pubs.acs.org/Langmuir © 2009 American Chemical Society
Hydroxyapatite Nanoparticles as Particulate Emulsifier: Fabrication of Hydroxyapatite-Coated Biodegradable Microspheres Syuji Fujii,*,† Masahiro Okada,*,‡ Hidekatsu Sawa,† Tsutomu Furuzono,‡ and Yoshinobu Nakamura† †
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 Institute5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan Received March 30, 2009. Revised Manuscript Received May 21, 2009
Hydroxyapatite (HAp) nanoparticle-coated micrometer-sized poly(L-lactic acid) (PLLA) microspheres were fabricated via a “Pickering-type” emulsion route in the absence of any molecular surfactants. Stable oil-in-water emulsions were prepared using 40 nm HAp nanoparticles as a particulate emulsifier and a dichloromethane (CH2Cl2) solution of PLLA as an oil phase. It was clarified that the interaction between carbonyl/carboxylic acid groups of PLLA and the HAp nanoparticles at the CH2Cl2-water interface played a crucial role to prepare the stable Pickering-type emulsion. The HAp nanoparticle-coated PLLA microspheres were fabricated by the evaporation of CH2Cl2 from the emulsion and characterized in terms of size, particle size distribution, and morphology using scanning/transmission electron microscopes. Scanning electron microscopy study and ultrathin cross section observation using transmission electron microscopy confirmed adsorption of the HAp nanoparticles only at the surface of the PLLA microspheres. Cell-adhesion experiments suggested the HAp nanoparticles on the surface of the PLLA microspheres promoted the cell adhesion and spreading.
1. Introduction Pickering emulsions are solid particle-stabilized emulsions in the absence of any molecular surfactant, where solid particles adsorbed to an oil-water interface.1,2 Although this area of research lay dormant for many years, there has been increasing interest recently. Inorganic particles such as silica,3 metals,4 quantum dots,5 apatite,6 and clays,7 and organic particles such as latex particles,8-10 microgels11 and shell cross-linked micelles12 have been used as a particulate emulsifier. The wettability of the particulate emulsifiers to oil-water interfaces decides the stability and type of emulsions. Recently, we have reported the preparation of oil-in-water Pickering-type emulsions using hydroxyapatite (HAp, Ca10(PO4)6(OH)2) nanoparticles as a particulate emulsifier in the *To whom correspondence should be addressed. Dr. Syuji Fujii: s.fujii@ chem.oit.ac.jp (tel./fax +81-6-6954-4274). Dr. Masahiro Okada: okada04@ ri.ncvc.go.jp (tel. +81-6-6833-5012 (ext. 2623); fax +81-6-6872-7485). (1) (a) Ramsden, W. Proc Roy Soc. 1903, 72, 156. (b) Pickering, S. U. J. Chem. Soc. 1907, 91, 2001. (2) (a) Binks, B. P.; Horozov, T. S. Colloidal Particles at Liquid Interfaces; Cambridge Univ. Press: Cambridge, 2006.(b) Aveyard, R.; Binks, B. P.; Clint, J. H. Adv. Colloid Interface Sci. 2003, 100-102, 503. Also see references shown in the book and journal. (3) (a) Binks, B. P.; Lumsdon, S. O. Phys. Chem. Chem. Phys. 1999, 1, 3007. (b) Binks, B. P.; Lumsdon, S. O. Langmuir 2000, 16, 2539. (4) Wang, D.; Duan, H.; M€ohwald, H. Soft Mater. 2005, 1, 412. (5) (a) Lin, Y.; Skaff, H.; B€oker, A.; Dinsmore, A. D.; Emrick, T.; Russell, T. P. J. Am. Chem. Soc. 2003, 125, 12690. (b) Lin, Y.; Skaff, H.; Emrick, T.; Dinsmore, A. D.; Russell, T. P. Science 2003, 299, 226. (6) Fujii, S.; Okada, M.; Furuzono, T. J. Colloid Interface Sci. 2007, 315, 287. (7) (a) Cauvin, S.; Colver, P. J.; Bon, S. A. F. Macromolecules 2005, 38, 7887. (b) Bon, S. A. F.; Colver, P. J. Langmuir 2007, 23, 8316. (8) (a) Velev, O. D.; Furusawa, K.; Nagayama, K. Langmuir 1996, 12, 2374. (b) Dinsmore, A. D.; Hsu, M. F.; Nikolaides, M. G.; Marquez, M.; Bausch, A. R.; Weitz, D. A. Science 2002, 298, 1006. (9) Amalvy, J. I.; Armes, S. P.; Binks, B. P.; Rodrigues, J. A.; Unali, G. F. Chem. Commun. 2003, 1826. (10) (a) Fujii, S.; Randall, D. P.; Armes, S. P. Langmuir 2004, 20, 11329. (b) Fujii, S.; Aichi, A.; Akamatsu, K.; Nawafune, H.; Nakamura, Y. J. Mater. Chem. 2007, 17, 3777. (11) (a) Fujii, S.; Read, E. S.; Armes, S. P.; Binks, B. P. Adv. Mater. 2005, 17, 10140. (b) Ngai, T.; Behrens, S. H.; Auweter, H. Chem. Commum. 2005, 331. (c) Tsuji, S.; Kawaguchi, H. Langmuir 2008, 24, 3300. (12) Fujii, S.; Cai, Y.; Weaver, J. V. M.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 7304.
