Fabrication of Phospholipid Vesicle-Interacted Calcium Phosphate

Jul 25, 2017 - The morphologies and physicochemical properties of the films depended on the film formation process as well as the mineralizing time. I...
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Fabrication of Phospholipid Vesicle-Interacted Calcium Phosphate Films with Sterilization Stability Yadong Chai, Tadashi Yamaguchi, and Motohiro Tagaya* Department of Materials Science and Technology, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan S Supporting Information *

ABSTRACT: The development of a calcium phosphate (CP) coating technique on tissue culture poly(styrene) (TCPS) is important in biomedical fields. In this study, the CP hybridized with L-α-phosphatidylcholine phospholipid vesicle (PV) was formed on TCPS as the film state for suggesting the unique CP/PV hybrid films. The film formation was conducted by the following two different processes: (i) the CP deposited on PV was prepared in a simulated body fluid (SBF) and subsequently casted on TCPS to form the films, and (ii) the CP was precipitated in SBF on the pre-prepared PV films on TCPS. These films were denoted as “CP/PV-Bef” and “CP/PV-Aft” films. The stability of the CP/PV hybrid films against the sterilization processes (ethanol, UV/ozone, and autoclave treatments) and subsequent immersion process in a phosphate buffer saline (PBS) was also demonstrated. As a result, the CP/PV hybrid films were successfully coated on TCPS through the mediation by PV with preserving the vesicle structure. The morphologies and physicochemical properties of the films depended on the film formation process as well as the mineralizing time. It is presumed that two functional groups of cationic choline and negative phosphate in the phosphatidylcholine molecule can act as the CP nucleation sites in SBF and the subsequent CP crystal growth would occur along the PV surfaces. Furthermore, it was confirmed that amorphous calcium phosphate and calcium pyrophosphate dihydrate were mainly formed in the initial mineralization stage and were eventually converted to the hydroxyapatite phase. The conversion was accelerated by the autoclave treatment. Therefore, these CP/PV films have transparency, unique structures, and good stability against sterilization treatments, suggesting the application for cell culture plates.

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INTRODUCTION

Based on the synthetic inorganic−organic interactions, the selfassembly formation of chemical entities has been increasingly formed on the basis of many biomineralization processes.15−20 The self-assembly structures of the surfactants with hydrophobic and hydrophilic parts have been applied for preparing the hierarchical inorganic/organic hybrid systems with the interesting properties,21−24 and the biofunctions.25−27 In the calcium phosphate (CP) nanostructures, the various shapes such as particles,28,29 and films,30,31 and their biofunctions32 have been investigated by our group. However, the fabrication process and investigation of the assembly structures on the

The biomineralization processes almost invariably generate species-specific and intricate hierarchical structures.1−3 It is worth noting that these are related to supramolecular selfassembly processes.4−6 The spatial topological structures effectively enhance the cell adhesion and subsequent activities.7,8 Thus, the biomimetic structure formation based on supramolecular assemblies by virtue of the mineralization process has been attracting much attention.9−12 The bioceramic structures have proven to be biologically important.8 For instance, the structured bioceramics such as calcium phosphate have enhanced biological performance, exhibiting promising orthopedic and dental implant formulations.13,14 The supramolecular-interacting technique using a structure-directing agent (SDA) can hybridize with the various inorganic phases to form the unique hierarchical structures. © XXXX American Chemical Society

