Article pubs.acs.org/Langmuir

Decoupling of Mass Transport Mechanisms in the Stagewise Swelling of Multiple Emulsions Jana Bahtz,*,† Deniz Z. Gunes,*,‡ Eric Hughes,‡ Lea Pokorny,† Francesca Riesch,† Axel Syrbe,‡ Peter Fischer,† and Erich J. Windhab† †

Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland

‡

S Supporting Information *

ABSTRACT: This contribution reports on the mass transport kinetics of osmotically imbalanced water-in-oil-in-water (W1/O/W2) emulsions. Although frequently studied, the control of mass transport in W1/O/W2 emulsions is still challenging. We describe a microfluidics-based method to systematically investigate the impact of various parameters, such as osmotic pressure gradient, oil phase viscosity, and temperature, on the mass transport. Combined with optical microscopy analyses, we are able to identify and decouple the various mechanisms, which control the dynamic droplet size of osmotically imbalanced W1/O/W2 emulsions. So, swelling kinetics curves with a very high accuracy are generated, giving a basis for quantifying the kinetic aspects of transport. Two sequential swelling stages, i.e., a lag stage and an osmotically dominated stage, with different mass transport mechanisms are identified. The determination and interpretation of the different stages are the prerequisite to control and trigger the swelling process. We show evidence that both mass transport mechanisms can be decoupled from each other. Rapid osmotically driven mass transport only takes place in a second stage induced by structural changes of the oil phase in a lag stage, which allow an osmotic exchange between both water phases. Such structural changes are strongly facilitated by spontaneous water-in-oil emulsification. The duration of the lag stage is pressure-independent but significantly influenced by the oil phase viscosity and temperature.

■

INTRODUCTION Multiple water-in-oil-in-water (W1/O/W2) emulsions consist of inner water-in-oil emulsion droplets (W1/O) which are further dispersed in an outer aqueous phase (W2). Because of their assembly, W1/O/W2 emulsions lead to fat reduction without reducing the oil droplet size or the phase fraction.1 Thus, they may find application in low calorie food products, especially in the case of high W1 fractions. Their additional capability of encapsulating hydrophilic drugs or other active components and subsequently releasing them in a controlled and slow manner2,3 extends their potential application beyond the food sector to pharmaceutic, 4−8 cosmetic, 3,9 and agriculture3,10 applications. Moreover, drugs with different physicochemical properties, such as hydrophilic and hydrophobic actives, may be encapsulated and delivered simultaneously with controlled release kinetics.11 However, the structural integrity of all W1/O/W2 emulsions is sensitive, and processing, particularly at elevated stresses such as high shear forces, can cause droplet rupture or coalescence,12,13 resulting in irreversible damage and stability loss.14,15 Such sensitivity is more pronounced in the case of high W1 fractions.16 Thus, direct processing of W1/O/W2 emulsions with high W1 fractions leads to partial leakage of the inner aqueous phase. Leakage causes a decrease of the emulsion viscosity and a loss of encapsulated components. The application of structure-destructive stress in W1/O/W2 © 2015 American Chemical Society

emulsions might be overcome by a controlled osmotic swelling step after production of an emulsion with initially low disperse phase ratio under gentle, structure-preserving conditions. In this case a positive chemical potential difference between the W1 and W2 phase will force the W1/O droplets to take up water and swell.5,17−21 As a consequence, the emulsion becomes more viscous due to, first, the viscoelastic properties of the inner W1/O emulsion increase in the absence of coalescence and, second, the total disperse phase fraction increases due to the decrease of the W2 fraction. However, progressive swelling leads to a strong thinning of the oil layer. Instability of the oil layer to coalescence between W1 and the outer water drop may follow leading to leakage, known as the “swelling−breakdown” mechanism.21,22 In order to control and adjust the osmotic swelling and/or drug release, the mechanism of water transport has to be fully understood. Up to now, four main mechanisms of water transport across the oil layer have been proposed in the literature21 covering water release as well as water uptake. The first mechanism is related to diffusion of single water molecules through a thin oil lamella.21 In the presence of an oil film direct diffusion of single water molecules through the oil is not very effective because of their low Received: September 4, 2014 Revised: April 27, 2015 Published: April 28, 2015 5265

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir solubility in oil.23 The second proposed mechanism is the transport of water entrapped in reverse micelles formed by lipophilic surfactant molecules in the oil phase.2,24−28 In contrast to this, the third mechanism is based on water transport in the form of tiny droplets which form spontaneously and diffuse through the oil layer.28−31 The water entrapment in either inverse micelles or droplets leads to an increased effective water solubility in oil.28 A fourth, but less frequently utilized, explanation is the transport of water carried by single hydrated surfactant molecules.32 In all these explanations the different mechanistic aspects of the mass transport in W1/O/W2 emulsions are not fully considered. Most studies do not differentiate between water uptake into the oil, water transport across the oil layer, and water release into either the W1 or the W2 phase. As release kinetics are often investigated over long time periods, early stage studies are lacking. For the time being only Sela et al.2 described different stages of release kinetics including a pronounced initial lag time, which they assumed to result from the time required to entrap water in reverse micelles. However, they emphasized that their interpretations of the stagewise release mechanism did not present direct evidence and were highly speculative. Our study shows that not only the water release but also the swelling obeys two sequential stages with different mechanisms caused by individual driving forces and which have to be decoupled from each other. In order to identify clearly the different processes and the parameters that are involved, we designed an experimental setup which favors one or the other. This was possible by investigating the water transport in microfluidic processed monodisperse double emulsions with only one inner droplet,33 called single drop-in-drop emulsions in the following. The experimental design allows differentiating between the water uptake into the oil phase and the inner water droplet. Further, microfluidic processing enables one to deliver a precise control of droplet size and composition.34−38 These simplifications allowed us to study single as well as combined contributions of several parameters, such as osmotic pressure gradient, disperse phase fraction, viscosity of the oil phase, and temperature on the swelling behavior in a systematic and comprehensive manner. The study assembles kinetic and mechanistic investigations, which allow adequate interpretation of the different stages of water transport. They thereby allow the quantification of the respective effects of the osmotic pressure gradient, nature and concentration of surfactants, and further structural parameters of the engineering of multiple emulsions on the swelling rate.

