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Langmuir 2008, 24, 1644-1647
Microcapsules from Self-Assembled Colloidal Particles Using Aqueous Phase-Separated Polymer Solutions Albert T. Poortinga* Friesland Foods KieVit, Oliemolenweg 4a, 7944 HX Meppel, The Netherlands ReceiVed NoVember 4, 2007. In Final Form: December 27, 2007 We report a new way of producing microcapsules consisting of a shell of aggregated colloids (also referred to as colloidosomes) using aqueous phase-separated polymer solutions (water-in-water emulsions) as a template. The extremely low interfacial tension of the template allows the production of reversible colloidosomes that spontaneously disintegrate depending on environmental conditions, making them ideal for controlled release purposes. Also, colloidosomes can have an elongated shape such that they may be used as microfluidic membranes or artificial arteries. Because the method described here does not use any organic solvents, it enables the use of sensitive materials such as cells and proteins.
Introduction Self-assembly is used to produce a wide range of structures. Recently, Dinsmore et al. used a self-assembly process to produce microcapsules referred to as colloidosomes1 that have sparked significant interest as a new microencapsulation tool.2-4 In short, colloidal particles were made to self-assemble at emulsion interfaces and subsequently aggregated to form shells. To obtain water-filled colloidosomes in an aqueous environment, particles were aggregated at the interface of a water-in-oil emulsion, the oil phase was washed away using an oil-soluble alcohol such as octanol, and then the capsules were transferred by centrifugation from the washing solution to an aqueous phase. This method has several drawbacks. First, great stresses act on the self-assembled structures during the washing and transfer step such that strong irreversible aggregation is needed.2 Second, the use of organic solvents does not allow sensitive ingredients such as cells and proteins to be used. Third, the high interfacial tension of emulsion droplets excludes the production of nonspherical structures. There is thus a need to replace the use of emulsions as a template in the production of colloidosomes. Solutions containing two different polymers at concentrations that are not too low generally demix into regions enriched in the first polymer and regions enriched in the second polymer. If the solvent is water and if one of the phases is dispersed in the other phase, then these systems are often referred to as water-in-water emulsions (or w/w emulsions).5 Among other things, these emulsions are characterized by an extremely low interfacial tension, generally between 10-4 and 10-6 N/m (i.e., 100-1000 times lower than the interfacial tension of typical oil-water interfaces6). Because of this and because they are all aqueous systems, w/w emulsions seem to be ideal templates for the production of water-filled colloidosomes in an aqueous phase having none of the disadvantages described above. Despite this, * E-mail:
[email protected]. (1) Dinsmore, A. D.; Hsu, Ming F.; Nikolaides, M. G.; Marquez, M.; Bausch, A. R.; Weitz, D. A. Science 2002, 298, 1006-1008. (2) Paunov, V. N.; Noble, P. F.; Cayre, O. J.; Alargova, R. G.; Velev, O. D. Mater. Res. Soc. Symp. Proc. 2005, 845, 279-283. (3) Gu, S.; Decker, E. A.; McClements, D. J. Langmuir 2005, 21, 5752-5760. (4) Kim, J.-W.; Fernandez-Nieves, A.; Dan, N.; Utada, A. S.; Marquez, M.; Weitz, D. A. Nano Lett. 2007, 7, 2876-2880. (5) Tolstoguzov, V. B. Thermodynamic Incompatibility of Food Macromolecules. In Food Colloids and Polymers: Stability and Mechanical Properties; Dickinson, E., Walstra, P., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1993; Vol. 113, pp 94-102. (6) Scholten, E.; Tuinier, R.; Tromp. R. H. Langmuir 2002, 18, 2234-2238.
