pubs.acs.org/Langmuir © 2010 American Chemical Society
Nanosized Emulsions Stabilized by Semisolid Polymer Interphase Yoon Sung Nam,*,†,‡ Jin-Woong Kim,*,‡ Jongwon Shim,‡ Sang Hoon Han,‡ and Han Kon Kim‡ †
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, and ‡Amore Pacific R&D Center, 314-1 Bora-dong Giheung-gu Yongin-si, Gyounggi-do, 449-729, Republic of Korea Received May 23, 2010. Revised Manuscript Received July 18, 2010
We introduce a new approach for stabilizing oil-in-water nanoemulsions using a semisolid interphase formed by the phase separation of amphiphilic block copolymers from the organic phase. This system is illustrated using an amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL), with commonly used oils. PEO-bPCL can be miscible with oil at elevated temperatures (70-80 °C); however, polymer/oil demixing occurs as the temperature drops below the melting temperature of PEO-b-PCL (∼55 °C). A homogeneous polymer/oil mixture was dispersed in water at 80 °C to generate embryonic emulsions, and then the emulsion size was reduced to a nanometer range through microfluidic homogenization. The structure of the generated nanoemulsions is irreversibly frozen as they are cooled down to ambient temperature. The nanoemulsions stabilized by PEO-b-PCL show the excellent colloidal stability against thermal and chemical stresses, exhibiting no significant changes in the size distribution during incubation for 4 months at ambient temperature or 10 days at 60 °C. This study demonstrates that PEO-b-PCL is an attractive emulsifying material for practical nanoemulsion formulations requiring structural stability under a broad range of conditions.
Nanoemulsions are heterogeneous mixtures of oil and water, where the dispersed droplets are confined to nanometer-scale dimensions. Oil-in-water nanoemulsions have been well studied for industrial applications, including pharmaceutical, food, personal care, and cosmetics products, to dissolve poorly soluble ingredients and enhance their transport properties.1-6 Nanoemulsions usually have better colloidal stability compared to the micrometer-sized emulsions because of several reasons: creaming or sedimentation is efficiently avoided because small droplet size reduces the gravitational force; flocculation is suppressed by effective steric stabilization; and coalescence is well prevented because nanosized droplets are not easily deformed. In addition, the relatively large ratio of the interfacial thickness to the droplet size reduces the thinning of the interdroplet liquid film, driving two droplets apart. However, there is still an issue on longterm colloidal stability of nanoemulsions most likely because of Ostwald ripening, which is the growth of larger droplets through the transport of oil from small droplets.7-9 This issue particularly matters when lipids and surfactants are used as emulsifiers.2,3,10-12 The physical stability and shelf life of nanoemulsion formulations prepared using such low molecular weight emulsifiers *To whom all correspondence should be addressed. E-mail: yoonsung@ mit.edu (Y.S.N.);
[email protected] (J.-W. K.).
(1) Fang, J. Y.; Hung, C. F.; Hua, S. C.; Hwang, T. L. Ultrasonics 2009, 49, 39. (2) Ichikawa, H.; Watanabe, T.; Tokumitsu, H.; Fukumori, Y. Curr. Drug Delivery 2007, 4, 131. (3) Liu, S.; Lee, C. M.; Wang, S.; Lu, D. R. Drug Delivery 2006, 13, 159. (4) Sarker, D. K. Curr. Drug Delivery 2005, 2, 297. (5) Teeranachaideekul, V.; Souto, E. B.; Junyaprasert, V. B.; Muller, R. H. Eur. J. Pharm. Biopharm. 2007, 67, 141. (6) Wang, L.; Li, X.; Zhang, G.; Dong, J.; Eastoe, J. J. Colloid Interface Sci. 2007, 314, 230. (7) Liu, W.; Sun, D.; Li, C.; Liu, Q.; Xu, J. J. Colloid Interface Sci. 2006, 303, 557. (8) Izquierdo, P.; Esquena, J.; Tadros, T. F.; Dederen, C.; Garcia, M. J.; Azemar, N.; Solans, C. Langmuir 2002, 18, 26. (9) Tadros, T.; Izquierdo, R.; Esquena, J.; Solans, C. Adv. Colloid Interface Sci. 2004, 108, 303. (10) Ganta, S.; Amiji, M. Mol. Pharmaceutics 2009, 6, 928. (11) Wang, L.; Tabor, R.; Eastoe, J.; Li, X.; Heenan, R. K.; Dong, J. Phys. Chem. Chem. Phys. 2009, 11, 9772. (12) Graves, S. M.; Mason, T. G. J. Phys. Chem. C 2008, 112, 12669.
