Preparation of High Internal Water-Phase Double Emulsions

Sep 29, 2014 - Herein we report a one-step method to prepare high internal water-phase double emulsions (W/O/W) via catastrophic phase inversion of ...
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Preparation of High Internal Water-Phase Double Emulsions Stabilized by a Single Anionic Surfactant for Fabricating Interconnecting Porous Polymer Microspheres Zichao Li,† Huarong Liu,*,† Lai Zeng,† Hewen Liu,† Song Yang,‡ and Yanmei Wang† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Jinzhai Road 96, Hefei, Anhui 230026, P. R. China ‡ Chemistry Department, Zhengzhou Tobacco Research Institute of CNTC, Fengyang Street 2, Zhengzhou, Henan 450001, P. R. China S Supporting Information *

ABSTRACT: Herein we report a one-step method to prepare high internal water-phase double emulsions (W/O/W) via catastrophic phase inversion of water-in-oil high internal phase emulsions (W/O HIPEs) stabilized solely by 12-acryloxy-9-octadecenoic acid (AOA) through increasing the content of water phase. This is the first time for double emulsions to be stabilized solely by a single small molecular surfactant, which are usually costabilized by both hydrophilic and hydrophobic surfactants. After neutralized with ammonia, AOA is confirmed to be capable of stabilizing both W/O emulsions and O/W emulsions, which may account for its unique ability to stabilize double emulsions. The effects of different conditions (including changing the concentrations of AOA and salt (NaCl), pH value, the polarity of oils, the addition interval of water and stirring rate, etc.) on the formation and the stability of double emulsions as well as the inversion point have been investigated by using optical microscopy and conductivity monitoring. Finally, porous polymer microspheres with high interconnection (polyHIPE microspheres) were fabricated by γ-ray initiated polymerization of the as-prepared double emulsions composed of different monomers (styrene, or n-butyl acrylate, or methyl methacrylate), which have been confirmed by scanning electron microscopy. Our method is facile and effective for preparing high interconnecting porous polymer microspheres without tedious post-treatment of the products in common emulsion polymerization due to the use of polymerizable surfactant.

1. INTRODUCTION Double emulsions are complex liquid dispersion systems known as “emulsions of emulsions”, in which the droplets of the dispersed phase themselves contain even smaller dispersed droplets, for example, water droplets-in-oil droplets-in-water (W/O/W) or oil droplets-in-water droplets-in-oil (O/W/ O).1−4 Because of their compartmentalized internal structure, double emulsions can provide advantages over normal emulsions in controlled release, active species protection, catalysis, and so on. 5−8 Hydrophilic and hydrophobic surfactants are both usually needed to prepare double emulsions. For example, in preparation of W/O/W emulsions, the primary W/O emulsion is first prepared with hydrophobic surfactants (with hydrophile−lipophile balance (HLB) value of 3−8), then the as-prepared emulsion is further dispersed into the outer water continuous phase with hydrophilic surfactants (HLB = 9 to 10) to form W/O/W double emulsions. Double emulsions stabilized by a single kind of surfactants have scarcely been reported due to the fact that the curvatures of the two types of interfaces in double emulsions are opposite and a single surfactant is difficult to stabilize the two types of © 2014 American Chemical Society

interfaces simultaneously. To our knowledge, only a few published papers have reported on the stabilization of double emulsions by single component surfactant until now, all of which used complicated diblock polymeric surfactants. Hanson et al.9 first used a synthetic amphiphilic diblock copolypeptide as the single surfactant to prepare double emulsions with both inner and outer droplets under 100 nm. Hong et al.10 successfully prepared double emulsions by using diblock polymer surfactants (PEG-b-PS) as the single surfactant. However, both of the double emulsions prepared in the previously above-mentioned works had a low volume fraction of inner water droplets and were not suitable for fabricating interconnecting porous microspheres. Emulsions with high internal volume fraction can be used as templates to prepare interconnecting porous materials11−14 with high loading capacity,1,7 which is beneficial to their practical application. Received: July 1, 2014 Revised: September 1, 2014 Published: September 29, 2014 12154

