pubs.acs.org/Langmuir © 2009 American Chemical Society
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Internally Self-Assembled Thermoreversible Gelling Emulsions: ISAsomes in Methylcellulose, K-Carrageenan, and Mixed Hydrogels Matija Tomsic,†,§ Samuel Guillot,†, Laurent Sagalowicz,‡ Martin E. Leser,‡ and Otto Glatter*,† Institute of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria, and ‡Nestl e Research Center, Department of Food Science, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland. § Permanent address: University of Ljubljana, Faculty of Chemistry and Chemical Technology, Asker ceva cesta 5, SI-1000 Ljubljana, Slovenia. Permanent address: Centre de Recherche sur la Mati ere Divis ee - UMR 6619, Universit e d’Orl eans -CNRS, 1b, rue de la F erollerie 45071 Orleans Cedex 2, France )
†
Received March 3, 2009. Revised Manuscript Received April 27, 2009 Self-assembled thermo-gelling emulsions were developed by blending internally self-assembled particles (ISAsomes) with thermoreversible polysaccharide hydrogels of methylcellulose (MC), κ-carrageenan (KC), and their 1:1 mixture. In this way, the hierarchical structure of ISAsome samples was successfully promoted. The gelified polymer network corresponds to the highest level of the hierarchical structure and as such represents the capturing matrix for the medium structural level, i.e., dispersed emulsion particles, which are further internally structured as the lowest level of structure. Utilizing small-angle X-ray scattering, differential scanning calorimetry, dynamic light scattering, and oscillatory rheological experiments in the temperature regime from 20 to 70 °C, we were able to show that the ISAsomes stay practically intact during such embedment into a hydrogel matrix retaining its internal self-assembled structure and its functionality. The characteristic sol-gel and gel-sol transition temperatures of the ISAsome-loaded hydrogel samples showed a slight shift in comparison to the unloaded hydrogel samples. Furthermore, we found that MC is actually able to stabilize the ISAsomes at higher temperatures (tests were conducted up to 90 °C). Gels made from MC and KC show quite different features in terms of rheology and differential scanning calorimetry. However, the most interesting results were obtained for the ISAsome-loaded MC-KC (1:1) gelifying system, which behaves as a low- and high-temperature gel with a narrow intermediate temperature window where it is a sol. This specific thermal behavior allows for easy temperature tuning of the system’s aggregate state as well as the internal self-assembled structure. As such, this system is suggested to be further tested as the potential media for a temperature-controlled burst/sustained release media of various hydrophilic, hydrophobic, or amphiphilic guest functional molecules.
1. Introduction Formulation, characterization, and specific performance of various gel and hydrogel systems are very important topics of investigation in various fields of research. In this sense, gels can be used as stabilizers of various systems,1-3 slow down dynamics of the systems,4,5 can facilitate sustained or controlled release of *Corresponding author. Tel: +43 316 380 5433. Fax: +43 316 380 9850. E-mail:
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various functional molecules,6-13 lead to some other specifically desired properties of the systems,14-23 or be simply used to yield the final product solid-like and in this way facilitate its handling.24-27 In this way, such gels are of special importance in the field of pharmaceutical, cosmetics, and food applications. Our interest lies in the polysaccharides methylcellulose (MC; E461) and κ-carrageenan (KC; E407) that are well-known additives used for gelation in various nutrition and other products. In our previous study, we investigated their water mixtures and were able to formulate a thermoreversible mixed gelling system with rather interesting thermoreversible gelling properties.28 However, with the present study we aim to explore the possibility of using (15) Braudo, E. E.; G, P. I.; Semenova, M. G.; Yuryev, V. P. Food Hydrocolloids 1998, 12, 253–261. (16) Kato, N.; Oishi, A.; Takahashi, F. Mater. Sci. Eng., C 1998, 6, 291–296. (17) Lucey, J. A.; Singh, H. Food. Res. Int. 1998, 30, 529–542. (18) Reynolds, T.; Dweck, A. C. J. Ethnopharm. 