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Jan 19, 2016 - Mesoporous silica formation on hydroxyapatite nanoparticles. Shota Yamada , Masami Nishikawa , Motohiro Tagaya. Materials Letters 2018 ...
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Effect of Cationic Surfactant Micelles on Hydroxyapatite Nanocrystal Formation: An Investigation into the Inorganic−Organic Interfacial Interactions Kota Shiba,†,‡ Satoshi Motozuka,§ Tadashi Yamaguchi,∥ Nobuhiro Ogawa,⊥ Yuichi Otsuka,# Kiyoshi Ohnuma,∇ Takuya Kataoka,∥ and Motohiro Tagaya*,∥,8 †

International Center for Young Scientists (ICYS) and ‡World Premier International Research Center Initiative (WPI), International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan § Department of Mechanical Engineering, Gifu National College of Technology, 2236-2 Kamimakuwa, Motosu, Gifu 501-0495, Japan ∥ Department of Materials Science and Technology, #Department of System Safety, ∇Department of Bioengineering, and 8Top Runner Incubation Center for Academica-Industry Fusion, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan ⊥ Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan S Supporting Information *

ABSTRACT: To clarify hydroxyapatite (HAp) nanocrystal formation based on the interfacial interactions with organic molecules is important for controlling the dispersion states/ shapes of HAp and understanding the biomineralization mechanism. In this study, the effects of cetyltrimethylammonium bromide (CTAB) micelles on the HAp nanocrystal formation process were investigated through both the morphosynthesis technique and the molecular orbital calculations. The HAp nanocrystals synthesized in the presence of CTAB exhibited a controlled rod-like shape and subsequently grew up by a thermal treatment to be larger-sized nanocrystals at a well-dispersed state. It was also found that the CTAB micelles strained and disordered at the higher temperature effectively induced heterogeneous nucleation to initiate the nucleation/growth processes. In contrast, HAp nanocrystals synthesized without CTAB exhibited irregular-shaped and aggregated nanocrystals, which are due to dominantly occurring homogeneous nucleation. According to the molecular orbital calculations, the cationic N atom of the CTAB molecule strongly interacted with the Ca ion in the a-plane as well as the hydroxyl/phosphate groups in the c-plane of HAp via ionic/covalent bonding (e.g., only ionic bonding at the closer interfacial distance at 1.0 Å), leading to effective nucleation on the micelle surfaces. Therefore, a possible reason for the rod-like and well-dispersed nanocrystal formation is due to the heterogeneous nuclei formation/growth on the N atoms of CTAB micelles and subsequent fusion growth among the CTAB micelle-directed inorganic−organic complexes in the confined spaces. The present results will be applicable for designing tailored HAp morphologies based on inorganic−organic hybrid interaction systems.

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INTRODUCTION

precise synthesis and biomedical applications have been developed.7−9 An important point to be considered when we use the hybrids is to investigate the HAp cluster formation under the presense of functional organic molecules such as lipids, proteins, and cells consisting of tissues and the hybrid interfaces. In other words, HAp forms gradually charged crystal a- and c-planes on the organic groups at the nanoscale.10 Then, the resultant shapes of the crystals should be controlled to have effective interaction with the organic molecules that contain a

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) has attracted much attention because of their potential uses in biomedical fields such as bone tissue engineering, implantation, and drug delivery.1−4 One of the reasons why HAp is frequently utilized for such applications is due to its chemical composition similar to that of human hard tissues, and the highly biocompatible surface properties can be effectively enhanced by inorganic/ organic hybrid interfaces.5,6 In addition, the degradation products of HAp are non-cytotoxic compared to those of other materials utilized in biomedical studies,6 and the physicochemical properties are also controlled by the hybrid states. The hybrid functions thus accelerate research, and © XXXX American Chemical Society

