Tb3+-poly(acrylic acid) hybrid microspheres

Biomaterials 33 (2012) 2583e2592

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pH-responsive drug delivery system based on luminescent CaF2:Ce3þ/Tb3þ-poly(acrylic acid) hybrid microspheres Yunlu Dai a, b, Cuimiao Zhang a, Ziyong Cheng a, *, Ping’an Ma a, Chunxia Li a, Xiaojiao Kang a, b, Dongmei Yang 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, China Graduate University of the Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2011 Accepted 6 December 2011 Available online 21 December 2011

In this study, we design a controlled release system based on CaF2:Ce3þ/Tb3þ-poly(acrylic acid) (PAA) composite microspheres, which were fabricated by filling the pH-responsive PAA inside CaF2:Ce3þ/Tb3þ hollow spheres via photopolymerization route. The CaF2:Ce3þ/Tb3þ hollow spheres prepared by hydrothermal route possess mesoporous structure and show strong green fluorescence from Tb3þ under UV excitation. Doxorubicin hydrochloride (DOX), a widely used anti-cancer drug, was used as a model drug to evaluate the loading and controlled release behaviors of the composite microspheres due to the good biocompatibility of the samples using MTT assay. The composite carriers provide a strongly pH-dependent drug release behavior owing to the intrinsic property of PAA and its interactions with DOX. The endocytosis process of drug-loaded microspheres was observed using confocal laser scanning microscopy (CLSM) and the in vitro cytotoxic effect against SKOV3 ovarian cancer cells of the DOX-loaded carriers was investigated. In addition, the extent of drug release could be monitored by the altering of photoluminescence (PL) intensity of CaF2:Ce3þ/Tb3þ. Considering the good biocompatibility, high drug loading content and pH-dependent drug release of the materials, these hybrid luminescent microspheres have potential applications in drug controlled release and disease therapy. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Drug carrier Poly(acrylic acid) Hollow structure Doxorubicin Luminescence

1. Introduction Nowadays, hollow materials with mesoporous shells have attracted much attention due to their high specific surface, cavity volumes and spherical morphology [1e11]. Hollow mesoporous materials have been designed as the multifunctional drug delivery systems (DDS) [12e24]. An ideal DDS should not only protect the drug from biological degradation before reaching the target organs or cells, but also provide a sustained release in a controlled manner by external stimuli in the physiological condition [19,25e28]. To realize the goal, much work has focused on preparation of polyelectrolyte polymers based on hollow microcapsules by the layer by layer (LBL) self-assembly procedure [29]. However, the procedure becomes quite tedious when many layers are required. Meanwhile, these hollow capsules lack the mechanical robustness [30e32]. Inorganic hollow materials are mechanically

* Corresponding authors. E-mail addresses: [email protected] (Z. Cheng), [email protected] (J. Lin). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.12.014

strong, but they do not show the stimuli-responsive controlled release behavior. Therefore, inorganic hollow materials modified with polymers have been well developed in order to introduce more functions for inorganic DDS. Liu and coworkers prepared poly(methacrylic acid)-grafted hollow silica by a grafting-from approach using atom transfer radical polymerization (ATRP) of sodium methacrylate [33]. Chen group developed inorganic/ organic hybrid composite based on mesopouous silica nanotubes and pH-responsive polyelectrolytes via LBL self-assembly technique [34]. Yan et al synthesized chitosan-poly (acrylic acid)/SiO2 hollow nanospheres as drug carriers which provide a pH-sensitive switch to release loaded guest materials [35]. A series of controlled release systems have been developed which are responsive to external stimuli, such as pH, temperature, light and electric field, magnetic field and ultrasound [36e47]. Among all the triggers, pH-responsive DDS are important for many applications [48]. Each segment of organ maintains its own characteristic pH level from the acidic stomach lumen (pH ¼ 1e3) for digestion to the alkaline duodenum and ileum (pH ¼ 6.6e7.5) for the neutralization of chyme. The cancer cells have a more acidic environment compared with the normal cells. Therefore,

