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Hydroxyapatite nanorod-assembled hierarchical microflowers: rapid synthesis via microwave hydrothermal transformation of CaHPO4 and their application in protein/drug delivery ⁎
⁎
Ya-Dong Yua,b, Ying-Jie Zhua,b, , Chao Qia, Jin Wua,
a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
A R T I C L E I N F O
A BS T RAC T
Keywords: Hydroxyapatite Nanorods Triethyl phosphate Drug delivery
Hydroxyapatite (HAP) nanostructured materials have attracted much attention due to their excellent biocompatibility and promising applications in various biomedical fields. In this study, a facile method has been developed to synthesize HAP with flower-like hierarchical nanostructures. The flower-like CaHPO4 precursor is firstly synthesized using triethyl phosphate (TEP) as the organic phosphorus source by the solvothermal method. The HAP hierarchical microflowers constructed with nanorods are then fabricated through rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. The as-prepared HAP nanorod-assembled hierarchical microflowers are explored to study the protein/drug loading and release properties using hemoglobin (Hb) and ibuprofen (IBU) as a model protein and drug, respectively. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have relatively large specific surface area, high biocompatibility, high protein/drug loading capacity and pH-dependent sustained release properties. Thus, the as-prepared HAP nanorod-assembled hierarchical microflowers are promising for the applications in protein/drug delivery.
1. Introduction Hydroxyapatite [Ca10(PO4)6(OH)2, HAP], as the main inorganic component of human hard tissues such as teeth and bones, has a wide range of applications due to its excellent properties such as high biocompatibility [1–3]. HAP biomaterials have been extensively investigated for applications in bone repair and tissue engineering [4–7], and as adsorbents [8–10]. Moreover, HAP biomaterials are excellent candidates as drug or protein delivery carriers [11–13] owing to the fact that their degradation products could be absorbed by the body [14]. In recent years, various nanostructured HAP biomaterials with different morphologies such as HAP nanowires [15], nanoworms [1], nanoflowers [16], and hierarchically nanostructured porous microspheres [17–20] have been synthesized. Among these nanostructured materials, the hierarchically flower-like HAP nanostructures are promising carrier materials because of their unique nanostructures, high biocompatibility, and superior drug/protein loading and release properties. Up to now, various strategies have been developed for the synthesis of flower-like HAP materials. For example, Zhao et al.
prepared HAP nanosheet-assembled flower-like hierarchical nanostructures by the rapid microwave-assisted hydrothermal method [21]. Zhang et al. prepared HAP microflowers consisting of microsheets by the hydrothermal method [22]. Wang et al. fabricated HAP nanosheetassembled flower-like hollow spheres by the microwave-assisted hydrothermal method [23]. However, these HAP flower-like materials usually have relatively small specific surface areas. To overcome this disadvantage, one smart strategy is to prepare complex hierarchical HAP nanostructured materials from a less complex precursor, during which process the change of chemical component and/or crystal phase contributes to the further building of the secondary structure. CaHPO4 has been used as a precursor for the preparation of HAP. For example, Ito et al. prepared oriented arrays of bundled nanoneedles of HAP through the hydrolysis of CaHPO4 in alkali solution [24]. Furuichi et al. prepared organized, nanotextured and nanofibrous HAP through CaHPO4 as the precursor [25]. Zou et al. fabricated biomimetic dental enamel-like HAP bundles through microwave treatment of CaHPO4 in NaOH aqueous solution [26]. In addition, other related works have also been reported [27–29].
⁎ Corresponding author at: State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China. E-mail addresses: [email protected] (Y.-J. Zhu), [email protected] (J. Wu).
http://dx.doi.org/10.1016/j.ceramint.2017.02.073 Received 3 January 2017; Received in revised form 1 February 2017; Accepted 16 February 2017 0272-8842/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Yu, Y.-D., Ceramics International (2017), http://dx.doi.org/10.1016/j.ceramint.2017.02.073
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2.3. Synthesis of HAP nanorod-assembled hierarchical microflowers
Among these methods, the microwave-assisted hydrothermal treatment of CaHPO4 in alkaline aqueous solution has several advantages over the others. For example, it is promising for rapid volumetric heating, thus resulting in time saving (usually minutes rather than hours or days), and low energy consumption. Flower-like hierarchical nanostructures of CaHPO4 are usually difficult to synthesize because CaHPO4 tends to grow with a single flake-like morphology. To prepare CaHPO4 flower-like hierarchical nanostructures, using an organic phosphorus source is a promising strategy. Organic phosphorus compounds such as triethyl phosphate (TEP) can hydrolyze to release PO43− ions under certain conditions, which can be used as organic phosphorus sources to avoid the fast nucleation and growth of calcium phosphate crystals [30–33]. To the best of our knowledge, there has been no report on the solvothermal synthesis of CaHPO4 microflowers constructed with nanosheets as the building blocks using TEP as an organic phosphorus source so far. Herein, the flower-like CaHPO4 precursor is synthesized using TEP as an organic phosphorus source by the solvothermal method. Then, HAP hierarchical microflowers constructed with nanorods as the building blocks are synthesized through rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. The as-prepared CaHPO4 precursor and HAP nanorod-assembled hierarchical microflowers are characterized. Additionally, the in vitro loading and release properties of the as-prepared HAP nanorodassembled hierarchical microflowers are also investigated using hemoglobin (Hb) and ibuprofen (IBU) as a model protein and drug, respectively. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have relatively a large specific surface area, high biocompatibility, superior protein/drug loading and pH-dependent sustained release properties. The HAP nanorod-assembled hierarchical microflowers reported herein are promising for various biomedical applications such as protein/drug delivery.
The HAP nanorod-assembled hierarchical microflowers were prepared via rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. In a typical synthesis procedure: the as-prepared CaHPO4 sample (100 mg) was immersed in 120 mL NaOH aqueous solution (1.0 M), which was then microwave heated at 120 °C for 5 min. The resultant product was centrifuged and washed three times with deionized water and ethanol, respectively, then freeze-dried. 2.4. In vitro protein adsorption and release The as-prepared HAP nanorod-assembled hierarchical microflowers (10 mg) were immersed in Hb aqueous solutions (5.0 mL) at variable concentrations (0.4–3.2 mg mL−1). The suspension was shaken at 37 °C with a constant rate (140 rpm) for 4 h. The amount of Hb in the centrifuged supernatant was measured by UV–vis absorption analysis at a wavelength of 405 nm. For the study of in vitro protein release performance, the as-prepared HAP nanorod-assembled hierarchical microflowers (100 mg) were immersed in 20 mL Hb aqueous solution with a concentration of 3.2 mg mL−1, and the solution was shaken at 37 °C with a constant rate (140 rpm) for 6 h for protein adsorption. The Hb-loaded drug delivery system was washed with deionized water, dried in a freeze dryer. Then the Hb-loaded drug delivery system (24 mg) was immersed in 24 mL phosphate buffered saline (PBS) with different pH values (pH 7.4 and 4.5) at 37 °C with constant shaking (140 rpm). The supernatant (1.0 mL) was withdrawn for UV–vis absorption analysis at the wavelength 405 nm at given time intervals and replaced with the same volume and pH value of fresh PBS. The data is representative as the mean value of three parallel measurements. 2.5. In vitro drug loading and release The as-prepared HAP nanorod-assembled hierarchical microflowers (120 mg) were added into 20 mL 40 mg mL−1 IBU hexane solution. The suspension was continuously shaken in a sealed vessel at 37 °C with a constant rate (140 rpm) for 6 h. The drug-loaded drug delivery system was centrifuged from the solution, dried, and compacted into disks (100 mg per disk) at a pressure of 4 MPa. Each disk was immersed into 24 mL PBS with different pH values at 37 °C with constant shaking (140 rpm). The drug release medium (1 mL) was withdrawn for UV–vis analysis at a wavelength of 264 nm at given time intervals and replaced with the same volume and pH value of fresh PBS. The data is representative as the mean value of three parallel measurements.
