Synthesis of Hollow CaCO3 Nanospheres Templated by Micelles of

Nov 30, 2010 - Shunsuke Tanaka , Yusuf Valentino Kaneti , Ripon Bhattacharjee , Md Nazmul Islam , Rina Nakahata , Nawfel Abdullah , Shin-ichi Yusa ...
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Synthesis of Hollow CaCO3 Nanospheres Templated by Micelles of Poly(styrene-b-acrylic acid-b-ethylene glycol) in Aqueous Solutions Bishnu Prasad Bastakoti,† Sudhina Guragain,† Yuuichi Yokoyama,‡ Shin-ichi Yusa,‡ and Kenichi Nakashima*,† †

Department of Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan, and ‡Department of Materials Science and Chemistry, University of Hyogo, 2167 Shosha, Himeji 671-2280, Japan Received September 13, 2010. Revised Manuscript Received October 22, 2010

An asymmetric triblock copolymer, poly(styrene-b-acrylic acid-b-ethylene glycol) (PS-b-PAA-b-PEG), was synthesized via reversible addition-fragmentation chain transfer controlled radical polymerization. Micelles of PS-b-PAAb-PEG with PS core, PAA shell, and PEG corona were then prepared in aqueous solutions, followed by extensive characterization based on dynamic light scattering, zeta-potential, and transmission electron microscopy (TEM) measurements. The well-characterized micelles were used to fabricate hollow nanospheres of CaCO3 as a template. It was elucidated from TEM measurements that the hollow nanospheres have a uniform size with cavity diameters of ca. 20 nm. The X-ray diffraction analysis revealed a high purity and crystallinity of the hollow nanospheres. The hollow CaCO3 nanospheres thus obtained have been used for the controlled release of an anti-inflammatory drug, naproxen. The significance of this study is that we have overcome a previous difficulty in the synthesis of hollow CaCO3 nanospheres. After mixing of Ca2þ and CO32- ions, the growth of CaCO3 is generally quite rapid to induce large crystal, which prevented us from obtaining hollow CaCO3 nanospheres with controlled structure. However, we could solve this issue by using micelles of PS-b-PAA-b-PEG as a template. The PS core acts as a template that can be removed to form a cavity of hollow CaCO3 nanospheres, the PAA shell is beneficial for arresting Ca2þ ions to produce CaCO3, and the PEG corona stabilizes the CaCO3/micelle nanocomposite to prevent secondary aggregate formation.

Introduction Calcium carbonate (CaCO3) is a scientifically and industrially important mineral system. In particular, the hollow structured calcium carbonates may serve as drug deliverers and diagnostic markers1 like hollow nanospheres of other materials2 because it has excellent biocompatible and biodegradable properties. Its crystallization control has attracted significant attention for decades, as recently reviewed by Colfen and several workers.3 Several strategies have been suggested for the preparation of hollow CaCO3 particle. Langmuir monolayers,4 polysaccharide,5 foam lamellae,6 self-assembled monolayers,7 and solid matrices8 have been used as templates for the controlled growth of CaCO3 crystals. Gower et al. used a polymer-induced liquid precursor *To whom correspondence should be addressed. E-mail: nakashik@ cc.saga-u.ac.jp. (1) Wei, W.; Ma, G.; Hu, G.; Yu, D.; Mcleish, T.; Su, Z.; Shen, Z. J. Am. Chem. Soc. 2008, 130, 15808. (2) (a) Caruso, F.; Caruso, R. A.; Mohwald, H. Science 1998, 282, 1111. (b) Caruso, F. Chem.;Eur. J. 2000, 6, 413. (c) Caruso, F. Adv. Mater. 2001, 13, 11. (d) Cai, Y.; Pan, H.; Xu, X.; Hu, Q.; Li, L.; Tang, R. Chem. Mater. 2007, 19, 3081. (e) Lee, H. J.; Kim, S. E.; Kwon, K.; Park, C.; Kim, C.; Yang, J.; Lee, S. C. Chem. Commun. 2010, 46, 377. (f) Rosenholm, J. M.; Peuhu, E.; Eriksson, J. E.; Sahlgren, C.; Linden, M. Nano Lett. 2009, 9, 3308. (3) (a) Meldrum, F. C.; Colfen, H. Chem. Rev. 2008, 108, 4332. (b) Colfen, H. Curr. Opin. Colloid Interface Sci. 2003, 8, 23. (c) Gower, L. B. Chem. Rev. 2008, 108, 4551. (d) Kato, T.; Sugawara, A.; Hosoda, N. Adv. Mater. 2002, 14, 869. (4) Xu, G.; Yao, N.; Aksay, I.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977. (5) Sugawara, A.; Ishii, T.; Kato, T. Angew. Chem., Int. Ed. 2003, 42, 5299. (6) Chen, B. D.; Cilliers, J. J.; Davey, R. J.; Garside, J.; Woodburn, E. T. J. Am. Chem. Soc. 1998, 120, 1625. (7) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495. (8) (a) Manoli, F.; Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 1997, 182, 116. (b) Dalas, E.; Koutsoukos, P. G. Langmuir 1988, 4, 907. (9) Patel, V. M.; Sheth, P.; Kurz, A.; Ossenbeck, M.; Shah, D. O.; Gower, L. B. In Concentrated Dispersions: Theory, Experiments,and Applications; Markovic, B., Somansundaran, P., Eds.; ACS Symposium Series 878; American Chemical Society: Washington, DC, 2004; pp 15-25.

