Double emulsions prepared by two–step emulsification: History, state-of-the-art and perspective

Accepted Manuscript Double emulsions prepared by two–step emulsification: History, state-of-the-art and perspective

Shukai Ding, Christophe A. Serra, Thierry F. Vandamme, Wei Yu, Nicolas Anton PII: DOI: Reference:

S0168-3659(18)30740-5 https://doi.org/10.1016/j.jconrel.2018.12.037 COREL 9587

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

6 September 2018 18 December 2018 19 December 2018

Please cite this article as: Shukai Ding, Christophe A. Serra, Thierry F. Vandamme, Wei Yu, Nicolas Anton , Double emulsions prepared by two–step emulsification: History, stateof-the-art and perspective. Corel (2018), https://doi.org/10.1016/j.jconrel.2018.12.037

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ACCEPTED MANUSCRIPT Double emulsions prepared by two–step emulsification: History, State-of-the-art and Perspective Shukai Ding,1,2 Christophe A. Serra,2,* Thierry F. Vandamme,3 Wei Yu,2 Nicolas Anton3,* 1

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Shaanxi University of Science & Technology, Materials Institute of Atomic and Molecular Science, CN-710021, Xi'an, Shaanxi, China 2 Université de Strasbourg, CNRS, ICS UPR 22, F-67000 Strasbourg, France 3 Université de Strasbourg, CNRS, CAMB UMR 7199, F-67000 Strasbourg, France

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To whom correspondence should be addressed: Dr. Nicolas Anton ([email protected]), Pr. Christophe A. Serra ([email protected])

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Abstract

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Attractive interest on double emulsions comes from their unique morphology, making them general multifunctional carriers able to encapsulate different hydrophilic and lipophilic molecules in the same particle. Over the past century, two different types of methods were followed to prepare double emulsions for pharmaceutics applications, so-called “one-step” and “two-step” processes. The two-step approach, consisting in two different emulsifications successively performed, allows the optimal and more efficient formulations due to simplicity of principle and controllability of the process. In this review, focused on the formulation of double emulsions by two-step process, we recount the historical development of this approach, along with the state-of-the-art, including a discussion on the role of the formulation parameters, surfactants, amphiphilic polymers, interface stabilization, volume fraction, and so forth, on the final formulation stability, morphology and properties as drug delivery system. Discussion was also extended to polymeric microparticles and nanoparticles made by solvent diffusion, on the basis of double emulsions made by two-step process, along with literature review on the impact of different formulation and processing parameters. In addition, the properties of the polymers used in the microparticles matrix (molecular weight, chemical nature) potentially impacting on the ones of the microparticles formed (drug release kinetics, stability, morphology), were also discussed. Finally, the future trends in double emulsions application were addressed, emphasizing some new advances made in the emulsifications method as potentially able to open the range of applications, for example to nanoscale with spontaneous emulsification or low energy microfluidic emulsification.

Keywords Double emulsions; Two-step emulsification; Polymeric particles; Drug release; Microfluidic system; Encapsulation of hydrophilic materials.

ACCEPTED MANUSCRIPT Introduction

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Double emulsions, so-called Water-in-Oil-in-Water emulsions (W/O/W emulsions), consist of water droplets dispersed in oil or oil/polymer globules, themselves ultimately dispersed in an aqueous phase (Figure 1). Such a structure appeared highly interesting to encapsulate very different and even incompatible molecules (e.g. hydrophobic and hydrophilic) in a single carrier. Over the past thirty years, double emulsions has known an important interest in the formulation of pharmaceutics, including drug carriers in cocktail therapy [1, 2], substitute blood [3], vaccines[4], vitamins [5], and enzymes [6]. Therefore, the design of double emulsions was extensively modified in function of specifications and the diversity of the aimed applications. Indeed, different types of double emulsions have been developed such as oil-based double emulsion [7], polymeric microparticles [8], nanoparticles [9], nanocapsules [10], double emulsions template for the generation of micelles and liposomes [11]. However, the common particularity of all these systems lies in the fact that they were prepared by a two-step emulsification process. It follows therefrom that a deep understanding of this “two-step” emulsification process appears crucial for preparation, adaptation and optimization of double emulsions. Literature reviewed several specific aspects related to double emulsions, such as their structural stability [12], the transport phenomena between the different phases [13], double emulsion based polymeric carrier [14], microfluidic formulation of double emulsions [15] and so forth. To our best knowledge, even if it is a fundamental aspect of the design of double emulsion based particulate carriers, the two-step emulsification method has never been specifically reviewed. This aspect will precisely be the main interest of the current review, which namely will focus on two aspects: (1) the effect of formulation parameters, nature of the phases, volume fractions, additives’ concentration, and (2) the effect of processing parameters of emulsification. These two aspects of the process will be discussed in relation with their impact on their physico-chemical properties, size, size distribution and stability. In addition, since they are quite different from classical W/O/W double emulsions, the other complex specific systems like oil-in-oil-in-water (O/O/W) or water-in-water-in-oil (W/W/O) emulsions, will not be included in the scope of the current review. In this context, the discussion will be organized around the evolution of two-step emulsification process, in relation with the types, sizes and nature of the emulsions, and type of encapsulated molecules. Specific aspects herein reviewed will include: the given advantages of two-step over one-step processes, the efforts undertaken regarding the stabilization of double emulsions, aspects related to the controlled drug release, surface modification, the precise control of the numbers of inner water droplets, and finally to microfluidic tools used to prepare polymeric particles or double emulsions.

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Figure 1 Schematic drawing of a double emulsion structure

History of Double emulsions by two-step emulsification

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Advantages of double emulsions and two-step emulsification

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The double emulsions were firstly presented in 1925 by William Seifriz [16], included in research works investigating the impact of oil density on the type of emulsions formed. For instance, an emulsion prepared with straw-oil (having a density of 0.882 kg.L-1) exhibits an atypical behavior since it can form a complex system consisting in a coarse oil-in-water emulsion made of oil globules (about 1 mm) that encapsulate a fine water-in-oil emulsion. This example was actually the first model of double emulsions reported. However, these double droplets rapidly destabilized and turned to single emulsions, thus were considered as a transitional and instable state between Water-in-Oil (W/O) and Oil-in-Water (O/W) emulsions [17]. The reasons of this intrinsic instability have been explained by physical reasons, i.e. originated from osmotic pressure and Laplace pressure [18]. The difference of solute concentration (related to osmotic pressure) between inner water and continuous aqueous phases induces a water migration between these two phases in order to re-equilibrate osmotic pressures. Higher osmotic pressure in external phase induces the swelling of inner droplets, while, lower osmotic pressure results in the shrinkage of inner droplets. Accordingly, concentration or osmotic pressure is a crucial parameter as regards to the double emulsion stability. As such the Laplace pressure (that depends on the droplets size and surface tension) induces a very high pressure inside inner droplets compared to the one of the oil globule. This difference leads to the pressure re-equilibration and ultimately to the collapse and rupture of double emulsions structure. Interestingly, these particularities of double emulsions, that make them fragile in some extent, were also taken beneficial as a way to control the release of encapsulated materials stimulated by osmotic pressure. Thanks to their versatile and instable structure, the first application was designed for encapsulating insulin as drug carrier in the late 1960s, in fact thirty-five years after discovery of double emulsions [19]. However, the main advantage of double emulsions lies in the possibility they offer to encapsulate several types of molecules simultaneously.

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In 1976, Matsumoto et al. [20] firstly reported the two-step emulsification standard procedure for preparing double emulsions (Figure 2). First, a W/O emulsion is prepared in accordance with the Bancroft rule, and then the double W/O/W emulsion is obtained by using the former W/O emulsion as the oil phase for the second emulsification step. Since then, the two-step emulsification method became the most prevalent method to get double emulsions [21]. On the other hand, the one-step emulsification process, in which the double emulsion spontaneously form, has always got attraction owing to the simplicity of the process [22]. This approach however suffers of limitations such as the encapsulation efficiency that only remains quite low (ratio between encapsulated and total amount of species).

