Synthesis of Hydroxyapatite Crystals Using Amino Acid-Capped Gold

We report here the use of surface-modified gold nanoparticles as templates for the ... (2D) and, therefore, do not provide significant control over cr...
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Langmuir 2005, 21, 5185-5191

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Synthesis of Hydroxyapatite Crystals Using Amino Acid-Capped Gold Nanoparticles as a Scaffold Debabrata Rautaray, Saikat Mandal, and Murali Sastry* Nanoscience Group, Materials Chemistry Division, National Chemical Laboratory, Pune 411008, India Received June 14, 2004. In Final Form: March 6, 2005 Inorganic composites are of special interest for biomedical applications such as in dental and bone implants wherein the ability to modulate the morphology and size of the inorganic crystals is important. One interesting possibility to control the size of inorganic crystals is to grow them on nanoparticles. We report here the use of surface-modified gold nanoparticles as templates for the growth of hydroxyapatite crystals. Crystal growth is promoted by a monolayer of aspartic acid bound to the surface of the gold nanoparticles; the carboxylate ions in aspartic acid are excellent binging sites for Ca2+ ions. Isothermal titration calorimetry studies of Ca2+ ion binding with aspartic acid-capped gold nanoparticles indicates that the process is entropically driven and that screening of the negative charge by the metal ions leads to their aggregation. The aggregates of gold nanoparticles are believed to be responsible for assembly of the platelike hydroxyapatite crystals into quasi-spherical superstructures. Control experiments using uncapped gold nanoparticles and pure aspartic acid indicate that the amino acid bound to the nanogold surface plays a key role in inducing and directing hydroxyapatite crystal growth.

Introduction There is increasing scientific interest in materials chemistry to learn about the architecture, morphology, and patterning of inorganic materials at all dimensions from the nanoscale to macroscopic scale by mimicking the process of biomineralization.1 Mineralized tissues are often found to contain polymorphs and individual minerals whose crystal morphology, size, and orientation are determined by local conditions and, in particular, the presence of matrix proteins or other macromolecules. The processes and materials that control such crystal nucleation and growth are of great interest to materials scientists for making composite materials analogous to those produced by nature.2-4 There are several approaches to explore the promoting effect of biological/biomimetic templates on crystal nucleation and growth such as proteins or glycoproteins,5 inorganic polymer surfaces,6 polyelectrolyte surfaces, etc.7 The precipitation of calcium phosphates has attracted the interest of many researchers because of its importance in water treatment processes, in catalysis as a supporting material, in agriculture as fertilizers, and in biomineralization processes.8-12 The * Author for correspondence: phone, +91 20 25893044; fax, +91 20 25893952/25893044; e-mail, [email protected]. (1) (a) Mann, S.; Ozin, G. A. Nature 1996, 382, 313. (b) Estroff, L. A.; Hamilton, A. D. Chem. Mater. 2001, 13, 3227. (2) Mann, S. Nature 1988, 332, 119. (3) Mann, S. Nature 1993, 365, 499. (4) Berman, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. Science 1995, 269, 515. (5) (a) Berman, A.; Hanson, J.; Leiserowitz, L.; Koetzle, J. F.; Weiner, S.; Addadi, L. Science 1993, 259, 776. (b) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67. (6) Wong, K. K. W.; Brisdon, B. J.; Heywood, B. R.; Hodson, A. G. W.; Mann, S. J. Mater. Chem. 1994, 4, 1387. (7) Fendler, J. H.; Meldrum, F. C. Adv. Mater. 1995, 7, 607. (8) Joris, S. J.; Amberg, C. H. J. Phys. Chem. 1971, 75, 3167. (9) Ellis, N.; Margaritis, A.; Briens, C. L.; Bergougnou, M. A. AIChE J. 1996, 42, 87. (10) Christoffersen, J.; Christoffersen, M. R. J. Cryst. Growth 1988, 87, 41. (11) (a) Nancollas, G. H. J. Cryst. Growth 1977, 42, 185. (b) Koutsoukos, P. G.; Nancollas, G. H. J. Phys. Chem. 1981, 85, 2403. (12) Koutsopoulos, S.; Demakopoulos, J.; Argiriou, X.; Dalas, E.; Klouras, N.; Spanos, N. Langmuir 1995, 11, 1831.

