Aqueous Two-Phase System Formation in Small Droplets by Shirasu

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Aqueous two-phase system formation in small droplets by SPG membrane emulsification followed by water extraction Kazuki Akamatsu, Rieko Kurita, Daichi Sato, and Shin-ichi Nakao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01320 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Aqueous two-phase system formation in small droplets by SPG membrane emulsification followed by water extraction Kazuki Akamatsu1,*, Rieko Kurita1, Daichi Sato1, Shin-ichi Nakao1,2 1Department

of Environmental Chemistry and Chemical Engineering, School of Advanced

Engineering, Kogakuin University, 2665-1 Nakano-machi, Hachioji-shi, Tokyo 192-0015, Japan 2Research

Institute for Science and Technology, Kogakuin University, 2665-1 Nakano-machi,

Hachioji-shi, Tokyo 192-0015, Japan *Corresponding

author: Kazuki Akamatsu

Tel: +81-42-628-4584 Fax: +81-42-628-4542 E-mail: [email protected]

Abstract By utilizing water transport phenomena between two different water-in-oil (W/O) emulsion droplets through continuous oil phase, we developed a novel method of aqueous two-phase system (ATPS) formation in small droplets prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. When we mixed W/O emulsion droplets containing polyethylene glycol (PEG) and dextran ACS Paragon Plus Environment

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(DEX), at concentrations below the threshold of the phase separation, with droplets containing other solutes at high concentrations, water extraction from the droplets containing PEG and DEX to those containing the other solutes occurred owing to the osmotic pressure difference. This effect increased the concentrations of PEG and DEX in the droplets above the phase separation threshold. We demonstrated the feasibility of the preparation method by varying the pore sizes of the SPG membranes, the solutes, and their concentrations. Only when the concentration of the solute was high enough to extract sufficient amounts of water did the homogeneous disperse phase consisting of PEG and DEX in droplets turn into a PEG-rich phase and DEX-rich phase, showing ATPS. This result was irrespective of the solute itself and pore size of the SPG membrane. In particular, we successfully demonstrated monodisperse ATPS droplets with diameters of approximately 10 μm under a certain condition.

Introduction An aqueous two-phase system (ATPS) involves two different phases spontaneously forming on mixing of two aqueous solutions two water-soluble polymers, or a salt and a water-soluble polymer. One of the most well-known ATPS forming combinations is polyethylene glycol (PEG) and dextran (DEX). A solution of PEG and DEX at high concentrations separates into a PEG-rich phase and a DEX-rich phase.1-3 Because partitioning of biomacromolecules and cells in ATPSs is affected by various factors such as their charges, molecular weights, concentrations, ionic compositions of the medium, there have been many reports on the use of ATPSs for biotechnological processes, such as separation.4-12 Over the last decade, the preparation of single or double emulsion droplets with the use of ATPSs has also attracted considerable attention.13-18 Water-in-water (W/W) emulsions are the simplest systems; however, these are not easy to prepare because the interfacial tension between the two immiscible water phases is much lower than that of the interface formed with oil and water. Additionally, kinetic stability is an issue for preparing W/W emulsions because amphiphilic surfactants ACS Paragon Plus Environment

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are generally not useful. Thus, various attempts to prepare a stable W/W emulsion have been reported with the use of triblock copolymers,19 protein particles,20-21 nanoplates,22 and liposomes23 as W/W emulsion stabilizers. When W/W emulsion droplets are dispersed in an oil phase, a water-in-water-in-oil (W/W/O) emulsion is formed. Successful preparation of monodisperse W/W/O emulsions with the use of a microfluidic device has been reported by Ono’s group.24 A water-in-water-in-water (W/W/W) double emulsion was also prepared by Shum’s group.25 However, these studies used microfluidic techniques, which are suitable for preparing monodisperse multiple emulsion droplets but are not generally effective for preparing smaller droplets of sizes less than several tens μm. In this study, we report the novel achievement of an ATPS in smaller droplets. A schematic illustration of the preparation is shown in Figure 1. First, W/O emulsion droplets containing a mixture of PEG and DEX at low concentrations were prepared. At this stage, an ATPS was not formed in the droplet. Another set of W/O emulsion droplets, containing solute at high concentrations, was also prepared, and the two kinds of droplets were mixed with each other. The osmotic pressure difference between the two aqueous droplets should induce water molecules in the PEG-DEX droplets to pass through the continuous oil phase into the other concentrated droplets until equilibrium is reached. Thus, an ATPS is formed in each droplet when sufficient amounts of water molecules are extracted from the PEG-DEX droplets to increase their concentrations above the thresholds for phase separation. Here, the use of Shirasu Porous Glass (SPG) membrane emulsification technique26-29 enables the production of monodisperse W/O emulsion droplets with average diameters that can be tuned by the pore sizes of the membranes in the range from 0.1 to 50 μm. This size range is smaller than that typically achieved by microfluidic techniques. Water permeates through immiscible oils between two different W/O emulsion droplets owing to the chemical potential gradients.30 Penetration through the immiscible middle oil layer between the innermost aqueous phase and the outermost aqueous phase in the W/O/W emulsion droplets31-35 has been reported; hence, the strategy proposed in Figure 1 should be feasible. This preparation strategy comprises two steps; namely, preparation of the W/O emulsion and water ACS Paragon Plus Environment

