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Self-activated luminescent and mesoporous strontium hydroxyapatite nanorods for drug delivery
Biomaterials 31 (2010) 3374–3383
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Self-activated luminescent and mesoporous strontium hydroxyapatite nanorods for drug delivery Cuimiao Zhang a, b, Chunxia Li a, Shanshan Huang a, Zhiyao Hou a, Ziyong Cheng a, Piaoping Yang a, Chong Peng a, b, Jun Lin a, * a b
State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Accepted 10 January 2010 Available online 1 February 2010
Multifunctional strontium hydroxyapatite (SrHAp) nanorods with luminescent and mesoporous properties have been successfully synthesized by a hydrothermal method. SEM and TEM images indicate that the mesoporous SrHAp samples consist of monodiperse nanorods with lengths of 120–150 nm, diameters of around 20 nm, and the mesopore size of 3–5 nm. The as-obtained SrHAp nanorods show an intense bright blue emission (centered at 432 nm, lifetime 11.6 ns, quantum efficiency: 22%), which might arise from CO2$ radical impurities in the crystal lattice under long-wavelength UV-light irradiation. Furthermore, the amount of trisodium citrate has an obvious impact on the particle size and the luminescence properties of the products, respectively. The drug storage/release test indicates that the luminescent SrHAp nanorods show a drug loading and controlled release properties for ibuprofen (IBU). Additionally, the emission intensity of SrHAp in the drug carrier system increases with the cumulative released amount of IBU, making the drug release might be easily tracked and monitored by the change of the luminescence intensity. This luminescent material may be potentially applied in the drug delivery and disease therapy fields. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Strontium hydroxyapatite Nanorod Luminescence Mesoporous Drug delivery
1. Introduction In recent years, much attention has been made to develop the new drug-delivery systems with many advantages comparing with the conventional forms of dosage, such as enhanced bioavailability, greater efficiency, lower toxicity, controlled release, and so on [1–7]. An ideal drug-delivery system should possess the following properties: (1) maximum biocompatibility and minimal antigenic properties [8]; (2) proper particle size, which is important for the particles to reach the given location in the body due to the size of the vessels of the human circulatory system [9]; (3) the ability to transport the desired drug molecules to the targeted cells or tissues and release in a controlled manner [1,3,10]. So far, different types of drug-delivery systems have been developed, such as biodegradable polymers [1], xerogels [11], hydrogels [12], mesoporous materials, and so on [4,6]. Among different drug-delivery systems, mesoporous materials (such as SBA-15, MCM-41, and mesoporous silica nanoparticles) have gained enhanced interest with particular
* Corresponding author. Fax: þ86 431 85698041. E-mail address: [email protected] (J. Lin). 0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.01.044
attention as drug storage and release hosts due to their unique surface and textural properties [9,13,14]. Moreover, nanoscale materials have been exploited as active components in a wide range of technological applications in biological field [10,15–18]. Particularly, in biomedicine field, nanoparticles can be used as drug-delivery vehicles that can target tissues or cells [8,17], and can be functionalized with special characteristics (such as magnetization, fluorescence, and near-infrared absorption) for qualitative or quantitative detection of tumor cells [16,18–20]. As we know, the nanoscale fluorescent materials have attracted much interest due to the increasing demand for efficient photosensitive materials not only for sophisticated optoelectronic and photonic devices but also for a broad range of biomedical application [21–27]. In biomedical areas, the luminescent materials, mainly including fluorescent organic molecules [28,29] and semiconductor nanoparticles [30,31], have been widely investigated in biological staining and diagnostics. However, some serious problems of photobleaching and quenching of fluorescent organic molecules and the toxicity of semiconductor quantum dots are critically pronounced, which have seriously limited their applications in biomedical areas [31,32]. Furthermore, high performance in function-specific biological applications requires that the
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composites possess some unique characteristics, such as uniform morphology, large surface areas, good dispersion, etc [32]. Recently, a class of the stable, efficient, and self-activated luminescent materials, whose emission is induced by the defects or impurities in the host lattices, has been prepared by various synthesis routes [33–37]. These novel self-activated inorganic materials may be a promising fluorescent material for biodetection due to their good optical properties and nontoxicity. However, as far as we know, there has no report on the study of self-activated luminescent materials for application in the biomedical fields. It is well-known that hydroxyapatite is a form of bioceramics material and has been widely used as a bone substitute due to its adequate mechanical properties and the similar composition to bone mineral [38–42]. Due to the bioactivity, biocompatibility, stability, nontoxic properties, hydroxyapatite with porous surface structure and –OH groups may serve as an ideal candidate drug carrier for the delivery of a variety of pharmaceutical molecules [19,43–45]. Moreover, it is worth noting that mesoporous materials functionalized with photoluminescence (PL) have potential applications in the fields of drug delivery, disease diagnosis, and therapy [19,46,47]. Therefore, the design and development of luminescence functionalized hydroxyapatite with nano-sized and mesoporous characteristics might be able to reach this application. Herein, we demonstrate a general strategy for the synthesis of luminescence functionalized mesoporous strontium hydroxyapatite [Sr5(PO4)3OH, SrHAp] nanorods via a hydrothermal route. XRD, SEM, TEM, XPS, FT-IR, N2 adsorption/desorption, PL spectra, and kinetic decay were used to characterize the samples. The assynthesized uniform SrHAp nanorods show a strong self-activated luminescence ranging from 360 to 570 nm. Additionally, the drug storage/release properties were also investigated on this system based on their mesoporous and luminescent properties using ibuprofen (IBU) as a model drug. 2. Materials and methods 2.1. Chemicals and materials Cetyltrimethylammonium bromide (CTAB, 99%) was purchased from Beijing Yili Fine Chemicals Co., Ltd. Ibuprofen (IBU) was purchased from Nanjing Chemical Regent Co., Ltd. Other chemicals were of analytical grade reagents and purchased from Beijing Chemical Corporation. All the initial chemicals in this work were used without further purification. 2.2. Synthesis of luminescence functionalized mesoporous SrHAp Luminescence functionalized mesoporous SrHAp nanorods were prepared by a hydrothermal process. In a typical procedure for the preparation of luminescent Sr5(PO4)3OH nanorods, 3 mmol of Sr(NO3)2, 0.5 g of CTAB, and 10 mL of ammonia solution (NH3$H2O) (used for adjusting the pH value as alkaline solution) were dissolved in deionized water to form 40 mL solution 1. Then, 6 mmol of trisodium citrate (labeled as Cit3, the molar ratio of Cit3/Sr2þ is 2:1) and 2 mmol of (NH4)2HPO4 were added into 20 mL H2O to form solution 2. After vigorously stirring for 30 min, solution 2 was introduced into solution 1. After additional agitation for 20 min, the as-obtained mixing solution was transferred into a Teflon bottle (80 mL) held in a stainless steel autoclave, sealed, and maintained at 180 C for 24 h. As the autoclave cooled to room temperature naturally, the precipitate was separated by centrifugation, washed with deionized water and ethanol in sequence. Then, the obtained product was redispersed in 150 mL of acetone and refluxed at 80 C for 48 h to remove the residual template CTAB. Finally, the precipitate was separated by centrifugation again and dried in vacuum at 70 C for 24 h to obtain the final SrHAp sample. Additionally, different molar ratios (0:1, 1:1, 4:1, 180 C, 24 h) of Cit3/Sr2þ were selected to investigate the influence on the morphological and luminescence properties of the samples. 2.3. Preparation of drug storage/release systems The drug storage/release system using luminescence functionalized mesoporous strontium hydroxyapatite as a carrier was prepared according to the previous reports [10,13,14]. Ibuprofen was selected as the model drug. Typically, 0.2 g of luminescent SrHAp sample was added into 30 mL of hexane solution with an IBU concentration of 60 mg mL1 at room temperature, and soaked for 24 h with stirring