Received: November 11, 2015 Revised: December 29, 2015

A

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CTAB solution under magnetic stirring at 40 °C. Then, the pH of the solution was adjusted to 12 using 1 N NaOH. After that, 60 mL of deionized water containing 4.44 g of CaCl2 (40 mmol) was added to the CTAB/K2HPO4 aqueous solution at 6 mL/min with a peristaltic pump (Masterflex, Cole-Parmer Instrument Co., Ltd.). The initial molar ratio of CTAB:PO43− and Ca/P ratios were set at 2:1 and 1.67, respectively. As the reference, the synthesis at the initial molar ratio of CTAB:PO43− = 0:1 (without adding CTAB) and 1:1 was conducted. The precursor solution was then refluxed at 40 or 120 °C for 24 h. The solution was centrifuged (10000g, 15 min, 4 °C) to sediment solid product and washed with ultrapure water three times. The washed product was dried in an oven at 100 °C for 24 h. Finally, the dried powder was calcined in a furnace under air at 550 °C for 6 h. The resulting samples are named as CTAB/HApX-Y, where X is “40” or “120” (to mean “the reflux temperature”) and Y is “Bef” or “Aft” (to mean “before” or “after” the calcination). The HAp nanocrystals synthesized without adding CTAB are named as HApX-Y. Characterization. Infrared (IR) spectra were recorded with an IRPrestige-21 (Shimadzu Co., Ltd.) through a KBr pellet method. Xray diffraction (XRD) patterns were measured using a Smart Lab (Rigaku Co., Ltd.) equipped with monochromatic Cu Kα radiation operated at 20 mA and 40 kV. Crystallite sizes were calculated with the Scherrer’s equation (K = 0.9) based on full width at half maxima (fwhm) of 300 and 002 reflection at 2θ = 32.80 and 25.92, respectively. Transmission electron microscope (TEM) images were taken with a JEM-1400 (JEOL Co., Ltd.) at an accelerating voltage of 120 kV. The HAp nanocrystal ethanoic suspension was dropped onto a carbon/Formvar film coated copper grid (Okenshoji Co., Ltd.), and the grid was dried in a desiccator under nitrogen atmosphere for 24 h before the observation. N2 adsorption/desorption isotherms of all the samples were measured at −196 °C on a TriStar II (Simadzu Co., Ltd.). Prior to the measurements, the samples were dried at 100 °C under a vacuum for 3 h. Specific surface area (SBET) values were calculated by means of the Brunauer−Emmett−Teller (BET) method33 using a linear plot over the range of P/P0 0.05−0.30. Pore size distributions were derived from desorption isotherms by the Barrett−Joyner−Halenda (BJH) method.34 Molecular Orbital Calculations of the Interfacial Interactions between HAp and CTAB. On the basis of the experimental results of the HAp nanocrystal formation in the presence/absence of a CTAB molecule, the possible reaction mechanism between the cationic N atom of CTAB molecule and a calcium (Ca2+) ion on the a-plane and hydroxyl/phosphate groups on the c-plane of HAp was deduced using a discrete variational (DV)-Xα molecular orbital calculation method, which has been utilized as a powerful tool to calculate the electronic states between the specific molecules and local components of the crystalline lattice.35−43 The details of the present DV-Xα calculation procedure are described in the Supporting Information, Experimental Procedure S1. Briefly, the net charge of the N atom of CTAB, the Ca2+ ion in the a-plane and hydroxyl/phosphate groups in the c-plane of the HAp cluster model and the bond overlap population (BOP) between the above-mentioned components were calculated with approximating between the CTAB and HAp at the distance 0.5−2.5 Å. The possible bonding types can be estimated on the basis of these values. The molecular orbitals (MOs) obtained by the calculation method as also described in the Supporting Information are represented by the eq 1.

