Current Opinion in Colloid & Interface Science

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1 Current Opinion in Colloid & Interface Science 17 (2012) Contents lists available at SciVerse ScienceDirect Current Opinion in Colloid & Interface Science journal homepage: Nano-emulsions: Formation by low-energy methods Conxita Solans, Isabel Solé 1 Institut de Química Avançada de Catalunya, Consejo Superior de Investigaciones Científicas (IQAC-CSIC) and CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi-Girona 18 26, Barcelona, Spain article info abstract Available online 20 July 2012 Keywords: Emulsion Nano-emulsion Low-energy emulsification Self-emulsification Phase inversion temperature (PIT) Phase inversion composition (PIC) Scale-up The main developments on nano-emulsion formation by low-energy methods in the last five years are reviewed. A general description on nano-emulsions, including issues such as size-range, terminology and classification of low-energy emulsification methods is given in the introduction. Low-energy methods, which use the internal chemical energy of the system to achieve emulsification, are classified depending on whether or not changes in the surfactant spontaneous curvature are produced during the process. Nano-emulsion formation triggered by the rapid diffusion of surfactant and/or solvent molecules from the dispersed phase to the continuous phase without involving a change in the spontaneous curvature of the surfactant is referred to as self-emulsification. When changes in the surfactant spontaneous curvature are produced during the emulsification process they are designated as phase inversion methods. These are classified as phase inversion temperature (PIT) and phase inversion composition (PIC) methods if emulsification is triggered by a change in temperature or composition, respectively. Investigations on nano-emulsion formation from O/W and W/O microemulsions using different dilution procedures has set light on the factors determining small droplet size and low polydispersity. Phase behaviour studies and characterization of the transient phases formed during the emulsification process have confirmed that the mechanism by which small droplets are formed is analogue in the PIT and PIC methods. Recent advances on nano-emulsion optimization and scale-up are also reviewed Elsevier Ltd. All rights reserved. 1. Introduction The interest in nano-emulsions has experienced a continuous increase in the last years, as evidenced by the numerous publications and comprehensive reviews [1 5] on the subject. This enormous interest is triggered by the wide range of applications, namely in the pharmaceutical [6 16], cosmetic [17 20], food [21 24], chemical [25 28], etc. industries. Nano-emulsions (submicrometer-size droplets) have advantages over conventional emulsions (micrometer-size droplets) due to their small droplet size which confers them stability against sedimentation or creaming and a transparent or translucent optical aspect (similar to that of microemulsions). However, nano-emulsions, in contrast to microemulsions which are thermodynamically stable, are non equilibrium systems which may undergo flocculation, coalescence and/or Ostwald ripening. Nevertheless, with an appropriate selection of system components, composition and preparation method, nano-emulsions with high kinetic stability can be achieved. It is generally accepted [2,3,29] that nano-emulsion main breakdown process is Ostwald ripening (diffusion of molecules of the disperse phase from small to big droplets, through the continuous phase, as a consequence of their different Laplace pressure). However, recent reports have shown flocculation as a possible breakdown mechanism for Corresponding author. Tel.: ; fax: addresses: csmqci@cid.csic.es (C. Solans), Isabel.sole@iqac.csic.es (I. Solé). 1 Present/Permanent address. nano-emulsions formulated with mixed nonionic-ionic surfactants [30,31]. The misconception in the scientific literature between nanoemulsions and microemulsions, pointed out in the first issue of Emulsions and Microemulsions [32], has been addressed in the last five years. Two recent reviews [33,34] are illustrative of the effort in clarifying the differences and similarities of these colloidal dispersions. However, there is still some confusion remaining. The term microemulsion in spite of its inappropriateness (as microemulsions are neither emulsions nor their size falls in the micrometer range) is well-established in the scientific community, since it was introduced more than 50 years ago. The use of other forms, as micro-emulsion [33], couldbemisleading. The term nano-emulsion or nanoemulsion (as first introduced by Nakajima [35]) has been increasingly adopted over other terms such as