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Cite this: DOI: 10.1039/c5nr06394a
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NaYF4:Yb/Er@PPy core–shell nanoplates: an imaging-guided multimodal platform for photothermal therapy of cancers† Xiaojuan Huang,‡a Bo Li,‡a Chen Peng,‡b Guosheng Song,a Yuxuan Peng,a Zhiyin Xiao,a Xijian Liu,a Jianmao Yang,c Li Yud and Junqing Hu*a Imaging guided photothermal agents have attracted great attention for accurate diagnosis and treatment of tumors. Herein, multifunctional NaYF4:Yb/Er@polypyrrole (PPy) core–shell nanoplates are developed by combining a thermal decomposition reaction and a chemical oxidative polymerization reaction. Within such a composite nanomaterial, the core of the NaYF4:Yb/Er nanoplate can serve as an efficient nanoprobe for upconversion luminescence (UCL)/X-ray computed tomography (CT) dual-modal imaging, the shell of the PPy shows strong near infrared (NIR) region absorption and makes it effective in photothermal ablation of cancer cells and infrared thermal imaging in vivo. Thus, this platform can be simultaneously
Received 16th September 2015, Accepted 25th November 2015
used for cancer diagnosis and photothermal therapy, and compensates for the deficiencies of individual imaging modalities and satisfies the higher requirements on the efficiency and accuracy for diagnosis and
DOI: 10.1039/c5nr06394a
therapy of cancer. The results further provide some insight into the exploration of multifunctional nano-
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composites in the photothermal theragnosis therapy of cancers.
Introduction Recent advances in theranostics have expanded our ability to design and construct multifunctional nanoparticles for their bioapplications. So far, an imaging agent (e.g., Fe3O4,1,2 CdS3), photothermal agent (e.g., Au,4 Cu2−xS5–7), and/or anticancer drug (e.g., doxorubicin (DOX)5) were combined within a single nanoscale complex, and such a complex can offer the potential to reduce common chemotherapy- or radiation-associated side effects and increase the effectiveness of therapy.8 Our previous study has developed core–shell nanoparticles of Cu9S5@mSiO2 5 and Fe3O4@Cu2−xS2 for the combined photothermal- and chemo-therapies with infrared thermal imaging
a State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected] b Department of Radiology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China c Research Center for Analysis and Measurement, Donghua University, Shanghai 201620, China d Ian Wark Research Institute, University of South Australia, Mawson Lakes 5095, Australia † Electronic supplementary information (ESI) available: Experimental details and other characterization methods including TEM, TG, FTIR, etc. See DOI: 10.1039/ c5nr06394a ‡ These authors contributed equally to this work.
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and magnetic resonance imaging (MRI) for cancer treatment. As we know, imaging agents provide real-time imaging and thus guide accurate diagnosis for treatment of tumors, e.g. X-ray computed tomography (CT) imaging,9 MRI,1,2 and fluorescence imaging.3,7,8 In these imaging modes, fluorescence imaging has advantages of high temporal resolution, noninvasive functional imaging, high sensitivity and low costs,10 and thus plays an important role in biomedical study, visualization, and understanding of specific tissues and tumors. For instance, dual magnetic and optical probes have been developed using iron oxide nanoparticles and fluorescent dyes fluorescein isothiocyanate or porphyrin.11 Nie and Gao et al. have designed multifunctional nanoparticle probes based on CdSe– ZnS quantum dots (QDs)12 and InAs/InP/ZnSe QDs13 for in vivo tumor imaging, respectively. Also, Au@SiO2@CdTe/CdS/ZnS composite nanostructures were prepared to integrate fluorescence imaging with photothermal therapy (PTT) for cancer.14 However, organic dyes suffer from poor photostability and significant autofluorescence, and QDs have been receiving great concerns for their harmful tissue photodamage and high potential cytotoxic risks. In addition, both of them are generally excited with ultraviolet and visible light, which can cause biological sample photodamage and mutation under prolonged exposure.15 So, these drawbacks from the organic fluorophores and QDs will prompt the development of other luminescent nanomaterials for better biological applications.
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Lanthanide-doped nanomaterials absorb near-infrared (NIR) light, and emit high energy visible photons, thus reducing significantly the autofluorescence background, improving the tissue penetration depth, and showing better photostability, non-photoblinking and lower phototoxicity during upconversion luminescence (UCL) imaging.16,17 At the same time, excellent MRI performance and CT imaging can be obtained due to the doping of Gd3+, Yb3+, or Ho3+ within single upconversion nanoparticles (UCNPs).18 These characteristics made the UCNPs an ideal candidate for developing multifunctional theranostic nanoplatforms for combining imaging, drug delivery, and photodynamic therapy.18,19 Liu and co-workers prepared a DOXloaded upconversion@polydopamine (PDA) nanoplatform, in which PDA is used as a PTT agent.20 This platform not only combines MRI, UCL and CT imaging, but also realizes chemophotothermal synergistic therapy. Although the PTT agent of the PDA can kill tumor cells under the laser irradiation, its absorption coefficient in the NIR region is low, and this negatively affects the PTT effect. To promote the photothermal conversion efficiency, a prerequisite is to obtain an efficient PTT agent with strong NIR absorption. Polypyrrole (PPy), one of the well-known organic polymers, exhibits a strong and broad NIR absorption band, and allows it to be a favourable PTT agent.21,22 Besides, compared with these conventional inorganic photothermal agents, PPy has outstanding stability, good biocompatibility, and excellent biodegradability, which makes it a desirable platform for preparing multifunctional PTT agents.22,23 Therefore, a combination of UCNPs and PPy integrates both multimodal imaging functions holding infrared thermal imaging, UCL imaging, or CT imaging, and PTT for cancer cells. Herein, a NIR-response multimodal imaging-guided photothermal agent, NaYF4:Yb/Er@PPy core–shell nanoplates, was designed and fabricated. Within a core–shell nanostructure, the core of NaYF4:Yb/Er operates as both CT and UCL contrast agents, as the La (Y, Yb, and Er)-based upconversion material not only shows high performance in X-ray attenuation due to the presence of high Z rare earth elements but also provides fluorescence imaging function;16–18 the shell of the PPy polymer formed by the oxidative polymerization of Fe3+ functions as a photothermal agent for ablation of tumors and an infrared thermal imaging agent. It is interesting that the morphology of the NaYF4:Yb/Er core will change from a disc-like plate to a regular hexagon with the increasing amount of Fe3+. These nanocomposites achieve three-modal imaging guided PTT for cancer, compensating for the deficiencies of individual imaging modalities and satisfying the higher requirements on the efficiency and accuracy for diagnosis and research, which will provide potential for simultaneous diagnostics, therapeutics, and monitoring of tumors.
Experimental section Materials Y2O3, Yb2O3 and Er2O3 were purchased from Ourchem. Oleylamine (OM), 1-octadecene (ODE > 90%) and polyvinyl alcohol
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were purchased from Aladdin Chemistry Co., Ltd. Oleic acid (OA, 90%) was purchased from Tokyo Chemical Industry. Nitrosonium tetrafluoroborate (NOBF4) was purchased from Alfa Aesar. Pyrrole and sodium dodecylbenzene sulfonate were purchased from Sinopharm Chemical Reagent Co., Ltd. TC71cells bearing mice were purchased from Shanghai Tenth People’s Hospital. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Synthesis of the oleic acid-modified NaYF4:Yb/Er nanoplates In a typical experiment, 0.2 mmol of La2O3 (Y : Yb : Er = 80 : 18 : 2 mol%) was first dispersed in 20 mL of diluted HCl upon heating to 80 °C under stirring until dissolved. Then the solution was heated to 160 °C for 1 h, and dried. The solid was dispersed in 2 mL of methanol, then transferred into 50 mL of a flask containing 8 mL of oleic acid and 12 mL of 1-octadecene and the formed solution was heated to 150 °C for 60 min, and then cooled down to 50 °C. Thereafter, 5 mL of methanol solution of NH4F (3.2 mmol) and NaOH (2 mmol) were added and stirred for 60 min at 50 °C. After methanol evaporated, the solution was heated to 300 °C under nitrogen for 1 h and cooled down to room temperature. Later, the resulting product was precipitated by the addition of ethanol, collected by centrifugation, and finally re-dispersed in cyclohexane. Ligand exchange In a typical process, 5 mL of a cyclohexane solution containing 20 mg of oleate-capped NaYF4:Yb/Er nanoplates was mixed with 5 mL of a dichloromethane solution of NOBF4 (20 mg) at room temperature, and then gently stirred for 10 min to yield white precipitates, which were collected by centrifugation at 12 000 rpm for 5 min, and re-dispersed in 10 mL of N,N-dimethylformamide (DMF) to form a transparent solution. Subsequently, 50 mg of PVA was added to the above transparent solution. After stirring for 60 min at room temperature, the PVA-functionalized NaYF4:Yb/Er nanoplates were washed with water several times to remove the excess PVA. The final products were dispersed in distilled water for the following use. Synthesis of NaYF4:Yb/Er@PPy nanoplates 5 mL dispersion of the NaYF4:Yb/Er nanoplates (2 mg mL−1), 10 mg of SDBS and 30 mg of PVA were mixed in 15 mL of water and stirred for 3 h at room temperature. After that, 2 μL of pyrrole was added to the dispersion and stirred overnight. Then 5 mL of FeCl3 solution (FeCl3·6H2O: 10, 20, 40 mg) was added to the dispersion and the polymerization was carried out for 24 h. The nanoplates were precipitated by adding 20 mL of acetone, and collected by centrifugation. Synthesis of PEG-grafted poly(isobutylene-alt-maleic anhydride) (PIMA-PEG) PIMA-PEG was synthesized by referring to the literature.6 In brief, 10 mg of poly(isobutylene-alt-maleic anhydride) (PIMA) and 143 mg of mPEG-NH2 (5 K) were dissolved in 5 mL of dichloromethane, and 6 μL of triethylamine (TEA) and 11 mg
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of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were added. After 24 h of reaction, the solvent was evaporated and the solid product was dissolved in water.