crystallization of calcium phosphate (mainly hydroxyapatite (Ca10(PO4)6(OH)2, HAP) has attracted much attention primarily due to its participation in the biological calcification process of teeth and bone formation.13,14 However, other phases, such as dicalcium phosphate (DCP), dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), and tricalcium phosphate (TCP), may also participate in the crystallization reaction. Earlier reports on the biomineralization of bone have involved the deposition of calcium phosphate crystals on a matrix rich in collagen fibrils, the structural macromolecules that create the scaffold within which the biological mineral is formed.15 Generally acidic proteins or macromolecules containing negatively charged groups control most of the biomineralization processes.15 These hydrophilic macromolecules interact specifically in a solution in which minerals of calcium are growing in vitro16 and in vivo17 suggesting that they are involved in the crystal formation process at the molecular level. These macromolecules are believed to control nucleation, polymorphism, and growth of the crystals.18 The presence of these acidic macromol(13) Dalas, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1989, 85, 2465. (14) (a) Christoffersen, J.; Christoffersen, M. R. J. Cryst. Growth 1981, 53, 42. (b) Hirai, T.; Hodono, M.; Komasawa, I. Langmuir 2000, 16, 955. (15) (a) Bigi, A.; Boanini, E.; Panzavolta, S.; Roveri, N. Biomacromolecules 2000, 1, 752.(b) Lee, S. L.; Veis, A.; Glonek, T. Biochemistry 1977, 16, 2971. (c) Aizenberg, J.; Hanson, J.; Koetzle, T. F.; Weiner, S.; Addadi, L. J. Am. Chem. Soc. 1997, 119, 881. (d) Sharma, V. K.; Johnsson, M.; Sallis, J. D.; Nancollas, G. H. Langmuir 1992, 8, 676. (e) Kato, T.; Suzuki, T.; Amamiya, T.; Irie, T.; Komiyama, M.; Yui, H. Supramol. Sci. 1998, 5, 411. (f) Beniasha, E.; Simmerb, J. P.; Margolisa, H. C. J. Struct. Biol. 2005, 149, 182. (16) (a) Addadi, L.; Moradian, J.; Shai, E.; Maroudas, N.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2732. (b) Albeck, S.; Aizenberg, J.; Addadi, L.; Weiner, S. J. Am. Chem. Soc. 1993, 115, 11691. (c) Rautaray, D.; Ahmed, A.; Sastry, M. J. Am. Chem. Soc. 2003, 125, 14656. (17) (a) Shenton, W.; Pum, D.; Sleytr, U. B.; Mann, S. Nature 1997, 389, 585. (b) Vaucher, S.; Dujardin, E.; Lebeau, B.; Hall, S. R.; Mann, S. Chem. Mater. 2001, 13, 4408. (c) Zaremba, C. M.; Belcher, A. M.; Fritz, M.; Li, Y.; Mann, S.; Hansma, P. K.; Morse, D. E.; Speck, J. S.; Stucky, G. D. Chem. Mater. 1996, 8, 679. (18) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: Oxford, England, 1989.

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ecules usually inhibits the nucleation and growth of calcium salts in aqueous solution, whereas they promote calcification when adsorbed on a substrate.19 Several synthetic macromolecules and polyelectrolytes are also known to influence the nucleation and growth of calcium salts, resembling the role that biological macromolecules play in vivo.15 Therefore, development of experimental processes for the synthesis of biocompatible surfaces that would induce and support mineral growth is important not only from a fundamental point of view but also in biomedical applications such as bone implants/grafting in bone surgery20 and manufacture of artificial tissues.21 The biocompatibility and osteoconductive properties of HAP crystals make it desirable as an implant material and drug delivery agent. In implant applications, the ability to control the morphology and size of hydroxyapatite crystals is important since it enables tailoring the mechanical properties of the implant. Recently, the use of well-ordered two-dimensional structures such as those provided by a self-assembled film on solid substrates such as Langmuir monolayers22 and lamellar phosphonate precursors23 as nucleating templates for growing hydroxyapatite crystals have been investigated. The biological/biomimetic templates for growing hydroxyapatites are predominantly two-dimensional (2D) and, therefore, do not provide significant control over crystal size/morphology. In addition to 2D templates, different types of threedimensional (3D) templates such as polymer scaffolds,24a biocompatible hydrogel scaffolds,24b polyelectrolyte capsules,25a and aluminum oxide25b have also been used for growing hydroxyapatite crystals. An exciting possibility is to use nanoparticles as templates for the growth of minerals, and for such an application, gold nanoparticles have emerged as the scaffold of choice.26 Development of a large number of recipes for synthesizing gold particles over a range of sizes and the ease with which surface modification can be achieved have contributed to this bias. The chemistry related to surface modification of gold nanoparticles is well understood, and in addition to the popular thiol-based ligand binding strategies,27 gold nanoparticles are being increasingly bioconjugated with amino acids,28 proteins,29 and DNA.30 Amino acids, with their gold nanoparticle binding capability and wide spectrum of chemical properties should be excellent (19) Heuer, A. H.; Fink, D. J.; Laraia, V. J.; Arias, J. L.; Calvert, P. D.; Kendal, K.; Messing, G. L.; Blackwell, J.; Rieke, P. C.; Thompson, D. H.; Wheeler, A. P.; Veis, A.; Caplan, A. I. Science 1992, 255, 1098. (20) Schnettler, R.; Alt, V.; Dingeldein, E.; Pfefferle, H. J.; Kilian, O.; Meyer, C.; Heiss, C.; Wenisch, S. Biomaterials 2003, 24, 4603. (21) Dolder, J. V. D.; Farber, E.; Spauwen, P. H. M.; Jansen, J. A. Biomaterials 2003, 24, 1745. (22) Zhang, L.-J.; Liu, H.-G.; Feng, X.-S.; Zhang, R.-J.; Zhang, L.; Mu, Y.-D.; Hao, J.-C.; Qian, D.-J.; Lou, Y.-F. Langmuir 2004, 20, 2243. (23) Milev, A. S.; Kannangara, G. S. K.; Wilson, M. A. Langmuir 2004, 20, 1888. (24) (a) Song, J.; Malathong, V.; Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 3366. (b) Song, J.; Saiz, E.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 1236. (25) (a) Shchukin, D. G.; Sukhorukov, G. B.; Mohwald, H. Chem. Mater. 2003, 15, 3947. (b) Zhang, Y.; Zhou, L.; Li, D.; Xue, N.; Xu, X.; Li, J. Chem. Phys. Lett. 2003, 376, 493. (26) (a) Lee, I.; Han, S. W.; Choi, H. J.; Kim, K. Adv. Mater. 2001, 13, 1617. (b) Lee, I.; Han, S. W.; Lee, S. J.; Choi, H. J.; Kim, K. Adv. Mater. 2002, 14, 1640. (27) (a) Templeton, A. C.; Cliffel, D. E.; Murray R. W. J. Am. Chem. Soc. 1999, 121, 7081. (b) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66. (c) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 3944. (28) (a) Selvakannan, PR.; Mandal, S.; Phadtare, S.; Gole, A.; Pasricha, R.; Adyanthaya, S.; Sastry, M. J. Colloid Interface Sci. 2004, 269, 97. (b) Selvakannan, PR.; Mandal, S.; Phadtare, S.; Pasricha, R.; Sastry, M. Langmuir 2003, 19, 3545. (c) Mandal, S.; Selvakannan, PR.; Phadtare, S.; Pasricha, R.; Sastry, M. Proc. Indian Acad. Sci. (Chem. Sci.) 2002, 114, 513. (d) Zhong, Z.; Patskovskyvy, S.; Bouvrette, P.; Loung, J. H. T.; Gedanken, A. J. Phys. Chem. B 2004, 108, 4046.