1999, 68, 3–37. (19) Totosaus, A.; Montejano, J. G.; Salazar, J. A.; Guerro, I. Int. J. Food Sci. Technol. 2002, 37, 589–601. (20) Sui, X.; Cruiz-Aguado, J. A.; Chen, Y.; Zhang, Z.; Brook, M. A.; Brennan, J. Chem. Mater. 2005, 17, 1174–1182. (21) Mitsumata, T.; Sakai, K.; Takimoto, J.-i. J. Phys. Chem. B 2006, 110, 20217–20223. (22) Stephan, A. M. Eur. Polym. J. 2006, 42, 21–42. (23) Daniel-da-Silva, A. L.; Pinto, F.; Lopes-da-Silva, J. A.; Trindade, T.; Goodfellow, B. J.; Gil, A. M. J. Colloid Interface Sci. 2008, 320, 575–581. (24) Avnir, D.; Braun, S.; Ottolenghi, M. ACS Symp. Ser. 1992, 499, 384–404. (25) Pandey, P. C.; Upadhyay, S.; Pathak, H. C.; Pandey, C. M. D. Electroanalysis 1999, 11, 950–956. (26) Huang, I. Y.; Huang, R. S.; Lo, L. H. Sens. Actuators, B 2003, 94, 53–64. (27) Zaggout, F. Mater. Lett. 2006, 60, 1026–1030. (28) Tomsic, M.; Prossnigg, F.; Glatter, O. J. Colloid Interface Sci. 2008, 322, 41–50.
Published on Web 06/08/2009
DOI: 10.1021/la900766c
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them as an entrapment media for colloidal particles and characterize the resulting loaded-gel systems. Internally self-assembled (ISA) aqueous dispersions were chosen as the loading colloidal system in the present study due to some of their intriguing properties. Such dispersions were first discovered in the 1980s through the fat digestion studies29 and were later designated as the systems of cubosome and hexosome particles (trademark of Camurus, Lund, Sweden), with their preparation method and several potential, mainly medically oriented uses patented for the first time by Landh and Larsson in 1996.30 Afterward, these and similar systems have aroused huge research interest from various perspectives31-49 that have been nicely reviewed by Spicer50 and Garg et al.51 Internally selfassembled emulsions are hierarchically organized colloidal systems, i.e., submicrometer-sized droplets, which are dispersed and kinetically stabilized as aqueous emulsions with self-assembled interior of various possible types (fluid isotropic w/o microemulsions or L2 phase, or liquid crystalline mesophases: bicontinuous cubic, hexagonal, and discontinuous micellar cubic).32,33,37-39 All of these internal phases are thermodynamic equilibrium structures, but in these systems, they appear inside the kinetically stabilized dispersed particles also known as ISAsomes.37 Internal structure of ISAsomes can be tuned by changing the temperature and/or the oil concentration in the system. Such systems are longterm stable and are excellent candidates for carrier systems of various functional molecules.50,51 Namely, due to a large interfacial area between the hydrophilic and hydrophobic regions they are able to solubilize hydrophilic, hydrophobic, and also amphiphilic guest molecules. Our present objective is to promote and characterize the structure of ISAsome systems by embedding them into a thermoreversible gelifying system of polysaccharides, and their mixtures.28 Utilizing small-angle X-ray scattering and dynamic light (29) Lindstrom, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstrom, B. Lipids 1981, 16, 749–754. (30) Landh, T.; Larsson, K. Particles, method of preparing said particles and uses thereof. US. Patent 5531925, 1996. (31) Neto, C.; Aloisi, G.; Baglioni, P. J. Phys. Chem. B 1999, 103, 3896–3899. (32) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611–4613. (33) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; larsson, K. Langmuir 1997, 13, 6964–6971. (34) Spicer, P. T.; Hayden, K. L. Langmuir 2001, 17, 5748–5756. (35) Borne, J.; Nylander, T.; Khan, A. J. Phys. Chem. B 2002, 106, 10492–10500. (36) Boyd, B. J. Int. J. Pharm. 2003, 260, 239–247. (37) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Garti, N.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254–5261. (38) Yaghmur, A.; De Campo, L.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2005, 21, 569–577. (39) Yaghmur, A.; De Campo, L.; Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2006, 22, 517–521. (40) Yaghmur, A.; De Campo, L.; Segalowicz, L.; Leser, M. E.; Glatter, O.; Michel, M.; Watzke, H. Oil-in-Water Emulsion for Delivery. European Patent Applilcation EP 1 598 060 A1, 24.11.2005, 2005. (41) Dong, Y.-D.; Larsson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512– 9518. (42) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharm. 2006, 318, 154–162. (43) Sagalowicz, L.; Michel, M.; Adrian, M.; Frossard, P.; Rouvet, M.; Watzke, H. J.; Yaghmur, A.; De Campo, L.; Glatter, O.; Leser, M. E. J. Microsc. (Oxf.) 2006, 221, 110–121. (44) Yaghmur, A.; De Campo, L.