2. Experimental section 2.1. Materials Hemoglobin (Hb, > 98%) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Ibuprofen (IBU, 99.99%) was purchased from Shanghai Yuanji Chemical Co., Ltd. 96-Well flat bottom polystyrene TC-treated microplates (individually wrapped, with lid, sterile) were purchased from Corning Inc., USA. CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega Co., USA. The MG-63 human osteosarcoma cells were obtained from the Cells Resource Center, Shanghai Institutes for Biological Sciences, Shanghai, China. Other chemicals used in the synthesis of the samples were purchased from Sinopharm Chemical Reagent Co., China. All chemicals are of analytical grade and used as received without further purification.
2.6. In vitro cytotoxicity tests The cytotoxicity of the as-prepared HAP nanorod-assembled hierarchical microflowers was measured by the CellTiter-Glo® Luminescent Cell Viability Assay. The MG-63 human osteosarcoma cells were seeded in the 96-well microplates at a density of 1×104 cells per well and cultured in 100 µL Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a 5% CO2 incubator for 24 h. Then, the supernatant of each well was drawn off carefully and 100 µL of the as-prepared HAP nanorod-assembled hierarchical microflowers with fixed concentrations (0–50 µg mL−1) in DMEM containing 10% fetal bovine serum were added into the wells. The cells were incubated at 37 °C for another 24 h and 48 h. Finally, the microplate was taken out and 100 µL CellTiter-Glo® reagent was added into each well. After gentle shaking for 30 min, the luminescence was recorded by Synergy™ 2 Multi-Mode Microplate Reader. Cell viability is expressed as the percentage of the number of viable cells compared to the total number of cells. The data
2.2. Synthesis of the CaHPO4 microflower precursor In a typical synthesis procedure of the CaHPO4 microflowers, 0.660g CaCl2 was dissolved in mixed solvent of 48 mL deionized water and 12 mL ethylene glycol (EG). Then, TEP (2 mL) was added to the above solution under magnetic stirring for 15 min at room temperature. NaOH aqueous solution (1.0 M, 4 mL) was added dropwise. After magnetic stirring for 15 min, the clear solution was transferred into a 100-mL Teflon-lined stainless steel autoclave and maintained at 200 °C for 24 h. After cooling to room temperature, ethanol was added, and the white precipitate was obtained by centrifugation, washed with ethanol and deionized water three times, respectively, and dried at 60 °C for 24 h. 2
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is representative as the mean value of five parallel measurements. 2.7. Characterization The samples were characterized using X-ray powder diffraction (XRD) (Rigaku D/max 2550V, Cu-Kα radiation, λ=1.54178 Å), scanning electron microscopy (SEM, Hitachi SU8220, Japan), transmission electron microscopy (TEM, Hitachi H-800, Japan) with selected-area electron diffraction (SAED), Fourier transform infrared (FTIR) (FTIR7600, Lambda Scientific, Australia), Brunauer–Emmett–Teller (BET) surface area, Barrett–Joyner–Halenda (BJH) pore-size distribution (VSorb 2800P, Gold APP, China), and thermogravimetric (TG) analysis (STA 409/PC, Netzsch, Germany, at a heating rate of 10 °C min−1 in flowing air). The IBU and Hb concentrations were analyzed using a UV–vis spectrophotometer (UV-2300, Techcomp) at wavelengths of 264 nm and 405 nm, respectively. The Zeta potential values were measured using a Zeta potential analyzer (ZetaPlus, Brookhaven Instruments Corporation). 3. Results and discussion The strategy for the synthesis of HAP nanorod-assembled hierarchical microflowers is concisely illustrated in Fig. 1. CaCl2 and TEP are used as sources of calcium and phosphorus, respectively, and NaOH is used as the hydrolysis initiator of TEP. Firstly, TEP molecules hydrolyze to produce H2PO4− ions and H3PO4 molecules under solvothermal conditions at high temperatures and high pressures and strong basicity reaction system. Then, Ca2+ ions react with H2PO4− ions and H3PO4 molecules to form CaHPO4 nuclei, and newly formed nuclei grow into CaHPO4 nanosheets, which self-assemble into CaHPO4 microflowers. Then, the CaHPO4 microflowers as the precursor transform to HAP nanorod-assembled hierarchical microflowers in NaOH aqueous solution under microwave hydrothermal conditions. The possible chemical reactions are listed as following Eqs. (1) and (2). 3O=P(OC2H5)3 + OH− + 8H2O → 9C2H5OH + 2H3PO4 + H2PO4− (1) 2Ca
2+
+ H3PO4 +
H2PO4−
→ 2CaHPO4 + 3H
+
Fig. 2. SEM and TEM micrographs (the insets are SAED patterns) of the as-prepared CaHPO4 microflowers as the precursor and HAP nanorod-assembled hierarchical microflowers: (a)-(d) CaHPO4 microflowers prepared using TEP as the organic phosphorus source by the solvothermal method; (e)-(h) HAP nanorod-assembled hierarchical microflowers prepared by rapid microwave hydrothermal transformation of the asprepared CaHPO4 precursor in NaOH aqueous solution.
