Article pubs.acs.org/crystal
Alginate-Intervened Hydrothermal Synthesis of Hydroxyapatite Nanocrystals with Nanopores Yanming Wang,‡ Xiaoxiang Ren,† Xiaomin Ma,† Wen Su,† Yaping Zhang,§ Xiaosong Sun,*,‡ and Xudong Li*,† †
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, China College of Materials Science and Engineering, Sichuan University, Chengdu 610064, P. R. China § College of Materials Science and Technology, Southwest University of Science and Technology, Mianyang 621010, P. R. China ‡
ABSTRACT: This study reports the preparation of dispersive hydroxyapatite (HA) nanocrystals with nanopores based on hydrothermal reactions of Ca- and phosphate-containing aqueous solutions in the presence of alginate. Compared with the conventional hydrothermal HA nanocrystals, very fine rodlike HA nanocrystals were obtained; e.g., the average measures were 13 nm for diameter and 34 nm for length when the nanocrystals were hydrothermally synthesized in the presence of 1.6 wt % alginate. Systematic X-ray diffraction, Fourier transform infrared, thermogravimetric analysis, transmission electron microscopy (TEM) and high-resolutuion TEM, and Brunauer−Emmer−Teller results indicate that alginate-intervened hydrothermal synthesis caused alginate concentration-dependent effects on HA crystallization, agglomeration, particle size, and nanopore formation. In contrast to the generally mentioned roles of polymers as growth modifiers, alginate as well as its depolymerized products under hydrothermal conditions was found to exert synergic and enhanced modulating effects on the preparation of dispersive HA rodlike nanoparticles with numerous nanopores. These HA nanocrystals are promising for applications for various biomedical purposes.
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INTRODUCTION
Alginate is a naturally occurring linear polysaccharide abundant in various species of brown seaweed and has been widely used in food, pharmaceutical, and tissue engineering purposes because of its excellent biocompatibility and biodegradability. In contrast to the vigorous efforts to develop alginate-based organic species for varied biomedical purposes such as drug carriers and tissue engineering scaffolds,1,2,23,24 very limited studies contributed to the use of alginate to modulate HA precipitation or even to prepare alginate−HA nanocomposites. Different protocols in the ambient environment were reported in these studies to evaluate the effect of alginate concentration on HA crystallization and emerged morphology,1,2,9,22,25−27 and the presence of nanosized HA crystals was revealed generally by transmission electron microscopic observation of collected precipitates and even calcined counterparts. However, most of these studies actually yielded aggregates, either HA or HA embedded in an alginate matrix. In this work, hydrothermal modulation of nano-HA crystallization in the presence of alginate was investigated to prepare dispersive HA nanocrysals. Alginate is an anionic copolymer consisting of (1→4)-linked β-D-mannuronic acid
Preparation of nanosized hydroxyapatite (HA) is of great interest in the fields of biomedical engineering, materials science, chemical engineering, and nanotechnology. As the major constituent of the mineral phase in hard tissues of mammals, HA has excellent biocompatibility and osteoconductivity, and synthetic HA nanostructures have already been associated with the development of various bone grafting substitutes.1−3 Furthermore, because of its pH-sensitive solubility and the complexing capability of its Ca2+-rich domains with the helical phosphates of nucleic acids, HA nanoparticles are also promising as carriers for the delivery of guests such as growth factors, anticancer drugs, and vaccines as well as for gene transfection.4−11 Today, different preparation methods for obtaining HA nanoparticles have been explored, such as hydrothermal reaction,12 sol−gel synthesis,13 chemical precipitation,14 microemulsion,15 etc. Among extensive endeavors, the use of organic matters especially acidic species as growth modifiers is demonstrated to be an efficient strategy for controlling emerging HA from solutions with tunable crystallinity, morphology, and size. Typical organic species include small organic molecules16−19 and macromolecules.12,14,20−22 For biomedical purposes, biocompatible organic species are a preferential selection for use as a growth modifier. © 2015 American Chemical Society
Received: January 25, 2015 Revised: February 24, 2015 Published: March 4, 2015 1949
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