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pH-dependent release behavior is very meaningful in drug delivery. Solid inorganic fluorides have attracted much attention due to its uncommon properties, such as low-energy phonons, high resistivity, and anionic conductivity [49e57]. CaF2 doped with lanthanide (III) ions have been reported to display unique luminescence properties arising from their 4f electron configuration [58e61]. Meanwhile, the CaF2 codoped with Ce3þ and Tb3þ show intense, bright green photoluminescence under ultraviolet irradiation with high quantum efficiency [62]. Therefore, these properties lead to a wide range of potential application in optics, biology and biosensors. Recently, our group has successfully demonstrated a simple, template-free method for the synthesis of hollow mesoporous CaF2 microspheres by a facile hydrothermal route [62]. This material has been attempted to be used as a drug carrier to load ibuprofen (IBU). Unfortunately, the rate of drug release was only governed by free diffusion, which led to a burst drug release that about 60% IBU liberated within 0.5 h and completed release after 2 h in simulated body fluid (SBF). The drug release profile is completely uncontrolled by using this kind of inorganic carriers. In order to tackle this problem, in this study, we polymerized acrylic acid in the hollow CaF2 microspheres through the UV radiation photopolymerization process. The strong green photoluminescence arising from CaF2:Ce3þ/Tb3þ microspheres was reserved and the quantum efficiency can still be as high as 45% after integrating of PAA. Doxorubicin hydrochloride (DOX), a widely used anti-cancer drug, was used as a model drug to evaluate the loading and controlled release behaviors of the composite microspheres. The mechanically strong hollow structure of CaF2:Ce3þ/Tb3þ could provide large fraction of void in their inner space. Therefore, the drug encapsulation efficiency could reach 50% and the drug loading content was 250.5 mg DOX/CaF2:Ce3þ/Tb3þ-PAA g (the loading efficiency was 20 wt %). The DOX-loaded CaF2:Ce3þ/Tb3þ-PAA exhibits a pH-dependent release behavior, that is, the lower pH, the faster the drug release. The PAA-modified CaF2:Ce3þ/Tb3þ microspheres can effectively prevent the sudden burst drug release and they have potential to selectively release at the disease sites with acid environment such as tumor or stomach pH conditions, which decreases the dose-dependent bio-toxicity and adverse effects of doxorubicin. Moreover, the cytotoxic effect of the DOX-loaded CaF2:Ce3þ/Tb3þ -PAA against human SKOV3 ovarian cancer cells were examined in vitro. The dependence between extent of drug release and the luminescent intensity of CaF2:Ce3þ/Tb3þ was also investigated.

2. Experimental section 2.1. Materials Ce2(CO3)3 (99.99%) and Tb4O7 (99.99%) were purchased from Science and Technology Parent Company of the Changchun Institute of Applied Chemistry. Ca(NO3)2 $ 4H2O (A.R.), NH4F (A.R.), NaF (A.R.) and Trisodium citrate (A.R.) were purchased from Beijing Chemical Regent Co., Ltd., NaBF4 (A.R.) Acrylic acid (AA), N,N0 methylenebisacrylamide(BIS) and 2,4,6-trimethylbenzoyldiphenyl phosphine oxide (TPO) were purchased from Aladdin. DOX was obtained from the Nanjing Duodian Chemical Limited Company. Poly(acrylic acid) (Mvw450,000) was purchased from Aldrich. All the above chemicals were used without further purification. AA were de-inhibited via a column of activated alumina. Ce(NO3)3 and Tb(NO3)3 stock solutions were prepared by dissolving respective rare earth carbonate and oxide in dilute HNO3, drying at elevated temperatures to evaporate water and the excessive HNO3, and dissolving in water.

2.2. Synthesis of CaF2:Ce3þ/Tb3þ hollow microspheres The CaF2:Ce3þ/Tb3þ hollow microspheres were prepared according to the our previous report [62]. Typically, a total of 2 mmol of Ca(NO3)2 $ 4H2O, Ce(NO3)3 and Tb(NO3)3 (molar ratio Ca:Ce:Tb ¼ 96:2:2) and 4 mmol of trisodium citrate (Cit3-) were dissolved in deionized water to form 40 mL of solution 1. Then, 4 mmol of NaBF4 was added to 20 mL H2O to form solution 2. After vigorously stirring for 15 min, solution 2 was added to solution 1. Ammonia solution was added to adjust the pH value to 7.0 and stirred for another 30 min at room temperature. The as-obtained mixing solution was transferred into a Teflon bottle held in a stainless steel autoclave, sealed and maintained at 180  C for 24 h, and then allowed to cool down to room temperature naturally. The obtained precipitate was separated by centrifugation and washed with deionized water and then ethanol. Finally, the precipitate was separated by centrifugation again and dried in air at 70  C for 24 h to obtain the CaF2:Ce3þ/Tb3þ hollow spheres. 2.3. Synthesis of CaF2:Ce3þ/Tb3þ-PAA CaF2:Ce3þ/Tb3þ-PAA hybrid microspheres were prepared by vacuum nano-casting route (VCR) with some modification [63,64]. 100 mg of CaF2:Ce3þ/Tb3þ was subjected to vacuum in a round flask for 30 min. AA (2 mL) was mixed with 116 mg of BIS and 105.2 mg of TPO for 12 h in the dark. Then, the above mixture solution was injected into the round flask contained CaF2:Ce3þ/Tb3þ under vacuum conditions. After 30 min, the vacuum pump was turned off and the mixture kept stirred overnight. The products were separated by centrifugation and washed with ethanol twice to remove the monomers and initiators at the external surface. Finally, Polymerization was carried out using UV radiation (200 W/cm2, LAMP, PHILIPS) for 5 min. The collected solid was washed with ethanol and dried in air at 70  C for 24 h. 2.4. In vitro DOX loading and release 4 mg of CaF2:Ce3þ/Tb3þ-PAA was dispersed in 1 mL deionized water, 2 mL of DOX (1 mg/mL) was added into the above solution. The mixture was shaken for 24 h at room temperature to reach the equilibrium state. Then the solution was centrifuged to collect the DOX-loaded CaF2:Ce3þ/Tb3þ-PAA sample. The supernatant solutions were collected, and the content of DOX was determined by UVeVis spectral measurement at the wavelength of 480 nm. DOXloaded CaF2:Ce3þ/Tb3þ-PAA sample was immersed in 1 mL of PBS (pH ¼ 7.4, 4.0 and 2.0) at 37  C, At selected time intervals, buffer solution was taken and replaced with fresh buffer solution. The amounts of released DOX in the supernatant solutions were measured by UVeVis spectrophotometer. 2.5. In vitro cytotoxicity of DOX-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres and cell viability In vitro cytotoxicity of CaF2:Ce3þ/Tb3þ-PAA composite microspheres was assayed against human SKOV3 ovarian cancer cells. Human SKOV3 ovarian cancer cells were seeded in a 96-well plate at a density of 8000 cells per well and cultured in 5% CO2 at 37  C for 24 h. Then free DOX, DOX-loaded CaF2:Ce3þ/Tb3þ-PAA and CaF2:Ce3þ/Tb3þ-PAA were added to the medium at pH ¼ 7.4, and the cells were incubated in 5% CO2 at 37  C for 48 h. The concentrations of the nanospheres were 3.125, 6.25, 12.5, 25, 50 mg/mL, respectively. The concentrations of DOX were 0.78125, 1.5625, 3.125, 6.25, 12.5 mg/mL, respectively. At the end of the incubation, the media containing the nanospheres was removed, and 20 mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