carboxylic acid group or amino group,11−17 leading to novel morphosynthetic techniques based on biomimetic interactions. Thus, interfacial phenomena occurring in the HAp formation process under the presence of various organic molecules, which contain the functional C, O, N, and H atoms, should be focused and investigated for the HAp/organic hybrid systems. The coexistence of the functional groups containing C, O, N, and S atoms in the HAp synthesis and formation has been reported, especially in the biological apatite formation system.12,18−20 HAp nucleation is effectively enhanced by various functional groups such as carboxyl, hydroxyl, and amine groups.21−24 In simulated biological fluids, it is known that negative surfaces such as PO4H2, COOH, and OH-terminated surfaces had a greater induction capability for the heterogeneous nucleation and growth, whereas nucleation did not occur on positive NH2 terminated surfaces.25 However, the investigation of the relationship between amine groups and HAp as well as the interactive natures (i.e., driving forces) has not been clear. In this paper, we report the coexistence effect of a cationic surfactant micelle of cetyltrimethylammonium bromide (CTAB), which has a quaternary ammonium cation, on HAp nanocrystal formation and morphology. Although the ammonium cation of the CTAB is very important for the clarification of the interactions and mineralization, the interfacial interactions have not been reported. The assembled CTAB molecules have been widely utilized to synthesize the surfactant-templated inorganic−organic hybrids based on the cationic N atom. Especially, the CTAB molecules are famous precursors for mesoporous silica−surfactant hybrids that are usually synthesized from the supramolecular assembly.26 Although the organic molecule-based approach has been applied to the preparation of nanosized HAp crystals for the in-growth of natural bones,10,27−32 the CTAB-based nanohybrid formation mechanism has been unclear. Thus, effects of additives such as CTAB and/or other chemicals containing cationic N atom on the size, shape, and crystallinity of HAp nanocrystals should be examined and investigated in detail. To newly design the biocompatible HAp-based nanohybrids depending on the purpose, further studies are required not only based on experiments but also based on theoretical calculation, making a guideline to obtain the nanosized HAp with optimized properties. Thus, we focused on the formation of the HAp/CTAB nanohybrid system and investigate how the N atom of CTAB micelles affects the HAp nucleation and subsequent crystal formation. Molecular orbital calculations were carried out to speculate the possible interfacial phenomena between the CTAB and HAp surface functional groups.

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EXPERIMENTAL SECTION

Materials. Cetyltrimethylammonium bromide ((C 16 H33 )N(CH 3 ) 3 Br, CTAB), potassium phosphate dibasic trihydrate (K2HPO4·3H2O, 99.0 wt %), and sodium hydroxide (NaOH) as special grade chemicals were purchased from Wako Chemical Co., Ltd. Calcium chloride (CaCl2, 95.0 wt %) as special grade chemical was purchased from Nacalai Tesque, Inc. All the reagents used in the present investigations were used without further purification. Synthesis of the Nanocrystals under the Existence of CTAB Micelles. Hydroxyapatite nanocrystals were prepared based on the previous reports.10,28,31 Typically, 17.49 g of CTAB (48 mmol) was added into a Teflon vessel containing 100 mL of deionized water under magnetic stirring for 60 min. After complete dissolution of CTAB, 5.48 g of K2HPO4·3H2O (24 mmol) was added to the aqueous

ϕl(rk) =

∑ Cilχi (rk) i

(1)

where χi(rk) denotes the atomic orbitals and rk is one of the sampling points in the calculation. Using eq 1, the self-consistent MO wave function, MO energy levels, and major orbital components were obtained by solving the secular equation in which the matrix elements of the Hamiltonian and overlap integrals are evaluated by the DV numerical integrations. The Mulliken population analysis was employed to obtain the overlap population (Qlij) and the orbital population (Qlj) at the lth MO,35,36,38 which are given by the eqs 2 and 3, respectively. B

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Q ijl = fl CilCjl ∑ ω(rk)χi (rk)χj (rk)