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Scheme 1a

a (a) Formulation-composition 2D map with region labeling around the phase-inversion frontier. (b) Schematic illustration of interfacial films by which water droplets were embraced (or stabilized); the films are composed of two monolayers with anionic surfactant and oil phase containing AOA micelles. (c) Schematic illustration of the formation of double emulsions via catastrophic phase inversion of W/O emulsions by increasing the water content. (d) Molecular structure of AOA.

inversion temperature (PIT) method and catastrophic inversion (CPI) method. The phase inversion in PIT method is due to the change in the affinity of surfactant to oil and water phase during heating or cooling20−23 (see the blue inversion line in Scheme 1a), while that in CPI method is caused by increasing the volume fraction of water or oil phase to a certain degree24−26 (see the red inversion lines in Scheme 1a). However, the surfactants used in these references were the mixtures of hydrophilic and hydrophobic surfactants. In our present work, W/O/W double emulsions with high internal water phase are prepared via CPI of W/O HIPEs, as illustrated in Scheme 1c. It is noteworthy that the double emulsion is solely stabilized for the first time by a small molecular surfactant 12-acryloxy-9-octadecenoic acid (AOA whose molecular structure is shown in Scheme 1d) that has not been found in the literature until now. Compared with polymeric surfactants, small molecular surfactants are easier to be synthesized and purified or to be obtained commercially. Moreover, highly interconnecting porous polyHIPE micro-

High internal-phase emulsions (HIPEs) with the internalphase volume fraction higher than 74% are good templates to produce interconnecting meso-macroporous monolithic polymeric materials known as polyHIPEs, which are attracting much interest in their application including scaffolds for tissue engineering and 3D cell culture, supports for catalysts, water purification, and so on.15−19 By dispersing HIPEs into a third phase, high internal-phase double emulsions can be prepared, which are good templates for fabricating interconnecting porous polyHIPE beads.11−14 However, the challenge of this method arises from the difficulty in well dispersing due to the high viscosity of HIPEs, which leads to irregular shapes of the prepared polyHIPE beads.11,13 Although monodisperse spherical particles can be achieved using microfluid techniques,14 however, the low production rate of this method limits its wide application. Therefore, efforts have still been exerted in searching facile methodologies for massively producing polyHIPE particles. Double emulsions can also be prepared by one-step emulsification via a phase-inversion process, such as phase12155

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Figure 1. Optical micrographs of samples with different content of AOA: (a) 25.0, (b) 20.0, (c) 16.7; (d) 14.3; and (e) 10.4% based on the oil phase after catastrophic phase inversion. (f) Inversion point of emulsions with different content of AOA. composed of styrene (1.50 g, 14.4 mmol), EGDMA (1.50 g, 7.6 mmol), and AOA (0.75 g, 2.1 mmol) neutralized with ammonia (pH 8) under stirring at 300 rpm. The distilled water was added in multiple steps with each addition of 5.0 g, followed by further stirring for 5 min to ensure the newly added water to be emulsified completely. The addition of distilled water was stopped when the sudden change occurred in the conductivity of emulsions, and the water weight fraction of the system at this point was defined as the “inversion point”. Before “inversion point”, the emulsions were W/O HIPEs (water volume fraction>74%); after phase inversion, W/O HIPEs changed to (W/O HIPEs)/W double emulsions. The as-prepared double emulsions were then transferred to a 100 mL glass jar, followed by bubbling with nitrogen for 15 min, and then sealed and irradiated by γ-ray in a field of a 1.30 × 1015 Bq 60Co source with an absorbed dose of 154.8 kGy at a dose rate of 107.5 Gy·min−1. The resulting porous polymer microspheres were collected by filtration and washed with ethanol repeatedly for three times and then dried under vacuum at 60 °C.

spheres are facilely obtained by the polymerization of these double emulsions initiated by γ-ray.