Figure 2 Schematic drawings of the two-step processe to produce double emulsions

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Choosing surfactants: 1. Impact on the double emulsions stability

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Especially for the two-step emulsification process, the choice of the surfactants is a crucial parameter to insure the stability of each independent emulsion. In principle, a lipophilic/hydrophobic surfactant characterized by a HLB (Hydrophilic Lipophilic Balance) value lower than 7 (commonly around 3-4) should be chosen for the preparation of the primary W/O emulsions. Reverse droplets are in general adhesive and subject to flocculation and coalescence, the use of very-low HLB or lipophilic macromolecules are preferred to prevent this destabilization of internal droplets. On the other hand, hydrophilic surfactant (HLB value higher than 10) is necessary to stabilize the external surface of double globules after second emulsification. Eventually, choice of surfactants is essential to formulate stable double emulsions, but also to play on their size, encapsulation efficiency. Historically, the stability of double emulsions was the main key parameter taken into account in the quality of double emulsions. As a result, an important research effort was dedicated to this aspect and resulted in several efficient formulation strategies to increase double emulsions stability. To this end, the so-called optimal HLB was defined and corresponded to the HLB of surfactant mixture giving the most stable emulsion. Garti et al. [23] reported the preparation of stable W/O emulsions obtained by tailoring the HLB value of surfactant mixture, the so-called required HLB related to the oil chosen in the formulation. In their work, W/O emulsion with the best stability was simultaneously obtained with value HLB close to 4-5, either using a single amphiphile brij 93 at 8 wt.% (polyethylene glycol oleyl ether), with HLB value equal to 4.9, or a mixture of Span 80 (sorbitan oleate) and Span 85 (sorbitane trioleate) at

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10 wt.% with a HLB value equal to 4. Any separation of inner water droplets from the oil phase was observed after 30 days, and regarding the water droplets size, only a slight increase from 0.5 µm to 3 µm was reported [23]. On the other hand, the stability of W/O emulsions can be also affected and strengthened with the addition of a large amount of hydrophobic surfactants. Thus, a stable W/O emulsion was obtained with Span 80 at a concentration as high as 30 wt.% [20] but it was believed, as a result of the migration of the Span 80 towards the external interface, that its global effective concentration was reduced. Consequently, the corresponding high concentration of hydrophilic surfactant appeared necessary to balance and stabilize the double structure, while the opposite effect was observed with high concentration of hydrophilic surfactant, i.e. low encapsulation efficiency, uncontrollable droplets size and stability. It thus seemed imperative to develop alternative methods to stabilize such a fragile structure. Garti et al. [24] proposed a pioneer study using polymeric surfactant to increase the stability of W/O emulsions, resulting in a more stable interface and lower desorption and migration of stabilizing molecules. A polymeric surfactant was synthesized through a reaction between polymerized soybean oil and polyglycerol, giving rise to molecular weights ranging from 881 to 3758 g/mol. Importantly, these authors showed that the stability of W/O emulsions increased with the molecular weight. In addition, an excellent stability of W/O emulsions can be obtained at weight fractions of internal phase as high as 50 wt.%, even at low concentration of polymeric surfactant, around 3 - 5 wt.%. In comparison, to reach a similar level of stability with non-polymeric (i.e. classical) surfactants, concentrations must be increased up to 20 -25 wt.% [24]. Additionally, the concentration of hydrophilic surfactant in the external phase can also play a significant role on the double emulsions stability. As a result of an increased concentration of hydrophilic surfactants, it has been observed that the double droplets were smaller with an increased stability, and in the same time the encapsulation efficiency was decreased [25]. This phenomenon was explained by the effects of these surfactant excess, even in the internal droplets interface (stabilized by hydrophilic surfactant) affecting the established balance among three phases. Solving this trade-off problem was found in using a mixture of ionic-surfactant and nonionic hydrophilic surfactants, and showed significant improvements in terms of encapsulation efficiency and stability [23, 26]. This result is likely due to the much more decreased solubility of the ionic surfactants in the lipophilic phases, along with, on the other hand, some intrinsic incompatibilities in term of potential interactions with encapsulated materials or toxicity. Playing a crucial role in the encapsulation efficiency and stability, the choice of hydrophobic surfactant is fundamental. Generally considered through its low HLB value (HLB ≤ 4), some other factors need to be considered in order to optimize the results [27, 28]: firstly the “rigid” molecular structure (for example with a high degree of unsaturation), and secondly, better compatibility or solubility of the surfactant in the oil phase (for example oil having a structure close to the aliphatic part of the surfactant), are important factors that will increase the stability of primary W/O emulsion. It follows that a lipophilic surfactant with an important degree of unsaturation, formulated with unsaturation oil made with a similar structure, could be optimized conditions to increase the double emulsion stability.

Choosing surfactants: 2. Impact on the encapsulation efficiency The second major challenge regarding the formulation of double emulsion is the encapsulation efficiency, actually intimately related to the stability. High encapsulation efficiency, according to literature [29], corresponds to an optimum HLB value between hydrophilic and lipophilic surfactants in external phase and in oil, respectively. In the cited example, the authors shows the similar encapsulation efficiency for different concentrations of surfactants (mixture of Span 20 and Tween 80) but formulated

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at the same global mixture HLB. The concept is actually similar to the one followed in the stabilization of emulsions so-called HLB method, that consists in equalizing the mixture HLB with the required HLB of the oil/water couple, and eventually revealed that a high encapsulation efficiency is a direct consequence of a good stability of the double emulsion. A second notable factor impacting on the double emulsion stability [30] is the molecular weight of the hydrophobic surfactant. Three different surfactants were employed and compared to stabilize W/O emulsions: 1) low molecular weight classical emulsifiers such as Span 80; 2) medium molecular weight macromolecules such as polyglycerol polyricinoleate (ETD or PGPR) and 3) high molecular weight grafted silicone lipophilic surfactant (Abil EM-90). As a result the higher the molecular weight, the higher the encapsulation efficiency: highest with Abil EM-90 then PGPR, and finally Span 80. The explanation proposed was that higher molecular weight will induce a higher effective concentration in oil at same concentration owing to the lower migration rates towards external interface and phase.

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Choosing Surfactants 3. Impact of the types of double emulsions

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The different types of double emulsions have been classified based on the number of water droplets in global droplets: 1) Microcapsules: the oil droplet includes only one inner aqueous droplet (Fig. 3A); 2) Multivesicular, droplets for which the oil droplets encapsulates numerous inner aqueous droplets (Fig. 3B); 3) Finally, microsphere, double droplets with a complex inner structure inside oil droplet (Fig. 3C). A. T. Florence’s group [31] demonstrated that the different types of double emulsions can be obtained by a proper selection of the hydrophilic surfactant. Three types of double emulsions were also prepared with three types of hydrophilic surfactant:1) Polyethylene glycol dodecyl ether (Briji 30, 2 wt.%) 2) Octyl phenol ethoxylate (Triton X-165, 2 wt.%) 3) Combined surfactant by span 80 and tween 80, in which span 80 (5 wt.%) was used as hydrophobic surfactant in middle phase. The three types of double emulsions are illustrated in Fig. 3. On the other hand, different concentrations of span 80 in middle phase were also used to obtain different types of double emulsions, tween 80 (1 wt.%) and additional stabilizing agent (bovine serum albumin, BSA) was used in external phase [32]. It was demonstrated that the type of double emulsions can also be impacted by the concentration of span 80 ranging from 1 to 10 wt.%, resulting in morphology modification, e.g. from microcapsules to microspheres. By now, three main types of double emulsions have been shown and potentially controlled by the nature of hydrophilic surfactants or concentration of hydrophobic surfactants.

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Figure 3 Three typical types of double emulsions Left: Schematic drawings of double emulsions (A) Microcapsule, (B) Multivesicular, (C) Microsphere; Right: Three types of double emulsions as seen by an optical microscope [31]; Scale bar is 10 µm.

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Regarding the selection and concentration of surfactants, it can be summarized as follows: 1) for the stabilization of internal W/O emulsions, low HLB, high surfactant rigidity, and similarity between the nature of oil and aliphatic part of the surfactant, are required. In addition, increasing the hydrophobic surfactant concentration gives rise to more stable double emulsion, higher encapsulation efficiency, and complex structure of double emulsion (microsphere); 2) Then for the formulation of the double structure of W/O/W emulsions, the main parameters that impact on the emulsion stability are high HLB and/or surfactant concentration of hydrophilic surfactants. However, increasing concentration makes the size and the encapsulation efficiency decrease at a trade-off problem. Emulsion stability and encapsulation efficiency are also related to the mixing HLB related to the optimum formulation determination and required HLB of the oil 3) Finally, the types of hydrophilic surfactant and the concentration of hydrophobic surfactants will significantly influence the type of double emulsions.