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candidates to impart crystal-growth-inducing functionality to gold nanoparticles. In this communication we report a new synthetic approach to the fabrication of composite materials via controlling crystal growth with amino acidprotected gold nanoparticles as a template. Recognizing that carboxylic acid derivatization of gold nanoparticles for complexation with Ca2+ ions is the first step in HAP growth, we have chosen aspartic acid as the ligand for complexation. We observe that amino acid-protected gold nanoparticles as a template resulted in the formation of HAP platelets self-assembled into quasi-spherical structures. Presented below are details of this investigation. Experimental Details In a typical experiment, 100 mL of 10-4 M aqueous solution of chloroauric acid (HAuCl4) was reduced by 0.01 g of sodium borohydride (NaBH4) at room temperature to yield colloidal gold particles (step 1 in Scheme 1). This procedure results in a ruby red solution (pH 8.5) containing gold nanoparticles of diameter 6.5 ( 1 nm. The colloidal gold particles were capped by addition of 10 mL of an aqueous solution of 10-3 M aspartic acid to 90 mL of the gold hydrosol, and then the solution was allowed to age for 12 h (step 2 in Scheme 1). Uncoordinated aspartic acid in solution was removed by thoroughly dialyzing the aspartic acidcapped gold nanoparticle solution against distilled water for 2 days, using a 12 kDa cutoff dialyzing bag. The dialyzed solution was extremely stable over time. To 18 mL of the dialyzed aspartic acid-capped gold nanoparticle solution, 2 mL of 10-2 M aqueous solution of CaCl2 was added under continuous stirring for 10 min and then allowed to age for 1 h (step 3 in Scheme 1). The solution changed color from ruby red to blue indicating slight aggregation of gold nanoparticles. Even though aggregation of the gold nanoparticles was indicated, the blue solution was exceptionally stable over a period of several weeks. Isothermal titration calorimetric (ITC) measurements were carried out to characterize the interaction between aspartic acid-capped gold nanoparticles and calcium ions in a MicroCal VP-ITC instrument at 300 K. The calorimeter consists of two cells: a reference cell filled with pure solvent (water) and a sample titration cell filled with 1.47 mL of aspartic acid-capped gold nanoparticle solution. 5 × 10-3 M aqueous solution of CaCl2 was added from the syringe into the titration cell containing the aspartic acid-capped gold nanoparticles solution in small steps of 10 µL. The heat evolved/ absorbed during reaction of calcium ions with aspartic acid bound on the gold nanoparticles surface was measured, the time between successive injections of aqueous solution of CaCl2 being 2 min. UV-vis spectroscopy measurements of the aspartic acidcapped gold nanoparticle solution at different stages of treatment (steps 1, 2, and 3) were carried out on a Jasco UV-vis spectrophotometer (V570 UV-vis-NIR) operated at a resolution of 2 nm. After step 3, to 18 mL of Ca2+-aspartic acid-capped gold nanoparticle solution, 2 mL of 10-2 M aqueous solution of diammonium hydrogen orthophosphate (NH4)2HPO4 was added (step 4), and as the reaction proceeded, an insoluble precipitate was formed. The overall reaction took place within 1 h and removal of the solvent (water) by rotovapping resulted in a dry, gray powder of calcium phosphate crystals. To remove the nonreacted Ca2+ and PO43- ions, the powder was repeatedly washed with distilled water and then redispersed in deionized water. Samples were prepared for Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy dispersive analysis of X-rays (EDX) measurements by drop-coating films of the redispersed powder on Si(111) wafers. FTIR measurements were carried out on a Perkin-Elmer Spectrum One FTIR spectrometer operated at a resolution of 4 (29) (a) Gole, A.; Dash, C.; Ramakrishnan, V.; Sainkar, S. R.; Mandale, A. B.; Rao, M.; Sastry, M. Langmuir 2001, 17, 1674. (b) Gole, A.; Dash, C.; Soman, C.; Sainkar, S. R.; Rao, M.; Sastry, M. Bioconjugugate Chem. 2001, 12, 684. (c) Gole, A.; Vyas, S.; Phadtare, S.; Lachke, A.; Sastry, M. Colloids Surf., B 2002, 25, 129. (d) Zhao, J.; O’Daly, J. P.; Henkens, R. W.; Stonehuerner, J.; Crumblis, A. L. Biosens. Bioelectron. 1996, 11, 493. (e) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1999, 102, 9404. (30) Park, S.; Taton, T. A.; Mirkin, C. A. Science 2001, 295, 1503.