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are highly susceptible to the fluctuations in temperature, pH, and ionic strength. This limitation often requires the time-consuming optimization of emulsion products for practical applications.6,11,13 The physical properties of the oil phase are important in preventing Ostwald ripening.9,14-17 In general, nanoemulsions of highly viscous oils (e.g., long chain triglycerides) have a long shelf life because of the large molar volume of the oil molecule. Such large molar volume can reduce the solubility of the oil in the aqueous phase and thus provide a kinetic barrier to Ostwald ripening.17 Using a mixture of two immiscible oils can also cause a gain in entropy with oil demixing, playing as a thermodynamic barrier to Ostwald ripening.17 It was also reported that the colloidal stability of nanoemulsions can be improved by adding a secondary oil having a low solubility in the aqueous phase.18 These studies suggest that both the molecular size and the miscibility are critically important factors in the selection of oils for nanoemulsion formulations. In addition to the properties of the organic phase, appropriate emulsifiers need to be carefully chosen in maintaining the structural integrity of nanoemulsions.19 Nonionic emulsifiers based on poly(ethylene oxide) (PEO) are widely used because of the wellknown steric effect of the polymer chain. The steric hindrance is highly affected by the molecular shape of the emulsifier at the interface and the extent to which the hydrophobic segment of the emulsifier diffuses to the oil phase.20,21 To stabilize oil-in-water emulsions, the emulsifier having a hydrophobic head smaller than (13) Shafiq-un-Nabi, S.; Shakeel, F.; Talegaonkar, S.; Ali, J.; Baboota, S.; Ahuja, A.; Khar, R. K.; Ali, M. AAPS PharmSciTech 2007, 8, 28. (14) Bouchemal, K.; Briancon, S.; Perrier, E.; Fessi, H. Int. J. Pharm. 2004, 280, 241. (15) Dai, L.; Li, W.; Hou, X. Colloids Surf., A 1997, 125, 27. (16) Mason, T. G.; Wilking, J. N.; Meleson, K.; Chang, C. B.; Graves, S. M. J. Phys.: Condens. Matter 2006, 18, R635. (17) Wooster, T. J.; Golding, M.; Sanguansri, P. Langmuir 2008, 24, 12758. (18) Solans, C.; Izquierdo, P.; Nolla, J.; Azemar, N.; Garcia-Celma, M. J. Curr. Opin. Colloid Interface Sci. 2005, 10, 102. (19) Hanson, J. A.; Chang, C. B.; Graves, S. M.; Li, Z. B.; Mason, T. G.; Deming, T. J. Nature 2008, 455, 85. (20) March, G. C.; Napper, D. H. J. Colloid Interface Sci. 1977, 61, 383. (21) Barnes, T. J.; Prestidge, C. A. Langmuir 2000, 16, 4116.