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Modification of NaYF4:Yb/Er@PPy nanoplates NaYF4:Yb/Er@PPy nanoplates were dispersed in water to obtain a clear solution. A solution of PIMA-PEG (2 mg mL−1) was added into the NaYF4:Yb/Er@PPy solution, and then ultrasonicated for 30 min. The final product was purified by centrifugation several times. Characterization Electron microscopy images were acquired on a transmission electron microscope (JEM-2010F). The content of metals released from the NaYF4:Yb/Er nanoplates in the solution was confirmed using an inductively coupled plasma atomic emission spectroscope (ICP-AES, Prodigy). FTIR spectra were recorded by using a Fourier transform infrared spectrometer (Nexus-670). Thermogravimetric analysis was carried out on a USA TA/Q5000IR thermal analyzer under nitrogen ranging from 50 °C to 800 °C at a rate of 20 °C min−1. UV-vis absorbance spectra were recorded using a UV-visible-NIR spectrophotometer operating from 200 to 1100 nm (UV-1902PC, Phoenix). Measurement of the photothermal effect To measure the photothermal effect of NaYF4:Yb/Er@PPy core–shell nanoplates induced by the NIR absorbance, 100 μL aqueous dispersions of the NaYF4:Yb/Er@PPy core–shell nanoplates with different concentrations (0–0.5 mg mL−1) were added into plastic tubes (250 μL), and then irradiated with a 915 nm laser at a power density of 0.5 W cm−2 (a laser intensity of 0.17 W with a spot area of 0.35 cm−2) for 5 min. A thermocouple with an accuracy of ±0.1 °C was inserted into the aqueous dispersion at such a position that the direct irradiation of the laser on the probe was avoided. The temperature was recorded by using an online type thermocouple thermometer every 5 s. To evaluate the photothermal conversion efficiency of NaYF4:Yb/Er@PPy core–shell nanoplates, the temperature of a dispersion of NPs (0.5 mg mL−1) was recorded every 5 s with continuous irradiation with a 915 nm laser at a power density of 0.5 W cm−2 for 5 min. After that, the laser was turned off and the temperature of the dispersion dropped to the initial temperature. Then the photothermal conversion efficiency was calculated by a modified method referring to the report by Roper et al. In vitro cytotoxicity assay In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the presence of 5% CO2, HeLa cells were seeded into a 96-well plate at 1 × 104 cells per well at 37 °C. After incubation for 24 h, the NaYF4:Yb/Er@PPy nanoplates dispersed in a PBS solution were then added into the wells at various concentrations (0, 62.5, 125, 250, 375, and 500 ppm) and incubated
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for another 24 h. 0.1 mL of MTT solution (5 mg mL−1) was added to each well of the microtiter plate and incubated in the CO2 incubator for 4 h. Then the optical density (OD) was measured at 490 nm using a microplate reader. All experiments were independently performed three times. In vitro CT imaging The dispersion of the NaYF4:Yb/Er@PPy nanoplates with different concentrations of La (Y, Yb, Er) (0–6.3 mg mL−1) was detected using a CT imaging system (SOMATOM Sensation Cardiac) with the following operating parameters: thickness, 0.6 mm; field of view, 20 cm; tube voltage, 80 kV; effective mAs, 110. CT values were acquired on the same workstation using the software supplied by the manufacturer. In vivo UCL imaging A modified Maestro in vivo imaging system was employed to image the NaYF4:Yb/Er@PPy nanoplate treated mice using a 980 nm optical fiber coupled laser as the excitation source. The laser power density was ∼0.2 W cm−2 during imaging. An 850 nm short-pass emission filter was used to prevent the interference of excitation light to the CCD camera. The green emission from the NaYF4:Yb/Er@PPy nanoplates was collected at 550 nm. In vivo CT imaging The mice bearing tumors (∼5 × 5 mm) were anesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight. In vivo CT scanning was performed both before/after intratumoral injection of the aqueous dispersion of the NaYF4:Yb/Er@PPy nanoplates (50 μL, La: 4.2 mg mL−1) at 1 h post-injection. All CT scans were performed using the CT system with the parameters similar to those for in vitro experiments. In vivo infrared thermal imaging and photothermal ablation The tumor-bearing mice were randomly allocated into a control group and a treatment group. Firstly, two groups were anaesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight. Then, mice in the treatment group were intratumorally injected with the NaYF4:Yb/ Er@PPy nanoplates dispersed in phosphate-buffered saline (PBS) solution (100 μL, 0.5 mg mL−1). For the control, mice were injected with 100 μL of saline solution. After 0.5 h postinjection, mice from both the groups were simultaneously irradiated with a 915 nm laser at a power density of 0.5 W cm−2 for 10 min. During the laser irradiation, infrared thermal images were captured using an IR camera of a photothermal therapy monitoring system GX-A300 (Shanghai Guixin Corporation). After laser treatment, the mice were sacrificed, tumors were harvested, and embedded in paraffin, and cryosectioned into 4 μm slices using a conventional microtome. Then slides were stained with hematoxylin/eosin. The slices were examined under a Zeiss Axiovert 40 CFL inverted fluorescence microscope, and images were captured using a Zeiss AxioCam MRc5
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digital camera. The experiments were independently performed three times.
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Results and discussion The NaYF4:Yb/Er@PPy core–shell nanoplates were synthesized by combining a thermal decomposition reaction and an in situ chemical oxidative polymerization reaction, as illustrated in Fig. 1. Firstly, the oil-soluble NaYF4:Yb/Er (Y : Yb : Er = 80 : 18 : 2 mol%) disc nanoplates were synthesized by a thermal decomposition reaction (see the Experimental section for details). Subsequently, the long hydrocarbon ligands on the surface of the oil-soluble NaYF4:Yb/Er nanoplates were removed with NOBF4, and then the nanoplates were modified with polyvinyl alcohol (PVA) to get water-solubility. Finally, PPy was coated on the surface of nanoplates to form core–shell nanostructures by oxidative polymerization of Fe3+. Interestingly, when the amount of Fe3+ increased, the shape of the NaYF4:Yb/Er nanoplate core would become a regular hexagon from the disc-like shape. Lastly, to enhance the stability of the nanoplates in physiological solution, the NaYF4:Yb/Er@PPy nanoplates were modified with PIMA-PEG. Fig. 2a shows a representative transmission electron microscopy (TEM) image of the as-obtained oil-soluble NaYF4: Yb/Er disc-like shaped nanoplates. As depicted, the disc-like plates have a uniform size, i.e. a diameter of ∼78 nm and a thickness of ∼25 nm. The phase structure of the NaYF4:Yb/Er nanoplates was confirmed by X-ray diffraction (XRD). As shown in Fig. 2b, it could be well indexed to the hexagonal phase (JCPDS no. 28-1192) of NaYF4. Prior to the PPy coating, the oil-soluble NaYF4:Yb/Er nanoplates have been modified with the PVA in order to endow them with hydrophilic properties and facilitate the PPy coating, the resulting size and shape of the PVA–NaYF4:Yb/Er nanoplates remained unchanged, while the modification process involved a change in the color of the suspension from yellow to white (Fig. S1†), which could be observed visually during the procedure of surface modification. Fourier transform infrared (FTIR) spectroscopy is used to prove the ligand exchange. At first, the most obvious absorption peaks located at 2923 and 2855 cm−1 are the characteristic stretching absorptions of –CH2– and –CH3 of oil acid, and the peak at 1709 cm−1 is induced by
Fig. 1 Schematic illustration of the synthetic route of NaYF4:Yb/Er@PPy core–shell nanoplates.