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Scheme 1. Cartoon Showing the Different Steps Involved in the Growth of Calcium Phosphate Crystals on Aspartic Acid Capped Gold Nanoparticlesa

a Step 1, functionalization of Au nanoparticles with aspartic acid; step 2, addition of CaCl2 solution to the aspartic acid-functionalized gold nanoparticles; step 3, formation of hydroxyapatite crystals after reaction with (NH4)2HPO4 solution.

cm-1 while SEM measurements were carried out on a Leica Stereoscan-440 scanning electron microscope equipped with a Phoenix EDX attachment. EDX spectra were recorded in the spot-profile mode by focusing the electron beam onto specific regions of the film. Samples for TEM analysis were prepared by drop-coating films of the redispersed powder in deionized water on carbon-coated copper transmission electron microscopy (TEM) grids, allowing the grid to stand for 2 min following which the extra solution was removed using a blotting paper. TEM analysis was performed on a JEOL model 1200EX instrument operated at an accelerating voltage at 120 kV. X-ray diffraction (XRD) analysis of drop-coated films of the redispersed powder in deionized water on glass substrates was carried out on a Phillips PW 1830 instrument operating at a voltage of 40 kV and a current of 30 mA with Cu KR radiation. The templating action of aspartic acid-capped gold nanoparticles on HAP formation was evaluated in various control experiments. In control experiment 1, Ca2+ ions were added to the borohydride reduced gold nanoparticle solution without surface modification. In control experiment 2, Ca2+ ions were added to the aspartic acid solution without gold nanoparticles. The two solutions were aged for 1 h followed by the addition of (NH4)2HPO4 solution, under the conditions described earlier. As the reaction proceeded, insoluble precipitates were formed and the powders were collected after rotavapping. In control experiment 3, (NH4)2HPO4 was added to the dialyzed aspartic acidcapped gold nanoparticle solution and then allowed to age for 1 h followed by addition of CaCl2 solution under the conditions described earlier. As the reaction proceeded, insoluble precipitates were formed and the powders were collected. After thorough washing with the deionized water, the resulting powders were characterized by FTIR, XRD, SEM, and EDX measurements.

Results and Discussion Figure 1A shows the UV-vis spectra of the aspartic acid-capped gold hydrosol at different stages of preparation. Curve 1 in the figure corresponds to the spectrum

Figure 1. (A) UV-vis spectra recorded from: curve 1, borohydride reduced gold nanoparticles; curve 2, aspartic acidcapped gold nanoparticles; curve 3, after addition of 10-2 M aqueous CaCl2 into the aspartic acid-capped gold nanoparticle solution (text for details). (B) Isothermal titration calorimetric data recorded during successive injections of 10 µL aqueous CaCl2 solution into the titration cell containing 1.47 mL of aqueous aspartic acid-capped gold nanoparticle solution. (C) Shows the binding isotherm obtained by integration of the raw data shown in Figure 1B plotted as a function of total volume of aspartic acid solution injected.

of gold colloidal solution obtained by borohydride reduction of aqueous chloroauric acid, curve 2 is the spectrum of gold colloidal solution after capping with aspartic acid, and curve 3 is the spectrum of aspartic acid capped gold colloidal solution after adding CaCl2 solution. A strong