miniemulsion, submicron-size emulsion or ultrafine emulsion. The authors prefer nano-emulsion to emphasize the emulsion nature of these systems. Although there is agreement in the submicrometer size of nanoemulsion droplets, no agreement exists in a size range to distinctly distinguish them from conventional emulsions. Most common sizes stated in the literature vary from 20 nm up to 500, 300, 200 or 100 nm and have been established on criteria based on optical properties and often on the intended application. Since there is not a drastic change in the physico-chemical properties when emulsion droplet size is decreased from micrometer to nanometer range, it appears that this issue will remain open /$ see front matter 2012 Elsevier Ltd. All rights reserved. doi: /j.cocis

2 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Nano-emulsions have been commonly prepared by high-energy methods using mechanical devices able to produce intense disruptive forces, namely, high-shear stirrers, high pressure homogenizers and ultrasound generators. Nano-emulsion formation by these methods is quite straightforward as the higher the energy input the smaller the droplet size. However, the level of energy required to obtain nanometer-scaled droplets is very high and therefore cost-inefficient, especially considering that only a small amount (around 0.1%) of the energy produced is used for emulsification [36]. In contrast,low-energy emulsification methods, making use of the internal chemical energy of the system, are often more energy efficient as only simple stirring is needed, and generally allow producing smaller droplet size than high-energy methods [37]. Nevertheless, depending on the system and composition variables, similar droplet sizes can be achieved by both types of methods [38]. It has been also claimed that high-energy methods allow preparing nano-emulsions at higher oil-to-surfactant ratios than low-energy methods [38]. However, nano-emulsions with high oil-to-surfactant ratios prepared by low-energy methods have also been reported [13]. It is worth noting that low-energy emulsification methods have been focussed considerable research interest in the last years. Classification of low-energy emulsification methods is also varied and often misleading. Classification based on whether or not phase inversion of the spontaneous curvature of the surfactant is produced during emulsification seems quite straightforward, and is that adopted in this review. Nano-emulsion formation triggered by the rapid diffusion of surfactant and/or solvent molecules from the dispersed phase to the continuous phase without involving a change in the spontaneous curvature of the surfactant is referred to as self-emulsification. The spontaneous emulsification method is used in the pharmaceutical industry to obtain O/W nano-emulsions as carriers for lipophilic drugs in an aqueous media, with low energy costs. They are usually referred to in the literature as self-nano-emulsifying drug delivery systems (SNEDDS) [7,9,10,16]. When changes in the surfactant spontaneous curvature are produced during the emulsification process from negative to positive (to obtain O/W emulsions) or viceversa (for W/O emulsions), they are designated as phase inversion methods. These are classified as phase inversion temperature (PIT) and phase inversion composition (PIC) methods if emulsification is triggered by a change in temperature or composition, respectively. In some publications [39,40], self-emulsification is considered as a low-energy method performed at constant temperature involving phase inversion. This definition is misleading since coincides with that of the PIC method and does not take into account those emulsification processes at constant temperature without phase inversion (e.g. in a free-surfactant system). This review reports the main developments on nano-emulsion formation by low-energy methods in the last five years. The attention is focussed first on self-emulsification methods; it follows a description on phase inversion (PIT and PIC) methods and finally on recent advances on nano-emulsion optimization and scale up. 2. Nano-emulsion formation by self-emulsification methods Self-emulsification or spontaneous emulsification methods make use of the chemical energy released due to a dilution process with the continuous phase, generally at constant temperature, without any phase transitions (no change in the surfactant spontaneous curvature) taking place in the system during emulsification. When diluting, diffusion of water-miscible component(s) (solvent, surfactant and/or cosurfactant) from the organic phase into the aqueous phase (to obtain O/W nano-emulsions) is produced, which results in a dramatic increase of the interfacial area, giving rise to the metastable