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in a vial that was sealed to prevent the evaporation of hexane. The IBU-loaded SrHAp sample, denoted as IBU–SrHAp, was separated by centrifugation, and then dried in vacuum at 60 C for 24 h. The in vitro delivery of IBU was performed by immersing 0.2 g of the sample in the release media of simulated body fluid (SBF) with slow stirring under the immersing temperature of 37 C. The ionic composition of the as-prepared SBF solution was similar to that of human body plasma with a molar composition of 2 2 142.0/5.0/2.5/1.5/147.8/4.2/1.0/0.5 for Naþ/Kþ/Ca2þ/Mg2þ/Cl/HCO 3 /HPO4 /SO4 (pH ¼ 7.4) [14]. The ratio of SBF to adsorbed IBU was kept at 1 mL mg1. The amount of IBU adsorbed onto the mesoporous SrHAp was monitored by thermogravimetry (TG) analysis. At predetermined time intervals, 1.0 mL of the sample was withdrawn and immediately replaced with an equal volume of fresh SBF buffer to keep the volume constant. The withdrawn samples were filtered, properly diluted and the amount of IBU released at certain set times was determined by UV–vis spectroscopy at a wavelength of 220 nm. Scheme 1 shows the experimental process for the mesostructure of SrHAp nanorods and subsequent loading and release of IBU. 2.4. Characterization The X-ray diffraction (XRD) patterns of the samples were carried out on a D8 Focus diffractometer (Bruker) using Cu Ka radiation. FT-IR spectra were performed on Perkin–Elmer 580B infrared spectrophotometer using the KBr pellet technique. The morphology and composition of the samples were inspected using a scanning electron microscope (SEM; S-4800, Hitachi) equipped with an energy-dispersive Xray spectrum (EDX; XFlash-Detector 4010, Bruker) Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) micrographs were obtained from an FEI Tecnai G2 S-Twin transmission electron microscope with a field emission gun operating at 200 kV. Nitrogen adsorption/ desorption analysis was measured using a Micromeritics ASAP 2020 M apparatus. The specific surface area was determined by the Brunauer–Emmett–Teller (BET) method using the data between 0.05 and 0.35. Thermogravimetry (TG) measurement (Pyris Diamond Perkin–Elmer Thermal Analysis) was used to determine the loading amount of IBU on the materials. The X-ray photoelectron spectra (XPS) were taken on a VG ESCALAB MK II electron energy spectrometer using Mg Ka (1253.6 eV) as the X-ray excitation source. The photoluminescence (PL) measurements were performed on a Hitachi F-4500 spectrophotometer equipped with a 150 W xenon lamp as the excitation source. The luminescence decay curve was obtained from a Lecroy Wave Runner 6100 digital oscilloscope (1 GHz) using a tunable laser (pulse width ¼ 4 ns, gate ¼ 50 ns) as excitation source (Continuum Sunlite OPO). The UV– vis adsorption spectral values were measured on a TU-1901 spectrophotometer. Electron paramagnetic resonance (EPR) spectra were taken on the JESFE3AX elelctronic spin resonance spectrophotometer. The quantum efficiency of the phosphor was performed by the quantum yield measurement system (C9920-02, Hamamatsu Photonics K.K., Japan). All the measurements were performed at room temperature.
3. Results and discussion 3.1. Phase structure, morphology, and possible formation process The mesoporous and luminescent strontium hydroxyapatite (SrHAp) nanorods were synthesized by hydrothermal treatment at 180 C for 24 h. Fig. 1 shows the XRD pattern of as-prepared SrHAp powder sample, as well as the standard data for SrHAp (JCPDS No. 33-1348), respectively. The diffraction peaks of the sample can be indexed as pure hexagonal phase, which coincide well with the standard data of SrHAp (JCPDS No. 33-1348, space group: P63/m, No. 176). No peak shift and other phase can be detected in the XRD patterns, indicating that the pure SrHAp crystals can be obtained by this simple method. The SEM and TEM images provide direct information about the size and typical shape of the as-prepared SrHAp sample grown under hydrothermal process. Fig. 2 illustrates the SEM, TEM, and HRTEM images of the as-prepared SrHAp sample. From the lowand high-magnification SEM images of Fig. 2a and b, we can see clearly that the high yield, monodisperse, and uniform nanorods can be prepared by this approach. Fig. 2c shows a typical TEM image of the prepared SrHAp with the average length of 120– 150 nm and diameter of around 20 nm, respectively. The highmagnification TEM image of the SrHAp nanorods is shown in Fig. 2d. From Fig. 2d, we can see that the numerous individual mesopores (3–5 nm, white nanodots) spread around the surfaces of SrHAp nanorods, indicating the mesoporous structure of the
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Scheme 1. Schematic for the experimental process for luminescence mesostructure of SrHAp nanorods and subsequent loading and release of IBU.
sample. The selected area electron diffraction (SAED) pattern (inset in Fig. 2d) indicates that the strong concentric ring patterns can be indexed to the (102), (002), (211), (112), and (300) planes of hexagonal phase of SrHAp, respectively, demonstrating its polycrystalline nature. As disclosed by the corresponding HRTEM, the interplanar distance between the adjacent lattice fringes is determined as 0.28 nm, as shown in Fig. 2e. This plane can be indexed as d-spacing value of the (300) plane of the strontium hydroxyapatite crystal. The EDX spectrum (Fig. 2f) of SrHAp confirms the presence of strontium (Sr), phosphor (P), oxygen (O) and carbon (C) in the asprepared SrHAp sample. To investigate the influence of trisodium citrate on the particle size and luminescence properties in our current synthesis, the
Fig. 1. XRD patterns of as-prepared SrHAp sample by hydrothermal process at 180 C for 24 h with trisodium citrate (Cit3/Sr2þ, 2:1), and the standard data of strontium hydroxyapatite (JCPDS No. 33-1348) as a reference.
control experiments were carried out with different molar ratios of Cit3/Sr2þ, while other parameters remained identical. It can be found that the crystalline phase of the as-prepared samples remain unchanged, as shown in Fig. 3a. The morphology of the SrHAp samples has no obvious change, but the particle size changes obviously by adding different amount of trisodium citrate. Without Cit3 ions, the typical SEM image of the SrHAp is shown in Fig. 3b, which indicates that the product is composed of uniform longer nanorods. The as-obtained nanorods have an average length of 300 nm and diameter of about 30 nm instead of short nanorods (length: 120–150 nm; diameter: 20 nm). The aspect ratio for product without Cit3 anions is about 10, which is higher than that of the SrHAp obtained in the presence of trisodium citrate. The SEM images of SrHAp samples synthesized with different molar ratios of Cit3/Sr2þ (1:1, 4:1) are shown in Fig. 3c and d. It can be concluded that the particle size of the SrHAp nanorods decreases by increasing the amount of trisodium citrate. The experimental result demonstrates that the Cit3 ions play an important role in determining the size of SrHAp products. The exact mechanism for the change of the morphology of SrHAp grown with and without trisodium citrate is not clear. It might be explained in terms of the previous work. In the past decade, some researches have been done for the growth mechanism of hydroxyapatite crystal [48–50]. Johnsson et al. [49] reported that the adsorption of citrate or phosphocitrate ions by hydroxyapatite surfaces influences the constant composition growth kinetics of hydroxyapatite. Citrate adsorbed to hydroxyapatite can inhibit crystal growth. Lo´pezMacipe et al. [50] also pointed out that citrate ions exhibit inhibitory activity. In our experiments, there may be formed a little phosphocitrate in the hydrothermal process (high temperature and pressure), and we expect that Cit3 ions adsorbed on the hydroxyapatite surface to inhibit crystal growth. With trisodium citrate, the surface adsorption properties of hydroxyapatite were different, and Cit3 ions selectively adsorbed on different crystal facet of the growing particles at various conditions. It might inhibit
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Fig. 2. (a, b) SEM, (c, d) TEM, (e) HRTEM images and (f) EDX spectrum of as-prepared SrHAp sample. Inset in part d is the corresponding SAED pattern.
the crystal growth of corresponding crystal facets and lead to a morphological variation of the products. In this work, the Cit3 may preferentially absorb on the crystal facets perpendicular to the anisotropic growth direction, resulting in the morphological change of the SrHAp samples. In addition, the second organic additive CTAB as template results in a CTAB/SrHAp mesophase in the hydrothermal-derived SrHAp nanorods. The subsequent treatment with refluxing acetone could remove CTAB template and lead to a mesoporous structure. The similar process has been successfully utilized to synthesize mesoporous SiO2 shell of core–shell structured Fe3O4 at SiO2 composite [51]. The possible formation process of mesoporous structures in SrHAp nanorods is schematically shown in Scheme 1. The unique nanostructure of as-prepared SrHAp nanorods would be very useful for application. Especially, the mesoporous structure not only offers high surface area for the derivation of numerous functional groups but also provides large accessible pore volume for the adsorption and encapsulation of biomolecules and even functional nanoparticles [21,51].