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Fig. 2 (a) TEM image of the NaYF4:Yb/Er nanoplates. (b) XRD patterns of as-prepared NaYF4:Yb/Er nanoplates (black line) and the standard NaYF4:Yb/Er powders (red bars) from JCPDS card (no. 28-1192). (c–e) TEM images of NaYF4:Yb/Er@PPy core–shell nanoplates with the disc core, irregular core and hexagon core, respectively. (f ) STEM image and EDS elemental maps of an individual NaYF4:Yb/Er@PPy nanoplate.
carbonyl (as shown in black line of Fig. S2†).24 After modification, the characteristic absorptions of oil acid disappeared, and the main peaks arose at 3410 and 1095 cm−1, which belong to –OH and C–O of PVA, respectively (the blue line of Fig. S2†). Fig. 2c–e show the TEM images of the final NaYF4:Yb/Er@PPy nanoplates after the PVA modification. The size of the nanoplates can be measured to be ∼100 nm, which is consistent with dynamic light scattering (DLS) diameter distribution shown in Fig. S3.† Viewing from the nanoplates standing or lying on the Cu grid (C film support) during the TEM sample processing, it demonstrates that PPy was present around whole individual NaYF4:Yb/Er nanoplates, forming a core–shell structure. Elemental maps in Fig. 2f recorded by using an energy dispersive spectrometer (EDS) display that Na, Y, F, Yb, Er, and N elements existed in the nanoplates and indicate that all these elements are uniformly distributed throughout the nanoplates. The FTIR spectrum (red line of Fig. S2†) of the NaYF4: Yb/Er@PPy nanoplates displays absorption peaks at 1630 and 1400 cm−1, which are induced by CvC and C–N on the pyrrole ring, respectively.25 The thermal gravity analysis (TGA) curve in Fig. S4† shows that the weight of the NaYF4:Yb/Er@PPy nanoplates began to reduce at about 200 °C under a nitrogen atmosphere and almost stopped reducing at above 500 °C. It is consistent with the TGA data of PPy, which is caused by the degradation of PPy (the black line in Fig. S4†). This further
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confirms the existence of the PPy and the content of the PPy in the NaYF4:Yb/Er@PPy nanoplates of ∼10% (wt%). During the polymerization process of pyrrole, it was interesting to find that if the concentration (or amount) of Fe3+ was increased, while the amount of pyrrole was unchanged, the NaYF4:Yb/Er core within the NaYF4:Yb/Er@PPy composite nanoplates would change from the disc plate to a hexagonal plate. Fig. 2c shows the NaYF4:Yb/Er@PPy nanoplates formed with a concentration of 0.41 mg per mL of Fe3+ (5 mL, the quality ratio of FeCl3·6H2O and NaYF4:Yb/Er is 1 : 1) added, and the NaYF4:Yb/Er core kept disc-shape unchanged and the PPy thickness was ∼7.5 nm. When the concentration of Fe3+ was increased to 0.82 mg mL−1, some of the edges of the disc cores were etched to irregular, as shown in Fig. 2d. A concentration of 1.65 mg per mL of Fe3+ (5 mL) added would further change the cores into regular hexagonal plates. At the same time, the thickness of the PPy shell also increases to 12.5 nm, as demonstrated in Fig. 2e. However, with the concentration of Fe3+ further increased, the nanoplates would finally become ear-like shaped (Fig. S5†). The phenomenon may be due to the etching of an excess of Fe3+. When the amount of Fe3+ is lower (the quality ratio of FeCl3·6H2O and NaYF4:Yb/Er is less than 1 : 1), Fe3+ is used as an oxidizing agent to oxidize the pyrrole monomer into PPy coating on the NaYF4:Yb/Er nanoplates’ surface. When the amount of Fe3+ is higher, apart from the usage as an oxidizing agent, the excess Fe3+ can reach the surface of the NaYF4:Yb/Er nanoplates through the outside layer of the PPy to carry out the etching process. The discshaped nanoplates started to change from their edges rather than from their facets and then transformed into hexagonalshaped plates. This may be an energetically favored route considering the fact that those atoms located on the edges have lower coordination and therefore possess higher chemical reactivities.26 Here, Fe3+ is used as an etchant to change the morphology of the NaYF4:Yb/Er core.27 The optical properties of the obtained NaYF4:Yb/Er@PPy core–shell nanoplates were then studied. Owing to the doping of Yb and Er, the NaYF4:Yb/Er nanoplates exhibit strong fluorescence.16 As revealed in Fig. 3a, the luminescence spectrum of the dispersion of the NaYF4:Yb/Er nanoplates displays three characteristic emission peaks located at 527 nm (2H11/2 → 4I15/2), 546 nm (4S3/2 → 4I15/2), and 660 nm (4F9/2 → 4I15/2) under an excitation at 980 nm.17 As the emission at 546 nm is the strongest, the UCL emission color of NaYF4:Yb/Er was almost green, as shown in the inserted image. After the PPy shell was coated, the fluorescence is partly quenched, as demonstrated by the fluorescence spectra and photographs. However, the disc-shaped NaYF4:Yb/Er@PPy nanoplates still show strong upconversion fluorescence, due to a thinner PPy coating compared with that of the hexagonal-shaped NaYF4:Yb/Er@PPy nanoplates, and thus could act as a contrast agent for UCL imaging. Fig. 3b demonstrates the UV-vis absorbance spectrum for the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates. It can be seen that the aqueous dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates exhibits a gradually
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Fig. 3 (a) The fluorescence spectra of the aqueous dispersions of the NaYF4:Yb/Er nanoplates and NaYF4:Yb/Er@PPy core–shell nanoplates, respectively. Insert: the fluorescence photographs of NaYF4:Yb/Er (left) and NaYF4:Yb/Er@PPy (right) dispersions when excited with a 980 nm laser. (b) UV-vis absorbance spectrum for the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates. (c) Temperature elevation of water and the dispersion of the NaYF4:Yb/Er@PPy nanoplates with different concentrations over a period of 5 min under exposure to NIR light (915 nm, 0.5 W cm−2) measured every 5 s. (d) Temperature elevation of the dispersions of the NaYF4:Yb/Er@PPy nanoplates with the increasing concentration.
increasing absorption from 700 to 1100 nm because of the strong NIR absorbance of PPy (Fig. S6†).21,22 This means the potential photothermal conversion effect of the NaYF4:Yb/ Er@PPy core–shell nanoplates upon laser irradiation. Fig. 3c shows the photothermal conversion performance of the NaYF4:Yb/Er@PPy core–shell nanoplates under the irradiation with a 915 nm NIR laser at a power density of 0.5 W cm−2. The temperature of the aqueous dispersion of the nanoplates of different concentrations (i.e., 0.1, 0.2, 0.3, 0.4 and 0.5 mg mL−1) can increase by 15.0, 20.3, 24.1, 28.4 and 29.3 °C, respectively, in the period of 300 s. This corresponds well to the absorbance of NaYF4:Yb/Er@PPy nanoplates, which showed a linear relationship at 915 nm versus concentrations (Fig. S7†). As a control experiment, pure water only increased by less than 5 °C. These demonstrate that the NaYF4:Yb/ Er@PPy core–shell nanoplates can rapidly and efficiently convert the 915 nm laser energy into thermal energy, which has the potential to be a promising PTT agent. In comparison, the dispersion (0.5 mg mL−1) of the hexagonal-shaped NaYF4: Yb/Er@PPy nanoplates increased by 30.3 °C under the same conditions, it has no much difference from the disc-shaped NaYF4:Yb/Er@PPy nanoplates, as shown in Fig. S8.† So, we choose the disc-shaped NaYF4:Yb/Er@PPy nanoplates for further biological test. We measured the photothermal conversion efficiency of the NaYF4:Yb/Er@PPy nanoplates by a modified method referring to the report by Roper et al.28 The temperature change of the NaYF4:Yb/Er@PPy dispersion was recorded as a function of
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time under continuous irradiation with a 915 nm laser for 5 min. Then the laser was shut off, and the temperature decrease of the aqueous dispersion was monitored to determine the rate of the heat transfer from the dispersion system to the environment. Fig. 4a shows temperature elevation of the dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates and water. Following Roper’s report and our previous work,7,28 the conversion efficiency of the dispersion of the NaYF4:Yb/ Er@PPy nanoplates can be calculated to be 44.7%. This means the favourable character of NaYF4:Yb/Er@PPy nanoplates to convert laser energy to heat. In order to investigate the NIR photothermal stability of the NaYF4:Yb/Er@PPy nanoplates’ dispersion, five LASER ON/OFF cycles of the NIR laser irradiation were examined. A solution (0.5 mg mL−1) of the NaYF4:Yb/Er@PPy core–shell nanoplates was irradiated with the NIR laser for 5 min (LASER ON), followed by naturally cooling to room temperature with the laser off. This process was repeated five times (Fig. 4b). The temperature elevation of 29 °C was achieved at the first cycle and no significant decrease from this temperature elevation was observed later. Meanwhile, the TEM images of the NaYF4:Yb/Er@PPy nanoplates after irradiation for 25 min showed no obvious change compared with that before the irradiation (Fig. S9†). These results indicate that the NaYF4:Yb/Er@PPy nanoplates exhibit excellent photothermal stability after a long period of the NIR laser irradiation. CT is an imaging method for detecting various degrees of attenuation of X-rays in different tissues when they come through the whole body. It is an inspection method with noninvasion and high-contrast resolution.9 La (Y, Yb, Er)-based upconversion materials were reported to possess high X-ray attenuation owing to their large atomic number.18 To verify the potential of the NaYF4:Yb/Er@PPy nanoplates as a contrast agent, we performed in vitro CT imaging test. Fig. 5a presents the CT images of iopromide (a kind of clinical contrast agent) and the aqueous dispersions of the NaYF4:Yb/Er@PPy core– shell nanoplates with different I or La concentrations. We can clearly observe positive contrast enhancement of the CT signals with the concentration increasing (i.e. 0, 0.525, 1.05, 2.1, 4.2, 6.3 mg mL−1). To quantitatively evaluate the X-ray
Fig. 4 (a) Temperature elevation of the dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates (the black line) and water (the red line), respectively, in which the irradiation (915 nm, 0.5 W cm−2) lasted for 5 min, and then the laser was shut off. (b) Temperature elevation of the dispersion of the NaYF4:Yb/Er@PPy nanoplates over 5 LASER ON/OFF cycles of the NIR laser irradiation.