emulsion state, as discussed early by Miller [41] and later by Bouchemal et al. [42]. The experimental conditions reported in the literature in order to obtain droplets in the nanoscale range with this spontaneous emulsification method are related to a very high solvent/oil ratio. The solvent diffusion is hence even quicker and the turbulence generated causes nano-scaled droplets to form. In a free-surfactant system, self-emulsification is produced by the so called Pastis or Ouzo effect [43 45]. These well-known anise-flavored alcoholic beverages are nano-emulsions produced when a large amount of water is added to a three-component homogeneous solution composed of water (about 55%), alcohol (e.g. ethanol, about 45%) and an anise-flavored oil, mainly trans-anethol (about 0.1%), which is soluble in ethanol, but insoluble in water. While diluting, some of the alcohol molecules move out of the organic phase into the aqueous phase, which causes the flavour oils to be no longer soluble, and small oil droplets spontaneously form in the solution. The Ouzo effect has recently been used to obtain nano-emulsions as templates for the preparation of nanoparticles in a variety of systems [8,25,26]. Both PLGA (poly(lactic-co-glycolic acid)) [8] and PMMA (polymethylmethacrylate) [25,26] nanoparticles have been obtained by addition of water to a solution of PLGA in acetone, or to a solution of PMMA in acetone or tetrahydrofuran, respectively. For specified concentrations, supersaturation allows the formation of stable nanoparticles. The formation of nano-emulsions by self-emulsification through dilution of O/W microemulsions with an alcohol as cosurfactant was first reported by Taylor and Ottewill [46] for the water/sds/pentanol/ dodecane system. The nano-emulsiondropletformationwasexplained as due to alcohol diffusion from the oil droplets into water upon dilution, being the microemulsion no longer thermodynamically stable as the surfactant concentration is not high enough to maintain the very low interfacial tension required (γb10-2 N m -1 ) for thermodynamic stability, becoming a nano-emulsion (Fig. 1). The relation between initial microemulsion structure (i.e. O/W and W/O) and dilution procedure with the properties of the resulting nano-emulsions after dilution with water has been recently reported [47] using the same surfactant system except for the alcohol (pentanol was substituted by hexanol). Fig. 2-A shows the dilution paths starting emulsification from both O/W (W m X) and W/O (O m X) microemulsions in the water/sds/hexanol/ dodecane system. The interest of this work lies in the fact that different dilution procedures were tested, which consisted on the addition of water (or microemulsion) over microemulsion (or water) stepwise or at once, at constant temperature, as shown in Fig. 2-B. Starting emulsification from O/W microemulsions, small droplet-size nano-emulsions are always obtained, independently on the microemulsion composition and the dilution procedure used, as exemplified in Fig. 2-C for a microemulsion with an oil-to-surfactant (O/S) ratio of 48/52 (W m 4in Fig. 2-A). It should be noted that no change in the surfactant spontaneous curvature is involved during the process. Therefore, the mechanism by which nano-emulsions are formed is self-emulsification: dilution with water induces part of the cosurfactant molecules to dissolve into water, the system becomes no longer thermodynamically stable giving rise to the nano-emulsion droplets. In contrast, starting emulsification from W/ O microemulsions, the results obtained depended on both dilution type and/or composition of the starting microemulsion. From microemulsions with low O/S ratios, such as O m 1inFig. 1-A (O/S ratio of 12/88), a turbid emulsion, rapidly separating into two phases, was obtained wathever the dilution procedure used. It should be pointed out that along this emulsification path no direct microemulsion domain (W m )iscrossedduring emulsification. In contrast, starting from W/O microemulsions with higher O/S ratios (e.g. O m 2-O m 5), small droplet-size nano-emulsions are obtained when the dilution procedure used allows reaching the equilibrium in an O/W microemulsion domain during emulsification. These conditions are achieved by stepwise addition of water over W/O microemulsions (dilution procedure I in Fig. 2-B) with O/S ratios at which a direct microemulsion domain is crossed during emulsification (O m 2-O m 5inFig. 2-A). Fig. 2-C illustrate this result for microemulsion O m 4. For the other dilution procedures used (dilution procedures II, III and IV in Fig. 2-B), bimodal distributions were obtained (Fig. 2-C). This strategy can be considered as a two-step emulsification process. In the first step, addition of water involves a change in the surfactant