Fig. 4 shows the survey XPS narrow scan spectra of as-prepared SrHAp nanorod sample. In Fig. 4, the main peaks at 134.60, 134.95, 531.40, and 284.55 eV can be readily assigned to the binding energy of Sr3d, P2p, O1s, and C1s, respectively. The detected carbon from the SrHAp sample can further indicate that the carbon-related impurities exist on the surface of the as-prepared SrHAp sample. The FT-IR spectra for SrHAp with trisodium citrate (a), SrHAp in the absence of trisodium citrate (b), IBU–SrHAp (c), and pure IBU (d) are shown in Fig. 5. As shown in Fig. 5a for the as-prepared SrHAp sample in the presence of trisodium citrate, the FT-IR result indicates the obvious absorption band at 3593 cm1 due to OH ions. The broadband at 3431 cm1 is ascribed to the O–H vibration of H2O absorbed in the sample. The presence of a large number of OH groups and H2O on the surface of the SrHAp is important for the bonding drug (IBU) molecules. The 1576 and 1437 cm1 peaks are attributed to the carbon-related impurities from the addition of Cit3 ions and tiny amount of CO2 3 caused by the CO2 in aqueous solution or air in the preparation process. The bands centered at 1080 and 1018 cm1 are ascribed to the asymmetric stretching
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Fig. 3. XRD patterns (a) and SEM images of the SrHAp samples prepared with different molar ratios of Cit3/Sr2þ in the prepare process: (b) 0:1 (without sodium citrate), (c) 1:1, (d) 4:1. All of the samples were prepared at 180 C for 24 h.
vibrations of the P–O and the band observed at 949 cm1 is assigned to the symmetric stretching mode of the P–O in PO3 4 groups. The two groups of bands in the low wavenumber ranging from 490 to 630 cm1 (centered at 560, 595 cm1) are assigned to the bending vibrations of the O–P–O in PO3 4 groups. In addition, the very small peak at 878 cm1 is caused by a few of HPO2 4 groups in the system [40,42,52]. The FT-IR spectrum (Fig. 5b) of SrHAp without trisodium citrate in the synthesis process is similar to the spectrum of SrHAp with trisodium citrate except that the intensity of the two bands (1576 and 1437 cm1). The two weak bands can be attributed to the CO2 3 groups, which might be from the CO2 in aqueous solution or air during the measurement process. For the IBU-loaded SrHAp (Fig. 5c), the band assigned to the vibration of –COOH at 1722 cm1 is obvious except for a slight decrease of intensity compared with that of IBU (Fig. 5d). Furthermore, the absorption bands assigned to the quaternary carbon atom located at 1463 and 1508 cm1, tertiary carbon atom at 1330 cm1, O–H bending vibration at 1421 cm1, and C–Hx bond at 2923 and 2956 cm1 can also be observed (Fig. 5c) [53], which confirms the successful adsorption of IBU onto the surface of the mesoporous strontium hydroxyapatite. Fig. 6 shows the N2 adsorption/desorption isotherms of as-prepared luminescent SrHAp (a) and the corresponding IBU-loaded SrHAp (IBU–SrHAp) sample (b), respectively. It can be seen that the SrHAp and IBU–SrHAp samples show similar N2 adsorption/desorption isotherms and the typical H1-hysteresis loops, which demonstrate the properties of typical mesoporous materials. The results reveal that the loading of IBU molecules has not changed the basic pore structure of mesoporous SrHAp sample. For SrHAp nanorods, the BET surface area is about 70.4 m2/g and the pore volume is about 0.373 cm3/g calculated from N2 isotherms. After loading IBU molecules, the BET surface area and pore volume decrease to 54.9 m2/g and 0.337 cm3/g, respectively.
3.2. Photoluminescence properties The hydrothermal-derived SrHAp samples exhibit a white color under sunlight. Under UV-light irradiation, SrHAp samples (prepared in the presence of trisodium citrate in the hydrothermal process) exhibit a strong blue emission. Fig. 7a shows the excitation (black line) and emission spectra (blue line) of the as-prepared SrHAp sample prepared with trisodium citrate (molar ratio of Cit3/ Sr2þ is 2:1). From Fig. 7a, we can see that SrHAp sample shows a strong emission consisting of a broadband (360–570 nm) with a maximum at 432 nm, and the corresponding excitation spectrum includes two broad bands: a weak band from 200 to 280 nm and a strong broadband from 280 to 410 nm with a maximum at 345 nm. The inset in Fig. 7a exhibits the photograph of the luminescent SrHAp nanorods dispersing in ethanol solution under UV lamp (365 nm) in dark. The photograph confirms the strong blue emission of the SrHAp nanorods. The chromaticity coordinates (CIE) of SrHAp nanorods are x ¼ 0.153 and y ¼ 0.094, located in the blue region, which agrees well with the luminescence photograph (inset in Fig. 7a). The PL quantum efficiency for the as-obtained SrHAp sample is 22% under the excitation of 345 nm. Fig. 7b shows the decay curve for the luminescence of SrHAp nanorods. The luminescence decay curve can be well fitted to a single-exponential function as I ¼ I0 exp (t/s) (s is lifetime), from which the lifetime s is determined to be 11.6 ns [36,54]. The short lifetime is typical value for the luminescence caused by defects [55]. In addition, the luminescence properties of SrHAp samples prepared with different molar ratios of Cit3/Sr2þ were also investigated (Fig. 8). It can be seen that the product without trisodium citrate in experiment shows no luminescence under UV excitation. The shape, profile, and maximum position for the emission spectra of SrHAP samples vary little by changing the molar ratios of Cit3/Sr2þ, but the emission intensity changes obviously. The result demonstrates that the luminescence of the as-synthesized SrHAp samples is induced
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Fig. 4. XPS narrow scan spectra of Sr3d (a), P2p (b), O1s (c), and C1s (d) for SrHAp sample prepared at 180 C for 24 h with trisodium citrate.
by the addition of trisodium citrate. At a higher molar ratio of Cit3/ Sr2þ (4:1, Fig. 8d), the emission intensity decreases greatly, which may be caused by the quenching effect of the luminescent centers [54,56]. Since neither the Sr2þ nor the PO3 4 group is able to show luminescence, the observed luminescence from SrHAp sample must be related to some impurities and/or defects in the host lattice, which can be confirmed by the short lifetime (11.6 ns). In addition, EPR spectra for luminescent SrHAp short nanorods (molar ratio of Cit3/ Sr2þ is 2:1) (a) and non-luminescent SrHAp long nanorods (b) are shown in Fig. 9. It can be observed that the luminescent SrHAp sample shows three obvious EPR signals, but the non-luminescent SrHAp sample exhibits no EPR signal. This indicates that the paramagnetic defects relating to the luminescence property exist in the luminescent SrHAp nanorods. Since no literature concerning the self-activated luminescence of Sr5(PO4)3OH sample can be found, the luminescent mechanism for SrHAp sample is not clearly at present. Therefore, we can only explain this luminescence phenomenon according to the existing models for other materials. Note that a very similar situation was reported by Angelov et al. [37], who found that the CO2$ radicals exhibiting three EPR signals in interstitials of the aragonite lattice of SrCO3 were most probably responsible for the self-activated luminescence. Compared with our present work, short lifetime, the similar PL results, EPR signals, together with the synthesis process, we can assume that the luminescence for SrHAp sample might be induced by CO2$ radicals in the SrHAp host latitice. Comparing the luminescent SrHAp short
nanorods with non-luminescent SrHAp long nanorods, the only difference is that the former has trisodium citrate as additives while the latter does not in the hydrothermal process. Therefore, we might conclude that defects are induced by trisodium citrate (Cit3) during the hydrothermal process. In the preparation of SrHAp sample with trisodium citrate, it is easy to form the Sr–citrate complex and adsorb Cit3 groups on the SHAp sample owing to the strong chelating ability of Cit3 groups and adsorption of SrHAp. Under the high pressure and hydrothermal conditions, some of R–C–COO (Cit3) occurs bond cleavages to form R–C (big group) and CO2$. Small amounts of CO2$ radicals resulting from the bond cleavages are trapped by the already formed SrHAp lattice or interstitial positions. The residual fragmented radicals (CO2$) are possible to form the luminescent centers. Furthermore, the carbon-related impurities have been detected by EDX (Fig. 2f), XPS (Fig. 4), and FT-IR (Fig. 5) spectra for luminescent SrHAp sample. 3.3. Drug adsorption and release properties The respective loading degree of IBU for SrHAp is 32.9 wt% determined by TG analysis. The TG curves of the as-prepared SrHAp (blue line) and IBU–SrHAp (black line) are shown in Fig. 10. The largest weight loss of ibuprofen occurs at 222 C in the IBU-loaded SrHAp sample (Fig. 10b, black line), while it is bare in the SrHAp sample (Fig. 10a, blue line). This result is similar with the previous report [57], which further confirms the loading of ibuprofen on the SrHAp sample.