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process to produce monodisperse CaCO3-coated oil core-shell particles.9 In recent years, a class of so-called double hydrophilic block copolymers has been developed as crystal growth modifiers for CaCO3 crystals.10 It is well known that amphiphilic block copolymers can form self-assembled nanostructures, such as rod, wire, lamella, sphere, and large compound micelles.11 There has been growing use of the micelle of the amphiphilic polymer as a template to fabricate hollow inorganic nanoparticles.12 It is because the size and morphology of the polymeric micelle can be easily tuned by adjusting the block length and solution properties.13 In these systems, it is designed that the corona-forming hydrophilic block strongly interacts with the precursor of the inorganic materials and the core-forming hydrophobic block acts as a template of the hollow.14 However, the template micelles become very unstable when the inorganic precursor is sorbed into the corona, leading to the formation of secondary or higher order aggregates in which (10) (a) Sedlak, M.; Antonietti, M.; Colfen, H. Macromol. Chem. Phys. 1998, 199, 247. (b) Colfen, H.; Antonietti, M. Langmuir 1998, 14, 582. (c) Colfen, H. Macromol. Rapid Commun. 2001, 22, 219. (11) (a) Zhang, L. F.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (b) Zhang, L. F.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (c) Cameron, N. S.; Corbierre, M. K.; Eisenberg, A. Can. J. Chem. 1999, 77, 1311. (d) Nakashima, K.; Bahadur, P. Adv. Colloid Interface Sci. 2006, 123-126, 75. (e) Nakashima, K.; Khanal, A. In Bottom-Up Nanofabrication; Ariga, K., Nalwa, H. S., Eds.; American Scientific Publishers: Stevenson Ranch, CA, 2009; Vol. 5, p 385. (f) Fustin, C. A.; Abetz, V.; Gohy, J. F. Eur. Phys. J. E 2005, 16, 291 . (12) (a) Ras, R. H. A.; Kemell, M.; Wit, J.; Ritala, M.; Brinke, G.; Leskel€a, M.; Ikkala, O. Adv. Mater. 2007, 19, 102. (b) Lee, H.; Char, K. ACS Appl. Mater. Interfaces 2009, 1, 913. (13) (a) Choucair, A.; Lavigueur, C.; Eisenberg, A. Langmuir 2004, 20, 3894. (b) Xu, C.; Fu, X.; Fryd, M.; Xu, S.; Wayland, B. B.; Winey, K. I.; Composto, R. J. Nano Lett. 2006, 6, 282. (c) Zhang, Y.; Lin, W.; Jing, R.; Huang, J. J. Phys. Chem. B 2008, 112, 16455. (14) (a) Jin, D.; Yu, X.; Yue, L.; Sun, P. Inorg. Mater. 2009, 45, 168. (b) Yue, L.; Zheng, Y.; Jin, D. Microporous Mesoporous Mater. 2008, 113, 538.

