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Tb3+ doped mesoporous bioactive glass nanofibers
Journal of Colloid and Interface Science 387 (2012) 285–291
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Electrospinning preparation and drug delivery properties of Eu3+/Tb3+ doped mesoporous bioactive glass nanofibers Shanshan Huang a,c, Xiaojiao Kang a,c, Ziyong Cheng a, Ping’an Ma a, Ye Jia b,⇑, Jun Lin a,⇑ a
State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China No. 2 Hospital, Jilin University, Changchun 130041, China c Graduate University of the Chinese Academy of Sciences, Beijing 100049, China b
a r t i c l e
i n f o
Article history: Received 10 June 2012 Accepted 1 August 2012 Available online 13 August 2012 Keywords: Electrospinning Mesoporous bioactive glass nanofibers Luminescence Biocompatibility Drug delivery
a b s t r a c t Luminescent Eu3+/Tb3+ doped mesoporous bioactive glass nanofibers (MBGNFs) with average diameter of 100–120 nm were fabricated by electrospinning method. Pluronic P123 and N-cetyltrimethylammonium bromide (CTAB) were used as co-surfactants to generate porous structure of the nanofibers. N2 adsorption–desorption measurement reveals that the MBGNF:Eu3+ have a surface area of 188 m2 g1, a pore volume of 0.246 cm3 g1 and average pore size of 4.17 nm, and the MBGNF:Tb3+ have a surface area of 171 m2 g1, a pore volume of 0.186 cm3 g1 and average pore size of 3.65 nm. Photoluminescence measurements reveal that the MBGNF:Eu3+ show strong red emission dominated by the 5D0 ? 7F2 transition of Eu3+ at 614 nm with a lifetime of 1.356 ms, and MBGNF:Tb3+ show strong green emission dominated by the 5D4 ? 7F5 transition of Tb3+ at 544 nm with a lifetime of 1.982 ms. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of MBGNF. These luminescent nanofibers show sustained release properties for ibuprofen (IBU) in vitro. The emission intensities of Eu3+ in the drug delivery system vary with the released amount of IBU, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bioactive glasses (BGs) and glasses ceramics have been widely studied and used since the pioneering work by Hench et al. in 1971 [1]. Because such materials have the ability to chemically bond with living bone tissue, they have been used in a variety of medical applications, such as implants in clinical bone repair and regeneration materials, bioactive coating of metallic implants in tissue engineering, tumor treatment, and protein and/or cell activation [2–10]. Zhao et al. synthesized highly ordered mesoporous bioactive glass (MBG) using nonionic block copolymers as structure-directing agents [11] through an evaporation-induced selfassembly (EISA) process. Compared with conventional BGs, MBGs show different structure and composition and have more specific surface area and pore volume, which may greatly accelerate the kinetic deposition process of hydroxycarbonate apatite and therefore enhance their bone-forming bioactivity [12]. Nanofibrous biomaterials have been intensively studied due to their large surface, which could enhance the ability to bond to the natural tissue [13]. The fabrication of bioactive glass fibers was mainly focused on the electrospinning techniques [14]. The electrospinning method was first explored in the 1930s [15], and ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Jia), [email protected] (J. Lin). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.08.004
now, it has been demonstrated that a variety of materials can be electrospun to form uniform fibers, such as organic, inorganic, and hybrid polymers (organic–inorganic composites) [16–22]. Kim et al. synthesized bioactive glass nanofibers using the electrospinning process and the bioactive glass nanofibers possessed excellent bioactivity and osteogenic potential in vitro [23]. Xia et al. studied the effect of electrospinning parameters on the diameter and morphology of bioactive glass fibers and the behavior of in vitro biomineralization [24]. However, these nanofibers prepared show no nanopores in their textures. Hong et al. designed and synthesized ultrathin mesoporous BG hollow fibers and nanopore-controllable BG nanofibers using electrospinning technique and P123-PEO as co-templates. These ultrathin porous bioactive glass fibers had high bioactivity in vitro assays and have potential applications for bone tissue engineering and drug delivery [25,26]. In recent years, much research attention has been paid to the rare earth ions since they bear unique electronic and optical characteristics arising from their 4f electrons. The luminescence features of rare earth ions include high luminescence quantum yield, narrow bandwidth, long-lived emission, large Stokes shifts, ligand-dependent luminescence sensitization, which have received startling interest because of the continuously expanding need for luminescent materials meeting the stringent requirements of telecommunication, lighting, electroluminescent devices, (bio-)
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2. Materials and methods
here. L929 fibroblast cells (5000–6000) in 200 lL media per well were plated in a 96-well plate for 24 h to allow the cells to attach and then exposed to different concentrations of the MBGNF (3.125, 6.25, 12.5, 25.0, 50.0, 100 and 200.0 lg mL1) for 24 h in 5% CO2 at 37 °C. At the end of the incubation time, the medium containing the MBGNF was removed and MTT solution (20 lL, diluted in a culture medium to a final concentration of 5 mg/mL) was added. After incubation at 37 °C in the dark for 4 h, 100 lL of acidified isopropanol was added to each well, and the absorbance was monitored with a microplate reader at a wavelength of 570 nm. Averages and standard deviations were based on four samples, and all tests were performed in triplicate. The cell viability was calculated using the following equation: cell viability (%) = [A]test/[A]control 100.
2.1. Chemicals and materials
2.4. Preparation of drug storage/delivery systems
All chemicals are of analytical reagents (A.R.) and used directly without further purification, including triethyl phosphate (TEP), calcium nitrate [Ca(NO3)24H2O, 99%], tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 99 wt.%, A.R., Beijing Yili Fine Chemical Co., Ltd.), N-cetyltrimethylammonium bromide (CTAB, Shanghai Chemical Reagent Co., Ltd.), polyvinylpyrrolidone (PVP, Mw = 1,300,000, Aldrich), pluronic P123 (EO20–PO70–EO20, Mw = 5800, Aldrich), Eu2O3, Tb4O7 (with purity > 99.99%, Science and Technology Parent Company of Changchun Institute of Applied Chemistry, China). Eu2O3 and Tb4O7 were dissolved in dilute HNO3, resulting in the formation of a colorless stock solution of Eu(NO3)3 (0.1 M) and Tb(NO3)3 (0.1 M), respectively.