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solution (diluted in a culture media with a final concentration of 0.8 mg/mL) was added into each cell and incubated for another 4 h. The supernatant in each well was aspirated. 150 mL of dimethyl sulfoxide (DMSO) was added to each well before the plate was examined using a microplate reader (Therom Multiskan MK3) at the wavelength of 490 nm. Meanwhile, cell viability was also determined using MTT assay, which was the same as the procedure for cytotoxicity assay for CaF2:Ce3þ/Tb3þ-PAA composite microspheres. 2.6. Confocal laser scanning microscopy (CLSM) observation of the CaF2:Ce3þ/Tb3þ-PAA microspheres

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was performed with a Micromeritics ASAP 2020 M apparatus. The specific surface area was determined by the Brunauer-EmmettTeller (BET) method. The UVeVis adsorption spectral values were measured on a U-3100 spectrophotometer (Hitachi). The photoluminescence (PL) spectra were taken on an F-7000 spectrophotometer (Hitachi) equipped with a 150 W xenon lamp as the excitation source. The quantum efficiency of the sample was determined on an absolute quantum yield measurement system (C9920-02, Hamamatsu Photonics K.K., Japan). Confocal laser scanning microscopy (CLSM) images were observed by confocal laser scanning microscope (Olympus, FV 1000). 3. Results and discussion

For CLSM, the SKOV3 ovarian cancer cells were seeded in 6-well culture plates (a clean cover slip was put in each well) and grown overnight as a monolayer, and were incubated with free DOX (20 mg equivalent DOX), DOX-loaded CaF2:Ce3þ/Tb3þ-PAA and CaF2:Ce3þ/ Tb3þ-PAA microspheres at 37  C for 10 min. Thereafter, the cells were rinsed with PBS three times, fixed with 2.5% formaldehyde (1 mL/well) at 37  C for 10 min, and then rinsed with PBS three times again. In order to nucleus labeling, the nuclei was stained with Hoechst 33,342 solution (from Molecular Probes, 20 mg/mL in PBS, 1 mL/well) for 10 min and then rinsed with PBS three times. The cover slips were placed on a glass microscope slide, and the samples were visualized using CLSM (FV10-ASW). 2.7. Characterization The X-ray diffraction (XRD) measure were performed on a D8 Focus diffractometer (Bruker) with CuKa radiation (l ¼ 0.15405 nm). The morphologies of the samples were obtained using a field emission scanning electron microscope (FE-SEM, XL30, Philips). Transmission electron microscopy (TEM) was obtained using FEI Tecnai G2 S-Twin with a field emission gun operating at 200 kV. Fourier-transform Infrared spectra were measured on a Vertex PerkineElmer 580BIR spectrophotometer (Bruker) with the KBr pellet technique. Thermogravimetry (TG) was carried out on a Netzsch Thermoanalyzer STA 409 instrument in an atmospheric environment with a heating rate of 10  C/min from room temperature to 800  C. Nitrogen adsorption/desorption analysis