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extraction by mixing the other W/O emulsion with a high solute concentration in the disperse phase. Another one-step preparation route might also be considered, for example an aqueous two-phase PEG-DEX solution could be simply emulsified into the oil phase. However, this procedure is not an option in principle, because it is impossible to pressurize the inhomogeneous solution into each pore to prepare emulsion droplets with constant inhomogeneity. The procedure would produce a mixture of droplets containing only PEG, those containing only DEX and those containing PEG and DEX with different compositions. We report herein that an ATPS can be formed in small droplets, having diameters less than 50 μm by our proposed strategy. In particular, we studied the effects of the solutes and their concentrations, and the pore sizes of the SPG membranes on the formation of the ATPS droplets.

Figure 1. Schematic illustration for ATPS formation in W/O emulsion droplets prepared by the SPG membrane emulsification technique followed by water extraction owing to the osmotic pressure difference.

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Experimental Section Materials. Sodium chloride, glucose and kerosene were purchased from Wako Pure Chemical Industries Ltd (Japan). Allura red, poly(ethylene glycol) (PEG, Mw : 8000), Fluorescein isothiocyanate–dextran (FITC-DEX, Mw : 20000) were purchased from Sigma-Aldrich. Dextran (DEX, Mw: 15000–20000) was purchased from Funakoshi Co., Ltd. (Japan). These materials were used without further purification. Tetraglycerol condensed ricinoleate (TGCR) was kindly supplied by Sakamoto Yakuhin Kogyo Co. (Japan). Preparation of small ATPS droplets. First of all, aqueous solutions containing PEG and DEX with various concentrations were prepared to judge visually whether ATPS was formed or not, and a simple phase diagram was constructed. Then as a disperse phase for achieving ATPS, aqueous solution containing 2.0 wt% PEG and 4.0 wt% DEX was prepared. This solution was below the thresholds of the phase separation and homogeneous. In some cases, FITC-labelled DEX was added. The weight ratio of DEX to FIDC-DEX was 8000:1. Aqueous solutions containing sodium chloride or glucose with known concentrations or no solute were prepared. In addition, 0.5 wt% Allura red was added to enable easy distinction from the PEG-DEX emulsion droplets. Two different W/O emulsions were prepared by the SPG membrane emulsification technique, where kerosene with 1.0 wt% TGCR was used as the continuous phase. The surfaces of the SPG membranes were treated to be hydrophobic for the emulsification,36 and the pore sizes were 15, 12, and 5.5 μm. The details in the preparation conditions are summarized in Table 1. The weight ratio of the disperse phase to the continuous phase was 3:10 in each case. Then the same amounts of these two different W/O emulsions were mixed in a beaker with stirring at 200 rpm. The droplets were observed with a digital microscope (VHX-2000, Keyence Corporation, Japan). The size distributions of the droplets directly after the emulsification were measured with a laser diffraction particle size distribution analyzer (LA-950V2; Horiba Ltd., Japan). When the DEX contained FITC-DEX, a fluorescent microscope (IX73, Olympus Corporation, Japan) was also used to observe the droplets. ACS Paragon Plus Environment

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Table 1. Experimental conditions for emulsion A (to achieve ATPS) and emulsion B (to extract water). 0.5 wt% Allura red was also added in every disperse phase in emulsion B for easy distinction. Condition

Pore size [μm]

Disperse phase in Emulsion A

(A)

Disperse phase in Emulsion B -

12

2.0wt% PEG, 4.0wt% DEX aq.

(B) 2.0wt% PEG, 4.0wt% DEX aq. (C)

3.0wt% NaCl aq.

15 (DEX : FITC-DEX = 8000 : 1)

(D)

0.30wt% NaCl aq. 12

(E) (F)

2.0wt% PEG, 4.0wt% DEX aq. 5.5

18.5wt% glucose aq. 3.0wt% NaCl aq.

Results and Discussion Effect of the solute concentration on water extraction. Figures 2(a)-(e) show the evolution of the droplets in the mixture under condition (A). The average diameters and coefficient of variations (CV) for the emulsion droplets containing PEG and DEX and for those containing only Allura red as prepared were 45.7 μm and 10.6%, and 43.7 μm and 12.3%, respectively. Although there was a small osmotic pressure difference between these two different emulsion droplets, no distinct changes in appearance or droplet size were observed within 6 h. The emulsion droplets prepared by the SPG membrane emulsification technique were stable and some of the changes that occurred in the droplets, as explained below for other conditions, are not attributed to their instability.

