Recent advances in multiple emulsions and their application as templates

Current Opinion in Colloid & Interface Science 25 (2016) 98–108

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Recent advances in multiple emulsions and their application as templates Bruno F.B. Silva a, Carlos Rodríguez-Abreu a,⁎, Neus Vilanova b a b

International Iberian Nanotechnology Laboratory (INL)., Av. Mestre José Veiga s/n., Braga 4715-330, Portugal Technische Universiteit Eindhoven, Institute for Complex Molecular Systems, Department of Chemical Engineering and Chemistry, P.O. Box, 513 5600 MB Eindhoven, The Netherlands

a r t i c l e

i n f o

Article history: Received 8 March 2016 Received in revised form 7 July 2016 Accepted 8 July 2016 Available online 16 August 2016 Keywords: Multiple emulsions Microfluidics Formulation Encapsulation Porous particles

a b s t r a c t We review recent developments on the preparation of multiple emulsions and their applications as templates for material fabrication. Emphasis is placed on microfluidic methods for accurate control of size and morphology and on new formulations that go beyond traditional surfactant systems for increased complexity. The straightforward applicability of multiple emulsions in the fabrication of multihollow particles or capsules of various materials is illustrated through representative examples. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Multiple emulsions are soft materials formed by dispersed droplets (called outer droplets herein) that contain in turn smaller droplets inside (called inner droplets herein). Their particular features as multicompartmental units with confined spaces in a continuum offer a wide range of possibilities for different applications. Following the conventional nomenclature for normal emulsions, multiple or double emulsions can be classified into water-in-oil-inwater (W/O/W) or oil-in-water-in-oil (O/W/O) depending on the phase sequence at different scales; in such a nomenclature, “water” actually means any aqueous or polar phase (even e.g. an ionic liquid [1]) whereas oil stands for any hydrophobic, i.e. water-insoluble phase. Such a phase sequence not only implies the necessity of selective stabilization of droplets of the different phases but also finding a proper method to “emulsify emulsions”, namely, to produce small droplets inside bigger ones. The two traditional and well known bulk methods to form multiple emulsions are the two-step method and the phase inversion method. These methods usually render polydisperse droplets. In the two-step method, briefly a simple W/O emulsion is produced first using a hydrophobic surfactant. Then this emulsion is poured into an aqueous phase containing a hydrophilic surfactant that helps to emulsify the initial emulsion so that a W/O/W multiple emulsion is obtained. Alternatively, an O/W/O emulsion can be produced if one starts with an O/W emulsion stabilized with a hydrophilic surfactant that is later emulsified in an oil phase containing a hydrophobic surfactant.

⁎ Corresponding author. E-mail address: [email protected] (C. Rodríguez-Abreu).

http://dx.doi.org/10.1016/j.cocis.2016.07.006 1359-0294/© 2016 Elsevier Ltd. All rights reserved.

The formation of multiple emulsions by phase inversion is driven by changes in the interfacial curvature and in the hydrophile–lipophile balance of the surfactant. Phase inversion can be transitional, as occurs at the phase inversion temperature (PIT), or catastrophic, as a result of a change in the volume fraction of one of the phases. Readers are referred to the abundant literature on the subject for in-depth description of these phenomena. Multiple emulsions have potential applications in encapsulation, template-assisted synthesis of materials or separation. In practice, however, their use in commercial applications remains challenging due to a lack of simple methods to create stable and reproducible multiple emulsions. Hence, studies on new emulsifiers or other stabilization strategies as well as on methods that combine high throughput and size and morphology control are continuously emerging. Herewith, we review recent advances on the preparation of multiple emulsions, particularly focusing on droplet microfluidics and new formulations, as well as the application of these multiple emulsions as templates for the fabrication of porous particles and capsules. 2. Double and multiple emulsion production by microfluidics 2.1. Microfluidics for droplet generation Microfluidics involve the flow and manipulation of fluids in channels with typical dimensions of tens to hundreds of micrometers [2•]. At such small length scales, the Reynolds number (Re) becomes small and inertial forces practically negligible. This leads to predictable, reproducible and controllable flow regimes that can be used with great advantage to manipulate materials for practical applications or studying natural phenomena. When two immiscible flowing fluids merge in a