(2)
The pH values of the precursor solution before and after the solvothermal treatment were measured, as shown in the histogram of Fig. 1. It can be seen that the reaction system changes from alkaline to acidic after the solvothermal treatment, which is consistent with the above reaction equations. Fig. 2 shows the SEM and TEM images of the CaHPO4 precursor and HAP nanorod-assembled hierarchical microflowers. As shown in Fig. 2a-d, it can be seen that the as-prepared CaHPO4 precursor consists of microflowers which are formed by self-assembled nanosheets, the sizes of microflowers are 10–15 µm. The SAED pattern
in Fig. 2d indicates that the as-prepared CaHPO4 microflowers are well crystallized. From Fig. 2e-h, one can see that the as-prepared product has a similar morphology and sizes to the CaHPO4 microflowers after the rapid microwave hydrothermal transformation process, and it is composed of HAP nanorod-assembled hierarchical microflowers. Apparently, the CaHPO4 microflowers play a role as the template in the formation of the HAP nanorod-assembled hierarchical microflowers. However, the building blocks of the microflowers change from nanosheets into nanorods (Fig. 2c and g), and the HAP nanorods have a preference for oriented attachment. The SAED in Fig. 2h shows that the as-prepared HAP nanorod-assembled hierarchical microflowers are in low crystallinity. The XRD patterns of the samples are shown in Fig. 3. The XRD pattern of the product prepared using TEP as the organic phosphorus source by the solvothermal method (Fig. 3a) is identified to be singlephase CaHPO4 (JCPDF Card No. 70-0359), and the high intensities of the diffraction peaks indicate a highly crystallized CaHPO4 [24,28,34]. After the rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution, the CaHPO4 precursor transforms into single-phase HAP (JCPDF Card No. 09–0432), as shown in Fig. 3b. The FTIR spectrum shown in Fig. 4a confirms the functional groups of the as-prepared CaHPO4 precursor. The peaks at 577, 899, 1003, 1068, and 1128 cm−1 are attributed to the characteristic bands of
Fig. 1. Illustration of the synthetic route to the CaHPO4 precursor and HAP nanorodassembled hierarchical microflowers. The inset histogram shows pH values of the precursor solution before and after the solvothermal treatment.
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Fig. 5. TG curves of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
Fig. 3. XRD patterns of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
Fig. 4. FTIR spectra of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
PO43−. The broad peaks at 3401 and 1653 cm−1 are assigned to the OH stretching mode and H-O-H bending mode of residual free water, respectively [24,27,35]. After rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution (Fig. 4b), the broad peaks at around 3401 cm−1 and 1653 cm−1 are assigned to the adsorbed water of HAP nanorod-assembled hierarchical microflowers. The peak at 1400 cm−1 is assigned to P-O-H scissoring vibration mode [26,36]. The characteristic bands of PO43− ions are located at about 1092, 1034, 604 and 563 cm-1 [32,37,38]. The thermal analysis represents one of convenient ways to investigate the thermal stability of materials. We investigated the TG curves of the as-prepared CaHPO4 precursor before and after rapid microwave hydrothermal treatment. Fig. 5a shows a rapid and relatively large weight loss at 100–400 °C, the main weight loss can be explained by that CaHPO4 transforms into dicalcium pyrophosphate (Ca2P2O7) by heating in air at the temperature range of 300–400 °C [39]. Fig. 5b shows the TG curve of HAP nanorod-assembled hierarchical microflowers, indicating that the weight loss is small due to the loss of adsorbed water in the HAP sample, and the residual weight is 94.3% at 650 °C. Nitrogen adsorption-desorption isotherms and the pore-size distributions of the as-prepared CaHPO4 microflowers and HAP nanorodassembled hierarchical microflowers were measured to analyse the specific surface area and porous structure of the samples. As shown in Fig. 6a, the BET specific surface area of HAP nanorod-assembled hierarchical microflowers is 72.1 m2 g−1, which is much higher than
Fig. 6. Nitrogen adsorption-desorption isotherms (a) and the pore-size distributions (b) of the as-prepared CaHPO4 microflowers and HAP nanorod-assembled hierarchical microflowers.
that of CaHPO4 microflowers (5.5 m2 g−1). From Fig. 6b, one can see that the as-prepared CaHPO4 microflowers and HAP nanorod-assembled hierarchical microflowers have similar average pore size of about 13 nm. However, the corresponding BJH desorption cumulative pore volume (VP) of HAP nanorod-assembled hierarchical microflowers is 0.363 cm3 g−1, which is much higher than that of CaHPO4 microflowers (0.088 cm3 g−1), and this result is in accordance with the analysis of the BET specific surface area. The relatively large specific surface area and cumulative pore volume of HAP nanorod-assembled hierarchical microflowers are favorable for the application in protein and drug delivery. We investigated the protein adsorption and release performance of the as-prepared HAP nanorod-assembled hierarchical microflowers using Hb as a model protein. By comparing the FTIR spectra of HAP nanorod-assembled hierarchical microflowers before and after Hb
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of 3.2 mg mL−1. Such a good Hb adsorption ability may be attributed to the relatively large specific surface area and pore volume of the HAP nanorod-assembled hierarchical microflowers, which provide available physical space and adsorption sites to load Hb molecules. However, the Hb loading efficiency decreases with increasing initial concentration of Hb. Langmuir isotherm model (Eq. (3)) is adopted to fit the experimental data [41],
Ce C 1 = + e qe qmb qm
(3)
where ce equals to the equilibrium concentration of Hb in aqueous solution, and qe equals to equilibrium adsorption quantity (mg Hb per gram adsorbent), qm and b represent the maximum monolayer adsorption capacity (mg g−1) and Langmuir adsorption constant, respectively. Fig. 8b shows the adsorption isotherms of Hb fitted by the Langmuir model. The correlation coefficient R2 (0.995) is high, implying that the Langmuir model is appropriate for interpreting the adsorption behavior of Hb molecules on HAP nanorod-assembled hierarchical microflowers. The good Hb adsorption properties of HAP nanorod-assembled hierarchical microflowers may also result from the electrostatic interactions (see below). We investigated the adsorbed amount of Hb on HAP nanorodassembled hierarchical microflowers in PBS with different pH values at a Hb concentration of 2 mg mL−1. TG analysis is employed to estimate the Hb adsorption capacity of HAP nanorod-assembled hierarchical microflowers. As shown in Fig. 9, a weight loss of approximately 5.7% of HAP nanorod-assembled hierarchical microflowers is observed without Hb adsorption, whereas a weight loss of about 12.0% with pH value 7.4, a weight loss of about 16.8% with pH value 6.0 and a weight loss of about 17.8% with pH value 4.5 were measured for Hb adsorption on HAP nanorod-assembled hierarchical microflowers in PBS. These experimental results implies that the adsorbed amount of Hb on HAP nanorod-assembled hierarchical microflowers increases with decreasing pH value. It can be explained by the electrostatic interactions between Hb molecules and HAP nanorod-assembled hierarchical nanoflowrers. In general, protein adsorption is attributed to surface interactions between protein and carriers. The isoelectric point (pI) of Hb is within the range 6.8–7.0 [41,42]. As shown in Fig. 10, when the pH value of PBS is below the pI value of Hb, the Hb molecules become positively charged, whereas when the pH value is higher than the pI value, the Hb molecules become negatively charged. The HAP nanorod-assembled hierarchical microflowers as the carrier are negatively charged in PBS at pH 7.4, 6.0 and 4.5. Thus, when the pH value of PBS is 7.4, the electrostatic repulsion effect decreases the adsorption of Hb on HAP nanorod-assembled hierarchical micro-
Fig. 7. FTIR spectra of the as-prepared HAP nanorod-assembled hierarchical microflowers, Hb-loaded HAP nanorod-assembled hierarchical microflowers, and pure Hb.
adsorption with that of pure Hb (Fig. 7), it is found that two obvious amide peaks at about 1652 and 1538 cm−1 appear after the Hb adsorption, which originate from the adsorbed Hb molecules [40,41]. As shown in Fig. 8a, the adsorption of Hb on the as-prepared HAP nanorod-assembled hierarchical microflowers was investigated at different initial concentrations of Hb aqueous solution. The Hb adsorption capacity of HAP nanorod-assembled hierarchical microflowers increases rapidly with increasing initial concentration of Hb aqueous solution, and reaches 137 mg g−1 at a Hb initial concentration
Fig. 9. TG curves of the as-prepared HAP nanorod-assembled hierarchical microflowers before and after Hb adsorption in PBS with different pH values. The initial Hb concentration is 2 mg mL−1.