3.1. Formation of CaF2:Ce3þ/Tb3þ-PAA hybrids The procedure for the synthesis of CaF2:Ce3þ/Tb3þ-PAA is shown in Scheme 1. Firstly, we prepared CaF2:Ce3þ/Tb3þ hollow microspheres through a facile hydrothermal method at pH ¼ 7.0 and 180  C for 24 h. In order to integrate the monomers in the hollow microspheres, we reduced the pressure of the round flask contained CaF2:Ce3þ/Tb3þ with the aid of vacuum pump and the monomers solution was injected into the round flask. The products were separated by centrifugation and washed with ethanol twice to remove the monomers absorbed on the external surface. Finally, poly(acrylic acid) was polymerized in the hollow microspheres through the UV radiation photopolymerization process. Fig. 1 shows the wide-angle XRD patterns of the standard data for CaF2 (JCPDS No. 35-0816, Fig. 1a), CaF2:Ce3þ/Tb3þ (Fig. 1b) and CaF2:Ce3þ/Tb3þ-PAA (Fig. 1c). The results of the XRD indicate that the as-synthesized CaF2:Ce3þ/Tb3þ samples are well crystallized, and the patterns are consistent with cubic phase structure of CaF2. Furthermore, the UV radiation photopolymerization of AA in the CaF2:Ce3þ/Tb3þ hollow microspheres has not changed the phase structure of the CaF2 microspheres. The SEM and TEM images of CaF2:Ce3þ/Tb3þ and CaF2:Ce3þ/Tb3þ-PAA microspheres are displayed in Fig. 2. The diameters of the hollow spheres are about 300 nm and the spherical shell is composed of nanoparticles with diameters of around 40 nm. The broken microsphere after ultrasound treatment confirms the hollow structure from the SEM

Scheme 1. Schematic illustration of the preparation process of CaF2:Ce3þ/Tb3þ-PAA composite microspheres and controlled release of anti-cancer drug of DOX.

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Fig. 1. The standard JCPDS card 35-0816 of CaF2 (a), wide-angle XRD patterns of the CaF2:Ce3þ/Tb3þ hollow microspheres (b) and CaF2:Ce3þ/Tb3þ -PAA composite microspheres (c).

image (Fig. 2a, inset) of the CaF2:Ce3þ/Tb3þ sample. The obvious lattice fringes in the high-resolution transmission electron microscopy (HRTEM) image (Fig. 2c, inset) imply the high crystallinity. The interplanar distances between adjacent lattice planes of about 0.315 nm is well coincident with the (111) plane of CaF2 (Fig. 1). We also used NH4F and NaF as the fluorine ion source to carry out contrastive experiments. The TEM images of CaF2 using NH4F and NaF as the fluorine ion source are shown in Fig. S1 (Fig. S1 in supplementary information). The shape of the as-obtained product using NH4F as the fluorine ion source is irregular nanoparticles with diameters of around 20 nm (Fig. S1a). When we use

NaF as the fluorine ion source, the similar product was obtained (Fig. S1b). Therefore, different fluorine source have a tremendous effect on the morphologies of the mesoporous shell. According to our previous report, when NaBF4 acted as a fluorine ion source,  NaBF4 was slowly hydrolyzed to produce BO33 and F anions [65].  Due to the very low F concentration, the particles growth of the CaF2 was very slow. In contrast, when NH4F or NaF as the F source, NH4F or NaF dissociates quickly to produce F ions, which is available immediately. Under the hydrothermal conditions, the Ca2þ reacts with a high concentration of F, which makes the growth very quickly. Therefore, NH4F or NaF as fluorine ion source can not form the hollow mesoporous shell. We can not find the difference between CaF2:Ce3þ/Tb3þ and CaF2:Ce3þ/Tb3þ-PAA from SEM or TEM in Fig. 2 due to the PAA polymerized in the hollow and low contrast of PAA compared with inorganic CaF2. However, we can confirm the existence of PAA by FT-IR and TGA. The FT-IR spectra of CaF2:Ce3þ/Tb3þ, CaF2:Ce3þ/ Tb3þ-PAA, DOX-loaded CaF2:Ce3þ/Tb3þ-PAA and pure DOX are given in Fig. 3aed, respectively. In Fig. 3a, the broad band at 3431 cm1 is ascribable to the OH vibration of H2O absorbed in the sample. The peaks appearing between 1400 and 1600 cm1 arise from the (C]O) stretching mode, which might be due to the addition of Cit3 ions in the hydrothermal process [66]. After polymerizing PAA in the hollow, several characteristic absorption peaks of PAA can be observed at 1718 cm1 and 2933 cm1, which could be assigned to the C]O stretching vibration in the carboxyl group, and the CeH stretching vibration [42], respectively. These results demonstrate that we have succeeded in polymerizing PAA inside CaF2:Ce3þ/Tb3þ hollow spheres. For the DOX-loaded CaF2:Ce3þ/Tb3þ-PAA (Fig. 3c), the absorption bands assigned to the stretching vibration of C]O at 1617 and 1579 cm1 from the anthraquinone ring of DOX are obvious except for slight decrease of

Fig. 2. SEM images of CaF2:Ce3þ/Tb3þ hollow microspheres (a) and CaF2:Ce3þ/Tb3þ -PAA composite microspheres (b), TEM images of CaF2:Ce3þ/Tb3þ hollow microspheres (c) and CaF2:Ce3þ/Tb3þ -PAA composite microspheres (d). The insets in panel (a) and (c) are the broken microsphere after ultrasound treatment and the HRTEM image of CaF2:Ce3þ/Tb3þ, respectively.