The drug storage/delivery system using the MBGNF as a carrier was prepared according to the previous reports [36,37]. Ibuprofen was selected as the model drug. Typically, 0.2 g of the MGBNF:Eu3+ was added into 50 mL of hexane solution with an IBU concentration of 60 mg mL1 at room temperature, and soaked for 24 h with slow stirring in a vial that was sealed to prevent the evaporation of hexane. The IBU-loaded sample was separated by centrifugation, and then dried in vacuum at 60 °C for 24 h, and denoted as IBU–MBGNF:Eu3+. The in vitro delivery of IBU was performed by immersing 100 mg of the drug-loading sample in the release media of simulated body fluid (SBF) with slow stirring under the immersion 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 142.0/5.0/2.5/1.5/147.8/ 2 2 4.2/1.0/0.5 for Na+/K+/Ca2+/Mg2+/Cl/HCO (pH7.4) 3 /HPO4 /SO4 [38,39]. The ratio of SBF to adsorbed IBU was kept at 1 mL mg1. At selected time intervals, a sample (0.5 mL) was removed and immediately replaced with an equal volume of fresh SBF. The solution removed was properly diluted and the amount of ibuprofen present was monitored at 264 nm using a UV–vis spectrophotometer.
analytical sensors, bioimaging set-ups, and solar cells [27–34]. To our knowledge, lanthanide (Ln3+) ions doped mesoporous luminescent bioactive glass nanofibers have not been reported so far. In this paper, we fabricated luminescent, mesoporous, and bioactive glass nanofibers doped with Eu3+/Tb3+ using the electrospinning technique. Pluronic P123 and CTAB were used as co-surfactants to generate porous structure of the nanofibers. The photoluminescence properties of the mesoporous bioactive glass nanofibers were studied. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of these systems. The drug-storage/release properties of the nanofibers were investigated by using ibuprofen as a model drug.
2.2. Synthesis of Eu3+/Tb3+ doped MBGNF A typical synthesis of Eu3+ doped 70S25C5P mesoporous bioactive glass nanofibers (70 mol% SiO2, 25 mol% CaO, 5 mol% P2O5) was as follows. Solution A: Ca(NO3)24H2O (1.535 g), 3.4 mL of 0.1 M Eu(NO3)3, TEOS (4.5 g), and triethyl phosphate (0.48 mL) were mixed with 0.5 mL of HCl solution (1 mol L1) and 40 mL of ethanol. The doping concentrations of Eu3+ ions are 5 mol% of Ca2+ in the system [35]. Solution B: 0.7 g PVP, 0.4 g P123 and 0.2 g CTAB were dissolved in 8 mL mixture of ethanol and deionized water (v/v = 3:5). After stirring for 2 h, 5 mL of solution A was added to 8 mL of solution B. Then another 4 h stirring was applied to obtain a homogeneous viscous solution for electrospinning. The above viscous solution was placed in a 5 mL hypodermic syringe. The anode of the high-voltage power supply was clamped to the syringe needle tip, and the cathode was connected to the grounded collector plate. The applied voltage was 10 kV, and the distance between the needle tip and the collector was 17 cm, and the flow rate of the spinning solution was controlled at 1 mL/h by a syringe pump (TJ-3A/W0109-1B, Baoding Longer Precision Pump Co, Ltd., China). The asspun nanofibers were placed in the oven at 37 °C for 1 day to allow complete hydrolysis of TEOS and TEP. Finally, the polymer and surfactant were removed from the nanofibers by calcining them at 600 °C for 4 h in air, the Eu3+ doped MBG nanofibers (denoted as MBGNF:Eu3+) were obtained. Tb3+ doped MBG nanofibers (with the doping Tb3+ concentration of 5 mol% to Ca2+, denoted as MBGNF:Tb3+) were prepared in the same manner except that the doped luminescent source Eu(NO3)3 was replaced by Tb(NO3)3. 2.3. In vitro cytotoxicity of the Eu3+/Tb3+ doped MBGNF The in vitro cytotoxicity of the MBGNF:Eu3+ and MBGNF:Tb3+ was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and the Vero cell line was used
2.5. Characterization The X-ray diffraction (XRD) patterns of the samples were recorded on a D8 Focus diffractometer (Bruker) with Cu Ka radiation (k = 0.15405 nm). Fourier transform infrared spectroscopy (FT-IR) spectra were measured with a Perkin–Elmer 580B infrared spectrophotometer with the KBr pellet technique. The UV–vis adsorption spectral data were measured on a U-3310 spectrophotometer. Nitrogen adsorption–desorption analysis was performed with a Micromeritics ASAP 2020 M apparatus. The morphology and structure of the samples were inspected using a field emission scanning electron microscopy (FE-SEM, S-4800, Hitachi) equipped with an energy-dispersive X-ray (EDX) spectrometer. Low- and high-resolution transmission electron microscopy (TEM) was performed using an FEI Tecnai G2 S-Twin instrument with a field emission gun operating at 200 kV. Images were acquired digitally on a Gatan multiple CCD camera. The ultraviolet–visible photoluminescence (PL) excitation and emission spectra were recorded with a Hitachi F-7000 spectrophotometer equipped with a 150 W xenon lamp as the excitation source under the same experimental conditions (this will decrease the experimental errors as far as possible). The luminescence decay curves were obtained from a Lecroy Wave Runner 6100 digital oscilloscope (1 GHz) using a tunable laser (pulse width = 4 ns, gate = 50 ns) as excitation source (Continuum Sunlite OPO). All measurements were performed at room temperature.