Fig. 8. (a) Hb adsorption curve and Hb adsorption efficiency of the as-prepared HAP nanorod-assembled hierarchical microflowers versus the initial concentrations of Hb aqueous solution; (b) the linear relationship between ce/qe and ce.
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Fig. 12. TG curves of the as-prepared HAP nanorod-assembled hierarchical microflowers before and after IBU loading.
Fig. 10. Zeta potentials of the as-prepared HAP nanorod-assembled hierarchical microflowers and Hb in PBS with different pH values.
flowers. When the pH values are 6.0 and 4.5, the electrostatic attraction effect increases the adsorption of Hb, and the adsorption amount of Hb at pH 4.5 is more than that at pH 6.0 owing to the stronger electrostatic attraction effect. Fig. 11 shows the Hb release performance of the as-prepared Hbloaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values. It shows that the release of Hb in PBS is pHdependent. The cumulative amount of released Hb reaches a plateau of 52.6% at pH 7.4 after 14 h (Fig. 11a), and 44.9% at pH 4.5 after 14 h (Fig. 11b). The amount of Hb released from HAP nanorod-assembled hierarchical microflowers in PBS at pH 7.4 is higher than that at pH 4.5. This result can be explained by the electrostatic interactions between Hb molecules and HAP nanorod-assembled hierarchical microflowers, which has been discussed above. We also investigated the drug loading and release performance of the as-prepared HAP nanorod-assembled hierarchical microflowers using IBU as the model drug. TG analysis is employed to estimate the IBU drug loading capacity. As shown in Fig. 12, the HAP nanorodassembled hierarchical microflowers and the corresponding IBUloaded sample have weight losses of about 5.6% and 25.1%, respectively. The IBU loading capacity of HAP nanorod-assembled hierarchical microflowers is about 260.3 mg g−1 (mg drug per gram carrier). This relatively high drug loading capacity may be explained by the relatively large specific surface area and pore volume of HAP nanorodassembled hierarchical microflowers. Fig. 13 shows the IBU drug release behavior of the HAP nanorod-assembled hierarchical microflower drug delivery system in PBS with different pH values, indicating
Fig. 13. IBU drug release profile of IBU-loaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values. The inset shows the natural logarithm of the cumulative IBU release amount as a function of the natural logarithm of the drug release time in the initial 24 h.
that the IBU drug release in PBS is dependent on the pH value. The cumulative IBU release amount reaches a plateau of 69.1% at pH 7.4 after 48 h, and 63.6% at pH 6.0 after 216 h, and 57.9% at pH 4.5 after 288 h. There is a carboxyl group existed in the IBU molecule. It can partially ionize to produce H+ ions in the aqueous solution. However, the H+ ions existed in the acidic solution (PBS, pH 6.0 or 4.5) can inhibit the ionization of the IBU molecules. Hence, the solubility of IBU at different pH values has an influence on the ultimate cumulative IBU release amount, because the solubility of IBU molecules increases with increasing pH value. The ultimate cumulative IBU release amount in PBS at pH 7.4 is obviously higher than that at pH 6.0 or 4.5, which is attributed to the larger solubility of IBU in more alkaline PBS at pH 7.4. The sustained IBU drug release at pH 6.0 and 4.5 is obvious because the IBU-loaded HAP nanorod-assembled hierarchical microflowers dissolve slowly to release IBU molecules under the weakly acidic condition. Moreover, the HAP nanorod-assembled hierarchical microflower drug delivery system exhibits a good linear relationship between the natural logarithm of the cumulative IBU release amount and the natural logarithm of drug release time in PBS with different pH values in the initial 24 h (the inset in Fig. 13). It is well acknowledged that the kinetics of drug release from carrier materials can be well described using the Higuchi model (C = k·t1/2), with a linear relationship between the cumulative amount of released drug (C), and the square root of drug release time (t1/2), where k is a constant, and the drug release is governed by a diffusion process [40,43]. However, a formula can be inferred for the HAP nanorod-assembled hierarchical
Fig. 11. Hb release curves of Hb-loaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values: (a) pH 7.4; (b) pH 4.5.
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[2]
[3] [4]
[5]
[6]
[7]
Fig. 14. Cytotoxicity tests of the as-prepared HAP nanorod-assembled hierarchical microflowers using MG-63 human osteosarcoma cells.
[8]
microflower drug delivery system in PBS:
[9]
n
C = k·t (t ≤ 24 h ) [10]
where n is a constant differing for different pH values of PBS. For the HAP nanorod-assembled hierarchical microflower drug delivery systems, the values of n are determined to be 0.5633 (pH 7.4), 0.5784 (pH 6.0), and 0.7724 (pH 4.5), respectively. The cytotoxicity tests of the as-prepared HAP nanorod-assembled hierarchical microflowers were performed using MG-63 human osteosarcoma cells. The CellTiter-Glo® Luminescent Cell Viability Assay shows no appreciable toxicity when the cells were co-cultured with the HAP nanorod-assembled hierarchical microflowers at concentrations of 0.1–50 µg mL−1 for 24 h and 48 h (Fig. 14). The high cytocompatibility may be explained by the chemical nature of HAP nanorodassembled hierarchical microflowers, which is similar to the inorganic constituent of the biological hard tissues [44,45]. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have promising applications in various biomedical fields.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
4. Conclusions
[18]
In summary, we have successfully synthesized HAP nanorodassembled hierarchical microflowers by rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution, and the CaHPO4 microflower precursor is prepared using TEP as an organic phosphorus source by the solvothermal method. The HAP nanorod-assembled hierarchical microflowers are explored for potential application in protein/drug delivery taking advantage of their high biocompatibility and relatively large specific surface area. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have a relatively high drug/protein loading capacity. Moreover, the protein/drug delivery system shows a pHdependent and sustained release behavior. Therefore, the as-prepared HAP nanorod-assembled hierarchical microflowers are promising for various biomedical applications such as pH-dependent protein/drug delivery.
[19]
[20]
[21]
[22]
[23]
[24]
Acknowledgements
[25]
The financial support from the National Natural Science Foundation of China (51302294) and the Science and Technology Commission of Shanghai (15JC1491001) is gratefully acknowledged.