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absorption in curve 1 at ca. 520 nm is observed that corresponds to excitation of surface plasmon vibrations in the gold nanoparticles.31 When the gold nanoparticles are capped with aspartic acid, a slight broadening and red shift of the surface plasmon band are observed (curve 2) indicating some aggregation of the gold nanoparticles consequent to surface modification. However, the aspartic acid-capped gold nanoparticle solution was stable for months with little evidence of further aggregation. After addition of CaCl2 solution to the aspartic acid capped gold nanoparticle solution, the absorption spectrum changes significantly with the appearance of an additional absorption band centered at ca. 620 nm (curve 3). This is a strong signature of aggregation of the particles into open structures and arises due to excitation of longitudinal (inplane) plasmon vibrations from the aggregates. It is known that amine groups bind to gold nanoparticles,32 and in the case of aspartic acid, this leads to the presence of free carboxylic acid groups on the gold surface. It is clear that Ca2+ ions bind strongly to the exposed -COO- groups present in the aspartic acid (solution pH 8.5, pI of aspartic acid ∼2.7) and that this process leads to screening of the repulsive electrostatic interaction between the negatively charged aspartic acid-capped gold nanoparticles and, therefore, to aggregation of the particles. Before addition of CaCl2 solution to aspartic acid-capped gold nanoparticle solution, the nanoparticles are stable due to electrostatic repulsion between negatively charged aspartic acid-capped gold nanoparticles. Isothermal titration calorimetric (ITC) measurements were performed to estimate the strength of the interaction between Ca2+ ions and aspartic acid bound to the surface of gold nanoparticles. ITC is an extremely powerful thermodynamic technique that has been used with much success in understanding biomolecular binding processes.33-37 However, this technique is yet to be used to study interactions between inorganic reactants and, to our knowledge, this study is one of the first in this direction. Figure 1B shows the ITC titration data recorded during injection of 10 µL of aqueous CaCl2 (5 × 10-3 M) from a syringe into 1.47 mL of aspartic acid-capped gold nanoparticle solution taken in the sample cell. It is seen from the figure that the reaction is endothermic, where each peak is the result of a single injection. The endothermicity of the first few injections is roughly the same since, during the initial stages of reaction between Ca2+ and gold nanoparticle bound aspartic acid, a number of binding sites (free carboxylate ions) are available for complexation. As the injections progress, the number of binding sites decreases continuously until saturation occurs and is mirrored as a monotonic fall in the endothermic calorimetric response (Figure 1B). Interestingly, a second maximum in the calorimetric response is observed (arrow, Figure 1B) and is interpreted below. The plot shown in Figure 1C is the binding isotherm determined from the raw data, where the total heat per injection (in kilocalories per mole of CaCl2 injected) is plotted against the total volume of CaCl2 solution added to the cell. We note here that in reactions where the concentrations of the reactants are known, the calorimetric data are normally plotted (31) Link, S.; Wang, Z. I.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 3529. (32) Kumar, A.; Mandal, S.; Selvakannan, PR.; Pasricha, R.; Mandale, A. B.; Sastry, M. Langmuir 2003, 19, 6277. (33) Jelesarov, I.; Bosshard, H. R. J. Mol. Recognit. 1999, 12, 3. (34) Dam, T. K.; Roy, R.; Page, D.; Brewer, C. F. Biochemistry 2002, 41, 1359. (35) Oda, M. J. Mol. Biol. 1998, 276, 775. (36) Wenk, M. R.; Seelig, M. J. Biochemistry 1998, 37, 3909. (37) Pierce, M. M.; Raman, C. S.; Nall, B. T. Methods 1999, 19, 213.

