POR ≫1. In particular, the variation of the normalized inner volume (insert of Figure 6) is in qualitative agreement with the experimental observations under the microscope, revealing a substantial decrease of the inner droplet fraction (Figure 4d), as well as with the evolution of the mean globule diameter (Table 2). To further probe the reliability of our interpretations, we fabricated an emulsion with 0.15 M lactose + 0.075 M CsCl in the inner phase and 0.15 M lactose in the external phase (emulsion 8). By dissolving lactose in the inners drops at t = 0, we intended to limit the transfer of water toward the external phase. The percentage of Cs+ released was measured, and the results are plotted in Figure 7. At t = 30 days,

Figure 5. Experimental evolution of the percentage of Cs+ released and percentage of glucose and Na+ incorporated for emulsion 7. Solid lines are theoretical predictions. Insert shows the theoretical evolution of the normalized inner droplet fraction. To carry on the calculations, the initial concentrations are provided in Table 1 and the following parameters were adopted: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; PXn+ = 5.0 × 10−11 m·s−1; POR = 25.0 × 10−11 m·s−1; PNa+ = 3.8 × 10−11 m·s−1. First, the measurements confirm that glucose is transferred more rapidly than Cs+. The model developed in section 3.1 was used to fit the experimental data, considering the permeation coefficients PXn+, POR, and PNa+ as the only free parameters (solid lines in Figure 5a). The best agreement (least-squares method) was obtained for PXn+ = 5.0 × 10−11 m·s−1, POR = 25.0 × 10−11 m·s−1, and PNa+ = 3.8 × 10−11 m·s−1. The variation of the normalized inner volume V1/V10 deduced from the model is provided in the insert of the same figure. The initial decay (20%) is due to the osmotic pressure difference between the two aqueous compartments because of the presence of Na+, N3−, and caseins in the external phase. The theoretical predictions for V1/V10 and for the mean globule diameter

dg =

⎛ V + V ⎞1/3 1 oil ⎟ 0 ⎝ V1 + Voil ⎠

dg0⎜

Figure 7. Experimental evolution of the percentage of Cs+ released in emulsion 8. Solid lines correspond to theoretical predictions. Insert shows the theoretical evolution of the normalized inner droplet fraction. Calculations were carried out considering the initial concentrations provided in Table 1 and the following parameters: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; values adopted for POR, PXn+, and PNa+ are given in Table 3. release is almost achieved (∼95%), and from microscope observations, we conclude that the internal droplet fraction remains elevated (see Figure 8a and 8b). Although comprising the same solutes, structural

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are compatible with our microscope observations (increase of the packing density, absence of internal droplet motion) as well as with the slight increase in the mean globule diameter from 10.2 to 12.3 μm. Indeed, the predicted value V1/V10 ≈ 2.8 at t = 30 days corresponds to a ∼20% increment of the mean globule diameter, consistent with our measurements. The model is thus correctly accounting for the experimental evolution of the solute concentrations and for the structural evolution of the double globules in the limit PXn+/POR ≪1 (in this case, PXn+/POR ≈ 0.2). We also measured the release kinetics of Cs+ in emulsion 6 containing lactose in the external phase (Figure 6). To fit the data, the Figure 8. Optical microscopy images showing the evolution of a W/ O/W emulsion initially containing CsCl + lactose in both aqueous compartments (emulsion 8). evolution of emulsions 6 and 8 is radically different. The fact that the globules in emulsion 8 remain filled confirms that the evolution observed in emulsion 6 (Figure 4c and 4d) was not due to coalescence but to diffusion phenomena. Data of Figure 7 were fitted considering the same values of the permeation coefficients as in Figure 6. The agreement between the model and the experimental points remains acceptable, and the normalized inner volume predicted at t = 30 days, V1/V10 ≈ 1.3 (insert of Figure 7), is in qualitative agreement with our microscope observations. Lactose dissolved in the droplets generates an osmotic mismatch that initially produces a nearly 1.5-fold increase of the inner volume. Owing to this initial swelling and the presence of lactose within the droplets, the deflation phenomenon that follows is considerably attenuated compared to emulsion 6.