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homogeneous blend, 2 mL of a 0.6 M NaH2PO4 aqueous solution was further added slowly while the mixture was being stirred. The pH value of the reaction blend was adjusted to 12.0 by using 2 M NaOH. Subsequently, the blend was transferred to a 200 mL Teflon-lined stainless autoclave container, and hydrothermal reaction was conducted in a preheated oven at 200 °C for 5 h. When the container cooled naturally to room temperature (RT), the synthetic products were centrifugally collected and rinsed with deionized water several times by monitoring with conductivity measurements. Finally, the collected precipitates were freeze-dried for further analyses. A series of HA hydrothermal experiments were conducted in the absence and presence of varying alginate concentrations of 0, 0.4, 0.8, and 1.6 wt %, thereafter designated as HA-control, HA-0.4%, HA-0.8%, and HA1.6%, respectively, where HA-control represents the control experiment in the absence of alginate. In addition, a hydrothermally depolymerized polymerized alginate aqueous solution was also used for HA synthesis, to understand the modulating effect of alginate intervention on HA crystallization under hydrothermal conditions. For this purpose, a 0.5 wt % alginate aqueous solution was prehydrothermally reacted at 180 °C for 0.5 h, and then the reactive supernatant was collected for hydrothermal synthesis of HA at 120 °C for 4 h in comparison with those in the absence and presence of 0.5 wt % alginate. X-ray diffraction (XRD) patterns were measured on a DX-1000 Xray diffractometer with Cu Kα radiation (λ = 1.5406 Å) to confirm the calcium phosphate phase. The working voltage and current were 40 kV and 25 mA, respectively. The data were recorded in the 2θ range of 10−70° at a scanning rate of 0.06° s−1. FT-IR measurements were performed with the KBr pellet method on a PerkinElmer Spectrum One B System to determine the molecular species of the samples. The spectrum was collected in the wavenumber range of 4000−400 cm−1 with a scanning resolution of 2 cm−1. Scanning electron microscopy (SEM) images were taken with a Hitachi S-4800 field emission scanning electron microscope to observe the morphology and size of the samples. The size distributions of the observed nanocrystals were statistically analyzed by counting 200 particles of each sample. Log-normal distributions were used to fit the scattering data, and the average sizes of these nanoparticles were therefore obtained. Thermogravimetric analysis (TGA) was performed on a Netzsch thermal analyzer TG (STA 449C), and the samples were examined under a nitrogen atmosphere at a constant rate of 15 °C min−1 in the scanning range of room temperature to 800 °C with an empty aluminum pan as a reference. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were examined with a Tecnai G2 F20 STWIN transmission electron microscope to reveal the microstructural morphology and to confirm the crystallographic structure together with fast Fourier transform (FFT) methods. The specific surface areas of hydrothermally synthesized samples were measured on a micromeritics Gemini instrument by using Brunauer−Emmer−Teller (BET) nitrogen absorption.
Figure 1. (A) Alginate molecular structure and its egg-box structure formed with Ca cations (inset). (B) G and M units showing the representative depolymerization of alginate under hydrothermal conditions. (C) FT-IR spectra of pure alginate and its carbonized product containing Ca2+ salt.
(M units) and its C-5 epimer α-L-guluronic acid (G units) with varying compositions and sequences and possesses unique complexation capability with divalent cations such as Ca2+ to form the so-called egg-box structure.28 Under hydrothermal conditions, alginate would depolymerize to oligosaccharides, monosaccharides, and decomposition products.29 The relevant molecular structures and interactions are depicted in Figure 1, together with Fourier transform infrared spectroscopy (FT-IR) spectra of pure alginate and its carbonized product containing Ca2+ salt. A series of HA hydrothermal experiments were conducted with different alginate concentrations. On the basis of structural characterization of hydrothermal products, alginate concentration-dependent formation of dispersive HA nanocrystals was presented, and the underlying formation mechanism was further elucidated.
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EXPERIMENTAL SECTION
RESULTS XRD. XRD patterns of the hydrothermally synthesized samples are given in Figure 2 together with the standard diffraction data of HA. It is evident that all the samples, HAcontrol, HA-0.4%, HA-0.8%, and HA-1.6%, have similar XRD traces. Their reflections coincide with the standard data of JCPDS Card No. 09-0432 for crystalline HA, confirming the formation of pure HA in all the samples rather than other calcium phosphates. For example, in the case of HA-1.6%, the strong reflections located at 2θ = 26.02°, 31.96°, 33.10°, 34.24°, 39.88°, 46.90°, 49.60°, and 53.38° can be attributed to (002), (211), (300), (202), (310), (222), (213), and (004) planes of HA, respectively. HA belongs to the p63/m space group, and the crystal lattice constants of the sample could be thus calculated simply by the d values of (002) and (300) planes.
