Aspartic Acid-Assisted Synthesis of Multifunctional Strontium

Jun 29, 2016 - We found out that CO2•− radicals in PASP template induced luminescence properties of HAP powders and this property is affected by S...
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Aspartic Acid-Assisted Synthesis of Multifunctional StrontiumSubstituted Hydroxyapatite Microspheres So Yeon Park,† Kyung-Il Kim,† Sung Pyo Park,*,‡ Jung Heon Lee,*,†,§ and Hyun Suk Jung*,† †

School of Advanced Materials Science & Engineering and §SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 16419, Republic of Korea ‡ Department of Ophthalmology, Kangdong Sacred Heart Hospital, Hallym University Medical Center, Seoul 05355, Republic of Korea S Supporting Information *

ABSTRACT: Strontium-substituted hydroxyapatite (SrHAP) microspheres with three-dimensional (3D) structures were successfully prepared via hydrothermal method using selfassembled poly(aspartic acid) (PASP) as a template. By controlling various parameters, including hydrothermal reaction time, amount of L-aspartic acid (L-Asp), and ratio of Sr ions, we were able to investigate the influences of the additive L-Asp on morphology and properties of final products as well as the role of self-assembled PASP template on the formation of HAP microspheres. The change in the amount of Sr substitution significantly affected the particle size, morphology, and concurrent surface area. This difference caused variation in the drug-release properties. In addition, substitution of Sr ions into Ca ion sites affected luminescence of HAP powders. Particularly, multifunctional SrHAP with molar ratios (Sr/[Ca+Sr]) of 0.25 possessed the strongest luminescence as well as superior drug-loading and sustained-releasing properties. These properties were associated with large surface area and large pore size of the SrHAP. This study suggests that the optical and structural properties of the HAP particles can be carefully tuned by controlling the amount of Sr ions doped into HAP particles during synthesis. This work provides new opportunities to synthesize HAP particles suitable for diverse applications including bone regeneration and drug delivery.

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lack of stimulation of the osteogenesis process.11,12 For example, mesoporous drug-releasing HAP microspheres, with high surface areas and optimal pore sizes, were synthesized using some biocompatible polymers, such as poly(lactide-coglycolide) (PLGA), cetyltrimethylammonium bromide (CTAB), and urea.13−15 Many important properties of HAP, such as bioactivity, biocompatibility, and chemical stability, are controlled via variation in morphology, crystallite size, composition, and structure. In addition, a few ions in bones, including strontium (Sr2+), magnesium (Mg2+), and chlorine (Cl−), are reported to stimulate bone growth and prevent bone loss.15 More importantly, HAP has attracted significant attention in biological staining and diagnostics as a luminescent material.13,16−19 Since its optical properties are also determined by its composition, morphology, and structure,13,18 a welldesigned HAP can be used to monitor or calibrate drug-release process through luminescence. Thus, HAP has great potential as a smart bone graft material, which can not only stimulate

INTRODUCTION A variety of synthetic bone graft materials have been developed over the last several decades, including hydroxyapatite (HAP), β-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP), calcium phosphate cements, bioactive glass, and biodegradable polymers.1−4 However, synthetic bone graft materials currently see limited clinical use because of their inferior in vivo performance and inflammation response of immune system against foreign materials. Thus, researchers are currently focused on loading antibiotic drugs on synthetic graft materials to solve these problems.5,6 As it is the main inorganic component of bone and tooth of humans and animals, HAP not only has favorable bioactivity and biocompatibility, but also is capable to chemically bond with natural bone.7,8 So, HAP has been widely used to replace or repair defected bone tissues in the clinical settings.9 In addition, HAP has great potential to be used as a drug delivery carrier because of the possibility to synthesize diverse nanostructured HAPs with large surface area, large pore volume (Vp), and excellent chemical stability.10 Thus, a lot of researchers have made significant effort to improve their drugloading and -releasing efficiencies and resolve their drawbacks, such as dumping release of loaded drug at an early stage and © XXXX American Chemical Society

