Oxygen Vacancy Enables Markedly Enhanced Magnetic Resonance

Mar 21, 2017 - Oxygen Vacancy Enables Markedly Enhanced Magnetic Resonance Imaging-Guided Photothermal Therapy of a Gd3+-Doped Contrast Agent...
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Oxygen Vacancy Enables Markedly Enhanced Magnetic Resonance Imaging-Guided Photothermal Therapy of a Gd3+-Doped Contrast Agent Dalong Ni,†,‡,§ Jiawen Zhang,⊥ Jing Wang,⊥ Ping Hu,† Yingying Jin,⊥ Zhongmin Tang,†,‡ Zhenwei Yao,⊥ Wenbo Bu,*,†,§ and Jianlin Shi*,† †

State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China ‡ University of Chinese Academy of Science, Beijing 100049, China § Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China ⊥ Department of Radiology, Huashan Hospital, Fudan University, Shanghai 200040, China S Supporting Information *

ABSTRACT: Gd3+-based contrast agents (CAs) are the most prevailing and widely used for enhanced magnetic resonance imaging (MRI). Numbers of approaches have been developed to regulate the key parameters in order to obtain high-relaxivity CAs, according to the classic Solomon−Bloembergen−Morgen theory. Herein, a method of controlling oxygen vacancies in inorganic nanosized CAs has been developed for largely accelerated proton relaxation to obtain a high r1 value. Such a strategy is verified on oxygen-deficient PEG-NaxGdWO3 nanorods, which exhibit a remarkable r1 value up to 80 mM−1 s−1 (at 0.7 T) and a high r1 value of 32.1 mM−1 s−1 on a clinical 3.0 T scanner, offering an excellent blood pool MRI performance at a low dose. Meanwhile, free electrons and/or oxygen-vacancy-induced small polarons can endow PEG-NaxGdWO3 nanorods with significant photothermal conversion for MRI-guided photothermal therapy. KEYWORDS: magnetic resonance imaging, oxygen vacancy, tungsten bronzes, photothermal therapy, theranostic agent Development of Gd3+-based CAs with a high r1 value will result in greatly improved diagnosis accuracy. According to the classic Solomon−Bloembergen−Morgen (SBM) theory,6−8 the strategies for enhancing relaxivity of Gd3+-based CAs mainly include prolonging the rotational correlation time (τr), optimizing the water residence time (τm), and increasing the number of bound water molecules (q). The relationship between τr and τm can be described by eq 2:9,10

M

agnetic resonance imaging (MRI) has evolved into routinely used tools in noninvasive diagnostic imaging due to its high spatiotemporal resolution, excellent soft tissue contrast, and nonionizing radiation. However, MRI is impaired by relatively low sensitivity, and thus, contrast agents (CAs) are needed to elevate the water proton relaxation rate for differentiating the anatomical changes of target areas.1−4 The most commonly used CAs are gadolinium (Gd3+) chelates by shortening longitudinal relaxation time (T1) of surrounding water protons. Relaxivity (r1) is used to evaluate CA’s efficiency, which is defined as the slope of a plot of 1/T1 versus Gd3+ concentration ([Gd]) as described by eq 1:5 1/T1 = (1/T1)sovlent + r1[Gd] © 2017 American Chemical Society

1/τc = 1/T1e + 1/τr + 1/τm

(2)

where τc is the correlation time parameter and T1e is the electronic spin relaxation time. Since T1e is hard to be predicted Received: February 23, 2017 Accepted: March 21, 2017 Published: March 21, 2017

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Figure 1. (a) Schematic diagram of PEG-NaxGdWO3 nanorods for MRI-guided photothermal therapy. (b) TEM image of PEG-NaxGdWO3 nanorods dispersed in water; high-resolution TEM image of a nanorod is shown in the inset. (c) SEM image of PEG-NaxGdWO3 nanorods. (d) Element mappings of PEG-NaxGdWO3 nanorods.

water proton relaxation by bonding oxygen atoms of water molecules to Gd3+ ions, which may, as we believe, substantially enhance the r1 value by changing the critical parameter in the SBM theory mentioned above. Herein, by simple regulation of oxygen vacancy, we envision that it is possible to achieve enhanced MR imaging and concurrently PTT treatment of tumors under the guidance of imaging, which is of great significance but has not been reported yet. It has been discovered that oxygen-deficient tungsten oxides (e.g., WO 2.83 , WO 2.9 , W 18 O 49 , etc.) 24−29 and tungsten bronzes30−32 are promising photothermal agents because of their strong NIR photothermal conversion. Their critical feature of tunable oxygen vacancy concentrations triggered our interest to develop Gd3+-doped tungsten bronzes for investigating the proton relaxation behavior and photothermal conversion properties. The oxygen-deficient PEGylated Gd3+doped NaxWO3 (PEG-NaxGdWO3) nanorods were synthesized to explore its probability of acting as an MRI-guided photothermal agent. Our results verified that the presence of oxygen vacancies could lead to significantly enhanced proton relaxation rates of the PEG-NaxGdWO3 nanorods, which has a high r1 value of 32.1 mM−1 s−1 on a clinical 3.0 T scanner. Both excellent blood pool and tumor MR imaging outcomes have