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Fig. 5. FT-IR spectra for SrHAp prepared with trisodium citrate (a), SrHAp in the absence of trisodium citrate (b), IBU–SrHAp (c), and IBU (d), respectively.
During the loading and release process, the IBU molecules can be adsorbed onto the surface of mesoporous materials in the impregnation process and liberated by diffusion-controlled mechanism. The OH groups on the surface should be the reaction sites to
Fig. 7. (a) PL excitation (black line) and emission (blue line) spectra and (b) the decay curve for the as-synthesized SrHAp sample. Inset in part (a) is the corresponding luminescence photograph for sample under UV lamp (365 nm) irradiation in dark.
Fig. 6. N2 adsorption/desorption isotherms for SrHAp nanorods (a) and IBU–SrHAp nanorods (b).
Fig. 8. PL emission spectra for the SrHAp samples prepared with different molar ratios of Cit3/Sr2þ in the prepare process: (a) 0:1 (without sodium citrate), (b) 1:1, (c) 2:1, and (d) 4:1. All of the samples were synthesized at 180 C for 24 h.
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Fig. 9. EPR spectra for (a) the luminescent SrHAp short nanorods prepared with trisodium citrate and (b) the SrHAp long nanorods obtained without trisodium citrate in the preparation process.
form hydrogen bonds with the carboxyl group of IBU when IBU is adsorbed on the surface. During the release process, the solvent enters the IBU-matrix phase through the pores. The drug is then slowly dissolved into SBF from the surface and diffuses from the system along the solvent-filled irregular capillary channels. The cumulative drug release profiles in IBU–SrHAp system as a function of release time in SBF are shown in Fig. 11. From Fig. 11, it can be seen that the amount released from IBU–SrHAp nanorods delivery system reaches about 50.5% after 3 h and 93.9% after 12 h. The system almost completes the release within 24 h. The initial quick release may be caused by the IBU molecules weakly adsorbed on the outer surface of mesoporous SrHAp nanorods, and the slow release of the rest of IBU can be due to the strong interaction between IBU molecules and the surface. It is worth noting that the luminescent property is still obvious in the emission spectrum for IBU–SrHAp (Fig. 12). From the typical PL spectra, it can be seen that the IBU–SrHAp sample (Fig. 12a) has a lower PL intensity than SrHAp sample (Fig. 12g). The PL quantum efficiency for the IBU–SrHAp sample also decreases to 11.7% under the excitation of 345 nm. The result provides additional evidence that IBU has been successfully loaded onto the surface of the mesoporous strontium hydroxyapatite. In addition, the PL emission
intensity of IBU–SrHAp nanorods is affected by the cumulative released IBU, which makes the drug loading system easily identifiable and monitorable by the luminescent properties. Fig. 12 shows the PL emission spectra of IBU–SrHAp samples at different release time. It can be seen that the PL intensity increases with increasing the release time. Due to the cumulative amount of IBU released from IBU–SrHAp nanorods is in direct ratio with the release time, in other words, the PL intensity increases with the cumulative released IBU and reaches a maximum when IBU is completely released. The reason might be that the IBU molecules can be adsorbed onto the surface of meseporous SrHAP nanorods and quench some luminescent centers. With the increase of release time, more and more IBU molecules can be released from IBU– SrHAp system and the quenched luminescent centers would be decreased, resulting in the increase of emission intensity. This relationship between the luminescence properties and drug release extent can be potential as a probe for monitoring or tracking the drug release during the drug release process.
Fig. 10. TG curves of the as-prepared SrHAp (a) and IBU–SrHAp (b).
Fig. 12. PL emission spectra of IBU–SrHAp nanorods at different release time: (a) 0 h, (b) 3 h, (c) 6 h, (d) 9 h, (e) 12 h, (f) 24 h, and (g) SrHAp nanorods.
Fig. 11. Cumulative ibuprofen release from IBU–SrHAp nanorods system as a function of release time in the release media of SBF.
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4. Conclusion In summary, via a simple hydrothermal synthesis route, luminescence functionalized mesoporous strontium hydroxyapatite nanorods were obtained, resulting in the formation of a multifunctional material. The as-prepared SrHAp sample conserves regular rodlike morphology, and exhibits mesoporous structure, which is suitable for controlled release as a drug carrier. Furthermore, a strong blue emission peaking at about 432 nm can be observed at room temperature under UV excitation. The CO2$ radicals in the interstitials of the SrHAp lattice may be responsible for self-activated luminescence. As the PL intensity of the IBU– SrHAp sample increases with the cumulative released amount of IBU, the drug release process might be tracked and monitored. These studies demonstrate the potential applications in the fields of luminescence, drug delivery, and disease therapy, based on its luminescent and mesoporous properties. Acknowledgements This project is financially supported by National Basic Research Program of China (2007CB935502, 2010CB327704), and the National Natural Science Foundation of China (NSFC 50872131, 20921002, 60977013, 20901074). Appendix Figures with essential color discrimination. Figs. 2, 3, 5–8, 10, and 12 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/ j.biomaterials.2010.01.044. References [1] Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev 1999;99:3181–98. [2] Fischer KE, Alema´n BJ, Tao SL, Daniels RH, Li EM, Bu¨nger MD, et al. Biomimetic nanowire coatings for next generation adhesive drug delivery systems. Nano Lett 2009;9:716–20. [3] Vivero-Escoto JL, Slowing II, Wu CW, Lin VSY. Photoinduced intracellular controlled release drug delivery in human cells by gold-capped mesoporous silica nanosphere. J Am Chem Soc 2009;131:3462–3. [4] Wei W, Ma GH, Hu G, Yu D, Mcleish T, Su ZG, et al. Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier. J Am Chem Soc 2008;130:15808–10. [5] Yang Q, Wang SC, Fan PW, Wang LF, Di Y, Lin KF, et al. pH-responsive carrier system based on carboxylic acid modified mesoporous silica and polyelectrolyte for drug delivery. Chem Mater 2005;17:5999–6003. [6] Zhao W, Chen H, Li Y, Li L, Lang M, Shi J. Uniform rattle-type hollow magnetic mesoporous spheres as drug delivery carriers and their sustained-release property. Adv Funct Mater 2008;18:2780–8. [7] Ro¨sler A, Vandermuelen GWM, Klok HA. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv Drug Deliv Rev 2001;53:95–108. [8] Chen FH, Gao Q, Ni JZ. The grafting and release behavior of doxorubincin from Fe3O4@SiO2 core–shell structure nanoparticles via an acid cleaving amide bond: the potential for magnetic targeting drug delivery. Nanotechnology 2008;19:165103. [9] Ritter J, Ebner A, Daniel K, Stewart K. Application of high gradient magnetic separation principles to magnetic drug targeting. J Magn Magn Mater 2004;280:184–201. [10] Slowing II, Trewyn BG, Giri S, Lin VSY. Mesoporous silica nanoparticles for drug delivery and biosensing applications. Adv Funct Mater 2007;17:1225–36. [11] Yang HH, Zhu QZ, Qu HY, Chen XL, Din MT, Xu JG. Flow injection fluorescence immunoassay for gentamicin using sol–gel-derived mesoporous biomaterial. Anal Biochem 2002;308:71–6. [12] Caliceti P, Salmaso S, Lante A, Yoshida M, Katakai R, Martellini F, et al. Controlled release of biomolecules from temperature-sensitive hydrogels prepared by radiation polymerization. J Control Release 2001;75:173–81. [13] Yang PP, Huang SS, Kong DY, Lin J, Fu HQ. Luminescence functionalization of SBA-15 by YVO4:Eu3þ as a novel drug delivery system. Inorg Chem 2007;46:3203–11. [14] Vallet-Regi M, Ra´mila A, Del-Real RP, Pe´rez-Pariente JA. New property of MCM-41: drug delivery system. Chem Mater 2001;13:308–11.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Self-activated luminescent and mesoporous strontium hydroxyapatite nanorods for drug delivery Cuimiao Zhang a, b, Chunxia Li a, Shanshan Huang a, Zhiyao Hou a, Ziyong Cheng a, Piaoping Yang a, Chong Peng a, b, Jun Lin a, * a b
State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Accepted 10 January 2010 Available online 1 February 2010
