Biomaterials 35 (2014) 7666e7678

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Mesoporous NaYbF4@NaGdF4 core-shell up-conversion nanoparticles for targeted drug delivery and multimodal imaging Liangjun Zhou a, c, Xiaopeng Zheng a, c, Zhanjun Gu a, *, Wenyan Yin a, Xiao Zhang a, Longfei Ruan a, Yanbo Yang a, Zhongbo Hu c, Yuliang Zhao a, b, ** a

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, PR China c College of Materials Science and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 April 2014 Accepted 20 May 2014 Available online 11 June 2014

We developed a facile strategy to obtain a new kind of mesoporous core-shell structured up-conversion nanoparticles (mUCNPs), composed of a NaYbF4:2%Er core and a mesoporous NaGdF4 shell. This mesoporous shell not only enhanced the up-conversion luminescence but also endowed many other functionalities of the nanoparticles such as drug delivery and bio-imaging capabilities. Moreover, after being conjugated with polyethylenimine (PEI) and folic acid (FA), core-shell mUCNPs exhibited good water dispersibility, enhanced drug delivery efficiency, and remarkable targeting ability to cancer cells. To certify the folate receptors (FR)-mediated targeted drug delivery, cell viability assay, cell up-conversion luminescence imaging and flow cytometry analysis were carried out. Furthermore, apart from the application for targeted drug delivery, the as-prepared core-shell mUCNPs could also be employed as the contrast agents for X-ray computed tomography (CT) and magnetic resonance (MR) imaging, because of the strong X-ray attenuation ability of Yb and high longitudinal molar relaxivity (r1) of Gd in the nanoparticles, providing the potential for simultaneously bio-imaging and cancer-targeting therapy. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Mesoporous Core-shell Up-conversion nanoparticles Targeted drug delivery Multimodality biomedical imaging

1. Introduction Among the broad spectrum of nanoscale materials investigated for biomedical applications, lanthanide (Ln) doped up-conversion nanoparticles (UCNPs) have been gaining enormous research attention for years, due to their unique up-conversion luminescence (UCL) property that enables the conversion of near infrared (NIR) photons into visible to ultraviolet photons via the multiphoton process.[1e6] However, when the bare UCNPs used for the biomedical applications, they usually suffer from the quenching effect caused by surface defects and vibrational deactivation from solvents in the colloidal dispersions.[7e9] A useful strategy to improve the luminescence intensity of UCNPs is the fabrication of core-shell structures that a shell with similar lattice constant with

* Corresponding author. ** Corresponding author. Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China. E-mail addresses: [email protected] (Z. Gu), [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.biomaterials.2014.05.051 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

the core is grown around the UCNPs cores to reduce the nonradiative decay losses.[10e12] Many core-shell UCNPs, such as NaYF4:Yb,Er@NaGdF4 [13,14], NaYbF4:Er/Tm@NaGdF4 [15] have been reported. Recently, the applications of UCNPs have been further expanded beyond bio-imaging by incorporating advanced features, such as specific targeting [16,17], multimodal imaging [18e22] and therapeutic delivery [16, 23e26]. A successful example is that UCNPs can be employed as drug carrier to realize simultaneously bio-imaging and disease therapy [27], which have the abilities to monitor the route of drug carriers and evaluate the efficiency of the drug release in living system by a simple and effective way. For this purpose, the UCNPs with mesoporous shells are preferable for drug loading because of its high specific surface area and cavity volumes.[28] Although many homogeneous/heterogeneous core-shell UCNPs have been prepared, few of them have the mesoporous shells.[10,11] Currently, coating mesoporous silica layer is the only way for the fabrication of UCNPs with such structure.[29,30] However, this process usually calls for perfect operation and complicated post-treatments, and always induces serious quenching of UCL, greatly limiting their further

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

applications. To overcome this problem, it is better to grow a mesoporous shell with similar lattice constant with the core, since the shell is easy to grow on the surface of UCNPs and has the ability to enhance the emission intensity as we mentioned above. However, no one has reported such core-shell mUCNPs and also no research has been done to evaluate their properties in biomedical field such as drug delivery and bio-imaging. In this study, as shown in Scheme 1, we firstly developed a simple but efficient strategy to grow a new kind of mUCNPs,

7667

composed of a NaYbF4:2%Er core and a mesoporous NaGdF4 shell, as a multifunctional theranostic platform. This mesoporous shell could not only enhance the intensity of UCL but also enable these nanoparticles (NPs) as drug delivery carrier, due to its high surface area and accessible pores for drug loading. To realize targeted drug delivery, we also conjugated FA to PEI coated mUCNPs, which enabled the drug carrier target many cancer cells.[31e34] Furthermore, since there are Gd element in the shell and Yb element in the core that are well known for enhancing the

PEI

FA

DOX

mUCNPs

UCL

CT CancerTargeting

MRI

PEI FA

Polyethylenimine (PEI)

DOX Folate Receptor

Doxorubicin hydrochloride (DOX) Folic acid (FA) Scheme 1. Synthesis of mUCNPs for multimodal imaging and cancer-targeting therapy.

7668

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

longitudinal relaxation in magnetic resonance (MR) imaging [35e38] and high intrinsic contrast in computed tomography (CT) imaging[15,19,39], respectively, the as-prepared core-shell mUCNPs are expected to be used as MR and CT imaging contrast agents. Therefore, the as-prepared mesoporous UCNPs offer a new possibility in exploring the multifunctional nano-platform for simultaneous multimodal imaging and targeted drug delivery. 2. Materials and Methods 2.1. Materials Ln oxides (Ln2O3, 99.9%, Ln: Y, Gd, Yb), Ln nitrate hydrates (Ln(NO3)3$6H2O, 99.9%, Ln: Yb, Er), trifluoroacetic acid (TFA), sodium trifluoroacetate (98%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), ammonium fluoride (NH4F, 96%), folic acid (FA), fluorescein isothiocyanate (FITC, 95%) were purchased from Alfa Aesar Ltd. Sodium oleate (98%) was provided by Aladdin Inc. Branched polyethylenimine(PEI, M.W. z25,000) was acquired from Sigma-Aldrich Co. LLC. N-hydroxysulfosuccinimide (NHS, 98%) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 99%) were bought from J&K Scientific Ltd. Doxorubicin hydrochloride (DOX, 99.9%) was purchased from Beijing Huafeng United Technology CO., Ltd. Cell counting kit-8 (CCK-8), Calcein-AM (CA) and propidium iodide (PI) were provided by

Dojindo Laboratories in Japan. Sodium hydroxide (NaOH, 98%), cyclohexane and ethanol were supplied from Beijing Chemical Reagent Company. All of the chemicals were analytical grade and used without further purification. Deionized water was used throughout experiments. 2.2. Preparation of Ln-oleate Complexes The Ln-oleate complexes were prepared by reacting Ln nitrates with sodium oleate, according to a similar report for the synthesis of iron oxide nanocrystals [40]. In a typical strategy, 20 mmol of Ln(NO3)3$6H2O (99.9%) and 60 mmol of sodium oleate were dissolved in a mixture solvent composed of ethanol (40 mL), deionized water (30 mL) and cyclohexane (70 mL). The resulting solution stirred at room temperature for 12 h. After that, the upper organic layer containing Ln-oleate complexes was washed three times with water: ethanol (1:1, v:v) in a separatory funnel. The remaining yellow solution suffered the reduced pressure distillation, leaving Ln-oleate complexes in a waxy solid form. For further use in later experiments, the complexes were re-dissolved in quantitative OA and ODE. 2.3. Preparation of Ln trifluoroacetate precursor In a typical process, reported by Lee JY[11] and Roberts JE [41], 10 mmol of Ln2O3 was added to a single-necked flask containing deionized water (2 mL) and TFA (5.5 mL). Reaction occurred immediately when the flask was put into water

Fig. 1. TEM/SEM images of (a) core: NaYbF4: 2%Er and (b), (c) mUCNPs: NaYbF4: 2%Er@NaGdF4. (d) XRD patterns with standard PDF card No. 27-1427 for b- NaYbF4, the peaks marked with rhombus indicate the existence of NaGdF4.(e) STEM image of mUCNPs. (feg) Elements maps of mUCNPs are colored by red and green, indicating gadolinium and ytterbium, respectively.