Additional Methods for stabilizing double emulsions

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Even though the stability of double emulsions was increased by adjustment of surfactant nature and concentration, in practice this approach could rapidly reach limitations. Florence et al. [33] proposed the transformation of aqueous phase (inner phase or external phase) to a polymeric gel for improving the stability of double emulsions. For instance, a polyoxyethylene-polyoxypropylene-polyoxyethylene ABA block copolymer (poloxamer) and acrylamide were added into inner phase or external phase, respectively. After emulsification, the gel is formed by crosslinking ABA block copolymer or polymerizing acrylamide with UV irradiations [34]. The formation of such hydrogel gel gave a more stable double emulsion, with however, a potential pitfall lying in the impact of UV irradiations on sensitive molecules encapsulated. Some other strategies were developed based on the formation of complexes at interface between macromolecules and nonionic surfactants [35]. A type of crosslinked polyacrylic acid or BSA was added in the inner phase to create a complex with hydrophobic surfactant (poloxamer), forming a strong interfacial film stabilizing the inner water droplets.

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Such a poloxamer/protein complex resulted in an important slowing down of the release kinetics, from 40% to 10% total released of encapsulated sulphane blue after 6h, without and with this interfacial stabilization, respectively [35]. A similar example was proposed with an interfacial stabilization with BSA and a small molecular weight lipophilic surfactant (Span 80), following the encapsulation efficiency of sodium chloride. The results evidenced an increase of the encapsulation efficiency around 20% to finally reach a value of 80% [32], and conserved over 60% after 30 days. Garti et al. [36] have shown that this BSA/Span 80 system significantly improved the stability of the double emulsions, giving release rate around 8% and 20% for 5h and 20 h, much smaller compared to the ones without stabilization about 35% to 52%% for 5h and 20 h. Another effect of the addition of BSA, was a smaller size of double droplet, along with a size stability not changing after 6 weeks. This effect was explained by the interfacial stability conferred by the closed-packed BSA layer at the external oil/water interface, likely improving the interfacial elasticity and resistance, thus preventing the double droplets coalescence and rupture of inner water droplets. In this context, double emulsion stabilization by such macromolecules have attracted an important interest. In this line, the use of colloidal microcrystal based of cellulose was associated to "mechanical stabilizers" of double emulsions [37]. Some examples have also described the formulation of double emulsions using different biopolymers to stabilize internal droplets, following different mechanisms such as gelation, caseins, whey protein, chitosan and cyclodextrins [24, 38-50]. Apart from molecules or macromolecules, double emulsions were also stabilized by nanoparticles, as Pickering double emulsions, comprehensively reviewed in literature by Clegg et al. [22]. A complementary approach have been shown, by Kawashima et al. [51], that the fact making hyperosmotic internal droplets improves the stability and encapsulation efficiency. Indeed, increasing sodium chloride or glucose in internal water, in a certain extent, induces the migration from external towards internal water phases, likely along with phenomena that induce the interfacial concentration and stabilization.

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Development of double emulsions by two-step emulsification

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Polymeric microparticles synthesized by the two-step emulsification-evaporation method

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Due to the real advantages of biocompatible polymers (such as Poly(lactide-co-glycolide) (PLGA), Poly(lactic acid) (PLA) and Poly--caprolactone (PCL)) in the encapsulation and controlled release of hydrophilic molecules (like protein or peptides), such microparticles were initially prepared with double emulsion as template according to a two-step emulsification method [52]. Ogawa et al. [53] have firstly described the standard experimental procedure as follows (schematically described in Fig. 4): 1) Polymer is dissolved in the organic solvent, immiscible with water in which are solubilized hydrophilic drugs. The W/O emulsion is fabricated with these two phases; 2) The double W/O/W emulsion is then formulated with (another) external water phase; 3) Last stage consists of the evaporation of the organic solvent, making precipitating the polymer in the form of solid microcapsules. Initially, Ogawa et al. [54] synthesized PLA or PLGA to be dissolved in dichloromethane used as oil phase, and a hydrophilic analogue of luteinizing hormone was encapsulated in the matrix of polymeric microparticles according to the above-described method. Since no hydrophobic surfactant was added into the oil phase, a broad size distribution of microparticles was obtained (from 30 to 125 µm), size was only controlled by the processing parameter (using sieves of different apertures).

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Figure 4 Schematic drawing of the two-step process to produce polymeric particles by the modified two-step emulsification method

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To optimize the properties of polymeric particles, all strategies described in the previous sections for oilbased double emulsions can be followed. For example, gelation of internal droplets using gelatin have shown the clear increase in the encapsulation efficiency, from 6.7 to 70.7%, without and with gelatin, respectively . Eventually, irrespective to the oil-based double emulsions, when polymers are used, the stability is much more increased, and the stability and drug release profiles will be more related to droplet morphology, degradation and destruction of the polymer [54]. Impact of the size of inner and middle phases in the case of polymeric microparticles

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Rosca et al. [55] proposed a study on the respective impact on the carrier properties, of the size of inner water droplets, global double droplets, and the morphology of the precipitated PLGA. These authors showed that in function of the protocol followed during evaporation, the size of the droplet may vary, and thus the resulting properties of the double globule, i.e. mainly depending on the inner droplets dimeter and (Di) and double globule diameter (Dg). When Di ≈ Dg, the polymeric layer was so thin that it could not resist to the inward forces originating from the Laplace pressure and osmotic pressure between the inner and external phases. Thus, this polymeric layer broke down during the solvent evaporation, which results in the impossibility to create the global particle as seen in Fig. 5 (I). When Di << Dg, a thicker polymeric barrier protects and stabilizes the double droplets (Fig. 5 (II)). These droplets properties were shown to be controllable by the processing and formulation parameters like emulsification energy input, polymer concentration, surface active agent concentration, phase volumes, phase viscosities and so forth [55].

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Figure 5 Evolution from double emulsions to solid PLGA microparticles and relation between the diameters of two phases and type of microparticles (I) a. Double emulsions synthesized for PLGA at 10% w/v, 1% w/v, 500 rpm by homogenizer for second step (II) a. Double emulsions synthesized for PLGA at 5% w/v, 1% w/v, 8000 rpm by homogenizer for second step. All SEM micrographs b. correspond to the same sample in a after solvent evaporation [55].

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Progress degradation and destruction of polymeric microparticles

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Langer et al. [52, 56] reported encapsulated proteins (BSA and tetanus toxoid) in PLGA microparticles (Fig. 6 (I)). In the case of PLGA, drug release typically comes from the PLGA degradation, as shown in Fig. 6 (II). Initially, the PLGA microparticles had a smooth surface. After one day, small micropores (diameter < 0.1 µm) appeared all over the surface. After 4 days in maintained in aqueous release medium, the size of the pores increased. After 14 days, the microspheres were highly porous, however, keeping their spherical shape until 76 days. The influence of hydrophobic surfactant, type and molecular weight of polymer, on the size and morphology, degradability of microparticles, has been investigated. For example, small and porous particles were formed when using hydrophobic surfactant (e.g. L-αphosphatidylcholine) in the organic solvent. On the other hand, bigger microparticles were obtained when using higher molecular weights polymer [52]. Degradability of microparticles was investigated in function of the PLGA/PLA molecular weight (Fig. 6 (III)), showing a higher stability for PLA, compared to PLGA. Regarding the release of model hydrophilic protein, PLGA microparticles exhibited a slower release rate, for which only 8% has been released, while it is 30% with PLA, after 3 days. It indicated that the release of protein strongly depended on the diffusion through small channels right from the initial stage, in which the reduction in molecular weight has a lower impact on the structure of microparticles. Along the degradation, a critical increase of porosity likely induced a higher release rate.