Published on Web 11/30/2010

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Bastakoti et al. Scheme 1. Synthesis of PS-b-PAA-b-PEG via RAFT-Controlled Radical Polymerization

two or more particles are bound to each other.15 This problem was recently overcome by our group by using a core-shellcorona-type micelle of asymmetric triblock copolymers.16 It was also possible to control the void space diameter and the inorganic wall thickness, respectively, by changing the chain lengths of the core-forming and shell-forming blocks.17 In this study, we fabricated hollow CaCO3 nanospheres by templating a polymeric micelle with core-shell-corona architecture. First, we synthesized poly(styrene-b-acrylic acid-b-ethylene glycol) (PS-b-PAA-b-PEG) triblock copolymer via reversible addition-fragmentation chain transfer (RAFT)-controlled radical polymerization. Second, we prepared the PS-b-PAA-b-PEG micelle to elucidate the physicochemical properties. The polymer was expected to form a polymeric micelle with the PS core, PAA shell, and PEG corona in an aqueous solution. The shape and size of the micelle are measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). We also tried to control the size of the micelle by utilizing external stimuli such as pH change and metal ion addition. We then incorporated Ca2þ ions into the PS-b-PAA-b-PEG micelle by interaction between the cationic metal ions and anionic PAA block of the polymer. The binding of Ca2þ ions to the micelle was confirmed by DLS, zetapotential, and TEM measurements. Third, we used the wellcharacterized micelle as a template for the fabrication of hollow CaCO3 nanospheres. In this synthesis, the PS core acts as a template of the hollow cavity, the PAA shell works as a nanocontainer and nanoreactor for the precursor (Ca2þ) of CaCO3, and the PEG corona prevents the polymer-CaCO3 hybrid nanospheres from forming secondary aggregates by steric repulsion between the PEG chains. The hollow CaCO3 nanospheres were obtained after removing the template polymer by calcination or solvent extraction. The hollow nanospheres thus prepared were characterized by various techniques including TEM and X-ray diffraction (XRD). Finally, we attempted to use hollow nanospheres of CaCO3 as a drug carrier because the biocompatible and biodegradable nature1 of CaCO3 is expected to make the hollow CaCO3 particles a smart drug carrier.

Experimental Section Materials. A poly(ethylene glycol) (PEG)-based chain transfer agent (PEG-CTA) with the number-average degree of polymerization (DP) of 47 was prepared as previously reported.18 2,20 Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol. Styrene was washed with an aqueous alkaline solution and distilled with calcium hydride under reduced pressure. Acrylic acid, N,N-dimethylformamide (DMF), and dioxane were dried over 4 A˚ molecular sieves and distilled under reduced pressure. (15) Rudloff, J.; Colfen, H. Langmuir 2004, 20, 991. (16) Khanal, A.; Inoue, Y.; Yada, M.; Nakashima, K. J. Am. Chem. Soc. 2007, 129, 1534. (17) Liu, D.; Khanal, A.; Inoue, Y.; Yada, M.; Nakashima, K. Chem. Lett. 2009, 38, 130. (18) Yusa, S.; Yokoyama, Y.; Morishima, Y. Macromolecules 2009, 42, 376.