Alginate (M/G ratio of 1.56, molecular weights of 120−190 kDa, viscosities of: 20−40 cps) was purchased from Sigma-Aldrich Co. LLC. CaCl2, NaH2PO4·2H2O, and NaOH were purchased from Kelong Chemical Co. (Chengdu, China). All chemicals were analytical grade and used without further purification. Deionized water (18.3 MΩ cm) was used throughout the experiments. For hydrothermal synthesis, the reaction either at a higher temperature or for a longer time interval is known to be beneficial for obtaining the products with a higher crystallinity. Accordingly, unless particularly specified, hydrothermal synthesis of HA at 200 °C for 5 h was used in solutions containing varying concentrations of alginate. In a typical procedure, 40 mL of a 0.4 wt % alginate aqueous solution was prepared by dissolving alginate powder into deionized water. Then, 2 mL of a 1 M CaCl2 aqueous solution was added drop by drop into the alginate aqueous solution. After the mixture was stirred using a vortex (IKA, Vortex, Genius 3) to obtain a 1950
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
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Figure 2. XRD patterns of the hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HAcontrol, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%.
Figure 5. TGA thermograms of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HAcontrol, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%.
The calculated values for HA-control, HA-0.4%, HA-0.8%, and HA-1.6% samples are 9.4008, 9.3953, 9.3677, and 9.3677 Å for a = b and 6.8590, 6.8590, 6.8434, and 6.8434 Å for c, respectively. Compared with those of HA-control, a slight decrease in a = b and c values was detected in both HA-0.8% and HA-1.6% samples that were obtained from two groups of the solutions containing a high alginate concentration. Furthermore, the sharpness of their reflections decreases from the top (A) sample (HA-control) down to the bottom (D) sample (HA-1.6%) (Figure 2), indicative of the gradually reduced crystallinity when a larger amount of alginate intervened in HA hydrothermal synthesis. This ascertainment is further validated by the steady increment in the half-heightwidth values of their (002) planes as well as by the more obvious halos observed around the main (211) reflections (marked with dotted lines). In the latter case, the more obvious halo indicates the lower crystallinity as this halo is related to the amorphous component of the sample. FT-IR. FT-IR spectra of the hydrothermally synthesized HAcontrol, HA-0.4%, HA-0.8%, and HA-1.6% samples are given in Figures 3 and 4. In the case of the HA-control sample, the adsorptions at 473, 567, 603, 962, 1037, and 1093 cm−1 correspond to PO43− stretching vibrations of HA whereas the adsorptions at 632 and 3575 cm−1 can be assigned to the stretching vibration of the hydroxyl group of HA.3 The formation of carbonated HA is indicated by the adsorptions at 875, 1420, and 1456 cm−1 that arose from the partial replacement of PO43− with CO32− anions.1,30 The broad adsorption centered at 3430 cm−1 and a small adsorption at 1644 cm−1 are due to the stretching and bending vibrations of absorbed water in HA. In contrast, alginate-intervening hydrothermal synthesis of HA led to altering the FT-IR spectrum of HA-control. With an increasing level of alginate, the relative intensity of adsorption at around 1619 cm−1 gradually increases and obviously widens from HA-0.4% to HA-0.8% to HA-1.6% (Figure 3). In fact, a minor shoulder at 1702 cm−1 appears in both HA-0.8% and HA-1.6% samples. In this range, pure alginate (Figure 1C) shows two characteristic adsorptions at 1614 and 1404 cm−1 due to asymmetric and symmetric stretching vibrations of the C−O bond from the carboxylate group, respectively. Meanwhile, hydrothermal carbonization of alginate containing Ca2+ salt caused the
Figure 3. FT-IR spectra of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HA-control, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%.
Figure 4. FT-IR spectra in the range of 1150−400 cm−1 of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HA-control, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%.