■

pressure gradient was established using different TMACl concentrations in the W1 and the W2 phase. 2 wt % poly(ethylene glycol) sorbitan monolaurate (Tween20 from Sigma-Aldrich, Switzerland) was added to the outer aqueous phase W2 as a water-soluble surfactant. The inner aqueous phase W1 contained 1 wt % sodium dodecyl sulfate (SDS from Sigma-Aldrich, Switzerland). The combination of SDS and PGPR was needed to access the jetting regime after the first junction of the microfluidics chip (see below) at high capillary numbers Ca. The capillary number Ca is defined as

Ca =

γη̇ d 4σ

(1)

with γ̇ the effective shear rate experienced by the droplet, η the viscosity of the oil phase O, d the diameter of the W1 jet, and σ the interfacial tension. At high capillary numbers Ca, it was possible to create a continuous jet of the inner aqueous phase over the whole channel distance in the microfluidic chip (Figure 1).

Figure 1. Sketches showing construction details of microfluidic chip for the preparation of monodisperse multiple W1/O/W2 emulsions: (a) jetting mode at the first junction; (b) dripping mode at the second junction. Swelling Kinetics of a Single W1 Droplet Sitting above a Planar O/W2 Interface. 1 mL of aqueous solution with the composition of the W2 phase (Milli-Q water with 2 wt % Tween 20 and 0.1 wt % TMACl) was filled into a 10 mL beaker and covered by 2 mL of MCT with 2 wt % PGPR and 10 wt % SAIB. In doing so, a planar O/W2 interface was generated. A water droplet, corresponding to the inner aqueous phase W1 (Milli-Q water with 1 wt % SDS and 5 wt % TMACl) was injected into the oil phase using a blunt needle (BN2505 from Brico Medical Supplies, Inc., Dayton, NJ). The droplet was injected (i) directly after filling the beaker with the W2 phase and the oil phase or (ii) after 1 h. The size of the droplet was microscopically monitored over time using an inverted Diaphot-TMD microscope (Nikon, Japan) with a high-definition color camera head DS-Fi1. The measurement started (t = 0 min) after the droplet sedimentation as soon as the water droplet was sitting above the O/ W2 interface, which took only a few seconds. Swelling Kinetics of Single Drop-in-Drop W1/O/W2 Emulsions. Single drop-in-drop emulsions were produced using a microfluidics glass chip purchased from Dolomite Centre Ltd. (30 × 15 × 4 mm) with two flow-focusing junctions aligned in series. The

EXPERIMENTAL SECTION

Materials. The oil phase contained either medium chain triglyceride (MCT Delios V from Impag AG, Switzerland), sunflower oil (SFO Florin, Switzerland), or a mixture of both oils, each with 2 wt % polyglycerol polyricinoleate (Grinsted PGPR 90 Kosher from Danisco, Switzerland). Additionally, sucrose acetate isobutyrate (SAIB from Eastman, Switzerland) was added in order to increase the oil density to a constant value of 0.960 g/cm3. In the case where the oil phase had to be enriched with water, it was mixed with water for 6 h under gentle magnetic stirring and centrifuged afterward to remove the excess water. The centrifugal treatment was performed at 20000g (g being the gravity acceleration) for either 1 or 3 h. The residual water concentration in the oil decreased with increasing centrifuge time and was measured by Karl Fischer titration. Both aqueous phases contained Milli-Q water and tetramethylammonium chloride (TMACl from Fluka, Switzerland). The osmotic 5266

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir detailed constitution of the chip and the droplet formation are illustrated in Figure 1 and described in detail elsewhere.23 The combination of one junction with hydrophobic walls and one flow focusing junction with hydrophilic walls was realized by a primary hydrophobic coating at both junctions and a subsequent removing of the coating at the first junction using an alkali treatment. The channel depth was constant equal to 100 μm. The width of the single-phase channels was 105 μm. It widened after the junctions to a width of 300 μm. Liquid flows were generated by three flow controlled syringe pumps (Harvard Pump Apparatus) with Hamilton gastight syringes (Hamilton, Switzerland). The pumps were connected to the chip by polymer tubing (Ercatech AG, Switzerland) with an inner diameter of 250 μm for the oil phase and the W2 phase and an inner diameter of 100 μm for the W1 phase. Upchurch Scientific PEEK Y connectors (Ercatech AG, Switzerland) were used to split the stream of the oil phase and the W2 phase, respectively. Comparable to the above-mentioned method, the swelling kinetics of the single drop-in-drop emulsions was analyzed microscopically by determining the increase in droplet size with time. In order to avoid squeezing of the droplets, microscope slides with a cavity (Marienfeld, Germany) were used. The temperature was controlled using a hot stage with system temperature controller (PE94 Linkam, UK). For each time point the diameters of five inner and outer droplets were measured by help of the program NIS-Elements D3.0. The diameters of the inner droplets (W1) were corrected to eliminate refractive effects derived from the refractive index between the outer W2 and the oil phase. Because of the density difference of 0.04 g/cm3 between the oil phase and the W1 phase, the inner W1 droplets sedimented toward the bottom of the oil droplet. Correspondingly, the correction was made by use of the following equation adapted from Erb et al.39 for the case of sedimented inner droplets using an inverted microscope: ⎡ dm − do( 1 − (dm/do)2 − 1) tan⎢sin−1(dm/do) − ⎣ di = ⎡ −1 n d 1 + tan⎢sin (dm/do) − sin−1 nw2 dm ⎣ o o