Published on Web 07/23/2010
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Figure 1. Formation of micrometer-sized oil-in-water emulsions. (a) Schematic description of the oil-in-water emulsification with PEO-bPCL. Amphiphilic polymer aggregates are formed in the organic phase and migrate toward the aqueous phase. The blue and red chains indicate the PEO and PCL blocks, respectively. (b) Optical micrograph of the oil-in-water embryonic emulsions containing the 1:10 mixture of PEO-b-PCL and PTM in the organic phase. The sample was taken from the emulsion after homogenization at 2000 rpm at 80 °C for 2 min and was quickly cooled down to ambient temperature on a glass slide. (c) Magnified version of an oil droplet in (b). Scale bar: 5 μm.
the PEO tail needs to be used to generate a large positive spontaneous curvature.22 In addition, the efficient emulsion stabilization through the steric repulsion mechanism requires the high solubility of the hydrophobic chain in the oil phase.23 The extension of the PEO chain to the aqueous phase is facilitated as the hydrophobic block is shielded from interactions with water molecules. This shielding effect can minimize the intermolecular association between the hydrophilic and hydrophobic chains of the emulsifier. The more extended conformation of PEO in the aqueous phase can allow more effective repulsion between neighboring hydrophilic chains. This report aims to demonstrate that oil-in-water nanoemulsions can be effectively stabilized even in the case where the hydrophobic part of a PEO-based amphiphilic polymer is longer than the PEO chain and not soluble in the oil phase. It was hypothesized that the formation of a physically robust interphase between oil and water would contribute to the suppression (22) Sela, Y.; Magdassi, S.; Garti, N. Colloids Surf., A 1994, 83, 143. (23) Sela, Y.; Magdassi, S.; Garti, N. Colloid Polym. Sci. 1994, 272, 684. (24) Savic, R.; Azzam, T.; Eisenberg, A.; Nedev, H.; Rosenberg, L.; Maysinger, D. Biomaterials 2009, 30, 3597. (25) Savic, R.; Azzam, T.; Eisenberg, A.; Maysinger, D. Langmuir 2006, 22, 3570. (26) Allen, C.; Han, J. N.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. J. Controlled Release 2000, 63, 275. (27) Nam, Y. S.; Kim, K. J.; Kang, H. S.; Park, T. G.; Han, S. H.; Chang, I. S. J. Appl. Polym. Sci. 2003, 89, 1631.
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of Ostwald ripening through the mechanical reinforcement of the interface.28-30 This approach is illustrated using poly(ethylene oxide)-poly(ε-caprolactone) block copolymers (PEO-b-PCL), which have been widely studied for biodegradable drug delivery nanoparticles.24-26 PCL was chosen as a hydrophobic block because it is a semicrystalline polymer that has a relatively low melting temperature (Tm = 50-60 °C).27 PEO-b-PCL is immiscible with commonly used oils at ambient temperature; however, above Tm, PEO-b-PCL can be dissolved in the organic phase, while it becomes recrystallized when it is cooled down to ambient temperature. In this work, it was hypothesized that, at elevated temperature, the amphiphilic nature of PEO-b-PCL can drive the migration of the polymer from the organic phase to the oil-water interface, generating the semisolid network of entangled polymer chains as an interphase, as schematically described in Figure 1a. This process can be effectively facilitated if the oil droplet size is reduced to a nanometer range because of the short path-length for the polymer migration. PEO-b-PCL was synthesized by the ring-opening polymerization of ε-caprolactone in the presence of methoxy PEO using (28) Anseth, J. W.; Bialek, A.; Hill, R. M.; Fuller, G. G. Langmuir 2003, 19, 6349. (29) Mehta, S. C.; Somasundaran, P. Langmuir 2008, 24, 4558. (30) Torres, L. G.; Iturbe, R.; Snowden, M. J.; Chowdhry, B. Z.; Leharne, S. A. Colloids Surf., A 2007, 302, 439.