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against the molar ratio of the reactants. In this study, the concentration of aspartic acid is not known accurately due to potential errors in estimating the total gold nanoparticle surface area, area occupied by surface-bound aspartic acid molecules, etc. We have used the calorimetric response merely to characterize the nature of reaction and have refrained from deriving thermodynamic parameters from the binding isotherms due to the above errors. The fact that the calorimetric response during the entire titration cycle of Ca2+ ions against aspartic acid-capped gold nanoparticles is endothermic (which opposes the reaction) implies that the reaction is an entropically driven one. The first monotonic decrease in the binding isotherm (Figure 1C) is attributed to Ca2+ binding with the gold nanoparticle bound-aspartic acid where neutralization of the negative charge of the carboxylate ions in aspartic acid occurs. The second enthalpic maximum (Figure 1B,C) is attributed to aggregation of the aspartic acid-capped gold nanoparticles as a consequence of neutralization of the negative surface charge by the Ca2+ ions. Bloomfield and co-workers have observed a similar two-step enthalpic binding isotherm in their ITC study of DNA binding and condensation with the trivalent cations cobalt hexamine and spermidine.38 The essential features of the binding isotherms could be understood in terms of a purely electrostatic model wherein the first stage of reaction consisted of Coulombic complexation of the cobalt hexamine/spermidine cations with the DNA leading to neutralization of the negative charge on DNA followed by DNA condensation.38 The aggregation of aspartic acidcapped gold nanoparticles in the presence of Ca2+ ions inferred from the ITC binding isotherms (arrow, Figure 1C) is in good agreement with the UV-vis data (curve 3, Figure 1A). FTIR spectra recorded from calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles, in the presence of uncapped gold nanoparticles and pure aspartic acid, showed the presence of absorption bands centered at 1115, 1010, and 960 cm-1 and are assigned to ν3 and ν1 stretching modes of PO43-, respectively39 (Supporting Information, S1). In all cases, an additional band at 602 cm-1 was also observed which arises due to excitation of the ν4 mode of PO43- groups. While the FTIR results indicate the presence of phosphate groups in the powder, further evidence is required to establish the formation of HAP. The X-ray diffraction pattern recorded from a dropcoated film of the calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles is shown as curve 1 in Figure 2A. The peaks at 32.8°, 33.1°, 47°, and 53° 2θ values (indicated by an asterisk) are consistent with the (112), (300), (132), and (004) Bragg reflections of hydroxyapatite, respectively.40 Curves 2, 3, and 4 in Figure 2A correspond to XRD patterns recorded from the powders obtained in solutions containing uncapped borohydride reduced gold nanoparticles (curve 2), pure aspartic acid (curve 3), and calcium phosphate grown on aspartic acidcapped gold nanoparticles by addition of (NH4)2HPO4 followed by CaCl2. Bragg reflections characteristic of HAP are not seen in the powders obtained in all above control experiments (curves 2, 3, and 4) (please note that the peak (38) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Mol. Biol. 2000, 296, 1053. (39) Ngankam, P. A.; Lavalle, P.; Voegel, J. C.; Szyk, L.; Decher, G.; Schaaf, P.; Cuisinier, F. J. G. J. Am. Chem. Soc. 2000, 122, 8998. (40) (a) Tarasevich, B. J.; Chusuei, C. C.; Allara, D. L. J. Phys. Chem. B 2003, 107, 10367. (b) Rodriguez- Lorenzo, L. M.; Vallet-Regi, M. Chem. Mater. 2000, 12, 2460.

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Figure 2. (A) XRD patterns recorded from drop-coated films on glass substrates of calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles (curve 1), on uncapped borohydride reduced gold nanoparticles (curve 2), in pure aspartic acid solution (curve 3), and in control experiment 3 (curve 4) (see text for details). (B) EDAX profiles recorded from calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles (curve 1) and on uncapped borohydride reduced gold nanoparticles (curve 2) and pure aspartic acid (curve 3).

at 44° arises from the sample holder of the instrument). The Bragg reflections in curve 3 (calcium phosphate crystals obtained using pure aspartic acid) show peaks that are characteristic of the other form of calcium phosphate (tricalcium phosphate). XRD patterns recorded from the calcium phosphate crystals grown on aspartic acid-modified gold (curve 1), uncapped gold nanoparticles (curve 2), and in control experiment 3 (curve 4), also show the presence of a strong diffraction peak at a 2θ value of 38.2° and is assigned to the (111) Bragg reflection from the gold nanoparticles in the calcium phosphate-nanogold composites. It is clear from the XRD data that HAP crystallization is promoted by the aspartic acid monolayer present on the surface of the gold nanoparticles. This correlates well with the nature of crystallites observed in the microscopy analysis. Figure 2B shows the spot-profile EDX spectra from specific regions of the calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles (curve 1), on the uncapped borohydride reduced gold nanoparticles (curve 2), and in the presence of pure aspartic acid (curve 3). The presence of strong Ca and P signals is clearly observed in all the EDX spectra indicating the formation of calcium phosphate. The Au signal from the gold nanoparticles seen clearly in curves 1 and 2 is absent as expected in curve 3 where HAP growth was attempted with pure aspartic acid. A quantitative analysis of the HAP crystals was carried out from the Ca and P signals in the EDX spectra after ZAF correction and yielded values of Ca/P ratios of 1.7:1, 1.5:1, and 1.5:1 for the aspartic acid capped nanogold, uncapped nanogold, and pure aspartic acid samples, respectively. These values are in good agreement with the expected stoichiometry based on the chemical structure of HAP [Ca10(PO4)6(OH)2]. Parts A and B of Figure 3 show representative SEM images recorded at different magnifications from a dropcoated film of HAP crystals grown on aspartic acid-capped gold nanoparticles as a template. The images show the presence of quasi-spherical structures of sizes ranging from 1 to 5 µm with significant surface texture that are in aggregated. At higher magnification (Figure 3B) the texture of the spherical particles is seen more clearly to be composed of thin platelets in a highly compact configuration. The HAP platelets appear to self-assemble into spheroidal structures. Such types of assemblies have been observed by Colfen and Antonietti during solution growth of CaCO3 in the presence of suitable double hydrophilic block copolymers (DHBCs).41a-c It was observed that these