Q il =

incorporated inside the HAp nanocrystals. On the other hand, the absorption band at around 1400−1500 cm−1 is due to the following two states: the carbonate ions incorporated inside the HAp nanocrystals and adsorbed on the surface. The former absorption band was affected by the presence of CTAB, whereas the latter was not, implying that CTAB would promote the incorporation of the carbonate ions into the HAp crystal structure. Taking account of the fact that the biopolymer assemblies such as collagen fibers can promote mineralization process through the surface functional groups to effectively accumulate ions,53 the present case is regarded as a phenomenon with the CTAB-assisted ion accumulation process. Since the detailed mechanism is still complicated, further investigation is required. In any case, the preparation under the presence of CTAB molecules would change the charge compensation states in/on the HAp crystal structure to effectively include the dianionic HPO42− ion and CO32− ions. The XRD patterns (shown in Figure 2) reveal that all the present nanocrystals are attributed to single phase HAp

(2)

k

∑ Q ijl

(3)

j

where ω(rk) is the weight of the sampling point at rk, and f l is the number of electrons at the lth MO level. The sum of Qlj on atom A becomes the effective charge of atom A. Thus, the net charge of the atom (nA) is represented by eq 4. nA = ZA −

∑ ∑ Q il l

(4)

i

Qlij

between atom A and where ZA is the atomic number. The sum of atom B determines the BOP (QA−B) which denotes the strength of the covalent bonding and it can be represented as follows: Q A−B =

∑ ∑ Q ijl , i ∈ A , j ∈ B l

i,j

(5)

Using these values, the net charge and BOP at the interfaces between the CTAB and HAp were quantified.

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RESULTS AND DISCUSSION Effects of CTAB Micelles on the HAp Nanocrystal Formation. On the basis of the previous reports for the FT-IR spectroscopic studies,28,31,44,45 the HAp nanocrystal formation was verified as shown in Figure 1. Several absorption bands

Figure 2. XRD patterns of the HAp40 and CTAB/HAp40 nanocrystals before and after the calcination.

(JCPDS No. 9−-432). The calculated lattice constant values (a = b = 0.943−0.946 nm and c = 0.686−0.688 nm) agreed well with those of the reference HAp data having lattice parameters a ≈ b ≈ 0.9418 nm, c ≈ 0.6884 nm and space group P63/m.31,46 Interestingly, the diffraction intensity of 300 slightly increased after the calcination in all the samples. In contrast, the diffraction intensity of 002 became smaller. Accordingly, the crystalline size ratio (d002/d300) values of the products after calcination were much lower than those before calcination (see Table 1), and the d002/d300 values of HAp40 products were lower than those of CTAB/HAp products, being supported by TEM results described in the next paragraph. These results suggest that the HAp nanocrystals preferentially grew along with the a-axis of its crystal plane, and this trend was more apparent in the case of the products synthesized in the presence of CTAB molecules. This might be because the different formation mechanism dominated by heterogeneous nucleation and growth environment provided the presence/absence of the strained CTAB micelles to effectively enhance the larger crystal formation. To discuss the effects of CTAB on the resultant products, we first examined their shapes by a TEM observation. As shown in Figure 3, HAp40-Aft mainly consists of spherical nanocrystals with the size of 10−50 nm. These nanocrystals form submicron-sized aggregates. In the case of CTAB/HAp40-Aft, the

Figure 1. FT-IR spectra of the HAp40 and CTAB/HAp40 nanocrystals before and after the calcination.

appeared at around 1090−1030, 600, and 560 cm−1 are attributed to P−O stretching of phosphate groups. Especially, the weak shoulder band at 960 cm−1 resulted from ν1 symmetric P−O(H) stretching vibrations, which is attributed to the presence of HPO42− ions. The characteristic band was enhanced in the samples prepared under the presence of the CTAB molecules. Two strong bands at around 2930 and 2850 cm−1 are only seen in the case of CTAB/HAp40-Bef because these bands originate from CH3− and −CH2− of the CTAB. Judging from the fact that no such absorption is seen in the spectrum of CTAB/HAp40-Aft, the CTAB molecules were removed by the calcination. A broad absorption at around 3400 cm−1 is due to the presence of OH groups on HAp and adsorbed water molecules. Carbonate ion (CO32−) bands were recognized at 870 cm−1 and 1400−1500 cm−1, which would come from the atmospheric carbon dioxide and would have then incorporated into the crystal structure. The absorption band at around 870 cm−1 is assigned to the carbonate ions C