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microchannel, the balance between the viscous forces (induced by the continuous phase) and the capillary forces (resulting from the interfacial tension between the two liquids) leads to different flow regimes or breakup modes [3••,4•], with the most common being dripping and jetting [5]. In the dripping regime, highly monodisperse droplets (coefficient of variation ~1–5%) of tunable size form right at the mixing point, while in the jetting regime, prevalence of viscous drag leads to the formation of a jet, which typically breaks into less monodisperse droplets further downstream due to the Rayleigh-Plateau instability [2•]. The prevalence of each mode can be easily controlled by means of adjusting the flow rates. For droplet production, many junction designs and combinations exist, but overall they can be grouped into three main types [4•,6]: Tjunctions [7], co-flowing [8••], and hydrodynamic flow-focusing [3••,9] – Fig. 1 (consult for instance Ref. [10•] for a more detailed review on different device designs). In a T-junction, fluids typically meet at an angle of 90°, and droplet formation arises from the pressure drop in the continuous phase and squeezing of the dispersible phase [11]. In a coflowing device the fluids meet in parallel streams, whereas in the flow-focusing design there is a geometric element that focuses/ squeezes the inner fluid thread [4•]. In both co-flowing and flowfocusing geometries, droplets of the dispersed phase typically form through the above-mentioned dripping or jetting modes [5]. Tjunctions are typically 2D devices, co-flow set-ups are made of 3D microcapillaries, while flow-focusing is available in both 2D and 3D geometries. 2D devices can be fabricated in glass by etching and in polymeric materials by soft lithography (e.g. poly(dimethylsiloxane) – PDMS, and poly(urethane)), thermal embossing and injection molding among other methods [10•]. Simple 3D devices are typically assembled out of glass capillaries but nowadays 3D devices can also be fabricated with polymeric materials, which allow not only mimicking the coaxial structure of capillaries [12•], but also the fabrication of complex 3D structures and networks [13,14•]. Soft lithography fabrication methods, e.g. for making 2D or 3D PDMS devices, are especially attractive due to the potential to include a high number of junction modules (e.g. T-junctions, flow-focusing, functions for sorting droplets, etc. [15,16]), ideal to achieve more complex assembly tasks such as a controlled production of multiple emulsions, or for parallelization of several identical processes for high-throughput. In addition, the possibility to design true 3D devices allows for an enhanced control and potentiates the use of additional functionalities on-chip. Nevertheless, the most typical co-flowing devices are still made of glass and are ideal also for one-step emulsification of multiple

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emulsions [17]. Like PDMS devices, they also allow cascading of multiple junctions to build ever-growing complex multi-emulsions, but one drawback is the required expertise to assemble such devices out of glass capillaries, and the difficulty in parallelization for a higher production rate of droplets. Recently, the possibility to use 3D-printers to fabricate polymeric devices with 3D features has also gained much deserved attention [14•]. These fabrication methodologies are still somewhat limited in the resolution that they offer, but they allow the facile design of very intricate 3D structures and networks that are fabricated in a single step, without the need of cleanroom facilities and fabrication expertise. As the technology is successfully reaching better and better resolution capabilities, 3D-printing device fabrication will allow a facile, accessible, and affordable way to make complex microfluidic devices. Another important parameter for droplet production in microfluidics is the wettability. Typically, the dispersed phase (i.e. droplets) should not wet the device walls [18]. This is challenging in the case of multiple emulsions since some different sections of the same device should wet the middle and outer phases which have different hydrophobicities. In glass microcapillary devices, the problem is avoided due to the 3D structure of the channels which help to engulf the inner and middle phases. Fortunately, fabrication of PDMS on-chip devices with channels of different depths (so that they become closer to the 3D approach) is now straightforward. Such channels help preventing the droplets from touching the walls, and under such conditions, wetting of the channels becomes less problematic [12•]. Also, now, functionalization of different sections of a polymeric device with different wetting properties is easily achieved, which renders them extremely versatile for most multiple emulsion fabrication applications [19–22]. 2.2. Device geometry and experimental considerations for the production of double and multiple emulsions The production of double and multiple emulsions through microfluidic manipulation can be achieved through two main approaches: (1) by cascading further droplet generator modules downstream, or (2) by variations of the modules design so that multiple emulsions are produced at a single step. In the first approach, by placing two T-junctions in series in a 2D glass device, double emulsions of W/O/W and O/W/O can be produced [23••] (Fig. 1E). Droplets of uniform size of the inner phase form reproducibly in the middle phase at the first T-junction and this resulting stream then breaks into droplets at the second junction, forming

Fig. 1. Typical microfluidic geometries and droplet formation modes (dripping and jetting). (A) T-junction [7]. (B) Co-flow [5]. (C) Flow-focusing through a constriction [9]. (D) Flowfocusing in a cross-junction [3••]. (E) Two T-junctions in series for double emulsion production in two steps [23••]. (F) Flow-focusing geometry for double emulsion production in one step [24••]. (G) Four flow-focusing cross-junctions in series for the production of quadruple emulsions [20•]. (H) Microcapillary coaxial flow-focusing device combining co-flow with flow-focusing for the production of double emulsions in a single step either in the dripping (I) or jetting (J) regimes [8••]. (I) Double emulsion droplet production in the dripping mode. (J) Coaxial jet before pinch off. Inset on the bottom: Double emulsion droplet pinch off from the coaxial jet. Panels (H-J) from Ref. [8••]. Reprinted with permission from AAAS http:// dx.doi.org/10.1126/science.1109164.