Received: June 30, 2017 Revised: July 21, 2017 Published: July 25, 2017 A

DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Preparation of Simulated Body Fluid. SBF containing 1.5 times ion concentrations (1.5SBF) was prepared. 1.5SBF (Na+, 213 mM; K+, 7.5 mM; Mg2+, 2.5 mM; Ca2+, 3.8 mM; Cl−, 222 mM; HCO3−, 6.3 mM; HPO42−, 1.5 mM; SO42−, 0.75 mM; Tris, 75 mM) was prepared according to a previous report,41 and the pH was adjusted to 7.4 with HCl. 75 mM of Tris buffer was prepared and adjusted to be the pH of 7.4 by HCl. Fabrication of CP/PV-Bef Films Based on Process 1. According to the previous reports on the PV preparation,42−44 the PV sedimentation was successfully dispersed into ultrapure water at the concentration of 1 mg/mL. 1 mg of the PV sedimentation was dispersed in 6 mL of 1.5SBF. The liquid was slowly stirred for 5 and 10 days at 37.5 °C, which were denoted as CP/PV-Bef-5d and CP/PVBef-10d particles, and then the sedimentary particles were washed and lyophilized by a freeze-dryer (EYELA FDU-1200) to give the CP/PVBef powders. Then, the powders were dispersed in 50 mL of ultrapure water (pH = 7.4), and 0.1 mL of each liquid was cast on silicon (Si), glass, and carbon/Formvar film coated copper grids (Okenshoji Co., Ltd.), TCPS surfaces at the particle concentrations of 0.03 mL/cm2, and the cast CP/PV-Bef-5d and CP/PV-Bef-10d films were lyophilized by the freeze-dryer for 1 day. Before the cast, the substrates were treated with a UV/ozone (ASM401N, Azumi GIGKEN Limited.) exposure. Fabrication of CP/PV-Aft Films Based on Process 2. A 0.3 mL aliquot of the liquid containing PV was cast on the substrates mentioned above at the particle concentrations of 0.03 mL/cm2. Then, the PV film was lyophilized by the freeze-dryer for 1 day. Before the cast, the substrate surfaces were treated by the UV/ozone exposure. The film was statically immersed into 3.0 mL of 1.5SBF for 5 and 10 days at 37.5 °C, and the films were washed with ultrapure water (pH = 7.4) and lyophilized to give the CP/PV-Aft-5d and CP/PV-Aft-10d films. Sterilization Processes and Subsequent Immersion into PBS. First, the films were cleaned by the UV/ozone exposure. Subsequently, the films were treated by an autoclave unit under 105 °C for 10 min, and were immersed into 3.0 mL of 50 vol % and 70 vol % of ethanol in water for 1 h. After all the sterilization, the films were denoted as CP/PV-Aft-5d/St and CP/PV-Aft-10d/St. Characterization. The particle size distribution was evaluated by a dynamic light scattering (DLS, SALD-7000, Shimazu, Japan) according to our previous report.29 The average particle size (Ave.) and coefficient of variation (Cv.) were calculated. The QCM-D (D300, Q-Sense AB) in situ measurements were performed at 37.5 °C for 1 h by detecting Δf and ΔD at 25 MHz in the QCM-D cell chamber having a volume of 100 μL. The solvent is the ultrapure water adjusted up to pH = 7.4 by a sodium salt. The measured Δf was divided by the harmonic overtone (n = 5) as the fundamental frequency of 5 MHz. The viscoelastic property of the adlayer was expressed as the saturated ΔD/Δf value from the ΔD−Δf plots according to our previous reports.30,31 The surface structures were observed by an atomic force microscope (AFM: Nanocute, SII Investments, Inc.). The surface roughness (Rrms) was calculated by the root mean squares of the height images. Transmission electron microscope (TEM) images were taken with a JEM-1400 (JEOL Co., Ltd.) at an accelerating voltage of 120 kV. The transmittances of the films on TCPS were measured on a UV−vis absorption spectroscopy (V-630, JASCO, Japan). The averaged transmittance values between 400 and 800 nm (Tv) were calculated. The elemental compositions were characterized by an X-ray fluorescence analysis (XRF: ZSX Primus II, Rigaku, Japan). Fourier transform infrared (FT-IR) spectra were obtained using an FTIR spectrometer (FT/IR-4100, JASCO, Japan). The spectra were measured with the films on the Si substrates, and recorded after subtracting a background spectrum of a pristine Si substrate. Thin film X-ray diffraction (XRD) patterns were recorded with a θ−2θ scanning out-of-plane X-ray diffraction (XRD) meter (Smart Lab, Rigaku, Japan) equipped with monochromatic CuKα radiation operated at 200 mA and 45 kV. The 2θ−ω oblique-incidence was recorded at the incident angle of X-ray in the geometry set at ω = 0.5°. The crystallite size (D) of the precipitated calcium phosphate was calculated with Scherrer’s equation (K = 0.9) based on full width at half-maxima

