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A Novel Method of Fabrication of Latex-Stabilized Water-Core Colloidosomes at Room Temperature Toshiyuki Nomura† and Alexander F. Routh* Department of Chemical Engineering and Biotechnology, BP Institute, University of Cambridge, Madingley Road, Cambridge CB3 0EZ, United Kingdom. †Present address: Department of Chemical Engineering, Osaka Prefecture University, Osaka, Japan Received August 20, 2010. Revised Manuscript Received November 1, 2010 Colloidosomes have attracted great interest in recent years because of the capability of storage and delivery of useful materials in various fields. In this article, a novel technique for formation of colloidosomes at room temperature suitable for encapsulation of biomaterials was examined. We demonstrate the formation of colloidosomes of 18.0 μm in size at room temperature by adding a small amount of ethanol into the continuous phase of sunflower oil. Poly(methyl methacrylate-co-butyl acrylate) latex particles of 185 nm in size, used in this study, were found to aggregate when ethanol was added to their suspension. We suggest that the shell of the water-core emulsions was locked by the aggregation of latex particles due to the diffusion of ethanol into the aqueous latex suspension.
1. Introduction Microencapsulation has attracted considerable attention in recent years because of promising potential applications in the field of medicine, biotechnology, agriculture, food and beverages, cosmetics, and catalysis, etc.1-4 Various methods have been employed to prepare microcapsules using self-assembly of colloidal particles,5-10 a sacrificial template,11,12 and template layer-by-layer polyelectrolyte self-assembly.13,14 In this paper, we focus attention on colloidosomes—formed from colloidal latex. The overall aim of our work is to develop a method suitable to encapsulate biomaterials. This necessitates the avoidance of any harsh chemicals or elevated temperatures. The technique of self-assembly of colloidal particles on the interface between oil and water phases was first reported by Ramsden15 and Pickering16 over 100 years ago. The particles stabilize the emulsion droplets, and these systems have been termed Pickering emulsions. Velev et al.5-7 used Pickering emulsions to make microcapsules, where the shell particles were locked into place. This type of hollow microcapsule was named “colloidosomes” by Dinsmore et al.8 due to the analogy with liposomes. In order to fabricate stable colloidosomes, the shell of self-assembled particles must be fixed. There are numerous possible methods: The simplest is to *Corresponding author. E-mail:
[email protected]. (1) Gibbs, B. F.; Kermasha, S.; Alli, I.; Mulligan, C. N. Int. J. Food Sci. Nutr. 1999, 50, 213. (2) Chang, T. M. S.; Prakash, S. Mol. Biotechnol. 2001, 17, 249. (3) Langer, R. Nature 1998, 392, 5. (4) Yow, H. Y.; Wu, X.; Routh, A. F.; Guy, R. H. Eur. J. Pharm. Biopharm. 2009, 72, 62. (5) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2374. (6) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2385. (7) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1997, 13, 1856. (8) Dinsmore, A. D.; Hsu, M. F.; Nikolaides, M. G.; Marquez, M.; Bausch, A. R.; Weitz, D. A. Science 2002, 298, 1006. (9) Laib, S.; Routh, A. F. J. Colloid Interface Sci. 2008, 317, 121. (10) Yow, H. N.; Routh, A. F. Langmuir 2009, 25, 159. (11) Antipov, A. A.; Shchukin, D.; Fedutik, Y.; Petrov, A. I.; Sukhorukov, G. B.; Mohwald, H. Colloid Surf. Physicochem. Eng. Aspects 2003, 224, 175. (12) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mohwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (13) Caruso, F.; Trau, D.; Mohwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (14) Mak, W. C.; Cheung, K. Y.; Trau, D. Chem. Mater. 2008, 20, 5475. (15) Ramsden, W. Proc. R. Soc. London 1903, 72, 156. (16) Pickering, S. U. J. Chem. Soc. 1907, 91, 2001.
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heat the system above the glass transition temperature of the shell latex and hence to form a film in the shell.8 An alternative is to use a chemical cross-linking reagent17 or to deposit polyelectrolytes5-7 or a further layer of another material.29 An alternative is to “lock” the core through a gelling or polymerization.18 These processing steps however may impose some application limits. Various materials and techniques have been developed to fabricate colloidosomes such as hairy colloidosomes,19 magnetic colloidosomes,20 carbon nanotubosomes,21 composite colloidosomes,22,23 temperature-responsive colloidosomes,24,25 and pH-responsive colloidosomes.26 In this work we demonstrate the locking of the shell through a colloidal instability. By making the shell latex particles transition to an unstable state after self-assembling into the shell, we establish a technique for fabrication, at room temperature, of water-core colloidosomes. To achieve this purpose, a small amount of ethanol in the continuous phase was found to be the most effective. In this study, the effects of the concentration of ethanol and of latex in the dispersed aqueous phase on the stabilization of colloidosomes were examined. The colloidosomes were characterized by optical, electron, and confocal microscopes.