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Figure 6 (I) Picture of typical PLGA microparticles prepared with the modified two-step emulsification method by phase contrast light microscopy [56]. (II) Typical degradation processes of PLGA microparticles prepared with the two-step emulsificationevaporation methods without hydrophobic surfactant by SEM, (A) immediately after preparation, (B) after 1 day, (C) after 4 days, (D) after 7 days, (E), after 14 days (F) and after 76 days [56]. (III) Typical polymer degradation rate of mciroparticles prepared with the two-step emulsification-evaporation methods between PLA and PLGA [52, 56]. (IV) Cumulative release of tetanus toxoid from microspheres prepared with different polymers, ○, PLA (Mw 50000); ▲, PLGA (Mw 100,000); ●, PLA (Mw 3000) [52, 56].

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Effect of parameters on polymeric microparticles 1. Interface stabilization by chelation

As discussed above for W/O/W double emulsions, the formation of chelates at the W/O interface has a strong stabilization effect on the resulting double emulsion. Similarly it has also been observed in the fabrication of polymeric double droplets, in addition to an important impact on the drug release kinetics. Nihant et al. [57] studied the influence of different parameters on the properties of PLA microparticles. BSA was chosen as stabilizer for the inner aqueous phase. The BSA concentration showed an important influence on the microparticle morphology, differentiating three types as depicted above in Fig. 3. Yang et al. [8] prepared PCL microparticles using PVA this time (instead of BSA) in order to strengthen the interface, owing to the higher hydrophobicity of PCL (compare to PLGA). The effect of PVA concentration in inner phase was investigated on the inner structure of microparticles. It was shown that low PVA concentration in inner phase (0.025%) induced a higher rate of internal droplet coalescence giving rise to a relatively low encapsulation efficiency around 42.8%. On the other hand, a

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Effect of parameters on polymeric microparticles 2. different volume ratios, concentration of polymer

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Different volume ratios of inner phases, middle phase and concentrations of polymer were studied. When a lower volume ratio and a higher concentrated polymer solution was applied, it appeared more difficult to break bigger global droplets into small ones during the second emulsification step. These experimental conditions resulted in an increase of the size of the microparticles. When BSA was used as a marker to investigate the encapsulation efficiency and loading rate of microparticles, higher concentrations of BSA were found to increase the loading rate (the ratio of mass of encapsulated molecule and mass of particles) while the encapsulation efficiency was decreased, which resulted from a higher loss of BSA during the emulsification. Finally, the size of microparticles was controlled between 60-120 µm, the encapsulation efficiency was modified from 40 to70 wt.% respectively [8]. Moreover, different volume ratios of inner and middle phase were investigated related to the morphology and inner structure of microparticles (Figure 7 (III)). The higher ratio of inner phase and middle phase, the higher porosity of the surface and inner network. Besides, increasing the volume ratio results in the increasing the globule size from 61.4 to 81.9 µm, slightly affecting the encapsulation efficiency, but highly impacting on the burst release that increased from 9.75 to 76.6 % [58, 59].

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Effect of parameters on polymeric microparticles: 3. evaporation temperature

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Yang et al. [58, 59] reported on the influence of solvent evaporation temperature on the property of PLGA microspheres during the solidification process. The whole process was recorded by microscope. The size and size distribution of microparticles increased with temperature. When the range of temperature was fixed from 5°C to 42°C, the size of the microparticles changed from 88 to 130 µm respectively (Figure 7 (IV)). It was explained that the global droplets kept the low viscosity solution during evaporation at low temperature until the critical concentration of polymer in organic solvent was reached. At this critical temperature, the low viscous solution transformed into solid state. Thus, low viscosity solution of droplets had ability to keep homogeneous size under shearing force. In contrast, as the microparticles rapidly transformed from a low viscosity solution to a high viscosity state at higher temperature and the rapidly evaporation of solvent, the size and size distribution of microparticles have been significantly influenced by shearing forces induced by stirring. In addition, the morphology of microparticles was similarly investigated with different temperature. It was observed that the prepared microparticles had a porous structure. It resulted from the low concentration of PLGA (3% w/v) and PVA (0.05% w/v) in inner phase. More porous and thicker polymeric layer were obtained at lower temperature, whereas the microparticles prepared at higher temperature presented a more homogeneous polymeric layer (Figure 7 (II)).

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Figure 7 (I) Effect of different concentrations of hydrophilic surfactant (PVA) in inner phase on the distribution of drug (BSA) inside PCL (Mn 10000) microparticles (A) 0.025 wt % (B) 0.1 wt % [8]; (II) cross-sectional of PLGA microparticles by SEM with different temperatures [58]; (III) Surface morphology and inner structure of PLGA microparticles with different ratio of inner phase and solvent phase [59];(IV) Size distribution of PLGA microspheres with respect to the temperature during solvent evaporation [58]

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Polymeric microcapsules

Though the polymeric microparticles have been produced by two-step emulsification method, the preparation of polymeric microparticles with single inner core remained quite complicated. Gao et al. [60] firstly adopted the coalescence of inner droplets after preparation of double emulsions to obtain the polymeric microcapsules by batch method, in which the method was denoted as "emulsion ripening" (Figure 8). To prevent the breaking of the middle phase during the ripening process of the emulsions, the middle phases were polymerized and crosslinked with methyl methacrylate as monomer, trimethylolpropane trimethacrylate as crosslinker and Azobis(2,4-dimethylvaleronitrile) as radical photoinitiator.

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Figure 8 (a) Flowchart of the preparation of templated double emulsion microcapsules, (b) evolution of transformation of double emulsion with different ripening times [60].

Double emulsions by microfluidic systems Microfluidic technologies have been widely developed over the past decades. They have been the cornerstone of intensive research for the production of particles owing to finely controlling segments of fluids. Due to a vigorous mixing in batch processes, the turbulent shear force resulted in a broad size distribution of the inner water droplets and double structure droplets. In addition, it is difficult to precisely control the number of inner droplets in the oil droplets by conventional methods [61]. However, the control of the number of inner droplets is very important to control the release rate of encapsulated molecular. Thanks to the precise manipulation of droplets and producing droplets of

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uniform size, there is an increasing interest to produce double emulsions by microfluidic methods [62, 63]. Nisisako et al. [64] firstly reported the formation of monodisperse double emulsions prepared by microfluidic systems with two T-shaped microchannels and Pyrex glass as chip materials, in which W/O emulsions and W/O/W emulsions were formed at a first T-shaped microchannels and second T-shaped microchannels, respectively (Figure 9 (I)). In this work, two types of microfluidic chips were fabricated to produce double emulsions. 1) A microfluidic chip composed of two T-shaped microchannels with different surface properties (Hydrophilic or hydrophobic surface); 2) Two T-shaped chips with different surface properties joined with Polytetrafluoroethylene (PTFE) tubes. To obtain a hydrophobic surface of T-junction, the surface was treated by a saline-coupling agent to prevent a phase inversion during the preparation of W/O emulsions (water phase flowing along the wall of the device). As results, the number of water droplets was controlled in one oil globule by modifying the flow rate of middle phase from 12 to 1 mL/h with such microfluidic system. The size of water droplets also was modified from 10 to 45 µm, and the oil globule size was controlled from 95 to 220 µm (Figure 9 (II)). Weitz et al. [7] designed a microfluidic system, which was based on flow focusing [65] and selective withdrawal technique [66], to synthesize double emulsions by assembling cylindrical glass capillary nested within a square glass tube (Figure 9 (III)). The inner and middle phases were pumped through the capillary tube and around the capillary respectively while the external phase was pumped through the square glass tube with opposite direction. The double emulsions were delicately formed at the entrance of the collection tube. Through the assembly of different sizes capillaries and collection tubes, different morphologies of double droplets can be prepared (Figure 9 (IV)). The thickness of middle phase in the double emulsion can be also modified. In addition, the design actually prevented the device from partial surface modification.

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Figure 9 (I) Basic concept for preparing double emulsions (W/O/W) using T-shaped microchannels [64]; (II) different types of double emulsion synthesized by T-junction microfluidic system [64]; (III) Schematic diagram of the coaxial microcapillary fluidic device [7]; (IV) different types of double emulsions synthesized by coaxial microcapillary fluidic device [7]; (V) Microcapsules prepared by microfluidic coaxial flow method. Arrows represent the dewetting phenomenon [67].