polymeric substrate such as poly(styrene) have not been achieved. Dental and orthopedic metallic implant surfaces have recently been modified by phospholipids, suggesting that the use of phospholipids can induce various kind of calcium interactive processes.33−36 The role of phospholipids has also been demonstrated in the viewpoint of the initiation of CP formation,37−40 and the phospholipid-coated bioceramics were fairly biocompatible.29 Thus, it is postulated that a nonbiocompatible surface such as a tissue culture poly(styrene) (TCPS) dish coated with phospholipids exhibited calciumphospholipid-phosphate complexes toward biomedical applications. However, the chemical and structural characteristics and sterilization stability of the CP-coated film on TCPS through the mediation by phospholipid have not been investigated. In this study, the CP hybridized with an L-α-phosphatidylcholine phospholipid vesicle (PV), which has two functional groups of cationic choline and negative phosphate which can attract the CP nucleation, was formed on TCPS as the film state for suggesting the unique CP/PV hybrid films in the biomedical fields. As shown in Scheme 1, the formation of the Scheme 1. Illustration of the Preparation Processes of CP/ PV-Bef and CP/PV-Aft Films, and Their Sterilization and PBS Immersion Processes

CP/PV hybrid films on TCPS was conducted by two different processes: (i) the CP deposited on the PV was prepared in a biological solution and subsequently casted on TCPS to form the films [Process 1], and (ii) the CP was precipitated on the pre-prepared PV films on TCPS in the biological solution [Process 2]. We also demonstrated the stability test of the CP/ PV hybrid films in sterilization processes (ethanol, UV/ozone, and autoclave treatments) and a subsequent immersion process in a phosphate buffer saline (PBS).

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EXPERIMENTAL SECTION

Materials. L-α-Phosphatidylcholine (see the Supporting Information, Scheme S1), tris(hydroxymethyl)aminomethane (Tris CNH2(CH2OH)3), chloroform, sodium chloride (NaCl), potassium chloride (KCl, dipotassium hydrogen phosphate (K2HPO4·3H2O), magnesium chloride (MgCl2·6H2O, calcium chloride (CaCl2), sodium sulfate (Na3SO4), hydrochloric acid (HCl, 1 N), and ethanol (99.5 vol %) as special grade chemicals were purchased from Wako Chemical Co., Ltd. Sodium hydrogen carbonate (NaHCO3) as a special grade chemical was supplied by Nacalai Tesque, Inc. The phosphate buffered saline (PBS) tablet was from DS Pharma Biomedical Co., Ltd. The oxidized poly(styrene) sensor (QSX-305) with the polymeric film thickness of 40 nm was purchased from Q-sense, Inc. The BD Falcon easy-grip cell culture dish (ϕ 35 mm) was purchased from Life Sciences Co., Ltd. The silicon(100) substrate was purchased from Ryoko Sngyo Co., Ltd. B

DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Figure 1. (a) TEM image of CP/PV-Bef-10d particles and (b) particle size distribution, and (c) AFM topographical image of the PV film on TCPS and (d) particle size distribution. (fwhm) of 300 and 002 reflections at 2θ = 32.80 and 25.92, respectively. The film surface morphologies were observed using a field emission scanning electron microscope (FE-SEM: SU8000, Hitachi High-Technologies, Japan) at an accelerating voltage of 5 kV and current of 10 μA. The calcium ion (Ca2+) concentration was measured on a D-73LAB equipped with the electrode (6583-10C, Horiba, Japan). The standard Ca2+ solution (conc.: 1000 mg/L) was prepared from CaCl2, and KCl (support salt) solution (conc.: 0.1 M) was prepared. Then, 100, 10, and 1.0 mg/L of the Ca2+ solution with admixture of the KCl solution was used for setting the calibration curve. The dissolved Ca2+ ratios from the sterilized hybrid films (CP/ PV-Aft-5d/St and CP/PV-Aft-10d/St films) were investigated by the immersion in PBS for 72 h under static condition. The dissolved Ca2+ concentration was measured on a D-73LAB equipped with the electrode (6583-10C, Horiba, Co., Ltd., Japan). The standard Ca2+ solution was prepared by the mixture of ultrapure water containing 1000 mg/L of CaCl2·2H2O and 0.1 M of KCl (support salt). 100, 10, and 1.0 mg/L of the Ca2+ solution with KCl (0.1 M) was also used for the calibration curve. The initial Ca2+ weight contained in the precipitated HAp films was calculated using the XRF results, and the dissolution Ca2+ weight was divided by the initial Ca2+ weight.