Figure 6. Experimental evolution of the percentage of Cs+ released in emulsion 6. Solid lines correspond to theoretical predictions. Inserts show the theoretical evolution of the normalized inner droplet fraction. Calculations were carried out considering the initial concentrations provided in Table 1 and the following parameters: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; values adopted for POR, PXn+, and PNa+ are given in Table 3. 17604

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4.5. Influence of the Nature of the Encapsulated Species (Same Osmotic Regulator). In the following set of experiments, we fabricated double emulsions with various chloride salts in the internal droplets at an initial concentration of 0.075 M and the same osmotic regulator, lactose, in the external phase (emulsions 6, 9, 10, and 11). Its initial concentration was set to match the osmotic pressure due to the inner electrolyte: 0.15 M for monovalent cations and 0.225 M for divalent cations. For each system the kinetics of release was measured by flame spectroscopy, and the results are given in Figure 9. The

storage and the obtained value was nearly equal to 80%. In Figure 10, the images reveal that the emulsions at t = 0 and 60 days are almost

Figure 10. Optical microscopy images showing the evolution of a W/ O/W emulsion initially containing MgCl2 in the inner droplets and lactose in the external aqueous phase (emulsion 11).

similar. The systems containing NaCl (emulsion 9) exhibited intermediate behavior: after 30 days, the apparent inner droplet fraction was significantly lower than the initial one. Overall, there is also a clear correlation between the structural evolution within the globules and the rate of delivery of the encapsulated species. Globules are prone to emptying in systems with a high release rate; conversely, the inner fraction tends to remain elevated in systems with a low rate of delivery. The experimental curves in Figure 9 were fitted using the mean-field model. We adopted one and the same value for the permeation coefficient of lactose, POR = 0.6 × 10−11 m·s−1, obtained in the previous section. To ensure consistency, for PNa+ we took the same value as that deriving from the fit in Figure 5. It is also worth noting for emulsion 9 that the asymptotic value of the percentage released is 85% and not 100% because of the contribution of Na+ ions initially present in the external phase. The solid lines in Figure 9 correspond to the best fit to the experimental data, and the corresponding values of the permeation coefficients PXn+ are reported in Table 3. Overall, the agreement is rather satisfactory. The insert shows the theoretical variation of the normalized inner fraction as a function of time. For CsCl, PXn+/POR is significantly larger than unity and the normalized inner fraction tends to vanish over time. The level of globule emptying becomes less significant as PXn+/POR approaches unity, and for MgCl2 there is almost no variation of the inner volume after the initial jump. Theoretical evolutions of the normalized inner fractions are in qualitative accord with our microscope observations. We thus conclude that the osmotic regulation effect is controlling the structural evolution of the systems via the ratio PXn+/POR which is much larger than unity for CsCl and NaCl and close to unity for CaCl2 and MgCl2 (Table 3). 4.6. Influence of the Osmotic Regulator (Same Encapsulated Electrolyte). Considering the findings from the previous sections, we expect the rate of delivery of a given encapsulated compound to be dependent on the chemical nature of the osmotic regulator. A series of experiments was thus conducted with the same encapsulated species, MgCl2, and with two different osmotic regulators: glucose and lactose (emulsions 12 and 13, respectively). In order to check that our conclusions are generic and do not depend on the oil chemical nature, Miglyol was replaced by sunflower oil. The microscope photographs of Figure 11 reveal the state of the systems at t = 0 and 30 days. In the emulsions containing glucose (Figure 11a and 11b), the globules undergo considerable swelling after 1 month storage. On one hand, the average globule diameter evolves from 10.1 ± 0.3 μm at t = 0 to 14.9 ± 0.3 μm at t = 30 days. On the other hand, the droplets within the globules become tightly packed and immobile after 1 month while they were submitted to slow Brownian motion right after fabrication of the double emulsion. These observations can be qualitatively interpreted assuming that glucose diffuses more rapidly than MgCl2, i.e., PXn+/POR ≪ 1. In contrast, no variation of the internal fraction and of the average globule size can be discerned in the double emulsion

Figure 9. Experimental evolution of the percentage released in W/O/ W emulsions initially containing various chloride salts in the inner droplets and lactose in the external aqueous phase (emulsions 6, 9, 10, 11). Solid lines are theoretical predictions. Insert shows the theoretical evolution of the normalized inner droplet fraction. Calculations were carried out considering the initial concentrations provided in Table 1 and the following parameters: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; values adopted for POR, PXn+, and PNa+ are reported in Table 3. following hierarchy in the rate of delivery is obtained: Cs+> Na+ > Ca2+ > Mg2+. Interestingly, there is a correlation between the rate of delivery and the enthalpy of hydration of the cations as can be deduced from Table 3: the lower the hydration enthalpy, the faster the release occurs.