[26] [27]
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Ceramics International journal homepage: www.elsevier.com/locate/ceramint
Hydroxyapatite nanorod-assembled hierarchical microflowers: rapid synthesis via microwave hydrothermal transformation of CaHPO4 and their application in protein/drug delivery ⁎
⁎
Ya-Dong Yua,b, Ying-Jie Zhua,b, , Chao Qia, Jin Wua,
a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
A R T I C L E I N F O
A BS T RAC T
Keywords: Hydroxyapatite Nanorods Triethyl phosphate Drug delivery
Hydroxyapatite (HAP) nanostructured materials have attracted much attention due to their excellent biocompatibility and promising applications in various biomedical fields. In this study, a facile method has been developed to synthesize HAP with flower-like hierarchical nanostructures. The flower-like CaHPO4 precursor is firstly synthesized using triethyl phosphate (TEP) as the organic phosphorus source by the solvothermal method. The HAP hierarchical microflowers constructed with nanorods are then fabricated through rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. The as-prepared HAP nanorod-assembled hierarchical microflowers are explored to study the protein/drug loading and release properties using hemoglobin (Hb) and ibuprofen (IBU) as a model protein and drug, respectively. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have relatively large specific surface area, high biocompatibility, high protein/drug loading capacity and pH-dependent sustained release properties. Thus, the as-prepared HAP nanorod-assembled hierarchical microflowers are promising for the applications in protein/drug delivery.
1. Introduction Hydroxyapatite [Ca10(PO4)6(OH)2, HAP], as the main inorganic component of human hard tissues such as teeth and bones, has a wide range of applications due to its excellent properties such as high biocompatibility [1–3]. HAP biomaterials have been extensively investigated for applications in bone repair and tissue engineering [4–7], and as adsorbents [8–10]. Moreover, HAP biomaterials are excellent candidates as drug or protein delivery carriers [11–13] owing to the fact that their degradation products could be absorbed by the body [14]. In recent years, various nanostructured HAP biomaterials with different morphologies such as HAP nanowires [15], nanoworms [1], nanoflowers [16], and hierarchically nanostructured porous microspheres [17–20] have been synthesized. Among these nanostructured materials, the hierarchically flower-like HAP nanostructures are promising carrier materials because of their unique nanostructures, high biocompatibility, and superior drug/protein loading and release properties. Up to now, various strategies have been developed for the synthesis of flower-like HAP materials. For example, Zhao et al.
prepared HAP nanosheet-assembled flower-like hierarchical nanostructures by the rapid microwave-assisted hydrothermal method [21]. Zhang et al. prepared HAP microflowers consisting of microsheets by the hydrothermal method [22]. Wang et al. fabricated HAP nanosheetassembled flower-like hollow spheres by the microwave-assisted hydrothermal method [23]. However, these HAP flower-like materials usually have relatively small specific surface areas. To overcome this disadvantage, one smart strategy is to prepare complex hierarchical HAP nanostructured materials from a less complex precursor, during which process the change of chemical component and/or crystal phase contributes to the further building of the secondary structure. CaHPO4 has been used as a precursor for the preparation of HAP. For example, Ito et al. prepared oriented arrays of bundled nanoneedles of HAP through the hydrolysis of CaHPO4 in alkali solution [24]. Furuichi et al. prepared organized, nanotextured and nanofibrous HAP through CaHPO4 as the precursor [25]. Zou et al. fabricated biomimetic dental enamel-like HAP bundles through microwave treatment of CaHPO4 in NaOH aqueous solution [26]. In addition, other related works have also been reported [27–29].
⁎ Corresponding author at: State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China. E-mail addresses: [email protected] (Y.-J. Zhu), [email protected] (J. Wu).
http://dx.doi.org/10.1016/j.ceramint.2017.02.073 Received 3 January 2017; Received in revised form 1 February 2017; Accepted 16 February 2017 0272-8842/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Yu, Y.-D., Ceramics International (2017), http://dx.doi.org/10.1016/j.ceramint.2017.02.073
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2.3. Synthesis of HAP nanorod-assembled hierarchical microflowers
Among these methods, the microwave-assisted hydrothermal treatment of CaHPO4 in alkaline aqueous solution has several advantages over the others. For example, it is promising for rapid volumetric heating, thus resulting in time saving (usually minutes rather than hours or days), and low energy consumption. Flower-like hierarchical nanostructures of CaHPO4 are usually difficult to synthesize because CaHPO4 tends to grow with a single flake-like morphology. To prepare CaHPO4 flower-like hierarchical nanostructures, using an organic phosphorus source is a promising strategy. Organic phosphorus compounds such as triethyl phosphate (TEP) can hydrolyze to release PO43− ions under certain conditions, which can be used as organic phosphorus sources to avoid the fast nucleation and growth of calcium phosphate crystals [30–33]. To the best of our knowledge, there has been no report on the solvothermal synthesis of CaHPO4 microflowers constructed with nanosheets as the building blocks using TEP as an organic phosphorus source so far. Herein, the flower-like CaHPO4 precursor is synthesized using TEP as an organic phosphorus source by the solvothermal method. Then, HAP hierarchical microflowers constructed with nanorods as the building blocks are synthesized through rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. The as-prepared CaHPO4 precursor and HAP nanorod-assembled hierarchical microflowers are characterized. Additionally, the in vitro loading and release properties of the as-prepared HAP nanorodassembled hierarchical microflowers are also investigated using hemoglobin (Hb) and ibuprofen (IBU) as a model protein and drug, respectively. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have relatively a large specific surface area, high biocompatibility, superior protein/drug loading and pH-dependent sustained release properties. The HAP nanorod-assembled hierarchical microflowers reported herein are promising for various biomedical applications such as protein/drug delivery.
The HAP nanorod-assembled hierarchical microflowers were prepared via rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution. In a typical synthesis procedure: the as-prepared CaHPO4 sample (100 mg) was immersed in 120 mL NaOH aqueous solution (1.0 M), which was then microwave heated at 120 °C for 5 min. The resultant product was centrifuged and washed three times with deionized water and ethanol, respectively, then freeze-dried. 2.4. In vitro protein adsorption and release The as-prepared HAP nanorod-assembled hierarchical microflowers (10 mg) were immersed in Hb aqueous solutions (5.0 mL) at variable concentrations (0.4–3.2 mg mL−1). The suspension was shaken at 37 °C with a constant rate (140 rpm) for 4 h. The amount of Hb in the centrifuged supernatant was measured by UV–vis absorption analysis at a wavelength of 405 nm. For the study of in vitro protein release performance, the as-prepared HAP nanorod-assembled hierarchical microflowers (100 mg) were immersed in 20 mL Hb aqueous solution with a concentration of 3.2 mg mL−1, and the solution was shaken at 37 °C with a constant rate (140 rpm) for 6 h for protein adsorption. The Hb-loaded drug delivery system was washed with deionized water, dried in a freeze dryer. Then the Hb-loaded drug delivery system (24 mg) was immersed in 24 mL phosphate buffered saline (PBS) with different pH values (pH 7.4 and 4.5) at 37 °C with constant shaking (140 rpm). The supernatant (1.0 mL) was withdrawn for UV–vis absorption analysis at the wavelength 405 nm at given time intervals and replaced with the same volume and pH value of fresh PBS. The data is representative as the mean value of three parallel measurements. 2.5. In vitro drug loading and release The as-prepared HAP nanorod-assembled hierarchical microflowers (120 mg) were added into 20 mL 40 mg mL−1 IBU hexane solution. The suspension was continuously shaken in a sealed vessel at 37 °C with a constant rate (140 rpm) for 6 h. The drug-loaded drug delivery system was centrifuged from the solution, dried, and compacted into disks (100 mg per disk) at a pressure of 4 MPa. Each disk was immersed into 24 mL PBS with different pH values at 37 °C with constant shaking (140 rpm). The drug release medium (1 mL) was withdrawn for UV–vis analysis at a wavelength of 264 nm at given time intervals and replaced with the same volume and pH value of fresh PBS. The data is representative as the mean value of three parallel measurements.