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droplets of the middle phase (which in turn contain the inner phase) in the continuous phase. The internal and external droplet size and number of encased droplets can be accurately controlled by manipulating the flow rates of the 3 phases, whereas two droplets of the inner phase can be incorporated within a single middle phase droplet by replacing the first T-junction with a cross-shaped module. Later, similar devices were also manufactured in PDMS with similar results [19]. For the second approach, double emulsions were obtained using a 2D flow-focusing device in poly(urethane), designed to form a coaxial jet of the inner and middle phases embedded in an outer fluid [24••] (Fig. 1F). Just by manipulation of the relative and absolute flow rates, the double jet would break into droplets in such a way that it allowed control over the inner and outer droplets' size (hence, middle phase thickness). Importantly, by adjusting the flow rates of the three fluids, jet breakup of the inner and middle fluids can be adjusted to incorporate a controlled number of inner droplets inside the outer one. Similar results were also achieved in coaxial flow-focusing glass microcapillary devices, which combine the co-flow and the flow-focusing geometries [8••] (Fig. 1H). The setup consists of two tapered cylindrical capillaries, aligned in the same axis and embedded within a square capillary. The inner fluid is injected through the injection capillary, whereas the middle fluid is injected through the interstices between the injection (cylindrical) and squared capillaries. This forms a coaxial flow at the tip of the tapered capillary. The outer fluid flows from the opposite side between the interstices of the collection and squared capillary, and focuses the coaxial stream of the inner and middle fluid into the collection capillary. Double emulsion droplets could be formed, with both inner and middle fluids breaking into droplets either in the dripping or jetting regime, depending on the individual fluids' flow rates [8••] (Fig. 1I–J). This configuration allowed not only control over the outer and inner droplets (hence the shell thickness), but also on the number of inner droplets. Interestingly, thin shells (~3% of the total droplet volume) could be produced, which allowed the formation of polymersomes, liposomes, polymer shells, and an array of other interesting materials [17,25]. Using combinations of these concepts, emulsions can grow in complexity either by cascading more junctions downstream of the channel [20•,26,27•] (first approach), or varying the design in order to allow the formation of more complex emulsions in a one-step fashion [28•, 29•] (second approach). Within the first strategy, by combining two or three coaxial co-flow junctions, monodisperse double or triple emulsions are formed in two or three steps respectively [26]. The separation of the multiple emulsification steps allows enhanced control over each, by allowing manipulation of the individual inner, middle and outer flow rates. However, the fact that the outer fluid at one junction becomes the middle fluid at the following junction, still brings some constraints into full control of the designer emulsion. An improvement for this strategy consists in incorporating two new modules to the previous design: a connector and a liquid extractor [27•] (Fig. 2A). The connector allows the combination of two coaxial co-flow droplet generation devices in such a way that two different droplets join in the same stream, flowing alongside and being co-encapsulated into an outer droplet further downstream. The connector also allows addition of more fluid to increase the spacing between the two droplet types, whilst the liquid extractor allows the removal of excess liquid to bring the droplets closer. These two new building blocks permit very different combinations to produce highly hierarchical multiple emulsions with tailored designs such as sextuple-component triple emulsions. The fabrication of these glass devices, including several aligned tapered microcapillaries, is difficult and labor intensive. In order to avoid the drawbacks associated with fabrication, the same concept could also be translated to PDMS devices, whose wettability can be manipulated by UV radiation through a patterned mask that allows only the regions of interest to be exposed [20•]. The shape of the junctions is adjusted for a triggered drop formation (each nozzle is slightly narrower than the incoming emulsion) in order to synchronize the

formation of drops and allow the production of triple, quadruple and quintuple emulsions. The control over the size and monodispersity of the individual droplets was excellent, but with the geometry in question it was not reported the incorporation of more than one droplet within the same hierarchical level as in the preceding example. As the continuous addition of further junctions downstream makes the device fabrication very difficult in glass microcapillary-based devices and fluid operation increasingly more complex, the introduction of variations on the droplet generation modules was also brought as an alternative. Hence, a variation of the combined coaxial flowfocusing setup introduced before, but now with a clever modification of the capillaries affinity for the different phases was proposed. This brings the ability to produce triple and quadruple emulsions in a single step [28•]. Now, instead of flowing a single fluid through the interstices of the square and collection/injection capillaries, two immiscible fluids are flowed together from each side (Fig. 2B). These fluids are stabilized against breakup by the confinement within the interstices, and by modification of the wettabilities of the square and circular capillaries. Hence, a flow scheme can be produced to generate monodisperse quadruple W/O/W/O/W and O/W/O/W/O emulsions. Droplet formation is achieved in the dripping and jetting regime and determined by the flow rates of the individual fluids. In the dripping regime, monodisperse highly regular “onion”-type structures are produced, whereas the jetting regime allows incorporation of several inner droplets in the core. Alternatively, by replacing the tapered inner fluid capillary of a coaxial flow-focusing setup with a dual, triple and even quadruple bore injection capillary, one-step production of double emulsion with multiple different inner droplets can be achieved [29•] (Fig. 2C). Importantly, the ability to independently change the flow rate and dripping to jetting regime of both the outer droplet and each of the individual inner droplets, allows the production of a wide range of interesting double emulsions. An application was demonstrated for the triggered coalescence of inner droplets, but applications for exquisite control of the structure of porous polymer particles could also be foreseen. In a slight modification of the device, by introducing smaller capillaries inside a two- and four-bore injection capillary to produce segmented flows of oil and water, and by hydrophobizing the inner surfaces of the injection capillary, very interesting nonspherical double emulsions with multiple distinct water cores enveloped by very thin shells could be produced [30]. One third approach to produce multiple emulsions using microfluidic methods is to revert to a single junction where a complex mixture specifically tailored for the application is emulsified in a suitable outer fluid [31•–36••]. As the emulsion forms, phase separation within the droplets occurs, leading to the formation of multiple emulsions (see more details in section 3.5). The role of the microfluidic device here is more to control the size and monodispersity of the outer droplets, while providing conditions for controlled mixing and diffusion (i.e. mass transport) of the different components in and out of the emulsion droplet. Hence, the microfluidic devices are here simplified, while the specific formulation (including the composition of the outer phase) has to be specifically tailored to provide the out-of-equilibrium phase transformations within the droplets. Due to device simplicity these methods are potentially more attractive for practical applications [6,31•]. 2.3. Challenges for the microfluidic technology Each of the described approaches have advantages and disadvantages, but to a wide extent, the current technology seems to be sufficient for a wide range of applications. However, two immediate limitations come presently to mind: achieving droplet dimensions of submicrometer size (e.g. nanoemulsions and nanoparticles) and output scalability for industrial production. Besides the flow conditions, droplet sizes typically also scale with the microchannel dimensions according to Rdrop ∝ d−1, with d being