Table 3. Table Providing Percentage Released after 30 Days, Enthalpies of Hydration of Different Cations, Permeation Coefficients Deduced from the Best Fit to the Data of Figure 9 (emulsions 6, 9, 10, 11), and Permeation Ratios PXn+/PORa ion type

Cs+

Na+

Ca2+

release (%) after 30 days ΔHhydration (kJ·mol−1)b PXn+ (m·s−1) POR (m·s−1) PXn+/POR

(100 ± 6)%

(87 ± 5)%

(72 ± 4)%

(65 ± 3)%

−264

−409

−1577

−1921

5.0 × 10−11 0.6 × 10−11 8.3

3.8 × 10−11

1.0 × 10−11

0. 75 × 10−11

6.3

1.7

a

Mg2+

1.3 b

Oil phase = Miglyol; osmotic regulator (OR) = lactose. From ref 35.

This supports the view that the ions cross the oil barrier in a dehydrated state. In addition, the fact that the permeation coefficient is different for each chloride salt suggests that the exchange rate is under kinetic control of the cations (their permeability is lower than that of the chloride). Otherwise, the exchange kinetics would have been almost similar whatever the cation. Emulsions were observed under the microscope to qualitatively assess the evolution of the inner droplet fraction. After 30 days, the globules undergo significant emptying in the emulsion based on CsCl (emulsion 6), as indicated above. Instead, we did not observe any significant variation of the inner fraction for the emulsions containing CaCl2 and MgCl2 (emulsions 10 and 11, respectively), and we did not measure any significant change in the mean globule size either. For emulsion 11, the percentage released was measured after a 60 day 17605

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Figure 13. Experimental evolution of the percentage released in W/ O/W emulsions initially containing MgCl2 in the inner droplets and glucose (emulsion 12) or lactose (emulsion 13) in the external phase. Solid lines are theoretical predictions. Insert shows the theoretical evolution of the normalized inner droplet fraction and of the surface area of the globules. Calculations were carried out considering the initial concentrations provided in Table 1 and the following parameters: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; values adopted for POR, PXn+ and PNa+ are given in Table 4.

Table 4. Permeation Coefficients of the Solutes Deduced from the Best Fit to the Data of Figures 12 and 13 (emulsions 12 and 13)a Figure 11. Optical microscopy images showing the evolution of W/O/ W emulsions initially containing MgCl2 in the inner droplets and glucose (emulsion 12) or lactose (emulsion 13) in the external phase. based on lactose (Figure 11c and 11d), most likely reflecting a situation such that PXn+/POR is close to unity. The concentrations of Mg2+ in the external phase and of glucose in the inner phase were measured at regular time intervals for emulsion 12 (see Figure 12). From the fits (solid lines), we deduce the

a

osmotic regulator

glucose

lactose

POR (m·s−1) PNa+ (m·s−1) PXn+ (m·s−1) PXn+/POR

20.0 × 10−11 3.0 × 10−11 0.5 × 10−11 0.03

0.3 × 10−11

1.7

Oil phase = sunflower oil; Xn+ = Mg2+; OR = osmotic regulator.

significant swelling in the presence of glucose and almost no variation after the initial jump in the presence of lactose, as experimentally observed (Figure 11). For emulsion 12, from the predicted value of the normalized inner volume at t = 30 days, i.e., V1/V10 ≈ 6, it is possible to calculate the mean globule diameter using eq 14 and the obtained value, dg ≈ 14.5 μm, is in close agreement with size measurements. The slower rate of delivery observed with glucose is a consequence of the dilution of the inner solute. The impact of swelling is partially compensated by the expansion of the surface area A involved in the permeation process (eq 2). Indeed, as underlined in section 3.2, parameter A evolves in a way that tends to counteract the evolution of the concentration gradient. The right-hand side insert in Figure 13 shows that the normalized surface area surface A/A0 progressively increases for emulsion 12 (thus accelerating the transport), while it remains almost constant (after the initial shift) in emulsion 13. Complete characterization of the delivery process was achieved for the system based on lactose (emulsion 13). For that purpose, we measured the concentrations of Mg2+ and Cl− ions in the two aqueous compartments. That way we could calculate the percentage released using eq 11 as well as the residual fraction of ions dissolved the inner droplets, defined as