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678 bath (85
C). The mixture was sealed and continuously stirred overnight until turned into a clear solution. Then, the clear filtrate, obtained from filtering by a microporous membrane, was stirred again at 70
C for 24 h to remove excess TFA and water. The final solid products were put into brown bottle and then stored in a desiccator. 2.4. Synthesis of UCNPs NaYbF4:2% Er NaYbF4:2% Er UCNPs were synthesized through a simplified process as follows. 0.98 mmol of Yb-oleate, 0.02 mmol of Er-oleate, 4 mmol of NH4F and 2.5 mmol of NaOH were put into a single-necked flask. Then, appropriate volume of OA and ODE were added, making sure that the volumes of OA and ODE were up to 6 mL and 15 mL, respectively. The mixture was heated up to 100
C and vacuumed for 30 min to remove oxygen and water. Afterward, the reaction occurred at 280
C in the presence of argon and kept for 1 h. Throughout the process, the mixture was continuously stirred. The products were collected by centrifugation at 12,000 rpm and washed for two times with ethanol to remove dissociative OA and ODE. 2.5. Synthesis of core-shell mUCNPs NaYbF4:2% Er@NaGdF4 Core-shell mUCNPs were synthesized in the way described below. 0.5 mmol of Gd (CF3COO)3 and 1 mmol of NaCF3COO were put into OA (6 mL) mixed with ODE (15 mL). After stirring for 10 min at room temperature, 4 mL of cyclohexane dispersion, containing 0.5 mmol of NaYbF4:2%Er, was added. Then, the mixture was kept at 100
C for 30 min under stirring to get rid of cyclohexane. After the oxygen and water were vacuumed out at room temperature, the mixture was kept at 280
C for 1 h. The products were collected by centrifugation at 12,000 rpm and washed with ethanol for three times to dislodge the residue. Finally, the white powders were obtained through lyophilization for 24 h. NaYbF4:2%Er@NaLnF4 (Ln: Y, Yb) could be prepared in the similar way. 2.6. Surface modification PEI, a cationic polymer with abundant free primary amine [42], was chose to modify the surface of core-shell mUCNPs. In a typical experiment, 250 mg of PEI was dissolved in ethanol (30 mL), and then 100 mg of core-shell mUCNPs dispersed in ethanol (10 mL) were added. After stirring for 24 h, the PEI-mUCNPs were gathered through centrifugation at 12,000 rpm and washed with ethanol and water. To target cancer cell, FA was chose to conjugate the PEI-UCNPs. Firstly, a solution was prepared by dissolving 30 mg of FA, 60 mg of NHS, 36 mg of EDC in 10 mL of water (pH ¼ 7~8) at room temperature in dark. Secondly, the solution (4 mL) was mixed with 20 mg of PEI- mUCNPs dispersed in 16 mL of water, and then stirred for 24 h without light. Finally, yellow powders, FA conjugated PEI-mUCNPs (FAmUCNPs), were obtained by centrifugation, washing with ethanol/water and lyophilization for 24 h.

7669

adsorptionedesorption isotherms were measured on a Micromeritics ASAP2020 (Micromeritics Instrument Ltd.) at 108 K. The Brunauer-Emmett-Teller (BET) method was utilized to calculate the specific surface area by using adsorption data in the range of the relative pressures (P/P0) from 0.01 to 1.00. The pore-size distribution was derived using the Barrett-Joyner-Halanda (BJH) method. Fourier transform infrared (FT-IR) spectra were recorded on an infrared spectrometer (iN10-IZ10, Thermo Fisher). The photoluminescence (PL) spectra were studied through fluorescence spectrophotometer (Horiba Jobin Yvon FluorolLog3). The UVeVis data were obtained by U-3900 spectrophotometer (Hitachi). All photographs were taken by Nikon D3100 digital camera. The contents of Yb and Gd in samples were measured by inductively coupled plasma mass spectrometry (ICPMS, Thermo Elemental X7, USA). 2.8. In vitro biocompability of PEI-mUCNPs and FA-mUCNPs 16HBE (a human bronchial epithelial cell line), HeLa (a human cervical carcinoma cell line) and KB (a human nasopharyngeal epidermal carcinoma cell line) cells were employed in the investigation. In vitro biocompabilities of PEImUCNPs and FA-mUCNPs with a series of concentration (0, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 mg/mL) were studied on all kinds of cells through the standard CCK-8 assay. The cells were pre-incubated in a 96-well plate (about 2000 cells/well, six wells for each concentration) with six blank-wells for 24 h in a humidified incubator (37
C, 5% CO2). After washing each well with phosphate buffer saline (PBS, 0.01 M, pH ¼ 7.4), coreeshell mUCNPs of different concentrations were added. Then the cells were continuously incubated in the same condition for 24 h. 100 mL of 10% CCK-8 solution was subsequently added to each well and the plate was kept in incubator for another 2 h. Finally, the absorbances at 450 nm were measured with a microplate reader (SpectraMax M2, MDC, USA), and the cell viabilities were calculated according to the control groups. Furthermore, inverted fluorescence microscopy (IX73, Olympus, Japan) was used to observed all kinds of cells, which were co-incubated with PEI-mUCNPs and FAmUCNPs (200 mg/mL) for 12 h and then stained by CA and PI via the stand protocol. 2.9. In vitro UCL imaging study 16HBE, HeLa and KB cells were all adopted for UCL imaging. The cells were cultured in 25 cm2 flasks (Corning) at 37
C under 5% CO2. After several days, the cells were put into quartz-bottom dishes, and pre-incubated for 24 h. Cells were washed with PBS (pH ¼ 7.4) and then co-incubated with FA-mUCNPs (100 mg/mL) dispersed in the corresponding medium for another 2 h. The unbound NPs were washed away with PBS, while cells were stained through co-incubation with hoechst 33258 (Beyotime) for no more than 10 min. Finally, all the solution was moved out while 20 mL of antifade mounting medium (Beyotime) was added to prevent hoechst from quenching. After that, the cells were imaged in bright field, under UV light and NIR excitation using an inverted fluorescence microscope (Olympus IX73) equipped with a 980 nm NIR laser.