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To increase the production rate, the parallelization of several single microfluidic system was commonly adopted [68]. Different types of microfluidic systems based on the aforementioned two microfluidic systems have been developed to address different drawbacks in microfluidic systems such as partial surface modification [69, 70] or low production rate [71-73]. Those microfluidic systems for the production of double emulsions have been reviewed by Chong et al.[15]. Moreover, due to the easy control of the fluid elements, the preparation of double emulsions showed exceptional flexibility in obtaining other types of particles [74], such as lipid vesicles [75], mesoporous hydroxyapatite [76], polymersomes [77], microgels [78, 79], gas-filled microparticles, non-spherical colloidosomes [80], coreshell particles [81], hollow particles [82] and high-order multiple emulsions [61]. To show the versatility of microfluidic systems, Weitz et al. [67] firstly prepared homogeneous polymeric microcapsules with single inner core using a microfluidic coaxial flow method (Figure 9(III)). Polymeric microcapsules have been produced with two types of diblock polymers: polystyrene-block-poly(ethylene oxide)(PS-PEO) and PBA-PAA poly(normal-butyl acrylate)-poly(acrylic acid) [83]. The research revealed the solidification process of microparticles by microscope, which help deeply understanding on the origin of transformation. The forming of microcapsules resulted from the polymeric shell, which slowly separates from the organic solvent, which was coined by dewetting phenomena (Figure 9(V)). The different concentrations of polymers in organic solvent resulted in different results, which have been classified into three categories: low concentration (0.01 wt.%), middle concentration (0.1 wt/%) and high concentration (1~1.5 wt.%). Firstly, it was not possible to form stable polymeric microcapsules at low concentrations. When the concentration of polymer was increased up to the middle concentration, stable microcapsules were obtained due to the dewetting phenomena along with the resolubilization of polymeric barrier. At high concentrations of polymer, the microcapsules will be breakup into smaller

ACCEPTED MANUSCRIPT microcapsules due to the dewetting instability, which have been applied to prepare anisotropic particles by double emulsions [84].

State-of-the-art and perspective of double emulsions Double emulsions in state-of-the-art for nanocarriers

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As delving into many diseases such as Cancer, Alzheimer’s disease and so on, drugs have been asked to be delivery into specific tissue even single cell. It brought the new challenge for pharmaceutists and biologists. Due to the potentiality of nanocarriers in these aspects, the encapsulation of hydrophilic molecules (such as insulin, protein and DNA) in nanocarriers has stimulated an extensive research activity. Blanco et al. firstly modified the two-step emulsification method to get nanocarriers [85, 86]. The typical picture of PLA nanoparticles prepared is presented in Figure 10(I). For preparation of nanoparticles, high-energy emulsification methods such as ultrasonication, microfluidic emulsification, were used instead of the homogenization or mechanical stirring. Thus, BSA-loaded PLGA nanoparticles have been successfully prepared from 320 nm to 530 nm depending on the molecular weight of PLGA. It showed us that the two-step emulsification has the ability to produce polymeric particles at the nanoscale. Compared to microparticles, the molecular weight and the structure of polymer have become the most important key on the properties of nanocarriers, which strongly relate to interaction between molecules. Thus, the researches have been focused on the influence of the polymer molecular weight. Tobío et al. [86] used poly(lactic acid)-b-poly(ethylene glycol) (PLA-PEG), a block copolymer, to encapsulate tetanus toxoid (TT, a model protein antigen) by the modified two-step emulsification method. PLA-PEG polymer nanoparticle size was controlled at 142.8 nm compared to PLA nanoparticle at 153 nm. Bonneaux et al. [87] used a two-step ultrasonication method to prepare double emulsions by using different molecular weights of PLA (from 1938 to 90000 Da) and varying different process parameters. In results, using rotating evaporator did not influence the size of the nanoparticles at room temperature. Finally, the size of PLA nanoparticles reached around 200 nm. Human Serum Albumin (HSA) was used as an encapsulated molecule to calculate encapsulation efficiency, which varied from 20 to 30% by increasing volume ratio of inner phase. As a result, different phenomena are observed for PLGA and PLA nanoparticles with respect to different molecular weights. The nanoparticles size increased with a decrease in the molecular weight of PLA and an increase in the molecular weight of PLGA. It was explained that the nanoparticle sizes depend on the property and the molecular weight of polymer at same time. PLA has more hydrophobic property than PLGA. Lowering the molecular weight of PLA or heightening the molecular weight of PLGA resulted in more hydrophilic property of polymer, which facilitated the faster exchange of polymer between global droplets. Ma et al. [88] conjugated bis(βcyclodextrin) (7-membered sugar molecular ring, commonly used as solubilizing agents to increase water-solubility of lipophilic compounds and enhance bioavailability of drug) into PLGA to encapsulate BSA by two-step emulsification method with sonication. It was found that the encapsulation efficiency, ranging from 80 to 90% wt., has apparently increased compared to the encapsulated HSA PLA nanoparticles (encapsulation efficiency of 20 to 30% wt.) and encapsulated HSA PLGA nanoparticle (encapsulation efficiency at around 60% wt.). However, the smallest size of nanoparticles was around 300 nm. It is to be noted that the nanoparticles were synthesized with sonication (30 W), bigger sizes might be resulting from the applied lower energy during the process. In addition, Doelker et al. [89] studied the influence of sonication parameters on the preparation of nanoparticles. The exposure time and energy of sonication were systematically investigated. Simultaneously, the nanoparticles were prepared by vortex mixing instead of sonication at each

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emulsification step. The results were compared with the nanoparticles prepared only with sonication. It was observed that the exposure time at the second emulsification step has a greater influence on the nanoparticle size than the one at the first step emulsification. The energy of sonication has two thresholds for the preparation of the nanoparticles. On one hand, if the applied energy is less than the low-energy threshold, this one cannot produce nanoparticles. On the other hand, if the applied energy is higher than the high-energy threshold, it will result in a high polydispersity index of nanoparticles due to the non-uniform energy dissipation. In this paper, the threshold was found between 20-65 W. Concerning the use of a vortex mixing during the first step emulsification, similar nanoparticle sizes to those produced with the sonicator were obtained. It suggested it is not necessary to use sonication for both emulsification steps. In contrast, replacing sonication by vortex mixing at second step cannot produce individual nanoparticles as polymer aggregation. To summarize the nanoparticle preparation mainly depends on the emulsification method and processing parameters [56, 90]. Fessi et al. [9] adopted PCL to synthesize nanoparticles by two-step of sonication (500 W, 20 kHz). They also studied the influence of the exposure time of sonication during the first step emulsification and the second step emulsification on the size of the obtained polymeric nanoparticles. These experimental studies allowed to conclude that the exposure time of sonication at the first step has not significant influence on the size of the obtained nanoparticles. Also, it was observed that, the size of the PCL nanoparticles decreased with the exposure time of sonication during the second step emulsification. Sonication frequency, PVA concentration in the external phase, PCL concentration in the middle phase and the volume of external phase have been investigated. All of these parameters in the two-step emulsification have a maximum limitation of influence on the size of particles. Over the threshold, the nanoparticle sizes cannot be further decreased. Finally, from this study, the smallest PCL nanoparticles were synthesized at 219 nm. Aforementioned polymeric nanoparticles were considered as nanospheres, which included different size of hydrophilic vesicles; not only one, dispersed into the matrix of the polymeric network. In contrast, Gao et al. [10] showed nanocapsules which have one hydrophilic core in the polymeric network. These nanocapsules were prepared by two-step emulsification methods (Figure 10(II)). The more complex structure of nanocapsules has been demonstrated by carefully choosing lipids as surfactants in double emulsions. Clearly, the double structure has been proven by negative staining TEM [91].

Figure 10 (I) TEM microphotograph of typical BSA-loaded PLGA nanospheres [85]; (II) TEM microphotograph of the nanocapsules prepared via the double emulsion approach [10].

