pubs.acs.org/Langmuir © 2010 American Chemical Society
Pickering-Type Water-in-Oil-in-Water Multiple Emulsions toward Multihollow Nanocomposite Microspheres Hayata Maeda,† Masahiro Okada,*,‡,§ Syuji Fujii,*,† Yoshinobu Nakamura,† and Tsutomu Furuzono‡,§ †
Department of Applied Chemistry, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka 535-8585, Japan, and ‡Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. § Present address: Department of Biomedical Engineering, School of Biology-Oriented Science and Technology, Kinki University, 930 Nishi-Mitani, Kinokawa, Wakayama 649-6493, Japan Received February 15, 2010. Revised Manuscript Received August 8, 2010
Multihollow hydroxyapatite (HAp)/poly(L-lactic acid) (PLLA) nanocomposite microspheres were readily fabricated by solvent evaporation from a “Pickering-type” water-in-(dichloromethane solution of PLLA)-in-water multiple emulsion stabilized with HAp nanoparticles. The multiple emulsion was stabilized with the aid of PLLA molecules used as a wettability modifier for HAp nanoparticles, although HAp nanoparticles did not work solely as particulate emulsifiers for Pickering-type emulsions consisting of pure dichloromethane and water. The interaction between PLLA and HAp nanoparticles at the oil-water interfaces plays a crucial role toward the preparation of stable multiple emulsion and multihollow microspheres.
1. Introduction Pickering-type emulsions are solid particle-stabilized emulsions, where solid particles are adsorbed onto oil-water interfaces.1 Inorganic particles such as silica and carbon black1 and organic particles such as latex2 and microgels3 have been used as particulate emulsifiers. Binks and Lumsdon showed that the type of emulsion (oil-in-water (O/W) or water-in-oil (W/O)) depends on the wettability of particulate emulsifiers at an oil-water interface: hydrophilic silica particles preferentially stabilize O/W emulsions, whereas hydrophobically modified silica particles preferentially stabilize W/O emulsions.4 Based on these results, Binks and co-workers succeeded in preparing a water-in-oilin-water (W/O/W) multiple emulsion with two kinds of silica particles having different hydrophobicity in the absence of any surfactant.5 There are a few reports stating that Pickering-type W/ O/W emulsions were used as a platform toward hollow microspheres.6,7 Mugel et al. fabricated polymer-particle-assembled hollow microspheres from Pickering-type W/O/W emulsions.6 Inorganic-particle-assembled hollow microspheres were fabricated
*Corresponding authors: (M.O.) E-mail:
[email protected]. Telephone: þ81-736-77-0345 ext 5205. Fax: þ81-736-77-4754. (S.F.) E-mail:
[email protected]. Telephone/fax: þ81-6-6954-4274.
(1) (a) Ramsden, W. Proc. R. Soc. 1903, 72, 156–164. (b) Pickering, S. U. J. Chem. Soc. 1907, 91, 2001–2021.(c) Binks, B. P.; Horozov, T. S. Colloidal Particles at Liquid Interfaces; Cambridge University Press: Cambridge, UK, 2006. (2) (a) Velev, O. D.; Furusawa, K.; Nagayama., K. Langmuir 1996, 12, 2374– 2384. (b) Fujii, S.; Randall, D. P.; Armes, S. P. Langmuir 2004, 20, 11329–11335. (3) (a) Fujii, S.; Read, E. S.; Armes, S. P.; Binks, B. P. Adv. Mater. 2005, 17, 1014–1018. (b) Ngai, T.; Behrens, S. H.; Auweter, H. Chem. Commum. 2005, 331–333. (4) (a) Binks, B. P.; Lumsdon, S. O. Phys. Chem. Chem. Phys. 1999, 1, 3007– 3016. (b) Binks, B. P.; Lumsdon, S. O. Langmuir 2000, 16, 2539–2547. (5) (a) Binks, B. P.; Dyab, A. K. F.; Fletcher, P. D. I.; Barthel, H. Multiple emulsions. German Patent assigned to Wacker-Chemie GmbH, DE10211313. (b) Binks, B. P.; Dyab, A. K. F.; Fletcher, P. D. I. Proceedings of 3rd World Congress on Emulsions. CME 2002, 1-10. (6) Miguel, A. S.; Scrimgeour, J.; Curtis, J. E.; Behrens, S. H. Soft Matter 2010, 6, 3163–3166. (7) (a) Lee, D.; Weitz, D. A. Adv. Mater. 2008, 20, 3498–3503. (b) Lee, D.; Weitz, D. A. Small 2009, 5, 1932–1935.
