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Biomaterials 29 (2008) 4341–4347

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Bioactive, luminescent and mesoporous europium-doped hydroxyapatiteas a drug carrier

Piaoping Yang a,b, Zewei Quan b, Chunxia Li b, Xiaojiao Kang b, Hongzhou Lian b, Jun Lin b,*

a College of Material Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, PR Chinab State Key laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China

a r t i c l e i n f o

Article history:Received 22 May 2008Accepted 30 July 2008Available online 19 August 2008

Keywords:HydroxyapatitePorosityLuminescenceIn vitro testDrug delivery

* Corresponding author. Fax: þ86 431 85698041.E-mail address: jlin@ciac.jl.cn (J. Lin).

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.07.042

a b s t r a c t

Bioactive, luminescent and mesoporous europium-doped hydroxyapatite (Eu:HAp) was successfullyprepared through a simple one-step route using cationic surfactant as template. The obtained multi-functional hydroxyapatite was performed as a drug delivery carrier to investigate the drug storage/release properties using ibuprofen (IBU) as a model drug. The structural, morphological, textural andoptical properties were well characterized by X-ray diffraction (XRD), scanning electron microscopy(SEM), transmission electron microscopy (TEM), X-ray photoelectron spectra (XPS), Fourier transforminfrared spectroscopy (FT-IR), N2 adsorption/desorption, and photoluminescence (PL) spectra, respec-tively. The results reveal that the multifunctional hydroxyapatites exhibit the typical orderedcharacteristics of the hexagonal mesostructure, and have rod-like morphology with the particle size of20–40 nm in width and 100–200 nm in length. The drug storage/release test indicates that the lumi-nescent HAp shows much similar drug loading amount and cumulative release rate to those of pure HAp.Interestingly, the IBU-loaded samples still show red luminescence of Eu3þ (5D0–7F1,2) under UV irradi-ation, and the emission intensities of Eu3þ in the drug carrier system vary with the released amount ofIBU, thus making the drug release be easily tracked and monitored by the change of the luminescenceintensity.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

During the past decade, a lot of efforts have been made todevelop novel drug storage/release systems, which exhibitnumerous advantages over the conventional forms of dosage, suchas enhanced bioavailability, greater efficacy and safety, controlledand prolonged release time, and predictable therapeutic response[1–7]. In general, an efficient delivery system should be able totransport the desired drug molecules to the targeted cells or tissues,and release in a controlled manner [8]. So far, a large number ofsystems have been employed as various drug delivery systems,such as biodegradable polymers [1], hydroxyapatite (HAp) [9–13],calcium phosphate cement (CFC) [14,15], xerogels [16], hydrogels[17,18], and mesoporous silica [8,19–29]. Recently, ordered meso-porous materials have gained enhanced interest with particularattention as drug storage and release hosts due to their uniquesurface and textural properties, including stable mesostructure,tunable pore size, and easily modified surface features for site-specific delivery. Several research groups have reported the drug

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delivery systems using the ordered mesoporous materials ascarriers [19–29].

Hydroxyapatite [HAp, Ca10(PO4)6(OH)2)] has been widely used asa bone substitute due to its adequate mechanical properties and thesimilar composition to bone mineral [30,31]. Moreover, HAp withvarious morphologies and surface properties have also been inves-tigated as drug carriers for the delivery of a variety of pharmaceuticalmolecules because of their biocompatible, osteoconductive,nontoxic, and noninflammatory properties [9–13]. Although somelanthanide-doped or complex hydroxyapatite-like compounds havebeen reported [32–36], these materials have never been really testedas drug storage/release systems to demonstrate their potentialapplication. The major limiting factors should be their conventionalmorphology and textural properties, which are not suitable for thestorage and release of drug molecules. It is well accepted that thechemical and biological properties of HAp are strictly linked to theirnanoscale dimensions [37,38], and the mesoporous structure of thematerials is usually necessary for drug storage and release in manycases. Therefore, the design and development of luminescencefunctionalized HAp with nano-sized and mesoporous characteris-tics should be able to reach this goal.