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Figure 2. Evolution of the droplets in the mixture in (a) 0 min, (b) 1 h, (c) 2 h, (d) 4 h, and (e) 6 h under condition (A). Figures 3(a)-(e) show the evolution of the droplets in the mixture under condition (B). No change was observed over 1 h, and the ATPS formation was observed in some droplets within 2 h. The ATPS formed in the droplets was a droplet-in-droplet structure, which means these emulsions are W/W/O. An ATPS with a Janus structure was also observed together with the droplet-in-droplet structure. Finally, within 6 h, an ATPS was formed in most droplets containing PEG and DEX. Notably, the droplets having an ATPS became smaller as the other droplets containing NaCl being larger, compared with their original states as shown in Figure 3(a). The water extraction from the droplets containing PEG and DEX owing to the osmotic pressure difference resulted in these changes, even though the water transport mechanism remains unclear at this stage. Throughout various experiments using W/O/W emulsion, some researchers have proposed several possible routes,

34, 37-41

including (i) transport via reverse

micelle, (ii) transport through thin lamella, (iii) transport via hydrated surfactant, and (iv) transport of dissolved water molecule itself. Two or more routes may be involved for the water transport. We attribute the coexistence of the two types of ATPS, droplet-in-droplet, and Janus structures to insufficient kinetic stability of the system tested. No stabilizers were used at the interface formed ACS Paragon Plus Environment

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between the two water phases. Throughout our careful observations, first the droplet-in-droplet structure was formed and then some droplets changed into Janus structures. The use of appropriate interface stabilizers might enable control over the ATPS structures.

Figure 3. Evolution of the droplets in the mixture in (a) 0 min, (b) 1 h, (c) 2 h, (d) 4 h, and (e) 6 h under condition (B). Figure 4 shows another evidence of the formation of ATPS in the droplets by using the strategy. This result was obtained under condition (C). One side of the droplets with a Janus structure is green, indicating a DEX-rich phase. The other side is the PEG-rich phase. Thus, these droplets consisted of a DEX-rich hemisphere and a PEG-rich hemisphere, which confirms that phase separation occurred in the droplets through the mechanism proposed in this study. The other larger droplet emulsion contained only NaCl.

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Figure 4. Photograph of the ATPS droplets prepared under condition (C), captured with a fluorescence microscope. Figures 5(a)-(e) show the evolution of the droplets in the mixture under condition (D). The concentration of NaCl was only one-tenth as low as that used under condition (B); however, the osmotic pressure was estimated to be 2.5 atm by the van’t Hoff equation in this case. This value is much higher than the osmotic pressure of other emulsion droplets containing 2.0 wt% PEG and 4.0 wt% DEX (~0.12 atm if the van’t Hoff equation is also applicable for the polymer solution owing to their lower concentrations); thus, water should extract from the droplets containing PEG and DEX. However, even after 6 h, no ATPS droplets were observed. We attribute this result to an insufficient difference in the osmotic pressures to extract sufficient amounts of water molecules and increase the concentrations of PEG and DEX above the thresholds of phase separation. On the basis of these results, the important factors for ATPS formation are the solute concentration, which drives the water extraction.

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Figure 5. Evolution of droplets in a mixture in (a) 0 min, (b) 1 h, (c) 2 h, (d) 4 h, and (e) 6 h under condition (D). Effect of the solute types for water extraction. Figures 6(a)-(e) show the evolution of the droplets in the mixture under condition (E). The osmotic pressure of the 18.5 wt% glucose aqueous solution was estimated to be 25 atm by the van’t Hoff equation, which is the same as for condition (B) with NaCl. The droplets behaviors were similar to those of condition (B), which formed an ATPS with a droplet-in-droplet structure together with a Janus structures. The diameters of the ATPS droplets decreased over time. In contrast, the diameters of the glucose droplets increased. Thus, sufficient water extraction from the droplets containing PEG and DEX occurred, owing to the osmotic pressure difference successfully raising their concentrations above the thresholds for phase separation. By comparing the results in the conditions (B) and (E), we conclude that the ATPS formation through the new method is independent of the solute type in terms of generating an osmotic pressure difference to extract water.