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by Lee and Weitz7 from W/O/W emulsions containing silica or magnetite nanoparticles in oil phases. Recently, we demonstrated the possibility of hydroxyapatite (HAp; Ca10(PO4)6(OH)2) nanoparticles as particulate emulsifiers to stabilize O/W emulsions.8 HAp is the main mineral of bones and teeth, and artificially synthesized HAp has been extensively used in a variety of applications, such as biomaterials, ion exchangers, adsorbents, and catalysts, by exploiting its biocompatibility and adsorbability with many compounds.9 In a previous article,8 we found that stable emulsions are readily prepared using oils with a carbonyl group such as methyl myristate; however, no stable emulsion was obtained using oils without a carbonyl group such as CH2Cl2. These results indicate that the interaction between HAp and the carbonyl groups of oils results in suitable wettability of the nanoparticles and promotes nanoparticle adsorption onto the oil-water interface, which is a prerequisite for efficient emulsification. Furthermore, we succeeded in stabilizing CH2Cl2 droplets with HAp nanoparticles by dissolving a polymer with carbonyl groups, poly(L-lactic acid) (PLLA), as a wettability modifier in CH2Cl2;10 this is the first demonstration of an O/W emulsion stabilized by interaction between nanoparticles in the aqueous phase and polymer molecules in the oil phase. We also succeeded in fabricating dense PLLA microspheres coated with HAp from HAp-stabilized CH2Cl2 droplets containing PLLA, and showed that HAp nanoparticles on microsphere surfaces promoted cell adhesion and spreading.10 In this study, we demonstrate the stabilization of a Pickeringtype W/O/W multiple emulsion by using HAp nanoparticles as particulate emulsifiers and PLLA molecules as wettability modifiers. Multihollow HAp/PLLA nanocomposite microspheres (8) Fujii, S.; Okada, M.; Furuzono, T. J. Colloid Interface Sci. 2007, 315, 287– 296. (9) (a) Aoki, H. Science and medical application of hydroxyapatite; Japanese Association of Apatite Science: Tokyo, 1991.(b) Brown, P. E.; Constanz, B. Hydroxyapatite and Related Materials; CRC Press: London, 1994. (10) Fujii, S.; Okada, M.; Sawa, H.; Furuzono, T.; Nakamura, Y. Langmuir 2009, 25, 9759–9766.
Published on Web 08/16/2010
DOI: 10.1021/la102529d
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were also fabricated by evaporation of CH2Cl2 from a waterin-(CH2Cl2 solution of PLLA)-in-water multiple emulsion stabilized with HAp nanoparticles. Hollow microspheres made from biodegradable polymers such as PLLA have been used as drug carriers for controlled release in medical fields.11 Although biodegradable multihollow microspheres are generally fabricated from W/O/W multiple emulsions stabilized with molecular surfactants,12 which involves the risk of cytotoxicity in medical applications,13 the synthetic method reported here requires no molecular surfactants. The multiple emulsion and the multihollow microspheres were characterized with respect to particle size, size distribution, and morphology. The interaction between PLLA and HAp was confirmed with a Fourier-transform infrared (FT-IR) study. As far as we are aware, this is the first example of the fabrication of inorganic/polymer composite multihollow microspheres from a Pickering-type emulsion.