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3. Results and discussion Fig. 1 shows the SEM images of as-formed precursory Eu3+/Tb3+ doped BG fibers, as well those samples annealed at 600 °C, respectively. From Fig. 1a and b, it can be seen that the as-formed precursor fibers are uniform with diameter ranging from 300 to 400 nm. After the PVP, P123, and CTAB were removed by calcining the precursor in air at 600 °C, the BG nanofiber retained its continual structure (Fig. 1c and d), while the average diameter was reduced to about 100–120 nm. Energy-dispersive X-ray (EDX) analysis in Fig. 1e and f shows that the nanofibers are composed of silicon (Si), calcium (Ca), phosphor (P), oxygen (O), europium (Eu) or terbium (Tb) and no other elements are present, demonstrating that the fabricated MBGNF are pure. The XRD pattern shown in Fig. 2 indicates that the calcined MBGNF:Eu3+ are still in the amorphous state. The morphology and structure of the nanofibers are further characterized by TEM techniques. The TEM images (Fig. 3) show that the calcined BG nanofibers are uniform with rough surface. There are dispersed nanopores with no long-range periodicity in the BG nanofibers, which provide volume for guest molecules. The removal of P123, CTAB, polymer surface tension, and strong electrostatic force pulling the fiber in the electrospinning process may contribute to the formation of the porous structure [40]. Fig. 4 shows the N2 adsorption–desorption isotherms of the MBGNF:Eu3+ and MBGNF:Tb3+. The isotherms of MBGNF:Eu3+ and MBGNF:Tb3+ display similar H3 type hysteresis loops, which can
Fig. 2. XRD spectrum of MBGNF:Eu3+.
be ascribed to the disordered nanopore system of the MBGNF. MBGNF:Eu3+ have a surface area of 188 m2 g1, a pore volume of 0.246 cm3 g1, and average pore size of 4.17 nm. MBGNF:Tb3+ have a surface area of 171 m2 g1, a pore volume of 0.186 cm3 g1, and average pore size of 3.65 nm. The differences of the surface area, pore volume and pore size between the MBGNF:Eu3+ and MBGNF:Tb3+ may be caused by the difference of their pore structure due to the calcination process. The pore sizes of the nanofibers are suitable for the adsorption of IBU molecules.
Fig. 1. SEM images of the precursor of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b), calcined MBGNF:Eu3+ (c), and MBGNF:Tb3+ (d). The EDX of calcined MBGNF:Eu3+ (e) and MBGNF:Tb3+ (f).
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Fig. 3. TEM images of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b).
Fig. 4. N2 adsorption–desorption curves of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b).
Fig. 5 shows the PL excitation and emission spectra of MBGNF:Eu3+, MBGNF:Tb3+, and IBU loaded samples, respectively. In the excitation spectra monitored by the Eu3+ 5D0 ? 7F2 transition at 614 nm, the broad band with a maximum near 275 nm may arise from the charge transfer transition between Eu3+ and O2 [41]. Upon excitation at 275 nm, the characteristic transition lines from the excited 5D0 level of Eu3+ can be detected in the emission spectra, and the locations of the emission lines with their assignments are labeled as well [42]. The two main characteristic peaks from 5 D0 ? 7F1 (590 nm) and 5D0 ? 7F2 (614 nm) are dominant. For the MBGNF:Tb3+ sample, the excitation spectrum consists of a strong band at 275 nm and a relatively weak band at 229 nm, which correspond to the spin-allowed (DS = 0) and spin-forbidden (DS = 1) components of 4f8 ? 4f75d transition, respectively [43]. The emission spectrum of MBGNF:Tb3+ is dominated by a green emission around 544 nm corresponding to the 5D4 ? 7F5 transition of Tb3+. Fig. 6 shows the respective decay curves for the luminescence of Eu3+ and Tb3+ in MBGNF:Eu3+ and MBGNF:Tb3+. It can be seen that both decay curves for 5D0?7F2 (614 nm) of Eu3+ and 5D4 ? 7F5 (544 nm) of Tb3+ can be well fitted into a double-exponential function as I = A1 exp(t/s1)+A2 exp(t/s2) (s1 and s2 are the fast and slow components of the luminescence lifetimes, A1 and A2 are the fitting parameters). The average lifetimes for 5D0 ? 7F2 (614 nm) of Eu3+ and 5D4 ? 7F5 (544 nm) of Tb3+ are calculated from the formula s ¼ ðA1 s21 þ A2 s22 Þ=ðA1 s1 þ A2 s2 Þ, and the fitting results are shown in the figure [44,45]. The calculated average lifetimes are 1.356 and 1.982 ms for 5D0 ? 7F2 (614 nm) of Eu3+ and 5 D4 ? 7F5 (544 nm) of Tb3+, respectively.