(

(

sin−1

n w2 no

dm do

electrolyte concentrations used, at least in the lag stages and the initial part of the osmotically dominated stage (defined later). Because of the fact that in this study the osmotic pressure gradients with up to ΔΠ = 13.3 bar were considerably higher than the Laplace pressure of approximately ΔpL ≈ 0.0001 bar, the contribution of ΔpL to Δp can be neglected, and thus Δp ≈ ΔΠ follows. Considering the interface of the inner W1 droplet Ai as the limiting interface for the osmotic flux, swelling in single dropin-drop emulsions can be described by the following simplified equation, which is based on Fick’s second law. dV ΔΠ * = PA i VW dt RT

with dV the volume change of the inner droplet, t the time, P ∼ sD/δ the permeation coefficient of water in oil, Ai the interfacial area of the inner droplet, ΔΠ the osmotic pressure gradient between the W1 and W2 phase, V*W the molar volume of pure water, D the diffusion coefficient of water in oil, δ the average effective value of oil layer thickness, and s the solubility of water in oil. For an established flux, the water transport rate dV/dt is inversely proportional to the oil layer thickness and proportional to the diffusion coefficient of the transported entities. With time, Ai and δ would change in a way to increase dV/dt, while ΔΠ decreases and thus lowers dV/dt. Figure 2a illustrates the swelling kinetics of the inner droplet (di(0) =59 ± 1 μm) in single drop-in-drop emulsions with varying initial osmotic pressure gradients. Droplet swelling S was defined as normalized volume increase of the inner water droplet with time:

)⎤⎦⎥

)⎤⎥⎦

⎛V ⎞ V (t ) V (0) S = Δ⎜ i ⎟ = i − i A i (t ) A i (0) ⎝ Ai ⎠

(2) where di is the corrected inner diameter, dm the measured inner diameter, do the measured outer diameter, and nw2 and no the refractive indices of the W2 (= 1.336) and the oil phase (= 1.446), respectively.

RESULTS AND DISCUSSION Demonstration of Stagewise Swelling. Water migration in W1/O/W2 emulsions is driven by the pressure gradient between W1 and W2 and leads either to droplet shrinking or swelling. As the osmotic pressure gradient ΔΠ is counterbalanced by the Laplace pressure ΔpL,20 the total pressure gradient Δp can be calculated by (3)

where Πw1 and Πw2 are the osmotic pressures of the W1 and W2 phase, respectively. For calculating the osmotic pressure of either water phase, the contribution of all components, including electrolytes and surfactants, has to be taken into account. Thus, Πw1 and Πw2 can be calculated by n

Π w1,2 =

n

∑ Πk = RT ∑ ikck k=1

k=1

(6)

with Vi(t) and Ai(t) the volume and the interfacial area of the inner droplet at time t and Vi(0) and Ai(0) the volume and the interfacial area at time t = 0, respectively. Thus, S describes the volume change independent of the initial size of the inner droplet. Since the solubility of MCT and SFO in water is very low, a volume change of the inner droplet is caused mainly by water migration. In general, any influence of neighboring droplets on the swelling kinetics, i.e., by steric effects, can be excluded. All samples were diluted in 1 mL of W2 phase. Thus, the droplets were well separated. Moreover, the volume of the W2 phase was much larger than the droplet volume. Consequently, any bursting of single droplets had no significant influence on the total osmotic pressure gradient. Figure 2a shows that the swelling curves can be divided into two characteristic sequential stages. The first stage is the lag stage, in which the water migration is very low. The lag time tlag describes the duration of the lag stage. The second stage is the osmotically dominated stage, characterized by a sudden accelerated water transport. In the beginning of the osmotically dominated stage, the droplet volume increases linearly with time. The slope in this part of the curve is referred to as “swelling rate” in the following. The numbers at the end of the curves give the theoretical equilibrium values of the normalized volume increase S based on eq 4. The calculation was possible under following assumptions: (i) only pure water is transported into the inner droplet W1, (ii) the osmotic pressure gradient is equilibrated when the osmotic pressure of the inner droplet ΠW1 becomes equal to the osmotic pressure of the outer water phase ΠW1, and (iii) neither water, salt, nor surfactant

■

Δp = ΔΠ − ΔpL = Π w1 − Π w2 − ΔpL

(5)

(4)

with R the universal gas constant, T the temperature, i the Van’t Hoff factor assumed to be one for the surfactants and two for TMACl, and c the molar concentration of the respective component. Eq 4 does not differentiate between free surfactant molecules and micelles and is only valid as long as intermolecular interactions can be neglected. Nevertheless, the contribution of the surfactants to the total osmotic pressure gradient is negligible compared to the contribution of the 5267

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir

the osmotic pressure gradients ΔΠ strongly decrease during swelling, following similar characteristic times. The permeation coefficient of water through oil has been determined to be P = 3.0−3.7 μm/s, which is in agreement with values stated in the literature (P = 1−10 μm/s).17,18 Figure 2c (unfilled symbols) shows a linear correlation between the osmotic pressure gradient ΔΠ and the initial swelling rate s = S/t of the osmotically dominated stage. Thus, it can be concluded that ΔΠ is the dominant driving force for the water transport in the early osmotically dominated stage. With time, s strongly decreases as ΔΠ is reduced as the system tends toward osmotic equilibrium. In contrast to the swelling rate, the osmotic pressure gradient ΔΠ had no measurable effect on the lag time tlag (Figure 2c, filled symbols). In conclusion, two sequential stages of water transport were observed, which render any model assuming single mechanisms for the mass transport futile. In particular, clear evidence of a lag stage was provided. The existence of a lag time before a fast, osmotically driven swelling may have two different reasons. It can be caused either by the time the inner water droplet W1 needs to sediment to the bottom of the oil droplets and/or by structural changes of the oil phase, which allow an osmotic exchange between both water phases. Both effects are further explained and evaluated in the following. Interpretation of the Lag Stage. Under the experimental conditions of the single drop-in-drop investigations, it has to be considered that the inner droplet W1 sediments to the bottom of the oil droplet due to the density difference of Δρ = 0.04 g/ cm3 between the two phases. According to Wen and Papadopoulos,29 the water transport mechanism depends on the oil layer thickness. They stated that in the case of a “visible minimum distance” water transport via inverse micelles or spontaneously formed droplets dominates. In contrast to that, in the case of visible contact of the inner water droplet W1 with the continuous water phase W2, they suggested water to be transported by hydrated lipophilic surfactant molecules. Moreover, they observed a much higher water transport rate in the case of visible contact (≈ 6.8 μm/min) compared to the case of a separating oil film (≈ 0.3 μm/min) without further specifying the corresponding length scales. Thus, the drastic change of the slope of the swelling curve illustrated in Figure 2a could be caused by a change of the water transport mechanism due to the W1 droplet sedimentation. The water transport rate w was defined as w=−