although the adsorption of colloidal particles at the interface of conventional emulsions has been frequently studied,7,8 adsorption at the interface of w/w emulsions has hardly been studied, and to our knowledge, the production of colloidosomes using w/w emulsions as templates has not been reported. Experimental Section We have used several phase-separating solutions containing either two polysaccharides or one polysaccharide and one protein. We tested several colloidal particles for their ability to adsorb at the interface in the tested water-in-water emulsions. The systems tested are summarized in Table 1. All experiments were carried out at room temperature. Materials. The following polymers (type, supplier) were used: dextran (D5376, Sigma-Aldrich), methylcellulose (Methocel A15LV, Akzo-Nobel), maltodextrin (6DE, Avebe), whey protein concentrate (Hiprotal 580HG, Friesland Foods DOMO), and alginate (29170, Caldic). All polymers were used in powder form. The following particles were used: fat particles in powder form (Vana Grassa 78) produced by Friesland Foods Kievit. Powders consist of mainly emulsified fat (78%) with a volume-based average particle size of 0.39 µm (d50), caseinate (6%), and lactose (16%). Quartz particles were reference particles no. 1123 from the European Community bureau of reference with a volume-based average particle size of 1.1 µm (d50). Monoglyceride particles were used as an emulsion prepared with an ultraturrax containing 10% of Dimodan U/J (unsaturated distilled monoglycerides produced by Danisco) in a 1% sodium caseinate solution at 60 °C. Thus-produced monoglyceride droplets had a droplet size of 0.21 µm (d50). Whey protein particles (Hiprotal 835MP) with a particle size of 1 µm in powder form produced by Friesland Foods DOMO were used. Probiotics (Lactobacillus paracasei CRL 431) were produced by Chr. Hanssen and consisted of at least 3 × 1010 CFU/g dried bacteria embedded in a maltodextrin matrix. Spherical Colloidosomes. We started from pure polymer solutions in demineralized water. Two solutions were added in the desired ratio to a total volume of 3 mL. Both solutions combined to form a phase-separated system with droplets of one phase predominantly containing the first polymer, but also containing the second polymer, dispersed in a continuous phase predominant in the second polymer. When whey protein was used as a polymer, the pH was set at 5.2 to facilitate phase separation. Particles were normally added to a particle concentration of 2%. Subsequently, the system was vortex (7) Clegg, P. S.; Herzig, E. M.; Schofield, A. B.; Egelhaaf, S. U.; Horozov, T. S.; Binks, B. P.; Cates, M. E.; Poon; Wilson, C. K. Langmuir 2007, 21, 59845994. (8) Binks, B. P. Curr. Opin. Colloid Interface Sci. 2002, 7, 21-41.
10.1021/la703441e CCC: $40.75 © 2008 American Chemical Society Published on Web 01/26/2008
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Langmuir, Vol. 24, No. 5, 2008 1645 Table 1. Experimental Systems Useda,b
system
phase 1
phase 2
1 2 3 4 5 6 7
10% dextran (20%) 90% dextran (20%) 50% MD6 (20%) 50% MD6 (20%) 20% WPC (30%) 20% WPC (30%) 10% dextran (20%)
90% MC (4%) 10% MC (4%) 50% MC (4%) 50% dextran (20%) 80% MC (4%) 80% MC (4%) 90% MC (4%)
8 9
10% dextran (20%) 90% MC (4%) 10% dextran (20%) 90% MC (4%)
10
90% dextran (20%) 10% MC (4%)
particles fat fat fat fat fat quartz wpp
particle location interface interface interface interface phase 1 phase 1 interface/ phase 2 interface interface
quartz monoglycerides probiotics interface
a
Percentages in parentheses denote weight percentage of polymer in solution. b MC, methylcellulose; MD6, maltodextrine 6DE; WPC, whey protein concentrate; WPP, whey protein particles.