It is worth noting that the characteristic emission lines are still obvious in the emission spectrum for IBU–MBGNF:Eu3+ and IBU–MBGNF:Tb3+ (Fig. 5), showing the potential application to be tracked or monitored by the luminescence. A detailed relationship between the emission intensity and extent of IBU drug release in the IBU–MBGNF:Eu3+ system are to be discussed in next section. In order to evaluate the biocompatibility of the Eu3+/Tb3+ doped MBGNF, an MTT assay was performed on these fibers. This method is based on the formation of dark red formazan by the metabolically active cells after their exposure to MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide). Cell viability is directly proportional to the amount of formazan produced monitored by the absorbance at 570 nm. The MBGNF were delivered over a wide range of dosages (3.125–200 lg mL1). Fig. 7 shows the cell viability of L929 fibroblast cells incubated with the sample. The difference in cell viabilities after 24 h of incubation is negligibly small, and there is little cytotoxicity after incubation at a high concentration of 200 lg mL1. These results suggest that these MBGNF have good biocompatibility. The FT-IR spectra for MBGNF:Eu3+, IBU–MBGNF:Eu3+, and IBU are displayed in Fig. 8, respectively. As shown in Fig. 8a for the MBGNF:Eu3+, the obvious broad absorption bands, assigned to OH (3430 cm1) and H2O (1630 cm1), indicate that a large number of –OH groups and H2O are present on the surface of the fiber, which are important for bonding drug (IBU) molecules. The peaks centered at 802, 461 cm1 are assigned to the Si–O–Si bands. The peaks centered at 1440 cm1 are assigned to the P–O bands [46]. For the IBU–MBGNF:Eu3+ (Fig. 8b), the sharp band centered at
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Fig. 5. Excitation spectra of (A): (a) MBGNF:Eu3+, (b) IBU–MBGNF:Eu3+, (C): (a) MBGNF:Tb3+, (b) IBU–MBGNF:Tb3+ and Emission spectra of (B): (a) MBGNF:Eu3+, (b) IBU– MBGNF:Eu3+, (D): (a) MBGNF:Tb3+, (b) IBU–MBGNF:Tb3+.
Fig. 6. The decay curves for the 5D0 ? 7F2 (614 nm) emission of Eu3+ in MBGNF:Eu3+ (a) and 5D4 ? 7F5 (544 nm) emission of Tb3+ in MBGNF:Tb3+ (b) (kex = 275 nm).
Fig. 7. Cell viability of MBGNF:Eu3+ (A) and MBGNF:Tb3+ (B).
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Fig. 8. FT-IR spectra of (a) MBGNF:Eu3+, (b) IBU–MBGNF:Eu3+ and (c) IBU.
1710 cm1 is attributed to the vibration of –COOH, which is same as the IBU (Fig. 8c). Intense carboxylate bands at 1559 cm1 are detected. The absorption bands assigned to the quaternary carbon atom located at 1465 and 1513 cm1, tertiary carbon atom at 1367 cm1, O–H bending vibration at 1414 cm1, and C–Hx bond at 2927 and 2955 cm1 can also be observed (Fig. 8b) [36,47]. These results confirm the successful adsorption of IBU onto MBGNF:Eu3+. The IBU loading amount determined by the TG analysis for IBU– MBGNF:Eu3+ and IBU–MBGNF:Tb3+ is 16 wt.% and 13 wt.%, respectively (Fig. 9). The MBGNF:Tb3+ owning relatively smaller surface area and pore volume shows lower ibuprofen loading amount compared with MBGNF:Eu3+. During the loading and release process, the IBU molecules can be adsorbed onto the surface of porous MBGNF in the impregnation process and liberated by diffusioncontrolled mechanism. The OH groups on the surface could form hydrogen bonds with the carboxyl groups 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 pore channels of the MBGNF:Eu3+. The cumulative drug release profiles for the IBU–MBGNF:Eu3+/ 3+ Tb systems as a function of release time in SBF are shown in Fig. 10. The systems show an initial burst release followed by the relatively slow release and completely release after 36 h. The IBU released in the first 0.5 h reaches 39% and 42% for IBU– MBGNF:Eu3+ and IBU–MBGNF:Tb3+, respectively. The initial burst
Fig. 9. TG curves of IBU–MBGNF:Eu3+ (a) and IBU–MBGNF:Tb3+ (b).
Fig. 10. The cumulative release of IBU from IBU–MBGNF:Eu3+ and IBU– MBGNF:Tb3+.
release may be attributed to the IBU weakly adsorbed on the outer surface of MBGNF, and the slow release of the rest of IBU can be due to the strong interaction between IBU molecules and the surface. The IBU release rate of the MBGNF was relatively faster than the previous reports [35], which can be ascribed to the disordered pore system of the nanofibers. The PL emission intensity of IBU–MBGNF:Eu3+ as a function of cumulative released amount of IBU is given in Fig. 11. The photoluminescence (PL) excitation and emission spectra were obtained on a Hitachi F-7000 spectrofluorimeter equipped with a 150 W xenon lamp as the excitation source under the same experimental conditions (this will decrease the experimental errors as far as possible). Peak area score were used to measure the change of luminescence. This method has good reproducibility and reliability for comparing the PL intensity of the rare earth doped luminescent materials [48–51]. It can be seen that the PL intensity (defined as the integrated area intensity of 5D0 ? 7FJ of Eu3+) increases with the cumulative released IBU, and reaches a maximum when IBU is completely released. It is well known that the emission of Eu3+ will be quenched to some extent in the environments where high phonon frequency is present, such as OH groups with a vibrational frequency near 3450 cm1 [52]. The organic groups in IBU with high vibration frequencies from 1000 to 3250 cm1 will greatly quench the emission of Eu3+ in IBU–MBGNF:Eu3+. The quenching effect will be weakened with the release of IBU, resulting in the
Fig. 11. PL emission intensity of Eu3+ in IBU–MBGNF:Eu3+ as a function of cumulative release amount of ibuprofen.