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Figure 2. Transmission electron micrographs of the nanoemulsions made from (a) PTM, (b) PDMS, (c) CEH, (d) ODO, (e) IPM, and (f) LP. The samples were prepared 1 day after the preparation. Scale bars: 150 nm.
stannous octoate as a catalyst. The weight ratio of PCL to PEO was 2.3, as measured from 1H nuclear magnetic resonance (NMR) spectroscopy, and the weight average molecular weight and the polydispersity were 16.6 kDa and 1.37, respectively, as determined by gel permeation chromatography (GPC). Nanoemulsions of six different oils were prepared: phenyl trimethicone (PTM), poly(dimethylsiloxane) (PDMS), cetyl ethylhexanoate (CEH), dioctanoyl-decanoyl-glycerol (ODO), isopropyl myristate (IPM), and liquid paraffin (LP). All of the selected oils are commonly used in various food and personal care products, and their chemical structures and sources are provided in Figure S1 in the Supporting Information. To examine the solubility of PEO-b-PCL in the oils, PEOb-PCL powder (1 g) was dispersed in the oils (100 g) by bath sonication for 3 h and was kept in an oven maintained at 80 °C for 24 h. PEO-b-PCL was not dissolved in any of the tested oils even after the procedures were repeated three times. However, the polymer was soluble in ethanol around 60 °C, and this ethanolic solution was readily miscible with all of the oils tested above. To prepare oil-in-water emulsions, PEO-b-PCL (1 g) was dispersed in absolute ethanol (3 mL) with magnetic stirring at 300 rpm at 60 °C. The polymer was quickly dissolved in ethanol. The ethanolic solution was then mixed with the oil (10 g) pre-equilibrated at 80 °C, resulting in a homogenously transparent solution. The oil/ polymer mixture was then emulsified in Milli-Q water (100 g, 18.2 Ω 3 cm) with homogenization at 2000 rpm at 80 °C for 2 min to produce micrometer-sized oil-in-water emulsions. For instance, the optical micrograph of the emulsions prepared using PTM shows that the size of the resulting emulsions was in the range of a few micrometers (Figure 1b). These embryonic emulsions were pumped through a microchannel, referred to as an interaction 13040 DOI: 10.1021/la102084f
chamber, of a M110EH microfluidizer (Microfluidics Corp., Newton, MA) at a pressure of about 1 kbar (Supporting Information Figure S2).19,31 The solidification of PEO-b-PCL occurred during the microfluidization as the emulsions flowed through the interaction chamber and channels, whose temperature was maintained at 15 °C with a circulating water jacket. The temperature of the final emulsion product was 24 ( 2 °C immediately after microfluidiation. As a control experiment, the emulsions were slowly cooled to ambient temperature after emulsification (without microfluidization) at 80 °C, and macroscopic phase separation of PEO-b-PCL from the organic phase resulted in the precipitation of irregular emulsion structures (Supporting Information Figure S3). Figure 2 shows the transmission electron microscopy (TEM) images of the nanoemulsions of various oils stabilized by PEOb-PCL. The average size and size distribution of the prepared nanoemulsions were highly dependent upon the oil types, as determined by dynamic light scattering (DLS) (Table 1). It was found that the physicochemical nature of the organic phase is critically important for the emulsification process. Six different oils were emulsified into nanoemulsions, and significant variations were found in the size distribution and morphology of nanoemulsions, though it is possible that the surface morphology of nanoemulsions in water might be affected by the drying process. Figure 2a and b shows that the emulsion surfaces of the silicone oils, PTM and PDMS, have distinctive, protruded structures, indicating the phase separation of PEO-b-PCL in the organic phase during the formation of nanoemulsions. In contrast, the nanoemulsions of CEH and ODO showed homogeneous, smooth (31) Kwon, S. S.; Nam, Y. S.; Lee, J. S.; Ku, B. S.; Han, S. H.; Lee, J. Y.; Chang, I. S. Colloids Surf., A 2002, 210, 95.