One of the most widely used non-steroidal analgesic and anti-inflammatory drugs, ibuprofen (IBU) has been extensively studied

Scheme 1. The experimental process for the loading and release of IBU on the as-synthesized luminescent Eu:HAp composite together with the corresponding pellet photographsunder the irradiation of 365 nm UV lamp.

P. Yang et al. / Biomaterials 29 (2008) 4341–43474342

as model drug for sustained and controlled drug delivery because ofits short biological half-life (2 h), good pharmacological activity andthe suitable molecule size of about 1.0� 0.6 nm, which make it freethrough the pore channels of host mesoporous materials[22,25,26]. Herein, we propose the one-step synthesis of novelluminescence functionalized mesoporous HAp materials by dopingEu3þ during the synthesis of mesoporous HAp, resulting in theformation of multifunctional material (bioactive, luminescent,mesoporous). The samples were fully characterized by means ofXRD, FT-IR, SEM, TEM, XPS, N2 adsorption, and photoluminescence(PL) spectra. Additionally, the drug storage/release properties werealso investigated on this system based on their mesoporous andluminescent properties using IBU as a model drug.

2. Materials and methods

2.1. Synthesis of luminescence functionalized mesoporous hydroxyapatite

All the reagents including cetyltrimethylammonium bromide (CTAB, ShanghaiHuishi Chemical Co., Ltd.), (NH4)2HPO4 (Beijing Chemical Regent Co., Ltd.), CaCl2(Beijing Chemical Regent Co., Ltd.), NaOH (A. R., Beijing Chemical Regent Co. Ltd.),Eu2O3 (99.999%, Science and Technology Parent Company of Changchun Institute ofApplied Chemistry), and HNO3 (A. R., Beijing Beihua Chemical Co., Ltd.) werereceived without further purification. The doping concentration of Eu3þ was 5 mol%to Ca2þ in Eu:Ca10(PO4)6(OH)2 (Eu:HAP). Eu(NO3)3 was obtained by dissolvingstoichiometric Eu2O3 in dilute HNO3 with vigorous stirring. The superfluous HNO3 inthe solution was driven off until the Eu(NO3)3 powders were obtained. The lumi-nescence functionalized mesoporous hydroxyapatite was prepared according to theliterature with some modifications [39]. In a typical process, 3.168 g of (NH4)2HPO4

and 8.740 g of cetyltrimethylammonium bromide (CTAB) were dissolved in 100 mLof deionized water and the pH was adjusted to 12 using 2 M NaOH. Then 4.218 g ofCaCl2 and 0.676 g of the as-prepared Eu(NO3)3 were dissolved into 60 mL ofdeionized water. Subsequently, the CaCl2 and Eu(NO3)3 mixed solution was addeddropwise to the former solution containing CTAB and (NH4)2HPO4, yielding a milkysuspension, which was refluxed at 120 �C for 24 h. The obtained precipitate wasthen filtered off and washed several times with deionized water and ethanol. Theresulting material was dried at 100 �C for 24 h, and then calcined at 550 �C for 6 h toremove the organic template.

2.2. Preparation of drug storage/release system

The drug storage/release system using luminescence functionalized mesoporoushydroxyapatite as a carrier was prepared according to the previous report [25].Ibuprofen (IBU, Nanjing Chemical Regent Co., Ltd.) was selected as the model drug.Typically, 0.4 g of the Eu:HAp sample was added into 50 mL of hexane solution withan IBU concentration of 60 mg/mL at room temperature, and soaked for 24 h withstirring in a vial which was sealed to prevent the evaporation of hexane. The IBU-loaded Eu:HAp sample, denoted as IBU–Eu:HAp, was separated by centrifugation,and then dried at 60 �C for 12 h.