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Figure 6. SEM images of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HA-control, (B) HA0.4%, (C) HA-0.8%, and (D) HA-1.6%. (E) Measured lengths (x-axis) and diameters (y-axis) of 200 particles from the respective SEM images (green dots for HA-control, pink dots for HA-0.4%, blue dots for HA-0.8%, and gray dots for HA-1.6%; the respective colored area highlights the predominant dots).
appearance of the adsorptions at 1698, 1619, and 1415 cm−1.31 Accordingly, the detection of the adsorption at 1619 cm−1 together with a minor shoulder at 1702 cm−1 suggests the incorporation of organic species into HA nanocrystals in HA0.8% and HA-1.6% samples. The simultaneous detection of the C−H stretching vibration at 2930 cm−1 further supports this finding. Concomitant with alginate incorporation, the extent of structural ordering of HA nanocrystals is found to decrease, as indicated by gradually reduced intensities in the stretching vibrations of the hydroxyl group of HA at 632 and 3575 cm−1 (Figures 3 and 4). The reduced level of ordering of HA nanocrystals is in agreement with the decrease in HA crystallinity revealed by XRD analysis (Figure 2). TGA. Figure 5 gives the TGA thermograms of HA-control, HA-0.4%, HA-0.8%, and HA-1.6% samples under a nitrogen flux of 22 mL min−1 with scans from room temperature to 800 °C. A multistage weight loss was recorded for all the samples. The weight losses in the range of RT to 160 °C and approximately 160−320 °C are due to the dehydration of
loosely absorbed and bound water and initial decomposition of organic species in the case of samples obtained with alginate, respectively. Further decomposition and evaporation of the organic components give rise to another stage of weight loss. The decomposition of carbonates incorporated into HA also occurred above 600 °C. It is evident that the samples obtained from solutions containing more alginate yield greater weight losses at different stages. At 800 °C, the residual weight percentage is 94.87% for HA-control, 92.33% for HA-0.4%, 88.75% for HA-0.8%, and 84.72% for HA-1.6%. SEM. Figure 6 displays the nanostructural features of hydrothermally synthesized samples. Their SEM images show that the synthetic samples are dispersive rodlike particles. The presence of hexagonal HA prisms is discernible, but the aggregation of very small nanocrystals gradually becomes prevalent in the rodlike particles synthesized with a higher alginate concentration. In contrast, the well-defined profile of rodlike particles in the HAP-control sample (Figure 6A) gradually becomes blurred in HA-0.4%, HA-0.8%, and HA-1.6% 1952
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
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dependent effect on the decrease in size (diameter and length) of these rodlike particles. The relevant statistical results are further shown in Figure 7. The diameters of these rodlike particles are narrowly distributed, relative to their lengths. Meanwhile, both the diameter and length distributions follow an alginate concentration-dependent narrowing fashion; i.e., HA-control yields the highest degree of dispersion, whereas HA-1.6% gives the best uniformity. In fact, the number-average distributions of both length and diameter of all the samples fit well with log-normal functions that are generally used to fit the scattering data.32,33 On the basis of the red fitted distribution curves, the average lengths of rodlike particles are 48, 44, 38, and 33 nm and the average diameters 27, 25, 17, and 12 nm for HA-control, HA0.4%, HA-0.8%, and HA-1.6%, respectively. Figure 7E depicts alginate concentration-dependent decremental effect on the size and its deviation in both length and diameter of rodlike particles. TEM. Representative TEM examinations are shown in Figure 8. TEM images of hydrothermally synthesized HA-control, HA0.8%, and HA-1.6% samples reveal that the rodlike nanoparticles are nanocrystals. The average diameter and length values are 24 and 52 nm for HA-control and 15 and 35 nm for HA-1.6%, respectively, fairly in agreement with the measured values from SEM images. HRTEM and FFT results indicate that the nanocrystals in the HA-0.8% sample are of the apatite type (Figure 8B). In addition, plenty of nanosized pores are distributed in these HA nanocrystals. In contrast to the HA control, more nanosized pores exist in HA-0.8% and HA-1.6% samples, and to an extent, some nanopores are arranged in alignment, probably because of the interstices between aggregates of nanocrystals. BET. Figure 9 shows the N2 adsorption/desorption isotherms of hydrothermally synthesized HA-control, HA0.4%, HA-0.8%, and HA-1.6% samples. The BET specific surface areas and pore volumes of different samples are summarized in Table 1. Both values are 48.0 m2/g and 0.411 cm3/g for the HA-control sample, respectively, almost the same as the reported values for hydrothermally synthesized HA.34 In contrast, with alginate intervention in hydrothermal synthesis, obvious elevation of BET specific surface areas and pore volumes was detected, especially for HAP-0.8% and HAP-1.6%.