2.7. Characterization The scanning electron microscope (SEM) images of nanoparticles (NPs) were taken on S-4800 (Hitachi), equipped with an energy-dispersive X-ray spectrum (EDX, HORIBA EMAX-250). The morphology and elements distribution of the NPs were observed on field emission transmission electron microscopes (TEM, Tecnai G2 F20 U-TWIN). X-ray diffraction (XRD) analysis was performed using Japan Rigaku D/max-2500 diffractometer with Cu Ka radiation (l 1.5418Å). Nitrogen

2.10. Flow cytometry analysis Flow cytometry analysis was used to determine cellular uptake of FA-mUCNPs in these three cells. The as-obtained FA-mUCNPs were linked with FITC through a simple method. [17] 30 mg of FA-mUCNPs, dispersed in ethanol (10 mL), were mixed with 10 mg of FITC and stirred overnight in dark. After that, FITC labeled FA-mUCNPs (FITC- FA-mUCNPs) were collected by centrifugation, washing with ethanol, and lyophilization. Cells were seeded in 6-well plate and pre-incubated at 37
C under 5% CO2 for 24 h. And then, after the cells were washed with PBS (pH ¼ 7.4), FITC-FAmUCNPs dispersion (100 mg/mL) were added into each well except the controlled ones. After another co-incubation for 1 or 2 h, cells were washed, detached and resuspended in PBS for flow cytometry analysis using a flow cytometer (accuri c6, BD, USA). 2.11. Drug loading and release study

Fig. 2. Nitrogen adsorptionedesorption isotherms and (inset) Barrette-JoynereHalenda (BJH) pore-size distribution curve of mUCNPs NaYbF4: 2%Er@NaGdF4.

DOX, a widely used anti-cancer drug for the treatment of many types of carcinoma [43,44], was selected as a model to study the drug loading and release capability of mUCNPs. For a typical drug loading experiment, 10 mg of mUCNPs were dispersed in 10 mL of PBS which contained DOX (20, 50, 100, 200, 500 and 1000 mM) in 20 mL brown vials. After stirring for 24 h in dark at room temperature, dark red complexes were obtained by centrifugation at 12,000 rpm for 3 min and then washed for several times. The UVeVis absorption spectra of the supernatant were measured. And according to the standard curve of DOX, the DOX loading amounts (w/w) were calculated by the absorbance changes of the characteristic absorption peak of DOX solution before and after the absorption by the mUCNPs. The products were endured freeze drying process and the resulting powders were kept out of the light for further experiment. The release of DOX from FA-mUCNPs@DOX was also studied as follows. FA-mUCNPs@DOX (10 mg) were dispersed in PBS (10 mL), and continuously vibrated in dark at 37
C. Then, at different time points (0.083, 0.25, 0.75, 1.5, 3.5, 4.5, 6, 8, 10, 12, 24, 36, 48 and 72 h), 4 mL of dispersion was taken out for centrifugation. After the UVeVis absorption of supernatant was measured, the dispersion was put into original mixture and the release ratio of DOX was calculated.

7670

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

Scheme 2. Schematic representation for the formation mechanism of mUCNPs NaYbF4:Er@NaGdF4 from the OA-capped cores.

In these above investigations, PBS with three different pH (5.5, 6.5 and 7.4) were all used to study the effect of pH on the DOX loading and release. 2.12. In vitro targeted drug delivery study of FA-mUCNPs@DOX 16HBE, HeLa and KB cells were all employed in the study. In order to make sure the cancer-targeting of FA-mUCNPs@DOX, after 24 h co-incubation, cytotoxicity of FA-mUCNPs@DOX was investigated via the CCK-8 assay, while free DOX and PEImUCNPs@DOX with the same series of concentrations (0, 1.05, 2.1, 4.2, 8.4, 16.8, 33.6 and 67.2 mg/mL of DOX) were taken as control groups. Furthermore, after 12eh co-incubation with PEI-mUCNPs@DOX and FA-mUCNPs@DOX at equivalent concentration of DOX (33.6 mg/mL), all the kinds of cells, staining with hoechst 33258 via the standard protocol, were observed by using inverted fluorescence microscopy (IX73, Olympus, Japan). 2.13. Investigation of in vitro MR and CT imaging The longitudinal relaxation time (T1) and T1-weighted images were obtained by using the 4.7 T magnetic resonance imaging instrument (Biospec; Bruker, Ettlingen, Germany). A series of mUCNPs with different concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1 mM of Gd3þ, which was measured to be ~34.95% in mUCNPs by ICP-MS) were prepared by dispersing them in 0.5% agarose gel solution, then these samples were placed in NMR tubes (1 mL for each tube) for measurement. The T1 values and T1weighted images were performed by the same multi-slice multi-echo sequence. The following parameters were adopted: the value of repetition time/echo time (TR/TE) was 400/11 ms (TI from 23 to 9000 ms), number of excitations (NEX) ¼ 8, and acquired images had a matrix size of 128  128, a field of view of 40  40 mm, and a slice thickness of 1.20 mm T1 values of each tube were calculated using the following formula: S(TI) ¼ S0*[12exp(TI/T1)] to fit the T1 recovery cure in the circular regions of interest for the sample [36]. The resulting T1 values were averaged and plotted as 1/T1 versus Gd3þ molar concentration. The slope of the graph provided the longitudinal molar relaxivity (r1). In vitro CT imaging was also studied as follows. mUCNPs with different concentrations (0, 1, 2, 5, 10, 20, 50, 100 mM of Yb3þ, which was measured to be ~41.2% in coreeshell mUCNPs by ICP-MS) were dispersed in 0.5% agarose gel solution and placed in 1.5 mL centrifuge tubes for phantom test. CT images were acquired using the CT system XM-Tracer-130. Imaging parameters were as follows: effective pixel size, 80 mm; 70 kV, 100 mA; field of view, 1024 pixels  1024 pixels. Images of phantom CT images, and Hounsfield units (HU) values were analyzed by Osiris software [15,45].

3. Results and discussions 3.1. Preparation and characterization of core-shell mUCNPs NaYbF4:Er@NaGdF4 The coreeshell mUCNPs NaYbF4:Er@NaGdF4 were prepared through a two-step process as mentioned above. The uniform