and affected experimentally, approaches for relaxation optimization commonly focus on regulation of τr and τm. Extensive fundamental research has been conducted to regulate these parameters for acquiring high-relaxivity nanosized CAs by controlling the nanoparticle’s size,11−13 changing their shape,10,14 or tuning surface modification (e.g., ligand group, Gd3+ shell, silica, etc.).15−19 However, these methods are only confined to the mediation of nanoparticles’ morphological features, and the r1 enhancement is limited at a clinical 3.0 T. For example, the NaGdF4-shell-coated strategy has achieved an r1 value of 6.18 mM−1 s−1,17 followed by a nearly 2-fold increase of the r1 value (12.38 mM−1 s−1) via further coating of the silica shell.18 Therefore, exploration of strategies on accelerating water proton relaxation is very valuable in order to obtain a high r1 value at a clinical 3.0 T.20,21 The oxygen vacancies’ own natural affinity to oxygen atoms are widely applied in designing heterogeneous catalysts especially for the catalytic redox reactions.22,23 Meanwhile, the free electrons and/or oxygen-vacancy-induced small polarons are responsible for the strong near-infrared (NIR) absorption and conversion to perform effective tumor photothermal therapy (PTT).24−26 However, so far, no attention has been paid to the possible effects of surface oxygen vacancies on 4257

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Figure 2. (a) UV−vis−NIR absorption spectrum of an aqueous dispersion of PEG-NaxGdWO3 nanorods (inset: photo of the aqueous dispersion). (b) Temperature evolution curves of pure water and aqueous dispersions containing PEG-NaxGdWO3 nanorods at varied W concentrations under continuous irradiation of a 980 nm laser for 5 min. (c) UV−vis−NIR absorption spectra of the PEG-NaxGdWO3 sample oxidized by H2O2 for multiple time periods. (d) X-ray diffraction patterns of PEG-NaxGdWO3 nanorods before and after oxidation by H2O2. (e) Plot of 1/T1 versus Gd3+ concentration for PEG-NaxGdWO3 nanorods oxidized with H2O2 for multiple time periods. (f) T1-weighted MR (3.0 T) phantom images obtained from PEG-NaxGdWO3 nanorods at varied Gd3+ concentrations.

existence of all expected essential chemical elements (Na, Gd, W, and O), which is further verified by element mappings (Figure 1d). As shown in Figure S4, the PEG-NaxGdWO3 nanorods are negatively charged (−8.46 mV) and their hydrated size is ∼171.4 nm with a low polydispersity index of 0.137 (Figure S5). Moreover, the PEG-Na x GdWO 3 nanorods present excellent stability under a range of salt, acid/alkali, and human serum conditions (Figures S6 and S7), which favors their applications in vivo. In Vitro Photothermal Evaluation. The aqueous dispersion of PEG-NaxGdWO3 nanorods exhibits a bright blue color (the inset of Figure 2a), and the UV−vis−NIR spectroscopy shows that the absorption becomes remarkably intensified from 510 to 1200 nm (Figure 2a), which is similar to the behavior of previously reported tungsten oxides (WO3−x).25 Under NIR (980 nm) laser irradiation, the temperature of the nanorods dispersed in aqueous solution increases rapidly from 25 °C to about 42 °C at a low laser density in 5 min, whereas pure water shows limited temperature increase (Figure 2b), demonstrating that the PEG-NaxGdWO3 nanorods can efficiently convert NIR laser energy into heat. Several physical mechanisms have been deemed to be responsible for NIR absorption of using these kinds of nanomaterials, including

been achieved at low Gd doses. In the meantime, the oxygen vacancies endow these nanorods with strong NIR photothermal conversion, enabling the efficient in vivo MRI-guided tumor photothermal therapy.

RESULTS AND DISCUSSION Synthesis and Characterization of PEG-NaxGdWO3 Nanoprobes. The Gd 3 + -doped tungsten bronzes (NaxGdWO3) were prepared by using a facile thermal decomposition approach with numerous modifications,33 along with poly(ethylene glycol)-thiol (PEG-SH) to improve the biocompatibility of the nanocrystals (Figure 1a).34 As demonstrated in Figure 1b,c, both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images indicate that PEG-NaxGdWO3 nanocrystals dispersed in water are rod-like in shape and are about 140 nm in length and 80 nm in width. By carefully controlling the nucleation and growth of the existing nuclei, the size and shape of inorganic nanoparticles can be well-controlled,35 leading to the size- and shape-dependent uptakes by cells,36 and the rod-like shape is believed to be more easily taken up by the cells.37 The energydispersive X-ray (EDX) spectrum (Figure S1) demonstrates the 4258

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Figure 3. Schematic diagram of oxygen vacancy’s affinity to the oxygen atom of a water molecule to affect the interaction between Gd3+ ions and water molecules.