2. Experimental Section Materials. Reagent grade (NH4)2HPO4 (Kishida Chemicals Co., Ltd., Osaka, Japan), 25% NH3 aq (Wako Pure Chemical Industries, Ltd., Osaka. Japan), and CH2Cl2 (Sigma Aldrich Co., St. Louis, MO) were used as received. PLLA with a weightaverage molecular weight (MW) of 800 g/mol was purchased from Taki Chemical Co., Ltd. (Hyogo, Japan), and PLLA with a weight-average MW of 150 000 g/mol was purchased from Sigma Aldrich Co. Water was purified with a Milli-Q system (Millipore Corp., Billerica, MA). Other chemicals were reagent grade and used as purchased from Nacalai Tesque Inc. (Kyoto, Japan). HAp Nanoparticles. Spherical HAp nanoparticles (SHAp) were prepared by adding 100 mM (NH4)2HPO4 aq (100 mL) rapidly into a mixture of 42 mM Ca(NO3)2 aq (400 mL) and 25% NH3 aq (2.5 mL) at 25 °C under N2.6 After the mixture was stirred for 10 h, the nanoparticles were centrifugally washed three times with water. Rod-shaped HAp nanoparticles (RHAp) were prepared under the same conditions described above except for the addition rate of (NH4)2HPO4 aq (0.33 mL/min) and temperature (40 °C).6 The RHAp were used after calcination at 800 °C to improve their crystallinity and thermal stability.14,15 PLLA Modification of RHAp. The aqueous dispersion of RHAp was centrifugally washed three times with ethanol followed by CH2Cl2 to substitute the dispersion medium with CH2Cl2. The CH2Cl2 dispersion of RHAp (RHAp, 0.5 g; total, 15 mL) and a CH2Cl2 solution of PLLA (Mw, 800 g/mol; PLLA, 1.16 g; total, 15 mL) were mixed, and then CH2Cl2 was removed by evaporation in a fume hood at rt. The dried mixture was heated at 100 or 200 °C for 20 h under reduced pressure. Unless otherwise noted, PLLA-modified RHAp (PLLA-RHAp) were used without purification after the heat treatment. PLLA-RHAp were analyzed with a FT-IR spectrometer (Spectrum One; Perkin-Elmer Inc., Waltham, MA) using a diffuse reflectance unit at a resolution of 4 cm-1 with 16 scans, after PLLA-RHAp were centrifugally washed six times with CH2Cl2 in order to remove free PLLA. The MW of PLLA was measured by gel permeation chromatography (GPC; HLC-8220GPC; TOSOH Co., Tokyo, Japan) using chloroform as the solvent, and calibrated with polystyrene standards (UBE Scientific Analysis Laboratory, Inc., Tokyo, Japan). For the GPC measurements of the PLLA component in PLLA-RHAp, the HAp component was removed by dissolving using a HNO3 aqueous solution (pH 3). The amount of PLLA (11) (a) Wada, R.; Tabata, Y.; Hyon, S.-H.; Ikada, Y. Bull. Inst. Chem. Res., Kyoto Univ. 1988, 66, 241–250. (b) Schugens, C.; Laruelle, N.; Nihant, N.; Grandfils, C.; Jerome, R.; Teyssie, P. J. Controlled Release 1994, 32, 161–176. (12) Perrin, P.; Prigent, F.; Hebraud, P. In Multiple Emulsions Technology and Applications; Aserin, A., Ed.; Wiley-Interscience: New York, 2008; p 29. (13) Arechabala, B.; Coiffard, C.; Rivalland, P.; Coiffard, L. J. M.; RoeckHoltzhauer, Y. D. J. Appl. Toxicol. 1999, 19, 163–165. (14) Okada, M.; Furuzono, T. J. Mater. Sci. 2006, 41, 6134–6137. (15) Okada, M.; Furuzono, T. J. Nanoparticle Res. 2007, 9, 807–815.