NaYbF4:2%Er cores were synthesized by the thermo-decomposition of Ln-oleates. As revealed in Fig. 1a, the as-obtained UCNPs are spherical, highly monodispersed, and narrow in size distribution. The average diameter of the UCNPs was estimated to be ca. 50 nm. Next, an undoped NaGdF4 shell was grown around the core NaYbF4:2%Er to enhance the UC emission and endow other functionality of the nanoparticles. It is worth noting that unlike the previous reported coreeshell UCNPs with smooth shell [1,7,11,12], the as-prepared shell possess mesoporous structure that composed of many ultra-small nanoparticles (ca. 5 nm), making it very useful for biological applications such as drug delivery. The shell thickness is about 10 nm and thus the average diameter of the coreeshell nanocrystals increase to ca. 70 nm. The successful coating of NaGdF4 shell on the NaYbF4 core was also verified by both EDX spectra (Fig. S1) and elements mapping (Fig. 1eeg). As shown in these colored maps, the Gd elements are highly concentrated in the shell area while the signals from Yb are only present in the core area, confirming the successful coating of NaGdF4 shell on the surface of core. The crystal structure and purity of the fabricated NaYbF4:Er cores and NaYbF4:Er@NaGdF4 coreeshell mUCNPs were measured by XRD. As shown in Fig. 1d, the cores are hexagonal phase of NaYbF4 (JCPDS No.27e1427), which is regarded as one of the most efficient host matrix for UC fluorescence [10]. After coating the NaGdF4 shell, the peaks from NaGdF4 in XRD pattern come out, which is in good agreement with TEM and EDX mapping results. In order to further investigate the specific surface area and porous nature of the as-prepared mesoporous NaYbF4:2% Er@NaGdF4, BET gas-sorption measurement was adopted. As revealed in the N2 absorption/desorption isotherms (Fig. 2), the mUCNPs present typical IV-typed isotherms and H1-hysteresis loops, which are the characteristics of typical mesoporous materials reported in the previous work [27,46,47]. The BET surface area and pore volume of the sample are about 46.8 m2/g and 0.124 cm3/ g, respectively. Moreover, as seen from the inset in Fig. 2, the mesopore size distribution calculated from the BJH model exhibits a narrow distribution apex centered at the mean value of 4.8 nm, indicating the uniform mesopores. In this case, cyclohexane played a key role in the formation of mesoporous shell. Cyclohexane was original used to disperse NaYbF4:Er cores, and then removed from the reaction system

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

7671

Fig. 3. (a) PL spectra of cores: NaYbF4: Er and mUCNPs: NaYbF4: Er@NaGdF4, and insets: images of these two dispersed in cyclohexane (1 mg/mL) irradiated by 980 nm laser (0.4 W). (b) Power dependence of the UC emission intensity of mUCNPs under 980 nm excitation. (c) Energy level diagrams of Yb3þ, Er3þ and UC mechanisms. (d) Remarkable photo-stability of these samples (lem. ¼ 654 nm) under the continuous irradiation with 980 nm laser (0.4 W) for 1 h. The bin time for each data point is 0.1 s.

before the further thermal-deposition process for the fabrication of shells. However, we found that the process of evaporating cyclohexane contribute to the fabrication of the mesoporous shell. Comparative experiment showed that, by dispersing cores in OA instead of cyclohexane, coreeshell UCNPs with dense and smooth shell were obtained (Fig. S2a), which was similar with the previous reported coreeshell UCNPs [7]. When cyclohexane was removed first, and then the shell precursors were added, the coreeshell UCNPs without mesoporous structure were also obtained (Fig. S2b). To shed light on the formation mechanism of these new coreeshell mUCNPs, we examined the intermediate products, which were collected after the evaporation of cyclohexane at 100
C. As shown in Fig. S2c, there are some ultra-small particles (black arrow) on the outer surface of NaYbF4 cores. In contrast, without cyclohexane while keeping other conditions unchanged, no small nanoparticles are observed on the surface of the core (Fig. S2d). Based on the above experimental results, the origin of formation the mesoporous shell is proposed to the contribution of cyclohexane bubbles around the core, due to the low boiling point of cyclohexane. It has been reported that gas bubbles could act as the aggregation centers [48e51]. As illustrated in Scheme 2, the monomers of NaGdF4 are formed at 100
C by the initial nucleation meanwhile abundant microbubbles of cyclohexane are coming out around the OA-capped cores (stage I). Then, at the stage II, driven by the minimization of interfacial energy [52,53], these nucleus have the attendance to aggregate around the bubbles, and grow into ultrasmall nanocrystals. At the stage III, under vacuum and the

ascending temperature, the escape of cyclohexane results in the disappearance of bubbles, and finally the mesoporous NaGdF4 shells are formed. Additionally, TEM images (Fig. S2eef) certify that this method can be extended for the synthesis of other coreeshell mUCNPs such as NaYbF4@NaLnF4 (Ln: Y, Yb). Fig. 3a illustrates the corresponding UC emission spectra of the core only and coreeshell mUCNPs. In these two curves, there are three major peaks observed at 521, 540 and 654 nm, which come

Fig. 4. (a) The FT-IR spectra of OA-capped mUCNPs, PEI-mUCNPs, FA-mUCNPs, and the corresponding pictures (inset). (b) Digital photograph of PEI-mUCNPs dispersion in physiological saline (2 mg/mL).

7672

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

Fig. 5. In vitro biocompability study of (a) PEI-mUCNPs and (b) FA-mUCNPs on 16HBE, HeLa and KB cells. Cell staining studies using Calcein-AM and PI on 16HBE, HeLa and KB cells co-incubated with (c) PEI-mUCNPs and (d) FA-mUCNPs of 200 mg/mL for 12 h, respectively.

from 2H11/2/4I15/2, 4S3/2/4I15/2 and 4F9/2/4I15/2 electron transition of Er3þ ions, respectively [2,36]. Notably, after the mesoporous shell was coated, the intensity of emission at 540 nm increased 6.8 times while the one at 654 nm was enhanced 9.1 times, resulting in the fluorescence change from grass green to yellow. The enhanced UCL from the mUCNPs undoubtedly originates from the effect of the mesoporous shell on the core NaYbF4: Er nanocrystals, which can restrain the UCL surface quenching by decreasing surface defects and alleviating the ligand influence. Additionally, in order to understand the UC mechanisms of these coreeshell mUCNPs, the laser power dependence of the aforementioned two UC emission bands are analyzed and the results are depicted in logelog plots of Fig. 3b. A linear behavior (log(Intensity) ∝ n  log(Powder), where n is the number of the absorbed photons) [18] are both noted with coefficient of determination (R-square) around 0.989 and n values are both around 2. These results show that the two-photon process is responsible for the green and red UC emission. And, the possible mechanisms for the emission bands under 980 nm laser could be explained in Fig. 3c. For further application in long-term UCL bioimaging, the photo-stability study of as-obtained coreeshell mUCNPs was investigated under the continuous 0.4 W laser excitation (980 nm). As shown in Fig. 3d, the fluorescence intensity remains more or less flat during 1-h irradiation, indicating their good photo-stability.

3.2. Surface modification The as-obtained coreeshell mUCNPs were hydrophobic as they were stabilized by oleic acid molecules. In order to meet the demands of biological applications, it was necessary to transfer the hydrophobic coreeshell mUCNPs to hydrophilic ones [3,12]. PEI was chose to transfer the hydrophobic coreeshell mUCNPs into aqueous phase, due to the abundant free amino groups of PEI that could be chemically modified for further applications [17,42]. The successful coating of PEI was verified by FT-IR (Fig. 4a). Compared to OA-capped mUCNPs, the curve of PEI-mUCNPs exists three characteristic peaks at 1653, 1453 and 1161 cm1, attributed to the NeH bending mode of amino group (eNH2) and the stretching vibrations of CeN bond, thereby supporting that the PEI successfully coats on the surface of mUCNPs [17,42]. After the surface modification, the PEI-mUCNPs could be well dispersed in physiological saline, as displayed in Fig. 4b. The significance of surface functionalization of mUCNPs lies in not only making them water-soluble, but also improving their targeting specificity of drug delivery by adding targeting agents on the surface of mUCNPs. Thus, we chose FA as the targeting molecules because the folate receptor is observed to be over expressed on the membrane of many human cancer cells. To realize the specificity of folate receptors (FR)-mediated targeting, we conjugated FA to PEI-mUCNPs based on the amide bond