380 DOI: 10.1021/la103660x

Na2CO3 (Wako), CaCl2 (Katayama), tris buffer (Katayama), and naproxen sodium (Sigma-Aldrich) were used without further purification. Preparation of PEG-b-PAA. Acrylic acid (2.18 g, 30.3 mmol), AIBN (12.5 mg, 0.08 mmol), and PEG-CTA (475 mg, 0.20 mmol) were dissolved in dioxane (30 mL). The solution was deoxygenated by purging with Ar gas for 30 min. The polymerization was carried out at 60 C for 40 h. The polymerization mixture was dialyzed against pure water for 1 week. The diblock copolymer (PEG-b-PAA) was recovered by a freeze-drying technique (1.70 g, 64.0%). The DP of the PAA block was estimated to be 90 from its 1 H NMR spectrum in DMSO-d6. The number-average molecular weight Mn(NMR) estimated from its 1H NMR and molecular weight distribution (Mw/Mn) determined by GPC for the block copolymer were 8.85  103 and 1.31, respectively. Preparation of PS-b-PAA-b-PEG. Styrene (10.4 g, 100 mmol), AIBN (8.23 mg, 0.05 mmol), and PEG-b-PAA (1.11 g, 0.13 mmol) were dissolved in DMF (100 mL). The solution was deoxygenated by purging with Ar gas for 30 min. The polymerization was carried out at 60 C for 24 h. The polymerization mixture was dialyzed against acetone for 3 days and pure water for 1 day. The obtained triblock copolymer (PS-b-PAA-b-PEG) was recovered by a freeze-drying technique (2.09 g, 18.2%). The DP of the PS block was 80, as estimated by 1H NMR in DMSO-d6. The Mn (NMR) and Mw/Mn for PS-b-PAA-b-PEG were 1.88  104 and 1.22, respectively. The synthetic procedure is shown in Scheme 1. The detailed characterization data are shown in the Supporting Information. Preparation of Polymeric Micelle. A known amount of PS-b-PAA-b-PEG was dissolved in water and gently agitated by a magnetic stirrer at room temperature until a clear solution was obtained. The solution was then transferred to a volumetric flask to obtain a stock solution with a concentration of 0.5 g L-1.

Titration of PS-b-PAA-b-PEG Micelle Solution with Ca2þ Ions. A known amount of PS-b-PAA-b-PEG solution

was titrated with Ca2þ ions. The amount of added Ca2þ ions is expressed in terms of the apparent degree of charge neutralization (DN), which is defined as DN% ¼

amount of metal ion in equivalent unit  100 amount of ionic group in the polymer in base equivalent unit

ð1Þ During titration, the samples were agitated by a magnetic stirrer for 1 min to accelerate the interaction between the micelle and the metal ions. The pH of the solution was maintained around 9. All samples were kept at room temperature for 1 day before the characterization measurements. Synthesis of Hollow CaCO3 Nanospheres. The hollow CaCO3 nanospheres were synthesized using the micelle of PSb-PAA-b-PEG as a template (Scheme 2). A specific amount of calcium chloride (10.6 mg) was added to 5 mL of a PS-b-PAA-bPEG micelle solution. An equimolar amount of sodium carbonate was added to the mixture to create CaCO3 in the PAA layer of the polymeric micelle. The molar ratio of acrylic acid in the polymer to Ca2þ ions was 1:8. The final concentration of the polymer was 0.5 g L-1. The pH was maintained around 10. The resulting mixture was stirred to accelerate the reaction between Ca2þ and Langmuir 2011, 27(1), 379–384

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Scheme 2. Synthesis of Hollow CaCO3 Nanospheres from PS-b-PAA-b-PEG Micelle Template

Figure 1. Hydrodynamic diameter (Dh) of PS-b-PAA-b-PEG micelles at pH 9 as a function of the polymer concentration. When the polymer concentration is 5.5), almost all carboxylic groups (22) (a) Colombani, O.; Ruppel, M.; Burkhardt, M.; Drechsler, M.; Schumacher, M.; Gradzielski, M.; Schweins, R.; Muller, A. H. E. Macromolecules 2007, 40, 4351. (b) Jacquin, M.; Muller, P.; Pabalan, R. T.; Cottet, H.; Berret, J. F.; Futterer, T.; Theodoly, O. J. Colloid Interface Sci. 2007, 316, 897. (c) Bastakoti, B. P.; Guragain, S.; Yoneda, A.; Yokoyama, Y.; Yusa, S.; Nakashima, K. Polym. Chem. 2010, 1, 347. (23) (a) Kajiwara, K.; Ross-Murphy, S. B. Nature 1992, 355, 16. (b) Shiga, T.; Fukumor, K.; Hiorse, Y. J. Polym. Sci., Part B: Polym. Phys. 1994, 32, 85. (24) Ludwigs, S.; Schmidt, K.; Krausch, G. Macromolecules 2005, 38, 2376.