3 248 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Fig. 1. Schematic representation of the proposed mechanism for self-emulsification by dilution of an O/W microemulsion: by dilution with water the cosurfactant difusses from the oil/water interface to the water phase, which makes the microemulsion no longer thermodynamically stable, obtaining a nano-emulsion. spontaneous curvature, from negative (W/O microemulsion) to positive (O/W microemulsion), while in the second step a self-emulsification process is taking place, as no change in the surfactant spontaneous curvature is produced. These results clarify previous reported conclusions [48], which indicated that starting emulsification from W/O microemulsions resulted always in bigger droplet size nano-emulsions than starting from O/W microemulsions. However, the effect of the dilution procedure had not been considered, as only one-step addition of water over microemulsion had been tested. The recent results by Sole et al. [47] have clearly shown that the same small-size nano-emulsions can be obtained starting emulsification from both W/O and O/W microemulsions by selecting an appropriate dilution procedure and composition. Nano-emulsions can also be formed by dilution of surfactant aggregates other than microemulsions. Indeed, the formation of O/W nanoemulsions by dilution from a direct cubic liquid crystalline phase in mixed non-ionic/ionic surfactant systems such as water/potassium oleate/oleic acid/c 12 E 10 /hexadecane and water/oleylammonium chloride/ oleylamine/c 12 E 10 /hexadecane has been reported [37,49]. Phase behaviour studies showed the presence of a direct cubic liquid crystalline phase with Pm3n structure in the range of co-surfactant(oleic acid or oleylamine)/c 12 E 10 ratios from 20/80 to 50/ 50, at water concentrations from 50% to 75%. In both systems, during dilution, the micelles that were packed into the cubic network separate, disrupting the cubic structure, and at the same time a part of the surfactant that was stabilizing the interface can migrate to the bulk aqueous phase, destabilizing a part of the interface, giving rise to the final nano-emulsion droplets (Fig. 3). It is worth noting that no change in surfactant spontaneous curvature is produced during the dilution step. Structural studies by SAXS (in the cubic structure) and DLS (in the final nano-emulsions) performed in both cationic and anionic surfactant systems confirmed that the size of the micelles that form the cubic structure is the same or slightly smaller than the size of the nano-emulsion droplets obtained (Fig. 4). 3. Nano-emulsion formation by phase inversion These methods make use of the chemical energy released by phase transitions taking place during the emulsification process. Although these phase transitions often involve the inversion of the surfactant film curvature from positive to negative or viceversa, it has been shown that transitions from structures having a surfactant film with an average zero curvature (e.g. bicontinuous microemulsions or lamellar liquid crystalline phases) are those playing a key role in nano-emulsion formation [2,30,31,50 59]. Knowledge on surfactant phase behaviour is important when these emulsification methods are used, since the phases involved in the emulsification process are determinant to obtain nanoemulsions with minimum droplet size and low polydispersity. Nevertheless, the kinetics of the emulsification process may play also an important role in the properties of the resulting nano-emulsions, especially if highly viscous phases (e.g. hexagonal or cubic liquid crystalline phases) are formed during emulsification. The phase transitions are triggered either by changing the temperature (Phase Inversion Temperature Method, PIT) or the composition (Phase Inversion Composition Methods, PIC). The PIT method, introduced by Shinoda in 1968 [60] is based on the changes in the surfactant spontaneous curvature induced by temperature. A schematic representation of the PIT method to form O/W nano-emulsions can be observed in Fig. 5. The PIT method can only be applied to surfactants sensitive to changes in temperature, i.e. polyoxyethylene-type nonionic surfactants in which changes in temperature induce a change in the hydration of the poly(oxyethylene) chains, and a consequent change of its curvature. In the PIC method, the phase transitions are induced by changes in the composition during emulsification,atconstanttemperature(schematically represented in Fig. 6). Consequently, it can be applied to surfactants other than ehtoxylated-type. Nevertheless, phase behaviour studies [51 56] as well as structural characterization, namely by SANS and NMR [30,31,57 59,61], have clearly shown that the phase transitions encountered (lamellar liquid crystalline phases or bicontinuous microemulsions) during the emulsification process leading to nano-emulsion formation are common for PIT and PIC methods, implying that they are governed by the same mechanisms. The procedure to obtain nano-emulsions by the PIT method consists on preparing the sample at its Phase Inversion Temperature (PIT) or Hydrophile-Lipophile Balance (HLB) temperature [62], where the hydrophilic and lipophilic properties of the system are balanced (e.g. the mean spontaneous curvature of the surfactant molecules is zero) and extremely low interfacial tensions ( mn m -1 ) are achieved [63] to promote emulsification. In such conditions, very small droplet sizes can be obtained. However, since the curvature of small droplets is very high and around the HLB-temperature the spontaneous surfactant curvature is near zero, the barriers that oppose coalescence processes are low and coalescence rate is extremely high [64,65].Consequently, at the HLB temperature, although emulsification in very small droplets is favoured, the emulsions are very unstable. The temperature has to be quickly moved away from the HLB-temperature by a rapid cooling or

4 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Fig. 2. (A) Phase diagrams (T=25 C) of water/sds/hexanol/dodecane system. Dashed and solid lines indicate the emulsification paths followed in the preparation of nano-emulsions starting from both O/W (W m X) and W/O (O m X) microemulsion domains. Final water concentration was kept constant at 98%. Adapted from reference [43]. (B) Scheme showing the emulsification procedures used to form nano-emulsions: (I) stepwise addition of water into microemulsion, (II) addition of water into microemulsion in one step, (III) stepwise addition of microemulsion into water, and (IV) addition of microemulsion into water in one step. W m refers to O/W microemulsions and O m to W/O microemulsions. (C) Nano-emulsion droplet sizes obtained as a function of the emulsification method used when dilution of an O/W microemulsion (W m 4) and a W/O microemulsion (O m 4) with the same O/S ratio. Adapted from Ref. [47]. heating (obtaining O/W or W/O emulsions, respectively) to obtain kinetically stable nano-emulsions. If the cooling or heating process is not fast enough, coalescence predominates and polydisperse emulsions are formed. Emulsification studies by the PIT method in several water/nonionic surfactant/aliphatic oil systems at constant W/O ratio as a function of surfactant concentration and temperature showed that the lowest droplet sizes were obtained starting emulsification from one-single microemulsion phase [52,54,56]. However, further emulsification studies in the water/c 16 E 6 /mineral oil, at constant O/S ratio as a function of water concentration and temperature revealed that nano-emulsions with minimum droplet size can be obtained independently on the initial phase equilibria (i.e. one bicontinuous microemulsion phase or two phases consisting of bicontinuous microemulsion and excess water) provided that all the oil and surfactant are in the same phase [52,56]. This was a clear indication that droplet formation is mainly controlled by the structure of the bicontinuous microemulsion phase, and the excess water acts as a dilution medium. In the last five years most of the literature dealing with nano-emulsion formation by the PIT method has been concerned with the use of nano-emulsions as carriers for different type of actives [11,21,23], and as templates for nanoparticle preparation [6,15]. Interesting contributions concerning the PIT emulsification process are of those by Roger et al. [58,66] which re-examined this process in the water/c 16 E 8 /hexadecane system. By keeping the W/O ratio constant, they observed that under gentle shear the system was homogeneous few degrees below the PIT, regardless the surfactant concentration, while at equilibrium the system might consist of one, two or three phases, depending on surfactant concentration. They designated this threshold temperature as clear boundary, and showed that cooling from the clear boundary (sub-pit method), nano-emulsions were of the same size as that obtained cooling from or above the PIT. They evidenced that shearing a bicontinuous microemulsion with excess oil gives rise to an O/W microemulsion (with all the oil solubilised) as a metastable structure along the clear boundary. An advantage of the sub-pit method over the PIT is the lower temperature at which the emulsification process is initiated by a given concentration of surfactant. The sub-pit method has been applied by other authors [67] using mixed nonionic-cationic surfactant systems to produce positively charged O/W nano-emulsions. It could be argued whether the so-called sub-pit