2. EXPERIMENTAL SECTION 2.1. Materials. Heptane (97%), toluene (analytical reagent), sodium chloride (NaCl), and ammonia (25%) were all purchased from Sinopharm Chemical Reagent (SCRC) and used without any further treatment. Styrene (St, 98%), butyl acrylate (BA, chemical pure), and methyl methacrylate (MMA, 98%) were purchased from SCRC and purified by passing through a basic alumina column to remove the inhibitor before use in a polymerization. Ethylene glycol dimethacrylate (EGDMA, 97%, Fluka) and 12-acryl-oxy-9-octadecenoic acid (AOA, Radiation Chemistry Corporation in Hefei of China) were used as received. 2.2. Preparation of Double Emulsions with High Internal Water Phase and Porous Polymer Microspheres. In a typical experiment, the starting W/O emulsions for a phase-inversion process were prepared by adding dropwise distilled water into the oil phase 12156

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2.3. Characterization. Optical micrographs were taken on Leica DM1000 optical microscope equipped with EC3 high-resolution digital CCD camera after diluting the double emulsions. The conductivities of emulsions were measured by DDS-307 conductivity meter (Shanghai Hongyi Instrument). Scanning electron microscopy (SEM) images were obtained on a JEOL JSM-6700 field-emission scanning electron microscope. Specimens of polyHIPE microspheres were coated with Au before observation. The average sizes of W/O droplets and microspheres were measured using Adobe Photoshop software. The interfacial tension was measured on CAM 200 (Finland, KSV) using pendant-drop method.

emulsions or W/O emulsions at pH >7 for at least 24 h, which agrees with the HLB value of neutralized AOA (6−8, Figure S2 in the Supporting Information) and its partition coefficient between oil and water (Coil/Cwater = 6.48). 3.1. Formation of W/O/W Double Emulsions via Phase Inversion of W/O HIPEs. The preparation of W/O/W double emulsions via phase inversion of W/O HIPEs is described in the Experimental Section. In short, primitive emulsions were prepared by intermittently adding dropwise of distilled water into the oil phase containing the surfactant under stirring. After the volume fraction of water was increased to just more than 74%, thick W/O HIPEs were formed without phase inversion for all samples. By further increasing the water content to a certain degree (sc. inversion point), W/O HIPEs eventually turned into high internal water-phase W/O/W double emulsions, as shown in Figure S3 in the Supporting Information. The process was monitored by conductivity tests and optical microscope. The effects of surfactant concentration, electrolyte concentration, the pH value, the polarity of oils, the addition interval of water, and stirring rate on the formation of double emulsions via the phase-inversion process were investigated. 3.1.1. Effect of the Surfactant Concentration. Because the surfactant AOA plays two roles simultaneously to stabilize both the W/O and O/W interfaces, the contents of AOA should be enough for distribution on the W/O and O/W interfaces, and thus the concentration of AOA may have an important impact on the formation of double emulsions. We first investigated the effect of AOA concentration on the formation and the average size of double emulsions via phase inversion of HIPEs, and the results are outlined in Figure 1 and Table 1. Learning from

3. RESULTS AND DISCUSSION We first test the ability of AOA to stabilize both W/O and O/ W emulsions under the same circumstance (see Supporting Table 1. Effect of AOA Concentration on Inversion Point (IP) and the Average Size of W/O Droplets sample

CAOA (%)a

WAOA (%)b

IP (%)

(μm)c

1 2 3 4 5

25.0 20.0 16.7 14.3 10.4

4.2 2.2 1.6 1.1 0.7

84.2 88.9 90.7 91.4 92.3d

19.3 54.8 191.7 328.3

a

Weight fraction of AOA based on the oil phase. bWeight fraction of AOA in the whole system at IP. cAverage size of W/O droplets. dNo phase inversion but a catastrophic change in conductivity. All of these samples: 3.0 g of styrene, pH 8, 300 rpm, interval = 5 min.

Information), and the results (Figure S1 in the Supporting Information) show that AOA can stabilize either W/O

Figure 2. (a) Conductivity of the sample 2 without NaCl at different weight fraction of water and after adding saturated NaCl solution to asprepared W/O/W double emulsion. (b) W/O HIPE formed by adding saturated NaCl solution to sample 2 after catastrophic phase inversion. (c) Inversion point of emulsions with various concentration of NaCl. (d) Change of interfacial tension of styrene containing 0.17 mg/mL of AOA and aqueous solution of different NaCl concentration with time. 12157

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Figure 3. Optical micrographs of emulsions at different pH values after phase inversion: (a) pH 7; (b) pH 7.5; and (c) pH 9. (d) Inversion point of emulsions at different pH values.

