Water-in-Water (W/W) Emulsions

Jordi Esquena

Institute of Advanced Chemistry of Catalonia, Spanish National Research Council (IQAC-CSIC) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona, 18-26, 08034 Barcelona, Spain

jordi.esquena@iqac.csic.es

Abstract

Water-in-water (W/W) emulsions are colloidal dispersions of an aqueous solution into another aqueous phase. Such dispersions can be formed in mixtures of at least two hydrophilic macromolecules, which are thermodynamically incompatible in solution, generating two immiscible aqueous phases. W/W emulsions are much less known than conventional oil-in-water or water-in-oil emulsions, despite the fact that phase separation in aqueous mixtures is highly common. The thermodynamics and the phase behavior of segregative phase separation in mixtures of hydrophilic polymers have focused a great attention, with many excellent scientific reports in the literature. However, the kinetic stability of water-in-water emulsions is generally difficult to control, since amphiphilic molecules do not adsorb on W/W interfaces. Consequently, surfactants are not good stabilizers for W/W emulsions, and until recently, only a limited number of scientific studies have dealt with the formation and stabilization of emulsions in aqueous two-phase systems. However, recent advances and successful results in the stabilization of these emulsions, by alternative mechanisms, have triggered a renewed interest. Nowadays, fast progress is being made in formation and stabilization methods, and new knowledge is rapidly acquired, opening a wide range of novel possibilities for practical applications. Interestingly, highly stable water-in-water emulsions can be formulated using fully biocompatible and edible components, and consequently, these emulsions can be used in food formulations, among many other interesting applications. This review describes the general background of research in the field, and focuses on recent scientific advances, including phase behavior, formation, stability and kinetic aspects, and applications such as formation of microgels, encapsulation and drug delivery.

1. Introduction

Water-in-water emulsions are very interesting colloidal dispersions consisting of two immiscible aqueous phases that are in thermodynamic equilibrium. These emulsions, which consist of droplets of an aqueous phase dispersed into another aqueous phase, are formed in aqueous mixtures of at least two water-soluble molecules, which are mutually incompatible in solution. The present review focuses on W/W emulsions resulting from mixtures of hydrophilic polymers, which can lead to a complex phase behavior [1]–[4] and stable emulsions with a very wide variety of potential applications [5]–[7]. Other water-in-water systems such as vesicles (liposomes or polymersomes), hexosomes or cubosomes, are colloidal dispersions of liquid crystals, and consequently they are not considered as emulsions. Therefore, these systems are not included in the present review.

Aqueous mixtures that separate in two immiscible phases are generally denoted in the literature as “Aqueous Two-Phase Systems” [8], [9], abbreviated as ATPS, or “Aqueous Biphasic Systems” [10], [11]. Water-in-water emulsions can be formed in such systems, and thus, are sometimes denoted as “ATPS emulsions”. Herein, the most common term “water-in-water emulsion” [12], [13], which can be abbreviated as “W/W emulsion”, is preferred because it is self-defining, non-ambiguous and already well accepted.

Water-water phase separation can be found in a large variety of systems, which include aqueous mixtures of polymers, polymers and surfactants, polymers and electrolytes and surfactant solutions. For instance, it is well-known that the addition of salting-out electrolytes to hydrophilic polymer solutions might induce separation in two immiscible aqueous solutions: a polymer-rich phase and a salt-rich phase. Another typical example of aqueous biphasic system is the phase separation commonly observed in micellar solutions of nonionic surfactants, at temperatures above the cloud point of the surfactant.

Often, phase separation in aqueous two-phase systems is very fast, varying between seconds and a few hours. The colloidal stability of droplets in such systems is poor because of the lack of significant interdroplet repulsion forces, and therefore emulsions in many aqueous biphasic systems are often highly unstable. However, this colloidal stability can be greatly controlled in solutions containing polymers, and consequently, the formation of water-in-water emulsions is feasible. Recently, water-in-water emulsions in aqueous mixtures of hydrophilic polymers have been the subject of a great renewed interest, with novel fundamental discoveries and new technological applications.

Water-in-water emulsions are formed due to the thermodynamic incompatibility between aqueous solutions of certain combinations of hydrophilic polymers [3]. This phenomenon has been known for a long time [14]–[16], and the most basic aspects of segregative phase separation in mixtures of long-chain molecules

were already described many years ago [4]. Water-in-water emulsions were first described by Beijerinck, who discovered these emulsions by serendipity in 1896 [14]. He was a microbiologist studying the growing of bacteria on starch, and he was preparing water-soluble starch by hydrolysis with HCl for culturing bacteria. He was surprised to observe the formation of droplets when mixing the starch solutions with gelatin [14], [15]. Beijerinck was a very curious scientist, and using simple experiments based on iodine dying of starch, he correctly described that the emulsions were composed of two different aqueous solutions. He also observed that gelatin-in-starch or starch-in-gelatin emulsions could be obtained depending on the polymer ratio, and that the droplet size could be controlled by agitation. Interestingly, he also described the observation of some multiple emulsions composed of small droplets of starch solution, located inside larger gelatin droplets, which were dispersed in the starch solution.