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

7673

Fig. 6. Cell UCL imaging using an inverted fluorescence microscope on (a) 16HBE, (b) HeLa and (c) KB cells under bright field, UV light and 980 nm laser, after being co-incubated with FA-mUCNPs (100 mg/mL) for 2 h.

formation between COOH carboxyl amino group of FA and NH2 group of PEI [31,32]. As displayed in the insets of Fig. 4a, the color of powders change from white to yellow after coating with FA, visually confirming the successful grafting of FA on the surface of mUCNPs. As observed from the FT-IR spectrum of FA conjugated PEI-mUCNPs (FA-mUCNPs) in Fig. 4a, there are two new peaks appearing at 1675 and 1580 cm1, corresponding to the C]O amide stretching of the a-carboxyl group from the FA molecules and the NeH bending vibration of the amide bond [16,33,34]. 3.3. Biocompability of modified core-shell mUCNPs For further applications in biomedical field, it was necessary to understand the biocompability in vitro and in vivo of these modified coreeshell mUCNPs. In vitro toxicity investigations were carried out on 16HBE, HeLa and KB cells. As illustrated in Fig. 5a and b, it is clearly observed that after 24-h co-incubation with PEI-mUCNPs and FA-mUCNPs of varied concentrations, the viabilities of all cells remain ca. 90%, indicating good biocompability in vitro of modified mUCNPs. For further confirming the low cytotoxicity of as-prepared particles, cell staining with CA and PI was also carried out. The images (Fig. 5c and d) present a visual impression that after 24 incubation with the PEI and FA modified mUCNPs, most of cells are alive (the green ones) while only a few of cells are dead (the red ones), which is in good agreement with the cell viability results, indicating that these two samples are low cytotoxic. Thereafter, to evaluate the in vivo biocompatibility of FA-mUCNPs, the in vivo toxic assay was carried out on female CD-1 mice. FA-mUCNPs dispersed in physiological saline (1 mg/mL) were injected via tail vein (5 mg/kg) at several time-points and the weights of mice are recorded. Compared with the control group injected with physiological saline, the FA-mUCNPs injected mice showed no abnormal performance in skin, appetite and behaviors. And, the mice were all alive after 21 days. Moreover, no obvious weight change was

observed between experimental and control groups (Fig. S3), suggesting no obvious toxicity in vivo of FA-mUCNPs. Thus, FAmUCNPs exhibit good biocompability in vitro and in vivo, providing the potential for biomedical applications. 3.4. Cell UCL imaging with FA-mUCNPs It is well known that FA is one of the most promising ligands for targeting a range of human carcinomas by the specific binding with FR via its gamma-carboxyl group. FR is a glycosylphosphatidylinositol (GPI) anchored high-affinity membrane protein, over-expressed on the cytomembrane in many types of cancer cells but highly restricted in the normal cells (such as

Fig. 7. Flow cytometry studies on 16HBE, Hela and KB cells co-incubated with FITC-FAmUCNPs 100 mg/mL for 1 or 2 h, with not treated ones as control group.

7674

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

were taken as control. The results of flow cytometry exhibited in Fig. 7 are in accordance with those of UCL imaging. After being coincubated with the FITC-FA-mUCNPs for 1 h, the amounts of 16HBE, HeLa and KB cells that take up the NPs are respectively detected to be 33.0%, 78.2% and 86.6%, showing a surpassed two times improvement in cellular uptake of FITC-FA-mUCNPs between normal and cancer cells, and an approximately 10 percent increment between HeLa and KB cells. These results could put down to the different amount of FR expression in normal (16HBE) and cancer (HeLa, KB) cells. Additionally, when the incubation time prolongs to 2 h, the ratios of labeled cells are up to 38.6%, 80.4% and 91.4%. The slight increments suggest that the cellular uptake of FITC-FA-mUCNPs is a fast-process and most composites are internalized by cells in 1 h. These flow cytometry results directly confirm the cancer targeting via specific binding between FA and FR, and cell uptake efficiency enhancement of FA-conjugated nanocarriers for cancer-targeting. 3.6. Drug loading, release and in vitro targeted drug delivery study

Fig. 8. The studies of (a) drug loading and (b) release in FA-mUCNPs platform at varied pH.

16HBE), making it a useful and common marker for targeted drug delivery to tumors. [16,33,34] To evaluate the cancer targeting ability of as-prepared FA-mUCNPs, UCL imaging in vitro was carried out on these three cells (16HBE, HeLa and KB cells). Cells treated with FA-mUCNPs (100 mg/mL) for 2 h were observed under inverted fluorescence microscope (IX73) equipped with mercury lamp and a 980 nm laser. It is displayed in Fig. 6 that a few of FA-mUCNPs (yellow) appear on cytomembrane or in cytoplasm except nucleus (blue) in cell, demonstrating that FA-mUCNPs enter into cytoplasm via endocytosis and have no ability to cross nuclear membranes. Comparing these three cells (Fig. 6) with each other, the FA-mUCNPs absorbed on the surface or internalized in the cytoplasm of KB cells are the most, while that of 16HBE are the fewest. The reason for these phenomena is that the FR expression is not same in different cancer cells. KB cells strongly over-express FR (FRþþ), while HeLa cells are FR positive (FRþ) and 16HBE cells are negative (FR-) [31,33,44]. These results not only certify their cancertargeting efficiency, but also indicate the potential of as-obtained FA-mUCNPs for optical imaging in cells. 3.5. Cellular uptake study We also studied cellular uptake of as-prepared FA-mUCNPs for further certificating their cancer cells targeting capability. After conjugating FA-mUCNPs with FITC through the typical method, flow cytometry assay was carried out to determine cellular uptake by counting the composites-labeled cells, through detecting the green fluorescence from the cells treated with FITC-FA-mUCNPs for different time. And, the ones not co-incubated with the composites

Seeing mesoporous structure of mUCNPs, DOX loading and release were both studied in PBS with different pH. According to the changes of the characteristic DOX optical absorbance at 480 nm, the DOX loading capability of FA-mUCNPs at varied pH (pH ¼ 5.5, 6.5 and 7.4) are exhibited in Fig. 8a, and the fitted curves well match data points with R-square around 0.99. The DOX loading capability of these mUCNPs at pH ¼ 7.4 is as high as 16.8% in weight. Additionally, the DOX loading capability declines to 9.3% and 5.7% as pH decreases to 6.5 and 5.5, respectively. This is visually proved by the products' photographs with obvious color variance (insets in Fig. 8). Such a phenomenon results from the protonation of the amine group, weakening the interaction between DOX and FA-mUCNPs [54]. In addition, as depicted in Fig. S4a, the green emission gradually weakened with the increase of DOX loading amount. And it is almost completely quenched when the loading amount of DOX reached maximum, leaving only red fluorescence remained (Fig. S4b). This could be used to monitor the drug loading and release behaviors of nanocarriers. In the next experiments, the mUCNPs with the highest DOX loading ratio (ca.16.8%) were selected for in vitro drug release study and cytotoxicity assay. The DOX release profiles in PBS with different pH values (5.5, 6.5 and 7.4) at 37
C are presented in Fig. 8b. Obviously, the DOX release rate is pH-sensitive and increases with the decrease of pH value. After 10-h oscillation in constant temperature shaking table, 75.7% of DOX release from FA-mUCNPs@DOX platform at pH 5.5, while 30.2% and 10.1% release at pH 6.5 and pH 7.4, respectively. This trend is ascribed to the deprotonation of the amino group, enhancing the interaction between of DOX and FA-mUCNPs and retarding drug release [31,43,44]. Since FA conjugated nanomaterials can be selectively uptaken by cancer cells via receptor-mediated endocytosis, the in vitro targeted drug delivery were studied on 16HBE, HeLa and KB cells via cell viability assay and cell staining. As shown in Fig. 9a-c, the targeted DOX delivery of FA-mUCNPs@DOX was investigated by testing their cytotoxicity. Free DOX and PEI-mUCNPs@DOX were taken as control. It is noteworthy that the concentrations of DOX were equivalent in DOX-loaded nanocarriers and free DOX. As expected, these three DOX-contained samples show the lowest toxicity in vitro on 16HBE cells (Fig. 9a) among 16HBE, HeLa and KB cells, resulting from that 16HBE cells are normal cells without strong proliferation ability and FR expression. Compared to nontargeted PEI-UCNPs@DOX, obvious decrement of cell viability can be observed on KB cells after being co-incubated with FAmUCNPs@DOX (Fig. 9c). This is attributed to the over-expression of FR, which mediate the endocytosis of FA conjugated