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double-hydrophilic block copolymers, due to their strong interaction with inorganic surfaces, resulted in the formation of spherical assembly of platelike calcite crystals. Spheroidal fluorapatite with prismatic needles have been observed by Kniep et al.41d in flourapatitegelatin composites where growth of fluorapatite starts with formation of elongated prismatic seeds followed by self-similar branching into anisotropic spherical aggregates, this process being mediated by local electric fields. Very recently, Sastry and co-workers have shown the formation of flat, platelike calcite crystals and their hierarchical assemble into uniform spherical aggregates by hydrophobic association, in an aqueous foam stabilized by the surfactant Aerosol OT.41e The above assembly processes mostly rely on hydrophobicity-driven association of individual crystallites into spheroidal structures. In this study, there are no HAP crystal bound surfactant molecules to assist in hydrophobic association of the HAP crystallites. As briefly discussed earlier, the ITC data indicate that consequent to addition of Ca2+ ions, the aspartic acid-capped gold nanoparticles aggregate into larger superstructures, which then on reaction with (NH4)2HPO4 act as templates for the growth of calcium hydroxyapatite crystals. We believe the regular, quasispherical HAP platelet structures formed are due to crystal growth on the gold nanoparticle aggregates rather than individual aspartic acid-capped gold nanoparticles. The process of surface modification of gold nanoparticles with aspartic acid (step 1), aggregation induced by calcium ions (step 2), and subsequent reaction with (NH4)2HPO4 to yield HAP crystal assemblies (step 3) is illustrated in Scheme 1. The aspartic acid molecules bind with gold nanoparticles through the amine groups,28c thereby leaving the carboxylic acid groups free to complex with Ca2+ions, as shown in the schematic. Parts C and D of Figure 3 show representative SEM images recorded from a drop-coated film of the calcium phosphate grown in the presence of uncapped gold nanoparticles and pure aspartic acid, respectively. The overall morphology of calcium phosphate in the control experiments is quite different from the highly regular, spherical assemblies observed with aspartic acid-capped gold nanoparticles as a template. In the case of HAP growth in the presence of uncapped gold nanoparticles, the calcium phosphate precipitated in the form of irregular structures (Figure 3C) while aspartic acid induces growth of highly irregular crystals with no particular morphology (Figure 3D). As shown by the XRD data, in these two control experiments, the crystalline HAP phase was not nucleated. It is clear from the above that aspartic acid bound to the surface of gold nanoparticles plays an important role in promoting the growth of crystalline HAP with a well-defined spherical morphology. To strengthen this aspect, another control experiment was performed wherein (NH4)2HPO4 was added into the aspartic acidcapped gold nanoparticle solution followed by addition of CaCl2 solution (control experiment 3). Parts E and F of Figure 3 show the SEM images at different magnifications from a drop-coated film of the calcium phosphate precipitate obtained from the control experiment 3. From the SEM images it is clearly seen that no specific morphology was obtained from the calcium phosphate precipitates. (41) (a) Colfen, H.; Antonietti, M. Langmuir 1998, 14, 582. (b) Yu, S. H.; Colfen, H.; Hartmann, J.; Antonietti, M. Adv. Funct. Mater. 2002, 12, 541. (c) Yu, S. H.; Colfen, H.; Antonietti, M. J. Phys. Chem. B 2003, 107, 7396. (d) Busch, S.; Dolhaine, H.; Duchesne, A.; Heinz, S.; Hochrein, O.; Laeri, F.; Podebrad, O.; Vietze, U.; Weiland, T.; Kniep, R. Eur. J. Inorg. Chem. 1999, 1643. (e) Rautaray, D.; Sinha, K.; Adyanthaya, S. D.; Sastry, M. Chem. Mater. 2004, 16, 1356.

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Figure 3. (A) and (B) Representative SEM images recorded at different magnifications from drop-cast films of calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles. (C) and (D) Representative SEM images from drop-cast films of calcium phosphate grown in the presence of uncapped borohydride reduced gold nanoparticles and pure aspartic acid, respectively. (E) and (F) Representative SEM images recorded at different magnifications from drop-cast films of calcium phosphate grown in the presence of aspartic acid-capped gold nanoparticles by adding (NH4)2HPO4 followed by CaCl2 (see text for details).