2. Experimental section 2.1. Materials Hemoglobin (Hb, > 98%) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Ibuprofen (IBU, 99.99%) was purchased from Shanghai Yuanji Chemical Co., Ltd. 96-Well flat bottom polystyrene TC-treated microplates (individually wrapped, with lid, sterile) were purchased from Corning Inc., USA. CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega Co., USA. The MG-63 human osteosarcoma cells were obtained from the Cells Resource Center, Shanghai Institutes for Biological Sciences, Shanghai, China. Other chemicals used in the synthesis of the samples were purchased from Sinopharm Chemical Reagent Co., China. All chemicals are of analytical grade and used as received without further purification.
2.6. In vitro cytotoxicity tests The cytotoxicity of the as-prepared HAP nanorod-assembled hierarchical microflowers was measured by the CellTiter-Glo® Luminescent Cell Viability Assay. The MG-63 human osteosarcoma cells were seeded in the 96-well microplates at a density of 1×104 cells per well and cultured in 100 µL Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a 5% CO2 incubator for 24 h. Then, the supernatant of each well was drawn off carefully and 100 µL of the as-prepared HAP nanorod-assembled hierarchical microflowers with fixed concentrations (0–50 µg mL−1) in DMEM containing 10% fetal bovine serum were added into the wells. The cells were incubated at 37 °C for another 24 h and 48 h. Finally, the microplate was taken out and 100 µL CellTiter-Glo® reagent was added into each well. After gentle shaking for 30 min, the luminescence was recorded by Synergy™ 2 Multi-Mode Microplate Reader. Cell viability is expressed as the percentage of the number of viable cells compared to the total number of cells. The data
2.2. Synthesis of the CaHPO4 microflower precursor In a typical synthesis procedure of the CaHPO4 microflowers, 0.660g CaCl2 was dissolved in mixed solvent of 48 mL deionized water and 12 mL ethylene glycol (EG). Then, TEP (2 mL) was added to the above solution under magnetic stirring for 15 min at room temperature. NaOH aqueous solution (1.0 M, 4 mL) was added dropwise. After magnetic stirring for 15 min, the clear solution was transferred into a 100-mL Teflon-lined stainless steel autoclave and maintained at 200 °C for 24 h. After cooling to room temperature, ethanol was added, and the white precipitate was obtained by centrifugation, washed with ethanol and deionized water three times, respectively, and dried at 60 °C for 24 h. 2
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is representative as the mean value of five parallel measurements. 2.7. Characterization The samples were characterized using X-ray powder diffraction (XRD) (Rigaku D/max 2550V, Cu-Kα radiation, λ=1.54178 Å), scanning electron microscopy (SEM, Hitachi SU8220, Japan), transmission electron microscopy (TEM, Hitachi H-800, Japan) with selected-area electron diffraction (SAED), Fourier transform infrared (FTIR) (FTIR7600, Lambda Scientific, Australia), Brunauer–Emmett–Teller (BET) surface area, Barrett–Joyner–Halenda (BJH) pore-size distribution (VSorb 2800P, Gold APP, China), and thermogravimetric (TG) analysis (STA 409/PC, Netzsch, Germany, at a heating rate of 10 °C min−1 in flowing air). The IBU and Hb concentrations were analyzed using a UV–vis spectrophotometer (UV-2300, Techcomp) at wavelengths of 264 nm and 405 nm, respectively. The Zeta potential values were measured using a Zeta potential analyzer (ZetaPlus, Brookhaven Instruments Corporation). 3. Results and discussion The strategy for the synthesis of HAP nanorod-assembled hierarchical microflowers is concisely illustrated in Fig. 1. CaCl2 and TEP are used as sources of calcium and phosphorus, respectively, and NaOH is used as the hydrolysis initiator of TEP. Firstly, TEP molecules hydrolyze to produce H2PO4− ions and H3PO4 molecules under solvothermal conditions at high temperatures and high pressures and strong basicity reaction system. Then, Ca2+ ions react with H2PO4− ions and H3PO4 molecules to form CaHPO4 nuclei, and newly formed nuclei grow into CaHPO4 nanosheets, which self-assemble into CaHPO4 microflowers. Then, the CaHPO4 microflowers as the precursor transform to HAP nanorod-assembled hierarchical microflowers in NaOH aqueous solution under microwave hydrothermal conditions. The possible chemical reactions are listed as following Eqs. (1) and (2). 3O=P(OC2H5)3 + OH− + 8H2O → 9C2H5OH + 2H3PO4 + H2PO4− (1) 2Ca
2+
+ H3PO4 +
H2PO4−
→ 2CaHPO4 + 3H
+
Fig. 2. SEM and TEM micrographs (the insets are SAED patterns) of the as-prepared CaHPO4 microflowers as the precursor and HAP nanorod-assembled hierarchical microflowers: (a)-(d) CaHPO4 microflowers prepared using TEP as the organic phosphorus source by the solvothermal method; (e)-(h) HAP nanorod-assembled hierarchical microflowers prepared by rapid microwave hydrothermal transformation of the asprepared CaHPO4 precursor in NaOH aqueous solution.
(2)
The pH values of the precursor solution before and after the solvothermal treatment were measured, as shown in the histogram of Fig. 1. It can be seen that the reaction system changes from alkaline to acidic after the solvothermal treatment, which is consistent with the above reaction equations. Fig. 2 shows the SEM and TEM images of the CaHPO4 precursor and HAP nanorod-assembled hierarchical microflowers. As shown in Fig. 2a-d, it can be seen that the as-prepared CaHPO4 precursor consists of microflowers which are formed by self-assembled nanosheets, the sizes of microflowers are 10–15 µm. The SAED pattern
in Fig. 2d indicates that the as-prepared CaHPO4 microflowers are well crystallized. From Fig. 2e-h, one can see that the as-prepared product has a similar morphology and sizes to the CaHPO4 microflowers after the rapid microwave hydrothermal transformation process, and it is composed of HAP nanorod-assembled hierarchical microflowers. Apparently, the CaHPO4 microflowers play a role as the template in the formation of the HAP nanorod-assembled hierarchical microflowers. However, the building blocks of the microflowers change from nanosheets into nanorods (Fig. 2c and g), and the HAP nanorods have a preference for oriented attachment. The SAED in Fig. 2h shows that the as-prepared HAP nanorod-assembled hierarchical microflowers are in low crystallinity. The XRD patterns of the samples are shown in Fig. 3. The XRD pattern of the product prepared using TEP as the organic phosphorus source by the solvothermal method (Fig. 3a) is identified to be singlephase CaHPO4 (JCPDF Card No. 70-0359), and the high intensities of the diffraction peaks indicate a highly crystallized CaHPO4 [24,28,34]. After the rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution, the CaHPO4 precursor transforms into single-phase HAP (JCPDF Card No. 09–0432), as shown in Fig. 3b. The FTIR spectrum shown in Fig. 4a confirms the functional groups of the as-prepared CaHPO4 precursor. The peaks at 577, 899, 1003, 1068, and 1128 cm−1 are attributed to the characteristic bands of
Fig. 1. Illustration of the synthetic route to the CaHPO4 precursor and HAP nanorodassembled hierarchical microflowers. The inset histogram shows pH values of the precursor solution before and after the solvothermal treatment.