Emulsification methods for the future of double emulsions In summary, different materials have been used for the middle phases such as mineral oil, vegetable oil or polymer. Different parameters in process and materials presented huge influence on the physicochemical and encapsulation properties, release profile of the encapsulated product. The two step emulsification process, thanks to the easy modulation of the formulation parameters (interfacial

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properties, volume ratio), will allow obtaining very controllable double emulsions. Among considerable properties of double emulsions, the suitable size and size distribution are prerequisite parameters for potential applications in pharmaceutics. These properties can influence many functions including encapsulation efficiency, degradation, flow properties, clearance and uptake mechanisms of carriers [92]. From our point of view, the emulsification method during two-step emulsification significantly affect the size and size distribution of the double emulsion. Based on this consideration, the development of emulsification methods should attract much attention from researchers, because it will definitively affect the characteristics of the final product. To clarify the development of emulsification, the relation between emulsification methods and size of carriers have been clearly shown in Figure 11. Besides, for more details information of formula, all of results are clearly shown in Table 1. The double emulsions, firstly, have been observed by William Seifriz [16]. Hand shaking was used to prepare double emulsions in (Figure 11(I)). The results presented a coarse emulsion. Thereafter, in the early stage, double emulsions were prepared by the simple mechanical stirring (Figure 11 (III)), in which the detail information on equipment are presented in Table 1. The size of such double emulsions ranged from 5 to 30 µm. The double emulsions size strongly depended on the stirring rate. Kawashima et al. used an extremely low stirring rate such as 390 rpm and 630 rpm to prepare W/O emulsion and W/O/W emulsions, respectively [93].In this last example, the size of double emulsions was large and polydisperse (8 to90 µm, Figure 11 (II)). On the contrary, Matsumoto et al. [20] declared that the size of synthesized double emulsions can achieve 2 µm by using a special pin-mixer and a high speed homogenization (Figure 11(VII)). However, these authors thought that the obtained results were originated from the incorporation of high concentrations of Span 80 (30% wt.). Further, Kawashima[93]et al. [93] introduced a method to decrease the polydispersity and the size of double emulsions, in which the double emulsions were firstly synthesized by a conventional method and then were extruded through a porous membrane. Finally, the obtained double emulsion was redispersed in an additional external phase (Figure 11(VI)). The size of double emulsion was decreased down to 3.64 µm. Kim et al. [94] reported double emulsions synthesized by two-step emulsification with sonication. The double emulsion size varied from 1.3 to 1.6 µm. Ohwaki et al. [95] claimed a size of W/O emulsions ranging from 100 to 200 nm when they were formulated by using a high pressure homogenizer. However, the prepared double emulsions after a second emulsification were still in the micron range (Figure 11(IV)). Okochi et al. [96] proposed the concept of the mixed emulsification for the formation of a single emulsion. The size of W/O emulsion was around 256 nm by using a combination of sonication and homogenizer. It was concluded that the production of fine and homogeneous emulsions can be reached by the combination of multi emulsification process, in which size and size distribution of emulsions can be reduced step by step. Besides, double emulsions were synthesized by using the membrane emulsification method. The results presented a size distribution profile of membrane method, which was sharper than the one obtained with the stirring method. Leal-Calderon et al. [25] used a new technology to get monodisperse 400 nm W/O emulsions after a coarse emulsification by mechanical mixing, which was called fractionated crystallization technique (Figure 11(V)). However, the method involved multi-purification [97]. Following the pioneering work of Talyor [98] , an efficient method for producing quasi-monodisperse double emulsions was proposed by using a couette mixer (Figure 11 (VIII)) [99]. When polyols and PGPR were incorporated in the middle phase, extremely small W/O emulsion sizes were produced by the combination of a rotor-stator emulsifier and a high-pressure homogenizer. The size of W/O emulsions (0.2 µm) obtained with this method was much lower compared to the W/O emulsions size of 1.8 µm produced without any polyols and that of 0.8 µm with high-pressure homogenizer without any polyols [100]. By high-pressure microfluidizer, a new type of emulsification called a dual-feed process was applied to a second step emulsification to obtain double emulsions. The size of the double emulsion was decreased down to 3-4 µm with an incorporation of polysaccharides and proteins in the external phase [39]. Yafei

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et al. [101] reported an extremely complicated process to obtain submicron size double emulsion (0.7 to 2 µm). In this process, the production rate seemed to be excessively low since a multi-fractionation centrifugation stage was applied after each step of sequential emulsifications.

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Figure 11 Evolution of the relationship between double emulsions and process. (‫ )׀‬Double emulsions prepared by one-step emulsification with hand-shaking [16]; (‫ )׀׀‬Double emulsions prepared by two-step emulsifications with mechanical stirring [93]; (‫ )׀׀׀‬Double emulsions prepared by two-step emulsifications with small vibrating mixer [31] (‫׀‬v) Double emulsions prepared by Ultraturrax (X1020, Ystral) [95] (v) Double emulsions prepared by a fractionated crystallization technique [97] and Ultraturrax [25]; (v‫ )׀‬Double emulsions prepared by extrusion of a primary emulsions prepared by method (‫ )׀׀‬through a porous membrane [93]. (v‫ )׀׀‬Double emulsions prepared with pin-mixer and Ultraturrax (Tokushu Kikakogyo Co., Japan) for W/O and W/O/W emulsions, respectively [20]. (v‫ )׀׀׀‬Double emulsions prepared bya couette mixer [99]. (‫׀‬x) Double emulsions synthesized by Microfludizer [102].

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In contrast, for polymeric particles, Ogawa et al. [54] firstly introduced the two-step emulsification to encapsulate a hydrophilic drug in the matrix of polymeric microparticle. Rotor-stator emulsifier and a turbine-shaped mixer were employed for the two-steps. The prepared microparticles had extremely broad size distribution so that the sieves had to be used to separate the different sizes of microparticles. As the development of emulsification methods, more advanced technologies were considered for the preparation of polymeric double particles which allowed to reduce and narrow the size and size distribution of microparticles respectively [52]. Encapsulated hydrophilic molecules in nanocarriers have been also considered. Alonso et al. synthesized nanoscale polymeric particles by two-step emulsification with ultrasonication [85]. It was found that the emulsification methods employed had a great influence on the properties of double emulsions during process. Recently, the advantages of microfluidic systems assembling T-type microchannel or co-axial capillary have completely solved the poor control drawback of the two-step emulsification method for the preparation of double emulsions at micron sizes [64]. The microfluidic systems stimulated the new prospective for a precise control of double emulsions, in which the number of water droplets included in one global droplet can be accurately controlled by the flow rate of the middle phase and the inner phase. Nevertheless, the biggest challenge still lies in the development of efficient methods that can produce nanoscale double emulsions. To date, for effectively preparation of nanoemulsions, two methods mainly were involved, namely emulsification of premixed emulsions by ultrasonic agitation and high pressure microfluidic system [103]. Yet, for the preparation of nano double emulsion, the situation became more complex due to the extra requirement for balancing Laplace-pressure and osmotic pressure between the inner phase and the middle phase. Thus, it is almost impossible to complete this mission, though they have been shown by means of special surfactants [102] (Figure 11 (IX)) or gelation of inner phase [104]. Besides, for controlling drug release or targeting, the involved high-energy processes will damage fragile molecules such as peptides, proteins, nucleic acids. So, the preparation of nano double emulsions by the moderate method is the other challenge we face in future. Flow-focusing [105] and spontaneous emulsification [106] are known as potential low energy methods, which should be paid attention to develop moderate two-step emulsification for nano double emulsions.

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Table 1. 1 Formulations, processes and properties for double emulsions in literatures Author

Year

Oil

Encapsulated molecule

Inner phase

Inter phase

External phase

Process

Size of W/O emulsio n

R.H. Engel [19]

1968

Trioctanoi n

Insulin

ZnCl2 (0.003 M) Insulin (100 u/ml) solution

Palmitic acid (0.03 M) in trioctanion

Sodium lauryl sulphate (1 w/v % ) solution

Sonifier

N/A

Sachio Matsumoto [20]

1976

Liquid paraffine (same as light mineral in wiki)

Glucose

Glucose 0.5 wt% aqueous phase

Span-80 (30 wt%) in liquid paraffin

Tween-20 0.5% aqueous solution

Pin-mixer for W/O emulsions; Ultraturrax for W/O/W emulsions.

A. T. Florence [31]

1981

Isopropyl myristate

N/A

Water

Oil containing Span 80 (5 wt% )

Water containing 2% wt/wt surfactant as follows: Brij 30, Triton X165, or a 3:1 Span 80:Tween 80 mixture.