DOI: 10.1021/acs.cgd.5b01599 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Table 1. Lattice Constant and Crystalline Sizes Calculated from the XRD Patterns of the Synthesized Nanocrystals, and the SBET and Pore Sizes Calculated from the Nitrogen Adsorption Isotherms lattice constant HAp40-Bef CTAB/HAp40-Bef HAp40-Aft CTAB/HAp40-Aft CTAB/HAp120-Aft

crystalline size

nitrogen adsorption/desorption

a (Å)

c (Å)

d300 (nm)

d002 (nm)

d002/d300

SBET (m2/g)

pore sizes (nm)

9.44 9.43 9.46 9.45 9.46

6.86 6.87 6.88 6.87 6.86

14.5 15.5 15.9 18.4 22.0

45.7 55.3 40.0 47.0 68.9

3.15 3.57 2.51 2.55 3.13

75.9 48.5 32.5 30.7 40.5

25−40 28−40 40−70 40−100 50−100

Figure 3. TEM images of the HAp40-Aft and CTAB/HAp40-Aft nanocrystals.

Figure 4. N2 adsorption/desorption isotherms and pore distributions of the HAp40 and CTAB/HAp40 nanocrystals before and after calcination.

nanocrystals with rod-like shape were significantly observed at the well-dispersed states. The size of the nanorods was approximately 20 nm in diameter and 50 nm in length. We also derived the crystallite size based on the XRD results using the Scherrer’s equation. As the Scherrer’s equation gives an averaged crystallite size, a single value is enough to be calculated based on a single diffraction in the case of spherical crystals. In the present case, however, the HAp nanocrystals are known to have an anisotropic shape as revealed by TEM. Thus, we calculated the crystallite sizes based on different crystal directions. The lattice constant, crystallite size, specific surface area, and pore size are summarized in Table 1. Since the crystallite size estimated from the 300 and 002 diffractions of XRD results (15.9 nm in diameter and 40 nm in length, respectively) corresponded well with that obtained by TEM, CTAB/HAp40-Aft should be composed of single crystals. As

compared to HAp40-Aft, the CTAB/HAp40-Aft seems to be well-dispersed and uniform in size, indicating that the CTAB micelle plays a significant role in the present nanocrystal formation in the solution process. CTAB molecule consists of two parts: a positively charged hydrophilic head with strong affinity for water molecules and a hydrophobic tail with less affinity for water molecules. When the surfactant concentration increases, the CTAB molecules form micelles through a self-assembly process with their hydrocarbon chains inside. The surface of micelles is positively charged. After adding phosphate solution, these anions (e.g., PO43−, HPO32−) are attracted to the micelle surfaces due to the strong electrostatic attraction. It was reported that both CTA+ and PO43− had the same tetrahedral structure, which make them stereochemically compatible to interact with each other.28 The CTA+−PO43− complex micelles act as a nucleation center. D

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Figure 5. TEM images (two different magnifications) of the CTAB/HAp120-Aft nanocrystals.

Figure 6. DV-Xα molecular orbital calculation of the interface between the N atom of CTAB and the atoms in the HAp cluster model surfaces.

Thus, when Ca2+ is added into the solution, it combines immediately with the surface of the complex micelles by the electrostatic attraction of PO43−. Therefore, the formation of the rod-like nanocrystals results from the crystallization of these complexes on the surface of the micelles, indicating the importance of CTAB concentration for the well-dispersed and uniform in sizes. Taking into account the aggregation form and insufficient shapes in the case of the CTAB/HAp40-Aft synthesized by the molar ratio of CTAB:PO43− = 1:1 in the