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Figure 6. Evolution of the droplets in the mixture in (a) 0 min, (b) 1 h, (c) 2 h, (d) 4 h, and (e) 6 h under condition (E). Effect of the pore sizes of the SPG membranes. Figures 7(a)-(e) show the evolution of the droplets in the mixture under condition (F). Emulsion size, when prepared by the SPG membrane emulsification, depends on the pore size of the membrane. In this case the pore size was 5.5 μm and the average diameters and CV of the emulsion droplets containing PEG and DEX and for those containing NaCl with Allura red were 19.9 μm and 10.4%, and 18.9 μm and 10.0%, respectively. The size-uniformity was comparable with other conditions where SPG membranes with larger pores were used; however, the average diameters were much smaller. The stability of these smaller emulsion droplets was also confirmed before mixing. The droplet behaviors were also similar to those under condition (B), and ATPS with droplet-in-droplet structure together with Janus structures was formed. In addition, we also observed that the diameter of the ATPS droplets decreased over time while the NaCl droplets grew larger. After 6 h, the ATPS droplet sizes were approximately 10 μm. Although the initial droplet sizes were smaller in this case, the osmotic pressure difference generated between the two emulsion droplets was the same as that in the condition (B); hence, the behaviors in this case were similar to those under condition (B). By comparing the results under conditions (B) and (F), we concluded that an ATPS was ACS Paragon Plus Environment

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formed, irrespective of the SPG membrane pore size used to prepare the emulsion droplets. The initial size of the droplet depended on the pore size; thus, the size of the resultant ATPS droplets also depended on the pore size.

Figure 7. Evolution of droplets in the mixture in (a) 0 min, (b) 1 h, (c) 2 h, (d) 4 h, and (e) 6 h under condition (F). Analysis by using phase diagram. As shown in Figure 8, we can see ATPS is formed when the concentrations of PEG and DEX exceeds the threshold. And the initial concentrations of PEG and DEX in the disperse phase were 2.0 wt% and 4.0 wt%, which was below the threshold. It was difficult to determinate the droplets sizes containing PEG and DEX in 6 h with the laser diffraction particle size distribution analyzer because it was impossible to separate them from the other droplets containing NaCl or glucose completely. However we estimated the average diameters of the droplets containing PEG and DEX in 6 h by analysis of more than 100 droplets in the microscope pictures. Then we estimated the concentrations of PEG and DEX in the final droplets under the assumption that all the water molecules extracted from the droplets containing PEG and DEX moved to the other droplets containing NaCl or glucose. The results are also shown in Figure 8. The weight ratio of PEG to DEX did not change under the assumption that only water molecules were extracted, thus the final ACS Paragon Plus Environment

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concentrations should be on the dotted line. Under the conditions (B), (E) and (F), the final concentrations of PEG and DEX exceeded the threshold. This estimation was consistent with the experimental results shown above. Under the condition (D), the final concentrations of PEG and DEX were estimated to be 4.4 wt% and 8.8 wt%, respectively, which was below the threshold and also consistent with the experimental result.

Figure 8. (a) Phase diagram of the ATPS formation. (b) Magnified diagram for easy distinction at lower concentration range.

Conclusion We successfully demonstrated a novel strategy to form ATPS in emulsion droplets having sizes less than 50 μm. This preparation consists of the two steps; both W/O emulsion droplets containing PEG and DEX with concentrations below the threshold of the phase separation and those containing NaCl or glucose with very high concentrations were prepared by SPG membrane emulsification. These two different droplets were then mixed to extract water from the PEG-DEX droplets into the other droplets through the continuous oil phase. This water extraction occurred owing to the osmotic pressure difference, and finally resulted in an increase of the concentrations of PEG and DEX in the droplets above the threshold of phase separation. To demonstrate the feasibility of this strategy, we initially ACS Paragon Plus Environment

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examined the effects of the solute concentration for the water extraction on the ATPS formation. By comparing the results obtained with 3.0 wt% and 0.30 wt% NaCl aqueous solution in the disperse phase used to mix with droplets containing 2.0 wt% PEG and 4.0 wt% DEX in the disperse phase, we showed that the droplets with a higher solute concentration are essential for ATPS formation because the water extraction is driven by the osmotic pressure difference. This result was also examined from the results obtained when 18.5 wt% glucose aqueous solution in the disperse phase was used, whose osmotic pressure was estimated as large as those containing 3.0 wt% NaCl. In this case, the ATPS formation was clearly observed. Finally, by comparing the results obtained when the SPG membranes with 12 or 5.5 μm of pore sizes were used, we demonstrated the ATPS formation is independent of the emulsion size even when the sufficient water extraction is guaranteed. In the case using the smaller pores, we obtained ATPS droplets having sizes of approximately 10 μm. Throughout this study, two types of ATPS droplets were observed; droplet-in-droplet and Janus structure. At this stage we cannot control these structures, but we consider that interfacial stabilizers might be useful for overcoming this issue in the future.

Acknowledgement TGCR was kindly supplied by Sakamoto Yakuhin Kogyo Co., Japan. This study was partly supported by a Grant-in-Aid for Challenging Exploratory Research (No. 15K14208) from the Japan Society for the Promotion of Science (JSPS).

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TOC graphic K. Akamatsu et al,

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