Received: March 16, 2016 Revised: June 20, 2016

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phenol red). After an additional 3 h of incubation at 37 °C, the solution was replaced with 100 μL of dimethyl sulfoxide (DMSO). The purple formazan crystals were dissolved in DMSO after vigorous shaking for 1 h. The absorbance was measured with a spectrophotometer (PerkinElmer) at the wavelength of 570 nm. Characterization. The morphology and composition of the samples were studied using a field emission scanning electron microscope (FE-SEM; JSM-7600F, JEOL). The morphology, crystal lattice, and composition of the samples were characterized using highresolution transmission electron microscopy (HR-TEM; JEM-3010, JEOL). The binding energies of the elements in the samples were analyzed by X-ray photoelectron spectrometer (XPS; ESCA 2000, VG Microtech). Elemental analyses of Sr and Ca in the solid samples were carried out by inductively coupled plasma optical emission spectrometry analysis (ICP-OES; Optima-4300DV). The structure of the samples was investigated by a D/max2550 X-ray diffractometer (XRD; D8 discover, Bruker). The Fourier transform infrared (FT-IR) spectrum of each sample was measured with a Bruker IFS-66/S infrared spectrophotometer with the KBr pellet technique. The surface area and total pore volume were determined by the Brunauer− Emmett−Teller (BET; SA3100, Bechman coulter) method. The photoluminescent (PL) excitation and emission spectra were monitored by a fluorescence spectrometer (LS55, PerkinElmer).

bone regeneration but also deliver drug with monitoring capability. In this study, strontium-substituted mesoporous HAP (SrHAP) microspheres were synthesized by hydrothermal method using self-assembled poly(aspartic acid) (PASP) as a template. The variation in the amount of Sr substitution turned out to influence the particle size, morphology, and surface area, which affect drug-loading as well as -releasing properties. In addition, we found out that CO2•− radicals in PASP template induced luminescence properties of HAP powders and this property is affected by Sr ions substituted into calcium ion sites. Our study shows that many essential properties of the HAP particles can be controlled simultaneously by simply doping certain amount of Sr ions into HAP particles during synthesis.

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EXPERIMENTAL SECTION

Synthesis of Hydroxyapatite (HAP) and Strontium-Substituted Hydroxyapatite (SrHAP) Microspheres. The HAP microspheres were synthesized by hydrothermal method using L-aspartic acid monomer (L-Asp) as a template. In a typical experiment, 0.294 g (2 mmol) of calcium chloride dihydrate (CaCl2·2H2O) and 1 g of LAsp were dissolved in 20 mL distilled water (solution 1). Next, 0.158 g (1.2 mmol) of (NH4)2HPO4 was added to 15 mL of distilled water to form another batch of solution (solution 2). After vigorous stirring for 30 min, transparent solution 2 was added to solution 1. After adjusting the pH of the mixed solution to pH 5.0 with diluted ammonia, the solution was sealed in a 50 mL Teflon bottle, placed in a stainless steel autoclave, and kept at 200 °C for 12 h. After cooling the samples in the autoclave down to room temperature, the precipitates were separated by centrifugation. After washing the samples with distilled water and ethanol in sequence, they were dried by placing in a freeze−dryer overnight. Additionally, to study the influence of process parameters and optimize the synthesis values, the HAP was synthesized following the same procedure at different synthesis times (20 min, and 3, 6, 12, and 24 h) and different amount of L-Asp (0, 0.5, 0.7, 1, or 1.5 g). The SrHAP microspheres with designed Sr/[Ca + Sr] molar ratios of 0, 0.25, 0.5, and 1 were hydrothermally synthesized using optimized parameters such as the amount of L-Asp, synthesis temperature, and time. In the synthesis process, the aqueous solution 1 containing 2 mmol (Ca2+ + Sr2+) and 1 g L-Asp were prepared by dissolving CaCl2· 2H2O (0.294, 0.221, 0.147 or 0 g) and SrCl2·6H2O (0, 0.133, 0.267, or 0.533 g) in 20 mL distilled water. Then, 1.2 mmol (NH4)2HPO4 was added to 15 mL of distilled water to form another batch of solution (solution 2). After additional vigorous stirring for 30 min, transparent solution 2 was added to solution 1. The resulting products were labeled as Sr0, Sr0.25, Sr0.5, and Sr1, respectively. In Vitro Study of Drug Loading and Release. The 10 mg of synthesized SrHAP particles (0.5 mg/mL) was directly added into 20 mL vancomycin (VCM) solution (3 mg/mL) under stirring at 37 °C for 24 h. The VCM-loaded SrHAP samples were separated by centrifugation and then dried under vacuum at 60 °C for 24 h. The amount of VCM loaded on SrHAP was calculated by comparing the concentration of the VCM in the solution before and after treatment with SrHAP by UV/vis spectroscopy at 280 nm. To monitor drugreleasing behavior of SrHAP particles, VCM-loaded SrHAP samples were transferred in 10 mL phosphate buffer solution (PBS) at pH 7.4. At predetermined intervals, the PBS solution was replaced with the same amount of fresh PBS. The relation between drug-release and luminescent properties of SrHAP particles was studied by monitoring the luminescent intensity of Sr0.25 sample, which has the highest luminescent intensity among all the SrHAP samples. Biocompatibility Test. Cytotoxicity was evaluated with MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Roche Diagnostics] assay. After seeding (104/well) MC3T3-E1 cells and adding 0.5 mg/mL SrHAP microspheres in 96-well culture plates, the plates were incubated in an incubator at 37 °C with 5% CO2 for 24, 48, and 72 h. Subsequently, the medium in each well was replaced with 100 μL of MTT solution (0.5 mg/mL in culture medium without