This publication in 1896 [14], can be considered the first scientific report of a water-in-water emulsion, albeit he did not mention this term. Only two years later, in 1898, Bütschli reported his studies on a similar system [16], which was also composed of water, gelatin and hydrolyzed starch. He reproduced the formation of stable liquid droplets, but neverthesless, he did not study it in detail. However, Beijerinck in another paper published in 1910, he described that aqueous mixtures of agar and gelatin also showed the formation of droplets. He correctly stated that one phase was agar-rich but nevertheless contained small amount of starch, and that the other phase was the opposite, rich in starch and depleted in agar [15]. Phase separation was observed in a wide range of polymer concentrations [15]. Coalescence of dispersed droplets was slow, and thus, droplets could be formed and remained in dispersion.

Nowadays, the phenomenon of phase separation in aqueous mixtures of two hydrophilic polymers is well documented, and comprehensive reviews have been published [3], [6], [7], [17], [18]. It is known that mixtures of hydrophilic polymers in water often have a rather complex phase behavior [1], [19], and two different types of phase separation can occur: segregative phase separation and associative phase separation; depending on the repulsion or attraction between the two different hydrophilic polymers, and also the hydration interactions between each polymer and water.

Segregative phase separation is observed in many mixtures of two hydrophilic components. At low polymer concentrations, the mixture remains a single phase, with the two components in solution. However, above some minimum concentrations, the two components separate into two immiscible aqueous phases in thermodynamic equilibrium. Each phase is enriched with one of the hydrophilic components, and is saturated with the other component. The thermodynamic incompatibility between the two components is generally due to

negative entropy of mixing, in conditions where either the two molecules have no electrostatic charge or they are bearing charges of the same sign.

This phase behavior is shown schematically in the ternary phase diagram of Fig. 1a, in which the immiscibility region is delimited by a binodal line. Compositions that are mutually in equilibrium are indicated by tie-lines [1], which converge into a critical point, where the two immiscible phases merge together becoming one single phase. Such behavior is commonly observed in mixtures of nonionic polymers, or in the cases of one ionic polymer mixed with a nonionic one. Moreover, this segregative phase separation can also occur in mixtures of a surfactant and a polymer [1]. The phase separation is caused by thermodynamic incompatibility of the two components, above certain concentrations.

Associative phase separation is a different phenomenon, where mixtures of two components and water separate in a solid-like precipitate, which contains high concentrations of the two hydrophilic polymers, and a supernatant liquid solution that only has a residual low amount of polymers. Associative phase separation is commonly produced by electrostatic complexation, due to the presence of oppositely charged polymers [20]. The tie-lines, represented in a ternary phase diagram, are approximately vertical, as indicated in Fig. 1b [1]. The precipitate, which can consist of amorphous non-crystalline particles, is usually denoted as a coacervate. This term is used to refer to either solid-like particles or highly viscous droplets, made of two oppositely charged macro-ions held together by strong electrostatic attraction. Coacervates will not be discussed further, since it is not in the scope of the present review.

Fig. 1. Schematic ternary phase diagrams, which illustrate segregative (a) and associative (b) phase separation in mixed hydrophilic polymer systems. S: solvent (water); P1: Polymer 1; P2: Polymer 2. (Reproduced from Reference [1], with permission).

The various phenomena that can lead to phase separation are summarized in Fig. 2. The figure illustrates the different colloidal systems that can be observed, depending mainly on the interactions between the two polymers.

Repulsive interactions between polymer molecules produce segregative phase separations (at high macromolecule concentrations) or cosolubilization (at low concentrations). This behavior is a general phenomenon commonly observed in mixtures of hydrophilic polymers [21]. As an illustration, a comprehensive review listed around 100 different combinations of proteins and polysaccharides that resulted in segregative phase separation [3]. Moreover, the same phase separation can also be found in many other combinations of macromolecules, and therefore, there is an almost unlimited number of possible different systems that could be used to form W/W emulsions.

Fig. 2. Scheme showing the four different phase situations, generated by either attraction or repulsion between macromolecules. The example refers to mixtures of proteins and polysaccharides, but the same behavior can be extrapolated and is found in many other combinations of two different macromolecules. (Reproduced from reference [19], with permission).

Interactions between the two polymers can be modulated as a function of pH and ionic strength, leading to a very rich phase behavior. The nature of the electrolyte is also of the utmost importance, since the hydration ability of electrolytes, in the context of Hofmeister series, can greatly influence interactions between polymers. Antonov, Moldenaers, Gonçalves and co-workers have studied in detail the phase behavior and phase transitions of hydrophilic polymers in aqueous mixtures [22]–[25]. They have also described the formation of W/W emulsions in various biopolymer mixtures, in absence and presence of polyelectrolytes. These investigations include studies of phase behavior in the casein/alginate system [24], studies of the BSA/dextran system

and its use in the formation and stability of W/W emulsions [23], [25], or phase behavior studies in mixtures of pectins, alginates and gelatin [26]. The phase separation in aqueous mixtures of poly(ethylene glycol) and dextran has also been studied in detail [27], [28].