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

7675

Fig. 9. Cell viability assays at different concentrations of free DOX, PEI-mUCNPs and FA-mUCNPs@DOX on (a) 16HBE, (b) HeLa and (c) KB cells after 24 h co-incubation. (d) Cell cytotoxicity of FA-UCNPs@DOX on 16HBE, HeLa and KB cells.

nanocarriers [33]. Moreover, compared to the HeLa cells (Fig. 9b), FA-mUCNPs@DOX possess higher suppression effect on KB cells. In contrast, PEI-mUCNPs@DOX exhibit no obvious change in anticancer cell effect on both cancer cells. This may be attributed to the fact that KB cells strongly over-express FR (FRþþ), while HeLa are FR-positive (FRþ). In addition, another fact is that free DOX exhibits the highest suppression effect on all cells among these three samples, ascribed to the faster diffusion of small molecules into cells than cell endocytosis of nanocarriers [55]. Based on these

results above, it turns out to be that FA-mUCNPs@DOX is a promising targeted drug delivery system. To further confirm the targeted drug delivery of FAmUCNPs@DOX platform, cell staining was employed. And, in order to avoid the interference of red fluorescence from DOX under UV light, hoechst 33258 was used to stain the cells treated with PEImUCNPs@DOX and FA-mUCNPs@DOX (equivalent in the concentration of DOX) instead of CA and PI. These images (Fig. 10) directly suggest that FA-mUCNPs@DOX cause more dead cells (the bright

Fig. 10. Cell staining studies using Hoechst 33258 on 16HBE, HeLa and KB cells after 12 h co-incubation with (a) PEI-mUCNPs@DOX and (b) FA-mUCNPs@DOX of equal DOX concentration (33.6 mg/mL).

7676

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

Fig. 11. Color-mapped (a) T1-weighted images and (b) CT images. (c) Relaxation rate (1/T1, R1) vs various molar concentrations of Gd3þ in mUCNPs (0, 0.05, 0.1, 0.2, 0.5, 1 mM of Gd3þ); (d) CT values (HU) as a function of the concentration of Yb in NPs and I in Iopromide.

blue ones) in cancer cells than in 16HBE cells, especially in KB cells. In contrast, there is no obvious change in the ones treated with PEImUCNPs@DOX. The above images clearly evidence high efficiency of the targeted drug delivery system (FA-mUCNPs@DOX). Furthermore, after co-incubating these three cells with FA-mUCNPs@DOX for 1 h, the DOX cellular accumulations and distributions are depicted in Fig. S5. The red fluorescence of DOX surrounds the cell nuclei, suggesting that DOX is released as free form, which tends to diffuse towards the nuclei [31,56]. Moreover, the fluorescence in KB cells is the highest but lowest in 16HBE cells, showing high selectivity on cell endocytosis of FA-mUCNPs@DOX via FR-mediated mechanism. 3.7. In vitro MR and CT imaging As one of diagnosis strategies, multimodality biomedical imaging combined UCL, MR and CT imaging have been one of research hotspots in recent years. UCL imaging is well known for good signal-to-noise ratio (SNR), high penetration depth and resolution in biological tissues but low spatial resolution and 3D tissue detail. However, CT imaging can provide high-resolution 3D structure details of tissues with differential X-ray absorption features, except soft tissue with limited density differences. Fortunately, MR imaging can overcome this shortage with excellent sensitivity to soft tissue without radio isotopes [3,29,45,47,57]. Hence, as-obtained mUCNPs own broad potential for further exploration beyond as drug carriers and optical imaging agents. Firstly, because that the existing Gd on the surface can efficiently enhance the longitudinal relaxation of water protons, these mUCNPs can serve as T1 contrast agent. Fig. 11a reveals a series of T1-weighted images obtained with 6 incremental concentration in the range of 0e1 mM. The same series of concentrations are also employed to investigate the r1 values, which are calculated from a fitting curve of the longitudinal relaxation rate (1/T1, R1) as a function of the Gd3þ concentration (Fig. 11c). As the concentration of mUCNPs gradually increased, it can be clearly seen that these NPs brighten the T1-weighted image and enhance the longitudinal relaxation of water protons. The r1 value is calculated to be 1.416 s1mM1. The good performance in MR imaging of mUCNPs demonstrates that they are promising candidate as T1 contrast agent. Besides the application for MR imaging, these mUCNPs could also be used as CT imaging contrast agents, since they contained

Yb ions with strong X-ray attenuation capability. Similar with the MR imaging experiment, varied concentrations of mUCNPs dispersed in 0.5% agarose gel solution were used to obtain the Xray phantom images in vitro. A commonly used CT contrast agent, Iopromide, were used as control. As shown in Fig. 11b and d, with the increase of Yb concentration, the enhancement of CT signal and continuous rise of the CT value, called Hounsfield units (HU), are clearly observed. It is worth noting that the slope of the linear fitting plot for the NPs' HU is calculated to be 12.45, which is 2.4 times as high as that of commercial iodine-based X-ray contrast agent (Iopromide). This possibly results from the fact that Yb3þ ions have a higher X-ray absorption efficiency than that of Iopromide (Yb, 3.88 cm2/g; I, 1. 94 cm2/g at 100 keV, respectively) [58]. Therefore, the mUCNPs are potential to be used as a contrast agent for both MR and CT imaging, which has many advantages for diagnose compared to the each single modality, since it is possible to obtain multiple imaging data with taking the advantages of both techniques. 4. Conclusions In this study, we developed a facile strategy to obtain coreeshell mesoporous up-conversion nanoparticles (mUCNPs) NaYbF4:2%Er@NaGdF4 as a multifunctional theranostic platform, simultaneously realizing multimodality biomedical imaging and cancer-targeting therapy. These as-obtained coreeshell mUCNPs were of enhanced emission, large specific surface and accessible pores. After being modified with PEI and FA, the mUCNPs showed good water-dispersibility and exhibit well drug loading and release capacity with varied pH. Moreover, different methods, including cell viability assays, cell fluorescent images, and flow cytometry analysis, were employed to verify the FA-mediated targeting ability to cancer cells. Furthermore, the mUCNPs displayed both high r1 value and strong X-ray attenuation, proving the potential for multimodality bio-imaging. In addition, the NPs showed good biocompatibility both in vivo and in vitro. As a result, the bio-friendly nature, multimodal contrast imaging functionality and cancer-targeting capability impel these mUCNPs to be potential and suitable agents for biomedical applications such as multimodality bio-imaging and cancertargeting therapy.