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Fig. 5. TG curves of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
Fig. 3. XRD patterns of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
Fig. 4. FTIR spectra of the as-prepared CaHPO4 microflowers (a) and HAP nanorodassembled hierarchical microflowers (b).
PO43−. The broad peaks at 3401 and 1653 cm−1 are assigned to the OH stretching mode and H-O-H bending mode of residual free water, respectively [24,27,35]. After rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution (Fig. 4b), the broad peaks at around 3401 cm−1 and 1653 cm−1 are assigned to the adsorbed water of HAP nanorod-assembled hierarchical microflowers. The peak at 1400 cm−1 is assigned to P-O-H scissoring vibration mode [26,36]. The characteristic bands of PO43− ions are located at about 1092, 1034, 604 and 563 cm-1 [32,37,38]. The thermal analysis represents one of convenient ways to investigate the thermal stability of materials. We investigated the TG curves of the as-prepared CaHPO4 precursor before and after rapid microwave hydrothermal treatment. Fig. 5a shows a rapid and relatively large weight loss at 100–400 °C, the main weight loss can be explained by that CaHPO4 transforms into dicalcium pyrophosphate (Ca2P2O7) by heating in air at the temperature range of 300–400 °C [39]. Fig. 5b shows the TG curve of HAP nanorod-assembled hierarchical microflowers, indicating that the weight loss is small due to the loss of adsorbed water in the HAP sample, and the residual weight is 94.3% at 650 °C. Nitrogen adsorption-desorption isotherms and the pore-size distributions of the as-prepared CaHPO4 microflowers and HAP nanorodassembled hierarchical microflowers were measured to analyse the specific surface area and porous structure of the samples. As shown in Fig. 6a, the BET specific surface area of HAP nanorod-assembled hierarchical microflowers is 72.1 m2 g−1, which is much higher than
Fig. 6. Nitrogen adsorption-desorption isotherms (a) and the pore-size distributions (b) of the as-prepared CaHPO4 microflowers and HAP nanorod-assembled hierarchical microflowers.
that of CaHPO4 microflowers (5.5 m2 g−1). From Fig. 6b, one can see that the as-prepared CaHPO4 microflowers and HAP nanorod-assembled hierarchical microflowers have similar average pore size of about 13 nm. However, the corresponding BJH desorption cumulative pore volume (VP) of HAP nanorod-assembled hierarchical microflowers is 0.363 cm3 g−1, which is much higher than that of CaHPO4 microflowers (0.088 cm3 g−1), and this result is in accordance with the analysis of the BET specific surface area. The relatively large specific surface area and cumulative pore volume of HAP nanorod-assembled hierarchical microflowers are favorable for the application in protein and drug delivery. We investigated the protein adsorption and release performance of the as-prepared HAP nanorod-assembled hierarchical microflowers using Hb as a model protein. By comparing the FTIR spectra of HAP nanorod-assembled hierarchical microflowers before and after Hb
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of 3.2 mg mL−1. Such a good Hb adsorption ability may be attributed to the relatively large specific surface area and pore volume of the HAP nanorod-assembled hierarchical microflowers, which provide available physical space and adsorption sites to load Hb molecules. However, the Hb loading efficiency decreases with increasing initial concentration of Hb. Langmuir isotherm model (Eq. (3)) is adopted to fit the experimental data [41],
Ce C 1 = + e qe qmb qm
(3)
where ce equals to the equilibrium concentration of Hb in aqueous solution, and qe equals to equilibrium adsorption quantity (mg Hb per gram adsorbent), qm and b represent the maximum monolayer adsorption capacity (mg g−1) and Langmuir adsorption constant, respectively. Fig. 8b shows the adsorption isotherms of Hb fitted by the Langmuir model. The correlation coefficient R2 (0.995) is high, implying that the Langmuir model is appropriate for interpreting the adsorption behavior of Hb molecules on HAP nanorod-assembled hierarchical microflowers. The good Hb adsorption properties of HAP nanorod-assembled hierarchical microflowers may also result from the electrostatic interactions (see below). We investigated the adsorbed amount of Hb on HAP nanorodassembled hierarchical microflowers in PBS with different pH values at a Hb concentration of 2 mg mL−1. TG analysis is employed to estimate the Hb adsorption capacity of HAP nanorod-assembled hierarchical microflowers. As shown in Fig. 9, a weight loss of approximately 5.7% of HAP nanorod-assembled hierarchical microflowers is observed without Hb adsorption, whereas a weight loss of about 12.0% with pH value 7.4, a weight loss of about 16.8% with pH value 6.0 and a weight loss of about 17.8% with pH value 4.5 were measured for Hb adsorption on HAP nanorod-assembled hierarchical microflowers in PBS. These experimental results implies that the adsorbed amount of Hb on HAP nanorod-assembled hierarchical microflowers increases with decreasing pH value. It can be explained by the electrostatic interactions between Hb molecules and HAP nanorod-assembled hierarchical nanoflowrers. In general, protein adsorption is attributed to surface interactions between protein and carriers. The isoelectric point (pI) of Hb is within the range 6.8–7.0 [41,42]. As shown in Fig. 10, when the pH value of PBS is below the pI value of Hb, the Hb molecules become positively charged, whereas when the pH value is higher than the pI value, the Hb molecules become negatively charged. The HAP nanorod-assembled hierarchical microflowers as the carrier are negatively charged in PBS at pH 7.4, 6.0 and 4.5. Thus, when the pH value of PBS is 7.4, the electrostatic repulsion effect decreases the adsorption of Hb on HAP nanorod-assembled hierarchical micro-
Fig. 7. FTIR spectra of the as-prepared HAP nanorod-assembled hierarchical microflowers, Hb-loaded HAP nanorod-assembled hierarchical microflowers, and pure Hb.
adsorption with that of pure Hb (Fig. 7), it is found that two obvious amide peaks at about 1652 and 1538 cm−1 appear after the Hb adsorption, which originate from the adsorbed Hb molecules [40,41]. As shown in Fig. 8a, the adsorption of Hb on the as-prepared HAP nanorod-assembled hierarchical microflowers was investigated at different initial concentrations of Hb aqueous solution. The Hb adsorption capacity of HAP nanorod-assembled hierarchical microflowers increases rapidly with increasing initial concentration of Hb aqueous solution, and reaches 137 mg g−1 at a Hb initial concentration
Fig. 9. TG curves of the as-prepared HAP nanorod-assembled hierarchical microflowers before and after Hb adsorption in PBS with different pH values. The initial Hb concentration is 2 mg mL−1.