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2006, 22, 9919–9927. (45) Guillot, S.; Moitzi, C.; Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Colloids Surf., Part A 2006, 291, 78–84. (46) Moitzi, C.; Guillot, S.; Fritz, G.; Salentinig, S.; Glatter, O. Adv. Mater. 2007, 19, 1352–1358. (47) Swarnakar, N. K.; Jain, V.; Dubey, V.; Mishra, D.; Jain, N. K. Pharm. Res. 2007, 24, 2223–2230. (48) Salentinig, S.; Yaghmur, A.; Guillot, S.; Glatter, O. J. Colloid Interface Sci. 2008, 326, 211–220. (49) Salonen, A.; Muller, F.; Glatter, O. Langmuir 2008, 24, 5306–5314. (50) Spicer, P. T. Curr. Opin. Colloid Interface Sci. 2005, 10, 274–279. (51) Garg, G.; Saraf, S.; Saraf, S. Biol. Pharm. Bull. 2007, 30, 350–353.
9526 DOI: 10.1021/la900766c
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scattering, we were already able to show that it is possible to successfully entrap ISAsomes into the aqueous KC system, which is a gel at low temperatures (room temperature) and melts into a sol at higher temperatures.52 However, a slight influence of the KC polymer on the internal structure of ISAsomes was evidenced. With the present study, we notably extend our research to hightemperature gels, i.e., to aqueous systems of polysaccharide MC (hydrophobically modified cellulose) and to MC/KC polysaccharide mixtures, which form gels at low and high temperatures. We characterize these MC and KC systems into detail on different structural levels utilizing various experimental techniques in the temperature regime from 20 to 70 °C. Small-angle X-ray scattering is used to investigate primarily the internal self-assembled structure of these hierarchically structured systems, dynamic light scattering reveals the information on the structure one level higher, i.e., ISAsomes as emulsion particles; in addition, DSC and rheology reveal the thermodynamic and viscoelastic information on these samples as macroscopically gelifying systems. At this point, we have to mention that MC shows the reverse thermo-gelling behavior in comparison to KC solutions;MC solutions are sols at low temperatures (room temperature) and gel at elevated temperatures. The two polymers exhibit independent gelling mechanisms; therefore, the detailed investigation of mixed thermoreversible gelifying systems with very intriguing properties was also possible. A very important new finding presented in this study is the fact that by incorporating ISAsomes into such gelling polymer systems their stability is increased. This is benefited especially at higher temperatures, when the plain ISAsome systems are subjected to a rather rapid creaming. This can be of great importance during formulation of more complex systems. Furthermore, as indicated from the first test results, arresting ISAsomes into a gel matrix actually provides an additional possibility to control these systems in terms of the release of various host molecules. Originally, ISAsomes like cubosomes were developed in order to use the special feature of the liquid crystalline phases but to allow for low-viscosity fluids. In our systems, we go to the reverse direction: we gelify the fluid system to make it more viscous and stable. However, as a major difference, the system has a continuous aqueous phase with a water content of 90% or more, while the liquid crystalline phase exists only inside the droplets in the gel. This situation can be advantageous for controlled release. The findings presented in this paper are very important for understanding the behavior and structural situation in these specific systems, which are planned to be used in future studies of the release phenomena from such systems. However, in addition, a rather general importance of these findings should be recognized by the scientific community, because the present study focuses on a self-assembly organization on three different structural levels. Namely, a traditional understanding of molecular self-assembly in a sense of the formation of liquid crystalline phases of ISAsomes, stabilization of ISAsomes via self-assembly of the secondary emulsifier on their surface, and eventually self-assembly phenomena of the polymers during the gelation process induced by the temperature changes.