these large W/O droplets. On the contrary, pure oil droplets are transparent under optical microscope, as shown in Figure S4(b,d) in the Supporting Information. When the AOA content is decreased to 10.4%, double emulsions cannot be formed when a catastrophic change in conductivity takes place, but the W/O emulsions are sustained with some large irregular waterdispersion zones instead. In addition, the average size of these double-emulsion droplets increases from 19.3 to 328.3 μm as lowering the contents of AOA from 25.0 to 14.3% based on the oil phase; meanwhile, “inversion point” is also postponed from 84.2 to 91.4% and reaches the highest water weight fraction of 92.3% as the content of AOA is only 10.4% (see Table 1). In this work, plenty of AOA is used because it is not only generally required in HIPEs to stabilize a great deal of oil-water interface, but also necessary for double emulsions to stabilize both the W/O and O/W interfaces simultaneously. Thus, redundant AOA should be dissolved in the continuous oil phase and form micelles.27 Before phase inversion, our system is W/O HIPEs when the volume fraction of water is increased to more than 74%, in which close-packed water droplets are

Table 2. Effect of pH Value on IP and the Average Size of W/O Droplets sample

pH

WAOA (%)a

IP (%)

(μm)b

9 10 2 11

7 7.5 8 9

1.5 1.9 2.2 2.6

92.3 90.3 88.9 86.9

277.9 170.8 54.8 32.6

a

Weight fraction of AOA in the system at IP bAverage size of W/O droplets. All of these samples: 3.0 g of St, 0.75 g of AOA, 300 rpm, interval = 5 min.

Figure 1, except for sample 5 with 10.4% AOA (Figure 1e), all other samples 1−4 (with AOA of 25.0, 20.0, 16.7, or 14.3%, respectively) form double emulsions after phase inversion. From enlarged optical micrographs of samples 1 and 2 in the inset of Figure 1a,b, we can clearly observe the obvious structure of double emulsions with lots of close-packed water droplets dispersed in the oil droplets. The dark droplets with faintly visible inner structure for samples 3 and 4 (Figure 1c,d) suggest that there are a large number of W/O interfaces in

Table 3. Effect of Oil Polarity on Inversion Point (IP) and the Average Size of W/O Droplets sample

oil

solubility (mg/L)a

WAOA (%)b

IP (%)

(μm)c

12 2 13 14

heptane styrene toluene BA

2.8 300 526 2000

1.3% 2.2% 2.6% 3.2%

93.6 88.9 87.1 84.2

101.7 54.8 17.7 6.2

a

Solubility of oil in water was cited from Chemoffice software. bWeight fraction of AOA in the system at IP. cAverage size of W/O droplets. All of these samples: 3.0 g of oil, 0.75 g of AOA, pH 8, 300 rpm, interval = 5 min. 12158

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Figure 4. Optical micrographs of emulsions of different oils after phase inversion: (a) heptane, (b) toluene, and (c) BA. (d) Inversion point of emulsions with various oil phases.

1.1% then to 0.7% along with the decrease in the weight fraction of AOA from 25.0 to 14.3% then to 10.4% based on oil phase, which should be responsible for the increase of the average size of W/O droplets with the decrease in the amount of AOA. However, sample 5 with 10.4% AOA cannot form double emulsion at the inversion point, which should be due to the shortage of transferable AOA. As the newly formed double emulsions are prepared via the phase inversion of W/O HIPEs, they maintain the characteristic of high internal water phase in oil droplets (see Figure S3b in the Supporting Information), which can be used as templates for making highly interconnecting porous polymer microspheres, as described later. 3.1.2. Effect of NaCl Concentration. Because the surfactant AOA has a carboxyl end group, the addition of salts such as NaCl can influence not only emulsifying performances of the anionic surfactant owing to a decline in charge repulsion30,31 but also the osmotic pressure of droplets,32 which is important for the stability of both W/O HIPEs and W/O/W double emulsions. Therefore, we studied the effect of NaCl concentrations ranging from 0 to 2% on the formation of double emulsions (see Figure 2 and Table S1 and Figure S4 in the Supporting Information). It is found that stable double emulsions could not be formed via catastrophic phase inversion of W/O HIPEs in the presence of NaCl (see Figure S4 in the Supporting Information). When the content of NaCl is 0.5% (sample 6) or 1.0% (sample 7) based on the water phase, although double emulsions are formed instantly after phase inversion of W/O HIPEs (see Figure S4a,c in the Supporting Information), these double emulsions are unstable and changed to single emulsions after standing for 24 h at room temperature (Figure S4b,d in the Supporting Information). When the content of NaCl is further increased to 2 wt % (sample 8), double emulsions cannot be formed at all after a catastrophic