63%) by the DLS measurement (Figure 1b), indicating the crystal growth on the PV surfaces. In contrast, the AFM topographical image of the PV film on TCPS and particle size distribution are shown in Figure 1c,d. The Rrms value of the film surface was 3.39 nm, and the vesicle-like shapes were preserved without spreading out by the particulate film formation (Figure 1c). Moreover, the Ave. value of the PV at film state on TCPS was 87 nm (Cv.: 16%) (Figure 1d). The particle size of the PV at film state was significantly smaller than that dispersed in ultrapure water. In this case, the strain occurred in the inner and outer vesicle structures during the water removal because of the shrinkage,46 indicating that the vesicle shapes were elliptically deformed at the freeze-dry process. In this study, the PV−substrate and PV−PV interactions during the lyophilization would be generated to form the closely packed states of the PV particles. The PV adsorption process onto TCPS in water was measured by QCM-D, which are shown in the Supporting Information (Figure S1). The Δf, ΔD, and saturated ΔD−Δf values were hardly changed,47 which was different from the case onto a hydrophilic silica, indicating that there was no specific interactions between poly(styrene) and PV in the ultrapure water. Therefore, it was found that the PV films were performed on the TCPS while maintaining the vesicle-like shapes. Figure 2a shows the photographs of the films prepared on TCPS. As observed, all of the films (PV, CP/PV-Bef-5d, CP/ PV-Aft-5d, CP/PV-Bef-10d, CP/PV-Aft-10d) formed on the TCPS surface were transparent. The Tv values were 82, 84, 67, and 74% for CP/PV-Bef-5d, CP/PV-Aft-5d, CP/PV-Bef-10d, and CP/PV-Aft-10d, respectively, which were lower than those of 89% and 86% of TCPS and PV (Figure 2b), suggesting that the hybrid films still have the transparency even if immersed in

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RESULTS AND DISCUSSION The particle size distribution of the PV dispersed in ultrapure water (pH = 7.4) in this study exhibited the Ave. value of 235 nm (Cv.: 63%) by the DLS measurement, indicating the larger particle size as compared that dispersed in 1.5SBF.29 The TEM image of the CP/PV-Bef-10d particles and particle size distribution are shown in Figure 1a,b. In the CP/PV-Bef-5d and CP/PV-Bef-10d particles, the spherical shapes consist of some platelike nanocrystals,45 suggesting that the platelike crystals grew on the PV surfaces to form the spheres with the size of 150−300 nm (Figure 1 a). The particle size distribution of CP/PV-Bef-10d exhibited the Ave. value of 250 nm (Cv.: C

DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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due to the PO43− stretching mode at 1090 and 1030 cm−1 were observed for the hybrid films, in which the bands increased with the increase in the immersion time, indicating the CP crystal growth.48 Furthermore, the band at 1240 cm−1 in the hybrid films suggests the bending mode in O3PO−H···O−PO3. It indicated the phosphate ions coordinated to the phosphate group of PV. The bands at 875, 1415, and 1460 cm−1 can be ascribed to carbonate ions, which would be in the adsorption or substitution state with PO43− of CP. The XRD patterns of the CP/PV films are shown in Figure 3b, in which the diffraction pattern of CP/PV-Bef-5d film indicated the amorphous calcium phosphate (ACP) and that of CP/PV-Bef-10d film was attributed to hydroxyapatite (HAp) with low crystallinity, suggesting transformation of ACP to HAp by the immersion time. In the CP/PV-Aft films, there were the mixture phases of calcium pyrophosphate (CPPD) and HAp. Moreover, the crystallite sizes (D200) of CP/PV-Aft5d and CP/PV-Aft-10d films were 1.9 and 7.1 nm, respectively, indicating that the crystal growth effectively occurred along with the a-axis direction by the immersion. Therefore, the ACP and subsequent HAp were formed in the CP/PV-Bef and the both CPPD and HAp were formed in the CP/PV-Aft. The AFM topographical images of the TCPS and CP/PV-Aft films are shown in the Supporting Information (Figure S2). The Rrms values of the TCPS surface with immersion in 1.5SBF for 0, 5, and 10 days were 1.1, 1.2, and 1.2 nm, respectively, and the fine particles were observed and were just sporadically distributed on the surface. It seems that these particles caused by the homogeneous nucleation in 1.5SBF were attached on the surface. In other words, it is difficult to directly form CP on TCPS by the immersion. The Rrms values of CP/PV-Aft-5d and CP/PV-Aft-10d films were 10 and 41 nm, respectively, suggesting that the CP crystal grew and subsequently covered the TCPS surface through the mediation of the PV film. The morphologies of the CP/PV-Bef and CP/PV-Aft films were observed by FE-SEM as shown in Figure 4. In the CP/PVBef films, the primary particles were aggregated at the several micrometers. The poor smoothness of the film was attributed to the aggregated CP/PV-Bef particles when coated on TCPS, indicating the irregular shapes due to the aggregation as shown in the possible scheme in Figure 4e. In the CP/PV-Aft films, the platelike crystals were gathered together to form the petalshaped crystals, which were similar to the HAp shape in vivo, suggesting the possible scheme in Figure 4f. In the following, the CP/PV-Aft films with the higher crystallinity and more homogeneous morphologies were investigated in detail. The FT-IR spectra and the XRD patterns of the CP/PV-Aft5d and CP/PV-Aft-10d film with the continuous different sterilization treatments are shown in Figure 5. There was no change in the absorption bands at 1090 and 1030 cm−1 derived from the phosphate groups in the remained CP, suggesting the stability of the CP/PV films. The intensity of the diffraction peak 100 at 2θ = 15° attributed to the CPPD significantly decreased, and the diffraction peak 101 at 2θ = 17° attributed to the HAp increased in CP/PV-Aft-5d, suggesting that the mixed phases of CPPD and HAp were converted to a single phase of HAp by the sterilization treatments. The Hap crystalline phase of the CP/PV-Aft-10d film was not changed with the sterilization treatments. The crystallite sizes of the CP/ PV-Aft-5d and CP/PV-Aft-10d films are shown in Table 1. In the CP/PV-Aft-5d film, the crystallite size of D100 due to HAp increased with the sterilization treatments, indicating that the crystal growth specifically occurs along with the a-axis direction.

Figure 2. (a) Photographs of the films prepared on TCPS, and (b) Ca/P molar ratio and Tv changes of the CP/PV films with the immersion time in 1.5SBF.

1.5SBF. The Ca/P molar ratio changes of the CP/PV films increased with the increase in the immersion time in 1.5 SBF. These Ca/P ratios of the hybrid films measured by the XRF measurement were lower than those of the stoichiometric CP, because the mineralized CP contained the ions such as Na and Mg, which would be substituted with calcium ion, and the P atoms in PV were additionally detected. Concretely, the P element in PV film was 0.59 mol %, and the P and Ca elements and the other metals (Na, Mg, K) were 0.46, 0.98, and 8.08 mol % for CP/PV-Bef-5d, 0.66, 1.62, and 1.12 mol % for CP/PVBef-10d, 1.20, 2.91, and 2.82 mol % for CP/PV-Aft-5d, and 1.27, 2.45, and 4.89 mol % for CP/PV-Aft-10d. The FT-IR spectra of the PV and CP/PV films (Figure 3a) exhibit the characteristic absorption bands attributable to

Figure 3. (a) FT-IR spectra and (b) XRD patterns of the PV and CP/ PV films. The XRD patterns of HAp (closed squares, JCPDS 00-0090432) and CPPD (closed triangles, JCPDS 01-075-2756) are marked.

phospholipids and phosphate groups. The stretching and bending vibration modes due to −CH2− and −CH3 appeared at 2850, 2900 and 1420, 1490 cm−1, respectively, and the stretching vibration mode due to CO group appeared at 1740 cm−1 in the PV and CP/PV films, suggesting the existence of fatty acids residues included in CP. The characteristic bands D

DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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In the CP/PV-Aft-10d film, the crystallite size gradually increased. Therefore, the stability against the sterilization treatments was confirmed and the crystallinity and phase of the CP/PV-Aft-5d film was significantly changed. The investigation into the film preparation and sterilization processes is shown in Scheme 2, suggesting the possible initial Scheme 2. Investigation into the Film Preparation and Sterilization Processes of This Study, Suggesting the Initial Ion Adsorption, Subsequent Nucleation/Growth, and Phase Transition

Figure 4. FE-SEM images of (a-1, a-2) CP/PV-Bef-5d, (b-1, b-2) CP/ PV-Bef-10d, (c-1, c-2) CP/PV-Aft-5d, and (d-1, d-2) CP/PV-Bef-10d at (a-1, b-1, c-1, d-1) low and (b-1, b-2, c-2, d-2) high magnifications, and their possible film illustrations of (e) CP/PV-Bef and (f) CP/PVAft films.

ion adsorption, subsequent nucleation/growth, and crystalline phase transition behaviors. From the above results, since the isoelectric point of the zwitterion-type PV molecule is 5.5, the cationic quaternary ammonium ion of the choline group would be charge-compensated by OH− in 1.5SBF (pH = 7.4) to be negatively charged at the phosphate group. Thus, the Ca2+ ion in 1.5SBF preferentially reacts to the negatively charged phosphate group of PV to generate the CP nucleation. Then, the crystal growth occurred while incorporating Ca2+, H2PO4−, HPO42, and CO32− around the nucleus. The CP/PV-Bef films indicated that the ACP cluster in 1.5 SBF was formed and covered on the PV surfaces, and subsequently converted to the HAp phase. Since the PV particles were dispersed in 1.5 SBF, it was presumed that the ACP was formed as a precursor. In the precipitation process of the CP/PV-Aft films, the CP phases (CPPD, HAp) were formed in 1.5SBF on the pre-prepared PV films on TCPS, indicating that the nucleation took place in the gap between PV particles to induce the effective crystal growth, resulting in the smooth CP formed on the PV films. The nucleation was carried out in the gap to induce the crystal growth occurred while including the phosphate groups of the PV molecules, so that the lower Ca/P ratio as well as the precursor CPPD formation was interestingly observed. Furthermore, the phase transition to HAp and stability in the sterilization processes were achieved in the CP/PV-Aft films.

Figure 5. (a, b) FT-IR spectra and (c, d) XRD patterns of (a) CP/PVAft-5d and (b) CP/PV-Aft-10d films with the continuous different sterilization treatments. The plane indices were attributed to HAp single phase (JCPDS 00-009-0432).

Table 1. Crystalline Sizes of the CP/PV-Aft-5d and CP/PV-Aft-10d Films with the Continuous Three Different Sterilization Treatments CP/PV-Aft-5d

CP/PV-Aft-10d

treatment

D100 (nm)

D002 (nm)

D211 (nm)

D002/D100

D100 (nm)

D002 (nm)

D211 (nm)

D002/D100

none UV irradiation autoclave 70 vol % ethanol

1.27 5.11 29.6 27.1

7.32 15.6 13.9 4.08

16.8 16.2 23.4 9.68

5.76 3.05 0.47 0.15

1.48 1.05 1.83 1.78

13.3 9.41 13.1 12.4

4.95 3.96 4.94 4.22

8.99 8.96 7.16 6.97

E

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treatment. Therefore, these CP/PV films have transparency, unique structures, and good stability against sterilization treatments, suggesting the application for novel cell culture plates.