Figure 2. Osmotic pressure dependent swelling characteristics of single drop-in-drop W1/O/W2 emulsions. (a) Swelling kinetics of inner droplet with characteristic swelling stages: I = lag stage; II = osmotically dominated stage. Droplet swelling was defined as S = Δ(Vi/Ai). (b) Decrease of osmotic pressure gradient ΔΠ during swelling calculated based on eqs 4 and 5. (c) Lag time (lag stage) and swelling rate s = S/t (osmotically dominated stage) as a function of osmotic pressure gradient. Temperature and initial oil phase viscosity were constant (T = 23 °C; η = 44.4 mPa·s).

ΔVi Δr =− i At t

(7)

with ΔVi the volume change of the W1 droplet, A the surface area, Δri the radius change of the W1 droplet, and t the time. In this study the water transport rates in the osmotically dominated stage calculated by eq 7 did not exceed a value of w = 0.5 μm/min even for a very high osmotic pressure gradient of ΔΠ = 13.3 bar. Thus, they are in the range of the water transport rates of systems with a “visible minimum distance” between the water droplet W1 and the continuous water phase W2 observed by Wen and Papadopoulos.29 The absolute values of the water transport rates in the lag stage were one order of magnitude lower. Differences in the absolute values of the water transport rate by a different sample composition can of course not be excluded. However, considering the inner droplet W1, the water transport in the lag stage (w < 0 μm/min, slight shrinkage) had the opposite direction compared to the water transport in the osmotically dominated stage (w > 0 μm/min,

molecules are transported from the inner W1 toward the outer W2 phase. It is clearly shown that the calculated equilibrium values are in the range of experimentally predictable equilibrium values. Figure 2b illustrates the evolution of the osmotic pressure gradient ΔΠ in the course of swelling, exemplarily shown for initial osmotic pressure gradients of ΔΠ(0) = 2.7, 7.1, and 13.3 bar and calculated either based on eq 4 or 5. For both equations 5268

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir

other. In fact, already in an early state of the measurements the spontaneous formation of tiny W/O emulsion droplets in the oil phase was observed (see Figure 5b), which accumulated close to the large W1 droplet. The number of spontaneously formed tiny water droplets increased with time. On the basis of this, we assume that the spontaneously formed tiny water droplets form a structure across the oil layer in the lag stage, which provokes the contact of both aqueous phases with each other and enables a directed accelerated osmotic exchange in the osmotically dominated stage. This hypothesis is additionally supported by Figure 4 (triangles), which illustrates a lag time reduction Δtlag with

strong swelling). Both water transport mechanisms, which were suggested by Wen and Papadopoulos, are directed equally. Mok and Kim40 developed a first-order estimation for calculating the sedimentation velocity of a sphere inside a concentric spherical liquid container, which can be found in the Supporting Information. Justification is given why a calculation of the sedimentation velocities and thus the theoretical sedimentation times is nonfeasible for the single drop-in-drop emulsions investigated in this work. Nevertheless, in our experiments sedimentation was observed to occur in less than 1 min and thus in a time frame much shorter than the measured lag times of several minutes. Consequently, sedimentation of the inner water droplet W1 and thus an oil film thinning cannot be the main reason for the appearance of the lag stage. The assumption that the appearance of the lag stage is not governed by droplet sedimentation is confirmed by the swelling curves illustrated in Figure 3. In contrast to the single drop-in-

Figure 4. Lag time reduction (lag stage) and swelling rate (osmotically dominated stage) as a function of the initial water content in the oil phase (T = 23°C; η = 44.4 ± 0.2 mPa·s; dashed lines are drawn to guide the eye).

increasing initial water content (t = 0 min) in the oil for single drop-in-drop emulsions with two different initial W1 droplet sizes. The initial water content in the oil was increased by enriching the oil phase with water in a preprocessing step before pumping the oil into the microfluidics chip. It has to be noted that during this preprocessing step water was not completely dissolved in the oil, but very tiny water droplets were formed, comparable to the spontaneously formed water droplets described before. An effect of the initially entrapped water on the oil phase density and viscosity can be neglected as the initial water content was in either case very low. The density difference Δρ was reduced by less than 0.3%. The viscosity had a constant value of η = 44.4 ± 0.2 mPa·s. The lag times were reduced for both initial W1 droplet sizes in a comparable manner. It must be concluded that the structural changes of the oil phase in the lag stage caused by the formation/presence of tiny water droplets strongly facilitate the rapid water transport in the osmotically dominated stage. In contrast to the lag times, the normalized swelling rates remained constant (Figure 4, circles). The fact that they did not depend on the initial water content in the oil shows that the transport elements in the oil layer, i.e., micelles or small droplets, have reached a size distribution that either does not vary anymore or at least not in a way that it affects the water transport rate. Figure 5a compares the swelling behavior of the inner and the outer droplet. The lag stage is characterized by a slight volume decrease of the inner droplet with time (−0.68 × 103 μm3/min), whereas the volume of the outer droplet increases (0.93 × 103 μm3/min). Thus, it must be concluded that in the