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absence of any molecular surfactant.6 HAp is one of the calcium phosphates and the main mineral of bones and teeth in vertebrate, and artificially synthesized HAp has been extensively used in a variety of applications, such as biomaterials, ion exchangers, adsorbents and catalysts by exploiting their biocompatibility and adsorbability with many compounds.13 The HAp exhibits excellent adhesion not only to cells but also to hard and soft tissues. In the previous article,6 we found that stable emulsions are readily prepared by homogenization of oils with carbonyl group (e.g., methyl myristate and methyl trimethyl acetate) and HAp aqueous dispersion; however, no stable emulsion was obtained using oils with no carbonyl group such as dichloromethane (CH2Cl2). Fourier-transform infrared (FT-IR) studies suggested that emulsion stabilization should be due to an interaction between calcium ion on the HAp particle surface and carbonyl group of the oils, which achieved the adsorption of the HAp nanoparticles to the oil-water interfaces. In this study, we first demonstrate stabilization of CH2Cl2 emulsion droplets with the HAp nanoparticles by dissolving polymer with carbonyl groups in CH2Cl2 (Figure 1). To the best of our knowledge, this is the first report of the stabilization of Pickering emulsion via interaction between polymer and nanoparticles at the oil-water interface. As a polymer with carbonyl groups, poly(L-lactic acid) (PLLA) was used in this study. PLLA is one of the most promising biodegradable polymers and have been used in the fields of orthopedic and reconstructive surgery14,15 and tissue engineering16 because it is not necessary to remove the polymeric materials after healing. (13) (a) Bett, J. A. S.; Christner, L. G.; Hall, W. K. J. Am. Chem. Soc. 1967, 89, 5535. (b) Brown, P. E.; Constanz, B. Hydroxyapatite and Related Materials; CRC Press: London, 1994. (c) Norton, J.; Malik, K. R.; Darr, J. A.; Rehman, I. Adv. Appl. Ceram. 2006, 105, 113. (14) Kohn, J.; Abramson, S.; Langer, R. Bioresorbable and Bioerodible Materials. In Biomaterials Science: An Introduction to Materials in Medicine, 2nd ed.; Ratner B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Elsevier Academic Press: CA, 2004; p 115. (15) Athanasiou, K. A.; Niederauer, G. G.; Agrawal, C. M. Biomaterials 1996, 17, 93. (16) (a) Langer, R.; Vacanti, J. P. Science 1993, 260, 920. (b) Seal, B. L.; Otero, T. C.; Panitch, A. Mater. Sci. Eng. R. Rep. 2001, 34, 147. (c) Rezwan, K.; Chen, Q. Z.; Blaker, J. J. Biomaterials 2006, 27, 3413.
Published on Web 06/10/2009
DOI: 10.1021/la901100z
9759
Article
Fujii et al.
Figure 1. Schematic representation of HAp nanoparticle-stabilized dichrolomethane (CH2Cl2) solution of poly(L-lactic acid) (PLLA)-inwater emulsion droplets, fabrication of HAp nanoparticle-coated PLLA microspheres and their high cell adhesiveness.