Table 4. Average Size of Various PolyHIPE Microspheres sample

oil

CAOA (%)

26 27 28 29

St/EGDMA (1:1) St/EGDMA (1:1) BA/St/EGDMA(1:1:2) MMA/St/EGDMA(1:1:2)

20 23 20 13

a

IP (%)

(μm)

88.9 84.2 80.0 84.2

collapsed 18.8 9.8 17.7

b

a

Weight fraction of AOA based on oil phase. bAverage size of porous polymer microspheres. All samples: 3.0 g of oil phase, pH 9, 300 rpm.

surrounded by films composed of surfactant AOA monolayer and styrene oil phase containing reverse AOA micelles27 (see the illustration of interfacial films in Scheme 1b). These reverse micelles and surfactant monolayer are considered to be channels for water transport during coalescence.3 According to the coalescence kinetics in emulsions, the essential step in the phase-inversion process is the rapid coalescence of droplets throughout the whole system to form a new continuous phase, and the drainage of the films around droplets is the ratedetermining step.28,29 Therefore, when lowering the content of AOA, the amount of AOA micelles acting as water transport channels is decreased in the oil films of emulsions, leading to a slow rate of coalescence and thus the postponement of “inversion point” (see Figure 1f). When the phase inversion happens, the redundant AOA micelles in the oil phase of W/O HIPEs would be immediately dissolved and adsorb to the newly formed outer O/W interfaces because AOA neutralized with ammonia is also able to stabilize O/W emulsions (see the Supporting Information), leading to the formation of double emulsions stabilized by a single surfactant if the concentration of AOA is enough (see the illustration of formation of double emulsions in Scheme 1c). From Table 1, we can see that the AOA content in emulsion system at inversion point (WAOA) is greatly reduced from 4.2 to 12159

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Figure 5. SEM images of polyHIPEs microspheres: (a1−a4) sample 27, (b1−b4) sample 28, and (c1−c4) sample 29, where a3, b3, and c3 show the surface of samples and a4, b4, and c4 show the cross-section of samples.

change in conductivity occurs, and the final structure of sample 8 is the same as that of sample 5 (see Figure S4e in the Supporting Information and Figure 1e). As previously mentioned, double emulsions with high internal water phase can be formed in the absence of NaCl (samples 1−4), even stable for at least 3 months without obvious change in the structure (see Figure S4f in the Supporting Information), and the average size of W/O droplets is only slightly reduced from 54.8 to 50.2 μm (Table S1 in the Supporting Information). Interestingly, the phase inversion from W/O HIPEs to W/O/ W double emulsions is reversible. By adding saturated NaCl aqueous solutions to double emulsions (sample 2), the conductivity is greatly decreased (Figure 2a), and the optical micrograph (Figure 2b) shows the formation of W/O HIPEs again. These results indicate that the addition of salts interferes with the formation of W/O/W double emulsions via the phase inversion of W/O HIPEs, although it is needed for stabilizing W/O HIPEs. The reason may be that the addition of salts will induce osmotic pressure that prevents the coalescence of water droplets.32 Thus, the addition of salts can greatly improve the stability of W/O HIPEs, leading to the difficulty in phase inversion (e.g., sample 8). Moreover, the addition of salts will not only weaken the charge repulsion between the hydrophilic head of anionic surfactant AOA30,31 but also reduce the ionization degree of carboxyl33,34 to make AOA more hydrophobic, resulting in the instability of outer O/W interface, thereby facilitating the formation of W/O emulsions. Thus, even if the phase inversion of W/O HIPEs in the presence of NaCl occurs to form double emulsions (e.g., samples 6 and 7), they would be unstable and finally turn into single O/W emulsion. Similarly, double emulsions prepared from the phase inversion of W/O HIPEs without NaCl (e.g., sample 2) will