Multifunctional strontium hydroxyapatite (SrHAp) nanorods with luminescent and mesoporous properties have been successfully synthesized by a hydrothermal method. SEM and TEM images indicate that the mesoporous SrHAp samples consist of monodiperse nanorods with lengths of 120–150 nm, diameters of around 20 nm, and the mesopore size of 3–5 nm. The as-obtained SrHAp nanorods show an intense bright blue emission (centered at 432 nm, lifetime 11.6 ns, quantum efficiency: 22%), which might arise from CO2$ radical impurities in the crystal lattice under long-wavelength UV-light irradiation. Furthermore, the amount of trisodium citrate has an obvious impact on the particle size and the luminescence properties of the products, respectively. The drug storage/release test indicates that the luminescent SrHAp nanorods show a drug loading and controlled release properties for ibuprofen (IBU). Additionally, the emission intensity of SrHAp in the drug carrier system increases with the cumulative released amount of IBU, making the drug release might be easily tracked and monitored by the change of the luminescence intensity. This luminescent material may be potentially applied in the drug delivery and disease therapy fields. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Strontium hydroxyapatite Nanorod Luminescence Mesoporous Drug delivery
1. Introduction In recent years, much attention has been made to develop the new drug-delivery systems with many advantages comparing with the conventional forms of dosage, such as enhanced bioavailability, greater efficiency, lower toxicity, controlled release, and so on [1–7]. An ideal drug-delivery system should possess the following properties: (1) maximum biocompatibility and minimal antigenic properties [8]; (2) proper particle size, which is important for the particles to reach the given location in the body due to the size of the vessels of the human circulatory system [9]; (3) the ability to transport the desired drug molecules to the targeted cells or tissues and release in a controlled manner [1,3,10]. So far, different types of drug-delivery systems have been developed, such as biodegradable polymers [1], xerogels [11], hydrogels [12], mesoporous materials, and so on [4,6]. Among different drug-delivery systems, mesoporous materials (such as SBA-15, MCM-41, and mesoporous silica nanoparticles) have gained enhanced interest with particular
* Corresponding author. Fax: þ86 431 85698041. E-mail address: [email protected] (J. Lin). 0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.01.044
attention as drug storage and release hosts due to their unique surface and textural properties [9,13,14]. Moreover, nanoscale materials have been exploited as active components in a wide range of technological applications in biological field [10,15–18]. Particularly, in biomedicine field, nanoparticles can be used as drug-delivery vehicles that can target tissues or cells [8,17], and can be functionalized with special characteristics (such as magnetization, fluorescence, and near-infrared absorption) for qualitative or quantitative detection of tumor cells [16,18–20]. As we know, the nanoscale fluorescent materials have attracted much interest due to the increasing demand for efficient photosensitive materials not only for sophisticated optoelectronic and photonic devices but also for a broad range of biomedical application [21–27]. In biomedical areas, the luminescent materials, mainly including fluorescent organic molecules [28,29] and semiconductor nanoparticles [30,31], have been widely investigated in biological staining and diagnostics. However, some serious problems of photobleaching and quenching of fluorescent organic molecules and the toxicity of semiconductor quantum dots are critically pronounced, which have seriously limited their applications in biomedical areas [31,32]. Furthermore, high performance in function-specific biological applications requires that the
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composites possess some unique characteristics, such as uniform morphology, large surface areas, good dispersion, etc [32]. Recently, a class of the stable, efficient, and self-activated luminescent materials, whose emission is induced by the defects or impurities in the host lattices, has been prepared by various synthesis routes [33–37]. These novel self-activated inorganic materials may be a promising fluorescent material for biodetection due to their good optical properties and nontoxicity. However, as far as we know, there has no report on the study of self-activated luminescent materials for application in the biomedical fields. It is well-known that hydroxyapatite is a form of bioceramics material and has been widely used as a bone substitute due to its adequate mechanical properties and the similar composition to bone mineral [38–42]. Due to the bioactivity, biocompatibility, stability, nontoxic properties, hydroxyapatite with porous surface structure and –OH groups may serve as an ideal candidate drug carrier for the delivery of a variety of pharmaceutical molecules [19,43–45]. Moreover, it is worth noting that mesoporous materials functionalized with photoluminescence (PL) have potential applications in the fields of drug delivery, disease diagnosis, and therapy [19,46,47]. Therefore, the design and development of luminescence functionalized hydroxyapatite with nano-sized and mesoporous characteristics might be able to reach this application. Herein, we demonstrate a general strategy for the synthesis of luminescence functionalized mesoporous strontium hydroxyapatite [Sr5(PO4)3OH, SrHAp] nanorods via a hydrothermal route. XRD, SEM, TEM, XPS, FT-IR, N2 adsorption/desorption, PL spectra, and kinetic decay were used to characterize the samples. The assynthesized uniform SrHAp nanorods show a strong self-activated luminescence ranging from 360 to 570 nm. Additionally, the drug storage/release properties were also investigated on this system based on their mesoporous and luminescent properties using ibuprofen (IBU) as a model drug. 2. Materials and methods 2.1. Chemicals and materials Cetyltrimethylammonium bromide (CTAB, 99%) was purchased from Beijing Yili Fine Chemicals Co., Ltd. Ibuprofen (IBU) was purchased from Nanjing Chemical Regent Co., Ltd. Other chemicals were of analytical grade reagents and purchased from Beijing Chemical Corporation. All the initial chemicals in this work were used without further purification. 2.2. Synthesis of luminescence functionalized mesoporous SrHAp Luminescence functionalized mesoporous SrHAp nanorods were prepared by a hydrothermal process. In a typical procedure for the preparation of luminescent Sr5(PO4)3OH nanorods, 3 mmol of Sr(NO3)2, 0.5 g of CTAB, and 10 mL of ammonia solution (NH3$H2O) (used for adjusting the pH value as alkaline solution) were dissolved in deionized water to form 40 mL solution 1. Then, 6 mmol of trisodium citrate (labeled as Cit3, the molar ratio of Cit3/Sr2þ is 2:1) and 2 mmol of (NH4)2HPO4 were added into 20 mL H2O to form solution 2. After vigorously stirring for 30 min, solution 2 was introduced into solution 1. After additional agitation for 20 min, the as-obtained mixing solution was transferred into a Teflon bottle (80 mL) held in a stainless steel autoclave, sealed, and maintained at 180 C for 24 h. As the autoclave cooled to room temperature naturally, the precipitate was separated by centrifugation, washed with deionized water and ethanol in sequence. Then, the obtained product was redispersed in 150 mL of acetone and refluxed at 80 C for 48 h to remove the residual template CTAB. Finally, the precipitate was separated by centrifugation again and dried in vacuum at 70 C for 24 h to obtain the final SrHAp sample. Additionally, different molar ratios (0:1, 1:1, 4:1, 180 C, 24 h) of Cit3/Sr2þ were selected to investigate the influence on the morphological and luminescence properties of the samples. 2.3. Preparation of drug storage/release systems The drug storage/release system using luminescence functionalized mesoporous strontium hydroxyapatite as a carrier was prepared according to the previous reports [10,13,14]. Ibuprofen was selected as the model drug. Typically, 0.2 g of luminescent SrHAp sample was added into 30 mL of hexane solution with an IBU concentration of 60 mg mL1 at room temperature, and soaked for 24 h with stirring