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increase of emission intensity. This relationship between the PL intensity and drug release extent can be a potential probe for monitoring or tracking the drug release during the drug delivery process. 4. Conclusions Eu3+/Tb3+ doped luminescent mesoporous bioactive glass nanofibers were fabricated using the electrospinning technique. The synthesized MBGNF show uniform diameter and dispersed nanopores. Photoluminescence measurements reveal that these nanofibers show strong luminescence under the ultraviolet irradiation. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of these systems. These luminescent nanofibers show sustained release properties using ibuprofen as the model drug. The emission intensities of Eu3+ in the drug carrier system vary with the released amount of IBU, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity. Acknowledgment
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Electrospinning preparation and drug delivery properties of Eu3+/Tb3+ doped mesoporous bioactive glass nanofibers Shanshan Huang a,c, Xiaojiao Kang a,c, Ziyong Cheng a, Ping’an Ma a, Ye Jia b,⇑, Jun Lin a,⇑ a
State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China No. 2 Hospital, Jilin University, Changchun 130041, China c Graduate University of the Chinese Academy of Sciences, Beijing 100049, China b
a r t i c l e
i n f o
Article history: Received 10 June 2012 Accepted 1 August 2012 Available online 13 August 2012 Keywords: Electrospinning Mesoporous bioactive glass nanofibers Luminescence Biocompatibility Drug delivery
a b s t r a c t Luminescent Eu3+/Tb3+ doped mesoporous bioactive glass nanofibers (MBGNFs) with average diameter of 100–120 nm were fabricated by electrospinning method. Pluronic P123 and N-cetyltrimethylammonium bromide (CTAB) were used as co-surfactants to generate porous structure of the nanofibers. N2 adsorption–desorption measurement reveals that the MBGNF:Eu3+ have a surface area of 188 m2 g1, a pore volume of 0.246 cm3 g1 and average pore size of 4.17 nm, and the MBGNF:Tb3+ have a surface area of 171 m2 g1, a pore volume of 0.186 cm3 g1 and average pore size of 3.65 nm. Photoluminescence measurements reveal that the MBGNF:Eu3+ show strong red emission dominated by the 5D0 ? 7F2 transition of Eu3+ at 614 nm with a lifetime of 1.356 ms, and MBGNF:Tb3+ show strong green emission dominated by the 5D4 ? 7F5 transition of Tb3+ at 544 nm with a lifetime of 1.982 ms. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of MBGNF. These luminescent nanofibers show sustained release properties for ibuprofen (IBU) in vitro. The emission intensities of Eu3+ in the drug delivery system vary with the released amount of IBU, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bioactive glasses (BGs) and glasses ceramics have been widely studied and used since the pioneering work by Hench et al. in 1971 [1]. Because such materials have the ability to chemically bond with living bone tissue, they have been used in a variety of medical applications, such as implants in clinical bone repair and regeneration materials, bioactive coating of metallic implants in tissue engineering, tumor treatment, and protein and/or cell activation [2–10]. Zhao et al. synthesized highly ordered mesoporous bioactive glass (MBG) using nonionic block copolymers as structure-directing agents [11] through an evaporation-induced selfassembly (EISA) process. Compared with conventional BGs, MBGs show different structure and composition and have more specific surface area and pore volume, which may greatly accelerate the kinetic deposition process of hydroxycarbonate apatite and therefore enhance their bone-forming bioactivity [12]. Nanofibrous biomaterials have been intensively studied due to their large surface, which could enhance the ability to bond to the natural tissue [13]. The fabrication of bioactive glass fibers was mainly focused on the electrospinning techniques [14]. The electrospinning method was first explored in the 1930s [15], and ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Jia), [email protected] (J. Lin). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.08.004
now, it has been demonstrated that a variety of materials can be electrospun to form uniform fibers, such as organic, inorganic, and hybrid polymers (organic–inorganic composites) [16–22]. Kim et al. synthesized bioactive glass nanofibers using the electrospinning process and the bioactive glass nanofibers possessed excellent bioactivity and osteogenic potential in vitro [23]. Xia et al. studied the effect of electrospinning parameters on the diameter and morphology of bioactive glass fibers and the behavior of in vitro biomineralization [24]. However, these nanofibers prepared show no nanopores in their textures. Hong et al. designed and synthesized ultrathin mesoporous BG hollow fibers and nanopore-controllable BG nanofibers using electrospinning technique and P123-PEO as co-templates. These ultrathin porous bioactive glass fibers had high bioactivity in vitro assays and have potential applications for bone tissue engineering and drug delivery [25,26]. In recent years, much research attention has been paid to the rare earth ions since they bear unique electronic and optical characteristics arising from their 4f electrons. The luminescence features of rare earth ions include high luminescence quantum yield, narrow bandwidth, long-lived emission, large Stokes shifts, ligand-dependent luminescence sensitization, which have received startling interest because of the continuously expanding need for luminescent materials meeting the stringent requirements of telecommunication, lighting, electroluminescent devices, (bio-)
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2. Materials and methods
here. L929 fibroblast cells (5000–6000) in 200 lL media per well were plated in a 96-well plate for 24 h to allow the cells to attach and then exposed to different concentrations of the MBGNF (3.125, 6.25, 12.5, 25.0, 50.0, 100 and 200.0 lg mL1) for 24 h in 5% CO2 at 37 °C. At the end of the incubation time, the medium containing the MBGNF was removed and MTT solution (20 lL, diluted in a culture medium to a final concentration of 5 mg/mL) was added. After incubation at 37 °C in the dark for 4 h, 100 lL of acidified isopropanol was added to each well, and the absorbance was monitored with a microplate reader at a wavelength of 570 nm. Averages and standard deviations were based on four samples, and all tests were performed in triplicate. The cell viability was calculated using the following equation: cell viability (%) = [A]test/[A]control 100.
2.1. Chemicals and materials
2.4. Preparation of drug storage/delivery systems
All chemicals are of analytical reagents (A.R.) and used directly without further purification, including triethyl phosphate (TEP), calcium nitrate [Ca(NO3)24H2O, 99%], tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 99 wt.%, A.R., Beijing Yili Fine Chemical Co., Ltd.), N-cetyltrimethylammonium bromide (CTAB, Shanghai Chemical Reagent Co., Ltd.), polyvinylpyrrolidone (PVP, Mw = 1,300,000, Aldrich), pluronic P123 (EO20–PO70–EO20, Mw = 5800, Aldrich), Eu2O3, Tb4O7 (with purity > 99.99%, Science and Technology Parent Company of Changchun Institute of Applied Chemistry, China). Eu2O3 and Tb4O7 were dissolved in dilute HNO3, resulting in the formation of a colorless stock solution of Eu(NO3)3 (0.1 M) and Tb(NO3)3 (0.1 M), respectively.