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RESULTS AND DISCUSSION

Synthesis of Mesoporous Spherical HAP. To confirm the composition and phase purity of the spherical HAP powders, we characterized crystallization behavior of HAP powders as a function of reaction time. Figure 1a presents the X-ray diffraction (XRD) patterns of the HAP powders synthesized for up to 24 h at 200 °C. The 20-min-synthesized powder shows X-ray reflections associated with HAP, which indicates that the evolution of HAP phase takes place at an earlier reaction stage. The intensity and sharpness of diffraction peaks significantly increases with longer hydrothermal reaction time, suggestive of improved crystallinity. However, there is negligible difference of crystallinities between the samples synthesized for 12 and 24 h. Thus, we optimized the synthesis condition as 200 °C for 12 h. The diffraction peaks of the sample are indexed as (002), (102), (210), (211), (112), (300), (202), and (310) planes of HAP. Given that peaks of other phases are not observed, the samples are identified as HAP (JCPDS card: No. 09−0432) with high purity. Among them, the (002) reflection is quite strong and narrow. This implies that the HAP crystallites are preferably oriented along the (002) c-axis orientation. Figure 1b−e shows the morphological change in HAP powders synthesized through hydrothermal treatment at 200 °C. After reaction time of 20 min, the HAP powders exhibit microspherical shape with relatively dense surface. On the other hand, HAP particles synthesized via 12 h hydrothermal process show porous microspheres with uniform size and morphology. The HAP microspheres with overall diameter of ∼4.5 μm are composed of needle-like primary nanoparticles. The high magnification transmission electron microscopy (TEM) images and selected area electron diffraction (SAED) images shown in Figure 1f,g indicate that each HAP primary nanoparticle is a well-crystallized single crystal. The interplanar spacing of the lattice fringes of primary nanoparticle is 0.34 nm, corresponding to the (002) planes of HAP. This demonstrates that the [001] direction is the favorable growth direction for the HAP needle-like primary nanoparticles during this synthesis process. This is in good consistency with preferred growth of HAP confirmed by X-ray diffraction. B