Supporting Information, Figure S1, we can suggest the ratio of CTAB:PO43− = 2:1 is the best for our purpose. CTAB is frequently utilized as the structure directing agent.26 Thus, we investigated the macroscopic structures confined among the HAp nanocrystals and the effect of micelles on the macroscopic shapes by means of N2 adsorption/desorption isotherms as shown in Figure 4. All the samples give typical type III adsorption isotherms with a slight H3 hysteresis loop as defined by IUPAC, 47 indicating the macropores and E

DOI: 10.1021/acs.cgd.5b01599 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Ca2+ ion in the a-plane of HAp. For the OH and phosphate groups generated on the c-plane, the OH group forms the ionic/covalent bonding, and the H−dissociated OH group forms only the ionic bonding. The P−OH group forms the ionic/covalent bonding, and PO43− ion forms only the ionic bonding. The net charge, BOP values, and possible bonding types are also summarized in Table 2 in the case that the

aggregation form by plate-like particles. In the samples before calcination as shown Table 1, SBET values of the HAp40-Bef and CTAB/HAp40-Bef nanocrystals were 75.9 m2/g and 48.5 m2/g, respectively, revealing that the present HAp nanocrystals possess macropores. These pores would come from the interstitial voids confined by each nanocrystal, and the larger nanocrystals were formed by the CTAB micelles. If the HAp nanocrystals were calcined, SBET values decreased to 32.5 m2/g for HAp40-Aft and 30.7 m 2/g for CTAB/HAp40-Aft, respectively. Corresponding pore size distributions became broader and peak values shifted to 40−100 nm. This could be reasonable since the HAp nanocrystals grew up to the larger sizes after the calcination. In any case, the CTAB micelle surfaces mainly worked as a shape controlling agent rather than a structure directing agent and there is no mesopores. Thermal Effects of CTAB Micelles on the HAp Nanocrystal Formation. We also investigated the enhanced thermal effects of the CTAB micelles on the HAp nanocrystal formation process. The CTAB/HAp nanocrystals (synthesized by the molar ratio of CTAB:PO43− = 2:1) were prepared through the same experimental procedure, whereas the reaction was conducted at 120 °C. An XRD pattern of CTAB/HAp120-Aft (synthesized by the molar ratio of CTAB:PO43− = 2:1) is shown in Figure S2a. We confirmed that all the diffraction peaks were attributed to single phase hydroxyapatite (JCPDS 9-432). Although crystallite size increased along with both 300 and 002 directions (Table 1), the growth in 002 direction was notable (d002/d300 value increased up to 3.13 from 2.55 of CTAB/HAp40-Aft). Figure 5 shows the TEM images of the resultant CTAB/HAp nanocrystals after the calcination (i.e., CTAB/HAp120-Aft). Similar to CTAB/HAp40-Aft, the CTAB/HAp120-Aft has a rod-like shape. As compared to CTAB/HAp40-Aft, CTAB/ HAp120-Aft seems to have grown much more. We preliminarily confirmed that the HAp nanocrystals did not grow up to the same size as CTAB/HAp120-Aft, indicating that the promoted growth is due to the presence of CTAB, not simply due to a higher temperature effect. The detailed mechanism will be reported elsewhere. The estimated crystallite sizes agree well with the values shown in Table 1. The magnified TEM image revealed that the crystal planes aligned periodically with the distance of approximately 0.64 nm, indicating the 001 observation. The shape of N2 adsorption/ desorption isotherms of CTAB/HAp120-Aft (Figure S2b) was almost the same as that of CTAB/HAp40-Aft shown in Figure 4, suggesting that there was no clear structural difference compared to CTAB/HAp40-Aft. Therefore, it is suggested that the CTAB micelles were strained and disordered at the higher temperature, and the resultant structurally changed CTA+ surfaces effectively induce the heterogeneous nucleation to efficiently cause the nucleation/growth process. Possible Formation Mechanism of the HAp Nanocrystals Interacted with CTAB Micelle Surfaces. Our hypothesis at the interfacial interactions mentioned above was theoretically verified by the DV-Xα molecular orbital calculations as shown in Figure 6. The initial interfacial interactions between the N atom in the focused trimethylammonium cation (TA+) structure of CTAB and all the surface atoms of HAp were calculated each other. As the surface atoms in the HAp formation process, Ca2+ ion, the H atom of OH, the O atom of H−dissociated OH, the H atom of P−OH, and the O atom of phosphate ion are very important. The N atom in the CTA+ structure could form only the ionic bonding with a