emulsification method should be regarded as an inversion emulsification method, since from the initial state, clear boundary (O/W microemulsion), to the final state, O/W nano-emulsion, no change in the spontaneous curvature of the surfactant is produced. The procedure to obtain nano-emulsions by the PIC method consists on progressively adding one of the components (water or oil) over a mixture of the other two components (oil-surfactant or water-surfactant, respectively) [15,55,57,68 70]. The PIC method is considered to have a greater potential for a large-scale production than the PIT because it is experimentally easier to add one component to a large volume of emulsion than to produce a sudden change in temperature. Moreover, PIC method is also preferred when dealing with components with temperature-stability problems. Furthermore, as discussed above, this method is not restricted to POE-type surfactants. Nevertheless, this type of surfactants have been widely used for the preparation of both O/W and W/O nano-emulsions by the PIC method. The initial system, when water is added progressively into the oil phase to form an O/W nano-emulsion, is generally a water-in-oil microemulsion. As the volume fraction of water increases, the hydration grade of the POE chains of the surfactant progressively increases, and the surfactant spontaneous curvature change from negative to zero. Around this transition composition, surfactant hydrophilic-lipophilic properties are balanced, as at the HLB-temperature. Consequently, bicontinuous or lamellar structures are formed. When the transition composition is exceeded, the structures with zero curvature separate into metastable small direct (O/W) droplets which still contain the oil, and have a very small diameter, which implies a very high positive curvature of the surfactant layer. Therefore, the mechanism by which small droplets are formed is analogue to that of the PIT method. Most of the published work on formation of nano-emulsions by the PIC method, as with the PIT, is related to their use as carriers for different types of actives [9,16] and also for nanoparticle preparation [6,13,14]. Concerning studies on the phase inversion process leading to O/W nano-emulsions by the PIC method, that of Sonneville-Aubrun et al. [57] using the water/polyethylene glycol 400 monoisostearate/hydrogenated polyisobutene system is of particular interest. A transient phase with zero mean curvature (identified as a lamellar liquid crystalline phase) at very short times after water addition, was detected by

5 250 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Fig. 3. Schematic representation of the proposed mechanism for self-emulsification by dilution of a cubic liquid crystal with Pm3n structure: dilution with water makes the micelles that form the cubic phase separate, while the cosurfactant difusses from the oil/water interface to the water phase. These makes the micelles no longer thermodynamically stable, obtaining a nano-emulsion. Adapted from Ref. [49]. SANS. Nano-emulsions with homogenenous size distribution and a mean diameter of 100 nm were reported in the studied system. The structural transformations taking place during emulsification by the PIC method have been also investigated by Roger et al. in the water/ C 16 E 8 /hexadecane system [59], which was previously used by the same authors to investigate the PIT method [58]. It was showed that the emulsification proceeds through the swelling of a reverse micellar phase, with the consequent formation of a bicontinuous sponge phase. Then, further water addition produces the nucleation of oil droplets in the sponge phase. However, it was pointed out that part of the surfactant remains adsorbed on the droplets, and the rest is expelled as micelles that coexist with the droplets, which leads to bimodal size distributions, in contrast to the PIT emulsification method in which monomodal distributions were obtained using the same system [58]. The different size distributions obtained by both methods were attributed to the fact that in the PIT (sub-pit) method, the initial state is an oil/ water droplet-like structure with all the oil solubilised while emulsification proceeds through expulsion of oil from an homogeneous state by the PIC method. It was concluded that PIT is a more efficient method than PIC to obtain small droplets and narrow size distributions [59]. O/ W nano-emulsions with bimodal size distributions prepared by the PIC method have been also reported by Heunemann el al [61]. In a systematic study, involving characterization of the system at different concentration of the dispersed phase and the final O/W nano-emusion state by SANS and cryo-tem, showed that at an intermediate water content a two-phase system of bicontinuous