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diffraction peaks of the samples shift to lower angles as the amount of Sr2+ incorporation increases (Table 1). Since the radius of a Sr ion (1.18 Å) is larger than that of a Ca ion (1.00 Å), incorporation of Sr ions leads to the change of crystallinity and broadening of the interplanar spacing.20 Moreover, we investigated the evidence of Sr substitution using X-ray photoelectron spectrometer (XPS) spectra and energydispersive X-ray spectroscopy (EDX) mapping images. In XPS spectra (Figure 2b), intensities of Ca 2s and Ca 2p peaks decrease with the increase of the amount of Sr. In EDX mapping (Figure 2c), we could detect Sr in SrHAP microspheres overall except for the Sr0 sample. These data clearly indicate that the Sr ions are substituted into HAP powders. Figure 2d−n shows the SEM and TEM images of the samples with the different Sr/[Ca+Sr] molar ratios. From the SEM images, significant difference in the morphology and size of SrHAP was observed with different molar ratios of Sr/[Ca +Sr]. Most of the synthesized SrHAP samples, except for the Sr1 sample, are composed of microspheres containing primary nanoparticulate structures. The size of synthetic microspheres increases with the molar ratios of Sr/[Ca+Sr]. The diameter of Sr0, Sr0.25, and Sr0.5 is about 4.5, 8.5, and 11 μm, respectively. The morphology of nanostructured primary nanoparticles, each of which constitute the secondary SrHAP microspheres, changed from needle-like to sheet-like structure with increasing Sr/[Ca+Sr] molar ratio. The lattice images shown in HRTEM (Figure 2l−n) demonstrate that the prepared SrHAP possesses high crystallinity and d-spacing changes with the different Sr molar ratio. The interplanar spacing of Sr0.25 is determined to be 0.34 and 0.28 nm, which can be indexed as d-spacing values of the (002) and (112) planes of Ca7Sr3(PO4)6(OH)2 crystal (JCPDS No. 34−0842). This result indicates that the Sr0.25 is sheet-like crystals and hexagonal columnar crystal, which is oriented growth along the c-axis. The interplanar spacings of Sr0.5 are 0.28, 0.29, and 0.35, which respectively correspond to d-spacing values of the (300), (211), and (002) planes of Ca5Sr5(PO4)6(OH)2 crystal (JCPDS No. 34−0479). The HRTEM data clearly demonstrates substitution of Sr ions for Ca ions, which is correspondent with XRD results. The (002), (300), and (211) lattice planes were all observed in the HRTEM image of Sr0.5 nanocrystal.30 It implies that Sr0.5 nanocrystal is formed by the growth along the c-axis as well as a- or b-axis. In result, we believe the primary nanocrystals of Sr0.5 were formed as a curved plate-shape. In general, the HAP grows preferably along the c-axis because the growth rate in the c-axis is higher than that in the a-axis. However, when Ca is substituted with Sr, the growth of apatite along the c-axis would be restrained, resulting in lower aspect ratio of the apatite crystal.21 Therefore, the shape of apatite primary particles changes from a needle-like to sheet-like structure as the content of Sr increases. In additional, introduction of Sr results in crystal grain distortion and dislocation. As the ionic radius of Sr2+ is larger than that of Ca2+, the distance between metal ion and a hydroxyl group becomes larger.31 We believe that is the reason the primary Sr0.5 nanocrystals have irregular morphology based on TEM image. In contrast, as observed from SEM and TEM images of Sr1 (Figure 2g,k,n), the crystal preferably grows along the c-axis when Ca ions were fully substituted by Sr ions. Mechanism of the Synthesis of Mesoporous SrHAP Microspheres. To understand the detailed synthesis mechanism and role of L-aspartic acid (L-Asp) on the formation of the HAP powder, we carried out some control experiments by altering the amount of L-Asp used for the synthesis of HAP

Figure 1. (a) XRD patterns of HAP microspheres as a function of synthesis time. (b−e) Low- and high-magnification SEM images of HAP microspheres synthesized on PASP template: (b,c) 20 min working time; (d,e) 12 h working time. (f,g) TEM image and SAED pattern of HAP microspheres.

Characterization of SrHAP Microspheres. We investigated the structural and morphological characteristics of strontium (Sr) ions-incorporated HAP microspheres. To find the effect of Sr ions in the Sr substituted HAP (SrHAP) microsphere formation, we characterized the samples with inductively coupled plasma optical emission spectrometry (ICP-OES) and X-ray diffractometer (XRD). The list of molar ratios of Sr and Ca ions of the particles synthesized at different reaction solution (labeled as Sr0, Sr0.25, Sr0.5, and Sr1) is shown in Table 1. From the ICP-OES results, the ratio of Sr in the particle increases proportionally with the amount of Sr in the solution, which suggests incorporation of Sr ions in HAP microspheres. The characteristic XRD peak of each SrHAP sample is shown in Figure 2a. It is remarkable that the Table 1. Sr/[Ca+Sr] Molar Ratios in Solution and Synthetic Particles and 2θ of (002) Reflection sample