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in a vial that was sealed to prevent the evaporation of hexane. The IBU-loaded SrHAp sample, denoted as IBU–SrHAp, was separated by centrifugation, and then dried in vacuum at 60 C for 24 h. The in vitro delivery of IBU was performed by immersing 0.2 g of the sample in the release media of simulated body fluid (SBF) with slow stirring under the immersing temperature of 37 C. The ionic composition of the as-prepared SBF solution was similar to that of human body plasma with a molar composition of 2 2 142.0/5.0/2.5/1.5/147.8/4.2/1.0/0.5 for Naþ/Kþ/Ca2þ/Mg2þ/Cl/HCO 3 /HPO4 /SO4 (pH ¼ 7.4) [14]. The ratio of SBF to adsorbed IBU was kept at 1 mL mg1. The amount of IBU adsorbed onto the mesoporous SrHAp was monitored by thermogravimetry (TG) analysis. At predetermined time intervals, 1.0 mL of the sample was withdrawn and immediately replaced with an equal volume of fresh SBF buffer to keep the volume constant. The withdrawn samples were filtered, properly diluted and the amount of IBU released at certain set times was determined by UV–vis spectroscopy at a wavelength of 220 nm. Scheme 1 shows the experimental process for the mesostructure of SrHAp nanorods and subsequent loading and release of IBU. 2.4. Characterization The X-ray diffraction (XRD) patterns of the samples were carried out on a D8 Focus diffractometer (Bruker) using Cu Ka radiation. FT-IR spectra were performed on Perkin–Elmer 580B infrared spectrophotometer using the KBr pellet technique. The morphology and composition of the samples were inspected using a scanning electron microscope (SEM; S-4800, Hitachi) equipped with an energy-dispersive Xray spectrum (EDX; XFlash-Detector 4010, Bruker) Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) micrographs were obtained from an FEI Tecnai G2 S-Twin transmission electron microscope with a field emission gun operating at 200 kV. Nitrogen adsorption/ desorption analysis was measured using a Micromeritics ASAP 2020 M apparatus. The specific surface area was determined by the Brunauer–Emmett–Teller (BET) method using the data between 0.05 and 0.35. Thermogravimetry (TG) measurement (Pyris Diamond Perkin–Elmer Thermal Analysis) was used to determine the loading amount of IBU on the materials. The X-ray photoelectron spectra (XPS) were taken on a VG ESCALAB MK II electron energy spectrometer using Mg Ka (1253.6 eV) as the X-ray excitation source. The photoluminescence (PL) measurements were performed on a Hitachi F-4500 spectrophotometer equipped with a 150 W xenon lamp as the excitation source. The luminescence decay curve was obtained from a Lecroy Wave Runner 6100 digital oscilloscope (1 GHz) using a tunable laser (pulse width ¼ 4 ns, gate ¼ 50 ns) as excitation source (Continuum Sunlite OPO). The UV– vis adsorption spectral values were measured on a TU-1901 spectrophotometer. Electron paramagnetic resonance (EPR) spectra were taken on the JESFE3AX elelctronic spin resonance spectrophotometer. The quantum efficiency of the phosphor was performed by the quantum yield measurement system (C9920-02, Hamamatsu Photonics K.K., Japan). All the measurements were performed at room temperature.
3. Results and discussion 3.1. Phase structure, morphology, and possible formation process The mesoporous and luminescent strontium hydroxyapatite (SrHAp) nanorods were synthesized by hydrothermal treatment at 180 C for 24 h. Fig. 1 shows the XRD pattern of as-prepared SrHAp powder sample, as well as the standard data for SrHAp (JCPDS No. 33-1348), respectively. The diffraction peaks of the sample can be indexed as pure hexagonal phase, which coincide well with the standard data of SrHAp (JCPDS No. 33-1348, space group: P63/m, No. 176). No peak shift and other phase can be detected in the XRD patterns, indicating that the pure SrHAp crystals can be obtained by this simple method. The SEM and TEM images provide direct information about the size and typical shape of the as-prepared SrHAp sample grown under hydrothermal process. Fig. 2 illustrates the SEM, TEM, and HRTEM images of the as-prepared SrHAp sample. From the lowand high-magnification SEM images of Fig. 2a and b, we can see clearly that the high yield, monodisperse, and uniform nanorods can be prepared by this approach. Fig. 2c shows a typical TEM image of the prepared SrHAp with the average length of 120– 150 nm and diameter of around 20 nm, respectively. The highmagnification TEM image of the SrHAp nanorods is shown in Fig. 2d. From Fig. 2d, we can see that the numerous individual mesopores (3–5 nm, white nanodots) spread around the surfaces of SrHAp nanorods, indicating the mesoporous structure of the
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Scheme 1. Schematic for the experimental process for luminescence mesostructure of SrHAp nanorods and subsequent loading and release of IBU.
sample. The selected area electron diffraction (SAED) pattern (inset in Fig. 2d) indicates that the strong concentric ring patterns can be indexed to the (102), (002), (211), (112), and (300) planes of hexagonal phase of SrHAp, respectively, demonstrating its polycrystalline nature. As disclosed by the corresponding HRTEM, the interplanar distance between the adjacent lattice fringes is determined as 0.28 nm, as shown in Fig. 2e. This plane can be indexed as d-spacing value of the (300) plane of the strontium hydroxyapatite crystal. The EDX spectrum (Fig. 2f) of SrHAp confirms the presence of strontium (Sr), phosphor (P), oxygen (O) and carbon (C) in the asprepared SrHAp sample. To investigate the influence of trisodium citrate on the particle size and luminescence properties in our current synthesis, the
Fig. 1. XRD patterns of as-prepared SrHAp sample by hydrothermal process at 180 C for 24 h with trisodium citrate (Cit3/Sr2þ, 2:1), and the standard data of strontium hydroxyapatite (JCPDS No. 33-1348) as a reference.
control experiments were carried out with different molar ratios of Cit3/Sr2þ, while other parameters remained identical. It can be found that the crystalline phase of the as-prepared samples remain unchanged, as shown in Fig. 3a. The morphology of the SrHAp samples has no obvious change, but the particle size changes obviously by adding different amount of trisodium citrate. Without Cit3 ions, the typical SEM image of the SrHAp is shown in Fig. 3b, which indicates that the product is composed of uniform longer nanorods. The as-obtained nanorods have an average length of 300 nm and diameter of about 30 nm instead of short nanorods (length: 120–150 nm; diameter: 20 nm). The aspect ratio for product without Cit3 anions is about 10, which is higher than that of the SrHAp obtained in the presence of trisodium citrate. The SEM images of SrHAp samples synthesized with different molar ratios of Cit3/Sr2þ (1:1, 4:1) are shown in Fig. 3c and d. It can be concluded that the particle size of the SrHAp nanorods decreases by increasing the amount of trisodium citrate. The experimental result demonstrates that the Cit3 ions play an important role in determining the size of SrHAp products. The exact mechanism for the change of the morphology of SrHAp grown with and without trisodium citrate is not clear. It might be explained in terms of the previous work. In the past decade, some researches have been done for the growth mechanism of hydroxyapatite crystal [48–50]. Johnsson et al. [49] reported that the adsorption of citrate or phosphocitrate ions by hydroxyapatite surfaces influences the constant composition growth kinetics of hydroxyapatite. Citrate adsorbed to hydroxyapatite can inhibit crystal growth. Lo´pezMacipe et al. [50] also pointed out that citrate ions exhibit inhibitory activity. In our experiments, there may be formed a little phosphocitrate in the hydrothermal process (high temperature and pressure), and we expect that Cit3 ions adsorbed on the hydroxyapatite surface to inhibit crystal growth. With trisodium citrate, the surface adsorption properties of hydroxyapatite were different, and Cit3 ions selectively adsorbed on different crystal facet of the growing particles at various conditions. It might inhibit
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Fig. 2. (a, b) SEM, (c, d) TEM, (e) HRTEM images and (f) EDX spectrum of as-prepared SrHAp sample. Inset in part d is the corresponding SAED pattern.
the crystal growth of corresponding crystal facets and lead to a morphological variation of the products. In this work, the Cit3 may preferentially absorb on the crystal facets perpendicular to the anisotropic growth direction, resulting in the morphological change of the SrHAp samples. In addition, the second organic additive CTAB as template results in a CTAB/SrHAp mesophase in the hydrothermal-derived SrHAp nanorods. The subsequent treatment with refluxing acetone could remove CTAB template and lead to a mesoporous structure. The similar process has been successfully utilized to synthesize mesoporous SiO2 shell of core–shell structured Fe3O4 at SiO2 composite [51]. The possible formation process of mesoporous structures in SrHAp nanorods is schematically shown in Scheme 1. The unique nanostructure of as-prepared SrHAp nanorods would be very useful for application. Especially, the mesoporous structure not only offers high surface area for the derivation of numerous functional groups but also provides large accessible pore volume for the adsorption and encapsulation of biomolecules and even functional nanoparticles [21,51].