mixed and observed microscopically to determine the location of the particles. After adsorption at the interface of the w/w droplets, fat particles were allowed to aggregate in several ways. Calciuminduced aggregation was carried out by adding 0.5% calcium chloride. Aggregation by acidification was performed by adding 1% gluconoδ-lactate (GDL), which made the pH drop to the isoelectric pH of the caseinate in about 5 min. Aggregating the particles by complex coacervation was done similarly to the pH-induced aggregation, but then 0.05% alginate was added to the system before adding GDL. After particle aggregation, the phase separation was removed by diluting the system 10 times. Dilution was done either with a 0.5% calcium chloride solution (in the case of calcium-induced aggregation) or a 1% GDL solution (pH-induced aggregation) to keep the particles in the aggregated state. Dilution left the great majority of the colloidosomes intact. Colloidosomes were also stable during vortex mixing or during storage for at least 3 days. The reversibility of the aggregation was checked by adding either 5% EDTA (for the calciumaggregated particles) or 0.1% NaOH (aggregation by acidification), which led to the disintegration of the colloidosomes. Elongated Colloidosomes. Experiments were done using 35% dextran solutions as the dispersed phase and 10% methylcellulose solution as the continuous phase. The polymer concentrations were increased as compared to the solutions used to produce spherical colloidosomes in order to further slow down the relaxation and break up of elongated structures. However, solutions were still pourable with a viscosity of around 1 Pa s. About 0.3 mL of dextran solution was injected through a 1 mm syringe needle into 3 mL of the methylcellulose phase with added fat particles while slowly moving the needle through the methylcellulose phase. This produced elongated structures covered with particles.
Results and Discussion We have used several phase-separating solutions containing either two polysaccharides or one polysaccharide and one protein and have tested several colloidal particles for their ability to adsorb at the interface of the tested water-in-water emulsions. Generally, when using a combination of protein and polysaccharide, colloidal particles favored the protein phase most likely because protein adsorbs at the particle surface such that the particle has a slightly lower interfacial energy in contact with the proteinenriched phase than with the polysaccharide-enriched phase. When two polysaccharides were used, the colloidal particles in most cases showed a strong affinity for the interface, and the interface became closely packed with adsorbed particles (Figure 1). It is likely that the interfacial energy of the particles against both polysaccharide phases will be dominated by the interfacial energy with water such that both interfacial energies are about equal, which leads to a decrease in the total interfacial energy upon particle adsorption. Interestingly, the adsorption of colloidal particles at the waterwater interface clearly increased the stability of the emulsion.
Figure 1. Microscopic images of several water-in-water emulsions containing different colloidal particles: (a) Dextran in a methylcellulose emulsion containing fat particles (system 1 in Table 1); (b) dextran in a methylcellulose emulsion containing monoglyceride particles (system 9 in Table 1); and (c) methylcellulose in a dextran emulsion containing microorganisms (system 10 in Table 1). The scale bars denote (a, b) 40 and (c) 10 µm. Note that “dextran in methylcellulose emulsion” means droplets of a solution containing mainly dextran in a continuous phase consisting of a solution containing mainly methylcellulose.
The size of the emulsion droplets in Figure 1a was hardly found to change during 1 week of storage. For comparison, if no particles had been added, then the system had completely phase separated into two separate layers. The interfacial stabilization of w/w emulsions may find practical application in food, where w/w emulsions can yield an emulsion-like structure without using fat.5 Particles adsorbed at the w/w interface may be aggregated to form colloidosomes in several ways. Adsorbed fat particles have been made to aggregate through acidification, the addition of calcium ions, and acidification in the presence of some added alginate. In the last case, particles are linked together through complex coacervation of the negatively charged alginate and the positively charged caseinate at the particle surface at low pH. It should be noted that these treatments aggregate only the particles at the interface and do not gel the fluid phases. To produce colloidosomes, aggregation needed to be carried out slowly (e.g.,