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adsorbed to RHAp was calculated from the carbon content measured via a CHN elemental analyzer (CHN-Corder MT-5; Yanaco New Science Inc., Kyoto, Japan) at 1100 °C in an oxygen stream. For elemental analysis, PLLA-RHAp were used after centrifugal washing six times with CH2Cl2. Multiple Emulsion and Multihollow Microspheres. A total of 80 mg of PLLA-RHAp was dispersed in a 5 wt % CH2Cl2 solution of PLLA (MW, 150 000; PLLA, 2.42 g) placed in a Teflon tube with the aid of ultrasound (US-2; SND Co., Ltd., Nagano, Japan) for 1 min. A drop of pure water (20 μL) was then homogenized in the dispersion of PLLA-RHAp with an ultrasonic homogenizer (Sonicator Model W-220F; Heatsystems Ultrasonics Inc., NY, USA) for 1 min to prepare a W/O emulsion. A W/O/W emulsion was prepared by emulsifying the W/O emulsion in a 25 g aqueous dispersion of SHAp (0.04 wt %) with a homogenizer (Ultra-Turrax T10; IKA Works, Inc., Wilmington, NC) at 20 500 rpm for 1 min. Multihollow HAp/PLLA composite microspheres were prepared by evaporating CH2Cl2 from the W/O/W emulsion at rt for 24 h. A drop of the emulsion or the microsphere dispersion was placed on a slide glass and viewed using an optical microscope (TE2000-U; Nikon Corp.), and the mean droplet or microsphere size was estimated from the micrographs (n=300). HAp nanoparticles and nanocomposite microspheres were observed with a scanning electron microscope (SEM; JSM-6301F; JEOL Ltd., Tokyo, Japan) operating at 5 kV. The number-average sizes of HAp nanoparticles and microspheres in the dried state (n = 300) were measured from the SEM photographs, and the resulting data were presented as mean size ( standard deviations. For the observation of the internal structure of the microspheres, ultrathin cross sections (thickness, 100 nm) of the microspheres buried in an epoxy matrix (Agar low viscosity resin kit; Agar Scientific Ltd., Essex, U.K.; cured at 60 °C for 24 h) were observed using a transmission electron microscope (TEM; CM120; Philips, Eindhoven, The Netherlands) operated at 80 kV. The weight percentage of HAp in the composite microspheres, which were purified by four sedimentation-redispersion cycles with water, was determined by using an SII TG-DTA 6300 instrument (Seiko Instruments Inc., Chiba, Japan; heating rate, 10 °C/min; temperature range, 30-700 °C; N2 atmosphere).
3. Results and Discussion HAp Nanoparticles. Figure 1 shows SEM photographs of SHAp and RHAp prepared by the wet chemical method. The number-average diameter of SHAp was 39 ( 7 nm, and the average lengths of RHAp were 234 ( 84 nm (long axis) and 111 ( 20 nm (short axis). SHAp were used without any surface modification to stabilize the O/W emulsions and to coat the outer surfaces of the microspheres. On the other hand, RHAp were used after PLLA modification to stabilize the W/O emulsions and to coat the inner (water domain) surfaces inside the multihollow microspheres. The PLLA modification of RHAp was conducted in order to increase its hydrophobicity. It should be noted that the pristine HAp prepared in this study could not be dispersed but only agglomerated in CH2Cl2 due to its hydrophilic surface character. There are several HAp surface modification methods, such as silane coupling treatment,16 esterification reaction with longchain alcohol,17 and grafting polymerization.18 These methods require nonbiodegradable modifiers, which remain in a living body for a long time. In this study, HAp was modified only with (16) Furuzono, T.; Sonoda, K.; Tanaka, J. J. Biomed. Mater. Res., Part A 2001, 56, 9–16. (17) Borum-Nicholas, L.; Wilson, O. C., Jr. Biomaterials 2003, 24, 3671–3679. (18) Lee, S. C.; Choi, H. W.; Lee, H. J.; Kim, K. J.; Chang, J. H.; Kim, S. Y.; Choi, J.; Oh, K.-S.; Jeong, Y.-K. J. Mater. Chem. 2007, 17, 174–180.