The drug storage/delivery system using the MBGNF as a carrier was prepared according to the previous reports [36,37]. Ibuprofen was selected as the model drug. Typically, 0.2 g of the MGBNF:Eu3+ was added into 50 mL of hexane solution with an IBU concentration of 60 mg mL1 at room temperature, and soaked for 24 h with slow stirring in a vial that was sealed to prevent the evaporation of hexane. The IBU-loaded sample was separated by centrifugation, and then dried in vacuum at 60 °C for 24 h, and denoted as IBU–MBGNF:Eu3+. The in vitro delivery of IBU was performed by immersing 100 mg of the drug-loading sample in the release media of simulated body fluid (SBF) with slow stirring under the immersion 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 142.0/5.0/2.5/1.5/147.8/ 2 2 4.2/1.0/0.5 for Na+/K+/Ca2+/Mg2+/Cl/HCO (pH7.4) 3 /HPO4 /SO4 [38,39]. The ratio of SBF to adsorbed IBU was kept at 1 mL mg1. At selected time intervals, a sample (0.5 mL) was removed and immediately replaced with an equal volume of fresh SBF. The solution removed was properly diluted and the amount of ibuprofen present was monitored at 264 nm using a UV–vis spectrophotometer.
analytical sensors, bioimaging set-ups, and solar cells [27–34]. To our knowledge, lanthanide (Ln3+) ions doped mesoporous luminescent bioactive glass nanofibers have not been reported so far. In this paper, we fabricated luminescent, mesoporous, and bioactive glass nanofibers doped with Eu3+/Tb3+ using the electrospinning technique. Pluronic P123 and CTAB were used as co-surfactants to generate porous structure of the nanofibers. The photoluminescence properties of the mesoporous bioactive glass nanofibers were studied. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of these systems. The drug-storage/release properties of the nanofibers were investigated by using ibuprofen as a model drug.
2.2. Synthesis of Eu3+/Tb3+ doped MBGNF A typical synthesis of Eu3+ doped 70S25C5P mesoporous bioactive glass nanofibers (70 mol% SiO2, 25 mol% CaO, 5 mol% P2O5) was as follows. Solution A: Ca(NO3)24H2O (1.535 g), 3.4 mL of 0.1 M Eu(NO3)3, TEOS (4.5 g), and triethyl phosphate (0.48 mL) were mixed with 0.5 mL of HCl solution (1 mol L1) and 40 mL of ethanol. The doping concentrations of Eu3+ ions are 5 mol% of Ca2+ in the system [35]. Solution B: 0.7 g PVP, 0.4 g P123 and 0.2 g CTAB were dissolved in 8 mL mixture of ethanol and deionized water (v/v = 3:5). After stirring for 2 h, 5 mL of solution A was added to 8 mL of solution B. Then another 4 h stirring was applied to obtain a homogeneous viscous solution for electrospinning. The above viscous solution was placed in a 5 mL hypodermic syringe. The anode of the high-voltage power supply was clamped to the syringe needle tip, and the cathode was connected to the grounded collector plate. The applied voltage was 10 kV, and the distance between the needle tip and the collector was 17 cm, and the flow rate of the spinning solution was controlled at 1 mL/h by a syringe pump (TJ-3A/W0109-1B, Baoding Longer Precision Pump Co, Ltd., China). The asspun nanofibers were placed in the oven at 37 °C for 1 day to allow complete hydrolysis of TEOS and TEP. Finally, the polymer and surfactant were removed from the nanofibers by calcining them at 600 °C for 4 h in air, the Eu3+ doped MBG nanofibers (denoted as MBGNF:Eu3+) were obtained. Tb3+ doped MBG nanofibers (with the doping Tb3+ concentration of 5 mol% to Ca2+, denoted as MBGNF:Tb3+) were prepared in the same manner except that the doped luminescent source Eu(NO3)3 was replaced by Tb(NO3)3. 2.3. In vitro cytotoxicity of the Eu3+/Tb3+ doped MBGNF The in vitro cytotoxicity of the MBGNF:Eu3+ and MBGNF:Tb3+ was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and the Vero cell line was used
2.5. Characterization The X-ray diffraction (XRD) patterns of the samples were recorded on a D8 Focus diffractometer (Bruker) with Cu Ka radiation (k = 0.15405 nm). Fourier transform infrared spectroscopy (FT-IR) spectra were measured with a Perkin–Elmer 580B infrared spectrophotometer with the KBr pellet technique. The UV–vis adsorption spectral data were measured on a U-3310 spectrophotometer. Nitrogen adsorption–desorption analysis was performed with a Micromeritics ASAP 2020 M apparatus. The morphology and structure of the samples were inspected using a field emission scanning electron microscopy (FE-SEM, S-4800, Hitachi) equipped with an energy-dispersive X-ray (EDX) spectrometer. Low- and high-resolution transmission electron microscopy (TEM) was performed using an FEI Tecnai G2 S-Twin instrument with a field emission gun operating at 200 kV. Images were acquired digitally on a Gatan multiple CCD camera. The ultraviolet–visible photoluminescence (PL) excitation and emission spectra were recorded with a Hitachi F-7000 spectrophotometer equipped with a 150 W xenon lamp as the excitation source under the same experimental conditions (this will decrease the experimental errors as far as possible). The luminescence decay curves were obtained from a Lecroy Wave Runner 6100 digital oscilloscope (1 GHz) using a tunable laser (pulse width = 4 ns, gate = 50 ns) as excitation source (Continuum Sunlite OPO). All measurements were performed at room temperature.