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

Acknowledgment This work was supported by National Basic Research Programs of China (973 program, No. 2012CB932504, 2011CB933403 and 2012CB934001), and National Natural Science Foundation of China (No. 21177128, 21303200 and 21101158). We would like to thank Wuhan Center of Magnetic Resonance for MRI test. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.051. References [1] Li Z, Zhang Y, Jiang S. Multicolor core/shell-structured upconversion fluorescent nanoparticles. Adv Mater 2008;20:4765e9. [2] Gorris HH, Wolfbeis OS. Photon-upconverting nanoparticles for optical encoding and multiplexing of cells, biomolecules, and microspheres. Angew Chem Int Ed 2013;52:3584e600. [3] Liu C, Gao Z, Zeng J, Hou Y, Fang F, Li Y, et al. Magnetic/upconversion fluorescent NaGdF4:Yb,Er nanoparticle-based dual-modal molecular probes for imaging tiny tumors in vivo. ACS Nano 2013;7:7227e40. [4] Mai H-X, Zhang Y-W, Si R, Yan Z-G, L-d Sun, You L-P, et al. High-quality sodium rare-earth fluoride nanocrystals: controlled synthesis and optical properties. J Am Chem Soc 2006;128:6426e36. [5] Wang F, Han Y, Lim CS, Lu Y, Wang J, Xu J, et al. Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping. Nature 2010;463:1061e5. [6] Gai S, Li C, Yang P, Lin J. Recent progress in rare earth micro/nanocrystals: soft chemical synthesis, luminescent properties, and biomedical applications. Chem Rev 2014;114:2343e89. [7] Wang Y, Tu L, Zhao J, Sun Y, Kong X, Zhang H. Upconversion luminescence of b-NaYF4: Yb3þ, Er3þ@b-NaYF4 core/shell nanoparticles: excitation power density and surface dependence. J Phys Chem C 2009;113:7164e9. [8] Zhu H, Chen X, Jin LM, Wang QJ, Wang F, Yu SF. Amplified spontaneous emission and lasing from lanthanide-doped up-conversion nanocrystals. ACS Nano 2013;7:11420e6. [9] Li C, Lin J. Rare earth fluoride nano-/microcrystals: synthesis, surface modification and application. J Mater Chem 2010;20:6831e47. [10] Chen G, Shen J, Ohulchanskyy TY, Patel NJ, Kutikov A, Li Z, et al. (alphaNaYbF4:Tm3þ)/CaF2 core/shell nanoparticles with efficient near-infrared to near-infrared upconversion for high-contrast deep tissue bioimaging. ACS Nano 2012;6:8280e7. [11] Zhang C, Lee JY. Prevalence of anisotropic shell growth in rare earth core-shell upconversion nanocrystals. ACS Nano 2013;7:4393e402. [12] Ren W, Tian G, Jian S, Gu Z, Zhou L, Yan L, et al. Tween coated NaYF4:Yb,Er/ NaYF4 core/shell upconversion nanoparticles for bioimaging and drug delivery. RSC Adv 2012;2:7037e41. [13] Zhang F, Che R, Li X, Yao C, Yang J, Shen D, et al. Direct imaging the upconversion nanocrystal core/shell structure at the subnanometer level: shell thickness dependence in upconverting optical properties. Nano Lett 2012;12: 2852e8. [14] Su Q, Han S, Xie X, Zhu H, Chen H, Chen C-K, et al. The effect of surface coating on energy migration-mediated upconversion. J Am Chem Soc 2012;134: 20849e57. [15] Jin S, Zhou L, Gu Z, Tian G, Yan L, Ren W, et al. A new near infrared photosensitizing nanoplatform containing blue-emitting up-conversion nanoparticles and hypocrellin a for photodynamic therapy of cancer cells. Nanoscale 2013;5:11910e8. [16] Chien Y-H, Chou Y-L, Wang S-W, Hung S-T, Liau M-C, Chao Y-J, et al. Nearinfrared light photocontrolled targeting, bioimaging, and chemotherapy with caged upconversion nanoparticles in vitro and in vivo. ACS Nano 2013;7: 8516e28. [17] Yang D, Kang X, Ma P, Dai Y, Hou Z, Cheng Z, et al. Hollow structured upconversion luminescent NaYF4:Yb3þ, Er3þ nanospheres for cell imaging and targeted anti-cancer drug delivery. Biomaterials 2013;34:1601e12. [18] Xia A, Chen M, Gao Y, Wu D, Feng W, Li F. Gd3þ complex-modified naluf4based upconversion nanophosphors for trimodality imaging of NIR-to-NIR upconversion luminescence, X-ray computed tomography and magnetic resonance. Biomaterials 2012;33:5394e405. [19] Xing H, Bu W, Zhang S, Zheng X, Li M, Chen F, et al. Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging. Biomaterials 2012;33:1079e89. [20] Zeng S, Yi Z, Lu W, Qian C, Wang H, Rao L, et al. Simultaneous realization of phase/size manipulation, upconversion luminescence enhancement, and blood vessel imaging in multifunctional nanoprobes through transition metal Mn2þ doping. Adv Funct Mater; 2014. http://dx.doi.org/10.1002/ adfm.201304270.