Interestingly, depending on pH and ionic strength, associative and segregative phase separation can occur simultaneously. In this case, three equilibrium phases (two immiscible aqueous solutions plus a coacervate precipitate) coexist in the same sample. This surprising behavior was described in 2006 in the system composed of water/gelatin/carrageenan [29] for temperatures below 25ºC, in the presence of moderate amounts of NaCl. More recent studies have demonstrated that this behavior might not be unusual, and it can be attributed to transitions in molecular conformation [30]. The electrostatic complexation of gelatin with carrageenan is affected by the conformational ordering of carragenan chains, leading to a coupling of both phenomena, which might result in simultaneous complexation and segregative phase separation. In conclusion, one could find particular conditions in which both association and segregation can coexist, especially in the case of polymers with large polydispersity in charge density and molecular weight.

The mechanisms behind associative and segregative phase separation are clearly different. Association is induced by electrostatic attraction between charges of opposite signs, whereas segregation arises from negative entropy of mixing, which is highly dependent on molecular conformation. Repulsion between polymer chains is mainly attributed to restrictions in the free conformational movement of the polymers. The conformational freedom depends very largely on molecular weight, and short molecules tend to restrict free conformation of long linear chain molecules. Therefore, repulsive interactions will always be present in mixtures of nonionic polymers with clearly distinct molecular weights.

The mechanisms of segregative phase separation have been studied from various theoretical points of view [17], and the simulation of phase diagrams can be attempted by various theoretical models. Currently, various different theoretical approaches are used to predict the thermodynamic incompatibility between two hydrophilic polymers in aqueous solution [17]. One approach is based on the Flory-Huggins interactions parameters [31], [32]. When the polymer1–polymer2 parameter is a large positive value, which accounts for a strong mutual repulsion between the two polymers, this repulsion predominates over solvent-polymer1 and solvent-polymer2 Flory-Huggins parameters. Therefore, the two polymers are segregated and the system is demixed, forming two separate aqueous phases. In 2000, A.H. Clark was able to describe [32], by fitting the tie-lines of phase diagrams to theoretical models, that the phase separation could be predicted according to theories based on Flory-

Huggins parameter. The results demonstrated the great importance of entropic contributions over enthalpic contributions.

However, there were discrepancies between experimental phase diagrams and theoretical fitting. Such discrepancies were attributed to the fact that Flory-Huggins parameters are significantly dependent on molecular weight of polymers [32]. Moreover, theoretical models do not usually take polydispersity into consideration, but this is known to be a factor with a large influence on intermolecular interactions [17].

Another theoretical approach is based on the theory of depletion flocculation, which can be considered valid in the case of large globular macromolecules in presence of a short flexible molecule. This theory has been successfully applied by various authors [33], [34] to mixtures of globular proteins (such as casein or BSA) and flexible elongated polymers (such as neutral or charged polysaccharides).

Polymer polydispersity is a key factor that influences polymer-polymer interactions and phase behavior [35]. Considering, that molecular weight is often ill-defined and many polymers are highly polydisperse, this is a backward that often impedes the use of theoretical modelling in phase behavior studies. Moreover, the lack of information about polymer-polymer and polymer-solvent interactions frequently impedes the use of theoretical modelling methods. Consequently, prediction is not an easy task, and trial-and-error experiments are often used to determine whether a certain combination of two polymers will form a water-in-water emulsion. Phase diagrams cannot be easily simulated in real practical systems, and empirical methods are still used to gain information, searching for immiscibility regions in phase diagrams of polymer mixtures. In conclusion, experimental methods are used to determine phase diagrams, and such diagrams are used to select the most appropriate compositions for the preparation of water-in-water emulsions.

Phase behavior in mixed biopolymer systems composed of maltodextrin with either -carrageenan or gelatine, which display segregative phase separation, were studied in detail by Lundin et al. [2]. These systems are of interest, since both gelatin and -carrageenan are gelifying agents used in food formulations. In both cases, segregative phase separation was shown to be very much dependent on temperature, pH and ionic strength, as expected. One of the interesting results is that, by controlling the rate of gelation by quickly cooling down to various temperatures it was possible to obtain stable water-in-water emulsions, because of the formation of gelled states that prevented coalescence. Therefore, colloidal dispersions of microgel particles could be obtained is such systems. This opened the door for the use of W/W emulsions as templates for the formation of microgels.

Water-in-water emulsions have a wide range of applications, greatly expanding over the years, taking advantage of their all-water nature, in absence of both oil and surfactant. Interestingly, W/W emulsions can be obtained in completely biocompatible systems, using mixtures of edible biopolymers, such as proteins and polysaccharides. Consequently, W/W emulsions might have many potential applications in food formulations. Moreover, they can be used to encapsulate hydrophilic active components for drug delivery.