The XRD pattern (curve 4 in Figure 2A) shows that the calcium phosphate (Figure 3E,F) is amorphous in nature. In control experiment 3, addition of (NH4)2HPO4 solution to aspartic acid-capped gold nanoparticle solution resulted in the aggregation of aspartic acid functionalized gold nanoparticles due to presence of NH4+ ions from the (NH4)2HPO4 salt. However (HPO4)2- ions would be free in the solution phase which then reacts with Ca2+ ions after addition of CaCl2 solutionsthis results in the formation of a calcium phosphate precipitate which settles down together with the aggregated gold nanoparticles. Separation of the calcium phosphate precipitate from the aggregated gold nanoparticles by washing is difficult, and for this reason, the XRD pattern of the calcium phosphate precipitate shows the diffraction peak of gold (curve 4 in Figure 2A). In this control experiment, calcium phosphate precipitate forms separately in solution without involving

the crystalline gold nanoparticles, whereas in our main experiment formation of HAP crystals involves the crystalline gold nanoparticles through electrostatic binding with the Ca2+ ions (Scheme 1). From all the control experiments, it is clear that the assembly of aspartic acidcapped gold nanoparticles due to Ca2+ ions play the crucial role in the formation of HAP crystals. From the ITC measurements, it is inferred that addition of Ca2+ ions to the aspartic acid-capped gold nanoparticle solution leads to aggregation of the nanoparticles. Reaction of these aggregates with (NH4)2HPO4 results in the formation of HAP crystals in a spherical aggregated structure. Thus, the spherical morphology of the HAP crystals assemblies is not due to crystal growth on individual nanoparticles but more likely to be due to crystal nucleation and growth on the Ca2+ ion-mediated assemblies of aspartic acid-capped gold nanoparticles.

Inorganic Crystals Grown on Nanoparticles

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Figure 4. (A) and (B) Representative TEM micrographs recorded at different magnifications from drop-cast films of calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles. The inset in A shows the selected area electron diffraction pattern recorded from the crystals shown in the main part of the figure.

Figure 4 shows representative transmission electron microscopy (TEM) images recorded at different magnifications from the calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles. At low magnification, the quasi-spherical morphology of the crystallite assemblies is clearly seen as the individual platelets (Figure 4A). At higher magnification, the calcium phosphate crystals are observed in much greater detail (Figure 4B). The individual platelets themselves are not very smooth and present an irregular, corrugated surface. Gafni and co-workers have previously observed a similar morphology of HAP crystals in their study of the effect of chondroitin sulfate and biglycan on the crystallization of hydroxyapatite under physiological conditions.42 The inset of Figure 4A shows the selected area electron diffraction (SAED) pattern of the calcium phosphate crystals shown in Figure 4A. The diffraction rings have been indexed in the figure based on the hydroxyapatite form structure of the crystals.40 The hydroxyapatite crystals were grown on the aspartic acid-capped gold nanoparticle aggregates which act as nucleation centers. From the SEM (Figure 3B) and TEM images (Figure 4A), it is observed that the HAP crystals completely cover the underlying gold nanoparticles and grow outward from this core. In the SAED analysis, the electron beam was focused along the edges of the quasi-spherical platelike hydroxyapatite crystals, and thus it is possible that the gold nanoparticle density is considerably smaller at these edge regions. It is also possible that due to the thickness of the hydroxyapatite crystallites, the electron beam penetration is not sufficient to sense the underlying gold nanoparticles. In any case, the XRD (curve 1 in Figure 2A) and EDAX results (curve 1 in Figure 2B) clearly indicate the presence of gold nanoparticles in the HAP crystallites. Taken together with the control experiments, these results underline the important role played by the aspartic acid monolayer capping the gold nanoparticles in directing the nucleation (42) Gafni, G.; Septier, D.; Goldberg, M. J. Cryst. Growth 1999, 205, 618.

and growth of the HAP crystallites. The various steps involved in the formation of HAP crystals on the aspartic acid capped-gold nanoparticles have been discussed earlier (Scheme 1). In conclusion, we have described the crystallization of HAP in the presence of aspartic acid-capped gold nanoparticles. Calcium phosphate crystal growth was achieved by binding Ca2+ ions to the carboxylate ions of the nanogold surface-bound aspartic acid molecules followed by reaction with (NH4)2HPO4. The morphology of the calcium phosphate crystals formed was found to be a strong function of the nanogold surface modifier. Platelike HAP crystals were observed to form in well-defined spherical assemblies. On the basis of the ITC and UV-vis spectroscopy measurements, it is believed that addition of Ca2+ ions to the aspartic acid-capped gold nanoparticle solution results in aggregation of the nanoparticles and that it is these aggregates that serve as a template for the growth of HAP crystals. These nanometal-organic-inorganic hybrid structures are expected to play an important role not only in crystal engineering but in areas such as catalysis and novel optical materials as well. Acknowledgment. D.R. and S.M. thank the Department of Science and Technology (DST) and the University Grants Commission (UGC), Government of India for financial assistance, respectively. We thank Mr. Pravin S. Shirude, Organic Chemistry (Synthesis) Division for assistance with the ITC measurements. Supporting Information Available: FTIR spectra recorded from drop-coated films on Si(111) wafers of calcium phosphate crystals grown on aspartic acid-capped gold nanoparticles (curve 1) and calcium phosphate crystals grown on borohydride reduced gold nanoparticles (curve 2) and in the presence of pure aspartic acid (curve 3). This material is available free of charge via the Internet at http://pubs.acs.org. LA048541F