The in vitro delivery of IBU was performed by immersing 0.2 g of the sample inthe release media of simulated body fluid (SBF) with slow stirring under theimmersing temperature of 37 �C. The ionic composition of the as-prepared SBFsolution was similar to that of human body plasma with a molar composition of142.0/5.0/2.5/1.5/147.8/4.2/1.0/0.5 for Naþ/Kþ/Ca2þ/Mg2þ/Cl�/HCO3

�/HPO42�/SO4

2�

(pH¼ 7.4) [25]. The ratio of SBF to adsorbed IBU was kept at 1 mL/mg. The amount ofIBU adsorbed onto the mesoporous HAp was monitored by thermogravimetry (TG)analysis. The amount of IBU released at certain set times was determined by UV–vis

spectroscopy at a wavelength of 220 nm. For comparison, the drug storage/releasesystem for pure HAP was also prepared through the same process.

The experimental process for the loading and subsequent release of the IBU onthe luminescent Eu:HAp carrier and the corresponding sample pellet photographsunder the irradiation of 365 nm UV lamp in dark are schematically shown inScheme 1.

2.3. Characterization

Powder X-ray diffraction (XRD) patterns were obtained on a Rigaku DMAX-2500diffractometer using Cu Ka radiation (l¼ 0.15405 nm). Fourier transform IR spectrawere recorded on a PerkindElmer 580B IR spectrophotometer using KBr pellettechnique. Field emission scanning electron microscope (FESEM) study was per-formed on an XL30 microscope (Philips) equipped with an energy-dispersive X-rayspectrum (EDS, JEOL JXA-840). Transmission electron microscope (TEM) and high-resolution transmission electron microscope (HRTEM) images were recorded on anFEI Tecnai G2 S-Twin with an acceleration voltage of 200 kV. The X-ray photoelec-tron spectra (XPS) were taken on a VG ESCALAB MK II electron energy spectrometerusing Mg Ka (1253.6 eV) as the X-ray excitation source. Nitrogen adsorption/desorption analysis was measured using a Micromeritics ASAP 2010 M apparatus.The specific surface area was determined by the Brunauer–Emmett–Teller (BET)method using the data between 0.05 and 0.35. The pore volume was obtained fromthe t-plot method. Thermogravimetry (TG) measurement (Netzsch ThermoanalyzerSTA 409) was used to determine the loading amount of IBU on the materials. Theexact doping amount of Eu3þ in the materials was determined by InductivelyCoupled Plasma (ICP) (ICP–PLASMA 1000). The UV–vis excitation and emissionspectra were obtained on a Hitachi F-4500 spectrofluorimeter equipped witha 150 W xenon lamp as the excitation source. The UV–vis adsorption spectral valueswere measured on a TU-1901 spectrophotometer. All the measurements were per-formed at room temperature.

3. Results and discussion

3.1. Structure, formation, morphology and luminescent propertiesof pure HAp, Eu:HAp, and IBU–Eu:HAp

Fig. 1A shows the wide-angle XRD patterns of pure HAp,Eu:HAp, and the standard data for the hexagonal hydroxyapatite,respectively. In Fig. 1A(a) for pure HAp, the typical diffraction peaksof hexagonal Ca10(PO4)6(OH)2 can be found, which can be indexedas the standard data (JCPDS No. 09–0432). For the Eu:HAp samplein Fig. 1A(b), the characteristic diffractions of HAp is still obvious,and no other phase related with the doped Eu3þ can be detected.The results indicate that Eu3þ has been successfully doped into theframework of HAp. Additionally, the XRD patterns reveal thatthe structure of pure HAp and Eu:HAp belongs to the hexagonalP63/m space group with lattice constants of a¼ 0.9418 nm andc¼ 0.6884 nm. The respective calculated lattice constants ofa¼ 0.9417 nm, c¼ 0.6881 nm and a¼ 0.9412 nm, c¼ 0.6879 nmare calculated for pure HAp and Eu:HAp respectively, which are ingood agreement with the standard data (JCPDS No. 09–0432).Furthermore, Table 1 shows that the respective doping amounts ofEu3þ in Eu:HAp and IBU-released IBU–Eu:HAp are 7.14 wt% and7.12 wt%, which are much close to the stoichiometric value(7.16 wt% or 5 mol% to Ca2þ in Eu:HAp). The low-angle XRD

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Fig. 1. Wide-angle XRD patterns (A) and low-angle XRD patterns (B) of pure HAp (a),Eu:HAp (b), and the standard data for hydroxyapatite (JCPDS No. 09–0432) (c).