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Letter Table 1. Properties of the Nanoemulsions Stabilized by PEO-b-PCL μ2/Γ2 c
d (nm)b oil name
molecular weight (g mol-1)
specific gravitya
oil viscosity (mPa 3 s)a
30 mind
24 he
30 mind
24 he
PTM 372 0.99 15 161.2 163.6 0.136 0.120 0.96 20 185.1 184.1 0.193 0.208 PDMS N/Af CEH 368 0.86 13 153.3 155.1 0.177 0.160 ODO 408 0.94 70 140.8 137.3 0.121 0.108 IPM 270 0.85 4 173.8 249.3 0.156 0.203 0.83 18 252.6 N/Ag 0.249 N/Ag LP N/Af a At 25 °C. b Hydrodynamic diameter determined by DLS analysis using the CONTIN algorithm. c The second order polydispersity index, where μ is the second moment about the mean and Γ is the average decay rate, determined by DLS using the method of cumulants. d Measured within 30 min after the preparation of emulsions at 25 °C. e Measured 24 h after the preparation of emulsions at 25 °C. f Not available because the oil is a mixture of molecules having different molecular weights. g Not measured because macroscopic phase separation was observed.
Figure 3. (a) Photographs of the nanoemulsions containing various oils incubated for 4 months (1, PTM; 2, PDMS; 3, CEH; 4, ODO; 5, IPM; and 6, LP). (b) Average diameter of the nanoemulsions measured by DLS measured after 1 day (black) and 4 months (red) of incubation at room temperature, and the size distributions of the ODO nanoemulsions after (c) 1 day of incubation at room temperature, (d) 4 months of incubation at room temperature, and (e) 10 days of incubation at 60 °C.
surface morphology (Figure 2c and d). PEO-b-PCL seems to have higher miscibility with CEH and ODO because they are composed of alcohol and fatty acids linked by an ester bond, which is the same chemical bond connecting the monomeric units of PCL. The average diameters of the nanoemulsions were 153.3 and 140.8 nm for CEH and ODO, respectively, which were the smallest sizes among the oils tested in this study. The nanoemulsions of IPM, which also comprises hydrocarbon chains with an ester bond, showed an average hydrodynamic diameter of 173.8 nm and a polydispersity of 0.156. Both of the values were initially smaller than those of PDMS, 184.1 nm and 0.208, when they were measured at 30 min after preparation. However, the emulsion size and the polydispersity of IPM rapidly increased to 249.3 nm and 0.203, respectively, after incubation at room temperature for 24 h (Table 1). The surface morphology of such nanoemulsions was very similar to that of silicone oil nanoemulsions, showing the appearance of small particle-like structures (Figure 2e). The Langmuir 2010, 26(16), 13038–13043
colloidal instability of the nanoemulsions of IPM, compared to other ester oils, might be caused by its low viscosity (4 mPa 3 s) and molecular weight (270 g mol-1). The small molar volume of IPM is also kinetically disadvantageous for the prevention of Ostwald ripening, which is known to be a major destabilization mechanism of nanosized emulsion droplets. In the case of LP, the average size of the nanoemulsions was about 252.6 nm in diameter and their polydispersity was 0.249, both of which were significantly higher than those of any other oils. The TEM image of the LP nanoemulsions exhibited quite irregular emulsion structures (Figure 2f), and the macroscopic phase separation began to occur 24 h after preparation, indicating that LP is not compatible with PEO-b-PCL for the stabilization of nanoemulsions; the phase separation between polymer and oil occurs too quickly, and PEO-b-PCL precipitates in the organic phase rather than migrates to the interface. Figure 3a shows the photograph of the nanoemulsions of different oils incubated at room temperature in the dark for DOI: 10.1021/la102084f
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Figure 4. TEM images of the nanoemulsions containing 10 wt % ODO incubated at room temperature for 24 h in the presence of 1 wt % SDS (a) and 1.25 wt % CTAB (b). Scale bars: 150 nm.