structure is formed. They also reported that the relative proportion of each population depends on the amount of added water and the smaller droplet size is consistent with the size of microemulsion droplets. However, there have been also reports on nano-emulsions obtained by the PIC showing small droplet sizes (as low as 20 nm) and low polydispersity values [51,55,57]. An aspect that should be taken into account in the emulsification by low-energy methods is the kinetics of the process, especially when transient viscous phases are formed. In this context, Sole et al showed [37,49] that when a cubic liquid crystalline phase was involved in the emulsification process either monomodal or bimodal size distributions were obtained depending on the agitation speed used and on the water addition rate. Dependence of droplet size distribution on stirring speed and surfactant concentration have been also reported [40]. In many investigations and practical applications, nano-emulsion formation is achieved by a combination of low-energy methods, (e.g. phase-inversion and self-emulsification). This is generally referred to in the literature as two- step emulsification processes. Wang et al. reported a two-step emulsification process at constant temperature [30,31,71]. Inthe first step, the system is brought to a fixed composition where, at equilibrium, a bicontinuous microemulsion or a lamellar liquid crystalline phase is present. In the second step, dilution with water resulted in the formation of nano-emulsions, involving a curvature change from almost zero to positive. These authors also compared the size of nano-emulsions obtained by this two-step method with other two methods [71]. One of the methods was dropwise addition of water to the oil-surfactant mixture and the other method consisted in mixing all the components at the final composition. The smallest droplet sizes were obtained by the two-step method and by dropwise addition Fig. 4. Comparison of the radius of the micelles that form the cubic liquid crystal with the radius of the nano-emulsion droplets obtained for several emulsification paths for the W/ potassium oleate-oleic acid-c 12 E 10 /hexadecane ionic system. Adapted from Ref. [49].

6 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Fig. 5. Schematic representation of the formation of nano-emulsions by the PIT method. water. The droplet sizes were coincident by both methods. It was pointed out that the ways the single-phase regions are reached are not relevant if equilibration when reaching these phases is assured. Moreover, if the initial concentrate in the two-step emulsification process is not a single phase, but an emulsion, opaque emulsions are generated [71]. The droplet sizes of nano-emulsions were determined both by dynamic light scattering (DLS) and small angle neutron scattering (SANS), and a discrepancy was observed, which is consistent with long-range droplet interaction occurring outside of the SANS sensitivity range [30,31]. It should be noted that when the concentrate reached in the first step of the emulsification process is an O/W microemulsion, no inversion takes place upon further dilution and consequently the second step consist on self-emulsification. The PIC method has also been recently used to form O/W nanoemulsions using as an oily phase a highly polar solvent, such as ethyl acetate, containing a preformed polymer (a hydrophobically modified polysaccharide) [13]. It was shown that the low-energy emulsification methods are not only valid for aliphatic and semi-polar oils, but also for polar solvent/preformed polymer mixtures. Conductivity determinations along the emulsification path confirmed that a phase inversion from W/O structures to an O/W nano-emulsion takes place. Nano-emulsion droplet sizes obtained at a water content of 90%wt. were around 200 nm and showed low polydispersity index. Nano-emulsion formation and stability has been reported [72] in a system with rapeseed oil where the presence electrolytes was found to have an important role. 4. Tunability and scale-up Application of nano-emulsions requires optimization studies for achieving the best properties. This optimization is required not only respect to formulation variables, but also regarding preparation variables (for example mixing rate or addition rate) to obtain the desired characteristics. This dependence of nano-emulsion characteristics on preparation variables is a direct consequence of them being thermodynamically unstable systems [1]. Also, a scale-up to a higher level of production is necessary for industrial applications. An accurate scale-up study allows predicting the properties of a system at different scales. Fig. 6. Schematic representation of the formation of nano-emulsions by the PIC method.