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Fig. 2. Multiple emulsion production with glass microcapillary devices. (A) Five-component triple emulsion formation using a multi-step microfluidic system. (a1) schematic of the microfluidic assembly made out of three different modules: connector, liquid extractor and droplet generator. (a2-a11) different multiple emulsion droplet combinations obtained with different scheme arrangements of the modules shown in a1. The scale bar represents 200 μm. Reproduced and adapted from Ref. [27•] with permission from The Royal Society of Chemistry http://dx.doi.org/10. 1039/c1lc20065h. (B) Microfluidic one-step production of quadruple emulsions. (b1) Scheme of the surface-modified microcapillary coaxial flow-focusing device. Orange walls are hydrophobic and blue walls are hydrophilic. (b2) Snapshot of quadruple emulsion formation in the microfluidic device. (b3) Collected monodisperse quadruple emulsions, along with a very small amount of single and double emulsions. Reproduced and adapted with permission from [28•]. Copyright 2011 John Wiley and Sons. (C) Microfluidic one-step production of multi-component double emulsions. (c1) Schematic of a coaxial flow-focusing device with a two-bore injection capillary for the production of double emulsions with a controlled number of multi-component inner drops. (c2) Snapshot of double emulsion droplet generation containing two different inner drops. (c3) Color optical micrograph of double monodispersed double emulsions produced in a2, each containing one red and one blue inner droplets. The scale bars represent 100 μm. Reproduced and adapted from Ref. [29•] with permission from The Royal Society of Chemistry http://dx.doi.org/10.1039/c2sm25953b.

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the channel diameter [2•]. Hence for production of submicrometer emulsions nano-sized channels are desired. However this is not an easy task. The fabrication of channels of submicrometer dimensions is not straightforward and accessible, and furthermore, pumping fluids through small nanometer sized channels becomes more challenging due to the huge hydrodynamic resistance that scales with Rhyd. ~ η·L·d− 4 (with Rhyd. being the hydrodynamic resistance, η the fluid viscosity, and L the channel length). New ways for fluid transport may then need to be realized. In addition, in nanosized channels the cross-section dimensions through which fluids flow become comparable to the screening lengths of double layer and other interfacial forces [37], making the overall emulsification processes more difficult to predict. One additional problem that cannot be overlooked is the difficulty for in-situ visualization of the nanochannels with conventional laboratory instruments, making the experiments much more complicated, and calls for the use of other in-situ characterization methods such as X-ray scattering [38,39] and TEM [40]. For all of these reasons, the use of nanofluidic devices for droplet production has been scarce. Recently one nanofluidic system was successfully used for the preparation of very small multiple emulsions. Yet, the minimum size reached was still around 1 μm [41••]. In that example, a clever use of a cross design, where the liquid is injected into wide microchannels that branch out into nanochannels and an open end kept at constant pressure, helps to control the pressure drop across the nanochannels and prevents clogging due to dust particles. The fluids within the nanochannels form a jet in a flow-focusing cross. The jet is stabilized by the nanosized channels, but breaks into droplets when it reaches a micronsized reservoir, in a process called step-emulsification [42], where the height of the nanochannel is the most important parameter controlling the size of the droplets. By manipulation of the fluid components, simple, multiple and Janus emulsions could be produced. With a related device, but by further decreasing the nanochannel dimensions, droplets of around 400 nm could be prepared, although in this case, no multiple emulsion preparation was reported [43]. Regarding practical applications, one of the most concerning drawbacks of microfluidic methods for emulsion preparation and particle synthesis is its low output, which typically does not exceed more than 1 mL/h per device, completely impractical for the industry. One way of partially overcoming this limitation is to scale-up by parallelizing several copies of the device within the same chip, so that a single set of pumps can be used to drive the fluids through the device array [44–46••]. Here, soft lithography methods provide an obvious advantage compared to microcapillary methods, since scaling-up many copies of the same device on a wafer is straightforward. Still, a few problems have to be overcome in order to make the procedure feasible and entirely scalable. One of these problems is that very minor differences in the several copies will increase the polydispersity of the droplets (e.g. even just a small tilt in the silicon wafer during the spin-coating process can provoke minor variations in channel height from device to device [45•]). Another problem is that since the different copies are fed by the same liquid source, droplet formation in one copy creates pressure oscillations that transmit to the source and influence the other copies. This cross-talk can be dampened by increasing the hydrodynamic resistance of the channels between the droplet makers and the common feeding channels [45•]. By increasing the hydrodynamic resistance of the droplet maker feeding channels and lowering the resistance of the common supply channels, it is also ensured that the pressure drop is the same in each channel, and therefore the flow rates and droplet sizes are also identical across the different copies [46••]. Another issue to overcome is the wettability of the channels. Typically the dispersed phase should not wet the walls of the device, which is challenging when producing double and multiple emulsions. As mentioned earlier, devices with sections with different wettabilities are easy to make nowadays, but in a scaled-up parallelized device the challenge is still considerable. One way to avoid this is to simply ignore the different wettabilities, and tune the size of the channels at each junction so that