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Fig. 3. FT-IR spectra of CaF2:Ce3þ/Tb3þ hollow microspheres (a), CaF2:Ce3þ/Tb3þ-PAA composite microspheres (b), DOX-loaded CaF2:Ce3þ/Tb3þ-PAA (c) and pure DOX (d).

intensity compared with the pure DOX (Fig. 3d), which confirms the successful loading of DOX in the CaF2:Ce3þ/Tb3þ-PAA. The content of PAA in the composite microspheres was estimated by TGA, as was given in Fig. 4. CaF2:Ce3þ/Tb3þ and CaF2:Ce3þ/Tb3þ-PAA showed a weight loss of 4.1% and 16.4% after heating to 800  C, respectively. Therefore, the content of PAA in CaF2:Ce3þ/Tb3þ-PAA hollow microspheres was determined to be about 12.3% (w/w). The N2 absorption/desorption isotherms of the hollow CaF2:Ce3þ/Tb3þ and CaF2:Ce3þ/Tb3þ-PAA are shown in Fig. 5A and Fig. 5B, respectively. It can be seen that both the samples exhibit typical IV-typed isotherms with H1- hysteresis loops, which are the properties of typical mesoporous materials. The BET surface area and total pore volume of CaF2:Ce3þ/Tb3þ are calculated to be 16.82 m2/g and 0.1823 cm3/g. After polymerizing PAA in the hollow, the respective BET surface area and pore volume are 13.21 m2/g and 0.1433 cm3/g, which are slightly declined compared to those of CaF2:Ce3þ/Tb3þ sample. The results indicates that we have successfully polumerized PAA in the hollow and occupied some part of space of the inner cavity, which lead to the decrease of BET surface area and pore volume. As the digital photo shown in Fig. 6, the water-dispersed CaF2:Ce3þ/Tb3þ-PAA hybrid microspheres exhibit strong and bright green luminescence under UV excitation with a wavelength

Fig. 4. TG curves of CaF2:Ce3þ/Tb3þ hollow microspheres and CaF2:Ce3þ/Tb3þ-PAA composite microspheres.

Fig. 5. N2 absorption/desorption isotherm of the CaF2:Ce3þ/Tb3þ hollow microspheres (A) and CaF2:Ce3þ/Tb3þ-PAA composite microspheres (B).

Fig. 6. Excitation (left) and emission (right) spectra of CaF2:Ce3þ/Tb3þ hollow microspheres and CaF2:Ce3þ/Tb3þ-PAA composite microspheres. The inset shows the luminescence photograph of the CaF2:Ce3þ/Tb3þ-PAA composite microspheres under 254 nm UV lamp in the dark.

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of 254 nm in the dark. Meanwhile, the PL properties of the samples were further characterized by excitation and emission spectra in Fig. 6. The excitation spectrum monitored at the 541 nm (5D4-7F5 transition of Tb3þ) contains a weak band at 219 nm, a moderate band at 258 nm, and a strong broad band with a maximum at 305 nm we can ascribe the band at 219 nm to the 4f8-4f75d transitions of Tb3þ ions, and the bands at 258 and 305 nm to 5d-4f transitions of Ce3þ ions. We have studied the energy transfer from Ce3þ to Tb3þ in CaF2:Ce3þ/Tb3þ hollow microspheres in detail [62,67,68]. The energy transfer process of Ce3þ and Tb3þ in CaF2:Ce3þ/Tb3þ is shown in Fig. S2. First, Ce3þ ions were excited with UV light excitation. Then, energy transfer takes place from Ce3þ to Ce3þ, and from 5d (Ce3þ) to the high excitation levels of Tb3þ (4f9) followed by cross relaxation to the 5D4 level of Tb3þ, which decays to various lower levels of 7FJ (J ¼ 0, 1, 2, 3, 4, 5, 6). The presence of the excitation bands of Ce3þ in the excitation spectrum monitored with Tb3þ emission indicates that energy transfer occurs from Ce3þ to Tb3þ in CaF2:Ce3þ/Tb3þ. Excitation into the Ce3þ band at 305 nm yields the strong emission of Tb3þ (5D4-7FJ at 491, 544, 580 and 620 nm; J ¼ 6, 5, 4, 3) is a further indication of energy transfer from Ce3þ to Tb3þ in CaF2:Ce3þ/Tb3þ hollow spheres. After polymerizing PAA in the CaF2:Ce3þ/Tb3þ hollow, the characteristic emission and excitation peaks are not changed except for only a slight decrease of intensity. In addition, the PL quantum efficiency of CaF2:Ce3þ/Tb3þ-PAA composite microspheres is as high as 45% under excitation at 305 nm.