Figure 7. Length (left) and diameter (right) distribution histograms of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HA-control, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%. The red curves correspond to a fit of the data with the log-normal function. (E) Graphical representation of the correlation of length and diameter with different samples (error bars representing the standard deviation).
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DISCUSSION Hydrothermal reaction is an efficient method for the synthesis of crystalline inorganic materials. In general, the grain size and crystallinity of hydrothermally synthesized inorganic materials increase with the elevated temperature and the prolonged reaction interval. In addition, it is very convenient to introduce different growth modifiers into the hydrothermal reaction for controlled synthesis of inorganic materials with desirable morphology, crystallinity, and particle size. Hydrothermal synthesis of HA, especially HA nanoparticles, has been extensively conducted in recent years under varying conditions such as different reaction temperatures and intervals in the absence or presence of organic modifiers.12,17,22 However, the introduction of alginate in hydrothermal synthesis of HA nanoparticles has not yet been reported, to the best of our knowledge. This study shows that alginate-intervening hydrothermal HA synthesis leads to a reduction in the crystallinity and size of rodlike HA nanocrystals in a concentration-dependent fashion. The existence of a higher alginate concentration during
samples (Figure 6B−D, respectively), which were hydrothermally synthesized with increasing concentrations of alginate. This phenomenon at least indicates that alginateintervening HA hydrothermal synthesis with smaller rodlike particles as the equivalent bar for 100 nm is given in Figure 6. To quantify the size of these rodlike particles, 200 particles of each sample were randomly selected to measure their diameters and lengths. The measured data are given by color dots in Figure 6E, and the predominating dots in each sample are highlighted by the respective annotated color. Both measured diameter and length values visually depict a range of distributions with the most disperse degree observed in HAcontrol sample. Meanwhile, these color dots together with their respectively colored areas show the alginate concentration1953
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
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Figure 8. Representative TEM images of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HAcontrol, (B) HA-0.8%, and (C) HA-1.6%. HRTEM image and fast Fourier transform (FFT) pattern are typically given on the particles of the HA0.8% sample.
hydrothermal reaction steadily decreases the crystallinity and size of synthetic rodlike nanocrystals. These conclusions are based on four groups of experimental data of the samples obtained with alginate concentrations of 0, 0.4, 0.8, and 1.6 wt %. In fact, hydrothermal experiments with a much higher alginate concentration up to 3.2 wt % were also conducted, and in the collected samples, inorganic particles were found to be embedded with the carbonized organic matrix. Thus, the relevant experimental data were not incorporated as one purpose of the present study is to synthesize disperse HA nanoparticles. Synthesis of HA nanoparticles is still considered as an intractable task because the immediate mixing feedstock of calcium-containing and phosphate-containing aqueous solutions would lead to the uncontrollable rapid growth of calcium phosphates. Thus, various organic species have been used as growth modifiers to obtain HA nanoparticles. Alginate is an anionic copolymer consisting of M and G units in alternating G/M units. Its molecular chains are rich in carboxylate groups, and G units contribute to forming the egg-box structures through stereocomplexation with divalent metallic cations. Recent studies in the ambient environment demonstrated the modulating effect of alginate on HA crystallization. Depending on the specific experimental protocols, different modulating results were presented in an ambient environment. Malkaj et al. reported that by the constant composition technique alginate was found to inhibit HAP crystal growth at low concentrations and reduced the crystal growth rates.27 Son et al. revealed that
Figure 9. N2 adsorption/desorption isotherms of hydrothermally synthesized samples from solutions containing various alginate concentrations: (A) HA-control, (B) HA-0.4%, (C) HA-0.8%, and (D) HA-1.6%.