the droplets formed do not “touch” the microchannel walls [12•,46••]. Having these criteria in mind, a device containing 15 parallelized double emulsion drop makers was able to produce drops at rates of 1 Kg/day with a polydispersity inferior to 6%, whereas continuous scaling to a device fulfilling 1 L of volume could produce around 30 tons of double emulsion droplets per year [46••]. Even so, this figure is relatively small for industrial standards, which makes material synthesis with microfluidics economically viable only for materials with very high added value, such as pharmaceuticals and cosmetics [17]. 3. Innovative formulations for multiple emulsions 3.1. New copolymer stabilizers for one-step preparation of multiple emulsions The preparation of multiple emulsions with bulk methods generally involves a time consuming two-step procedure and the combination of hydrophilic and hydrophobic stabilizers, as indicated at the beginning of this paper. In this context, amphiphilic block copolymers or biopolymers are increasingly being used as emulsifiers. They do not only decrease the interfacial tension, but also increase emulsion stability by multiple anchoring which translates into a higher energy of adsorption at the interface. Moreover, the high efficiency of polymers means that they perform at relatively low concentrations, which can have positive impacts on industrial costs. With this in mind, major efforts are dedicated on exploring new copolymers that allow preparing stable multiple emulsions in one step. Nowadays, studies are focused on understanding the correlation between the copolymer structure and its emulsification performance, as the interactions with the other emulsion components were shown to be decisive [47]. For instance, Poly(ethylene glycol)/ Polypropylene (PEG/PPG)-glycol-dimethicone copolymers with different hydrophilic-hydrophobic length segments have been used to prepare silicone multiple emulsions [48,49], because of their dual compatibility with aqueous and PDMS phases. There are also few examples where a unique copolymer is used to stabilize inner and outer droplets, thus ruling out possible destabilization pathways that might be present in surfactant mixtures. Promising formulations were found using pH-responsive polystyrene-2-(dimethylamino)ethyl methacrylate (PS-DMAEMA) based copolymers with different asymmetric architectures [50,51]. Upon increasing the pH, the system reaches a sufficiently low interfacial tension to form transient bicontinuous structures. The asymmetric fragmentation of this structure, caused by the asymmetric partition of the copolymer, results in the formation of a discontinuous oil phase with nested water droplets, subsequently evolving to a multiple emulsion. Similarly, asymmetric block PEGlysine copolymers mixed with various electrolytes at different concentrations were also investigated. In this particular case, the interactions between the electrolyte anion and the cation residues of the lysine aminoacid seemed to be the main responsible for the long-term stability [52]. Additionally, the hydrogen-bonds between the peptide segments provided an extra mechanical stabilization. Contrary to these examples, a symmetric PEG-b-PS based copolymer showed to be a superior stabilizer compared to its asymmetric counterparts [53]. Here, the stability was ascribed to the high surfactant coverage in both interfaces, providing at the same time steric repulsion. Despite the advantages that the one-step processes offer in terms of processing, they still do not allow a control over the emulsion morphology as accurate as the two-step processes. 3.2. Multiple Pickering emulsions The use of particles as emulsifiers is a well-established strategy to produce highly stable emulsions, coined as Pickering emulsions. Inspired by results on simple emulsions, research on multiple Pickering emulsions has developed. Actual formulations include both emulsions co-stabilized by surfactants and particles or solely by particles. In the