Paraffinic oil containing 8-10 wt%, of oil soluble emulsifiers

Brij 92 in light mineral oil

Nissim Garti [23]

1983

Paraffinic oil

Chloropromaz in hydrochloride

Nissim Garti [27]

1984

Light mineral oil

NaCl

Aqueous drug solution (55 mg/ml of the drug)

T P

E C

C A

D E

NaCl solution (1 wt%)

Inner phase: Inter phase (v/v) 7.2:10.8

Size of W/O/W emulsion

W/O emulsions :Ext ernal phase (v/v)

Encapsulatio n efficiency (wt.%)

N/A

4.5:13.5l

N/A

2 µm

72:28

about 2 µm

51:49

90

Small vibrating mixer

2~3 µm

1:1

8~25 µm

1:1

N/A

Water(3-5 wt%, emulsifier) Span 20 + Tween 80, Triethanolamin e + Oleic acid (1:1 wt)

Magnetic stirring

0.5~3 µm

3:7

5~15 µm

3:7

10

Span 20 + Tween 80 (HLB=11) glucose solution (5.5 wt%)(equalizin g the osmotic pressure caused by the NaCl.)

Homogenizati on (Silverson homogenizer) for W/O emulsions, Magnetic strring for W/O/W emulsions

N/A

30 :70-x (Here, x represent the concentrati on of surfactant )

N/A

20:80-X (wt%) Here, x represent the concentration of surfactant

30

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C S

U N

A

I R

T P

ACCEPTED MANUSCRIPT A.T. Florence [32]

1986

Isopropyl myristate

NaCl

BSA (0.05~2.0 wt%) NaCl 1.25 wt% solution

Span 80 (1~10 wt%) in isopropyl myristate

Water containing 1% w/v Tween 80 adding sorbitol to the external aqueous phase

Whirlimixer

2~3 µm

Kamlesh P. Oza [37]

1989

Heavy mineral oil

NaCl

Avicel RC591 colloidal microcrystallin e cellulose (CMCC 2 wt% or 1 wt% ) solution

Span 80 (2 wt%) or Span 85 in heavy oil phase

CMCC (2 wt% or 1 wt% ) dispersion

Polytron Homogenizati on

N/A

liquid paraffin

NaCl

NaCl 0.02 M aqueous solutions

70 wt% liquid paraffin and 30 wt% Span 80

Tween 20 (0.5 % w/v) aqueous solution

Mechanical stirring (390 rpm) for W/O emulsions, 630 rpm for W/O/W emulsions. Prepared W/O emulsions or W/O/W emulsions is extruded by porous membranes

Yoshiaki Kawashima [93]

1991

A

Takayuki Ohwaki [95]

Nissim Garti [30]

1993

1994

liquid paraffin

Paraffinic oil

Secretin

Sodium alkylsulfonate in buffer(PH 6.33), sodium chloride(0~1.8 % w/v) coccine (0.2 % w/v) or Secretin (0.064 % w/v) solution NaCl solution

D E

M

C A NaCl

1:1

50 ~80

1:9 or 4:6

24~30 µm

1:4 or 1:1

N/A

2.25 µm or 2.5 µm

5:3

3.64 µm or 4.41µm

1:1

67~98

I R

T P

Span 80 (10% w/v ) in liquid paraffin

PSML 20 (1.5% w/v) buffer solution

Ultraturrax with Ushaped blade

100~20 0 nm

8:5 (v/v)

14~20µm

1:1

N/A

Span 80, ETD, PGPR Or Grafted silicone lipophilic surfactant (Siliconebased poly(ethyleneglycol) copolymer in paraffinic oil, concentration of surfactant set at 10 wt%

Hydrophilic poly (siloxane)graftpoly(oxyethyle ne) solution, concentration of surfactant set 2 wt%

Ultraturrax homogenizer (8000~ 24500) rpm for W/O emulsions, Microfluidize r for W/O/W emulsions

Below 0.5 µm

3:7

4.5 µm

1:4

N/A

T P

E C

8~25 µm

C S

U N

1:1

ACCEPTED MANUSCRIPT Chong-Kook Kim [94]

1995

Liquid paraffin

Cytarabine

Cytarabine solution

Span (30 wt%, Span 20, 80) in liquid paraffin

Tween (0.5 wt% Tween 20, 80 ) solution

Ultrasonicator

N/A

2:5

1.3~1.6 µm

1:4

60~80

F. LealCalderon [25]

1998

Dodecane

NaCl

NaCl (0.1 M) in water

Span 80 in the dodecane (1:1 Weight mixture).

SDS , TTAB (0.0008 mol/l) and Tween 80 in water

Mechanical stirring then Fractionated crystallization technique for W/O emulsions, Ultraturrax for W/O/W emulsions

400 nm

1:1 After diluted in dodecane to 10 % by volume

10 µm

1:9

N/A

Pluronic F-88 (0.5 wt% ~5 wt%) solution

Complex of homogenizati on and Ultrasonicator for W/O emulsions, Homogenizati on for W/O/W emulsions

250~50 0 nm

1:4

20 ~100 µm

1:1

55~90

Hideaki Okochi [96]

2000

The mixture of several oils

VCM (5 % w/v) saline solution

Vancomycin (VCM)

HCO-40 (5 %) in the mixture of soybean oil (70 wt%) and Lipiodol (30 wt%)

Nissim Garti [39]

2001

2002

Dodecane or Commerci al sunflower oil (Stora)

NaCl

Mixture of mono glyceride oleate (GMO) and medium chain fatty acid trglycerid es (MCT, C8~C10)

Vitamin B1

NaCl (0.2M ) in water

M

I R

C S

U N

A

F. LealCalderon [99]

T P

Admul Wol 1403 (10 wt%, Polyrycinoleate of polyglycerol) , Arlacel p135 (polyethylene-30 dipolyhydroxystearat e) ; Arlacel 186 (glyceryl monooleate) in oil phase

NaCl solution or glucose ( 0.4M ).

Couette-type mixer

0.3µm

2:3~3:1

2 ~7 µm

7:3 or 3:2

95

PGPR (11.4 wt%) in Mixture of mono glyceride oleate (GMO 2.8 wt%) and medium chain fatty acid trglycerides (MCT, C8~C10)

Whey protein isolate (WPI 2~6 wt%) and Xanthan gum (0.1 or 0.5 wt%) water solution

Complex of homogenizati on and High pressure homogenizer for W/O emulsions, High pressure Homogenizer for W/O/W emulsions

N/A

3:47 w/w

3~4 µm

1:5 w/w

60~90

D E

T P

C A

E C

Glucose (16.7 wt%) aqueous phase

ACCEPTED MANUSCRIPT Hu Gang [101]

2006

Dodecane

N/A

NaCl (0.1 M ) Dextran (9*105 M) water solution

Arlacel P 135 dodecane solution

NaCl (0.1 M) solution

Homogenizer or sonication, polycarbonate membrane emulsification

0.1 µm

1:1 or 1:5

0.7 ~ 2 µm

1:10

N/A

T P

I R

C S

Table 1. 2 Formulations, processes and properties for polymeric microparticles in literatures Author

Year

Organic solvent

Polymer

Process of emulsificati on

Encapsula ted molecule

Inner phase

Yasuaki Ogawa [53,54]

1988

Dichloro methane

PLA (Mw 22500), PLGA (Mw 14000)

leuprolide acetate

Leuprolide acetate(8 % w/v) water or gelatin solution

Robert Langer [56]

1991

Methyle ne chloride

PLGA (75:25 Mw 5000, 10000, 14000 )

Homogenize r for W/O emulsions, turbineshaped mixer for W/O/W emulsions Vortex mixer or probe sonicated for W/O emulsions. Magnetic stirring for W/O/W emulsions

T P

E C

C A

D E

Fluorescein isothiocyan ate-lablled bovine serum albumin and FITClabelled horseradish peroxidase

External phase

Diluted phase

PLA or PLGA dichloromet hane solution

Polyvinyl alcohol (0.5 %) solution

N/A

PLGA (100 % w/v) methylene chloride solution

PVA (1 %) solution

PVA (0.1 %) soluti on

A

M

Protein (20 % w/v) solution

U N

Inter phase

Inner phase: Inter phase (v/v)

W/O emulsions: External phase (v/v)

Double emulsion/ Diluted emulsion (v/v)

Size of polymeric particles

Encapsulati on efficiency ( wt%)