7677

[21] Zeng S, Tsang M-K, Chan C-F, Wong K-L, Hao J. PEG modified BaGdF5:Yb/Er nanoprobes for multi-modal upconversion fluorescent, in vivo X-ray computed tomography and biomagnetic imaging. Biomaterials 2012;33:9232e8. [22] Zeng S, Wang H, Lu W, Yi Z, Rao L, Liu H, et al. Dual-modal upconversion fluorescent/X-ray imaging using ligand-free hexagonal phase NaLuF4:Gd/Yb/ Er nanorods for blood vessel visualization. Biomaterials 2014;35:2934e41. [23] Jiang S, Zhang Y, Lim KM, Sim EKW, Ye L. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA. Nanotechnology 2009;20:155101. [24] Wang C, Xu H, Liang C, Liu Y, Li Z, Yang G, et al. Iron oxide @ polypyrrole nanoparticles as a multifunctional drug carrier for remotely controlled cancer therapy with synergistic antitumor effect. ACS Nano 2013;7:6782e95. [25] Wang F, Banerjee D, Liu Y, Chen X, Liu X. Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 2010;135:1839e54. [26] Dai Y, Xiao H, Liu J, Yuan Q, Pa Ma, Yang D, et al. In vivo multimodality imaging and cancer therapy by near-infrared light-triggered trans-platinum pro-drugconjugated upconverison nanoparticles. J Am Chem Soc 2013;135:18920e9. [27] Yang Y, Velmurugan B, Liu X, Xing B. NIR photoresponsive crosslinked upconverting nanocarriers toward selective intracellular drug release. Small 2013;9:2937e44. [28] Hou Z, Li C, Ma P, Li G, Cheng Z, Peng C, et al. Electrospinning preparation and drug-delivery properties of an up-conversion luminescent porous NaYF4: Yb3þ, Er3þ@silica fiber nanocomposite. Adv Funct Mater 2011;21:2356e65. [29] Zhang X, Yang P, Dai Y, Pa Ma, Li X, Cheng Z, et al. Multifunctional upconverting nanocomposites with smart polymer brushes gated mesopores for cell imaging and thermo/pH dual-responsive drug controlled release. Adv Funct Mater 2013;23:4067e78. [30] Yang J, Deng Y, Wu Q, Zhou J, Bao H, Li Q, et al. Mesoporous silica encapsulating upconversion luminescence rare-earth fluoride nanorods for secondary excitation. Langmuir 2010;26:8850e6. [31] Yoo HS, Park TG. Folate-receptor-targeted delivery of doxorubicin nanoaggregates stabilized by doxorubicinePEGefolate conjugate. J Control Release 2004;100:247e56. [32] Zhu Y, Fang Y, Kaskel S. Folate-conjugated Fe3O4@SiO2 hollow mesoporous spheres for targeted anticancer drug delivery. J Phys Chem C 2010;114:16382e8. [33] Fan NC, Cheng FY, Ho JA, Yeh CS. Photocontrolled targeted drug delivery: photocaged biologically active folic acid as a light-responsive tumor-targeting molecule. Angew Chem Int Ed 2012;51:8806e10. [34] Setua S, Menon D, Asok A, Nair S, Koyakutty M. Folate receptor targeted, rareearth oxide nanocrystals for bi-modal fluorescence and magnetic imaging of cancer cells. Biomaterials 2010;31:714e29. [35] Yin W, Zhou L, Gu Z, Tian G, Jin S, Yan L, et al. Lanthanide-doped GdVO4 upconversion nanophosphors with tunable emissions and their applications for biomedical imaging. J Mater Chem 2012;22:6974e81. [36] Zhou L, Gu Z, Liu X, Yin W, Tian G, Yan L, et al. Size-tunable synthesis of lanthanide-doped Gd2O3 nanoparticles and their applications for optical and magnetic resonance imaging. J Mater Chem 2012;22:966e74. [37] Ren W, Tian G, Zhou L, Yin W, Yan L, Jin S, et al. Lanthanide ion-doped GdPO4 nanorods with dual-modal bio-optical and magnetic resonance imaging properties. Nanoscale 2012;4:3754e60. [38] Yin W, Zhao L, Zhou L, Gu Z, Liu X, Tian G, et al. Enhanced red emission from GdF3:Yb3þ,Er3þ upconversion nanocrystals by Liþ doping and their application for bioimaging. Chem Eur J 2012;18:9239e45. [39] Lusic H, Grinstaff MW. X-ray-computed tomography contrast agents. Chem Rev 2012;113:1641e66. [40] Park J, An K, Hwang Y, Park JG, Noh HJ, Kim JY, et al. Ultra-large-scale syntheses of monodisperse nanocrystals. Nat Mater 2004;3:891e5. [41] Roberts JE. Lanthanum and neodymium salts of trifluoroacetic acid. J Am Chem Soc 1961;83:1087e8. [42] Wong H-T, Tsang M-K, Chan C-F, Wong K-L, Fei B, Hao J. In vitro cell imaging using multifunctional small sized KGdF4:Yb3þ,Er3þ upconverting nanoparticles synthesized by a one-pot solvothermal process. Nanoscale 2013;5: 3465e73. [43] Basuki JS, Duong HT, Macmillan A, Erlich RB, Esser L, Akerfeldt MC, et al. Using fluorescence lifetime imaging microscopy to monitor theranostic nanoparticle uptake and intracellular doxorubicin release. ACS Nano 2013;7:10175e89. [44] Chang Y, Liu N, Chen L, Meng X, Liu Y, Li Y, et al. Synthesis and characterization of DOX-conjugated dendrimer-modified magnetic iron oxide conjugates for magnetic resonance imaging, targeting, and drug delivery. J Mater Chem 2012;22:9594. [45] Lee N, Choi SH, Hyeon T. Nano-sized CT contrast agents. Adv Mater 2013;25: 2641e60. [46] He Q, Ma M, Wei C, Shi J. Mesoporous carbon@silicon-silica nanotheranostics for synchronous delivery of insoluble drugs and luminescence imaging. Biomaterials 2012;33:4392e402. [47] Li C, Yang D, Pa Ma, Chen Y, Wu Y, Hou Z, et al. Multifunctional upconversion mesoporous silica nanostructures for dual modal imaging and in vivo drug delivery. Small 2013;9:4150e9. [48] Chen Z, Chen H, Hu H, Yu M, Li F, Zhang Q, et al. Versatile synthesis strategy for carboxylic acid-functionalized upconverting nanophosphors as biological labels. J Am Chem Soc 2008;130:3023e9. ~ a M, Rodriguez-Clemente R, Serna CJ. Uniform colloidal particles in so[49] Ocan lution: formation mechanisms. Adv Mater 1995;7:212e6. [50] Peng Q, Dong Y, Li Y. ZnSe semiconductor hollow microspheres. Angew Chem Int Ed 2003;42:3027e30.

7678

L. Zhou et al. / Biomaterials 35 (2014) 7666e7678

[51] Jiang ZY, Xie ZX, Zhang XH, Lin SC, Xu T, Xie SY, et al. Synthesis of singlecrystalline ZnO polyhedral submicrometer-sized hollow beads using laserassisted growth with ethanol droplets as soft templates. Adv Mater 2004;16:904e7. [52] Peruffo M, Mbogoro MM, Edwards MA, Unwin PR. Holistic approach to dissolution kinetics: linking direction-specific microscopic fluxes, local mass transport effects and global macroscopic rates from gypsum etch pit analysis. Phys Chem Chem Phys 2013;15:1956e65. [53] Hulett GA. The solubility of gypsum as affected by size of particles and by different crystallographic surfaces. J Am Chem Soc 1905;27:49e56. [54] Wang C, Cheng L, Liu Z. Drug delivery with upconversion nanoparticles for multi-functional targeted cancer cell imaging and therapy. Biomaterials 2011;32:1110e20.

[55] Guo M, Que C, Wang C, Liu X, Yan H, Liu K. Multifunctional superparamagnetic nanocarriers with folate-mediated and pH-responsive targeting properties for anticancer drug delivery. Biomaterials 2011;32:185e94. [56] Di W, Ren X, Zhao H, Shirahata N, Sakka Y, Qin W. Single-phased luminescent mesoporous nanoparticles for simultaneous cell imaging and anticancer drug delivery. Biomaterials 2011;32:7226e33. [57] Liu Y, Ai K, Lu L. Nanoparticulate X-ray computed tomography contrast agents: from design validation to in vivo applications. Acc Chem Res 2012;45: 1817e27. [58] Liu Z, Li Z, Liu J, Gu S, Yuan Q, Ren J, et al. Long-circulating Er3þ-doped Yb2O3 up-conversion nanoparticle as an in vivo X-ray CT imaging contrast agent. Biomaterials 2012;33:6748e57.