2. Experimental Methods 2.1. Materials. Gelifying polymer methylcellulose Methocel A4C FG was purchased from Dow Chemical Company, gelifying polymer κ-carrageenan and oil tetradecane from Fluka (Buchs, Switzerland), and distilled monoglyceride mixture Dimodan (52) Guillot, S.; Tomsic, M.; Sagalowicz, L.; Leser, M. E.; Glatter, O. J. Colloid Interface Sci. 2009, 330, 175–179.
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U/J (DU) from Danisco A/S (Braband, Denmark). Triblock copolymer Pluronic F127 (F127) was a gift from BASF (Mount Olive, New Jersey, USA). All materials were used without any further purification. Water was deionized using Millipore Water systems. Dimodan U/J is a sample of distilled gycerides. It consists of 96% monoglycerides (62% linoleate and 25% oleate), the rest are diglycerides and free fatty acids. In the studied samples, Dimodan U/J was used as a lipophilic additive (LPA) and F127 as the secondary stabilizer of the aqueous dispersions. ISAsome samples were prepared by ultrasonication of the mixture of LPA, tetradecane, F127, and water. The concentration of secondary stabilizer was given by β ¼
mF127 100 mLPA þ moil
ð1Þ
where m represents mass. β was kept constant at 8.1% for all samples. Similarly, the concentration of the dispersed phase was given by φ ¼
mF127 þ mLPA þ moil 100 mF127 þ mLPA þ moil þ mwater
ð2Þ
and the concentration of LPA by δ ¼
mLPA 100 moil þ mLPA
ð3Þ
The ISAsome samples contained 10% of the dispersed phase and were prepared with three different values of δ corresponding to three different types of the internal structure at room temperature: ISAsomes with reverse hexagonal H2 phase at δ=85% (hexosomes), ISAsomes with the discontinuous cubic structure Fd3m at δ=70% (micellar cubosomes), and ISAsomes with emulsified liquid isotropic L2 microemulsion phase at δ=55% (EMEs; as “emulsified microemulsions”). In the next step, these ISAsome samples were mixed with the gelifier/water samples in mass ratio 1:1 (diluted by a factor of 2). The concentration of gelifier in the final sample was given by ε ¼
mgelifier 100 mgelifier þ mF127 þ mLPA þ moil þ mwater
ð4Þ
All concentrations in the text are thus given in wt %, as also the polysaccharide MC-KC mixture was prepared in a 1:1 weight ratio. 2.2. Small-Angle X-ray Scattering. Small-angle X-ray scattering (SAXS) intensities were measured with a SAXSess camera53 (Anton-Paar, Graz, Austria). This high-flux camera with low background scattering was attached to the X-ray generator Philips PW 1730/10, which was operating at 40 kV and 50 mA. A sealed tube with Cu anode and Gobel mirror was used to obtain a focused monochromatic line-shaped beam of Cu KR radiation (λ=0.154 nm). The samples were measured in a standard quartz capillary centered in the X-ray beam and carefully thermostatted ((0.1 °C) using a Peltier element. Temperature was changed in a stepwise manner;the samples were equilibrated at a specific temperature for 30 min, and then, three subsequent 5 min measurements followed, which were averaged afterward. The 2D scattering pattern was recorded by a PI-SCX fused fiberoptic taper CCD camera (pixel size of 2424 μm2) from Princeton Instruments, a division of Roper Scientific, Inc. (Trenton, NJ, USA). Cosmic-ray correction and CCD background subtraction was performed on the 2D image, which was further reduced into one-dimensional scattering function I(q), where q represents the length of the scattering vector q=4π/λ sin(ϑ/2), with ϑ being the (53) Bergmann, A.; Orthaber, D.; Scherf, G.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 869–875.
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scattering angle. SAXS data were recorded for the regime 0.07 nm-1