Sr/(Ca+Sr) molar ratio in solution

Sr/(Ca+Sr) molar ratio in particle

2θ (deg) (002 reflection)

Sr0 Sr0.25 Sr0.5 Sr1

0 0.25 0.5 1

0 0.23 0.48 1

25.9 25.68 25.48 24.7 C

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Figure 2. (a) XRD patterns of SrHAP with different Sr/[Ca+Sr] molar ratios (Sr0, Sr0.25, Sr0.5, and Sr1). (b) XPS spectra of SrHAP with different Sr/ [Ca+Sr] molar ratios. (c) EDX mapping of Ca, P, Sr elements. (d−n) Low- and high-magnification SEM images of SrHAP (Sr0 (d,h), Sr0.25 (e,i), Sr0.5 (f,j), Sr1 (h,k)) and HRTEM images and SAED pattern of Sr0.25 (l), Sr0.5 (m), Sr1 (n).

microspheres. The morphologies and sizes of the samples prepared with different amount of L-Asp (0 to 1.5 g) were analyzed by FE-SEM (Figure S1). Samples synthesized without L-Asp had an irregular sheet-like morphology. By increasing the amount of L-Asp monomer from 0 to 0.7 g, the samples started to have spherical structures. By increasing the amount of L-Asp up to 1 g, the synthesized microspheres became smaller with better uniformity. However, as the amount of L-Asp became larger than 1.5 g, the microspheres started to become irregular. This implies that L-Asp is an important factor determining the structure and surface morphology of HAP microspheres. Generally, one of the well-known disadvantages of the hydrothermal method is poor controllability of the morphology and size distribution of nanoparticles.22 However, these problems were solved in this study by optimizing the amount of L-Asp monomers during synthesis. In order to investigate how L-Asp exist in HAP microspheres, we first compared FT-IR spectra of L-Asp with HAP. The of LAsp peaks at 2900 to 3400 cm−1, associated with −NH3+ and −CH− groups, were not observed in the spectra of HAP microspheres. This suggests that the conformation of L-Asp changed significantly in HAP microspheres during the synthesis process. Instead, we wondered whether the self-assembled

formation of L-Asp, e.g., PASP, would have similar structure to HAP microspheres. So, we compared FT-IR spectra between PASP and HAP microspheres. The FT-IR spectra of HAP microspheres was quite similar to PASP with −COO− peaks at 1427 and 1596 cm−1 and H2O peak at 3500 cm−1. This strongly suggests that L-Asp exists in HAP microspheres in a selfassembled formation as PASP. In addition, we observed the bands corresponding to PO43− (1094(v3), 1032(v1), 608(v4), and 564(v2) cm−1), HPO42− (634 cm−1), and −OH (3570 and 633 cm−1) chains. This proves the formation of HAP. Furthermore, the asymmetric and symmetric bands of COO− groups (1596 and 1427 cm−1) appeared on HAP microspheres as well. These bands have been reported to be observed in the Ca-PASP complex.23,24 In addition, Raman spectra of HAP microspheres synthesized on self-assembled PASP template were investigated (Figure S3). We observed phosphate group peaks at 1100, 960, 610, and 430 cm−1, amide III peak at 1430 cm−1, and −COO− peak at 1250 cm−1 from HAP microspheres. Those amide III and −COO− peaks of HAP microspheres match quite well with those peaks of PASP. In consistent with what we observed with FT-IR, this Raman spectrum verifies that synthetic HAP contains PASP template. D

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Figure 3. (a) FT-IR spectra of synthetic HAP, PASP, and commercial L-Asp monomer (Sigma-Aldrich A9256). (b−d) Schematic illustration for the formation mechanism of SrHAP with PASP self-assembly (b), SrHAP nucleation (c), and process of SrHAP synthesis (d).