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Fig. 2. SEM images of pure HAp (a), Eu:HAp (b), and the EDS spectrum of HAp (c).

P. Yang et al. / Biomaterials 29 (2008) 4341–4347 4343

patterns for pure HAp and Eu:HAp are depicted in Fig. 1B. It can beseen that both samples show one sharp low-angle reflection atabout 2q¼ 1.3�, indicating the presence of their relatively uniformmesostructure nature. The d100 (7.1 nm and 7.2 nm) values calcu-lated from small-angle XRD patterns are well consistent with thoseobserved from the TEM images for the corresponding samples(see Fig. 3).

The SEM images of the pure HAp and Eu:HAp samples are dis-played in Fig. 2, respectively. It is found that both pure HAp andEu:HAp consist of relatively uniform rod-like particles with thewidth of 20–40 nm and the length of 100–200 nm. The resultssuggest that the doping of Eu3þ has little influence on themorphology of pure HAp. The EDS spectrum (Fig. 2c) of Eu:HApconfirms the presence of calcium (Ca), phosphor (P), oxygen (O),and europium (Eu) in the Eu:HAp sample (the Au signal is from theAu coating for measurement).

Fig. 3 displays the TEM images of pure HAp and Eu:HAp withlow resolution, the TEM image of Eu:HAp along the [110] direction,and the HRTEM image of Eu:HAp, respectively. As shown in Fig. 3a

Table 1Textural parameters of calcined HAp, Eu:HAp, IBU–Eu:HAp, and the IBU-releasedIBU–Eu:HAp sample

Samples VP (cm3/g) SBET (m2/g) DP (nm) IBU loading(wt%)

Eu3þ loading(wt%)

HAp 0.227 57.9 12.1Eu:HAp 0.253 54.8 10.3 46 7.14IBU–Eu:HAp 0.122 28.4 8.9 44IBU-releasedIBU–Eu:HAp

0.240 44.1 11.3 7.12

and b, both pure HAp and Eu:HAp exhibit a rod-like morphology,which is consistent with the SEM results. From the TEM image ofEu:HAp along the [110] direction (Fig. 3c), the typical characteristicsof hexagonally packed mesostructure are present. The observeddistance (7 nm) between the two adjacent fringes agrees well withthose calculated from the low-angle XRD patterns. It can be seenfrom the HRTEM image of Eu:HAp (Fig. 3d) that the crystallinephase of hydroxyapatite with well-resolved lattice fringes can beobserved. The distances (0.28 nm and 0.27 nm) between the adja-cent lattice fringes agree well with the d211 and d112 spacing of theliterature values (0.2814 nm and 0.2778 nm) (JCPDS No. 09–0432).

The XPS spectrum (Fig. 4) of Eu:HAp shows the binding energyof Eu (3d, 1132.3 eV), Ca (2p, 347.5 eV), O (1s, 532.1 eV), and P (2p,133.9 eV), respectively. By combination of previous XRD results, itcan be deduced that these signals arise from Eu3þ and HAp. XPS

Fig. 3. TEM images of pure HAp (a), Eu:HAp (b), along [110] direction for Eu:HAp (inset is the enlarged image) (c), and the HRTEM image of Eu:HAp (d).

P. Yang et al. / Biomaterials 29 (2008) 4341–43474344

results provide the additional evidence for the successful crystal-linity of Eu:HAp.

The FT-IR spectra for IBU, Eu:HAp, and IBU–Eu:HAp are dis-played in Fig. 5, respectively. As shown in Fig. 5A(a) for the Eu:HAp,the obvious absorption bands, assigned to OH (3430 cm�1) and

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P2p

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Fig. 4. XPS spectrum of the Eu:HAp sample.