Table 2. DV-Xα Calculation Results with Approaching N Atom of CTA+ onto the HAp Cluster Model Surface Atoms at the Interfacial Distance of 1.0 Å net charge in the NX distance at 1.0 Å X for the bond between N atom of CTAB and surface X atom of HAp

N atom (CTAB)

X (HAp)

BOP

possible bonding

Ca ion OH group O of the H-dissociated OH group P−OH group

−0.69 −0.53 −0.57

+2.18 +0.45 +0.02

−0.07 −0.11 +0.34

ionic ionic covalent

−0.40

+1.37

+0.40

O of phosphate ion

+0.20

−0.87

−0.09

covalent ionic ionic

interfacial distance was much closer to be 1.0 Å. From the table, we can understand that the Ca2+ ion and phosphate groups dominantly form the interfacial ionic bonding with the CTA+ structure, while the O atom of H−dissociated OH and P−OH group do not seem to have the interfacial ionic bonding because of their suggested covalency. Therefore, it is possible to say that the heterogeneous nucleation, which comes from the CTA+−PO43−− Ca2+ complex, was induced by the N atoms in the strain-induced CTA+ micelle surfaces under the heating solution. Since the N atoms regularly align in the micelle structure surfaces, the possible ionic/covalent interactions between each HAp planes should constrain the growth direction, resulting in the successful formation of a rod-like shape. These results suggest that the N atom of the micelle can act as the mineralization medium. We have demonstrated the results of HAp nanocrystal syntheses in the presence/absence of CTAB. A possible formation mechanism of the HAp nanocrystals is shown in Scheme 1. As described, the HAp nanocrystals easily formed aggregation states in the absence of CTAB, whereas the CTAB micelle induced the formation of well-dispersed rod-like HAp nanocrystals. The growth of the rod-like nanocrystals was accelerated at 120 °C, resulting in the formation of longer rodlike nanocrystals. In the case of the HAp nanocrystal formation without the CTAB micelles, the homogeneous nucleation occurred and the resultant nanocrystals aggregated because of the absence of any stabilizer such as CTAB. In contrast, the HAp nanocrystals were formed by heterogeneous nucleation in the presence of CTAB since CTAB molecules could form a micellar structure in the present synthesis condition. The critical micelle concentration (CMC) of CTAB in aqueous solution was 0.9−1 mM.48 Above the CMC, a transition from spherical micelles to rod-like micelles occurred. Taking into account that CTAB molecules are dissociated into CTA+ and Br− in this system, the ammonium group in the CTA+ can work as a heterogeneous nucleation site.29,31,32,49 Accordingly, the phosphate ions are electrostatically interact with the CTA+ micelles to form CTA+−PO43− complex, and when calcium F

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Scheme 1. Illustration of the Possible Coexistence Effect of the Cationic Surfactant (CTAB) Micelles on the HAp Nanocrystal Formation Process

the presence of the CTAB micelles, whereas the aggregates composed of irregular-shaped HAp nanocrystals formed without the CTAB micelles. Furthermore, the growth of the rod-like nanocrystals was accelerated at the higher synthetic temperature, indicating the effective nucleation on the strained micelle surfaces. Thus, it is suggested that the cationic N atom of CTAB micelles worked as the nucleation center for forming the CTA+−PO43−−Ca2+ complex and subsequent crystalline formation. The well-dispersed and rod-like HAp nanocrystal shape was formed in the confined spaces by the sufficient CTAB micelles. The possible interactions between the N atom of a cationic CTAB molecule and each component of the HAp nanocrystals (such as Ca ion and hydroxyl/phosphate groups) were clearly estimated by the DV-Xα molecular orbital calculation. For the HAp formation process, the N atom of the CTAB molecule interacts with the Ca ion via ionic bond, whereas the phosphate groups effectively have both ionic and covalent interactions with the N atom, suggesting the efficient HAp nucleation on the CTAB micelles. Therefore, the interfacial interactions between the HAp nanocrystals and organic molecules containing N atom were experimentally and theoretically demonstrated to understand the HAp formation mechanism. This result will be applicable for the clarification of the biomineralization mechanism.