2. Experimental Section 2.1. Materials. Sunflower oil (Sainsbury’s) was used as a continuous phase without purification. Ethanol (Fisher Scientific, 99.5%) was used without purification. Milli-Q water (18.2 MΩ 3 cm) (17) Thompson, K. L.; Armes, S. P. Chem. Commun. 2010, 46, 5274. (18) Bon, S. A. F.; Colver, P. J. Langmuir 2007, 23, 8316. (19) Noble, P. F.; Cayre, O. J.; Alargova, R. G.; Velev, O. D.; Paunov, V. N. J. Am. Chem. Soc. 2004, 126, 8092. (20) Duan, H.; Wang, D.; Sobal, N. S.; Giersig, M.; Kurth, D. G.; Mohwald, H. Nano Lett. 2005, 5, 949. (21) Panhuis, M.; Paunov, V. N. Chem. Commun. 2005, 13, 1726. (22) He, Y.; Li, T.; Yu, X..; Zhao, S.; Lu, J.; He, J. Appl. Surf. Sci. 2007, 253, 5320. (23) Kim, S. H.; Heo, C. J.; Lee, S. Y.; Yi, G. R.; Yang, S. M. Chem. Mater. 2007, 19, 4751. (24) Lawrence, D. B.; Cai, T.; Hu, Z.; Marquez, M.; Dinsmore, A. D. Langmuir 2007, 23, 395. (25) Kim, J. W.; Fernandex-Nieves, A.; Dan, N.; Utada, A. S.; Marquez, M.; Weitz, D. A. Nano Lett. 2007, 7, 2876. (26) San Miguel, A.; Scrimgeour, J.; Curtis, J. E.; Behrens, S. H. Soft Matter 2010, 6, 3163.
Published on Web 11/18/2010
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Figure 2. Photographs of latex stabilized water-in-oil emulsions prepared at various ethanol concentrations in sunflower oil.
Figure 1. SEM image of latex particles. was produced by a Pure Lab Ultra apparatus from Elga. Poly(methyl methacrylate-co-butyl acrylate) latex particles of 185 nm diameter (by DLS), with a low-temperature glass transition of 34.0 °C, have been previously reported.27 Figure 1 shows an SEM image of the latex particles used in study. Fluorescent probe SYTO 9 was purchased from Molecular Probes. 2.2. Formation of Water-in-Oil Colloidosomes. The formation technique for water-in-oil colloidosomes is essentially simple: A mixture of sunflower oil and ethanol is mixed with an aqueous latex suspension to form emulsions. In this paper, the effect of ethanol dissolved in the oil phase on the fixation of the shell of colloidosomes stabilized by the latex particles was investigated. A typical experimental procedure for fabrication of colloidosomes is as follows: ethanol (0.16 mL) was mixed with sunflower oil (3.8 mL) in an 8 mL glass vial with a vortex mixer (Fisher Scientific, TopMix FB15024). Approximately 10 wt % of ethanol can be dissolved in the sunflower oil.28 After any air bubbles have disappeared from the mixture, the aqueous latex suspension (0.04 mL) was added. The latex suspension typically has a solids fraction of 15 wt %. Immediately after addition, the lid of the vial was shut, and the mixture was homogenized with a vortex mixer for 1 min to form a stable emulsion. The total volume was constant at 4 mL. To check the fixation of the colloidosome shell, Milli-Q water (2 mL) was added to the emulsion solution and mixed with a vortex mixer for 1 min. 2.3. Characterization. A Leica DME transmitted light microscope equipped with an XLICap color digital camera and capture software (XLI Imaging Ltd., version 12.0) was used to observe water-in-oil microcapsules. They were also observed under a JEOL-6340F scanning electron microscope (SEM) at 5.0 kV. The microcapsules were air-dried on a stainless steel SEM stub overnight and then platinum-coated using an Emitech K575 sputter coater (argon environment, 1 10-3 mbar, 40 mA, 1 min). They were also observed under a Leica TCS SP2-AOBS confocal laser scanning microscope (CLSM) using a 1.4 numerical aperture 63 oil immersion objective at a wavelength of 488 nm for excitation and 510-540 nm for emission. To achieve fluorescence, SYTO 9 (2 μL) was added to the additional Milli-Q water (2 mL), which was added for checking the shell fixation. The glass vial was stored in the dark at room temperature for ∼15 min prior to the use of the confocal microscope. (27) Yow, H. N.; Beristain, I.; Goikoetxea, M.; Barandiaran, M. J.; Routh, A. F. Langmuir 2010, 26, 6335. (28) Da Silva, C. A. S.; Sanaiotti, G.; Lanza, M.; Follegatti-Romero, L. A.; Meirelles, A. J. A.; Batista, E. A. C. J. Chem. Eng. Data 2010, 55, 440. (29) Pan, Y.; Gao, J. H.; Zhang, B.; Zhang, X. X.; Xu, B. Langmuir 2010, 26, 4184–4187.