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by using GDL as an acidifier) in order not to start the aggregation while the aggregation-inducing agent is not yet homogeneously distributed throughout the system. After the aggregation step, colloidosomes are generally less spherical than before. Also, they are less spherical than previously described colloidosomes.1 This is because the particles used here are far from monodisperse such that stresses will develop during the aggregation step. The interfacial tension tends to keep the template droplets spherical, but this effect is relatively small when using w/w emulsions. Using monodisperse particles is expected to yield more spherical colloidosomes. Gelling the droplets at which the particles are adsorbed before aggregating the particles may also be an option. After aggregation, the droplets at which the particles are adsorbed may be removed by gently diluting the system such that the phase-separating polymers become completely miscible again. Interestingly, the aggregated particle shells can withstand the small interfacial stresses that will develop during this step such that the great majority of the aggregated particle shells remain intact (Figure 2). In contrast to this, Clegg et al., studying shells of silica particles adsorbed around droplets in partially miscible alcohol/oil mixtures, found that many shells crumbled when phase separation was removed by a temperature quench.9 Because in the system used by Clegg et al. the interfacial tension in between the phase-separated phases will be much larger than in the systems that we use, greater stresses are expected to act on the shells during the removal of the phase separation, which may explain the great number of crumbled shells. In addition, Clegg et al. do not explicitly make the adsorbed particles aggregate before removing the phase separation, in contrast to what is reported here. The colloidosomes described here will, for example, be applicable in low fat foods, in which they may give a full fat impression while they substitute for only a very small part of the total fat. In addition, they may be used for encapsulation purposes. In this case, it may be necessary to further decrease the size of the pores in the particle shell to trap small actives. This may be done by having additional finer particles adsorb to the particle shells or by sintering the particles together. Both the sintering and the adsorption of multiple layers have been applied before to colloidosomes based on oil-water emulsions.1,3 For encapsulation purposes, the self-assembled microcapsules will have to be loaded with the material to be encapsulated. To this end, use can be made of the fact that the partitioning of molecules and particles over the dispersed phase and the continuous aqueous phase can be steered by the proper choice of the phase-separating polymers or by using additives. For example, Franssen et al. showed that liposomes or the protein IgG could be made to partition almost completely in the dextran phase of a phaseseparated aqueous methacrylated dextran/poly(ethylene glycol) solution.10 What characterizes the colloidosomes described here is that they are made using reversible particle aggregation methods. If, for example, a calcium chelator such as EDTA is added to the calcium-aggregated colloidosomes, then they rapidly disintegrate. Also, raising the pH to 10 makes the acid-aggregated colloidosomes unstable. This is a very useful property for controlled release applications, where one wants to be able to release the encapsulated actives at the right place or time. To date, a great (9) Clegg, P. S.; Herzig, E. M.; Schofield, A. B.; Horozov, T. S.; Binks, B. P.; Cates, M. E.; Poon, W. C. K. J. Phys. Condens. Matter 2005, 17, S3433S3438. (10) Franssen, O.; Stenekes, R. J. H.; Hennink, W. E. J. Controlled Release 1999, 59, 219-228. (11) Langer, R. Nature 1998, 392, 5-10. (12) Gouin, S. Trends Food Sci. Technol. 2004, 15, 330-347. (13) Hyuk Im, S.; Jeong, U.; Xia, Y. Nat. Mater. 2005, 4, 671-675.
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Figure 2. Microscopic images of several colloidosomes produced from dextran in methylcellulose emulsions containing fat particles (system 1 in Table 1) after aggregation of the particles and removal of the phase separation. (a) Confocal scanning laser microscope (CSLM) picture of calcium aggregated particle shells (green ) fat). (b) CSLM picture from a complex coacervated particle shell (red ) fat). (c) Microscopic picture of complex coacervated particle shells. The scale bars denote 10 µm.
variety of encapsulates exist.11-13 Among these are encapsulates with a permeability that is responsive to environmental stimuli. Examples are normally based on molecular interactions that depend on the environment14,15 (e.g., polymer networks that swell or shrink depending on the redox potential or the temperature of the surrounding solution16,17). The use of colloidal interactions as described here is ideal for changing the capsule permeability
Letters
Figure 3. (a) Microscopic image of an elongated structure consisting of fat particles adsorbed at the dextran/methylcellulose solution interface. (b) Microscopic image of an elongated structure (at the left) consisting of fat particles adsorbed at the dextran/methylcellulose solution interface that is stable for hours without having aggregated the particles. The scale bars denote 10 µm.