ions are added into the solution, Ca9(PO4)6 clusters are formed on the rod-like micelle surface as an intermediate state.50,51 Then, the nanocrystals would grow along with the micelles and also grow in a confined space surrounded by micelles, leading to the formation of rod-like shape. In the present study, water molecules interacting with CTA+ micelles at the surface would become much more active (i.e., hydrophobic interaction becomes weaker) to randomly move the molecules when reaction temperature increases. The surfactant molecularassembly structure (e.g., vesicle) then becomes unstable, forming a strain in its structure by the temperature which would be similar to that in the case of the reported vesicle system.52 To compensate the strain, the Ca2+ and/or PO43− ions in the synthesis system effectively interact with the CTA+ micelle surfaces. In other words, since the number of nuclei heterogeneously formed on the surface of the CTA+ micelles increases, the formation of the anisotropic structure becomes dominant as shown in the TEM images of this study. This is supported by the data shown in the Supporting Information, Figure S3, which were the CTAB/HAp120-After synthesized at the molar ratio of CTAB:PO43− = 1:1. In this case, however, a number of characteristic smaller spherical nanoparticles with the size of approximately 10 nm were clearly observed on the HAp nanocrystal surfaces (Figure S3, Supporting Information). Taking into account that there were no small nanoparticles in the magnified image of the CTAB/HAp120-Aft synthesized at the molar ratio of CTAB:PO43− = 2:1, we suggested that this should be due to lack of micelles, i.e., nucleation sites, inducing homogeneous nucleation. To be more precise, the small nanoparticles are formed on the surface of the micelles during synthesis. Although many small nanoparticles assemble to form the rod-like nanocrystals along with the shape of the micelles, the hydrocarbon tails of CTAB micelles trap some small nanoparticles inside the core. Accordingly, the calcination would release the organic surfactant molecules and the small nanoparticles in the core leave on the rod-like nanocrystals. Therefore, the shape of nanocrystals synthesized by the molar ratio of CTAB:PO43− = 2:1 was optimized to have the welldispersed state and uniform size. On the basis of the viewpoints of CTAB micelles reactions, it is suggested that the HAp nanocrystals do not fuse together during the calcination process because the CTA+ micelles effectively separate each HAp nanocrystal.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01599. TEM image of the CTAB/HAp40-Aft synthesized at the molar ratio of PO43−:CTAB = 1:1. XRD pattern, nitrogen adsorption/desorption isotherms and TEM images of the CTAB/HAp120-Aft nanocrystals synthesized at the molar ratio of PO43−:CTAB = 1:1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-258-47-9345. Fax: +81-258-47-9300. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid for Young Scientists (A) (Grant No. 26709052) from MEXT/JSPS KAKENHI and was partially supported by Izumi Science and Technology Foundation (Grant No. H26-J-028). Collaterally, this research was also supported by the World Premier

CONCLUSIONS The effects of the CTAB micelles on the HAp nanocrystal formation process were investigated based on the morphosynthesis technique and molecular orbital calculations. The rodlike and well-dispersed HAp nanocrystals successfully formed in G

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International Research Center Initiative on Materials Nanoarchitectonics (WPI-MANA) and was partially supported by a Grant-in-Aid for Young Scientists (B) (Grant No. 26870836) from MEXT/JSPS KAKENHI and Kazuchika Okura Memorial Foundation.

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DOI: 10.1021/acs.cgd.5b01599 Cryst. Growth Des. XXXX, XXX, XXX−XXX