Next, we describe the first synthesis of HAp nanoparticlecoated biodegradable PLLA microspheres by the evaporation of the oil from the CH2Cl2 solution of PLLA-in-water emulsions and evaluate their cell adhesiveness (Figure 1). There are strong needs for synthesis of PLLA in a form of microsphere17 because they have found their applications as carriers for controlled drug delivery system18 and 3D porous scaffold and cell carrier for tissue engineering 19. Although PLLA found various applications, there is a disadvantage to be overcome: low cell adhesion to PLLA because of its hydrophobic surface character.20 In this study, the HAp nanoparticles were employed as a particulate emulsifier in order to prevent flocculation of emulsion droplets/microspheres as well as to give high cell adhesive property to the PLLA microspheres obtained. The synthetic method described in this study needs neither molecular surfactant nor polymeric stabilizer, which is usually used to synthesize/stabilize the microspheres in media and has possibilities to cause allergy-like reactions and carcinogenicity21 at the same time. No adhesion proteins derived from animals (e.g., collagen and gelatin, which are intractable in terms of sterilization and storage) were used in this method to give high cell adhesive property.
2. Materials and Methods 2.1. Materials. Unless otherwise stated, all materials were guaranteed reagent grade and used as received from Nacalai Tesque Inc. Calcium nitrate (Ca(NO3)2 3 4H2O), diammonium hydrogen phosphate ((NH4)2HPO4), 25% ammonia aqueous solution, and 60% HNO3 were used as received. Dichloromethane (CH2Cl2) was purchased from Sigma-Aldrich, Inc. Milli-Q water (Millipore Corp., MA) with a specific resistance of 18.2 106 Ω 3 cm was used for the synthesis of the HAp nanoparticles and for the preparation of emulsion. PLLA with molecular weights of 5000, 12 500 g/mol were purchased from Nacalai Tesque Inc. and was used as received. PLLA of (17) (a) Grandfils, C.; Flandroy, P.; Nihant, N.; Barbette, S.; Jerome, R.; Teyssie, P.; Thibaut, A. J. Biomed. Mater. Res. 1992, 26, 467. (b) Lemperle, G.; Morhenn, V.; Charrier, U. Aesthetic Plast. Surg. 2003, 27, 354. (c) Lemperle, G.; Morhenn, V. B.; Pestonjamasp, V.; Gallo, R. L. Plast. Reconstr. Surg. 2004, 113, 1380. (18) (a) Cohen, S.; Yoshioka, T.; Lucarelli, M.; Hwang, L.; Langer, R. Pharm. Res. 1991, 8, 713. (b) Kohane, D. S.; Tse, J. Y.; Yeo, Y.; Padera, R.; Shubina, M.; Langer, R. J. Biomed. Mater. Res. A 2006, 77A, 351. (c) Malafaya, P. B.; Silva, G. A.; Reis, R. L. Adv. Drug Delivery Rev. 2007, 59, 207. (19) (a) Borden, M.; Attawia, M.; Laurencin, C. T. J. Biomed. Mater. Res. 2002, 61, 421. (b) Borden, M.; Attawia, M.; Khan, Y.; Laurencin, C. T. Biomaterials 2002, 23, 551. (c) Choia, Y. S.; Parka, S. N.; Suh, H. Biomaterials 2005, 26, 5855. (d) Hong, Y.; Gao, C.; Xie, Y.; Gong, Y.; Shen, J. Biomaterials 2005, 26, 6305. (20) Nakagawa, M.; Teraoka, F.; Hamada, Y.; Kibayashi, H.; Takahashi, J.; Fujimoto, S. J. Biomed. Mater. Res. Part A 2006, 77, 112. (21) (a) Wong, J.; Brugger, A.; Khare, A.; Chaubal, M.; Papadopoulos, P.; Rabinow, B.; Kipp, J.; Ning, J. Adv. Drug Delivery Rev. 2008, 60, 939. (b) Fong, J. W. French Patent No. 2484281, 1981.