Fig. 4 shows the survey XPS narrow scan spectra of as-prepared SrHAp nanorod sample. In Fig. 4, the main peaks at 134.60, 134.95, 531.40, and 284.55 eV can be readily assigned to the binding energy of Sr3d, P2p, O1s, and C1s, respectively. The detected carbon from the SrHAp sample can further indicate that the carbon-related impurities exist on the surface of the as-prepared SrHAp sample. The FT-IR spectra for SrHAp with trisodium citrate (a), SrHAp in the absence of trisodium citrate (b), IBU–SrHAp (c), and pure IBU (d) are shown in Fig. 5. As shown in Fig. 5a for the as-prepared SrHAp sample in the presence of trisodium citrate, the FT-IR result indicates the obvious absorption band at 3593 cm1 due to OH ions. The broadband at 3431 cm1 is ascribed to the O–H vibration of H2O absorbed in the sample. The presence of a large number of OH groups and H2O on the surface of the SrHAp is important for the bonding drug (IBU) molecules. The 1576 and 1437 cm1 peaks are attributed to the carbon-related impurities from the addition of Cit3 ions and tiny amount of CO2 3 caused by the CO2 in aqueous solution or air in the preparation process. The bands centered at 1080 and 1018 cm1 are ascribed to the asymmetric stretching
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Fig. 3. XRD patterns (a) and SEM images of the SrHAp samples prepared with different molar ratios of Cit3/Sr2þ in the prepare process: (b) 0:1 (without sodium citrate), (c) 1:1, (d) 4:1. All of the samples were prepared at 180 C for 24 h.
vibrations of the P–O and the band observed at 949 cm1 is assigned to the symmetric stretching mode of the P–O in PO3 4 groups. The two groups of bands in the low wavenumber ranging from 490 to 630 cm1 (centered at 560, 595 cm1) are assigned to the bending vibrations of the O–P–O in PO3 4 groups. In addition, the very small peak at 878 cm1 is caused by a few of HPO2 4 groups in the system [40,42,52]. The FT-IR spectrum (Fig. 5b) of SrHAp without trisodium citrate in the synthesis process is similar to the spectrum of SrHAp with trisodium citrate except that the intensity of the two bands (1576 and 1437 cm1). The two weak bands can be attributed to the CO2 3 groups, which might be from the CO2 in aqueous solution or air during the measurement process. For the IBU-loaded SrHAp (Fig. 5c), the band assigned to the vibration of –COOH at 1722 cm1 is obvious except for a slight decrease of intensity compared with that of IBU (Fig. 5d). Furthermore, the absorption bands assigned to the quaternary carbon atom located at 1463 and 1508 cm1, tertiary carbon atom at 1330 cm1, O–H bending vibration at 1421 cm1, and C–Hx bond at 2923 and 2956 cm1 can also be observed (Fig. 5c) [53], which confirms the successful adsorption of IBU onto the surface of the mesoporous strontium hydroxyapatite. Fig. 6 shows the N2 adsorption/desorption isotherms of as-prepared luminescent SrHAp (a) and the corresponding IBU-loaded SrHAp (IBU–SrHAp) sample (b), respectively. It can be seen that the SrHAp and IBU–SrHAp samples show similar N2 adsorption/desorption isotherms and the typical H1-hysteresis loops, which demonstrate the properties of typical mesoporous materials. The results reveal that the loading of IBU molecules has not changed the basic pore structure of mesoporous SrHAp sample. For SrHAp nanorods, the BET surface area is about 70.4 m2/g and the pore volume is about 0.373 cm3/g calculated from N2 isotherms. After loading IBU molecules, the BET surface area and pore volume decrease to 54.9 m2/g and 0.337 cm3/g, respectively.
3.2. Photoluminescence properties The hydrothermal-derived SrHAp samples exhibit a white color under sunlight. Under UV-light irradiation, SrHAp samples (prepared in the presence of trisodium citrate in the hydrothermal process) exhibit a strong blue emission. Fig. 7a shows the excitation (black line) and emission spectra (blue line) of the as-prepared SrHAp sample prepared with trisodium citrate (molar ratio of Cit3/ Sr2þ is 2:1). From Fig. 7a, we can see that SrHAp sample shows a strong emission consisting of a broadband (360–570 nm) with a maximum at 432 nm, and the corresponding excitation spectrum includes two broad bands: a weak band from 200 to 280 nm and a strong broadband from 280 to 410 nm with a maximum at 345 nm. The inset in Fig. 7a exhibits the photograph of the luminescent SrHAp nanorods dispersing in ethanol solution under UV lamp (365 nm) in dark. The photograph confirms the strong blue emission of the SrHAp nanorods. The chromaticity coordinates (CIE) of SrHAp nanorods are x ¼ 0.153 and y ¼ 0.094, located in the blue region, which agrees well with the luminescence photograph (inset in Fig. 7a). The PL quantum efficiency for the as-obtained SrHAp sample is 22% under the excitation of 345 nm. Fig. 7b shows the decay curve for the luminescence of SrHAp nanorods. The luminescence decay curve can be well fitted to a single-exponential function as I ¼ I0 exp (t/s) (s is lifetime), from which the lifetime s is determined to be 11.6 ns [36,54]. The short lifetime is typical value for the luminescence caused by defects [55]. In addition, the luminescence properties of SrHAp samples prepared with different molar ratios of Cit3/Sr2þ were also investigated (Fig. 8). It can be seen that the product without trisodium citrate in experiment shows no luminescence under UV excitation. The shape, profile, and maximum position for the emission spectra of SrHAP samples vary little by changing the molar ratios of Cit3/Sr2þ, but the emission intensity changes obviously. The result demonstrates that the luminescence of the as-synthesized SrHAp samples is induced
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Fig. 4. XPS narrow scan spectra of Sr3d (a), P2p (b), O1s (c), and C1s (d) for SrHAp sample prepared at 180 C for 24 h with trisodium citrate.
by the addition of trisodium citrate. At a higher molar ratio of Cit3/ Sr2þ (4:1, Fig. 8d), the emission intensity decreases greatly, which may be caused by the quenching effect of the luminescent centers [54,56]. Since neither the Sr2þ nor the PO3 4 group is able to show luminescence, the observed luminescence from SrHAp sample must be related to some impurities and/or defects in the host lattice, which can be confirmed by the short lifetime (11.6 ns). In addition, EPR spectra for luminescent SrHAp short nanorods (molar ratio of Cit3/ Sr2þ is 2:1) (a) and non-luminescent SrHAp long nanorods (b) are shown in Fig. 9. It can be observed that the luminescent SrHAp sample shows three obvious EPR signals, but the non-luminescent SrHAp sample exhibits no EPR signal. This indicates that the paramagnetic defects relating to the luminescence property exist in the luminescent SrHAp nanorods. Since no literature concerning the self-activated luminescence of Sr5(PO4)3OH sample can be found, the luminescent mechanism for SrHAp sample is not clearly at present. Therefore, we can only explain this luminescence phenomenon according to the existing models for other materials. Note that a very similar situation was reported by Angelov et al. [37], who found that the CO2$ radicals exhibiting three EPR signals in interstitials of the aragonite lattice of SrCO3 were most probably responsible for the self-activated luminescence. Compared with our present work, short lifetime, the similar PL results, EPR signals, together with the synthesis process, we can assume that the luminescence for SrHAp sample might be induced by CO2$ radicals in the SrHAp host latitice. Comparing the luminescent SrHAp short
nanorods with non-luminescent SrHAp long nanorods, the only difference is that the former has trisodium citrate as additives while the latter does not in the hydrothermal process. Therefore, we might conclude that defects are induced by trisodium citrate (Cit3) during the hydrothermal process. In the preparation of SrHAp sample with trisodium citrate, it is easy to form the Sr–citrate complex and adsorb Cit3 groups on the SHAp sample owing to the strong chelating ability of Cit3 groups and adsorption of SrHAp. Under the high pressure and hydrothermal conditions, some of R–C–COO (Cit3) occurs bond cleavages to form R–C (big group) and CO2$. Small amounts of CO2$ radicals resulting from the bond cleavages are trapped by the already formed SrHAp lattice or interstitial positions. The residual fragmented radicals (CO2$) are possible to form the luminescent centers. Furthermore, the carbon-related impurities have been detected by EDX (Fig. 2f), XPS (Fig. 4), and FT-IR (Fig. 5) spectra for luminescent SrHAp sample. 3.3. Drug adsorption and release properties The respective loading degree of IBU for SrHAp is 32.9 wt% determined by TG analysis. The TG curves of the as-prepared SrHAp (blue line) and IBU–SrHAp (black line) are shown in Fig. 10. The largest weight loss of ibuprofen occurs at 222 C in the IBU-loaded SrHAp sample (Fig. 10b, black line), while it is bare in the SrHAp sample (Fig. 10a, blue line). This result is similar with the previous report [57], which further confirms the loading of ibuprofen on the SrHAp sample.