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Figure 3. Photographs of the solutions in Figure 2 with added Milli-Q water.
3. Results and Discussion Figure 2 shows photographs of latex stabilized water-in-oil emulsions prepared at various concentrations of ethanol in sunflower oil. 0.04 mL of aqueous latex suspension was added to the sunflower oil with different ethanol concentrations (0-10 vol %). The solution was emulsified using a vortex mixer for 1 min and then left at rest for 1 day. In the case of no ethanol, white sediment was observed at the bottom of the vial. This means that colloidosomes formed from the latex particles cannot be stabilized without some assistance. The unstable colloidosomes coalesce and sediment. When 2 vol % ethanol was dissolved in the oil phase, the emulsions were still not stabilized, and white sediment was observed at the bottom of the vial. By contrast, with ethanol contents of more than 4 vol %, the sediment was not observed. This result suggests that the water-core emulsions were stabilized by the ethanol in the sunflower oil. To check the stability of watercore emulsions, Milli-Q water was added to the mixture solution containing water-core emulsions, and the solution was vortexed for 1 min. They were then left standing for 1 day. If the shell of water-core emulsions is not locked efficiently, the colloidosomes will simply coalesce and fall apart on transfer into the water phase. Figure 3 shows photographs of the solutions in Figure 2 with added Milli-Q water. When the ethanol concentration was either 0 or 2 vol %, the latex particles reside in the water phase. By contrast, the water phase did not include the latex particles when the concentration of ethanol was more than 4 vol %. In addition, huge droplets were observed at the interface between the oil and water phases. They were formed by the colloidosomes and the excess latex particles; however, they were not observed in the mixture before adding water to check the fixation of the shell (Figure 4a). This result confirms that the shell of the water-core emulsions was irreversibly fixed by adding ethanol to the oil phase. Figure 4 shows characterization of the colloidosomes formed at typical experimental conditions with an ethanol concentration in the oil phase of 4 vol %: (a) optical microscope image, (b) size distribution, (c) CLSM image, and (d, e) SEM images. The watercore emulsions were found to have a hollow structure. This is suggested because dense structures would be colored black (Figure 4a), DOI: 10.1021/la103331e
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Figure 4. Characterization of the colloidosomes formed at typical experimental conditions with an ethanol concentration in the oil phase of 4 vol %: (a) optical microscope image, (b) size distribution, (c) CLSM image, and (d, e) SEM images.