Figure 3. Swelling kinetics curve of W1 droplets sitting above the interface between oil and bulk water W2 dependent on the age of the O/W2 interface. The oil phase contained MCT, 2 wt % PGPR, and 10 wt % SAIB. The W1 phase contained 1 wt % SDS and 5 wt % TMACl. The W2 phase contained 2 wt % Tween 20 and 0.1 wt % TMACl.

drop emulsions produced by microfluidics, the inner water droplet W1 was injected into bulk oil, which covered a bulk water phase W2. The droplets had a size of d > 1 mm. Thus, droplet sedimentation was clearly visible. Figure 3 shows that the swelling kinetics of W1 droplets, which sedimented to a freshly generated O/W2 interface, is comparable to the characteristic swelling kinetics of single drop-in-drop emulsions illustrated in Figure 2a. The droplet size decreased slightly in the beginning, before it strongly increased after a certain lag time. In contrast to this, the initial lag stage did not appear in the swelling kinetics of W1 droplets, which sedimented to an O/W2 interface of an age of 60 min. The droplets directly started to swell. Thus, with time the oil phase properties have to change in a way eliminating the requirement of the lag stage. The water transport across very thin liquid layers, such as surfactant bilayers as present in droplets with visible contact, has shown to be linked to the bilayer fluidity.41 However, the above results indicate that although the W1 droplets sink to the O/W2 interface, i.e., the bottom of the oil droplet in our systems, there is still an oil layer of a certain thickness left, which establishes a border between both aqueous phases. The osmotic pressure gradient between the W1 and W2 phase can only act if both aqueous phases come into contact with each 5269

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir

Correlation of Spontaneous Emulsification and the Osmotically Induced Water Transport. In general, water might migrate either as single water molecules,21 entrapped in inverse micelles,2,24−27 or water droplets.29−31 With respect to the short experimental time frame observed here, water migrates more likely in the form of inverse micelles or spontaneously formed tiny droplets than by diffusion of single water molecules as it is weakly soluble in oil (saturation solubility at 20 °C in MCT = 0.22 wt % and in SFO = 0.135 wt % measured by Karl Fischer titration, values comparable with Hilder43). This is also supported by the observation that the lag time clearly depends on the surfactant concentrations, an effect which will be further addressed in a future paper. The entrapment of water in inverse micelles and droplets can be considered as increasing water solubility in the oil phase.28 Figure 5b,c reveals the capability of the oil phase to spontaneously entrap water in the presence of PGPR. Figure 5b shows the time-dependent formation of tiny water droplets in the oil phase close to a planar water−MCT interface. Figure 5c shows details of the microscopic images of Figure 5a, which confirm the same effect of a spontaneous water droplet formation in the oil phase in single drop-in-drop emulsions. A comparable behavior has also been observed for other oil soluble surfactants, such as sorbitan monooleate (Span 80)28−31 or amphiphilic block copolymers.44 It has to be noted that for the spontaneous W/O emulsification the presence of PGPR in the oil phase was required, whereas there was no need of a water-soluble surfactant in the aqueous phase. The spontaneous droplet formation occurred without any external energy input such as stirring or shearing. Thus, the spontaneous formation of water droplets in the oil phase stabilized by PGPR is comparable to the formation of a microemulsion. Nevertheless, it has to be noted that in contrast to microemulsions, the spontaneously formed W/O emulsions observed in this study were only kinetically stable but not thermodynamically. The droplet number and size increased with time. During spontaneous emulsification the total interfacial area is expanded, resulting in an increase of the total free energy, which seems to contradict basic thermodynamic principles.45 A notional negative interfacial tension of a hypothetical planar interface could be the reason for such spontaneous expansion of the interface.46−50 Although very low interfacial tensions (γ < 1 mN/m) were obtained with 2 wt % PGPR in combination with 2 wt % Tween20 and 1 wt % SDS, this explanation is oversimplified. It ignores other causes for destabilizing the interface at low but positive interfacial tensions such as entropy contributions,51−53 which would needed to be quantified in bulk and at the interfaces. Other authors propose spontaneous emulsification to be an out-of-equilibrium phenomenon caused by interfacial turbulences or diffusion and stranding of a cosolvent, which is oil- and water-soluble.54 In the case of amphiphilic block copolymers, the spontaneous formation of water droplets in oil is suggested to be an impurity effect, i.e., caused by residual salt species originating from the polymer synthesis.44 The salt is assumed to provide an osmotic driving force for the formation and growth of water droplets in oil. However, due to the high reproducibility of our results under various conditions, we can conclude that impurities play only a minor role during the spontaneous emulsification. Nevertheless, further work is needed to provide a concrete mechanistic explanation of the water-transport structure. In summary, evidence is provided that there is a correlation between the spontaneous formation of tiny W/O emulsion

Figure 5. Water migration into the oil phase. (a) Typical swelling kinetics of inner and outer droplet in the lag stage (I) and the osmotically dominated stage (II). The oil phase contained 41 wt % MCT, 41 wt % SFO, 16 wt % SAIB, and 2 wt % PGPR. The microscopic images show the droplets at 0, 10, 15, and 25 min (from the left to the right). Temperature and initial oil phase viscosity were constant (T = 23 °C; η = 69.8 mPa·s. (b) Spontaneous timedependent formation of water droplets in MCT with 2 wt % PGPR close to the planar water−oil interface. The microscopic images were taken after 30, 60, 90, and 120 min (from the left to the right) (scale bar = 100 μm). (c) Details of microscopic images of (a) (in the same order) showing the spontaneous formation of water droplets in the oil phase (scale bar = 10 μm).