H2O (1631 cm�1), indicate that a large number of OH groups andH2O are present on the surface of HAp, which are important forbonding drug (IBU) molecules. Additionally, the typical bandsattributed to PO4

3� can be obviously found. The bands at1091 cm�1 and 1037 cm�1 may be assigned to the triply

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Fig. 5. FT-IR spectra of Eu:HAp (a), IBU–Eu:HAp (b), and IBU (c).

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Fig. 6. N2 adsorption/desorption isotherms for pure HAp (a), Eu:HAp (b), IBU–Eu:HAp(c), and IBU-released IBU–Eu:HAp (d).

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659 5D

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590 5D

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650 5D

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c

Fig. 7. Excitation (a,c) and emission (b,d) spectra for Eu:HAp (a,b) and IBU–Eu:HAp(c,d).

P. Yang et al. / Biomaterials 29 (2008) 4341–4347 4345

degenerated n3 anti-symmetric stretching of P–O band, and the960 cm�1 band can be due to the n1 non-degenerated symmetricstretching of P–O bond. The bands at 606 cm�1 and 561 cm�1 canbe attributed to the triply degenerated n4 vibration of O–P–O bond,and the bands in the range of 460–473 cm�1 may be related withthe doubly degenerated n2 O–P–O bending [40]. For the IBU-loadedIBU–Eu:HAp (Fig. 5b), the band assigned to the vibration of –COOHat 1720 cm�1 is obvious except for a slight decrease of intensitycompared with that of IBU (Fig. 5c). Furthermore, the absorptionbands assigned to the quaternary carbon atom located at1462 cm�1 and 1520 cm�1, tertiary carbon atom at 1340 cm�1, O–Hbending vibration at 1421 cm�1, and C–Hx bond at 2919 cm�1 and2962 cm�1 can also be observed (Fig. 5b) [41], confirming thesuccessful adsorption of IBU onto the surface of the mesoporoushydroxyapatite.

The respective N2 adsorption/desorption isotherms of pure HAp,Eu:HAp, IBU–Eu:HAp, and IBU-released IBU–Eu:HAp are shown inFig. 6. It can be seen that all the samples show similar VI isothermsand the typical H1-hysteresis loops, demonstrating the propertiesof typical mesoporous materials. The results reveal that the dopingof Eu3þ and loading of IBU molecules have not altered the basic porestructure of mesoporous HAp. The textural parameters of the cor-responding materials are summarized in Table 1. As shown, thepure HAp has BET surface area of 57.9 m2/g, pore volume (VP) of0.227 cm3/g, and average pore size (DP) of 12.1 nm. For the Eu3þ

doped sample (Eu:HAp), the respective BET surface area, porevolume, and average pore size are 54.8 m2/g, 0.253 cm3/g, and10.3 nm, which is much similar to those of pure HAp. Afterincorporating of IBU molecules, all the BET surface area, pore sizeand pore volume are markedly reduced compared with Eu:HAp.Significantly, the sample after complete release of IBU still repre-sents a mesoporous hysteresis loop, and the BET surface area, porevolume, and average pore size can almost be recovered (Table 1),indicating the good stability of this drug storage/release system.

Under UV lamp irradiation (365 nm), the Eu:HAp, IBU–Eu:HAp,and IBU-released IBU–Eu:HAp samples show strong red lumines-cence, as shown in Scheme 1. Fig. 7 shows the PL excitation andemission spectra of Eu:HAp and IBU–Eu:HAp, respectively. In theexcitation spectra monitored by the Eu3þ 5D0–7F2 transition at612 nm (Fig. 7a,c), the broad band with a maximum at 250 nm mayarise from the charge transfer transition between Eu3þ and O2�

(CTB of Eu–O), and some sharp peaks originating from the f–ftransitions of Eu3þ can also be observed in the longer wavelength

region [42]. Upon excitation at 250 nm, the characteristic transitionlines from the excited 5D0 level of Eu3þ can be detected in theemission spectra [43], as shown in Fig. 7b,d, and the locations of theemission lines with their assignments are labeled as well. The twomain characteristic peaks from 5D0 / 7F1 (590 nm) and 5D0 / 7F2

(612 nm) are dominant. It is worth noting that the characteristicemission lines are still obvious in the emission spectrum for IBU–Eu:HAp (Fig. 7d), showing the potential application to be tracked ormonitored by the luminescence. A detailed relationship betweenthe emission intensity and extent of IBU drug release in the IBU–Eu:HAp system are discussed in Section 3.2.