Table 1. BET Specific Surface Areas and Pore Volumes of Different Samples sample
Vp (cm3/g)
SBET (m2/g)
HA-control HA-0.4% HA-0.8% HA-1.6%
0.411 0.380 0.327 0.335
48.0 54.6 65.1 69.3
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Figure 10. SEM images of hydrothermally synthesized samples at 120 °C for 5 h: (A) without alginate, (B) with 0.5% alginate, and (C) by using the supernatant after a prehydrothermally treated 0.5 wt % alginate solution at 180 °C for 0.5 h.
saccharides, and decomposition products imposed upon HA synthesis. The modulating effects of alginate-depolymerized components in terms of reducing the extent of agglomeration and particle size of HA crystals are demonstrated in Figure 10. Accordingly, the enhanced modulating effects occur under the circumstance presented here. The synergic contributions by alginate and its depolymerized components result in alginate concentration-dependent inhibitory effects on HA crystallinity, agglomeration, and size. In terms of the size of synthetic products, the statistical data in Figure 7 further reveal that the more coherent reduced effect on the diameter exists relative to the length of these rodlike nanoparticles, This tendency suggests the occurrence of preferential adsorption of organic species onto the active growth sites perpendicular to the [001] direction along which the rodlike HA nanocrystal grows (Figure 8). The adsorption of alginate or other derived products into the structure of HA nanocrystals leads to the formation of poorly defined aggregates of much smaller nanocrystals, thus modifying the morphology of discernible hexagonal prisms in the HA-control sample (Figure 6). The incorporation of organic species into rod-like nanoparticles was also verified by FT-IR and TGA analyses. In addition to the reduced crystallinity, XRD analyses reveal a decrease in the a, b, and c cell parameters of HA crystals in the samples hydrothermally synthesized with the increase in the level of alginate in aqueous solution. HRTEM and FFT results for HA0.8% nanocrystals further exclude the possible presence of any other crystalline phase except HA. Accordingly, XRD results indicate that alginate-related species not only are adsorbed on the growing surfaces of HA, thus modifying the formation of hexagonal HA prisms in the HA-control sample, but also can be absorbed in the HA lattice, thereby changing the XRD reflections in both 2θ position and intensity. Similar results were also reported in additive-intervened synthesis of inorganic materials, including HA.19,35 Meanwhile, the FT-IR detection and assignment of carbonated HA and alginate concentrationdependent BET results together with TEM observation of nanosized pores further correspond to the occurrence of alginate depolymerization under hydrothermal conditions. It is believed that the prevalent aggregation of nanocrystals in the samples synthesized with a higher concentration of alginate also contributes to the elevated BET results because of the interstices between the aggregates of nanocrystals.
the reaction of calcium-containing solutions in the presence of alginate contents of 0.01, 0.02, 0.05, and 0.1% (w/v) with phosphate-containing solutions yielded HA agglomerates with the steadily reduced secondary particle sizes.25 Rajkumar et al. found that the HA crystallite size and degree of crystallinity increased with the elevation of the alginate concentration to 1.5 wt % but decreased with a further increase in the concentration of alginate.26 Wang et al. showed that with the involvement of alginate from 10 to 40 wt % the crystallinity of HA decreased and the crystallites tended to elongate along the c-axis with an increase in alginate content.1 Obviously, almost all the reported modulation effects of alginate on the size of HA nanoparticles were actually based on examining either the secondary structure of HA aggregates or the HA nanocrystals embedded in the alginate matrix, instead of being based on the as-synthesized dispersive HA nanoparticles. Under hydrothermal conditions, the role of alginate is expected to be different from that in the synthesis