lag stage water migrates into the oil from direction of both aqueous phases. Moreover, the water migration from direction of the W2 phase is faster than from the direction of the W1 phase. Depending on the surfactant and salt concentrations, migration from both sides may occur at different rates.42 Thus, at different osmotic pressure gradients, the total water uptake into the oil varies slightly during the lag stage, but we only access the total effective rate in the present experiments. The fact that in the initial linear part of the osmotically dominated stage the swelling rate of the outer droplet is larger than of the inner droplet (Figure 5a) indicates that the oil phase accumulates water. In conclusion, the lag stage can be described by a combined effect of W1 droplet sedimentation and structural changes of the oil phase caused by the spontaneous formation of tiny water droplets across the oil layer. Because of the facts that (i) the droplet sedimentation is expected to occur within a very short time and (ii) the lag stage appeared also for already sedimented droplets, the major contribution to the lag stage requirement is imputed from the spontaneous W/O emulsification. 5270

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir

such as for instance the addition of thickeners or structure forming agents.

droplets and the appearance of a lag stage. We assume that the structure formation across the oil layer during the lag stage caused by water entrapped in reverse micelles or spontaneously formed water droplets allows a contact of both water phases and thus strongly facilitates the osmotically induced water transport. Consequently, the spontaneous emulsification determines the duration of the lag stage and is a precondition for the subsequent osmotically dominated stage. Variation of Kinetic Transport Parameters. Figure 6 illustrates the lag time tlag and the swelling rate s as a function of

■

CONCLUSIONS

This study provides a robust methodology to validate and, for the first time, quantify the mechanisms, which contribute to the water transport through the oil phase of osmotically imbalanced multiple W1/O/W2 emulsions. The production of well-defined monodisperse single drop-in-drop emulsions by microfluidics permits a very precise and comprehensive study of the swelling kinetics of osmotically imbalanced W1/O/W2 emulsions. In addition, it allows not only investigations of the long-term evolution, in common for many previous release studies, but also early stage investigations. The actual swelling, which is caused by the osmotic pressure difference between W1 and W2, started after a certain lag time. We showed that a complete description and thus control of the water transport are only possible by mechanistically differentiating between the lag stage and the osmotically dominated stage. We provided evidence that an experimental decoupling of the transport mechanisms in these stages is needed to determine their nature and related parameters. The lag time was precisely controlled by the oil phase viscosity and the temperature, while the osmotic pressure difference had no significant influence on the duration of the lag time. Thus, the osmotic imbalance drives the water transport in W1/O/W2 emulsions, but it does not initiate it as widely assumed. In fact, the osmotic pressure difference can only act if W1 and W2 are in full thermodynamic contact. This study provided evidence of structural changes of the oil layer during the lag stage by migration of water into the oil phase without the prerequisite of an osmotic pressure gradient. Together with an oil film thinning caused by sedimentation of the inner W1 droplet toward the bottom of the oil droplet, these structural changes were responsible for the duration of the lag stage. In this context the entrapment of water in reverse micelles formed by PGPR and the spontaneous formation of W/O emulsion droplets were discussed. The spontaneous entrapment of water can be considered as increasing water permeability through the oil film. Although we cannot exclude one of the other water transport mechanisms suggested in the literature, such as molecular water transport, our results show clearly that spontaneous W/O emulsification has a primary impact on the water transport rate through the oil layer. Most important is that the initial lag stage of swelling sets the structures within the oil layer, which facilitate the water transport between W1 and W2 and determine the swelling rate. The properties of these structures were not completely investigated here. On the basis of the present results, we assume the formation of a “water transport structure” involving PGPR and the spontaneously formed W/O droplets. Further work for providing evidence for this assumption is needed, i.e., by using appropriate scattering techniques. As a final remark, although our work concentrated on the water transport into the inner droplets, the conclusions can be transferred to the opposite direction as a stagewise water mass transfer was also observed in release kinetics.2 Thus, the findings of this study provide a promising approach for precisely controlling or inhibiting mass uptake and release in multiple W1/O/W2 emulsions without changing the concentration of entrapped active components.

Figure 6. Dependence of the lag time (lag stage) and the swelling rate (osmotically dominated stage) in single drop-in-drop W1/O/W2 emulsions on the oil phase viscosity. The viscosity varied by using different oil mixtures at T = 23 °C (rhombi) or by applying different temperatures to emulsions with MCT in the oil phase (triangles). The droplets had constant initial droplet sizes (ri = 32 μm; ro = 44 μm). k = 19; c = 0.12.

the oil phase viscosity. First, the oil phase viscosity was varied using different mixing ratios of MCT and SFO with SAIB and PGPR constant at 2 wt % (rhombi). Second, different oil phase viscosities were obtained investigating emulsions with MCT, SAIB, and 2 wt % PGPR at different temperatures (triangles). For the different MCT:SFO mixtures, tlag increased proportionally with increasing viscosity, whereas the swelling rate decreased. The dependence of the swelling rate on the viscosity was less pronounced at higher viscous oil mixtures. Assuming validity of the Stokes−Einstein equation, the water migration rate should be inversely proportional to the viscosity. In this study this was only the case for low viscosities η < 80 mPa·s. With increasing oil phase viscosity the deviation from the reciprocal curve k1/η + c increased slightly. Diffusivity of water in complex systems is not necessarily inversely proportional to the viscosity. With increasing temperature, tlag decreased linearly and the swelling rate increased. The trends of both curves match the slopes of the viscosity curves using oil mixtures at constant temperature. Consequently, the effect of the temperature on the decrease in lag time and increase in swelling kinetics can be explained by the accompanied viscosity reduction. In summary, the lag time is strongly determined by the oil phase viscosity. Thus, the start of the osmotically driven accelerated water transport in the osmotically dominated stage can be triggered by targeted adaptations of the oil composition 5271