Figure 12. Experimental evolution of the percentage of Mg2+ released and of the percentage of glucose incorporated in emulsion 12. Solid lines are theoretical predictions. Calculations were carried out considering the initial concentrations provided in Table 1 and the following parameters: V10 = 0.04 L; V20 = 0.90 L; d0g = 10 μm; values adopted for POR, PXn+, and PNa+ are given in Table 4. permeation coefficients of glucose, Mg2+, and Na+ across sunflower oil. The fit quality is rather insensitive to PNa+ because of the relatively small concentration of this ion. Since the inward diffusion of Na+ ions was not experimentally measured, the proposed value of PNa+ has to be considered as a rough estimate. In Figure 13, we compare the experimental evolutions of the percentage of Mg2+ released in the presence of the two osmotic regulators. The differences between the two plots are beyond experimental uncertainty, and it can thus be stated that lactose is provoking the most rapid delivery. The experimental plot corresponding to emulsion 13 (OR = lactose) was fitted taking the same values of PXn+ and PNa+ as for emulsion 11 and with POR as the only adjustable parameter. The whole set of permeation coefficients is reported in Table 4. The left-hand side insert of Figure 13 shows the theoretical evolution of the normalized inner volume. The model predicts that the inner droplets undergo

Fs(t ) = 100

C1,s(t ) C10

(s = Mg 2 +or Cl−)

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All data are gathered within a single graph on Figure 14. The decrease of the inner concentrations is a clear indication that the release is at least partially due to diffusion. Otherwise (if coalescence was the main release mechanism), the inner concentrations would have remained almost constant. Assuming that the release involves both coalescence and diffusion, the following relation holds between the residual fraction, Fs(t), and the released one, Rs(t) R s(p , t ) = 17606

[1 − Fs(t )]VT V2 + p[V1 − VTFs(t )]

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evolution of double emulsions and bring new insights into the design and development of these materials. Finally, the phenomena reported here are also of relevance to other encapsulation systems like, for instance, simple W/O or O/W emulsions comprising several droplet types loaded with different solutes, for applications such as emulsion polymerization, droplet-based microfluidics, storage of chemical compound libraries,37 etc.

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AUTHOR INFORMATION

Corresponding Author

Figure 14. Experimental evolution of the residual and released fractions of Mg2+ and Cl− ions in emulsion 13. (Insert) Released vs residual percentage plot. Black line is the theoretical prediction when the release is purely diffusive (p = 0, no coalescence); colored lines are predictions assuming that a fraction p of the percentage released occurs through coalescence.

*Phone: +33 5 40 00 68 38 (L.-C.F.); +216 71 537 659 (E.W.). E-mail: fl[email protected] (L.-C.F.); essafi[email protected] (E.W.). Notes

The authors declare no competing financial interest.

■

The parameter p indicates the molar percentage of solute released through coalescence (p = 0, no coalescence; p = 1, full coalescence). To obtain eq 16, it was assumed that volume changes, if any, are only due to coalescence. Indeed, since the permeation coefficients of lactose and Mg2+ ions are close to each other (Table 4), diffusional fluxes are not provoking significant volume variations. The terms V1 and V2 were calculated from eqs 3 and 4 and correspond to the state of the system right after the initial osmotic jump. In the absence of coalescence, the previous equation can be simplified to

V R s(p = 0, t ) = [1 − Fs(t )] T V2

ACKNOWLEDGMENTS This work was supported by a DGRS/CNRS cooperation grant (no. 09/R11-37). The authors acknowledge the Tunisian Ministry of Higher Education for its financial support through a Ph.D. research grant for S.H.