and Sr2+ would significantly affect the morphology of the particles.26 Especially, the morphology of Sr1 samples with needle-like primary nanoparticles is very similar to the HAP synthetic product synthesized without L-Asp. Furthermore, this hypothesis agrees with the FT-IR spectra of Sr1 sample (Figure S2) with no band corresponding to organic functional groups in PASP template appearing in other Sr0, Sr0.25, and Sr0.5 samples. Luminescent Properties of SrHAP Microspheres. Although Sr-substituted HAP (SrHAP) is known to have luminescence properties, most of the investigation on its optical properties are carried out with the particles synthesized with CTAB.15,17 Since the mechanism of the luminescence property is known to be due to the formation of CO2•− radicals from CTAB, we became curious whether SrHAP microspheres based on PASP template has luminescence property as well. Figure 4a shows the excitation and emission spectrum of SrHAP samples. All the SrHAP samples show a similar trend in excitation and emission spectra with maximum peaks appearing at 335 nm and at 395 nm, respectively. The fluorescence of SrHAP particles exhibits a maximum intensity at Sr/[Ca+Sr] molar ratio of 0.25. Further increase in Sr molar ratio

We infer the principle of PASP template formation in this study as the following (Figure 3b−d). Since L-Asp is an amino acid with a carboxylic acid side chain, it has two −COO− groups and one −NH3+ group at pH 5−6. So, both ionic interaction and hydrogen bonding can be formed between −COO− group of one L-Asp molecule and the −NH3+ group of another L-Asp molecule in water (Figure. 3b). Self-assembly of − L-Asp occurs via the chemical reaction between the −COO group and the −NH3+ group, forming a peptide bond on PASP template.25 As each L-Asp contains two −COO− groups, the remaining −COO− groups in PASP template can chelate Ca2+ ions and become a nucleation site for the synthesis of HAP crystal. In additional, the chelated Ca2+ ions act as a binder among L-Asp monomers. That is, the Ca ions and −COO− on PASP template are bound when Ca-PASP complex are formed. Thus, the nucleation and initial growth of HAP can occur inside of PASP template and surface of PASP template during hydrothermal synthesis. Subsequently, HAP crystal continues to grow along the c-axis (see Figure 1f,g). Importantly, as the chelating effect of carboxyl group is much stronger for Ca ions than Sr ions, the relative amount of Ca2+ E

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Figure 4. (a) Photoluminescence excitation and emission spectra of SrHAP with different Sr/[Ca+Sr] molar ratios. (b) XPS deconvolution for P 2s, C 1s, Ca 2s, and O 1s peaks.

deteriorates the fluorescence property of SrHAP. Zhang C. et al. reported that the luminescence of the SrHAP is induced by CO2•− radicals in the host lattice, which act as the luminescence centers.17 The decrease of CO2•− radical by increasing doping ratio of Sr2+ can induce the deterioration of luminescence of SrHAP powders. In contrast, as Sr2+ possesses more outer electrons than Ca2+, the electrons donated from Sr2+ to CO2•− radicals can boost up the emission of the CO2•− radicals more efficiently in the SrHAP.17,19 Consequently, there should be an optimal amount of Sr required for efficient fluorescence property of SrHAP. From the luminescence data, we found that Sr0.25 had the highest luminescence among all samples (see Figure. 4a). Figure. 4b shows the XPS spectra of the particles prepared with different molar ratios of Sr/[Ca+Sr] (Sr0, Sr0.25, Sr0.5, and S1). C 1s peaks, especially those of CO, CO, and CH bonds, are detected in all of the samples. This suggests that carbon-related impurities, such as the CO2•− radical, exist on

the surface of these samples. The carbon-related impurities might be formed by binding of Ca ions on −COO− groups in SrHAP crystal during the synthesis process. The C 1s peaks shift to higher binding energies with increasing molar ratios of Sr/[Ca+Sr]. This verifies electron donation from Sr2+ ions, producing higher electron density in CO2•− radicals. Other peaks related to PO43− ions shift to higher binding energies, which is in good consistency with C 1s peaks. However, the intensity of C 1s peaks decreases with Sr incorporation, implying the decrease of the amount of CO2•− radicals. As Sr/ [Ca+Sr] molar ratio become higher than 0.25, the PL intensity starts to decrease. This is because the amount of −COO− on PASP template interacting with Ca ions decrease as the ratio of Ca ions decreases. As shown in Figure S2, the peaks at 2700 to 3400 cm−1, related to −NH3+−, −NH−, and −CH− groups, appeared most significantly on the Sr0.5 sample. This suggests that there are substantial amount of unreacted L-Asp molecules, resulting in incomplete formation of PASP. Thus, the efficiency F