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir

■

(15) Van der Graaf, S.; Schroën, C. G. P. H.; Boom, R. M. Preparation of double emulsions by membrane emulsificationa review. J. Membr. Sci. 2005, 251, 7−15. (16) Schuch, A.; Deiters, P.; Henne, J.; Köhler, K.; Schuchmann, H. P. Production of W/O/W (water-in-oil-in-water) multiple emulsions: droplet breakup and release of water. J. Colloid Interface Sci. 2013, 402, 157−164. (17) Matsumoto, S.; Kohda, M. The viscosity of W/O/W emulsions: An attempt to estimate the water permeation coefficient of the oil layer from the viscosity changes in diluted systems on aging under osmotic pressure gradients. J. Colloid Interface Sci. 1980, 73, 13. (18) Matsumoto, S.; Inoue, T.; Kohda, M.; Ikura, K. Water permeability of oil layers in W/O/W emulsions under osmotic pressure gradients. J. Colloid Interface Sci. 1980, 77, 555. (19) Yan, J.; Pal, R. Osmotic swelling behavior of globules of W/O/ W emulsion liquid membranes. J. Membr. Sci. 2001, 190, 79−91. (20) Jiao, J.; Rhodes, D. G.; Burgess, D. J. Multiple emulsion stability: Pressure balance and interfacial film strength. J. Colloid Interface Sci. 2002, 250 (2), 444−450. (21) Mezzenga, R.; Folmer, R.; Hughes, E. Design of double emulsions by osmotic pressure tailoring. Langmuir 2004, 20 (9), 3574−3582. (22) Florence, A. T.; Whitehill, D. Some features of breakdown in water-in-oil-in-water multiple emulsions. J. Colloid Interface Sci. 1981, 79 (1), 243−256. (23) Hughes, E.; Maan, A. A.; Acquistapace, S.; et al. Microfluidic preparation and self diffusion PFG-NMR analysis of monodisperse water-in-oil-in-water double emulsions. J. Colloid Interface Sci. 2013, 389 (1), 147−156. (24) Kita, Y.; Matsumoto, S.; Yonezawa, D. Permeation of water through oil layer in W-O-W-type multiple-phase emulsions. Nippon Kagaku Kaishi 1978, 1, 11−14. (25) Garti, N.; Magdassi, S.; Whitehill, D. Transfer phenomena across the oil phase in water-oil-water multiple emulsions evaluated by Coulter counter: 1. Effect of emulsifier 1 on water permeability. J. Colloid Interface Sci. 1985, 104 (2), 587−591. (26) Cheng, J. S.; Chen, J. F.; Zhao, M.; Luo, Q.; Wen, L. X. Transport of ions through the oil phase of W1/O/W2 double emulsions. J. Colloid Interface Sci. 2007, 305 (1), 175−182. (27) Wen, L. X.; Papadopoulos, K. D. Effects of osmotic pressure on water transport in W1/O/W2 emulsions. J. Colloid Interface Sci. 2001, 235 (2), 398−404. (28) Koroleva, M. Y.; Yurtov, E. V. Water mass transfer in W/O emulsions. J. Colloid Interface Sci. 2006, 297 (2), 778−784. (29) Wen, L. X.; Papadopoulos, K. D. Visualization of water transport in W1/O/W2 emulsions. Colloids Surf., A 2000, 174 (1−2), 159−167. (30) Wen, L. X.; Papadopoulos, K. D. Effects of surfactants on water transport in W1/O/W2 emulsions. Langmuir 2000, 16 (29), 7612− 7617. (31) Yan, J.; Pal, R. Effects of aqueous-phase acidity and salinity on isotonic swelling of W/O/W emulsion liquid membranes under agitation conditions. J. Membr. Sci. 2004, 244 (1−2), 193−203. (32) Colinart, P.; Delepine, S.; Trouve, G.; Renon, H. Water transfer in emulsified liquid membrane processes. J. Membr. Sci. 1984, 20, 167−187. (33) Datta, S. S.; Abbaspourrad, A.; Amstad, E.; Fan, J.; Kim, S.; Romanowsky, M.; Shum, H. C.; Utada, A. S.; Windbergs, M.; Zhou, S.; Weitz, D. A. Double emulsion templated solid microcapsules: Mechanics and controlled release. Adv. Mater. 2014, 26, 1−14. (34) Okushima, S.; Nisisako, T.; Torii, T.; Higuchi, T. Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices. Langmuir 2004, 20, 9905−9908. (35) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Droplet microfluidics. Lab Chip 2008, 8, 198−220. (36) Dendukuri, D.; Doyle, P. S. The synthesis and assembly of polymeric microparticles using microfluidics. Adv. Mater. 2009, 21, 1− 16. (37) Zhang, H.; Tumarkin, E.; Peerani, R.; Nie, Z.; Sullan, R. M. A.; Walker, G. C.; Kumacheva, E. Microfluidic production of biopolymer

ASSOCIATED CONTENT

S Supporting Information *

(I) Comment on theoretical expression of droplet sedimentation in a sphere with immobile boundaries; (II) Swelling kinetics of multiple drop-in-drop W/O/W emulsions prepared by microfluidics. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.5b01138.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (J.B.). *E-mail [email protected] (D.Z.G.). Present Address

E.H.: Department of Chemistry, Durham University, South Road, Durham, United Kingdom. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support provided by the Nestlé Research Center Lausanne. REFERENCES