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Figure 3. (a) Dh and (b) zeta-potential of the Ca2þ/PS-b-PAA-bPEG chelated particles as a function of DN. The polymer concentration is 0.02 g L-1 in all of the samples in both Figures.

of the PAA chain get deprotonated, and the PAA chains are extended to produce the maximum size of the micelle. Complexation of Ca2þ Ions with the PS-b-PAA-b-PEG. The complexation of the PAA homopolymer anions with alkaline earth metal divalent cations in an aqueous solution has been widely investigated.25 The same discussion seems to be applied to the PAA block of PS-b-PAA-b-PEG. When alkaline earth metal ions are introduced into PS-b-PAA-b-PEG micellar solutions under basic conditions, the PAA block will be bound to the metal ions, which leads to a conformational change in the PAA block from an extended to shrunken form due to cancellation of the negative charge of the PAA block by the positive alkaline earth metal ions. To examine the incorporation of metal ions into the micelles, we performed titration of CaCl2 into the PS-b-PAA-bPEG solutions at pH 9 and monitored the particle size, zetapotential, and shape of the micelles. Figure 3a shows a plot of the Dh of the Ca2þ/PS-b-PAA-b-PEG chelated particles as a function of the DN with Ca2þ ions. As the DN is increased, the Dh of the Ca2þ chelated particles continues to decrease and attains a minimum value (∼43 nm) at 100% DN. After 100% DN, the Dh of the Ca2þ chelated particles remains almost constant. This fact indicates that the added Ca2þ ions are bound to the carboxylate anions of the PAA block, shielding the effective charges of the carboxylate anions. The electrostatic repulsion among the anionic PAA chains, therefore, is weakened, and the PAA blocks undergo a conformational change from an extended to shrunken form, resulting in a decrease in the total micellar size. The binding of Ca2þ ions to the polymer was also checked by zeta-potential (25) (a) Molnar, F.; Rieger, J. Langmuir 2005, 21, 786. (b) Sinn, C. G.; Dimova, R.; Antonietti, M. Macromolecules 2004, 37, 3444. (c) Bulo, R. E.; Donadio, D.; Laio, A.; Molnar, F.; Rieger, J.; Parrinello, M. Macromolecules 2007, 40, 3437. (d) Schweins, R.; Goerigk, G.; Huber, K. Eur. Phys. J. E 2006, 21, 99.

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Figure 4. TEM pictures of (a) Ca2þ/PS-b-PAA-b-PEG chelated particles and (b) hollow CaCO3 nanospheres.

measurements. Figure 3b shows the dependence of the zetapotential of the Ca2þ chelated particles on the DN. The successive addition of Ca2þ ions increases the zeta-potential from -57 mV to almost zero. Above a DN of 100%, the value of the zeta-potential is not increased. This fact shows that the binding of Ca2þ ions to the polymer is mainly driven by an electrostatic interaction. The morphologies of the Ca2þ chelated particles were investigated by TEM measurements (Figure 4a). We can see spherical particles with a dark periphery. This dark periphery is the PAA block of PS-b-PAA-b-PEG stained with Ca2þ ions. These images show the effective binding of the Ca2þ ions to the polymer. The average diameter obtained from the TEM images is ∼35 nm for Ca2þ chelated particles. The nanoparticles in the TEM pictures basically correspond to glassy PS cores surrounded by a compact Ca2þ/PAA shell. The PEG corona seems to be less stained compared with the PAA shell, giving an invisible image in the TEM picture. Fabrication of Hollow Nanopheres of Calcium Carbonate. One of the most interesting applications of the PS-b-PAAb-PEG micelles in this study is a template for the synthesis of inorganic hollow nanospheres. Therefore, using the wellcharacterized PS-b-PAA-b-PEG micelles as a template, we attempted to fabricate hollow CaCO3 nanospheres. The fabrication of hollow CaCO3 nanospheres is generally difficult because after initial mixing of Ca2þ ions and CO32- ions, the growth of CaCO3 is uncontrollably rapid to induce large crystal. However, the use of PS-b-PAA-b-PEG overcomes this issue of crystal overgrowth, allowing the formation of hollow nanospheres with uniform void space. The addition of Ca2þ ions forms a bidentate chelating complex26 with the PAA block resulting in colloidal droplets of the Ca2þ chelated particles. To the suspension of Ca2þ chelated particles, carbonate ions were added to start the CaCO3 formation. The TEM image of CaCO3/PS-b-PAA-b-PEG nanocomposite particles prior to removal of the core is shown in Figure S6 (Supporting Information). We obtained hollow CaCO3 nanospheres by removing the polymeric template with THF. Calcination at 500 C was carried out for complete removal of the template polymer. It was confirmed by FTIR spectra that the template polymer was completely removed from the hollow CaCO3 nanospheres (Figure S7, Supporting Information). Figure 4b illustrates a TEM image of the hollow CaCO3 nanospheres, which were fabricated under the condition that the concentration of PS-b-PAA-b-PEG was 0.5 g L-1 and the molar ratio of the acrylic acid unit in the PAA block to CaCl2 was 1:8. We can see a clear hollow structure of the individual CaCO3 nanospheres. The outer diameter of the sphere is ∼30 nm, whereas the void space diameter is ∼20 nm. We examined the effect of the CaCl2 concentration on the structure of the hollow particles. When the molar ratio of the acrylic acid unit in the PAA block to CaCl2 is increased to 1:15, we obtained irregular particles instead of separate hollow particles. The corona-forming PEG block (26) Fantinel, F.; Rieger, J.; Molnar, F.; H€ubler, P. Langmuir 2004, 20, 2539.