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Fig. 5 (a) In vitro CT images of iopromide and the dispersions of the NaYF4:Yb/Er@PPy nanoplates with different I or La concentrations. (b) CT value (HU) of iopromide (black line) and the NaYF4:Yb/Er@PPy nanoplates (red line) as a function of the concentration of I or La. (c) CT images of mice before and after intratumoral injection of the dispersion of the NaYF4:Yb/Er@PPy nanoplates. The position of tumors is marked by dotted circles.
attenuation capability of these nanoplates, we measured the CT values at different concentrations. As shown in Fig. 5b, the CT value increased linearly with the concentration of La. The slope of the linear equation for NaYF4:Yb/Er@PPy core–shell nanoplates is about 53.6 HU L g−1, a little higher than that of iopromide, indicating a high contrast efficiency of the NaYF4: Yb/Er@PPy nanoplates. This suggests that the NaYF4:Yb/ Er@PPy core–shell nanoplates can be used as a CT contrast agent. A high biocompatibility is required as a precondition for further applications in cancer treatment. Herein, the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates with various concentrations (0.0625, 0.125, 0.250, 0.375 and 0.500 mg mL−1) was incubated with HeLa cells for 24 h and then the cell viabilities were tested by using the MTT assay. The viabilities of cancer cells were used to assess the biocompatibility in vitro. No obvious adverse effect on cell viabilities was observed, as shown in Fig. S10,† suggesting a low cytotoxicity of the NaYF4:Yb/Er@PPy core–shell nanoplates. Encouraged by the negligible cytotoxicity and favorable performance of the nanoplate in vitro results, we further investigated the feasibility of the CT imaging in vivo by injecting the NaYF4:Yb/Er@PPy dispersion in tumor-bearing mouse. Fig. 5c shows in vivo tumor CT images before and after the intratumoral injection with the NaYF4:Yb/Er@PPy dispersion (50 μL, La: 4.2 mg mL−1). It shows that the corresponding injection site exhibited a much brighter contrast after injection than before. This proves the potential of the NaYF4:Yb/Er@PPy nanoplates as a CT contrast agent. Then UCL imaging was performed using a modified Maestro in vivo fluorescence imaging system with a 980 nm optical fiber coupled laser as the excitation source. Herein, the mice bearing TC71 tumors were intratumorally injected with the NaYF4:Yb/Er@PPy dispersion
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(50 μL, 1 mg mL−1). A significant UCL signal of the tumor region was observed clearly with almost no autofluorescence elsewhere, as shown in Fig. 6, while the blank group showed no UCL signal. The experimental result indicates the ability of the UCNPs to penetrate tissue and the function of NaYF4:Yb/
Fig. 6 UCL images of the blank control and experiment group with intratumoral injection of the NaYF4:Yb/Er@PPy dispersion.
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Er@PPy nanoplates as a promising probe for in vivo UCL imaging. Finally, we designed animal experiments to demonstrate the photothermal effect of the NaYF4:Yb/Er@PPy core–shell nanoplates. Two groups of mice bearing TC71 tumors were used in our experiment. After being anaesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight, one group was injected with the NaYF4:Yb/ Er@PPy dispersion (0.5 mg mL−1) of PBS into the tumors, and the other was injected with PBS. Then both the mice were simultaneously exposed to the 915 nm laser (0.5 W cm−2) for 10 min. We used an IR camera to capture the full-body infrared thermal images and monitor the temperature change of the irradiated area. The photograph of the experimental setup is shown in Fig. S11.† In the infrared thermal image in Fig. 7a, there is no distinction between regions 11 and 12 (regions 11 and 12 are from the treatment group and the control group, respectively.). After a period of laser irradiation, a strong image contrast was observed between the tumor regions (Fig. 7b), exhibiting different temperature changes. As seen in Fig. 7c, the tumor region of the mice in the treatment group heated up very quickly with the temperature reaching 47.9 °C from 25.9 °C. As for the control mice, the temperature of the irradiated area only increased by about 3 °C under the same condition. These confirm that the platform can be an infrared thermal imaging contrast agent. After the laser irradiation, the hematoxylin and eosin stained histological examination was performed, as shown in Fig. 7d, e and S12.† The treatment
Fig. 7 (a, b) Infrared thermal images of two tumor-bearing mice after injecting with the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates (the left mouse, indicated region 11) or PBS (the right mouse, indicated region 12) via the intratumoral injection respectively, irradiated with a 915 nm laser (0.5 W cm−2) at a time point of 0 min and 5 min. (c) The temperature profiles in regions 11 and 12 as a function of the irradiation time. (d, e) The representative hematoxylin and eosin stained histological images of ex vivo tumor sections treated by irradiation over a period of 10 min injected with: PBS only and an aqueous dispersion of the NaYF4:Yb/Er@PPy nanoplates, respectively.
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(Fig. 7e) shows severe cellular damage, such as shrinkage of the malignant cells, nuclear condensation, fragmentation and lysis compared with mice from the control group (Fig. 7d). It suggests that the higher temperature of 47.9 °C can kill cancer cells in expectation, and the NaYF4:Yb/Er@PPy core–shell nanoplates still have an excellent photothermal effect in vivo. Simultaneously, the NaYF4:Yb/Er@PPy nanoplates allow realtime monitoring of the temperature dynamics of a PTT process by infrared thermal imaging.
Conclusion In summary, an imaging-guided multimodal platform of the NaYF4:Yb/Er@PPy core–shell nanoplates was successfully designed and prepared for PTT of cancers. The shape of the NaYF4:Yb/Er core could be adjusted by the amount of Fe3+ in the polymerization process of pyrrole, which is an interesting phenomenon. The NaYF4:Yb/Er@PPy core–shell nanoplates can be an efficient photothermal theragnosis platform for in vitro and in vivo photothermal ablation of cancer cells guided by CT imaging, UCL imaging and infrared thermal imaging, simultaneously. Moreover, owing to the delocalized π-electrons of the PPy shell, it is believed that aromatic molecules, such as DOX, may be bound on the shell for multifunctional applications. Overall, our work demonstrates a straightforward route to develop a multifunctional platform for imagingguided therapy of cancer, and encourages further exploration of imaging-guided cancer therapy for better properties and enhanced efficacy.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No. 51472049, 51302035, and 21171035), the National 863 Program of China (Grant No. 2013AA031903), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), Ph.D. Programs Foundation of Ministry of Education of China (Grant No. 20110075110008 and 20130075120001), the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Shanghai Leading Academic Discipline Project (Grant No. B603), and the Program of Introducing Talents of Discipline to Universities (Grant No. 111-2-04).