Fig. 8. (a) Hb adsorption curve and Hb adsorption efficiency of the as-prepared HAP nanorod-assembled hierarchical microflowers versus the initial concentrations of Hb aqueous solution; (b) the linear relationship between ce/qe and ce.
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Fig. 12. TG curves of the as-prepared HAP nanorod-assembled hierarchical microflowers before and after IBU loading.
Fig. 10. Zeta potentials of the as-prepared HAP nanorod-assembled hierarchical microflowers and Hb in PBS with different pH values.
flowers. When the pH values are 6.0 and 4.5, the electrostatic attraction effect increases the adsorption of Hb, and the adsorption amount of Hb at pH 4.5 is more than that at pH 6.0 owing to the stronger electrostatic attraction effect. Fig. 11 shows the Hb release performance of the as-prepared Hbloaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values. It shows that the release of Hb in PBS is pHdependent. The cumulative amount of released Hb reaches a plateau of 52.6% at pH 7.4 after 14 h (Fig. 11a), and 44.9% at pH 4.5 after 14 h (Fig. 11b). The amount of Hb released from HAP nanorod-assembled hierarchical microflowers in PBS at pH 7.4 is higher than that at pH 4.5. This result can be explained by the electrostatic interactions between Hb molecules and HAP nanorod-assembled hierarchical microflowers, which has been discussed above. We also investigated the drug loading and release performance of the as-prepared HAP nanorod-assembled hierarchical microflowers using IBU as the model drug. TG analysis is employed to estimate the IBU drug loading capacity. As shown in Fig. 12, the HAP nanorodassembled hierarchical microflowers and the corresponding IBUloaded sample have weight losses of about 5.6% and 25.1%, respectively. The IBU loading capacity of HAP nanorod-assembled hierarchical microflowers is about 260.3 mg g−1 (mg drug per gram carrier). This relatively high drug loading capacity may be explained by the relatively large specific surface area and pore volume of HAP nanorodassembled hierarchical microflowers. Fig. 13 shows the IBU drug release behavior of the HAP nanorod-assembled hierarchical microflower drug delivery system in PBS with different pH values, indicating
Fig. 13. IBU drug release profile of IBU-loaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values. The inset shows the natural logarithm of the cumulative IBU release amount as a function of the natural logarithm of the drug release time in the initial 24 h.
that the IBU drug release in PBS is dependent on the pH value. The cumulative IBU release amount reaches a plateau of 69.1% at pH 7.4 after 48 h, and 63.6% at pH 6.0 after 216 h, and 57.9% at pH 4.5 after 288 h. There is a carboxyl group existed in the IBU molecule. It can partially ionize to produce H+ ions in the aqueous solution. However, the H+ ions existed in the acidic solution (PBS, pH 6.0 or 4.5) can inhibit the ionization of the IBU molecules. Hence, the solubility of IBU at different pH values has an influence on the ultimate cumulative IBU release amount, because the solubility of IBU molecules increases with increasing pH value. The ultimate cumulative IBU release amount in PBS at pH 7.4 is obviously higher than that at pH 6.0 or 4.5, which is attributed to the larger solubility of IBU in more alkaline PBS at pH 7.4. The sustained IBU drug release at pH 6.0 and 4.5 is obvious because the IBU-loaded HAP nanorod-assembled hierarchical microflowers dissolve slowly to release IBU molecules under the weakly acidic condition. Moreover, the HAP nanorod-assembled hierarchical microflower drug delivery system exhibits a good linear relationship between the natural logarithm of the cumulative IBU release amount and the natural logarithm of drug release time in PBS with different pH values in the initial 24 h (the inset in Fig. 13). It is well acknowledged that the kinetics of drug release from carrier materials can be well described using the Higuchi model (C = k·t1/2), with a linear relationship between the cumulative amount of released drug (C), and the square root of drug release time (t1/2), where k is a constant, and the drug release is governed by a diffusion process [40,43]. However, a formula can be inferred for the HAP nanorod-assembled hierarchical
Fig. 11. Hb release curves of Hb-loaded HAP nanorod-assembled hierarchical microflowers in PBS with different pH values: (a) pH 7.4; (b) pH 4.5.
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[2]
[3] [4]
[5]
[6]
[7]
Fig. 14. Cytotoxicity tests of the as-prepared HAP nanorod-assembled hierarchical microflowers using MG-63 human osteosarcoma cells.
[8]
microflower drug delivery system in PBS:
[9]
n
C = k·t (t ≤ 24 h ) [10]
where n is a constant differing for different pH values of PBS. For the HAP nanorod-assembled hierarchical microflower drug delivery systems, the values of n are determined to be 0.5633 (pH 7.4), 0.5784 (pH 6.0), and 0.7724 (pH 4.5), respectively. The cytotoxicity tests of the as-prepared HAP nanorod-assembled hierarchical microflowers were performed using MG-63 human osteosarcoma cells. The CellTiter-Glo® Luminescent Cell Viability Assay shows no appreciable toxicity when the cells were co-cultured with the HAP nanorod-assembled hierarchical microflowers at concentrations of 0.1–50 µg mL−1 for 24 h and 48 h (Fig. 14). The high cytocompatibility may be explained by the chemical nature of HAP nanorodassembled hierarchical microflowers, which is similar to the inorganic constituent of the biological hard tissues [44,45]. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have promising applications in various biomedical fields.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
4. Conclusions
[18]
In summary, we have successfully synthesized HAP nanorodassembled hierarchical microflowers by rapid microwave hydrothermal transformation of the CaHPO4 precursor in NaOH aqueous solution, and the CaHPO4 microflower precursor is prepared using TEP as an organic phosphorus source by the solvothermal method. The HAP nanorod-assembled hierarchical microflowers are explored for potential application in protein/drug delivery taking advantage of their high biocompatibility and relatively large specific surface area. The experimental results indicate that the as-prepared HAP nanorod-assembled hierarchical microflowers have a relatively high drug/protein loading capacity. Moreover, the protein/drug delivery system shows a pHdependent and sustained release behavior. Therefore, the as-prepared HAP nanorod-assembled hierarchical microflowers are promising for various biomedical applications such as pH-dependent protein/drug delivery.
[19]
[20]
[21]
[22]
[23]
[24]
Acknowledgements
[25]
The financial support from the National Natural Science Foundation of China (51302294) and the Science and Technology Commission of Shanghai (15JC1491001) is gratefully acknowledged.
[26] [27]
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