2. Emulsion formation and stabilization

The present section focuses on formation and stabilization of water-in-water emulsions originated from segregative phase separation. These emulsions can be prepared by applying mechanical agitation in the two-aqueous phase systems. The phase with smaller volume fraction becomes the internal phase, and phase inversion occurs approximately at compositions in which the volume fraction of the two phases is approximately equal. Bicontinuous and/or multiple emulsions can be formed at compositions near to the inversion point at 50:50 volume ratio. This is illustrated in Fig. 3, which shows examples of various W/W emulsions prepared in the water/gelatin/maltodextrin system [36]. The two-phase region appears at high concentrations, where gelatin-in-maltodextrin, maltodextrin-in-gelatin, and even bicontinuous emulsions can be formed, depending on compositions.

Fig. 3. Scheme of W/W emulsion formation in the gelatin/maltodextrin system. (reproduced from Reference [36], with permission).

[Maltodextrin] (%W/W)

[Gelatin] (%W/W)

In the boundary line, denoted as binodal line, the chemical potentials of the two polymers are the same in both phases. The pairs of phases in thermodynamic equilibrium are indicated by tie-lines. It is interesting to remark that phase inversion occurs at approximately 50/50 volume ratio, in the center of the tie-lines (as illustrated in the examples of Fig.3), because the phase with larger volume fraction tends to be the external continuous phase. The tie-lines converge at the critical point, in which the composition difference between phases disappears. Beyond the critical point, the two immiscible phases vanish, forming one single phase. The tie-lines can be accurately determined by a simple method, based on measurements of volume ratio and densities, as reported by Atefi et al [9].

It is well-known that interfacial tensions at w/w interfaces are very low, often below the below the 10-2 mN/m range [37]–[42]. For example, in the system composed of H20/dextran/poly(ethylene glycol), which has been studied in detail [28], [42], [43], interfacial tension is only 0.07 mN/m at 8wt% dextrane and 6wt% PEG [42], and it is even lower near the critical point. For comparison, one should consider that the typical values of tension in oil/water interfaces, in absence of surfactant, are in the range of 30 mN/m.

Ultralow values around 1x10-3 mN/m have been measured in gelatin/dextran and gelatin/arabic gum systems, using the spinning drop technique [37], [38]. Antonov and coworkers have been able to measure interfacial tensions as low as 1x10-5 mN/m by a rheo-optical technique [41], near to the critical point of the sodium caseinate / sodium alginate aqueous system. These values of interfacial tension are so low that spinning drop technique cannot be used for such measurements. It has been demonstrated that interfacial tension depends on the difference in composition between the two aqueous phases, along the binodal line. Interfacial tension reaches extremely low values near the critical point [41], where tension becomes virtually zero.

Moreover, it has been described that electrostatic charges at the W/W interface, in the case of mixtures of polyelectrolytes and neutral polymers, phase separation reduces even more the already extremely low interfacial tension [39], [40]. The results suggested that an electrostatic potential difference spontaneously appears at the interface, producing a reduction in interfacial tension, which can be explained by the Poisson-Boltzmann theory.

The main backward of water-in-water emulsions is their usual lack of stability, especially in compositions near the critical point. Fast coalescence or flocculation tend to occur, leading to rapid and irreversible phase separation, despite the fact that W/W emulsions do not undergo Ostwald Ripenning. In W/W emulsions, the interfaces between the two phases are ill-defined, and are usually thicker in comparison to O/W interfaces. W/W interfaces have length scales larger than the correlation length of the polymer solutions [44].

Therefore, small hydrophilic molecules do not encounter an interface when they move from one polymer phase to the other. As a consequence, conventional surfactants do not adsorb on water/water interfaces, and thus, poor stability has been the main disadvantage for using W/W emulsions in practical applications. In conclusion, the stabilization of W/W emulsions is of the utmost technological importance, and finding new methods for the effective stabilization of emulsions is an important challenge for physical chemists. Consequently, much scientific effort is currently devoted to deal with this issue.

Poortinga reported in 2008 the first results that describe W/W emulsions stabilized by adsorption of particles at the water-water interface [45]. Later, Firoozmand, Murray and Dickinson reported that addition of particles was able to stop spinodal phase separation by adsorption of particles at the interface [46], and moreover, they clearly demonstrated that Pickering emulsions can be prepared in water/water systems. These works opened a new approach for achieving colloidal stability, and this subject has been studied in great detail by Nicolai, Murray and their coworkers [7], [47]–[52].

They prepared W/W emulsions by mixing immiscible solutions of dextran and poly(ethylene oxide) in the presence of either latex particles or globular proteins. They observed that particles were able to adsorb on the interface, despite of the very low interfacial tension. Adsorption was weak in the case of latex particles, but better results were obtained when using a globular whey protein (-lactoglobulin), which adsorbed stronger on the interface. Good interface coverage was achieved, as shown in Fig. 5a. The presence of protein particles on the droplet surface did not inhibit macroscopic phase separation, but nevertheless stability was certainly increased, with a much slower phase separation.