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Cite this: DOI: 10.1039/c5nr06394a
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NaYF4:Yb/Er@PPy core–shell nanoplates: an imaging-guided multimodal platform for photothermal therapy of cancers† Xiaojuan Huang,‡a Bo Li,‡a Chen Peng,‡b Guosheng Song,a Yuxuan Peng,a Zhiyin Xiao,a Xijian Liu,a Jianmao Yang,c Li Yud and Junqing Hu*a Imaging guided photothermal agents have attracted great attention for accurate diagnosis and treatment of tumors. Herein, multifunctional NaYF4:Yb/Er@polypyrrole (PPy) core–shell nanoplates are developed by combining a thermal decomposition reaction and a chemical oxidative polymerization reaction. Within such a composite nanomaterial, the core of the NaYF4:Yb/Er nanoplate can serve as an efficient nanoprobe for upconversion luminescence (UCL)/X-ray computed tomography (CT) dual-modal imaging, the shell of the PPy shows strong near infrared (NIR) region absorption and makes it effective in photothermal ablation of cancer cells and infrared thermal imaging in vivo. Thus, this platform can be simultaneously
Received 16th September 2015, Accepted 25th November 2015
used for cancer diagnosis and photothermal therapy, and compensates for the deficiencies of individual imaging modalities and satisfies the higher requirements on the efficiency and accuracy for diagnosis and
DOI: 10.1039/c5nr06394a
therapy of cancer. The results further provide some insight into the exploration of multifunctional nano-
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composites in the photothermal theragnosis therapy of cancers.
Introduction Recent advances in theranostics have expanded our ability to design and construct multifunctional nanoparticles for their bioapplications. So far, an imaging agent (e.g., Fe3O4,1,2 CdS3), photothermal agent (e.g., Au,4 Cu2−xS5–7), and/or anticancer drug (e.g., doxorubicin (DOX)5) were combined within a single nanoscale complex, and such a complex can offer the potential to reduce common chemotherapy- or radiation-associated side effects and increase the effectiveness of therapy.8 Our previous study has developed core–shell nanoparticles of Cu9S5@mSiO2 5 and Fe3O4@Cu2−xS2 for the combined photothermal- and chemo-therapies with infrared thermal imaging
a State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected] b Department of Radiology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China c Research Center for Analysis and Measurement, Donghua University, Shanghai 201620, China d Ian Wark Research Institute, University of South Australia, Mawson Lakes 5095, Australia † Electronic supplementary information (ESI) available: Experimental details and other characterization methods including TEM, TG, FTIR, etc. See DOI: 10.1039/ c5nr06394a ‡ These authors contributed equally to this work.
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and magnetic resonance imaging (MRI) for cancer treatment. As we know, imaging agents provide real-time imaging and thus guide accurate diagnosis for treatment of tumors, e.g. X-ray computed tomography (CT) imaging,9 MRI,1,2 and fluorescence imaging.3,7,8 In these imaging modes, fluorescence imaging has advantages of high temporal resolution, noninvasive functional imaging, high sensitivity and low costs,10 and thus plays an important role in biomedical study, visualization, and understanding of specific tissues and tumors. For instance, dual magnetic and optical probes have been developed using iron oxide nanoparticles and fluorescent dyes fluorescein isothiocyanate or porphyrin.11 Nie and Gao et al. have designed multifunctional nanoparticle probes based on CdSe– ZnS quantum dots (QDs)12 and InAs/InP/ZnSe QDs13 for in vivo tumor imaging, respectively. Also, Au@SiO2@CdTe/CdS/ZnS composite nanostructures were prepared to integrate fluorescence imaging with photothermal therapy (PTT) for cancer.14 However, organic dyes suffer from poor photostability and significant autofluorescence, and QDs have been receiving great concerns for their harmful tissue photodamage and high potential cytotoxic risks. In addition, both of them are generally excited with ultraviolet and visible light, which can cause biological sample photodamage and mutation under prolonged exposure.15 So, these drawbacks from the organic fluorophores and QDs will prompt the development of other luminescent nanomaterials for better biological applications.
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Lanthanide-doped nanomaterials absorb near-infrared (NIR) light, and emit high energy visible photons, thus reducing significantly the autofluorescence background, improving the tissue penetration depth, and showing better photostability, non-photoblinking and lower phototoxicity during upconversion luminescence (UCL) imaging.16,17 At the same time, excellent MRI performance and CT imaging can be obtained due to the doping of Gd3+, Yb3+, or Ho3+ within single upconversion nanoparticles (UCNPs).18 These characteristics made the UCNPs an ideal candidate for developing multifunctional theranostic nanoplatforms for combining imaging, drug delivery, and photodynamic therapy.18,19 Liu and co-workers prepared a DOXloaded upconversion@polydopamine (PDA) nanoplatform, in which PDA is used as a PTT agent.20 This platform not only combines MRI, UCL and CT imaging, but also realizes chemophotothermal synergistic therapy. Although the PTT agent of the PDA can kill tumor cells under the laser irradiation, its absorption coefficient in the NIR region is low, and this negatively affects the PTT effect. To promote the photothermal conversion efficiency, a prerequisite is to obtain an efficient PTT agent with strong NIR absorption. Polypyrrole (PPy), one of the well-known organic polymers, exhibits a strong and broad NIR absorption band, and allows it to be a favourable PTT agent.21,22 Besides, compared with these conventional inorganic photothermal agents, PPy has outstanding stability, good biocompatibility, and excellent biodegradability, which makes it a desirable platform for preparing multifunctional PTT agents.22,23 Therefore, a combination of UCNPs and PPy integrates both multimodal imaging functions holding infrared thermal imaging, UCL imaging, or CT imaging, and PTT for cancer cells. Herein, a NIR-response multimodal imaging-guided photothermal agent, NaYF4:Yb/Er@PPy core–shell nanoplates, was designed and fabricated. Within a core–shell nanostructure, the core of NaYF4:Yb/Er operates as both CT and UCL contrast agents, as the La (Y, Yb, and Er)-based upconversion material not only shows high performance in X-ray attenuation due to the presence of high Z rare earth elements but also provides fluorescence imaging function;16–18 the shell of the PPy polymer formed by the oxidative polymerization of Fe3+ functions as a photothermal agent for ablation of tumors and an infrared thermal imaging agent. It is interesting that the morphology of the NaYF4:Yb/Er core will change from a disc-like plate to a regular hexagon with the increasing amount of Fe3+. These nanocomposites achieve three-modal imaging guided PTT for cancer, compensating for the deficiencies of individual imaging modalities and satisfying the higher requirements on the efficiency and accuracy for diagnosis and research, which will provide potential for simultaneous diagnostics, therapeutics, and monitoring of tumors.
Experimental section Materials Y2O3, Yb2O3 and Er2O3 were purchased from Ourchem. Oleylamine (OM), 1-octadecene (ODE > 90%) and polyvinyl alcohol
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were purchased from Aladdin Chemistry Co., Ltd. Oleic acid (OA, 90%) was purchased from Tokyo Chemical Industry. Nitrosonium tetrafluoroborate (NOBF4) was purchased from Alfa Aesar. Pyrrole and sodium dodecylbenzene sulfonate were purchased from Sinopharm Chemical Reagent Co., Ltd. TC71cells bearing mice were purchased from Shanghai Tenth People’s Hospital. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Synthesis of the oleic acid-modified NaYF4:Yb/Er nanoplates In a typical experiment, 0.2 mmol of La2O3 (Y : Yb : Er = 80 : 18 : 2 mol%) was first dispersed in 20 mL of diluted HCl upon heating to 80 °C under stirring until dissolved. Then the solution was heated to 160 °C for 1 h, and dried. The solid was dispersed in 2 mL of methanol, then transferred into 50 mL of a flask containing 8 mL of oleic acid and 12 mL of 1-octadecene and the formed solution was heated to 150 °C for 60 min, and then cooled down to 50 °C. Thereafter, 5 mL of methanol solution of NH4F (3.2 mmol) and NaOH (2 mmol) were added and stirred for 60 min at 50 °C. After methanol evaporated, the solution was heated to 300 °C under nitrogen for 1 h and cooled down to room temperature. Later, the resulting product was precipitated by the addition of ethanol, collected by centrifugation, and finally re-dispersed in cyclohexane. Ligand exchange In a typical process, 5 mL of a cyclohexane solution containing 20 mg of oleate-capped NaYF4:Yb/Er nanoplates was mixed with 5 mL of a dichloromethane solution of NOBF4 (20 mg) at room temperature, and then gently stirred for 10 min to yield white precipitates, which were collected by centrifugation at 12 000 rpm for 5 min, and re-dispersed in 10 mL of N,N-dimethylformamide (DMF) to form a transparent solution. Subsequently, 50 mg of PVA was added to the above transparent solution. After stirring for 60 min at room temperature, the PVA-functionalized NaYF4:Yb/Er nanoplates were washed with water several times to remove the excess PVA. The final products were dispersed in distilled water for the following use. Synthesis of NaYF4:Yb/Er@PPy nanoplates 5 mL dispersion of the NaYF4:Yb/Er nanoplates (2 mg mL−1), 10 mg of SDBS and 30 mg of PVA were mixed in 15 mL of water and stirred for 3 h at room temperature. After that, 2 μL of pyrrole was added to the dispersion and stirred overnight. Then 5 mL of FeCl3 solution (FeCl3·6H2O: 10, 20, 40 mg) was added to the dispersion and the polymerization was carried out for 24 h. The nanoplates were precipitated by adding 20 mL of acetone, and collected by centrifugation. Synthesis of PEG-grafted poly(isobutylene-alt-maleic anhydride) (PIMA-PEG) PIMA-PEG was synthesized by referring to the literature.6 In brief, 10 mg of poly(isobutylene-alt-maleic anhydride) (PIMA) and 143 mg of mPEG-NH2 (5 K) were dissolved in 5 mL of dichloromethane, and 6 μL of triethylamine (TEA) and 11 mg
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of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were added. After 24 h of reaction, the solvent was evaporated and the solid product was dissolved in water.