7 252 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) Fig. 7. (A) Vessel and stirrer geometries used in both scales of a 100 g production and 644 g production, (B) Response surface showing the dependence of nano-emulsion droplet size on both addition rate and mixing rate in the system water/potassium oleat-oleic acid-c 12 E 10 /hexadecane for the two scales. Adapted from Ref. [73]. Systematic studies of the influence of formulation and preparation variables on the properties of the final nano-emulsions, using the experimental design technique, have been reported [1,37,49,73,74]. These studies use the response surface methodology (RSM) based on a Central Composite Design (CCD) in order to avoid the traditional methodology that consists on maintaining all the variables constant during test runs except for the one being studied. This type of experimentation assumes that variables are independent of each other, and that the effect of changing one variable would be the same at other levels of the remaining variables, not being very adequate for observation of interactions between them. The use of experimental design seems, then, to be more adequate. In a recent study [74], nano-emulsions obtained by the PIC method in the system water/tween 80-Span 80/paraffin oil were optimized with respect to the temperature of preparation, the O/S ratio, and the Hydrophilic-Lipophilic Balance (HLB). The use of the experimental design technique allowed illustrating the cross-interactions between these parameters through response surface maps. Regarding the scale-up of nano-emulsions to an industrial level of production, some studies have been published using high-energy methods [75], however, less information is available concerning low-energy methods. This issue has been addressed by Sole et al. in nano-emulsions prepared by the PIC method in different systems [73]. The production of nano-emulsions was scaled-up from lab scale (100 g) to medium scale (644 g). In all studied systems, nano-emulsions were produced by adding the aqueous phase over the oil/surfactants mixture. It was considered that inbothscalestheremustbeageometricsimilarity,thisis,theformfactors of both the stirrer and the vessel used have to be the same at both scales (Fig. 7-A). Also, mixing and addition conditions must be equivalent in both scales, in order to maintain the same mixing level at any point and any time into the vessel, allowing the proper transition of phases during emulsification. In order to assure a proper mixing level, the emulsification process is modelled, assimilating the system to an isotherm mixture of two fluids, A (water) and B (oil+surfactant mixture), in an agitated tank [76]. The variables addition and mixing rates are converted into total time (t t,t t =vessel volume/addition rate) and linear mixing rate (v c,v c =mixing rate*stirrer diameter) in both scales. Fig. 7-B shows the response surfaces obtained by using the experimental design technique for optimizing the preparation variables (mixing and addition rate) at a fixed composition for two different scales (100 g and 644 g) in the system water/potassium oleate-oleic acid-c 12 E 10 /hexadecane. It can be seen a nearly perfect correlation between both scales. Treating results in both scales together, droplet size was modelled to be predicted as a function of total time (t t ) and linear mixing rate (v c ) in the range of variables tested, and geometries (form factors) used. The resulting equation obtained predicted data really well, with an adjusted statistic R 2 indicating that the model as fitted explains 95.3% of the variability in droplet size. These results showed that the method of scale-up through the two dimensional variables total time and linear mixing rate is suitable for the studied system. 5. Conclusions Nano-emulsions have experienced a continuous and growing interest as, due to their characteristic properties (namely small size, high interfacial area and transparent optical properties), they are advantageous over other colloidal systems for a wide range of applications. It is worth noting the interest on nano-emulsions for pharmaceutical applications as the number of publications on this subject outweights those dealing with cosmetic, agrochemical, food, chemical, etc. applications. Lowenergy emulsification methods have focussed considerable research interest in the last years as small droplet sizes and narrow size distributions can be obtained using simple equipment. Review on formation of nano-emulsions by low-energy, self-emulsification and phase inversion (PIT and PIC) methods, has evidenced the progress in the knowledge of the factors leading to nano-emulsions with minimum size and low polydispersity. The importance of the presence of surfactant aggregates with an average zero curvature (e.g. bicontinuous microemulsions and lamellar liquid crystalline phases) during emulsification by phase inversion methods, which had been earlier predicted from equilibrium phase behaviour studies, has been confirmed by characterization of the transient structures that form during the process. For this characterization, SANS has been a valuable tool. The results gathered so far evidence that both the PIT and PIC are governed by the same mechanisms. However, there are still issues to be solved. One of them concerns the possibility

8 C. Solans, I. Solé / Current Opinion in Colloid & Interface Science 17 (2012) to obtain nano-emulsions with minimum droplet size and low polydispersity by the PIC method. It is likely that the kinetics of the emulsification process play an important role in this emulsification method which has not been taken sufficiently into account. Therefore, more research effort needs to be done on this subject. A more comprehensive knowledge on the mechanisms involved in nano-emulsion formation by low-energy methods, will allow their optimization and consequently will span their applications. Acknowledgements Financial support from Spanish Ministry of Economy and Competitivity, MINECO (grant CTQ CO3-01) and Generaltit de Catalunya grant 2009SGR-00961) is acknowledged. 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