The protein solutions were treated by heat at 85ºC, inducing aggregation of protein molecules, and obtaining stable suspensions of soft hydrogels based on proteins. These hydrogel particles were able to adsorb on the interface much stronger than native untreated protein. As a result, remarkable stable water-in-water emulsions could be obtained, because of Pickering mechanism [48]. This experimental result can be easily understood, considering that the energy of adsorption of a particle strongly depends on particle size [53], as:

∆G=π R2 γ (1−cosθ)2 (Eq. 1)

Where G is the energy of adsorption, R is the radius of particles, is the interfacial tension and is the contact angle of adsorbed particles on the interface. Considering that the interfacial tension is very low in water-in-water emulsions, the energy of adsorption remains low except for large particles. Native proteins were not able to stabilize the emulsions because they were too small, and partial aggregation resulted in larger particles able to successfully adsorb and stabilize.

Fig. 5. Examples of W/W emulsions stabilized by particles. (a) Image observed by confocal laser scanning microcopy with fluorescent labeling of droplets that contain dextran, dispersed into a solution of poly(ethylene oxide). Protein particles adsorbed on the interface appear as orange. (b) Amylopectin-in-xyloglucan emulsions, stabilized by β-lactoglobulin microgels surface-coated with xyloglucan. (Images reproduced from References [47], [50] with permission).

The adsorption contact angle of spherical latex particles was measured by Balakrishnan et al, [7], [47], observing fluorescently labeled particles at the interface of labeled dextran, in the PEG/dextran system. In this system, the contact angle is 145º. Therefore, the energy of adsorption, calculated according Eq. 1, is approximately 7kT [47]. This is not a very large energy of adsorption, but nevertheless, it if high enough to anchor the particles at the interface, against thermal motion. In any case, the energy of particle adsorption greatly depends on particle size, increasing with R2 (Eq. 1). Balakrishnan used latex particles with sizes around 1 m, and it could be presumed that this is an appropriate size for achieving good stabilization.

A systematic study on the influence of particle hydrophobicity was performed on silica particles [52], varying its surface hydrophobicity by controlling the percentage of –OH silanol groups remaining on silica surface. These particles were tested on emulsions prepared in the system composed of water, waxy corn starch and locus bean gum. In the presence of particles, phase separation was significantly slowed down. Observations under confocal microscopy showed that particles tended to form aggregates, and the inhibition of emulsion phase separation was attributed to the presence of large aggregates adsorbed at the W/W interface.

Very recently, Gonzalez-Jordan et al. have shown the importance of particle morphology and its preferential affinity for a given phase [49]. He studied W/W

(b)(a)

emulsion formation in mixtures of poly(ethylene oxide) and dextran, stabilized by β-lactoglobulin protein particles, which were pretreated by heating under different conditions, in order to control particle morphology and surface properties. These protein particles partition preferably into the PEO phase at pH 3, in which particles have a net positive charge. However, these protein particles prefer dextran phase at pH 7, where they have a net negative charge [49]. Consequently, dextran-in-PEO emulsions are obtained at pH 3, whereas PEO-in-dextran emulsions are formed at pH 7. These results clearly demonstrate that the nature of the emulsion (dextran-in-PEO or PEO-in-dextran) depend on the preferential affinity of the adsorbed particles. The layer of adsorbed particles forms an effective barrier only if it is solvated preferentially by the external continuous phase [7], [49]. Actually, this is not surprising, and it is a common behavior observed in conventional Pickering emulsions. Regarding particle morphology, the results reported by Gonzalez-Jordan et al. indicate that fractal aggregates produce a significantly better stability, in comparison to fibrils and hydrogels [49]. This can be attributed to the fact that fractals can cover a large interfacial area with a relatively low mass.

Other recent studies by Freitas, Nicolai et al [50] have demonstrated the possibility of improving the long-time stability of W/W emulsions by stabilizing with functionalized particles. They have studied the use of -lactoglobulin, in aqueous mixtures of xyloglucan and amylopectin. Amylopectin constituted the dispersed phase and xyloglucan was forming the external phase. The emulsions were stabilized with β-lactoglobulin microgel particles that were surface-functionalized with xyloglucan, and consequently, the particles had stronger affinity for the xyloglucan solution, which constituted the external phase. An example is shown in Fig. 5b. These interesting results have demonstrated that the emulsion nature (for example, amylopectin-in-xyloglucan or xyloglucan-in-amylopectin) can be controlled by functionalizing the particles.

In the last few years, the stabilization of W/W emulsions has become a hot topic and is the focus of a great scientific interest. Many different recent papers describe various strategies for enhancing emulsion stability. Vis et al. have reported that nanoplates are excellent stabilizers for W/W emulsions [54]. He used ultra-thin Gibbsite nanoplates, which have demonstrated the advantages that they can cover a large interface with a minimum mass, and the covered area is quite independent of the adsorption contact angle. Consequently, plate particles are more efficient stabilizers than spherical particles in W/W Pickering emulsions. Nanorods have also good stabilizing properties. Peddireddy et al. have demonstrated that hydrophilic nanorods, like cellulose nanocrystals, can efficiently stabilize water-in-water emulsions [55]. Another interesting work, reported by Dewey et al, shows that liposomes can also adsorb on W/W interfaces, being able to stabilize the emulsions [56]. Moreover, it has also been reported that O/W emulsion droplets can enhance the stability of W/W emulsions, by adsorbing on the water-water interface [57], [58].