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Figure 1. SEM images of (a) spherical and (b) rod-shaped hydroxyapatite (HAp) nanoparticles. Table 1. Summary of Microanalytical Data for Pristine RHAp, PLLA Homopolymers (MW, 800), and PLLA-RHAp Treated at Different Temperatures Followed by Washing with CH2Cl2 to Remove Free PLLA C (%)a H (%)a PLLA content (%)b Pristine RHAp 0.48 0.17 0 PLLA homopolymer 48.36 5.73 100 PLLA-RHAp treated at r.t. 0.72 0.27 0.5 PLLA-RHAp treated at 100 °C 1.01 0.23 1.1 PLLA-RHAp treated at 200 °C 1.15 0.24 1.4 a Determined by elemental analyses at 1100 °C in an oxygen stream. b Percentage mass, calculated from the following equation: 48.36(X/100) þ 0.48(1 - X/100) = C, where X is PLLA content (%) and C is carbon content (%).
biodegradable PLLA to avoid containing such nonbiodegradable modifiers. RHAp was used for a PLLA modification, because RHAp has larger a-plane surfaces, which have calcium ion sites,19 compared with spherical SHAp.14 It is expected that PLLA molecules carrying carboxyl groups can be adsorbed on HAp surfaces through ionic interaction with calcium ions.20,21 In this study, low-molecular-weight PLLA (800 g/mol) was used for the modification of RHAp because of its high content of carboxyl end (19) (a) Kawasaki, T. J. Chromatogr. 1978, 151, 95–112. (b) Kawasaki, T. J. Chromatogr. 1978, 157, 7–42. (20) (a) Misra, D. N. J. Dent. Res. 1989, 68, 42–47. (b) Yoshida, Y.; Meerbeek, B. V.; Nakayama, Y.; Yoshioka, M.; Snauwaert, J.; Abe, Y.; Lambrechts, P.; Vanherle, G.; Okazaki, M. J. Dent. Res. 2001, 80, 1565–1569. (21) Qiu, X.; Chen, L.; Hu, J.; Sun, J.; Hong, Z.; Liu, A.; Chen, X.; Jing, X. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5177–5185.
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groups as compared with general PLLA, used as drug carriers for controlled release in medical fields, with a MW of several tens or hundreds of thousands.11 CHN elemental analysis studies were conducted for PLLARHAp after washing with CH2Cl2 to remove unadsorbed PLLA (Table 1). Carbon microanalysis data indicate that PLLA molecules remained after washing with CH2Cl2 (good solvent for PLLA) and that the amount of PLLA increased with an increase in the heat treatment temperature. It is expected that the chance for PLLA end groups to migrate and interact with RHAp surfaces increased above the melting point of PLLA (around 145 °C),22 which led to an increase in the PLLA adsorption amount. A small amount of carbon detected in the pristine RAHp should be due to the incorporation of CO32- in the HAp lattice (see Figure 2; CO2 and H2O are eliminated from CO32--substituted HAp by heating between 700 and 1000 °C to form oxyapatite, Ca10(PO4)6O23). Figure 2 shows the FT-IR spectra of pristine RHAp, PLLARHAp after washing with CH2Cl2, and PLLA homopolymers. In the spectrum of pristine RHAp, the adsorptions at 1456/1413 cm-1 are attributed to CO32- ions substituting the phosphate positions in the HAp lattice, due to the reaction with CO2 during the wet chemical process and calcination.15 In the spectrum of PLLA homopolymers, a peak at 1760 cm-1 due to carbonyl main chains and carboxyl end groups was observed. In the spectra of three kinds of PLLA-RHAp treated at different temperatures, new broad peaks appeared at around 1590 cm-1, which should be assigned to carboxylate groups (originating from carboxyl end groups) interacting with calcium ions on the HAp surfaces.21 A peak due to carbonyl main chains in PLLA-RHAp treated at rt was small compared with that of carboxylate end groups, and a red-shift from 1760 to 1720 cm-1 was observed. These results, respectively, suggest that lower-molecular-weight PLLA was preferentially adsorbed on the RHAp surface and that