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latter case, solid particles of opposite wettability are required, analogous to surfactants with different hydrophile–lipophile balance (HLB) values for conventional multiple emulsions. As expected, the morphology, rheological properties and hence stability, of multiple Pickering emulsions can be regulated by adjusting the particles/surfactant ratio or the ratio between different types of particles. Particles used as stabilizers include inorganic particles (SiO2 [55], Fe3O4 [56,57]) or hybrid [58•], food-grade particles such as cellulose derivatives [59] or quinoa [60], or even soft particles as PNIPAM nanogels [61••]. Given that the use of SiO2 particles is the most extended strategy, several more sophisticated silica-based particles have emerged. For instance, the wettability of hydrophilic poly(ethylene imine)-functionalized silica particles (silica@PEI) can be modified in-situ (within the emulsion), by coupling an hydrophobic aldehyde dissolved in the oil phase. Through such in-situ interfacial reaction, the hydrophobicity of the particles increases enough to stabilize the W/O interface and yield multiple emulsions that are stable for months [54•] (Fig. 3A). Similar to surfactant-stabilized multiple emulsions, Pickering multiple emulsions via one-step processes have also been produced by using anisotropic cellulose particles [62] or stimuli-responsive styrene-acrylic acid particles [63]. However, as for surfactant-stabilized emulsions, the two-step method is still the most adequate to produce stable multiple Pickering emulsions with a certain control over their structure (droplet and globule sizes).

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release system. Differences are attributed to a different arrangement of the biopolymers at the interface. 3.4. Gelation routes Emulsion stabilization has also been achieved by adding a thickener or a gelling agent such as whey protein [66], sodium caseinate [67], starch [68], xanthan gum [69], alginate [70] or carrageenan [71] into one or more of the phases. By reducing the mobility of the droplets, its coalescence rate is decreased. This also allows to reduce the concentration of surfactant or even to avoid its use. In addition, when gelling the inner phase, the resulting multiple emulsion appears to be less sensitive towards an imbalance in the osmotic pressure. As a function of the thickener or gelling agent used, multiple emulsions with a wide variety of rheological properties can be prepared. Such capacity to form different gel structures is attractive for the cosmetic and food industries, where textures are particularly important. 3.5. Multiple emulsion formation assisted by an induced-phase separation Even though in most of the cases a phase separation seems to be a detrimental process, an induced-phase separation of a simple droplet can also be a powerful tool to form multiple emulsions. Normally, the dispersed phase contains a cosolvent that is more soluble in the continuous phase than in the dispersed phase. Hence when the dispersed phase-cosolvent mixture gets in contact with the outer fluid, a mass transport from the cosolvent into the outer fluid and vice versa occurs, inducing a phase separation within the droplet mixture [31•,34,35••]. That is why current multiple emulsion advances also comprise the study of the underlying mechanisms of phase separation processes. By understanding and mapping these processes, the formation of complex systems such as onion-like multiple emulsions is simplified and can even be predicted [35••] (Fig. 3B). For instance, hexane-perfluorohexane multiple emulsion showed to have transitions from a multiple emulsion to a Janus droplet or even a phase inversion

3.3. Interfacial complexation In the food industry, multiple emulsions are thoroughly investigated as promising reduced-fat formulations. Unfortunately, the allowed surfactant concentrations in this field are relatively low and there is only a limited number of food emulsifiers approved. To solve this problem, current studies are focused on expanding the range of edible stabilizers in the market including, besides the edible particles, protein-polysaccharide mixtures [64,65]. A better degree of food structuring is obtained when the biopolymers are added individually, whilst the addition of the preformed complex yields a much more effective oil

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Fig. 3. Formation of multiple emulsions via different pathways. A) Schematic representation of the in-situ functionalization of silica@PEI nanoparticles, placed at the interfaces of an emulsion, with an aldehyde dissolved in the organic phase (left) and a microscopy image of the resulting Pickering multiple emulsion (right). Adapted with permission from [54•]. Copyright 2014 American Chemical Society. B) Evolution in time (in seconds) of a diethyl phthalate/ethanol/water (0.41/0.42/0.17 vol%) mixture from a singlet droplet to a quintuple droplet. Scale bar is 100 μm. Adapted with permission from [35••]. Copyright 2014 Wiley-VCH. C) Single droplets of a hexane–perfluorohexane mixture in water. Above 22.65 °C (Tc), both organic solvents are miscible, below the Tc a phase separation is induced, which yields a double emulsion. Scale bar is 200 μm. Reprinted with permission from [36••]. Copyright 2015 Macmillan Publishers Ltd.: Nature.