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Figure 5. XRD patterns of hollow CaCO3 nanospheres.

may not stabilize the Ca2þ chelated particles at a higher CaCl2 concentration because an excess amount of Ca2þ ions dehydrates the PEG block.27 Therefore, it is important to adjust the molar ratio of the acrylic acid unit in the PAA block to the CaCl2. The crystalline characteristics of the obtained hollow CaCO3 nanospheres were confirmed by an XRD analysis. A typical XRD pattern is shown in Figure 5. The XRD pattern of the hollow CaCO3 nanospheres shows the formation of calcite, CaCO3, if compared with a standard diffraction pattern of calcite (JCPDS 5-0586). The peak with the maximum intensity at 2θ = 29.45 corresponds to the reflections from the (104) planes. No characteristic peaks due to other phase of CaCO3 have been detected in Figure 5, which indicates that the hollow CaCO3 nanospheres are of calcite phase. The TG-DTA curves for CaCO3/PS-b-PAA-b-PEG nanocomposites are shown Figure S8 of the Supporting Information. The initial part of the TG curves slightly declines. This may imply desorption of absorbed water molecules. The strong exothermic peak on DTA curves around 400 C shows decomposition of the template polymer. The much higher weight loss after 600 C together with large endothermic peak on the DTA curve is possibly attributed to the decomposition of the CaCO3.28 Release of Naproxen from Hollow CaCO3 Nanospheres. There has been increased interest in inorganic nanoparticles as drug carriers. Among many inorganic materials so far used for this purpose,29 CaCO3 is exceedingly suitable because of its excellent biocompatible and biodegradable properties.1 Therefore, we investigated kinetics of the release of naproxen from hollow CaCO3 nanospheres to obtain basic information on the hollow nanospheres as a building unit of a controlled release system. The experimental setup is schematically shown in Figure 6. First, the drug (naproxen) is released from the hollow nanospheres to the bulk aqueous phase inside the dialysis tube. Then, it diffuses to the aqueous phase outside the dialysis tube. This process can be written as k1

k2

Dh sf DW1 sf DW2

ð2Þ

where Dh denotes a naproxen molecule incorporated in the hollow nanospheres and DW1 and DW2 denote the naproxen molecule in aqueous bulk phase inside and outside the dialysis tube, respectively. k1 is a rate constant for the drug to exit from the nanospheres (27) Tjandra, W.; Yao, J.; Ravi, P.; Tam, K. C.; Alamsjah, A. Chem. Mater. 2005, 17, 4865. (28) Frost, R. L.; Hales, M. C.; Martens, W. N. J. Therm. Anal. Calorim. 2009, 95, 999. (29) Ajima, K.; Yudasaka, M.; Murakami, T.; Maigne, A.; Shiba, K.; Iijima, S. Mol. Pharmaceutics 2005, 2, 475.