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Modification of NaYF4:Yb/Er@PPy nanoplates NaYF4:Yb/Er@PPy nanoplates were dispersed in water to obtain a clear solution. A solution of PIMA-PEG (2 mg mL−1) was added into the NaYF4:Yb/Er@PPy solution, and then ultrasonicated for 30 min. The final product was purified by centrifugation several times. Characterization Electron microscopy images were acquired on a transmission electron microscope (JEM-2010F). The content of metals released from the NaYF4:Yb/Er nanoplates in the solution was confirmed using an inductively coupled plasma atomic emission spectroscope (ICP-AES, Prodigy). FTIR spectra were recorded by using a Fourier transform infrared spectrometer (Nexus-670). Thermogravimetric analysis was carried out on a USA TA/Q5000IR thermal analyzer under nitrogen ranging from 50 °C to 800 °C at a rate of 20 °C min−1. UV-vis absorbance spectra were recorded using a UV-visible-NIR spectrophotometer operating from 200 to 1100 nm (UV-1902PC, Phoenix). Measurement of the photothermal effect To measure the photothermal effect of NaYF4:Yb/Er@PPy core–shell nanoplates induced by the NIR absorbance, 100 μL aqueous dispersions of the NaYF4:Yb/Er@PPy core–shell nanoplates with different concentrations (0–0.5 mg mL−1) were added into plastic tubes (250 μL), and then irradiated with a 915 nm laser at a power density of 0.5 W cm−2 (a laser intensity of 0.17 W with a spot area of 0.35 cm−2) for 5 min. A thermocouple with an accuracy of ±0.1 °C was inserted into the aqueous dispersion at such a position that the direct irradiation of the laser on the probe was avoided. The temperature was recorded by using an online type thermocouple thermometer every 5 s. To evaluate the photothermal conversion efficiency of NaYF4:Yb/Er@PPy core–shell nanoplates, the temperature of a dispersion of NPs (0.5 mg mL−1) was recorded every 5 s with continuous irradiation with a 915 nm laser at a power density of 0.5 W cm−2 for 5 min. After that, the laser was turned off and the temperature of the dispersion dropped to the initial temperature. Then the photothermal conversion efficiency was calculated by a modified method referring to the report by Roper et al. In vitro cytotoxicity assay In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the presence of 5% CO2, HeLa cells were seeded into a 96-well plate at 1 × 104 cells per well at 37 °C. After incubation for 24 h, the NaYF4:Yb/Er@PPy nanoplates dispersed in a PBS solution were then added into the wells at various concentrations (0, 62.5, 125, 250, 375, and 500 ppm) and incubated
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for another 24 h. 0.1 mL of MTT solution (5 mg mL−1) was added to each well of the microtiter plate and incubated in the CO2 incubator for 4 h. Then the optical density (OD) was measured at 490 nm using a microplate reader. All experiments were independently performed three times. In vitro CT imaging The dispersion of the NaYF4:Yb/Er@PPy nanoplates with different concentrations of La (Y, Yb, Er) (0–6.3 mg mL−1) was detected using a CT imaging system (SOMATOM Sensation Cardiac) with the following operating parameters: thickness, 0.6 mm; field of view, 20 cm; tube voltage, 80 kV; effective mAs, 110. CT values were acquired on the same workstation using the software supplied by the manufacturer. In vivo UCL imaging A modified Maestro in vivo imaging system was employed to image the NaYF4:Yb/Er@PPy nanoplate treated mice using a 980 nm optical fiber coupled laser as the excitation source. The laser power density was ∼0.2 W cm−2 during imaging. An 850 nm short-pass emission filter was used to prevent the interference of excitation light to the CCD camera. The green emission from the NaYF4:Yb/Er@PPy nanoplates was collected at 550 nm. In vivo CT imaging The mice bearing tumors (∼5 × 5 mm) were anesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight. In vivo CT scanning was performed both before/after intratumoral injection of the aqueous dispersion of the NaYF4:Yb/Er@PPy nanoplates (50 μL, La: 4.2 mg mL−1) at 1 h post-injection. All CT scans were performed using the CT system with the parameters similar to those for in vitro experiments. In vivo infrared thermal imaging and photothermal ablation The tumor-bearing mice were randomly allocated into a control group and a treatment group. Firstly, two groups were anaesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight. Then, mice in the treatment group were intratumorally injected with the NaYF4:Yb/ Er@PPy nanoplates dispersed in phosphate-buffered saline (PBS) solution (100 μL, 0.5 mg mL−1). For the control, mice were injected with 100 μL of saline solution. After 0.5 h postinjection, mice from both the groups were simultaneously irradiated with a 915 nm laser at a power density of 0.5 W cm−2 for 10 min. During the laser irradiation, infrared thermal images were captured using an IR camera of a photothermal therapy monitoring system GX-A300 (Shanghai Guixin Corporation). After laser treatment, the mice were sacrificed, tumors were harvested, and embedded in paraffin, and cryosectioned into 4 μm slices using a conventional microtome. Then slides were stained with hematoxylin/eosin. The slices were examined under a Zeiss Axiovert 40 CFL inverted fluorescence microscope, and images were captured using a Zeiss AxioCam MRc5
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digital camera. The experiments were independently performed three times.
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Results and discussion The NaYF4:Yb/Er@PPy core–shell nanoplates were synthesized by combining a thermal decomposition reaction and an in situ chemical oxidative polymerization reaction, as illustrated in Fig. 1. Firstly, the oil-soluble NaYF4:Yb/Er (Y : Yb : Er = 80 : 18 : 2 mol%) disc nanoplates were synthesized by a thermal decomposition reaction (see the Experimental section for details). Subsequently, the long hydrocarbon ligands on the surface of the oil-soluble NaYF4:Yb/Er nanoplates were removed with NOBF4, and then the nanoplates were modified with polyvinyl alcohol (PVA) to get water-solubility. Finally, PPy was coated on the surface of nanoplates to form core–shell nanostructures by oxidative polymerization of Fe3+. Interestingly, when the amount of Fe3+ increased, the shape of the NaYF4:Yb/Er nanoplate core would become a regular hexagon from the disc-like shape. Lastly, to enhance the stability of the nanoplates in physiological solution, the NaYF4:Yb/Er@PPy nanoplates were modified with PIMA-PEG. Fig. 2a shows a representative transmission electron microscopy (TEM) image of the as-obtained oil-soluble NaYF4: Yb/Er disc-like shaped nanoplates. As depicted, the disc-like plates have a uniform size, i.e. a diameter of ∼78 nm and a thickness of ∼25 nm. The phase structure of the NaYF4:Yb/Er nanoplates was confirmed by X-ray diffraction (XRD). As shown in Fig. 2b, it could be well indexed to the hexagonal phase (JCPDS no. 28-1192) of NaYF4. Prior to the PPy coating, the oil-soluble NaYF4:Yb/Er nanoplates have been modified with the PVA in order to endow them with hydrophilic properties and facilitate the PPy coating, the resulting size and shape of the PVA–NaYF4:Yb/Er nanoplates remained unchanged, while the modification process involved a change in the color of the suspension from yellow to white (Fig. S1†), which could be observed visually during the procedure of surface modification. Fourier transform infrared (FTIR) spectroscopy is used to prove the ligand exchange. At first, the most obvious absorption peaks located at 2923 and 2855 cm−1 are the characteristic stretching absorptions of –CH2– and –CH3 of oil acid, and the peak at 1709 cm−1 is induced by
Fig. 1 Schematic illustration of the synthetic route of NaYF4:Yb/Er@PPy core–shell nanoplates.