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3. Results and discussion Fig. 1 shows the SEM images of as-formed precursory Eu3+/Tb3+ doped BG fibers, as well those samples annealed at 600 °C, respectively. From Fig. 1a and b, it can be seen that the as-formed precursor fibers are uniform with diameter ranging from 300 to 400 nm. After the PVP, P123, and CTAB were removed by calcining the precursor in air at 600 °C, the BG nanofiber retained its continual structure (Fig. 1c and d), while the average diameter was reduced to about 100–120 nm. Energy-dispersive X-ray (EDX) analysis in Fig. 1e and f shows that the nanofibers are composed of silicon (Si), calcium (Ca), phosphor (P), oxygen (O), europium (Eu) or terbium (Tb) and no other elements are present, demonstrating that the fabricated MBGNF are pure. The XRD pattern shown in Fig. 2 indicates that the calcined MBGNF:Eu3+ are still in the amorphous state. The morphology and structure of the nanofibers are further characterized by TEM techniques. The TEM images (Fig. 3) show that the calcined BG nanofibers are uniform with rough surface. There are dispersed nanopores with no long-range periodicity in the BG nanofibers, which provide volume for guest molecules. The removal of P123, CTAB, polymer surface tension, and strong electrostatic force pulling the fiber in the electrospinning process may contribute to the formation of the porous structure [40]. Fig. 4 shows the N2 adsorption–desorption isotherms of the MBGNF:Eu3+ and MBGNF:Tb3+. The isotherms of MBGNF:Eu3+ and MBGNF:Tb3+ display similar H3 type hysteresis loops, which can
Fig. 2. XRD spectrum of MBGNF:Eu3+.
be ascribed to the disordered nanopore system of the MBGNF. MBGNF:Eu3+ have a surface area of 188 m2 g1, a pore volume of 0.246 cm3 g1, and average pore size of 4.17 nm. MBGNF:Tb3+ have a surface area of 171 m2 g1, a pore volume of 0.186 cm3 g1, and average pore size of 3.65 nm. The differences of the surface area, pore volume and pore size between the MBGNF:Eu3+ and MBGNF:Tb3+ may be caused by the difference of their pore structure due to the calcination process. The pore sizes of the nanofibers are suitable for the adsorption of IBU molecules.
Fig. 1. SEM images of the precursor of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b), calcined MBGNF:Eu3+ (c), and MBGNF:Tb3+ (d). The EDX of calcined MBGNF:Eu3+ (e) and MBGNF:Tb3+ (f).
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Fig. 3. TEM images of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b).
Fig. 4. N2 adsorption–desorption curves of MBGNF:Eu3+ (a) and MBGNF:Tb3+ (b).
Fig. 5 shows the PL excitation and emission spectra of MBGNF:Eu3+, MBGNF:Tb3+, and IBU loaded samples, respectively. In the excitation spectra monitored by the Eu3+ 5D0 ? 7F2 transition at 614 nm, the broad band with a maximum near 275 nm may arise from the charge transfer transition between Eu3+ and O2 [41]. Upon excitation at 275 nm, the characteristic transition lines from the excited 5D0 level of Eu3+ can be detected in the emission spectra, and the locations of the emission lines with their assignments are labeled as well [42]. The two main characteristic peaks from 5 D0 ? 7F1 (590 nm) and 5D0 ? 7F2 (614 nm) are dominant. For the MBGNF:Tb3+ sample, the excitation spectrum consists of a strong band at 275 nm and a relatively weak band at 229 nm, which correspond to the spin-allowed (DS = 0) and spin-forbidden (DS = 1) components of 4f8 ? 4f75d transition, respectively [43]. The emission spectrum of MBGNF:Tb3+ is dominated by a green emission around 544 nm corresponding to the 5D4 ? 7F5 transition of Tb3+. Fig. 6 shows the respective decay curves for the luminescence of Eu3+ and Tb3+ in MBGNF:Eu3+ and MBGNF:Tb3+. It can be seen that both decay curves for 5D0?7F2 (614 nm) of Eu3+ and 5D4 ? 7F5 (544 nm) of Tb3+ can be well fitted into a double-exponential function as I = A1 exp(t/s1)+A2 exp(t/s2) (s1 and s2 are the fast and slow components of the luminescence lifetimes, A1 and A2 are the fitting parameters). The average lifetimes for 5D0 ? 7F2 (614 nm) of Eu3+ and 5D4 ? 7F5 (544 nm) of Tb3+ are calculated from the formula s ¼ ðA1 s21 þ A2 s22 Þ=ðA1 s1 þ A2 s2 Þ, and the fitting results are shown in the figure [44,45]. The calculated average lifetimes are 1.356 and 1.982 ms for 5D0 ? 7F2 (614 nm) of Eu3+ and 5 D4 ? 7F5 (544 nm) of Tb3+, respectively.