4 months. The emulsions of IPM and LP exhibited macroscopic phase separation (e.g., creaming) while the other nanoemulsions did not show any apparent phase separation under the same conditions. The arrows indicate the boundaries of two distinctive phases formed through the coarsening processes, presumably coalescence and Ostwald ripening. The average diameters of the other nanoemulsions were not significantly changed at room temperature for 4 months (Figure 3b), indicating the excellent physical stability of the prepared nanoemulsions. For instance, Figure 3c and d shows that the size distributions of the ODO nanoemulsions were very similar, with a slight increase of the average diameter, despite the long-term incubation. In addition to the long-term shelf stability, the effects of thermal and chemical stresses on the physical stability of the nanoemulsions stabilized by PEO-b-PCL were examined. To determine the thermal effects on the emulsion stability, the ODO nanoemulsions were incubated at 60 °C for 10 days and the emulsion size distribution was determined by DLS. ODO was chosen because the nanoemulsions of this oil had the smallest size and polydispersity and it was one of the most stable emulsions as described above. The average diameter and the polydispersity of the ODO nanoemulsions slightly increased to 138.1 nm and 0.121, respectively; however, their size distribution was not significantly affected (Figure 3e), indicating the maintenance of the structural integrity of the nanoemulsions under this condition. Chemical stresses were also applied to the ODO nanoemulsions by adding low molecular weight surfactants, 1 wt % sodium dodecylsulfate (SDS) and 1.25 wt % cetyl trimethylammonium bromide (CTAB), followed by incubation at room temperature for 24 h. A drop of each of the nanoemulsion samples was placed on a carbon-coated copper TEM grid. After incubation for 30 min, the droplet was very carefully removed using a micropipet and the grid was rinsed with fresh water three times. This procedure was repeated five times. The TEM images of the nanoemulsions showed that the original nanoemulsion structures were maintained, indicating their physical resistance against the stresses by SDS and CTAB (Figure 4). The oil concentration of the final emulsion formulations is an important parameter for practical applications. To determine the maximum oil content of the nanoemulsions stabilized by PEO-bPCL, the ODO nanoemulsions were prepared using different amounts of ODO (Figure 5a). Figure 5b shows the effect of the oil concentration on the emulsion size when the concentration of PEO-b-PCL was fixed to be 1 wt %. The size of nanoemulsions 13042 DOI: 10.1021/la102084f
Figure 5. (a) Photographs of the nanoemulsions for different oilcontent ratios labeled in the image below the tubes and (b) the average diameter and second order polydispersity index (μ2/Γ2) of the nanoemulsions as a function of the oil content.
increased with increasing the oil content and reached a plateau value of 205 ( 5 nm in the range of 2-10 wt %. The average size of the nanoemulsions remained the same when the oil content was increased, though the thickness of the PEO-b-PCL layer may have decreased. These results imply that PEO-b-PCL is very effective for the stabilization of nanoemulsions because nanoemulsions are generally prepared at the surfactant concentration of 4-8 wt %, which is much higher than the concentration of PEO-b-PCL (1 wt %) used in this study.8 Langmuir 2010, 26(16), 13038–13043
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In conclusion, this study has demonstrated that various oilin-water nanoemulsions can be stabilized using an amphiphilic block copolymer, PEO-b-PCL. The block copolymer, which was originally dissolved in the organic phase, was reorganized as a semisolid interphase at the oil/water interface. The morphology and colloidal stability of the prepared nanoemulsions were highly dependent upon the oil type: the emulsions of PTM, PDMS, ODO, and CEH were very stable and thus exhibit no significant structural changes over 4 months, while phase separation occurred in the emulsions of IPM and LP within 1 day. The polymer-rich interphase seems to be physically very robust and can prevent oil from diffusing out to the aqueous phase. Accordingly, the prepared nanoemulsions maintained structural integrity against thermal and chemical stresses. We expect that this
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approach of emulsion stabilization will be of practical use for applications in pharmaceutical, food, and personal care products. Acknowledgment. We thank Katherine Choi for assistance with manuscript preparation and Seung Jae Baik for helpful discussion. This work was performed at Amore Pacific R&D Center (Yongin, Republic of Korea) and supported by Amore Pacific Corporation (Seoul, Republic of Korea). Supporting Information Available: Detailed experimental procedures, chemical structures of the oils used, schematic description of the microfluidic process, and optical micrograph of crude emulsions. This material is available free of charge via the Internet at http://pubs.acs.org.
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