Doxorubicin hydrochloride (DOX) was selected as a model drug to evaluate the loading and controlled release behaviors of the composite microspheres. Because of the large cavity inside the hollow microspheres and the numerous mesopores presented in the shell, the drug encapsulation efficiency could reach 50% and the drug loading content was 251 mg DOX/CaF2:Ce3þ/Tb3þ-PAA g (the loading efficiency was 20 wt %). Fig. 8A shows in vitro release profiles of DOX from the hollow microspheres in PBS buffer solutions of different pH values (7.4, 4.0 and 2.0) at 37  C. The drug release rate of DOX-loaded CaF2:Ce3þ/Tb3þ-PAA was obviously pHdependent and increased with the decrease of pH. Only 6.5% of DOX was released from the composite microspheres even after 48 h at pH ¼ 7.4. With an increased acidity to pH ¼ 4.0, the DOX release rate reach to 52.5% after 48 h and more than 70% of DOX was released within 4 h in PBS of pH ¼ 2.0. As for an alternately changed pH environments, the carriers present an alternately switch-on/off effect of drug release. As shown in Fig. 8B, the DOX release was very slow at pH ¼ 7.4 at the beginning 24 h, but an abrupt increase was observed when adjusting pH to 2.0. From the inset image of Fig. 8B，the slop of DOX release at pH ¼ 7.4 was much lower than at pH ¼ 2.0, which means fewer DOX was released from the drug carriers at pH ¼ 7.4 stages. The stairstepping-up profiles of drug release shows that CaF2:Ce3þ/Tb3þ-PAA microspheres can quickly respond to the environment variation. The pKa values of DOX and

3.2. In vitro cytotoxicity and controlled drug release The stability and cytotoxicity would be important to be considered for the actual application as a potential drug carrier in biomedical fields. To evaluate the biocompatibility of CaF2:Ce3þ/ Tb3þ-PAA composite microspheres, the standard MTT cell assay were used on the L929 fibroblast cells. As show in Fig. 7, the CaF2:Ce3þ/Tb3þ-PAA hollow microspheres do not show apparent cytotoxicity against the L929 fibroblast cells. More than 97% cells viabilities were observed even at a high concentration of CaF2:Ce3þ/ Tb3þ-PAA composite microspheres of 100 mg/mL after incubation for 24 h. The MTT results indicate that the hybrid CaF2:Ce3þ/Tb3þPAA microspheres have good biocompatibility as drug carriers in biomedical applications. We further examined the drug loading and controlled release abilities of the CaF2:Ce3þ/Tb3þ-PAA hollow microspheres.

Fig. 7. The L929 fibroblast cells viability after incubating with CaF2:Ce3þ/Tb3þ-PAA composite microspheres for 24 h and quantitative assays by standard MTT method.

Fig. 8. (A) Cumulative DOX release from CaF2:Ce3þ/Tb3þ-PAA composite microspheres at pH ¼ 7.4 (a), pH ¼ 4.0 (b) and pH ¼ 2.0 (c) PBS buffer. (B) Release profile of CaF2:Ce3þ/Tb3þ-PAA composite microspheres by alternately changing the pH between 7.4 and 2.0.

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Fig. 9. The emission intensity of DOX-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres as a function of release time at pH ¼ 4.0 and 37  C PBS buffer.

PAA were 8.6 and 4.8, respectively. At pH ¼ 7.4, most of the carboxylic acid groups on the PAA chains should be deprotonated to form carboxylate anions in PBS solution and the amino group of DOX was positively charged. The electrostatic interaction between negatively charged PAA and positively charged DOX restricted the release of DOX in the polyelectrolyte. In contrast, at pH ¼ 4.0 and 2.0, the carboxylic acid groups on the PAA should be protonated and there is no electrostatic interaction between DOX and CaF2:Ce3þ/ Tb3þ-PAA that lead to a fast release of DOX. On the other hand, the pH-responsive polymer maybe plays a role of switch for DOX