9760 DOI: 10.1021/la901100z
300 000 g/mol was purchased and was used as received from Polysciences, Inc., Warrington, PA. 2.2. Hydroxyapatite Nanoparticles Synthesis. Spherical HAp nanoparticles were prepared via wet chemical process using the same protocol as described in our early study.6 Ca(NO3)2 aqueous solution (42 mN, 800 mL), whose pH was adjusted to pH 12.0 by addition of 25% ammonia solution, was poured into a 1 L reactor equipped with an inlet of N2, a reflux condenser and a halfmoon type stirrer. After a temperature in the reactor had been equilibrated at 25 °C, (NH4)2HPO4 aqueous solution (100 mN, 200 mL), whose pH was adjusted to pH 12.0 with ammonia solution, was added into the reactor at one batch within 10 s, and the mixture was stirred for another 10 h at 25 °C. The resulting HAp particles were centrifugally washed using Milli-Q water until the pH value of the supernatant decreased to approximately 7.
2.3. Hydroxyapatite Nanoparticles Characterization. 2.3.1. Fourier-Transform Infrared (FT-IR) Measurement. FT-IR spectra were obtained with a Spectrum One (Perkin-Elmer Inc., MA) using diffuse reflectance unit at a resolution of 4 cm-1 with 16 scans at room temperature. In the FT-IR spectrum of the spherical HAp, peaks at 1456/1413 and 877 cm-1 were observed (Figure S1, Supporting Information), which indicates the substitution of carbonate ions in phosphate positions of HAp lattice.22 The carbonate ions seemed to substitute during the wet chemical process and/or the reaction with CO2 in air at dry state. Interaction between the HAp nanoparticles and PLLA molecules was also estimated by measuring FT-IR spectrum of the PLLA molecules adsorbed on the HAp nanoparticles. The spherical HAp nanoparticles (0.1 g) were dispersed in the PLLA solution (0.1 g/10 mL CH2Cl2) at room temperature for 24 h, and PLLA molecules were adsorbed on the HAp nanoparticles. These HAp nanoparticles with PLLA on their surface were collected by filtration, washed with 100 mL CH2Cl2 and then dried at room temperature under reduced pressure. The FT-IR spectrum of the HAp nanoparticles was compared with those of pure PLLA and the HAp nanoparticles. 2.3.2. X-Ray Diffraction (XRD). To investigate the crystal phase of the HAp particles, XRD measurement was conducted using a RAD-X (Rigaku International Co., Tokyo, Japan) with Cu KR radiation.
2.4. Fabrication of HAp Nanoparticle-Stabilized Emulsion and HAp Nanoparticle-Coated PLLA Microspheres. Stock aqueous dispersions of the HAp nanoparticles with a solid content of 0.04 wt % were prepared by serial dilution; the pH of the dispersion was measured to be 6.5 using a pH meter. Aliquots of these dispersions (25 g) were then hand-shaken with the CH2Cl2 solution of PLLA (total 2.5 g, 1.0-9.1 wt % solid contents) at (22) Emerson, W. H.; Fisher, E. E. Arch. Oral Biol. 1962, 7, 671. (23) (a) Ishikawa, K.; Ishikawa, Y.; Kuwayama, N. Chem. Express 1991, 6, 463. (b) Cheng, Z. H.; Yasukawa, A.; Kandori, K.; Ishikawa, T. J. Chem. Soc. Faraday Trans. 1998, 94, 1501.
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Fujii et al. 25 °C for a period of 30 s by the same operator.24 The HAp nanoparticle-coated PLLA microspheres were prepared via in situ evaporation of CH2Cl2 from the emulsion at 25 °C. Bare PLLA microspheres were prepared by removal of the HAp nanoparticles from the HAp nanoparticle-coated PLLA microspheres using HNO3 aqueous solution (pH, 4.0), which can dissolve the HAp component. These bare PLLA microspheres were used after washing with water to remove ionic species generated by dissolution of the HAp nanoparticles.
2.5. Emulsion Characterization. 2.5.1. Conductivity Measurements. The conductivity of the emulsions immediately after preparation was measured using a digital conductivity meter (Hanna model Primo 5). The conductivities of aqueous nanocomposite dispersion ranged from 40 to over 2000 μS cm-1, depending on the solution pH. The emulsions were classified according to their conductivities. A high conductivity indicated an oil-in-water emulsion and a low (