and the confocal image suggests a fluorescent ring like shell. In addition, the fluorescent ring was not observed in the water-core emulsions without latex particles. Therefore, this result indicates that the ethanol in the continuous phase causes the latex particles to fuse and to fix the shell of the water-core colloidosomes at room temperature without heating. In addition, the water-core colloidosomes were not observed when the additional ethanol concentration was either 0 or 2 vol %. The size distribution of colloidosomes was analyzed by measuring over 500 particles using optical microscope images (Figure 4b). The median diameter and the standard deviation were 18.0 μm and 1.39, respectively. The fluorescent green ring in Figure 4c was observed due to the absorption of the probe SYTO 9 on the shell of the colloidosomes, confirming the hollow structure and that the shell was fused (Figure 4d,e). This is similar to the locked shell of colloidosomes formed by heating above the glass transition temperature.9,10 Next, the reason why the additional ethanol in the oil phase leads to a fixing of the shell was investigated. Figure 5 shows photographs of mixtures of the latex suspension and ethanol. 15 wt % of latex aqueous suspension was added to ethanol and mixed with a vortex mixer for 1 min. The total volume of the mixture in each vial was 4 mL with the ratio of water and ethanol being the same as in Figure 2. The mixture was then left to stand for 1 day after mixing. It was found that the latex particles aggregated when ethanol was added to the latex suspension. The boundary of stability is found to be a function of both the latex concentration in the aqueous phase and the amount of ethanol; in all cases the time required to aggregate was just a few minutes. Figure 6 shows the typical SEM images of latex particles in (a) aggregated ([latex] = 3 wt %, [EtOH] = 80 vol %) and (b) stable dispersions ([latex]=3 wt %, [EtOH] = 60 vol %). The latex aqueous suspension and ethanol were mixed with a vortex mixer for 1 min. When the latex particles were aggregated, they fused three dimensionally. By contrast, the latex particles were assembled and not fused when they were dispersed in the less ethanol-rich solvent mixture. These results indicate that the aggregation of the latex particles causes the stabilization of the water-core colloidosomes. Therefore, experiments were carried out, changing the concentration of ethanol and latex. Figure 7 shows photographs of the experiments where the ethanol concentration is varied from 40 to 85 vol % and the latex 18678 DOI: 10.1021/la103331e
Figure 5. Photographs of latex-water-ethanol suspensions. The concentration ratio of latex particles and ethanol was the same as in Figure 2.
concentration in the aqueous phase is held constant at 3.0 wt %. Image (a) shows that the system starts to aggregate when the ethanol concentration reaches 75 vol %. Image (b) shows the effect of using the same ethanol to water ratio as shown in image (a) to make latex stabilized water-in-oil emulsions. The ethanol was initially dissolved in the oil phase, but as will be discussed later it will readily partition into the aqueous phase. Image (c) shows the previous solutions with Milli-Q water (2 mL) added to check the fixation of the shell and vortexed for 1 min. All images were taken after 1 day. From Figure 7b, it can be seen that ethanol concentrations which result in a stable latex dispersion do not form stable emulsions. Conversely, the ethanol concentrations that cause latex destabilization result in stable colloidosomes. This result is confirmed by Figure 7c where the lower ethanol concentrations do not form stable emulsions and the latex particles partition into the aqueous phase. For the higher ethanol concentrations the colloidosome shells are fused irreversibly, and the capsules remain in the oil phase. These results indicate that the shell of colloidosomes was fixed by aggregation of latex particles. Figure 8 shows photographs of the experiments where the latex concentration in the aqueous phase is varied from 1 to 3 wt % and the ethanol concentration is held constant at 80 vol %: (a) the Langmuir 2010, 26(24), 18676–18680
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Figure 6. Typical SEM images of latex particles in (a) aggregated ([latex] = 3 wt %, [EtOH] = 80 vol %) and (b) stable dispersions ([latex] = 3 wt %, [EtOH] = 60 vol %).
Figure 7. Photographs showing the effect of varying the ethanol concentration while the latex concentration in the aqueous phase is held at 3 wt %: (a) mixture of latex suspension and ethanol, (b) the latex stabilized water-in-sunflower oil emulsions using the same ethanol to water ratio in (a), (c) the previous solutions with added Milli-Q water, to check the fixation of the shell. All images were taken after 1 day.
mixture of the latex suspension and ethanol, (b) the latex stabilized water-in-oil emulsions using the same ethanol-water ratios as shown in image (a) but with the ethanol initially in the oil phase, and (c) the previous solutions with Milli-Q water added and the mixture then vortexed for 1 min. From Figure 8a, the latex particles were dispersed in the ethanolwater mixture at 1 and 1.5 wt %, partly aggregated at 2 wt %, and strongly aggregated at 2.5 and 3 wt %, respectively. Figure 8b suggests successful emulsion formation irrespective of the latex concentration; however, Figure 7c shows that stable colloidosomes are only formed when the aqueous phase is colloidally unstable. Langmuir 2010, 26(24), 18676–18680
Figure 8. Photographs showing the effect of varying the aqueous latex concentration from 1 to 3 wt % and keeping the ethanol concentration fixed at 80 vol %: (a) mixture of latex suspension and ethanol, (b) the latex stabilized water-in-sunflower oil emulsions using the same ethanol to water ratio in (a), (c) the previous solutions with added Milli-Q water, to check the fixation of the shell. All images were taken after 1 day.