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release. At pH ¼ 7.4, the electrostatic repulsion between the negative-charged carboxylic acid groups enable the PAA network to swell, which plug up the pore channels on microspherical shell and block the out-diffusion of drug molecules. When pH decrease to below 4.8, the PAA network deswell with protonating of carboxylic acid groups and liberate the DOX molecules inside the microspheres. Due to the different pH values in blood plasma (pH ¼ 7.4), in extracellular tumor matrix (pH ¼ 5.8e7.2), in the endocytic compartments such as endosomes (pH ¼ 5-6) and lysosomes (pH ¼ 4-5) and in stomach (pH ¼ 1.5e3.5), the CaF2:Ce3þ/Tb3þ-PAA composite microsphere with pH-triggered drug-release property is promising to be used as a carrier for releasing anti-cancer drug after endocytosis by cancer cells. In addition, we can monitor the cumulative release of DOX through the relationship between the PL emission intensity of the DOX-CaF2:Ce3þ/Tb3þ-PAA composite microspheres. It is well known that the emission of rare earth ions will be quenched to some extent in the environments that have a high phonon frequency. According to Schuurmans and Van Dijk and Blasse, it is found that the radiative rate is approximately equal to the non-radiative rate if the energy gap in the non-radiative transition equals five times the maximum phonon frequency [69,70]. In DOX-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres, the maximum phonon energy of DOX and PAA are about 3528 and 2933 cm1 (determined by the IR spectra shown in Fig. 3), respectively. The energy gaps between the 5D4 and 7 F4/7F3/7F2/7F1/7F0 levels in Tb3þ amount to 16,957/16,291/15,437/ 15,166/14,900 cm1 [71], respectively. These energy gaps are smaller than five times of the maximum phonon energy of DOX (3528 cm1). Therefore, the relaxation of the excited electrons from 5 D4 level to the lower 7F4/7F3/7F2/7F1/7F0 levels is liable to occur via five vibrations of DOX molecules in our system, resulting in the extreme decrease of green emission from 5D4 level. In contrast, the lowest energy gap from 5D4 to 7F0 (14,900 cm1) is larger than five

Fig. 10. In vitro human SKOV3 ovarian cancer cell viabilities after incubation 48 h with free DOX, DOX-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres and bare CaF2:Ce3þ/ Tb3þ-PAA composite microspheres at 37  C and pH ¼ 7.4. The concentrations of the microspheres were 3.125, 6.25, 12.5, 25, 50 mg/mL, respectively. The concentrations of DOX were 0.78125, 1.5625, 3.125, 6.25, 12.5 mg/mL, respectively.

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times of the maximum phonon energy of PAA (14,665 cm1). The absorbance spectra of pure PAA is showed in Fig. S3. The maximum absorbance of PAA is at about 203 nm. And there is no absorbance when the wavelength is over 250 nm. Therefore, the lack of spectra overlap between the excitation of CaF2:Ce3þ/Tb3þ and the absorbance of PAA indicates that the absorption of PAA has no effect on the excitation of CaF2:Ce3þ/Tb3þ. Hence, after polymerizing PAA in the hollow, the luminescent intensity of PAA-loaded CaF2:Ce3þ/ Tb3þ has only slight decrease compared to the pure CaF2:Ce3þ/Tb3þ microspheres (as shown in Fig. 6). In addition, we have also calculated the intensity ratio of two strong emissions at 544 nm and 491 nm, as shown in Table S1. We found that the intensity ratio between 544 nm and 491 nm gradually decreased along with the time of drug release and have a recover trend to the pristine CaF2:Ce3þ/Tb3þ-PAA. This may be attributed to the spectra overlap between emission of CaF2:Ce3þ/Tb3þ-PAA and absorbance of DOX. As shown in Fig. S4, the absorbance of DOX at 491 nm is higher than that at 544 nm, which resulting in quenching degree of CaF2:Ce3þ/ Tb3þ-PAA emission intensity at 491 nm is stronger. The PL emission intensity of DOX-loaded microspheres increases with the release of DOX. This allows the carriers can be used as a bioprobe for tracking and monitoring drug release by PL intensity (as shown in Fig. 9).

3.3. In vitro cytotoxic effect on human cancer cells and cell uptake To test the pharmacological activity of the DOX-loaded hollow microspheres, the cytotoxic effect of DOX-loaded CaF2:Ce3þ/Tb3þPAA on human SKOV3 ovarian cancer cells is evaluated in vitro via MTT assay. Human SKOV3 ovarian cancer cells are commonly used in cancer research. Free DOX, DOX-loaded CaF2:Ce3þ/Tb3þ-PAA and CaF2:Ce3þ/Tb3þ-PAA were added to the Human SKOV3 ovarian cancer cells medium at pH ¼ 7.4, and the cells were incubated in 5% CO2 at 37  C for 48 h. The concentrations of the microspheres were 3.125, 6.25, 12.5, 25, 50 mg/mL, respectively and the concentrations of the DOX were 0.78125, 1.5625, 3.125, 6.25, 12.5 mg/mL, respectively. Fig. 10 shows the cell viabilities against free DOX, DOX-loaded CaF2:Ce3þ/Tb3þ-PAA, and blank CaF2:Ce3þ/ Tb3þ-PAA at different concentration after incubation for 48 h. Accordingly, the concentrations of CaF2:Ce3þ/Tb3þ-PAA in the cell culture medium were equivalent to the CaF2:Ce3þ/Tb3þ-PAA concentrations used in DOX-CaF2:Ce3þ/Tb3þ-PAA medium. The blank CaF2:Ce3þ/Tb3þ-PAA without DOX have no obvious cytotoxic effect on cancer cells even after 48 h of treatment with the samples at the concentration as high as 50 mg/mL. In contrast, both free DOX and DOX-loaded CaF2:Ce3þ/Tb3þ-PAA exhibited an