(1) Pawlik, A.; Cox, P. W.; Norton, I. T. Food grade duplex emulsions designed and stabilised with different osmotic pressures. J. Colloid Interface Sci. 2010, 352, 59−67. (2) 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. (3) Ficheux, M. F.; Bonakdar, L.; Leal-Calderon, F. L.; Bibette, J. Some stability criteria for double emulsions. Langmuir 1998, 14, 2702−2706. (4) Marti-Mestres, G.; Nielloud, F. Emulsions in health care applications − An overview. J. Dispersion Sci. Technol. 2002, 23 (1− 3), 419−439. (5) Florence, A. T.; Whitehill, D. The formulation and stability of multiple emulsions. Int. J. Pharm. 1982, 11 (4), 277−308. (6) Florence, A. T.; Jenkins, A. W.; Loveless, A. H. Effect of formulation of intramuscular injections of phenothiazines on duration of activity. J. Pharm. Sci. 1976, 65 (11), 1665−1668. (7) Benoy, C. J.; Schneide, R.; Elson, L. A. Multiple emulsions, a suitable vehicle to provide sustained release of cancer chemotherapeutic agents. Br. J. Pharmacol. 1972, 45 (1), 135−136. (8) Frankenfeld, J. W.; Fuller, G. C.; Rhodes, C. T. Potential use of liquid membranes for emergency treatment of drug overdose. Drug Dev. Commun. 1976, 2, 405. (9) Tadros, T. F. Future developments in cosmetic formulations. Int. J. Cosmet. Sci. 1992, 14 (3), 93−111. (10) Matsumoto, S.; Kita, Y.; Yonesawa, D. An attempt at preparing water-in-oil-in-water multiple-phase emulsions. J. Colloid Interface Sci. 1976, 57, 353−361. (11) Windbergs, M.; Zhao, Y.; Heyman, J.; Weitz, D. A. Biodegradable core-shell carriers for simultaneous encapsulation of synergistic actives. J. Am. Chem. Soc. 2013, 135, 7933−7937. (12) Gunes, D. Z.; Clain, X.; Breton, O.; Mayor, G.; Burbidge, A. S. Avalanches of coalescence events and local extensional flows − Stabilisation or destabilisation due to surfactant. J. Colloid Interface Sci. 2010, 343 (1), 79−86. (13) Gunes, D. Z.; Bercy, M.; Watzke, B.; Breton, O.; Burbidge, A. S. A study of extensional flow induced coalescence in microfluidic geometries with lateral channels. Soft Matter 2013, 9 (31), 7526−7537. (14) Vladisavljevic, G. T.; Williams, R. A. Recent developments in manufacturing emulsions and particulate products using membranes. Adv. Colloid Interface Sci. 2005, 113, 1−20. 5272

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273

Article

Langmuir microcapsules with controlled morphology. J. Am. Chem. Soc. 2006, 128, 12205−12210. (38) Shah, R. K.; Shum, H. C.; Rowat, A. C.; Lee, D.; Agresti, J. J.; Utada, A. S.; Chu, L. Y.; Kim, J. W.; Fernandez-Nieves, A.; Martinez, C. J.; Weitz, D. A. Designer emulsions using microfluidics. Mater. Today 2008, 11 (4), 18−27. (39) Erb, R. M.; Obrist, D.; Chen, P. W.; Studer, J.; Studart, A. R. Supplementary material: Predicting sizes of droplets made by microfluidic flow-induced dripping. Soft Matter 2011, 7, 1−10. (40) Mok, L. S.; Kim, K. Motion of a gas bubble inside a spherical liquid container with a vertical temperature gradient. J. Fluid Mech. 1987, 176, 521−531. (41) Thiam, A. R.; Bremond, N.; Bibette, J. From stability to permeability of adhesive emulsion bilayers. Langmuir 2012, 28, 6291− 6298. (42) Ruckenstein, E.; Chi, J. C. Stability of microemulsions. J. Chem. Soc., Faraday Trans. 2 1975, 71, 1690−1707. (43) Hilder, M. H. The solubility of water in edible oils and fats. J. Am. Oil Chem. Soc. 1968, 45, 703−707. (44) Bae, J.; Russell, T. P.; Hayward, R. C. Osmotically driven formation of double emulsions stabilized by amphiphilic block copolymers. Angew. Chem., Int. Ed. 2014, 53, 8240−8245. (45) Zhu, J.; Hayward, R. C. Spontaneous generation of amphiphilic block copolymer micelles with multiple morphologies through interfacial instabilities. J. Am. Chem. Soc. 2008, 130, 7496−7502. (46) Okazawa, T.; Bron, J. On thermodynamically stable emulsions. J. Colloid Interface Sci. 1979, 69, 86−96. (47) Schulman, J. H.; Stoeckenius, W.; Prince, L. M. Mechanism of formation and structure of micro emulsions by electron microscopy. J. Phys. Chem. 1959, 63 (10), 1677−1680. (48) Shah, D. O.; Tamjeedi, A.; Falco, J. W.; Walker, R. D. Interfacial instability and spontaneous formation of microemulsions. AIChE J. 1972, 18, 1116. (49) Prince, L. M. A theory of aqueous emulsions I. Negative interfacial tension at the oil/water interface. J. Colloid Interface Sci. 1967, 23, 165. (50) Granek, R.; Ball, R. C.; Cates, M. E. Dynamics of spontaneous emulsification. J. Phys. II 1993, 3 (6), 829−849. (51) Miller, C. A.; Neogi, P. Thermodynamics of microemulsions: Combined effects of dispersion entropy of drops and bending energy of surfactant films. AIChE J. 1980, 26, 212−220. (52) Aoki, K. Size-distribution of droplets in emulsions by statistical mechanics calculation. J. Colloid Interface Sci. 2011, 360, 256−261. (53) Miller, C. A.; Scriven, L. E. Interfacial instability due to electrical forces in double layers. 1. General considerations. J. Colloid Interface Sci. 1970, 33, 360−371. (54) Leal-Calderon, F.; Schmitt, V.; Bibette, J. Emulsion Science: Basic Principles; Springer: Berlin, 2007.

5273

DOI: 10.1021/acs.langmuir.5b01138 Langmuir 2015, 31, 5265−5273