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that could be triggered by changing the temperature (Fig. 3C), irradiating the system or adding a surfactant [36••]. The key of this approach was to play with the solubility of the components and adjust the interfacial tension by using stimuli-responsive surfactants. Janus emulsions have been also prepared with other oil mixtures [72••,73]. Along the same rational, through the use of different immiscible polymers, formulations were prepared for the production of W/W/W emulsions [32,33••]. Applying similar dewetting-wetting concepts but following a different strategy, Deng and coworkers [74•,75] were able to produce multiple emulsions from lower-order emulsion by a drop-engulfing approach within a simple microfluidic device. 3.6. Aerated multiple emulsions Another appealing multiple emulsion-based system is that in which either the inner phase [76,77] or the intermediate phase (antibubble) [78] is a gas. Such structures present many potential and varied applications, from aerated food products to ultrasound contrast agents. Unfortunately, the traditionally used ultrasonic methods lack control over the size of the air bubbles and the resulting systems present short lifetimes, as gas is prone to be dissolved in one of the phases. Recently, researchers began to use microfluidic technology to control the flow patterns to form highly monodisperse encapsulated air bubbles. For these complex architectures, a noticeably enhanced stability has also been observed when colloids are used as stabilizers. 3.7. Multiple nano-emulsions Multiple emulsions with outer droplet sizes below one micrometer, i.e. within the nanometer range, are rare, due to the intrinsic difficulties in producing outer droplets of such small size either by microfluidics or by bulk methods. However, there are a few examples of multiple nanoemulsions in the literature. One of the first reports concerned the stabilization of nanoscale double W/O/W emulsions by single-component block copolypeptides [79•]. The double emulsions were prepared by ultrasonication followed by high pressure homogenization; outer droplets with size below 200 nm were obtained, as evidenced by cryogenic transmission electron microscopy. Sub-micrometer multiple W/O/W emulsions have also been prepared by membrane emulsification using a two-step process [80]; these emulsions proved to enhance intestinal absorption of biopharmaceutics. Multiple nano-emulsions have been tested as drug delivery systems [81,82] and as vehicles for Magnetic Resonance Imaging (MRI) contrast agents [83]. 4. Multiple emulsions as templates for porous particles and capsules 4.1. Hardening multiple emulsions and their evolution during the process Among the different ways to produce multi-compartmentalized particles, the multiple emulsion templating route is one of the most widely used. As multiple emulsions already have a hierarchical architecture, the additional use of porogen agents is not needed. The ability to generate multiple emulsions with different compositions and morphologies paved the way to form a wide range of different porous particles by simply solidifying the middle phase. Moreover, the fact that the inner phase is separated by a physical barrier from the external medium makes this type of particles potentially useful for the encapsulation and isolation of active agents. Encapsulation can be accomplished either within the inner phase only, or within the inner and the middle phase, which allows encapsulating incompatible active agents within the same vehicle without cross-contamination. The most established routes to solidify the middle phase of multiple emulsions are polymerization, solvent-evaporation and temperature-induced solidification. In the polymerization route, the middle phase contains a monomer solution, whose chemical or physical crosslinking can be triggered at a specific time by UV irradiation [84,85], temperature or adding an initiator

[57,86–89]. Interestingly, the rigidity of the material can be easily tuned by regulating the crosslinking degree [90]. Although most of the research performed is focused onto polymer particles, the synthesis of inorganic particles is also feasible by using silica [91,92] or zirconium dioxide [93•] precursors. In the solvent evaporation-based method, the middle phase is a solution of normally a polymer or a lipid in a volatile solvent. Upon removing the solvent, the material precipitates creating the solid matrix. The most used polymers are the polyesters (poly(lactic acid) [94], poly(lactic-co-glycolic acid) [95,96]) and polyacrylates, which in some cases might even provide some stability without the need of surfactants [97]. When using templates produced with a microfluidic device, challenging structures with ultrathin shells such as vesicles [98•] or polymersomes [99] can also be fabricated. In the third approach, the emulsion is prepared using as an intermediate phase a material above its melting temperature. When the system is cooled down, the material crystalizes yielding the porous particle. As this is a solvent-free route, it has been widely used to prepare lipid vehicles for pharmaceutical drug delivery purposes [100,101]. It is obvious that in all cases the morphology of the final particle closely resembles the emulsion template, especially when the polymerization or the temperature-induced solidification routes are used. In the case of the solvent-evaporation route, the resulting particles are significantly smaller than the template, given that a fraction of the middle phase is evaporated. Such shrinkage could in turn modify the distribution and size of the pores, so that the final morphology is less predictable. Nevertheless, when the template is produced by bulk emulsification methods, the particle and pore size as well as the pore structure (isolated or interconnected) can be adjusted by playing with several formulation parameters, such as the stabilizer concentration or the ratio between the phases (Fig. 4A) [57,102,103]. However, their morphologies can only be controlled up to some extent. Difficulties might arise when a much more well-defined morphology is required. Fortunately, the fine control over the liquid flow rates afforded by the microfluidic technology can be exploited to tackle this problem. For instance, capsules can be synthesized from multiple emulsions with a single inner droplet. The mechanical properties and permeability of these capsules can be tuned by varying the wall thickness and the chemical composition of the middle phase (Fig. 4B–C) [104–107]. However, sometimes the structure of the final particle not only depends on the morphology of the template but also on its evolution during the solidification process. When a single droplet is generated, it tends to sink because of a density mismatch with the middle phase. Polymerization of this eccentric emulsion results in an open structure. Several strategies have been used to avoid this phenomenon, such us the use of highly viscous middle phases or generating the emulsion in the jetting regime [84]. Solidification processes could also be accompanied by a phase separation, but as pointed out above, this can be used to synthesize exotic particles with open structures. Different solid particles with shapeand/or composition-anisotropy have been prepared by controlling the degree of phase separation within the droplet (from a complete separation to partial separation) during the solidification process (Fig. 4D) [34,100,108,109]. Control over dewetting can be achieved by using surfactants or changing the composition of the middle phase, which governs the interfacial tension or adhesion force between the phases. Recently, a more sophisticated route based on the use of a poly(4-vinylpyridine)-oleic acid supramolecular complex, whose amphiphilicity can be modulated upon changing the pyridine-acid ratio, has been described [110]. Phase separation in double emulsion can also be used to prepare vesicles, circumventing the problem of the presence of nondesired organic solvent remnants [111•]. Interconnecting porous polymer microspheres were prepared using high internal water-phase double emulsions (W/O/W) generated via catastrophic phase inversion [112]. Interestingly, these emulsions were stabilized by a single surfactant (12-acryloxy-9-octadecenoic acid). These particles are designated as poly(HIPE) microspheres in