as a function of the environment because nowadays colloidal interactions can be rigorously controlled not only by selecting the proper particle surface chemistry but also by using external fields.18 Because the method described here does not use organic solvents, sensitive ingredients such as cells and proteins can be used without damaging them. In fact, aqueous-phase-separated polymer systems have been frequently used for cell separation. The mildness of the method proposed here may also lead to the production of capsules from completely new wall materials such as cells. As observed here for microbial cells, cells have been seen to accumulate in the water-water interface before.19 In nature, layers of cells such as those in the skin and intestines often form a barrier that selectively permeates certain substances. Thus, it seems valuable to mimic these systems in encapsulation or other technological areas. Recently, Helfrich et al. showed that DNA-derivatized nanowires adsorbed at the interface between two aqueous polymer solutions retained the ability to bind complementary DNA, unlike when using conventional emul-
Langmuir, Vol. 24, No. 5, 2008 1647
sions.20 It thus seems possible to use the wide variety of available biological interactions to have the colloidal particles constituting the shell of the colloidosome bind or unbind in response to very specific environmental triggers. w/w emulsions have an interfacial tension that is much lower than that of conventional emulsions. Therefore, nonspherical structures are easily obtained within phase-separated polymer solutions, and these slowly break up into droplets or relax back to spherical structures. To exploit this, we produced threads with a diameter of several micrometers of the dispersed phase in the continuous phase. An example is given in Figure 3a. When the adsorbed particles are not made to aggregate, these threads are eventually seen to break up into droplets. This takes several minutes, so there is enough time to perform a particle aggregation step to fix the elongated structures. Even when no aggregation is induced, often elongated structures are found that are stable (Figure 3b). This indicates that the driving force for break up and/or contraction is low enough to be successfully opposed by a close-packed layer of adsorbed particles. We envisage that the elongated structures shown here can find several applications, for example, as very thin membranes with an extremely large specific area or in the production of artificial arteries. This method allows the direct assembly of cells into a closely packed tubular layer without the use of scaffolds, which is not possible with current methods.21,22 Edmond et al. reported structures similar to the ones described here by producing confined jets of a viscoelastic aqueous solution in oil and having colloidal PMMA particles adsorb to the surfaces of the jets.23 Unlike in our system, where because of the extremely low interfacial tension simple stirring is enough to produce elongated structures with enough stability to have particles adsorb to their surface, Edmond et al. have to stabilize their jets by confining them in a capillary with a dimension slightly greater than the jet. Although scientifically very interesting, practical application may be difficult because of scalability problems.
Conclusions Using w/w emulsions as a template for the self-assembly of colloidal particles, microencapsulates can be produced with several important properties. First, encapsulates can be produced with a shell that can be responsive to the environment, thus allowing controlled release. Second, the absence of organic solvents allows the use of delicate ingredients, most notably, biological material, both as active and as shell material. This further extends the possibility of producing capsules with an environmental responsiveness by making use of the many available biological ligand-receptor interactions to aggregate the particles forming the shell. Third, the low interfacial tension of w/w emulsions allows the formation of tubular structures. LA703441E
(14) Ishida, T.; Okada, Y.; Kobayashi, T.; Kiwada, H. Int. J. Pharm. 2006, 309, 94-100. (15) Ma, Y.; Dong, W.-F.; Hempenius, M. A.; Moehwald, H.; Vancso, G. J. Nat. Mater. 2006, 5, 724-729. (16) Huang, S. L.; MacDonald, R. C. Biochim. Biophys. Acta 2004, 1665, 134-141. (17) Cheng, C.-J.; Chu, L.-Y.; Ren, P.-W.; Zhang, J.; Hu, L. J. Colloid Interface Sci. 2007, 313, 383-388. (18) Yethiraj, A.; Van Blaaderen, A. Nature 2003, 421, 513-517. (19) Walter, H.; Anderson, J. L. FEBS Lett. 1981, 131, 71-76.
(20) Helfrich, M. R.; El-Kouedi, M.; Etherton, M. R.; Keating, C. D. Langmuir 2005, 21, 8478-8486. (21) Isenberg, B. C.; Williams, C.; Tranquillo, R. T. Circ. Res. 2006, 98, 25-35. (22) Goldberg, M.; Langer, R.; Xinqiao, J. J. Biomater. Sci., Polym. Ed. 2007, 118, 241-268. (23) Edmond, K. V.; Schofield, A. B.; Marquez, M.; Rothstein, J. P.; Dinsmore, A. D. Langmuir 2006, 22, 9052-9056.