in the ambient environment as alginate would depolymerize to oligosaccharides, monosaccharides, and decomposition products.29 To specify the difference, a 0.5 wt % alginate solution was prehydrothermally treated at 180 °C for 0.5 h, and then hydrothermal synthesis of HA in the pretreated alginate supernatant was conducted at 120 °C for 5 h, with HA synthesis in the absence and presence of alginate as controls. The SEM images of the collected products are given in Figure 10. These poorly defined nanoparticles correspond to less incomplete crystallization achieved at 120 °C than those in Figure 6 obtained at 200 °C. However, compared with relatively integral HA rods (Figure 10a), the intervention of alginate as well as its prehydrothermally treated solution in HA synthesis both yielded better dispersive and smaller HA nanoparticles as shown in panels B and C of Figure 10. It is worth noting that the hydrothermal depolymerization of polymers as growth modifiers and the enhanced/auxiliary modulating effects were scarcely mentioned in the relevant literature. Herein, we tentatively propose the following mechanisms. Without using alginate as a growth modifier, the immediate mixing of Ca- and phosphate-containing solutions initiates the formation of CaP nuclei, which then hydrothermally grow, following the crystallographic growth habit, into well-defined HA rodlike nanocrystals generally as hexagonal prisms. When alginate intervenes, two kinds of modulating interactions would be considered. One is alginate-dominated interactions, generally described in the ambient environment, such as those forming the so-called egg-box structure, adsorption, and further blocking of the active growth sites.1,9,22,25−27 The other is hydrothermal process-initiated interactions that arise from the anionic groups of deploymerized oligosaccharides, mono-
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CONCLUSIONS Hydrothermal reactions of Ca- and phosphate-containing aqueous solutions in the absence and presence of alginate were investigated in this study to obtain HA nanoparticles. The intervention of alginate was found to exert a concentrationdependent modulating effect on the crystallinity, size, and 1955
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
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(17) Wu, Y. J.; Tseng, Y. H.; Chan, J. C. C. Cryst. Growth Des. 2010, 10, 4240. (18) Tao, J.; Pan, H. H.; Zeng, Y. W.; Xu, X. R.; Tang, R. K. J. Phys. Chem. B 2007, 111, 13410. (19) Matsumoto, T.; Uddin, M. H.; An, S. H.; Arakawa, K.; Taguchi, E. Mater. Chem. Phys. 2011, 128, 495. (20) Zhi, L.; Tao, W.; Sun, Y.; Wei, X.; He, C.; Wang, D. CrystEngComm 2014, 16, 4202. (21) Zhang, Y.; Lu, J. Cryst. Growth Des. 2008, 8, 2101. (22) Coleman, R. J.; Jack, K. S.; Perrier, S.; Grøndahl, L. Cryst. Growth Des. 2013, 13, 4252. (23) Tønnesen, H. H.; Karlsen, J. Drug Dev. Ind. Pharm. 2002, 28, 621. (24) Sang, L.; Luo, D. M.; Xu, S. M.; Wang, X. L.; Li, X. D. Mater. Sci. Eng., C 2011, 31, 262. (25) Son, K. D.; Yang, D. J.; Kim, M. S.; Kang, I. K.; Kim, S. Y.; Kim, Y. J. Mater. Chem. Phys. 2012, 132, 1041. (26) Rajkumar, M.; Meenakshisundaram, N.; Rajendran, V. Mater. Charact. 2011, 62, 469. (27) Malkaj, P.; Pierri, E.; Dalas, E. J. Mater. Sci.: Mater. Med. 2005, 16, 733. (28) Donati, I.; Morch, Y. A.; Strand, B. L.; Skjåk-Bræk, G.; Paoletti, S. J. Phys. Chem. B 2009, 113, 12916. (29) Aida, T. M.; Yamagata, T.; Watanabe, M.; Smith, R. L., Jr. Carbohydr. Polym. 2010, 80, 296. (30) Ren, F.; Leng, Y.; Ding, Y.; Wang, K. CrystEngComm 2013, 15, 2137. (31) Chen, J. Z.; Chen, Z. H.; Wang, C. H.; Li, X. D. Mater. Lett. 2012, 67, 365. (32) Renaud, G.; Lazzari, R.; Revenant, C.; Barbier, A.; Noblet, M.; Ulrich, O.; Leroy, F.; Jupille, J.; Borensztein, Y.; Henry, C. R.; Deville, J. P.; Scheurer, F.; Mane-Mane, J.; Fruchart, O. Science 2003, 300, 1416. (33) Wang, F.; Richards, V. N.; Shields, S. P.; Buhro, W. E. Chem. Mater. 2014, 26, 5. (34) Reddy, M. P.; Venugopal, A.; Subrahmanyam, M. Appl. Catal., B 2007, 69, 164. (35) Chen, Z.; Wang, C.; Zhou, H.; Li, X. Cryst. Growth Des. 2010, 10, 4722.