Fig. 11. Laser scanning confocal microscopy (LSCM) images of human SKOV3 ovarian cancer cells incubated with DOX-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres ([DOX] ¼ 20 mg/mL) for 10 min (aec), 1 h (def), 6 h (gei) at 37  C. Each series can be classified to the nuclei of cells (being dyed in blue by Hoechst 33324 for visualization), DOXloaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres, and a merge of the two channels of both above, respectively. All scale bars are 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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increasing inhibition against human SKOV3 ovarian cancer cells with an increased concentration. It was found that the anti-cancer efficacy of DOX-loaded CaF2:Ce3þ/Tb3þ-PAA is close to that of free DOX when the concentration of DOX is over 3.125 mg/mL. This may be attributed to the fact that small molecules like DOX can be diffused into cells rapidly whereas the microspheres have to be endocytosed to enter the cells. Therefore, free DOX was spread faster than the DOX-loaded CaF2:Ce3þ/Tb3þ-PAA microspheres by cellular uptake. When the concentration is higher, the more and more DOX-loaded CaF2:Ce3þ/Tb3þ-PAA microspheres can be endocytosed to enter the cancer cells and release DOX inside to introduce cell death. Once the DOX-loaded CaF2:Ce3þ/Tb3þ-PAA microspheres are internalized by the cell through the endocytosis process, they will encounter early endosomes, late endosomes and finally fuse with lysosomes [72]. Both endosomes (pH ¼ 5.0w6.0) and lysosomes (pH ¼ 4.0w5.0) have an acidic micro environment, which are distinct with the normal physiological environment (pH ¼ 7.4) [73]. In addition, compared with the normal tissues, solid tumors have a weakly acidic extracellular environment of pH < 7 due to the hypoxia-induced coordinated upregulation of glycolysis [74]. The result indicated that CaF2:Ce3þ/Tb3þ-PAA microspheres can also release the drug quickly to induce apoptosis in endosomes (pH ¼ 5.0w6.0) and lysosomes (pH ¼ 4.0w5.0) acidic micro environment after the DOX-loaded CaF2:Ce3þ/Tb3þPAA microspheres are internalized by the cell through the endocytosis process. To facilitate the observations of cell uptake of the microspheres, the confocal laser scanning microscopy (CLSM) photographs of human SKOV3 ovarian cancer cells incubated with DOX-loaded CaF2:Ce3þ/Tb3þ-PAA for 10 min, 1 h and 6 h at 37  C are showed in Fig. 11. In the first 10 min (Fig. 11aec), only a few of microspheres could be uptake by SKOV3 cells. After incubation 1 h (Fig. 11def), the redeemitting particles accumulated near the nucleus are apparently increased. If the incubation time was prolonged to 6 h (Fig. 11gei), the red fluorescence from DOX was observed in both the cytoplasm and the cell nucleus. The time course CLSM images suggest that the intracellular pathway that the DOX-loaded CaF2:Ce3þ/Tb3þ-PAA used to deliver DOX comprised rapid internalization, microspheres localization in the cytoplasm and DOX localization in the cell nucleus.

4. Conclusion In summary, CaF2:Ce3þ/Tb3þ-PAA composite spheres have been successfully prepared through the hydrothermal synthesis and UV radiation photopolymerization of PAA in the hollow mesoporous CaF2:Ce3þ/Tb3þ microspheres. The hybrid microspheres show bright green fluorescence under UV excitation coming from the inorganic CaF2:Ce3þ/Tb3þ shells and exhibit a significantly higher storage capacity of drug. The luminescence intensity is changed with the drug loading and release process, which has potential for the tracking and monitoring applications. Importantly, the integrating of PAA in the hollow microspheres can efficiently prevent the drug burst release from the carriers and introduce a pHresponsive drug release property. The MTT assay for L929 fibroblast cells viability demonstrates good biocompatibility of the carriers. Meanwhile, in vitro cell cytotoxicity tests on cancer cells verified that anti-cancer drug-loaded CaF2:Ce3þ/Tb3þ-PAA composite microspheres exhibited comparable cytotoxicity compared with free drug at the same concentration. These results show that luminescent CaF2:Ce3þ/Tb3þ-PAA microsphere with pHtriggered drug-release property is promising to be used as a carrier for releasing anti-cancer drug after endocytosis by cancer cells.

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