3.2. Drug loading and release properties

During the loading and release process, the IBU molecules canbe adsorbed onto the surface of mesoporous materials in theimpregnation process and liberated by diffusion-controlled mech-anism. The OH groups on the surface should be the reaction sites toform hydrogen bonding with the carboxyl group of IBU when IBU isadsorbed on the surface. During the release process, the solvententers the IBU-matrix phase through the pores. The drug is thenslowly dissolved into SBF from the surface and diffuses from thesystem along the solvent-filled capillary channels. It can be seenfrom Table 1 that the respective IBU loading for IBU–HAp and IBU–Eu:HAp are 46 wt% and 44 wt% determined by TG analysis. Thesimilar drug loading may be attributed to their much similar

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U (%

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Fig. 8. Cumulative ibuprofen release from IBU–Eu:HAp (a) and IBU–HAp (b) systems asa function of release time in the release media of SBF.

P. Yang et al. / Biomaterials 29 (2008) 4341–43474346

surface area and pore volume for the two samples. The cumulativedrug release profiles for the IBU–HAp and IBU–Eu:HAp systems asa function of release time in SBF are shown in Fig. 8. As shown in thefigure, the two release profiles are similar and all exhibit sustainedproperties. In addition, both systems show a burst release of about50% within 1 h followed by the relatively slow release and completerelease after 24 hours. The initial burst release may be attributed tothe IBU weakly adsorbed on the outer surface of mesoporoushydroxyapatite, and the slow release of the rest of IBU can be due tothe strong interaction between IBU molecules and the surface. Itshould be noted that the luminescence functionalized HAp showsmuch similar release characteristic in comparison with that of pureHAp due to their similar surface and textural properties for the twosamples.

The PL emission intensity of IBU–Eu:HAp as a function ofcumulative released amount of IBU is given in Fig. 9. It can be seenthat the PL intensity (defined as the integrated area intensity of5D0–7F2 and 5D0–7F1 of Eu3þ) increases with the cumulativereleased IBU, and reaches a maximum when IBU is completelyreleased. It is well known that the emission of Eu3þ will bequenched to some extent in the environments where high phononfrequency is present, such as OH groups with a vibrationalfrequency near 3450 cm�1 [42]. The organic groups in IBU withhigh vibration frequencies from 1000 cm�1 to 3250 cm�1 will

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Fig. 9. The PL emission intensity of Eu3þ in IBU–Eu:HAp as a function of cumulativerelease amount of ibuprofen.

greatly quench the emission of Eu3þ in IBU–Eu:HAp. The quenchingeffect will be weakened with the release of IBU, resulting in theincrease of emission intensity. This relationship between the PLintensity and drug release extent can be potential as a probe formonitoring or tracking the drug release during the drug releaseprocess.

4. Conclusion

In summary, a simple one-step synthesis route for makinga luminescence functionalized mesoporous hydroxyapatitecomposite is proposed, resulting in the formation of a multifunc-tional material. This material is hierarchically structured andexhibits a nanoscale rod-like, mesoporous, crystalline structure,which is suitable for drug (IBU) release as a drug carrier. Especially,it exhibits strong red luminescence even after loading of drugmolecules, and the PL intensity increases with the cumulativereleased amount of IBU, which can be easily tracked and monitoredin the drug release process by the change of PL emission intensity.This system demonstrates a potential application in the fields ofdrug delivery and disease therapy based on its bioactive, lumines-cent, and mesoporous properties.

Acknowledgements

This project is financially supported by the foundation of ‘‘BairenJihua’’ of Chinese Academy of Sciences, National Basic ResearchProgram of China (2003CB314707, 2007CB935502), and theNational Natural Science Foundation of China (NSFC 50572103,20431030, 00610227).

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