reverse to W/O HIPEs again after adding the saturated NaCl aqueous solutions. Figure 2c shows the inversion point of the systems with different NaCl concentrations by monitoring conductivity. It can be seen that with increasing the concentration of NaCl the inversion point is first advanced and then postponed, reaching the lowest value at 0.5 wt % of NaCl. By measuring interfacial tension of AOA-absorbed W/O interfaces, it is found that the interfacial tension has a sharp decline in the first 15 seconds, followed by a slow fall reaching a balanced value (Figure 2d). Among the samples with different NaCl concentration, the balanced interfacial tension at 0.5 wt % of NaCl is the lowest, which is consistent with the previous report in the literature.35 The lower the interfacial tension, the more easily the phase inversion and thus the earlier the inversion point. Although the balanced interfacial tensions with 1 to 2 wt % of NaCl are slightly lower than that without NaCl, the addition of salts will induce osmotic pressure to counter the coalescence of water droplets, as previously discussed,32 leading to the slowdown of the coalescence of water droplets in W/O HIPEs. According to the coalescence kinetics in emulsions, phase inversion is associated with the coalescence of droplets. By increasing the content of NaCl, the inversion point is postponed as the coalescence rate is reduced. (See Figure 2c.) 3.1.3. Effect of the pH Value. The pH value could also affect the ionization equilibrium of carboxyl groups and thus emulsification performances of AOA. If not neutralized, AOA is highly hydrophobic and unable to stabilize emulsions. As previously mentioned, after being neutralized with ammonia, AOA was able to stabilize both W/O emulsions and O/W emulsions, even W/O/W double emulsions. Figure 3 illustrates that all inverse emulsions at different pH values ranging from 7 12160

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the Supporting Information, and thus the average size of W/O droplets is decreased. 3.1.6. Effect of Stirring Rate. Previous literature indicated that the stirring rate had an important influence on phase inversions.38,39 So we also study the effect of stirring rate on the inversion point and the average size of double emulsions. As shown in Table S3 and Figures S6 and S7 in the Supporting Information, the stirring rate has almost no effect on the phase inversion of W/O HIPEs at the addition interval of 5 min (samples 2 and 19−21), but does affect the phase inversion of W/O HIPEs at the addition interval of 1 min (samples 22−25) in our system. When the stirring rate is increased from 250 to 600 rpm there is a slight difference in the inversion point (Figure S6d in the Supporting Information) and the average size of W/O droplets (Table S3 in the Supporting Information) for samples 2 and 19−21. However, the average size of W/O droplets in samples 22−25 decreases from 646.4 to 117.4 μm with the increase of the stirring rate from 250 to 600 rpm. These results elucidate that the phase inversion is closely related to the adsorption amount of AOA in the interface. If the addition interval of water is long enough for AOA to adsorb at the interface to approximate balance, the effect of stirring intensity on phase inversion will be weakened. Otherwise, the stirring rate will strongly influence the diffusion rate of AOA to interface, so as to affect the phase transition. 3.2. Preparation of Interconnecting Porous Polymer Microshperes. Highly interconnecting porous microspheres can be obtained through the polymerization of monomers in the oil phase of the as-prepared W/O/W double emulsions induced by γ-ray at room temperature, which is beneficial for thermodynamically unstable double emulsions to keep their structures.1,2,40 A cross-linker ethylene glycol dimethacrylate (EGDMA) is added to the oil phase to accelerate the polymerization reaction of emulsions and to obtain interconnecting porous structure. However, as-prepared porous microspheres of sample 26 collapsed during drying, which may be caused by the low concentration of monomers of sample 26 with high inversion point (Table 4). From Figure 5, it is found that samples 27−29 have all interconnecting porous structure, and the pores at the surface of samples 27 and 28 are circular, while those in sample 29 are irregular. Moreover, the internal open-cell structures of sample 27 (Figure 5a4) and sample 28 (Figure 5b4) are the same as that of polyHIPE monoliths,12,13 just sample 28 is more empty. However, the internal holes of as-prepared microspheres of sample 29 are big with irregular interconnecting pores (Figure 5c4). This may be due to a higher solubility of MMA in water, which leads to the coalescence of inner water droplets and water diffusion between the inner and outer water phases. The structures of polyHIPE microspheres can also support the formation mechanism of high internal water-phase double emulsions proposed in this work. It is worth noting that the average diameters of these polyHIPE microspheres are all