It is worth noting that the characteristic emission lines are still obvious in the emission spectrum for IBU–MBGNF:Eu3+ and IBU–MBGNF:Tb3+ (Fig. 5), showing the potential application to be tracked or monitored by the luminescence. A detailed relationship between the emission intensity and extent of IBU drug release in the IBU–MBGNF:Eu3+ system are to be discussed in next section. In order to evaluate the biocompatibility of the Eu3+/Tb3+ doped MBGNF, an MTT assay was performed on these fibers. This method is based on the formation of dark red formazan by the metabolically active cells after their exposure to MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide). Cell viability is directly proportional to the amount of formazan produced monitored by the absorbance at 570 nm. The MBGNF were delivered over a wide range of dosages (3.125–200 lg mL1). Fig. 7 shows the cell viability of L929 fibroblast cells incubated with the sample. The difference in cell viabilities after 24 h of incubation is negligibly small, and there is little cytotoxicity after incubation at a high concentration of 200 lg mL1. These results suggest that these MBGNF have good biocompatibility. The FT-IR spectra for MBGNF:Eu3+, IBU–MBGNF:Eu3+, and IBU are displayed in Fig. 8, respectively. As shown in Fig. 8a for the MBGNF:Eu3+, the obvious broad absorption bands, assigned to OH (3430 cm1) and H2O (1630 cm1), indicate that a large number of –OH groups and H2O are present on the surface of the fiber, which are important for bonding drug (IBU) molecules. The peaks centered at 802, 461 cm1 are assigned to the Si–O–Si bands. The peaks centered at 1440 cm1 are assigned to the P–O bands [46]. For the IBU–MBGNF:Eu3+ (Fig. 8b), the sharp band centered at
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Fig. 5. Excitation spectra of (A): (a) MBGNF:Eu3+, (b) IBU–MBGNF:Eu3+, (C): (a) MBGNF:Tb3+, (b) IBU–MBGNF:Tb3+ and Emission spectra of (B): (a) MBGNF:Eu3+, (b) IBU– MBGNF:Eu3+, (D): (a) MBGNF:Tb3+, (b) IBU–MBGNF:Tb3+.
Fig. 6. The decay curves for the 5D0 ? 7F2 (614 nm) emission of Eu3+ in MBGNF:Eu3+ (a) and 5D4 ? 7F5 (544 nm) emission of Tb3+ in MBGNF:Tb3+ (b) (kex = 275 nm).
Fig. 7. Cell viability of MBGNF:Eu3+ (A) and MBGNF:Tb3+ (B).
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Fig. 8. FT-IR spectra of (a) MBGNF:Eu3+, (b) IBU–MBGNF:Eu3+ and (c) IBU.
1710 cm1 is attributed to the vibration of –COOH, which is same as the IBU (Fig. 8c). Intense carboxylate bands at 1559 cm1 are detected. The absorption bands assigned to the quaternary carbon atom located at 1465 and 1513 cm1, tertiary carbon atom at 1367 cm1, O–H bending vibration at 1414 cm1, and C–Hx bond at 2927 and 2955 cm1 can also be observed (Fig. 8b) [36,47]. These results confirm the successful adsorption of IBU onto MBGNF:Eu3+. The IBU loading amount determined by the TG analysis for IBU– MBGNF:Eu3+ and IBU–MBGNF:Tb3+ is 16 wt.% and 13 wt.%, respectively (Fig. 9). The MBGNF:Tb3+ owning relatively smaller surface area and pore volume shows lower ibuprofen loading amount compared with MBGNF:Eu3+. During the loading and release process, the IBU molecules can be adsorbed onto the surface of porous MBGNF in the impregnation process and liberated by diffusioncontrolled mechanism. The OH groups on the surface could form hydrogen bonds with the carboxyl groups 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 pore channels of the MBGNF:Eu3+. The cumulative drug release profiles for the IBU–MBGNF:Eu3+/ 3+ Tb systems as a function of release time in SBF are shown in Fig. 10. The systems show an initial burst release followed by the relatively slow release and completely release after 36 h. The IBU released in the first 0.5 h reaches 39% and 42% for IBU– MBGNF:Eu3+ and IBU–MBGNF:Tb3+, respectively. The initial burst
Fig. 9. TG curves of IBU–MBGNF:Eu3+ (a) and IBU–MBGNF:Tb3+ (b).
Fig. 10. The cumulative release of IBU from IBU–MBGNF:Eu3+ and IBU– MBGNF:Tb3+.
release may be attributed to the IBU weakly adsorbed on the outer surface of MBGNF, and the slow release of the rest of IBU can be due to the strong interaction between IBU molecules and the surface. The IBU release rate of the MBGNF was relatively faster than the previous reports [35], which can be ascribed to the disordered pore system of the nanofibers. The PL emission intensity of IBU–MBGNF:Eu3+ as a function of cumulative released amount of IBU is given in Fig. 11. The photoluminescence (PL) excitation and emission spectra were obtained on a Hitachi F-7000 spectrofluorimeter equipped with a 150 W xenon lamp as the excitation source under the same experimental conditions (this will decrease the experimental errors as far as possible). Peak area score were used to measure the change of luminescence. This method has good reproducibility and reliability for comparing the PL intensity of the rare earth doped luminescent materials [48–51]. It can be seen that the PL intensity (defined as the integrated area intensity of 5D0 ? 7FJ of Eu3+) increases with the cumulative released IBU, and reaches a maximum when IBU is completely released. It is well known that the emission of Eu3+ will be quenched to some extent in the environments where high phonon frequency is present, such as OH groups with a vibrational frequency near 3450 cm1 [52]. The organic groups in IBU with high vibration frequencies from 1000 to 3250 cm1 will greatly quench the emission of Eu3+ in IBU–MBGNF:Eu3+. The quenching effect will be weakened with the release of IBU, resulting in the
Fig. 11. PL emission intensity of Eu3+ in IBU–MBGNF:Eu3+ as a function of cumulative release amount of ibuprofen.
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increase of emission intensity. This relationship between the PL intensity and drug release extent can be a potential probe for monitoring or tracking the drug release during the drug delivery process. 4. Conclusions Eu3+/Tb3+ doped luminescent mesoporous bioactive glass nanofibers were fabricated using the electrospinning technique. The synthesized MBGNF show uniform diameter and dispersed nanopores. Photoluminescence measurements reveal that these nanofibers show strong luminescence under the ultraviolet irradiation. The biocompatibility tests on L929 fibroblast cells using MTT assay reveal low cytotoxicity of these systems. These luminescent nanofibers show sustained release properties using ibuprofen as the model drug. The emission intensities of Eu3+ in the drug carrier system vary with the released amount of IBU, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity. Acknowledgment
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