For stabilization of W/W emulsions by Pickering mechanisms, there is much interest in the use of biocompatible particles that allow the use of such emulsions in biomedical applications and food formulations. For this reason, scientific efforts focus on stabilization using edible and/or low-toxicity particles, made of polysaccharides or proteins. Examples are the use of liposomes, cellulose nanorods or whey proteins, as mentioned above.

Soft microgel particles made of biopolymers are indeed excellent stabilizers, very well known in food industry for oil-in-water emulsions [59]. Then, a question arises whether microgels could also be useful in stabilization of W/W emulsions. This issue has been dealt recently by Murray at al., who have investigated in detail the use of soft particles made of aggregated whey proteins, as emulsion stabilizers [51]. Food-grade microgel particles with sizes around 150 nm, made of a whey protein isolate, were studied. The isoelectric point of such particles is reached at around pH=6, and thus, these particles have a low electrostatic charge at neutral pH. At 15wt% particle concentration and pH, the stability of emulsions composed by waxy corn starch and locust bean gum was remarkable good, inhibiting phase separation for a period of up to one year. Observations by confocal laser scanning microcopy indicated that stability has enhanced by aggregation of the particles on the interface, similarly as in a previous work [52] with silica particles.

W/W Pickering emulsions, stabilized with microgel particles, can be highly sensitive to pH and salinity, and thus, emulsions can be designed to be pH-responsive. Nguyen and Nicolai [44] have prepared emulsions by mixing solutions of dextran and PEG, which were stabilized by microgel particles made of ethyl acrylate and methacrylic acid, crosslinked with 1,4-butanediol diacrylate. The particle adsorption greatly depends on pH, and these emulsions were stable only in a narrow pH range between pH 7.0 and 7.5. Therefore, the emulsions could be stabilized or destabilized by fine changes in pH [44].

Buzza et al. [42] reported an interesting method for the stabilization of W/W emulsions, by using biblock and triblock copolymers. They synthesized copolymers with many different chains lengths, searching for the optimal stability. In the triblocks, the outer chains of copolymers are both hydrophilic, and are separated though a hydrophobic spacer chain. The two endblocks also possess different affinities for the two liquids of the aqueous two-phase systems. The results showed that this polymer configuration provided adsorption on the interface and improved emulsions stability. The authors hypothesized that a monolayer was formed on W/W droplets. This model is shown, schematically in Fig. 4.

Fig. 4. Scheme of the triblock copolymer with two hydrophilic end chains with different affinity, and scheme of the adsorbed monolayer. (Reproduced by Ref. [42], with permission).

However, the formation of monolayers by such triblock copolymers is an hypothesis that remains under discussion, since some diblock copolymers can provide better stability than many triblock copolymers. Nicolai and Murray suggest a different mechanism [7], proposing that the triblock copolymers might form aggregates, which adsorb on the interface in a similar way as other types of particles. This alternative mechanism would explain why stability increased with copolymer size, whereas it did not depend much on chain length ratios [7]. In any case, the question still remains open whether it is possible to stabilize W/W emulsions by diblock copolymers, in which the two hydrophilic chains possess different affinities for the two aqueous phases. This is a very interesting subject, that remains to be solved, and it could be the subject of future papers.

3. Control of droplet size and morphology

As a consequence of the low values of interfacial tension in W/W emulsions, it can be possible to control the droplet size of the emulsions by modulating the shear rate. For example, Fig. 6a shows that maltodextrin-rich droplets, dispersed into a continuous phase enriched in gelatin, can be obtained with a droplet size around 7 m, achieved by applying a shear rate of 100 s -1 in a 100 mm gap between two parallel plates [60]. The droplet size is increased to around 30 m by slowing shear rate down to 10 s-1 and increasing the gap to 500 m (Fig. 6b). In any case, as mentioned before, these emulsions are quite unstable in absence of a stabilizing agent, unless shear is continuously applied, or gelification and/or crosslinking takes place in the internal phase.

Fig. 6. Examples of water-in-water emulsions prepared in gelatin/maltodextrin mixtures. The emulsions, with 30wt% of disperse maltodextrin phase, have been obtained by applying shear between parallel plates at (a) 100 s -1 and (b) 10 s-1 shear rates. (Reproduced from Fig [60], with permission)

Norton and coworkers [61], [62] have dealt with the issue of controlling morphology by applying shear to gelifying systems. An interesting work showed that anisotropic elongated microgel particles, with controlled shape ratio could be obtained by applying shear simultaneously to gelation. Viscosity of some biopolymers, such as gelatin, greatly depends on temperature, and tuning carefully this parameter, while applying controlled shear, allows obtaining non-spherical particles under a kinetically arrested state. The influence of shear on particle shape is illustrated by Fig. 7, showing gelled structures obtained in aqueous blends of gellan and -carrageenan [62].