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Fig. 2 (a) TEM image of the NaYF4:Yb/Er nanoplates. (b) XRD patterns of as-prepared NaYF4:Yb/Er nanoplates (black line) and the standard NaYF4:Yb/Er powders (red bars) from JCPDS card (no. 28-1192). (c–e) TEM images of NaYF4:Yb/Er@PPy core–shell nanoplates with the disc core, irregular core and hexagon core, respectively. (f ) STEM image and EDS elemental maps of an individual NaYF4:Yb/Er@PPy nanoplate.
carbonyl (as shown in black line of Fig. S2†).24 After modification, the characteristic absorptions of oil acid disappeared, and the main peaks arose at 3410 and 1095 cm−1, which belong to –OH and C–O of PVA, respectively (the blue line of Fig. S2†). Fig. 2c–e show the TEM images of the final NaYF4:Yb/Er@PPy nanoplates after the PVA modification. The size of the nanoplates can be measured to be ∼100 nm, which is consistent with dynamic light scattering (DLS) diameter distribution shown in Fig. S3.† Viewing from the nanoplates standing or lying on the Cu grid (C film support) during the TEM sample processing, it demonstrates that PPy was present around whole individual NaYF4:Yb/Er nanoplates, forming a core–shell structure. Elemental maps in Fig. 2f recorded by using an energy dispersive spectrometer (EDS) display that Na, Y, F, Yb, Er, and N elements existed in the nanoplates and indicate that all these elements are uniformly distributed throughout the nanoplates. The FTIR spectrum (red line of Fig. S2†) of the NaYF4: Yb/Er@PPy nanoplates displays absorption peaks at 1630 and 1400 cm−1, which are induced by CvC and C–N on the pyrrole ring, respectively.25 The thermal gravity analysis (TGA) curve in Fig. S4† shows that the weight of the NaYF4:Yb/Er@PPy nanoplates began to reduce at about 200 °C under a nitrogen atmosphere and almost stopped reducing at above 500 °C. It is consistent with the TGA data of PPy, which is caused by the degradation of PPy (the black line in Fig. S4†). This further
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confirms the existence of the PPy and the content of the PPy in the NaYF4:Yb/Er@PPy nanoplates of ∼10% (wt%). During the polymerization process of pyrrole, it was interesting to find that if the concentration (or amount) of Fe3+ was increased, while the amount of pyrrole was unchanged, the NaYF4:Yb/Er core within the NaYF4:Yb/Er@PPy composite nanoplates would change from the disc plate to a hexagonal plate. Fig. 2c shows the NaYF4:Yb/Er@PPy nanoplates formed with a concentration of 0.41 mg per mL of Fe3+ (5 mL, the quality ratio of FeCl3·6H2O and NaYF4:Yb/Er is 1 : 1) added, and the NaYF4:Yb/Er core kept disc-shape unchanged and the PPy thickness was ∼7.5 nm. When the concentration of Fe3+ was increased to 0.82 mg mL−1, some of the edges of the disc cores were etched to irregular, as shown in Fig. 2d. A concentration of 1.65 mg per mL of Fe3+ (5 mL) added would further change the cores into regular hexagonal plates. At the same time, the thickness of the PPy shell also increases to 12.5 nm, as demonstrated in Fig. 2e. However, with the concentration of Fe3+ further increased, the nanoplates would finally become ear-like shaped (Fig. S5†). The phenomenon may be due to the etching of an excess of Fe3+. When the amount of Fe3+ is lower (the quality ratio of FeCl3·6H2O and NaYF4:Yb/Er is less than 1 : 1), Fe3+ is used as an oxidizing agent to oxidize the pyrrole monomer into PPy coating on the NaYF4:Yb/Er nanoplates’ surface. When the amount of Fe3+ is higher, apart from the usage as an oxidizing agent, the excess Fe3+ can reach the surface of the NaYF4:Yb/Er nanoplates through the outside layer of the PPy to carry out the etching process. The discshaped nanoplates started to change from their edges rather than from their facets and then transformed into hexagonalshaped plates. This may be an energetically favored route considering the fact that those atoms located on the edges have lower coordination and therefore possess higher chemical reactivities.26 Here, Fe3+ is used as an etchant to change the morphology of the NaYF4:Yb/Er core.27 The optical properties of the obtained NaYF4:Yb/Er@PPy core–shell nanoplates were then studied. Owing to the doping of Yb and Er, the NaYF4:Yb/Er nanoplates exhibit strong fluorescence.16 As revealed in Fig. 3a, the luminescence spectrum of the dispersion of the NaYF4:Yb/Er nanoplates displays three characteristic emission peaks located at 527 nm (2H11/2 → 4I15/2), 546 nm (4S3/2 → 4I15/2), and 660 nm (4F9/2 → 4I15/2) under an excitation at 980 nm.17 As the emission at 546 nm is the strongest, the UCL emission color of NaYF4:Yb/Er was almost green, as shown in the inserted image. After the PPy shell was coated, the fluorescence is partly quenched, as demonstrated by the fluorescence spectra and photographs. However, the disc-shaped NaYF4:Yb/Er@PPy nanoplates still show strong upconversion fluorescence, due to a thinner PPy coating compared with that of the hexagonal-shaped NaYF4:Yb/Er@PPy nanoplates, and thus could act as a contrast agent for UCL imaging. Fig. 3b demonstrates the UV-vis absorbance spectrum for the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates. It can be seen that the aqueous dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates exhibits a gradually
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Fig. 3 (a) The fluorescence spectra of the aqueous dispersions of the NaYF4:Yb/Er nanoplates and NaYF4:Yb/Er@PPy core–shell nanoplates, respectively. Insert: the fluorescence photographs of NaYF4:Yb/Er (left) and NaYF4:Yb/Er@PPy (right) dispersions when excited with a 980 nm laser. (b) UV-vis absorbance spectrum for the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates. (c) Temperature elevation of water and the dispersion of the NaYF4:Yb/Er@PPy nanoplates with different concentrations over a period of 5 min under exposure to NIR light (915 nm, 0.5 W cm−2) measured every 5 s. (d) Temperature elevation of the dispersions of the NaYF4:Yb/Er@PPy nanoplates with the increasing concentration.
increasing absorption from 700 to 1100 nm because of the strong NIR absorbance of PPy (Fig. S6†).21,22 This means the potential photothermal conversion effect of the NaYF4:Yb/ Er@PPy core–shell nanoplates upon laser irradiation. Fig. 3c shows the photothermal conversion performance of the NaYF4:Yb/Er@PPy core–shell nanoplates under the irradiation with a 915 nm NIR laser at a power density of 0.5 W cm−2. The temperature of the aqueous dispersion of the nanoplates of different concentrations (i.e., 0.1, 0.2, 0.3, 0.4 and 0.5 mg mL−1) can increase by 15.0, 20.3, 24.1, 28.4 and 29.3 °C, respectively, in the period of 300 s. This corresponds well to the absorbance of NaYF4:Yb/Er@PPy nanoplates, which showed a linear relationship at 915 nm versus concentrations (Fig. S7†). As a control experiment, pure water only increased by less than 5 °C. These demonstrate that the NaYF4:Yb/ Er@PPy core–shell nanoplates can rapidly and efficiently convert the 915 nm laser energy into thermal energy, which has the potential to be a promising PTT agent. In comparison, the dispersion (0.5 mg mL−1) of the hexagonal-shaped NaYF4: Yb/Er@PPy nanoplates increased by 30.3 °C under the same conditions, it has no much difference from the disc-shaped NaYF4:Yb/Er@PPy nanoplates, as shown in Fig. S8.† So, we choose the disc-shaped NaYF4:Yb/Er@PPy nanoplates for further biological test. We measured the photothermal conversion efficiency of the NaYF4:Yb/Er@PPy nanoplates by a modified method referring to the report by Roper et al.28 The temperature change of the NaYF4:Yb/Er@PPy dispersion was recorded as a function of
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time under continuous irradiation with a 915 nm laser for 5 min. Then the laser was shut off, and the temperature decrease of the aqueous dispersion was monitored to determine the rate of the heat transfer from the dispersion system to the environment. Fig. 4a shows temperature elevation of the dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates and water. Following Roper’s report and our previous work,7,28 the conversion efficiency of the dispersion of the NaYF4:Yb/ Er@PPy nanoplates can be calculated to be 44.7%. This means the favourable character of NaYF4:Yb/Er@PPy nanoplates to convert laser energy to heat. In order to investigate the NIR photothermal stability of the NaYF4:Yb/Er@PPy nanoplates’ dispersion, five LASER ON/OFF cycles of the NIR laser irradiation were examined. A solution (0.5 mg mL−1) of the NaYF4:Yb/Er@PPy core–shell nanoplates was irradiated with the NIR laser for 5 min (LASER ON), followed by naturally cooling to room temperature with the laser off. This process was repeated five times (Fig. 4b). The temperature elevation of 29 °C was achieved at the first cycle and no significant decrease from this temperature elevation was observed later. Meanwhile, the TEM images of the NaYF4:Yb/Er@PPy nanoplates after irradiation for 25 min showed no obvious change compared with that before the irradiation (Fig. S9†). These results indicate that the NaYF4:Yb/Er@PPy nanoplates exhibit excellent photothermal stability after a long period of the NIR laser irradiation. CT is an imaging method for detecting various degrees of attenuation of X-rays in different tissues when they come through the whole body. It is an inspection method with noninvasion and high-contrast resolution.9 La (Y, Yb, Er)-based upconversion materials were reported to possess high X-ray attenuation owing to their large atomic number.18 To verify the potential of the NaYF4:Yb/Er@PPy nanoplates as a contrast agent, we performed in vitro CT imaging test. Fig. 5a presents the CT images of iopromide (a kind of clinical contrast agent) and the aqueous dispersions of the NaYF4:Yb/Er@PPy core– shell nanoplates with different I or La concentrations. We can clearly observe positive contrast enhancement of the CT signals with the concentration increasing (i.e. 0, 0.525, 1.05, 2.1, 4.2, 6.3 mg mL−1). To quantitatively evaluate the X-ray
Fig. 4 (a) Temperature elevation of the dispersion of the disc-shaped NaYF4:Yb/Er@PPy nanoplates (the black line) and water (the red line), respectively, in which the irradiation (915 nm, 0.5 W cm−2) lasted for 5 min, and then the laser was shut off. (b) Temperature elevation of the dispersion of the NaYF4:Yb/Er@PPy nanoplates over 5 LASER ON/OFF cycles of the NIR laser irradiation.