1:4

1:8

N/A

125,88, 74, 44, and 37 µm

2~70

1:20

1:2

1:100

55-99 µm

60 ~90

ACCEPTED MANUSCRIPT Robert Langer [52]

Nicole Nihant [57]

M.J. Alonso [84]

1993

1994

1997

Methyle ne chloride

Methyle ne chloride

Ethyl acetate

PLA (Mw 3000, 50000) and PLGA (50:50 Mw 100000 Resomer RG 506)

PLA (Mn 51000)

Sonication (50 W 10 sec) for W/O emulsion, Homogeniza tion at 15000 rpm for 10 sec

Tetanus toxoid

Tetanus toxoid solution

Ultraturrax for W/O emulsions, A fourpitched blade impeller (800 rpm) for W/O/W emulsions

BSA

PLGA (50:50 Mw 15000 Resomer RG 502, Mw 43000 RG 503 and Mw 50000 503 H)

Sonication (15 W 15 s)

FITC-BSA

Sonication (15 W 15 s)

M.J. Alonso [85]

1998

Ethyl acetate

PLA (Mn 50000) PLAPEG (PLA Mn 45000 PEG Mn 5000)

F. Bonneaux [87]

1998

Methyle ne chloride

PLA (Mw 90 000 , 50000 , 17400, 1938)

C A

Tetanus toxoid

HSA

PVA(1%) solution

PVA (0.1 %) soluti on

1:20

1:1

1:100

10~50µm

N/A

T P

I R

C S

PVA (2.5 wt%) solution

U N

A

M

FITC-BSA (4 % w/v) solution

PVA (0.1 %) PBS soluti on

1:5(v/ g)

1:25 (v/g/v)

N/A

>100µm

N/A

PLGA (20 % w/v) ethyl acetate solution

PVA (1 % w/v)

PVA (0.3 % w/v)

1:20

1:2

1:50

320 ~ 530 nm

20 ~70 (with PVA obtained 20 wt%)

Tetanus toxoid (10 % w/v) solution or gelatine (2 %) added as stabilizer

PLA or PLA-PEG (5 % w/v) ethyl acetate solution

Sodium cholate (1 % w/v) solution

1:20

1:2

1:50

130~150n m

30 ~35

Water or Human serum albumin (HSA 2 % ) solution used as a 20% injectable solution)

PLA (2.5 wt%) methylene chloride solution

PVA (5 % w/v) solution

Sodiu m cholat e (0.3 % w/v) PVA (0.1 % w/v) soluti on

1:20

1:2

1:20

200±5 nm

20~30

D E

T P

E C Sonication

BSA solution

Polymer (PLA 20~50 % w/v PLGA 10~20 % w/v) ethylene chloride solution or Polymer Lαphosphatid ylcholine (0.3 % w/v) in chloroform PLA (9 wt %) methylene chloride with or without Pluronic F68

ACCEPTED MANUSCRIPT Yi-Yan Yang [58,59]

2000

Dichloro methane

PLGA(65:35 Mw 40 000– 75 000)

Sonication for W/O emulsions, Mechanic stirring for W/O/W emulsions

BSA

Eric Doelker [89]

2003

Ethyl acetate

PLGA (50:50 Mw 34,000 Da , Resomer RG 503)

Sonicator ( 20~65 W 2~20 s) for W/O emulsions, Sonicator/V ortex for W/O/W emulsions

Methylene blue

Iosif Daniel Rosca [55]

2004

Dichloro methane

PLGA(50:50 Mw 45000– 75000)

Homogenize r for W/O emulsion, or Homogenize r or Mechanic stirring W/O/W

N/A

Omid C. Fraokhzad [91]

2011

Dichloro methane

PLGA (Viscosity of 0.26~0.54 dL/g )

Sonication for W/O emulsions, W/O/W emulsions

Small interferenc e RNA (siRNA)

XiaoHu Gao[10]

2012

Chlorofo rm

Poly(styreneallyl alcohol), PS16-PAA10, Mw 2200)

C A

PVA (1% w/v ) water solution

PLGA (3 % w/v) methylene chloride solution

PVA (0.05 wt%) PBS solution

640 ml PBS with 0.05 % PVA

1:24

PLGA (20 % w/v ) ethyl acetate solution

PVA (2 %) solution

PVA (0.3 %) soluti on

1:20

D E

M

Dyelabelled plasmid DNA , Watersoluble quantum dots, Doxorubici n

PVA, MW 9000

25:64

88~130 µm

58

1:2

1:5

250~350 nm

16~20

1:6

1:16

10µm

N/A

T P

I R

C S

U N

1:20

PLGA dichloromet hane solution

PVA solution

PLGA , 1,2Dimyristole oyl-snglycero-3ethylphosp hocholine(EP C 14:1) dichloromet hane solution

1,2Distearoylsn-glycero-3phosphoetha nolamine-Nmethoxy(pol yethylene glycol) (DSPE-PEG) and Lecithin solution

DSPE PEG m and Lecith in soluti on

N/A

N/A

N/A

225nm

78~82

PS16PAA10, Oleic acid Solution

PVA solution

N/A

N/A

N/A

N/A

260~290 nm

60 for DNA, 20~53 for Doxorubicin

A

Water

T P

E C N/A

BSA (4 % w/v) phosphatebuffered saline (PBS) (pH 7.4) solution containing PVA (0.05% w/v) Water

500 ml of 0.1% w/v PVA soluti on

1:10

ACCEPTED MANUSCRIPT Table 1. 3 Formulations, processes and properties for polymeric microparticles prepared by microfluidic system in literatures Author

Year

Oil

Process of emulsification

Encapsulated molecule

Inner phase

Inter phase

External phase

Size of W/O emulsion

Size of W/O/W emulsion

Takasi Nisisako [64]

2004

Corn oil

Micro-chips which based on T-junction: One-chip and two chip One-chip module (Number represent width*depth) Type1:first junction:60*25 µm Second juntion:130*65µm;type2: 40*10 180*75,type3: 85*35 225*100 Two-chip module:80*40 (130 220)*90 Microcapillary device, Based on coaxial flow and flow focusing, (Typical inner diameters of the tapered end of the injection tube range from 10 to 50 µm, and the orifice in the collection tube of the injection tube range from 50 to 500 µm)

N/A

Water

Lecithin (0.5 wt%) or tetraglycerincondensed ricinoleic acid ester (1.0 wt %, CR-310) in corn oil

Sodium dodecyl sulfate or decaglycerol monostearate (0.5 wt%) water solution

52 µm ( including 4.7~11.9 droplets in one oil globule )

83 µm

water

10 ~50 µm

50~500 µm

D. A. Weitz [83]

2005

N/A

N/A

D E

T P

C A

E C

water

C S

U N

M

A

I R

T P

Polymer (70 % can be photopolymerized Norland Optical Ahesiive ) 30 % acetone or Poly(butyl acrylate)-bpoly(acrylic acid) (PBA-PAA) (2 wt%) toluene and tetrahydrofuran (7:3 v/v)

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Conclusion

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Preparation of stable double emulsions with highly controllable properties has become the development direction of two-step emulsification methods. However, microfluidic systems have well solved the broadness of the size distribution and non-controllable structures’ problems at microscale. Furthermore, the complex of protein, polysaccharides and polymeric surfactant or pickering stabilizing agents allowed obtaining stable microscale double emulsions. In contrast, polymeric materials tend to be the privileged strategy to obtain nanoscale carriers although some researchers still tried to find methods to get double nanoemulsions without polymer. To avoid the destabilization of the W/O emulsion during the second step emulsification, low energy methods could be considered in conjunction with an efficient emulsification method for the first step (like the high pressure microfluidizer). Among low energy methods, the spontaneous emulsification presents some advantages. The emulsification is promoted by the addition of an external compound dissolved in the external phase (water) that will trigger the nanoemulsification and thus can be carried out under gentle stirring. Thus, it is believed that the next breakthrough in the production of double nanoemulsions may come from the breakthrough of emulsification methods and complex for stabilization.

AN

Acknowledgments

ED

M

SD would like to acknowledge the China Scholarship Council for his Ph.D fellowship. Natural Science Foundation of ShaanXi Province in China (Grant No. 2018JQ5164 supported this work). Natural Science Foundation of Educational Department in ShaanXi Province, China (Grant No. 18JK0114 supported this work).

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