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REFERENCES

(1) Engel, R. H.; Riggi, S. J.; Fahrenbach, M. J. Insulin: Intestinal Absorption as Water-in-Oil-in-Water Emulsions. Nature 1968, 219, 856−857. (2) Weiss, J.; Scherze, I.; Muschiolik, G. Polysaccharide Gel with Multiple Emulsion. Food Hydrocolloids 2005, 19, 605−615. (3) Benichou, A.; Aserin, A.; Garti, N. Double Emulsions Stabilized with Hybrids of Natural Polymers for Entrapment and Slow Release of Active Matters. Adv. Colloid Interface Sci. 2004, 108−109, 29−41. (4) Sela, Y.; Magdassi, S.; Garti, N. Release of Markers from the Inner Water Phase of W/O/W Emulsions Stabilized by Silicone Based Polymeric Surfactants. J. Controlled Release 1995, 33, 1−12. (5) Tedajo, G. M.; Bouttier, S.; Fourniat, J.; Grossiord, J. L.; Marty, J. P.; Seiller, M. Release of Antiseptics from the Aqueous Compartments of a W/O/W Multiple Emulsion. Int. J. Pharm. 2005, 288, 63−72. (6) Higashi, S.; Setoguchi, T. Hepatic Arterial Injection Chemotherapy for Hepatocellular Carcinoma with Epirubicin Aqueous Solution as Numerous Vesicles in Iodinated Poppy-Seed Oil Microdroplets: Clinical Application of Water-in-Oil-in-Water Emulsion Prepared Using a Membrane Emulsification Technique. Adv. Drug Delivery Rev. 2000, 45, 57−64. (7) Shima, M.; Morita, Y.; Yamashita, M.; Adachi, S. Protection of Lactobacillus Acidophilus from the Low pH of a Model Gastric Juice by Incorporation in a W/O/W Emulsion. Food Hydrocolloids 2006, 20, 1164−1169. (8) Pays, K.; Giermanska-Kahn, J.; Pouligny, B.; Bibette, J.; LealCalderon, F. Double Emulsions: a Tool for Probing Thin Film Metastability. Phys. Rev. Lett. 2001, 87, 178304−1−178304−4. (9) Pays, K.; Giermanska-Kahn, J.; Pouligny, B.; Bibette, J.; LealCalderon, F. Coalescence in Surfactant-Stabilized Double Emulsions. Langmuir 2001, 17, 7758−7769. (10) Pays, K.; Giermanska-Kahn, J.; Pouligny, B.; Bibette, J.; LealCalderon, F. Double Emulsions: How Does Release Occur? J. Controlled Release 2002, 79, 193−205. (11) Wen, L.; Papadopoulos, K. D. Visualization of Water in W1/O/ W2 Emulsions. Colloids Surf. A 2000, 174, 159−167. (12) Wen, L.; Papadopoulos, K. D. Effects of Osmotic Pressure on Water Transport in W1/O/W2 Emulsions. J. Colloid Interface Sci. 2001, 235, 398−404. (13) Garti, N. Double Emulsions-Scope, Limitations and New Achievements. Colloids Surf. A 1997, 123−124, 233−246.

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In this limit, we predict a linear relationship between the two experimentally measured fractions with a slope very close to −1 (VT/ V2 ≈ 1.03). The experimental data relative to both Mg2+ and Cl− ions are recast in Rs(t) vs Fs(t) coordinates in the insert of Figure 14. We obtain a linear variation whose slope is very close to −1. For the sake of comparison, the blue, green and red lines in the insert correspond to p = 0.5, 0.9 and 1, respectively. This result, combined with all the experimental observations and measurements previously reported, make us fairly certain that the release is mainly due to diffusive phenomena and that coalescence is marginal.

5. CONCLUSIONS In this paper, the structural evolution of W/O/W double emulsions induced by diffusion of hydrophilic species was theoretically and experimentally investigated. The diffusion process involved simultaneous outward permeation of the initially encapsulated species and inward diffusion of the osmotic regulator. We provided a body of evidence that the evolution scenario (swelling or deswelling) mainly depends of the permeation ratio PXn+/POR, whatever the chemical nature of the species involved in the transfer, thus proving the generality of our approach. In particular, a regime leading to a progressive emptying of the droplets through passive diffusion was identified in the limit PXn+/POR ≫ 1. Thus far, globule emptying has always been described in the literature as being due to coalescence of the inner droplets on the globule surface.8−10 Here, we demonstrated that the emptying may occur through a diffusive process. In the limit PXn+/POR ≪ 1, the inner droplets undergo swelling. The transient, two way-diffusion process is inherent in double emulsions and may affect some macroscopic properties. For instance, water transport between the compartments may alter the globule as well as the inner droplet fractions, which both influence the rheological properties of double emulsions.36 We hope this article will allow better control of the kinetic 17607

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