Fig. 7. Morphology of gelled mixtures of gellan and k-carrageenan. The top sample was preconditioned at 60 °C and 1 Pa was applied for 10 min followed by 0.1 Pa for 110 min, and the bottom sample was preconditioned at 60 °C and 1 Pa for 10 min, followed by 0.05 Pa for 110 min. The volume fraction was 0.01 in both samples. Image widths corresponds to 500 m. (Reproduced from Reference [62], with permission)

Structures in two-aqueous phase mixtures decrease their interfacial area by minimizing the free energy of the system, with the final morphology dependent on temperature, molecular ordering and the relative phase volume of the equilibrium phases [63], [64]. As a consequence, many different transient structures, which are inherently unstable, can be observed. For example, bicontinuous structures can be formed during fast decrease of temperature that induces spinodal decomposition [64]. Interestingly, non-spherical structures can be kinetically arrested, and thus preserved, by crosslinking in the dispersed phase [63]. It should be remarked that non-spherical shapes are not unfavorable, given that interfacial energy is very low, thanks to the extremely low values of interfacial tension, and thus allowing a large interfacial area. In conclusion, a very wide variety of different shapes can be obtained (spheres, ellipsoids, rods, fibrils, etc.). There is an almost unlimited number of different morphologies, and authors are investigating different methods for controlling

size and shape, which are usually based on shearing and arresting phase separation [62], [65], [66].

A high degree of control in the droplet size of W/W emulsions can be achieved by microfluidic techniques [63], [67]–[70]. Various authors have studied in detail the application of microfluidic techniques, for the formation of water-in-water emulsions with controlled morphologies [69]. As an illustrative example, the formation of monodisperse droplets is displayed in Fig. 8. The main difference, respect to previous microfluidic methods for production of monodisperse O/W or W/O emulsions, is that the interfacial tension is very low. For this reason, the droplets are generated by applying a quite small hydrostatic pressure that induces flowing at relatively low speeds.

Fig. 8. Example of a microfluidic device that produces rather monodisperse water-in-water droplets. A small hydrostatic pressure is required, because of the ultralow interfacial tension in W/W aqueous two-phase systems. (Reproduced from Ref. [70], with permission).

4. Applications of water-in-water emulsions

For a long time, aqueous mixtures that separate in two immiscible phases (denoted as Aqueous Two-Phase Systems, ATPS, as mentioned above) have been used for the extraction of biomolecules, in a similar approach as other biliquid systems. An example is the separation of metals (cobalt and nickel) in ionic liquid based aqueous two-phase systems [11]. ATPS systems are highly useful for the separation and extraction of biomacromolecules in complex biological mixtures, and many examples can be found in the literature [8]. For example, the PEG/phosphate buffer two-phase system can be used for the extraction and purification of lipases [71].

ATPS are particularly present in mixtures of proteins and polysaccharides, which separate in two immiscible phases, one protein-rich phase and another polysaccharide-rich phase. Consequently, these systems allow the separation and extraction of proteins from mixtures of polysaccharides.

W/W emulsion droplets can be very appropriate in food formulations and functional food, as reported in a recent review on functional food formulations [72]. There are already food products that are based on W/W emulsions, mainly containing mixtures of gelatin and polysaccharides [73], despite the fact that often the colloidal nature of these products has not been stablished.

One of the most interesting applications of W/W emulsions is their use for the design of novel encapsulation and delivery systems for labile molecules [45], [74], [75]. The interesting point is that W/W emulsions can be prepared using food-grade components, and thus, the biocompatibility can be ensured. Biopolymers, such as polysaccharides and proteins, can be used to create a diverse range of delivery systems suitable for encapsulating, protecting, and controlled delivery of active components [19]. These delivery systems can be formulated by using simple processing operations (e.g., mixing, homogenizing, and thermal processing). The permeability and dynamics or release of encapsulation systems based on W/W emulsions has been reported [75], [76].

Water-in-water emulsions can be very useful as microreactors for synthesis of particles and/or products of high added value. W/W emulsions have the advantages that they maintain reaction-relevant internal environments, while allowing entry/exit of substrates and products in mild conditions. Dewey et al have demonstrated that this can be achieved in emulsions prepared in the dextran/PEG system [56], consisting of dextran droplets stabilized with liposomes. These droplets were used for performing a ribozyme cleavage reaction, using a two-piece hammerhead ribozyme. The reaction was followed by gel electrophoresis and confocal fluorescence microscopy. Another example of the use of W/W emulsions droplets as microreactors is the enzymatic synthesis of CaCO3 nanoparticles [77]. Interestingly, W/W emulsions droplets can be regarded as fully biomimetic microreactors, allowing the reproduction of biological reactions, as in this case of enzymatic mineralization processes.

One of the most studied applications of W/W emulsions is their use as templates for the preparation of microgel particles with controlled particle size. Microgels have found applications as carriers of therapeutic drugs and as diagnostic agents [67], [78]. Micro/nanogels are crosslinked polymer particles, which usually possess interesting swelling properties, dependent on external stimuli (pH, temperature, ionic strength, etc). Micro/nanogels also possess biocompatibility with an interior environment highly appropriate for the incorporation of biomolecules.