agglomeration of hydrothermal HA products, according to XRD, FT-IR, TG, SEM, TEM/HRTEM, and BET analyses. With alginate concentrations of 0, 0.4, 0.8, and 1.6 wt %, the diameter and length of obtained rodlike HA particles steadily decreased. The average values for HA particles without using alginate were 28 nm for diameter and 53 nm for length. In contrast, both values for HA particles obtained in the presence of 1.6 wt % alginate were only 13 and 34 nm, respectively. This study further revealed that alginate as well as its depolymerized components under hydrothermal conditions played very important modulating roles in inhibiting HA crystallization, particle size decrement, and the formation of nanosized pores. Incorporation of alginate organic species was also confirmed. The very fine HA nanoparticles distributed with numerous nanosized pores are expected to have potential applications in tissue engineering and controlled delivery.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
Y.W. and X.R. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the National Basic Research Program of China (2012CB933600), the National Natural Science Foundation of China (30970729), and the Engineering Research Center of Biomass Materials (Southwest University of Science and Technology) of the Ministry of Education of China (10zxbk04).
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REFERENCES
(1) Wang, L.; Li, Y.; Li, C. J. Nanopart. Res. 2009, 11, 691. (2) Hong, R. L.; Yu, J. Y. J. Biomed. Mater. Res., Part B 2004, 71, 52. (3) Wang, X. M.; Wang, X. L.; Ma, J. F.; Jiang, J. M.; Zheng, G. Q.; Chen, Z. H.; Li, X. D. Adv. Appl. Ceram. 2009, 108, 384. (4) Kakizawa, Y.; Miyata, K.; Furukawa, S.; Kataoka, K. Adv. Mater. (Weinheim, Ger.) 2004, 16, 699. (5) Wang, G. C.; Cao, R. Y.; Chen, R.; Mo, L. J.; Han, J. F.; Wang, X. Y.; Xu, X. R.; Jiang, T.; Deng, Y. Q.; Lyu, K.; Zhu, S. Y.; Qin, E. D.; Tang, R. K.; Qin, C. F. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 7619. (6) Kevin, C. W.; Ya, H. Y.; Yung, H. L.; Hui, Y. C.; Eric, S.; Yusuke, Y.; Feng, H. L. Curr. Nanosci. 2011, 7, 926. (7) Bishnu, P. B.; Yin, C. H.; Shih, H. L.; Kevin, C. W.; Masamichi, I.; Shin, Y.; Kenichi, N.; Yusuke, Y. Chem.Asian J. 2013, 8, 1301. (8) Ya, H. Y.; Chia, H. L.; Yung, H. L.; Feng, H. L.; Kevin, C. W. J. Mater. Chem. B 2013, 1, 2447. (9) Liang, Y. H.; Liu, C. H.; Liao, S. H.; Lin, Y. Y.; Tang, H. W.; Liu, S. Y.; Wu, K. C. W. ACS Appl. Mater. Interfaces 2012, 4, 6720. (10) Yang, P. P.; Quan, Z. W.; Li, C. X.; Kang, X. J.; Lian, H. Z.; Lin, J. Biomaterials 2008, 29, 4341. (11) Ribeiro, C. C.; Barrias, C. C.; Barbosa, M. A. Biomaterials 2004, 25, 4363. (12) Zhang, H. F.; Liu, M.; Fan, H. S.; Zhang, X. D. Cryst. Growth Des. 2012, 12, 2204. (13) Kim, I. S.; Kumta, P. N. Mater. Sci. Eng., B 2004, 111, 232. (14) Chen, Z. H.; Zhou, H. H.; Wang, X. L.; Sang, L.; Wang, C. H.; Ma, J. F.; Li, X. D. Chem. Commun. 2010, 46, 1278. (15) Shum, H. C.; Bandyopadhyay, A.; Bose, S.; Weitz, D. A. Chem. Mater. 2009, 21, 5548. (16) Zhao, H.; Zhu, Y. D.; Sun, J.; Wei, D.; Wang, K. F.; Liu, M.; Fan, H. S.; Zhang, X. D. Chem. Commun. 2014, 50, 12519. 1956
DOI: 10.1021/acs.cgd.5b00113 Cryst. Growth Des. 2015, 15, 1949−1956