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Figure 5. (a) N2 adsorption−desorption isotherm and pore size distributions (insets) of synthetic SrHAP samples. (b) Cumulative vancomycin release ratio from different synthetic SrHAP samples in PBS. (c) PL emission intensity of Sr0.25 as a function of release time. (d) PL excitation intensity of Sr0.25 as a function of release time.

of SrHAP crystal synthesis and generation of CO2•− radical will decrease in SrHAP crystal at this condition. Additionally, the intensity of PL spectra of the Sr1 sample is much lower than other samples because of the absence of CO2•− radicals (see Figure S2.). Drug Loading and Release Measurements. To investigate the specific surface area and pore size of synthesized product, we measured the N2 adsorption−desorption isotherm and the pore size distribution of the SrHAP samples. Figure 5a shows that Sr0, Sr0.25, and Sr0.5 particles exhibit type H4 hysteresis loop. This hysteresis loop is typically observed in mesoporous structures composed of aggregates of nanocrystals with slit-shaped pores.27 The pore size distribution of SrHAP was measured from the adsorption branches of the isotherms using the Barrett−Joyner−Halenda (BJH) method (see inset image in Figure 5a and Table 2). Although the pore sizes of the SrHAP are mostly in 2−200 nm ranges, the pore distribution depends significantly on the amount of Sr substituted in the SrHAP. Sr0 and Sr0.5 mostly have pores smaller than 100 nm. Sr1 only has pores larger than 50 nm. In contrast, Sr0.25 not only has pores smaller than 100 nm but also has those larger than 100 nm. The Brunauer−Emmett−Teller (BET) surface areas of the Sr0, Sr0.25, and Sr0.5 samples are 3 to 5.6 times higher than that of the Sr1 sample due to the difference in their shape and size. In addition, BJH total pore volumes of Sr0, Sr0.25, and Sr0.5 samples are approximately 2.4 times larger than that of the Sr1

Table 2. BET Surface Area, BJH Total Pore Volume, Average Pore Size, DLA, and DLE of Samplesa sample

BET surface area (m2/g)

BJH total pore volume (cm3/g)

average pore size (nm)

DLA (mg/g)

DLE (%)

Sr0 Sr0.25 Sr0.5 Sr1

135.99 133.38 79.64 24.44

0.82 0.87 0.78 0.35

19.79 29.29 22.40 25.52

6.89 7.09 5.80 3.84

57.4 59.1 48.3 32

a

Drug-loading amount (DLA: mloaded drug/mcarrier, mg/g) and drugloading efficiency (DLE: mloaded drug/mtotal drug amount, %).

sample as well. The surface area, pore volume, and average pore size of a particle directly affect the drug-loading ability. The drug-loading amount (DLA: mloaded drug/mcarrier, mg/g) and drug-loading efficiency (DLE: mloaded drug/mtotal drug amount, %) were calculated to evaluate the capacity of the synthetic SrHAP samples as a drug carrier by using vancomycin (VCM) as a model drug (see Table 2). Since VCM have strong absorbance at 280 nm (Figure S4), it was possible to calculate DLA and DLE by comparing the UV−vis spectra of media before and after drug loading. Sr0.25 sample had the highest DLA and DLE among all the samples (Table 2). This means that the Sr0.25 sample exhibits not only high BET surface area but also better pore accessibility than other samples. VCM is adsorbed onto the surface of particles via hydrogen bonding interaction between carboxyl (COOH) groups of VCM and G