B.F.B. Silva et al. / Current Opinion in Colloid & Interface Science 25 (2016) 98–108

A

5%

18%

30 µm

30 µm

C C1

100 µm

C2

100 µm

B

30%

C3

5% PAA-b-PMMA

20% PAA-b-PMMA

80 µm

30 µm

D D1

105

80 µm

D2

D3

50% ETPTA

70% ETPTA

100 µm 100% ETPTA

Fig. 4. Internal-structured particles formed from multiple emulsions. A) Scanning Electron Microscopy (SEM) images of silicone particles with different inner structures obtained from W/O/W multiple emulsions formulated with 5, 18 and 30% of inner aqueous phase. Adapted with the permission from [102]. Copyright 2013 American Chemical Society. B) The permeability of methacrylate capsules can be tuned by adding different amounts of polyacrylic acid-methyl methacrylate (PAA-b-PMMA, a pH-responsive material), what results in a different hole density and size. Adapted with permission from [104]. Copyright 2013 American Chemical Society. C) Fluorescent microscopy pictures of lipid capsules with different shell thicknesses (C1–2). The red and the green colors denote the encapsulation of a hydrophilic and a hydrophobic drug, respectively. A SEM image of a broken capsule is also shown (C3). Adapted with permission from [100]. Copyright 2013 American Chemical Society. D) Optical microscopy pictures (inset) of diverse emulsion templates with different ethoxylated trimethylolpropane triacrylate (ETPTA) concentrations in the middle phase and SEM images of the resulting particles. By varying the amount of ETPTA, the adhesion of the inner droplet with the outer phase changes, hence resulting in particles with fishbowl-, bowl- or truncated-sphere-shapes. Scale bar is 200 μm. Adapted with permission from [61••]. Copyright 2013 Wiley-VCH.

analogy to bulk porous polymers derived from high-internal-phase ratio (HIPE) emulsions. 4.2. Pickering-based porous particles The growing research on Pickering multiple emulsions has also evolved towards their use as scaffolds for particle synthesis. The reaction (usually a polymerization) can occur within the particlestabilized droplet, leading to formation of a porous particle decorated with the stabilizing particles. Highly porous particles without throat pores can be obtained [55], which is difficult to achieve using surfactant-stabilized emulsions. One of the main advantages of using Pickering emulsions as templates is the possibility to endow a specific performance to the system by simply using functional particles as stabilizers. For instance, some studies have demonstrated the possibility of adding magnetic properties to the system by using Fe3O2 nanoparticles as stabilizers [56–58•]. Alternatively, capsules can be obtained by crosslinking the particles adsorbed at the interface. This requires the use of chemically modified colloids. An example of this is the use of amine-functionalized silica colloids that can subsequently react with an epoxy-derivate molecule dissolved in one of the phases [113•]. The resulting particle film showed to be strong enough to withstand the drying process, hence, yielding colloidosomes-in-colloidosomes, which might offer extra encapsulating advantages compared to the conventional colloidosomes. 5. Summary and outlook The broad spectrum of potential applications of multiple emulsions is constantly catalyzing the development of better preparation methods and stabilization strategies. Concerning preparation, microfluidic devices have attracted much interest due to their capability to produce droplets with extremely fine control over size and structure. However, droplet production throughput to meet the industrial scale is still a significant challenge, making the synthesis of materials viable only for high value products, such as pharmaceuticals and cosmetics. On a different note, the advent of new fabrication methods such as 3D printing, will become accessible to a much wider range of scientists due to affordability and automated single-step fabrication. This promises to bring even more innovative ideas to the field of multiple emulsion production and microfluidics in general.

Advances on the formulation of multiple emulsions, particularly those related with the use of block copolymers and particles for emulsion stabilization, have expanded the field beyond traditional surfactant mixtures, with uses that range from edible multiple emulsions to systems for environmental purposes. The availability of multiple emulsions with controlled size and structure have helped to consolidate them as robust and versatile soft templates for the fabrication of porous particles and capsules with complex morphologies through several strategies for the hardening of the intermediate phases. New matrix and wall materials, with their associated functionalities, are expected to come.

Acknowledgments N.V. acknowledges The Netherlands Organization for Scientific Research (NWO-ECHO-STIP Grant 717.013.005) for financial support. C.R-A is grateful to the European Regional Development Fund (ON.2 – O Novo Norte Program).

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