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Fig. 5 (a) In vitro CT images of iopromide and the dispersions of the NaYF4:Yb/Er@PPy nanoplates with different I or La concentrations. (b) CT value (HU) of iopromide (black line) and the NaYF4:Yb/Er@PPy nanoplates (red line) as a function of the concentration of I or La. (c) CT images of mice before and after intratumoral injection of the dispersion of the NaYF4:Yb/Er@PPy nanoplates. The position of tumors is marked by dotted circles.
attenuation capability of these nanoplates, we measured the CT values at different concentrations. As shown in Fig. 5b, the CT value increased linearly with the concentration of La. The slope of the linear equation for NaYF4:Yb/Er@PPy core–shell nanoplates is about 53.6 HU L g−1, a little higher than that of iopromide, indicating a high contrast efficiency of the NaYF4: Yb/Er@PPy nanoplates. This suggests that the NaYF4:Yb/ Er@PPy core–shell nanoplates can be used as a CT contrast agent. A high biocompatibility is required as a precondition for further applications in cancer treatment. Herein, the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates with various concentrations (0.0625, 0.125, 0.250, 0.375 and 0.500 mg mL−1) was incubated with HeLa cells for 24 h and then the cell viabilities were tested by using the MTT assay. The viabilities of cancer cells were used to assess the biocompatibility in vitro. No obvious adverse effect on cell viabilities was observed, as shown in Fig. S10,† suggesting a low cytotoxicity of the NaYF4:Yb/Er@PPy core–shell nanoplates. Encouraged by the negligible cytotoxicity and favorable performance of the nanoplate in vitro results, we further investigated the feasibility of the CT imaging in vivo by injecting the NaYF4:Yb/Er@PPy dispersion in tumor-bearing mouse. Fig. 5c shows in vivo tumor CT images before and after the intratumoral injection with the NaYF4:Yb/Er@PPy dispersion (50 μL, La: 4.2 mg mL−1). It shows that the corresponding injection site exhibited a much brighter contrast after injection than before. This proves the potential of the NaYF4:Yb/Er@PPy nanoplates as a CT contrast agent. Then UCL imaging was performed using a modified Maestro in vivo fluorescence imaging system with a 980 nm optical fiber coupled laser as the excitation source. Herein, the mice bearing TC71 tumors were intratumorally injected with the NaYF4:Yb/Er@PPy dispersion
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(50 μL, 1 mg mL−1). A significant UCL signal of the tumor region was observed clearly with almost no autofluorescence elsewhere, as shown in Fig. 6, while the blank group showed no UCL signal. The experimental result indicates the ability of the UCNPs to penetrate tissue and the function of NaYF4:Yb/
Fig. 6 UCL images of the blank control and experiment group with intratumoral injection of the NaYF4:Yb/Er@PPy dispersion.
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Er@PPy nanoplates as a promising probe for in vivo UCL imaging. Finally, we designed animal experiments to demonstrate the photothermal effect of the NaYF4:Yb/Er@PPy core–shell nanoplates. Two groups of mice bearing TC71 tumors were used in our experiment. After being anaesthetized with trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg per kg body weight, one group was injected with the NaYF4:Yb/ Er@PPy dispersion (0.5 mg mL−1) of PBS into the tumors, and the other was injected with PBS. Then both the mice were simultaneously exposed to the 915 nm laser (0.5 W cm−2) for 10 min. We used an IR camera to capture the full-body infrared thermal images and monitor the temperature change of the irradiated area. The photograph of the experimental setup is shown in Fig. S11.† In the infrared thermal image in Fig. 7a, there is no distinction between regions 11 and 12 (regions 11 and 12 are from the treatment group and the control group, respectively.). After a period of laser irradiation, a strong image contrast was observed between the tumor regions (Fig. 7b), exhibiting different temperature changes. As seen in Fig. 7c, the tumor region of the mice in the treatment group heated up very quickly with the temperature reaching 47.9 °C from 25.9 °C. As for the control mice, the temperature of the irradiated area only increased by about 3 °C under the same condition. These confirm that the platform can be an infrared thermal imaging contrast agent. After the laser irradiation, the hematoxylin and eosin stained histological examination was performed, as shown in Fig. 7d, e and S12.† The treatment
Fig. 7 (a, b) Infrared thermal images of two tumor-bearing mice after injecting with the aqueous dispersion of the NaYF4:Yb/Er@PPy core–shell nanoplates (the left mouse, indicated region 11) or PBS (the right mouse, indicated region 12) via the intratumoral injection respectively, irradiated with a 915 nm laser (0.5 W cm−2) at a time point of 0 min and 5 min. (c) The temperature profiles in regions 11 and 12 as a function of the irradiation time. (d, e) The representative hematoxylin and eosin stained histological images of ex vivo tumor sections treated by irradiation over a period of 10 min injected with: PBS only and an aqueous dispersion of the NaYF4:Yb/Er@PPy nanoplates, respectively.
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(Fig. 7e) shows severe cellular damage, such as shrinkage of the malignant cells, nuclear condensation, fragmentation and lysis compared with mice from the control group (Fig. 7d). It suggests that the higher temperature of 47.9 °C can kill cancer cells in expectation, and the NaYF4:Yb/Er@PPy core–shell nanoplates still have an excellent photothermal effect in vivo. Simultaneously, the NaYF4:Yb/Er@PPy nanoplates allow realtime monitoring of the temperature dynamics of a PTT process by infrared thermal imaging.
Conclusion In summary, an imaging-guided multimodal platform of the NaYF4:Yb/Er@PPy core–shell nanoplates was successfully designed and prepared for PTT of cancers. The shape of the NaYF4:Yb/Er core could be adjusted by the amount of Fe3+ in the polymerization process of pyrrole, which is an interesting phenomenon. The NaYF4:Yb/Er@PPy core–shell nanoplates can be an efficient photothermal theragnosis platform for in vitro and in vivo photothermal ablation of cancer cells guided by CT imaging, UCL imaging and infrared thermal imaging, simultaneously. Moreover, owing to the delocalized π-electrons of the PPy shell, it is believed that aromatic molecules, such as DOX, may be bound on the shell for multifunctional applications. Overall, our work demonstrates a straightforward route to develop a multifunctional platform for imagingguided therapy of cancer, and encourages further exploration of imaging-guided cancer therapy for better properties and enhanced efficacy.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No. 51472049, 51302035, and 21171035), the National 863 Program of China (Grant No. 2013AA031903), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), Ph.D. Programs Foundation of Ministry of Education of China (Grant No. 20110075110008 and 20130075120001), the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Shanghai Leading Academic Discipline Project (Grant No. B603), and the Program of Introducing Talents of Discipline to Universities (Grant No. 111-2-04).
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