The most common method for the preparation of microgels is O/W emulsion polymerization [79], but W/W emulsion droplets might present enormous advantages, allowing the use of hydrophilic monomers and initiators in a completely mild aqueous system. Kasapis and coworkers have carried out a systematic research in the formation of gelatin microgels in the

gelatin/maltodextrin aqueous two-phase system [4], [18], [80]–[83]. This system has been deeply studied in detail because of the easy formation of both gelatin-in-maltodextrin or maltodextrin-in-gelatin emulsions, which can be kinetically trapped thanks to the gelling properties of gelatin, allowing the formation of microgels with controlled size and morphology detail [2], [19], [60], [63]. The viscosity of gelatin is highly temperature-dependent, and it gelifies below approx. 35ºC, which allows obtaining gelatin microgels by decreasing the temperature in gelatin-in-maltodextrin W/W emulsions. Other authors have focused their attention more specifically on drug release from microgels obtained in W/W emulsions, with the ability to control the kinetics of release [84], [85].

W/W emulsions can allow the design of smart particles for the encapsulation of active components. An interesting example is the formation of oil-in-water-in-water (O/W/W) emulsions [86]. First, a mixture of pectin and sodium caseinate was phase separated through a segregative mechanism. Then, casein-coated lipid droplets were added into the casein-rich phase. The application of shear lead to the formation of oil droplets (O), contained within a casein-rich aqueous phase, which was suspended in a pectin-rich aqueous external continuous phase. These O/W/W particles might be useful for the encapsulation and delivery of lipophilic components in the food, cosmetics and pharmaceutical industries [86].

As illustrated by the above examples, recent research on preparation and stabilization of W/W has opened a new field that could lead to many potential applications. The range of possible applications of W/W emulsions is expanding very rapidly, and many potential applications are being suggested and investigated, as an area of great interest. W/W emulsions can be formulated for functional food applications, or as biomimetic microreactors for biochemical processes. Another highly interesting application of W/W systems is in designing novel encapsulation and delivery systems for labile molecules. Probably scientist will focus more their attention on W/W systems, gaining new knowledge and resulting in novel technological and biomedical applications.

Conclusions

Water-in-water emulsions are very interesting colloidal dispersions consisting of droplets of an aqueous phase dispersed into another aqueous phase. These emulsions are obtained in mixtures that contain at least two incompatible polymers that form two equilibrium phases, where each phase is enriched in one of the two polymers. W/W emulsions have a wide range of applications, greatly expanding over the years, because of their all-water nature in absence of oil. Interestingly, these emulsions can be obtained in completely biocompatible systems, using mixtures of edible biopolymers, such as proteins

and polysaccharides. Consequently, W/W emulsions might have many potential applications in food formulations. Moreover, they can be used to encapsulate hydrophilic active components for drug delivery.

Interfacial tensions between the two aqueous immiscible phases are very low, and depend on the difference in composition between the two aqueous phases, along the binodal line. Interfacial tension is virtually zero at the critical point where tie-lines converge, and immiscibility vanishes, producing a merging of the two phases into a single aqueous phase. Surfactant molecules poorly adsorb on W/W interfaces, and thus, the stabilization of W/W emulsions is an important challenge for physical chemists. Often in the past, lack of stability has been the main disadvantage for using these emulsions in practical applications, and much scientific effort is currently devoted to study, control and improve the stability of W/W emulsions.

Recent discoveries have shown that W/W emulsions can display excellent stability by adsorption of particles at the water-water interface. There is much interest in the use of biocompatible particles, allowing applications in food formulations and also in biomedicine. For this reason, most of current scientific efforts are devoted to stabilization using edible and/or low-toxicity particles, made of polysaccharides or proteins. Pickering W/W emulsions in such systems have shown to be highly stable, opening many interesting possibilities for the design of novel colloidal systems.

For example, water-in-water emulsions can be very useful as templates for the formation of microgel particles with controlled particle size, morphology and responsiveness to external stimuli. These microgel particles can be highly useful in drug encapsulation and controlled release. Non-spherical W/W structures can be obtained by kinetically arresting W/W phase separation, thanks to crosslinking the dispersed phase. A very wide variety of different shapes can be obtained (spheres, ellipsoids, rods, fibrils, etc.), and much research is focused to control size and shape, usually by methods based on arrested phase separation combined with shearing.

The formation of emulsions stabilized with functionalized particles and/or microgels has opened a wide field for the smart design of functional systems, which are sensitive to external stimuli, allowing the controlled release of active components. Many interesting new developments can be expected to be published in the near future.

Acknowledgements

The author greatly acknowledges the financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2014-52687-C3-1-P project)

and Marie Sklodowska Curie Initial Training Networks (FP7-PEOPLE-2013-ITN, BIBAFOODS project). The author also acknowledges support from Generalitat de Catalunya (2014SGR1655 and TECCIT15-1-0009) and Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN).

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In this outstanding work, surface-functionalized microgels were used to properly stabilize W/W emulsion droplets. The results showed that modification of particle surface by coating with polysaccharides can be a highly potential strategy for controlling the stabilization of water-in-water emulsions.

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