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Figure 6. Schematic illustration for the drug release experiments.

to the aqueous bulk phase inside dialysis tube, and k2 is a rate constant for the drug to diffuse from the dialysis tube to the outside aqueous phase across the membrane. We expect that the CaCO3 shell of the hollow particle is porous enough for the drug to pass through, although it is difficult to detect such a small pore by usual TEM measurements. As can be seen in Scheme 2, CaCO3 is formed mainly in the PAA domain of the template micelle. When PAA is removed, pores should be left after that. Because the polymer/CaCO3 nanocomposite particles were heated at the rate of 8.3 C/min in the calcination process, the gradual combustion of polystyrene core can produce carbon dioxide and water vapor. They could also make pores in the CaCO3 shell when they effused out. We assume that the drug was exclusively incorporated in the hollow interior. Although the drug might be equally distributed on both sides of the CaCO3 shell on loading, most of the drug on the outer surface was removed after washing several times. The drug loading capacity of hollow CaCO3 nanospheres is 246 mg of drug for 1 g of CaCO3. The absorbance of naproxen in the outside of the dialysis tube is plotted against dialysis time in Figure 7. Here At and A¥ are absorbance of DW2 at time t and ¥, respectively. It is obvious from eq 2 that the whole process is conformed to well-known consecutive reaction kinetics. Thus, we calculated k1 and k2 by a nonlinear least-squares method. The rate constants obtained are k1 = 0.037 h-1 and k2 = 0.79 h-1. The details of the method are explained somewhere else.19 Here we also define a retention time (tR) of the drug in the nanospheres as a reciprocal of k1. We obtain tR = 27 h. This value indicates that naproxen is retained in the nanospheres on this time scale. In our previous study,19 we measured a retention time of cloxacillin sodium in a polymeric micelle of poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) and obtained the values of 22 h at pH 3 and 14 h at pH 7. We realize that the polymeric micelle19 and the hollow silica nanoparticle30 have a retention time with a similar scale, although the drugs tested were different. Many nano- and microparticles have been examined as drug carriers.31 It is pointed out that particle size (30) Zhu, Y.; Shi, J.; Shen, W.; Dong, X.; Feng, J.; Ruan, M.; Li, Y. Angew. Chem., Int. Ed. 2005, 44, 5083. (31) (a) Son, S. J.; Bai, X.; Lee, S. B. Drug Discovery Today 2007, 12, 650. (b) Cevc, G.; Vierl, U. J. Controlled Release 2010, 141, 277.

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Figure 7. Plot of the absorbance of naproxen released from the hollow CaCO3 nanospheres as a function of dialysis time.

significantly affects cellular and tissue uptake, and only the nanosized particles can be taken up efficiently. In this regard, it is expected that the present hollow CaCO3 nanoparticle can be easily taken into cell and tissue because the size (ca. 30 nm) is much smaller than that of the particles previously reported. Hence, the hollow CaCO3 nanoparticles obtained in this study could be a useful drug carrier.

Conclusions A triblock copolymer, PS80-b-PAA90-b-PEG47, was synthesized via the RAFT-controlled radical polymerization. The PSb-PAA-b-PEG triblock copolymer self-assembled into micelles in aqueous media. The size of the micelles could be tuned by changing the pH, which results from the conformational change in the PAA block of the triblock copolymer. The complexation of Ca2þ ion with the PS-b-PAA-b-PEG micelle was induced under basic conditions to give monodispersed Ca2þ chelated particles. This is based on the electrostatic interaction between the cationic Ca2þ ion and anionic PAA block. The monodispersed Ca2þ chelated particles were successfully used as a template for the synthesis of well-defined hollow CaCO3 (calcite) nanospheres. The kinetics of release of a drug from the hollow nanospheres was also examined using naproxen as a model medicine because CaCO3 is well known to be an excellent biocompatible inorganic material. It turned out that naproxen is retained in the hollow nanospheres on a time scale of 27 h. Acknowledgment. We thank Mr. Toshini Tabata for his help with the TEM measurements and Professor T. Watari and Dr. Hom Nath Luitel for the XRD and TG-DTA measurements. The present study was supported by a Grant-in-Aid for Scientific Research (20310054) from the Japan Society for the Promotion of Science (JSPS). Supporting Information Available: NMR spectra, GPC elution curve, fluorescence spectra, DLS data, FTIR spectra, and TG-DT analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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