Figure 9 shows the typical optical microscope images of watercore colloidosomes at various ethanol and latex concentrations in Figures 7b and 8b. Spherical aggregates of latex particles were observed in Figure 9a when the latex particles were dispersed in the ethanol-water mixture (Figure 7a, third vial from the left). By contrast, a lot of latex-stabilized colloidosomes were observed in Figure 9b when the latex particles were partly aggregating (Figure 7a, third vial from the left). When the latex concentration was lowered to result in a stable aqueous phase, not only latex-stabilized colloidosomes but also unstabilized droplets attached on the slide glass were seen (Figure 9c,d). These results indicate that the shell locking is inefficient when the system is colloidally stable. DOI: 10.1021/la103331e
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Figure 9. Optical microscope images of various concentrations: (a) [latex in water] = 3 wt %, [EtOH] = 70 vol % (Figure 7b, third vial from the left), (b) [latex in water] = 3 wt %, [EtOH] = 75 vol % (Figure 7b, fourth vial from the left), (c) [latex in water] = 1 wt %, [EtOH] = 80 vol % (Figure 8b, first vial from the left), (d) [latex in water] = 2 wt %, [EtOH] = 80 vol % (Figure 8b, third vial from the left).
The aggregated latex particles due to the additional ethanol were found to be irreversibly stuck even when transferred into pure water. Equally the results above indicate the colloidosome shell was fixed by aggregation. The mechanism of the fixation of the shell of colloidosomes is thought to be as follows: Approximately 10 wt % of ethanol can be dissolved in sunflower oil. By contrast, ethanol can be freely mixed in water. Therefore, when the latex suspension is added to the organic phase, the ethanol partitions to the water phase, presumably by a diffusive mechanism. The concentration of ethanol in the water phase increases, and the latex particles eventually aggregate. For successful colloidosome formation the assembly of particles at the emulsion interface must occur before a significant amount of ethanol diffuses into the aqueous phase, thereby causing destabilization of the latex and shell locking. The shell is formed and fixed as the latex particles aggregate. Addition of ethanol to the aqueous latex suspension caused formation of aggregated latex particles. The reason for this destabilization is that the dielectric permittivity of ethanol-water mixtures decreases with increasing ethanol concentrations. This leads to a decrease in the surface potential of the latex particles, and consequently the electrostatic repulsive force between the particles decreases. However, colloidal instability alone is not enough for colloidosome formation. The latex particles must be able to move to the emulsion interface before encountering a destabilizing environment. Hence, locating the ethanol in the oil phase allowed the correct staging of events and successful colloidosome formation. The mixing order was one of the important factors. When the ethanol is added directly to the aqueous phase, the latex simply aggregated prior to forming the shell. In a further experiment 15 wt % of latex aqueous suspension was added to sunflower oil without ethanol and mixed with a vortex mixer for 1 min. After 18680 DOI: 10.1021/la103331e
that, ethanol was added to the water-in-sunflower emulsions and mixed for 1 min. Because the mixing caused emulsion breakup and re-formation, aggregates of latex particles were observed, and they were similar to the image of Figure 9a. The use of the ethanol allows mild processing conditions to be employed. As we will demonstrate in a subsequent paper, this allows encapsulation of biologically active materials. The ethanol concentration must remain small enough so as to retain the biological activity, while it must be above 4 vol % to allow shell stabilization. As well as keeping the absolute concentration low, it is also necessary to clean the microcapsules fairly quickly so as to remove the ethanol.
4. Conclusions In this study, we fabricate latex-stabilized water-core colloidosomes at room temperature without heating. To enable fabrication of colloidosomes, a range of organic solvents were added to the sunflower oil. It was found that a small amount of ethanol was the most effective. The effects of the ethanol concentration in the continuous phase of sunflower oil on the stabilization of colloidosomes were examined as well as the effect of latex concentration. The shell of the water-core emulsions was found to be fixed by adding more than 4 vol % ethanol in the oil phase with the latex concentration in water phase at 15 wt %. These results show that colloidal aggregation of particles on the surface of an emulsion droplet is sufficient to create an irreversible shell for water-core emulsion droplets. Acknowledgment. The authors thank Dr. Grace Yow for her help with lab work, Dr. Kamran Yunus for help with CLSM, and Mr. Simon Griggs for help with SEM measurements. T.N. was supported for a year in Cambridge by a grant from Osaka Prefecture University. Langmuir 2010, 26(24), 18676–18680