The dissolution rate of Ca2+ ion from the sterilized CP/PVAft/St films with the immersion time in PBS is shown in Figure 6. In the first 6 h, 17 wt % of the initial Ca2+ in the CP/PV-Aft-

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CONCLUSIONS The CP hybridized with PV was successfully formed on TCPS as the film. The CP/PV hybrid film formation was conducted by the following two different processes. The stability of the films against the sterilization processes and subsequent immersion process in PBS were also demonstrated. As a result, the CP/PV hybrid films were successfully coated on TCPS through the mediation by PV with preserving the vesicle structure. The morphologies and physicochemical properties of the films depended on the film formation process as well as the mineralizing time. We suggested that two functional groups of cationic choline and negative phosphate in the phosphatidylcholine molecule can act as the CP nucleation sites in SBF, and the subsequent CP crystal growth would occur along the PV surfaces. Furthermore, the CP phases of ACP and CPPD were mainly formed in the initial mineralization stage and were eventually converted to a HAp phase. The conversion was accelerated by the autoclave treatment. Therefore, these CP/ PV films have transparency, unique structures, and good stability against sterilization treatments, suggesting the application for novel cell culture plates.

Figure 6. Dissolution rate of Ca2+ from the ion in the sterilized CP/PV films (●: CP/PV-Aft-5d/St, ○: CP/PV-Aft-10d/St) with the immersion in PBS for 72 h.

5d/St film was dramatically dissociated and then the dissolution rate slightly increased up to 18−19 wt % from the HAp phase at the immersion time between 24 and 72 h. In the CP/PV-Aft10d/St film, the dissolution rate of Ca2+ gradually increased from 1.2 to 6.8 wt % for 72 h. The dissolution rate of the CP/ PV-Aft-10d/St film was significantly lower than that of the CP/ PV-Aft-5d film, indicating the good stability of the CP/PV-Aft10d/St film in PBS for 72 h. The FT-IR spectra and XRD patterns of the CP/PV-Aft-5d/ St and CP/PV-Aft-10d/St films with the immersion treatment in PBS are shown in the Supporting Information (Figure S3a− d). There was no change in the absorption bands at 1090 and 1030 cm−1 derived from the phosphate groups of the HAp remaining in the both CP/PV-Aft-5d/St and CP/PV-Aft-10d/ St films. Combining with the crystalline size results of CP/PVAft-5d/St and CP/PV-Aft-10d/St films with the different immersion times (Supporting Information, Table S1), the slight increase and decrease in the crystallite size, it indicates that the dissolution and recrystallization at the surface repeatedly occurred. Therefore, the stability in the both CP/ PV-Aft-5d/St and CP/PV-Aft-10d/St by the immersion in PBS was confirmed. In this study, the CP/PV hybrid films were successfully coated on TCPS through the mediation by PV with preserving the vesicle structure. The morphologies and physicochemical properties of the films depended on the film formation process as well as the mineralizing time. It is presumed that two functional groups of cationic choline and negative phosphate in the phosphatidylcholine molecule (i.e., zwitterion-type) can act as the CP nucleation sites in SBF and the subsequent CP crystal growth would occur along the PV surface structures. The biological apatite formation on the films containing C, O, N, and S atoms has been invesitgated.49−51 The negatively charged −COOH and −OH groups efficiently induce the heterogeneous nucleation.52−55 Especially, the −PO4H2 group exhibits the effective nucleation induction.56 Thus, the SBF successfully gave the mild reaction process as well as the preserved vesicle structures in this study. Furthermore, it was confirmed that ACP and CPPD were mainly formed in the initial mineralization stage and were eventually converted to HAp phase. The conversion was accelerated by the autoclave

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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.7b00918. Molecular structure of L-α-phosphatidylcholine used in this study. QCM-D analyses of the PV adsorption process onto TCPS in water. AFM topographical images of the TCPS with the different immersion in 1.5 SBF as compared with those of the CP/PV-Aft-5d and CP/PVAft-10d surfaces. FT-IR spectra and XRD patterns of the CP/PV-Aft-5d/St and CP/PV-Aft-10d/St films with the different immersion times in PBS. Crystalline sizes of CP/PV-Aft-5d and CP/PV-Aft-10d films with the different immersion times (PDF)

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AUTHOR INFORMATION

Corresponding Author

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

Motohiro Tagaya: 0000-0003-3695-7253 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant-in-Aid for Young Scientists (A), Grant No. 17H04954, and Challenging Research (Exploratory), Grant No. 17K19027). The authors F

DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

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thank the Analysis and Instrumentation Center in Nagaoka University of Technology for providing the facilities.

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DOI: 10.1021/acs.cgd.7b00918 Cryst. Growth Des. XXXX, XXX, XXX−XXX