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either hydroxyl (OH) or COOH groups of apatite crystals.28 Thus, the OH and COOH groups present on surface help to increase the amount of drug molecules loaded to the SrHAP. In vitro release studies were performed in PBS (pH 7.4) at 37 °C. Figure 5b shows the cumulative drug release ratio of each sample. The release rate is affected by many factors, including the surface properties and the morphologies of the drug carriers. The kinetics of drug release proceeds in two stages: the initial fast release followed by sustained slow release. The initial fast release (the first 6 h) occurs through the breakage of the hydrogen bonding of VCM on the outer surface of SrHAP particle.17 As carboxyl groups present on the surface of HAP originate from L-Asp, we believe Sr1 to have the smallest amount of carboxyl groups on the surface among all samples. In contrast, as the amount of hydroxyl groups on the surface of a sample depends on its surface area, Sr1 will have the smallest amount of OH groups among the samples. The VCM release ratio is relatively slow for Sr0 and Sr0.25 in the early stages due to their relatively large surface area and large amount of OH and COOH groups present on the surface. The strong binding of VCM on OH and COOH on the particles will prevent burst drug release. In contrast, VCM release of Sr1 occurs quickly in the early stages because of the small amount of OH and COOH groups present on the surface of the sample. After initial release, the drug release rate decreased after 6 h and saturated after 72 h for all samples. However, the samples produced different sustained drug-release behaviors. Sr1 sample released most of the drug loaded on the particle after approximately 72 h. We think this complete drug release is due to the absence of the mesoporosity in Sr1 particle. The amount of drug released becomes smaller for Sr0.5 and Sr0 samples than Sr1. As expected, Sr0.25 had the biggest amount of sustained VCM on the powders. The amount of total drug release is controlled by the porosity and surface area of the drug carriers. The Sr0.25 sample has maximum pore volume of 0.87 cm3/g, largest average pore size of 29.29 nm, and the most complex pore size distribution among all samples. Thus, Sr0.25 has optimal structure for controllable and sustained drug release. Furthermore, the PL intensity of VCM-SrHAP (Sr0.25 sample) is influenced by the cumulative release of VCM as shown in Figure 5c,d. The results show that the emission and excitation intensity of the luminescent sample changes as a function of drug release time. VCM is reported to quench the PL intensity when loaded on SrHAP microsphere because it prevents the electron−hole recombination on the surface of SrHAP.29 This quenching effect become weakened as VCM is released from the SrHAP surface, resulting in the gradual increase of PL intensity. This indicates that SrHAP can work as a smart drug delivery system, capable of monitoring drug release via luminescence. Biocompatibility Test. Biocompatibility is an important property required for a material for drug delivery, implantation, and other biomedical applications. In order to investigate the cytotoxicity of the synthesized particles, we conducted MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay after treating MC3T3-E1 cells with the particles. We treated the cells with 0.5 mg/mL particles for 3 days before conducting MTT assay. The MTT viability assay used in cytotoxicity testing is shown in Figure 6. The viabilities of the cells treated with the SrHAP particles are similar to those of the control sample with no particle treatment. This suggests that the synthesized SrHAP particles do not have significant

Figure 6. Results of an MTT assay using MC3T3-E1 cells with different synthetic SrHAP samples.

cytotoxicity issues regardless of the Sr/[Ca+Sr] molar ratio of the samples.

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CONCLUSIONS In summary, mesoporous SrHAP (strontium substituted hydroxyapatite) microspheres were successfully prepared via hydrothermal method using PASP (poly(aspartic acid)) as a template by controlling various parameters, including hydrothermal reaction time, amount of L-Asp (L-aspartic acid), and ratio of Sr ions. The amount of Sr substitution turned out to affect the size, morphology, and surface area of the particle, which crucially determine its drug delivery efficiency. In particular, the multifunctional SrHAP with molar ratios (Sr/ [Ca + Sr]) of 0.25 showed the strongest luminescence as well as superior drug loading and sustained-release properties due to its large surface area and large quantity of OH and COOH groups present on the surface. Our study suggests that the fabricated multifunctional SrHAP microspheres can be very useful as a next generation bone graft material with drug delivery and monitoring functionality.

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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.6b00420.

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SEM images, FT-IR spectra, Raman spectra, UV−vis spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], Tel: +82-2-2224-2274; Fax: +822-470-2088. *E-mail: [email protected], Tel: +82-31-290-7404; Fax: +82-31290-7410. *E-mail: [email protected], Tel: +82-31-290-7403; Fax: +8231-290-7410. Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acs.cgd.6b00420 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Article

(31) Geng, Z.; Cui, Z.; Li, Z.; Zhu, S.; Liang, Y.; Lu, W. W.; Yang, X. J. Mater. Chem. B 2015, 3 (18), 3738−3746.

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2014R1A4A1008474 and 2014R1A2A2A01007722).

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DOI: 10.1021/acs.cgd.6b00420 Cryst. Growth Des. XXXX, XXX, XXX−XXX