carbonyl main chains also interacted with calcium ions on the RHAp surface.24 The peak due to carbonyl main chains in PLLA-RHAp increased and the red shifting of the peak decreased with an increase in the treatment temperature. These results might be due to the elongation of PLLA molecules on RHAp by condensation reactions among PLLA molecules.25 It is worth noting that GPC studies indicate that the weight-average MW of PLLA, which included adsorbed and free PLLA, in PLLA-RHAp increased from 800 g/mol to 1100 g/mol (100 °C) and to 8700 g/mol (200 °C) after the heat treatments. Stabilization of W/O Emulsion. In order to stabilize the W/O emulsion, RHAp modified with PLLA molecules was used as a particulate emulsifier. No other nonbiocompatible or nonbiodegradable chemicals such as molecular surfactants were used in this study. Figure 3a-c shows digital photographs of the mixtures of pure water and the CH2Cl2 solution containing the PLLA-RHAp and PLLA homopolymers. Before adding water into the CH2Cl2 phase, PLLA-RHAp were well dispersed in the CH2Cl2 phase in all cases. However, RHAp migrated and formed aggregates in the aqueous phases after adding water in the cases of PLLA-RHAp treated at rt and 100 °C (Figure 3a and b), and water droplets were not dispersed in continuous CH2Cl2 phases. On the other hand, lots of tiny water droplets (less than 2 μm) and some large water droplets were stabilized with PLLA-RHAp treated at 200 °C, as shown in Figure 3d. These results indicate (22) Cam, D.; Hyon, S.; Ikada, Y. Biomaterials 1995, 16, 833–843. (23) Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates; Elsevier: Amsterdam, 1994; Chapter 4. (24) Walsh, D.; Furuzono, T.; Tanaka, J. Biomaterials 2001, 22, 1205–1212. (25) Achmad, F.; Yamane, K.; Quan, S.; Kokugan, T. Chem. Eng. J. 2009, 151, 342–350.
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Figure 2. FT-IR spectra of (a) pristine RHAp nanoparticles, (b-d) PLLA-RHAp nanoparticles, and (e) PLLA homopolymers. PLLA-RHAp nanoparticles were treated at (b) room temperature, (c) 100 °C, and (d) 200 °C.
Figure 3. (a-c) Digital photographs and (d) an optical micrograph showing mixtures of pure water and CH2Cl2 containing PLLA-RHAp and PLLA homopolymers. PLLA-RHAp were treated at different temperatures: (a) room temperature, (b) 100 °C, and (c, d) 200 °C. Arrows in photographs (a) and (b) indicate aggregates of RHAp in aqueous phases. The inset in (d) shows a magnified image.
that PLLA-RHAp treated at 200 °C were hydrophobic enough to stabilize water droplets in the CH2Cl2 medium, and hereafter PLLA-RHAp treated at 200 °C was used for the preparation of W/O/W emulsion. Stabilization of W/O/W Emulsion and Multihollow Microspheres. Figure 4a shows optical micrographs of the W/O/W emulsion stabilized with SHAp and PLLA-RHAp treated at 200 °C. The water domains were observed in the oil droplets. In our previous study,10 it was clarified that CH2Cl2 droplets containing PLLA were stabilized in a HAp aqueous dispersion via interactions between calcium ions on the unmodified HAp nanoparticles in the aqueous phase and carbonyl/carboxyl groups of PLLA in the oil phase. In this study, the oil droplets containing water domains were not stabilized in the absence of free PLLA in the oil phase or unmodified HAp in the aqueous phase, indicating that the oil droplets containing water domains were also stabilized via interactions between unmodified SHAp and free PLLA at the interfaces between the aqueous and the oil phases. After evaporation of CH2Cl2, the number-average diameter of the droplets decreased from 14.9 ( 12.8 μm to 9.1 ( 8.8 μm (Figure 4b), and residual CH2Cl2 could not be detected after 13730 DOI: 10.1021/la102529d
evaporation for 24 h at 25 °C (