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Fig. 5. FT-IR spectra for SrHAp prepared with trisodium citrate (a), SrHAp in the absence of trisodium citrate (b), IBU–SrHAp (c), and IBU (d), respectively.
During the loading and release process, the IBU molecules can be adsorbed onto the surface of mesoporous materials in the impregnation process and liberated by diffusion-controlled mechanism. The OH groups on the surface should be the reaction sites to
Fig. 7. (a) PL excitation (black line) and emission (blue line) spectra and (b) the decay curve for the as-synthesized SrHAp sample. Inset in part (a) is the corresponding luminescence photograph for sample under UV lamp (365 nm) irradiation in dark.
Fig. 6. N2 adsorption/desorption isotherms for SrHAp nanorods (a) and IBU–SrHAp nanorods (b).
Fig. 8. PL emission spectra for the SrHAp samples prepared with different molar ratios of Cit3/Sr2þ in the prepare process: (a) 0:1 (without sodium citrate), (b) 1:1, (c) 2:1, and (d) 4:1. All of the samples were synthesized at 180 C for 24 h.
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Fig. 9. EPR spectra for (a) the luminescent SrHAp short nanorods prepared with trisodium citrate and (b) the SrHAp long nanorods obtained without trisodium citrate in the preparation process.
form hydrogen bonds with the carboxyl group of IBU when IBU is adsorbed on the surface. During the release process, the solvent enters the IBU-matrix phase through the pores. The drug is then slowly dissolved into SBF from the surface and diffuses from the system along the solvent-filled irregular capillary channels. The cumulative drug release profiles in IBU–SrHAp system as a function of release time in SBF are shown in Fig. 11. From Fig. 11, it can be seen that the amount released from IBU–SrHAp nanorods delivery system reaches about 50.5% after 3 h and 93.9% after 12 h. The system almost completes the release within 24 h. The initial quick release may be caused by the IBU molecules weakly adsorbed on the outer surface of mesoporous SrHAp nanorods, and the slow release of the rest of IBU can be due to the strong interaction between IBU molecules and the surface. It is worth noting that the luminescent property is still obvious in the emission spectrum for IBU–SrHAp (Fig. 12). From the typical PL spectra, it can be seen that the IBU–SrHAp sample (Fig. 12a) has a lower PL intensity than SrHAp sample (Fig. 12g). The PL quantum efficiency for the IBU–SrHAp sample also decreases to 11.7% under the excitation of 345 nm. The result provides additional evidence that IBU has been successfully loaded onto the surface of the mesoporous strontium hydroxyapatite. In addition, the PL emission
intensity of IBU–SrHAp nanorods is affected by the cumulative released IBU, which makes the drug loading system easily identifiable and monitorable by the luminescent properties. Fig. 12 shows the PL emission spectra of IBU–SrHAp samples at different release time. It can be seen that the PL intensity increases with increasing the release time. Due to the cumulative amount of IBU released from IBU–SrHAp nanorods is in direct ratio with the release time, in other words, the PL intensity increases with the cumulative released IBU and reaches a maximum when IBU is completely released. The reason might be that the IBU molecules can be adsorbed onto the surface of meseporous SrHAP nanorods and quench some luminescent centers. With the increase of release time, more and more IBU molecules can be released from IBU– SrHAp system and the quenched luminescent centers would be decreased, resulting in the increase of emission intensity. This relationship between the luminescence properties and drug release extent can be potential as a probe for monitoring or tracking the drug release during the drug release process.
Fig. 10. TG curves of the as-prepared SrHAp (a) and IBU–SrHAp (b).
Fig. 12. PL emission spectra of IBU–SrHAp nanorods at different release time: (a) 0 h, (b) 3 h, (c) 6 h, (d) 9 h, (e) 12 h, (f) 24 h, and (g) SrHAp nanorods.
Fig. 11. Cumulative ibuprofen release from IBU–SrHAp nanorods system as a function of release time in the release media of SBF.
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4. Conclusion In summary, via a simple hydrothermal synthesis route, luminescence functionalized mesoporous strontium hydroxyapatite nanorods were obtained, resulting in the formation of a multifunctional material. The as-prepared SrHAp sample conserves regular rodlike morphology, and exhibits mesoporous structure, which is suitable for controlled release as a drug carrier. Furthermore, a strong blue emission peaking at about 432 nm can be observed at room temperature under UV excitation. The CO2$ radicals in the interstitials of the SrHAp lattice may be responsible for self-activated luminescence. As the PL intensity of the IBU– SrHAp sample increases with the cumulative released amount of IBU, the drug release process might be tracked and monitored. These studies demonstrate the potential applications in the fields of luminescence, drug delivery, and disease therapy, based on its luminescent and mesoporous properties. Acknowledgements This project is financially supported by National Basic Research Program of China (2007CB935502, 2010CB327704), and the National Natural Science Foundation of China (NSFC 50872131, 20921002, 60977013, 20901074). Appendix Figures with essential color discrimination. Figs. 2, 3, 5–8, 10, and 12 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/ j.biomaterials.2010.01.044. References [1] Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev 1999;99:3181–98. [2] Fischer KE, Alema´n BJ, Tao SL, Daniels RH, Li EM, Bu¨nger MD, et al. Biomimetic nanowire coatings for next generation adhesive drug delivery systems. Nano Lett 2009;9:716–20. [3] Vivero-Escoto JL, Slowing II, Wu CW, Lin VSY. Photoinduced intracellular controlled release drug delivery in human cells by gold-capped mesoporous silica nanosphere. J Am Chem Soc 2009;131:3462–3. [4] Wei W, Ma GH, Hu G, Yu D, Mcleish T, Su ZG, et al. Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier. J Am Chem Soc 2008;130:15808–10. [5] Yang Q, Wang SC, Fan PW, Wang LF, Di Y, Lin KF, et al. pH-responsive carrier system based on carboxylic acid modified mesoporous silica and polyelectrolyte for drug delivery. Chem Mater 2005;17:5999–6003. [6] Zhao W, Chen H, Li Y, Li L, Lang M, Shi J. Uniform rattle-type hollow magnetic mesoporous spheres as drug delivery carriers and their sustained-release property. Adv Funct Mater 2008;18:2780–8. [7] Ro¨sler A, Vandermuelen GWM, Klok HA. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv Drug Deliv Rev 2001;53:95–108. [8] Chen FH, Gao Q, Ni JZ. The grafting and release behavior of doxorubincin from Fe3O4@SiO2 core–shell structure nanoparticles via an acid cleaving amide bond: the potential for magnetic targeting drug delivery. Nanotechnology 2008;19:165103. [9] Ritter J, Ebner A, Daniel K, Stewart K. Application of high gradient magnetic separation principles to magnetic drug targeting. J Magn Magn Mater 2004;280:184–201. [10] Slowing II, Trewyn BG, Giri S, Lin VSY. Mesoporous silica nanoparticles for drug delivery and biosensing applications. Adv Funct Mater 2007;17:1225–36. [11] Yang HH, Zhu QZ, Qu HY, Chen XL, Din MT, Xu JG. Flow injection fluorescence immunoassay for gentamicin using sol–gel-derived mesoporous biomaterial. Anal Biochem 2002;308:71–6. [12] Caliceti P, Salmaso S, Lante A, Yoshida M, Katakai R, Martellini F, et al. Controlled release of biomolecules from temperature-sensitive hydrogels prepared by radiation polymerization. J Control Release 2001;75:173–81. [13] Yang PP, Huang SS, Kong DY, Lin J, Fu HQ. Luminescence functionalization of SBA-15 by YVO4:Eu3þ as a novel drug delivery system. Inorg Chem 2007;46:3203–11. [14] Vallet-Regi M, Ra´mila A, Del-Real RP, Pe´rez-Pariente JA. New property of MCM-41: drug delivery system. Chem Mater 2001;13:308–11.
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