localized surface plasmon resonances,26 small polaron absorption,38 and intervalence charge transfer.39,40 In fact, based on the above three models, the free electrons and/or oxygenvacancy-induced small polarons are considered to be closely related to the NIR absorption.25 The concentration of oxygen vacancies was adjusted by adding the hydrogen peroxide (H2O2).24 As shown in Figure 2c, the NIR absorption intensity decreases at a prolonged H2O2 treatment duration and no NIR absorption tail is present after it is oxidized with H2O2 for 3 h (marked as 4# W-H2O2-3h). The spectral difference indicates that the oxygen vacancy concentration on the surface of the nanorods is largely reduced after oxidization by H2O2, which is caused by the valence increase of partial W5+ to W6+.24 The electron spin resonance (ESR) technique was further employed to monitor the concentration change of oxygen vacancy. As shown in Figure S8, the PEGNaxGdWO3 before oxidation presents a strong response, but the signal weakens after oxidation at the g value of 2.03,41 indicating the decreased oxygen vacancy concentration at extended oxidation time. The XRD patterns of PEGNaxGdWO3 remain almost unchanged before and after oxidization (Figure 2d), demonstrating the entire crystalline structure of the nanorods by oxidization. In addition, no leakage of Gd3+ from PEG-NaxGdWO3 nanorods during H2O2 oxidation was found, as detected using a previously reported method.42 In Vitro MR Imaging. Then, a series of samples with various oxygen vacancy concentrations were measured with r1 values on a 3.0 T clinical MRI scanner. As shown in Figure 2f, the aqueous samples containing higher concentrations of PEGNaxGdWO3 nanorods appear brighter in the T1-weighted MR images. For PEG-NaxWO3 without Gd3+ doping, no MRI contrast effects were found even at elevated concentrations of tungsten ions (Figure S9), indicating that tungsten ions are not responsible for enhanced MRI contrast, and the high MRI performance of PEG-NaxGdWO3 nanorods can be solely attributed to the doped Gd3+ ions. The r1 value of PEGNaxGdWO3 nanorods has been calculated to be 32.1 mM−1 s−1, which is a high value among the reported relaxivities of nanosized Gd3+-based CAs (e.g., the highest r1 value of 8.93 mM−1 s−1 for NaGdF4 NPs and 9.9 mM−1 s−1 for Gd2O3 NPs) at a clinical field strength of 3.0 T,43−47 and is 8-fold larger than that of commercial Gd3+ chelates (r1 = 3.8 mM−1 s−1). Accordingly, the r1 value decreases with the reduced oxygen

vacancy concentration (Figure 2e). Our results verify that the oxygen vacancy will lead to efficient enhancement of MR relaxivity of PEG-NaxGdWO3 nanorods. According to the SBM theory, prolonging τr to significantly improve r1 can be realized by immobilizing Gd3+ to a nanoparticle. Another strategy to obtain a high r1 value focuses on the rapid and efficient water exchange with the Gd3+ (τm) due to the relatively slow tumbling rates of nanoparticulate CAs. The Gd3+ complex formed or conjugated nanoparticles can be well-fitted using the traditional SBM theory due to the excellent work from Meade’s group.10 However, for those CAs that have Gd3+ doped into the crystal lattice of the nanoparticles (Gd3+-in-lattice NPs), it is totally different from the Gd3+-complex-conjugated NPs, so the SBM theory may need major amendments. For example, the q of Gd3+-in-lattice NPs may not have a particular value because of the various situations of thousands of Gd3+ ions in the matrix of one nanoparticle.11 The q distribution is defined here to be a more accurate description of the number of bound water molecules in a nanoparticle. The r1 increment may not originate from τr changes because the TEM and dynamic light scattering showed that the morphology and size of PEG-NaxGdWO3 had not been changed after H2O2 oxidation (Figure S10). Our previous research has validated that the τm can only be changed by a dense SiO2 shell coating for Gd3+-in-lattice NPs.18 Therefore, the r1 value appears to increase by changes of q distributions either in second or outer sphere effects, as shown by the nuclear magnetic relaxation dispersion (NMRD) profiles (Figure S11). Remarkably, the PEG-NaxGdWO3 nanorods exhibit a remarkable r1 value up to ∼80 mM−1 s−1 at 0.7 T. Unfortunately, it is hard to measure the q and τm values alone for fitting the NMRD using the traditional SBM theory. Herein, one reasonable explanation is proposed that the high r1 of PEGNaxGdWO3 nanorods may be attributed to the oxygen vacancy’s natural affinitive binding to oxygen atoms in water molecules bonded to Gd3+ ions (Figure 3), thus resulting in increased q distributions. At the much lowered oxygen vacancy concentrations, as indicated by decreased ESR signal (g = 2.03) (Figure S9), the above interaction is weakened, leading to the decreased relaxivity. In Vivo MR Imaging. Before using PEG-NaxGdWO3 nanorods for in vivo imaging and therapy, we first evaluated their cytotoxicity by the standard MTT assay. It is found that PEG-NaxGdWO3 nanorods exhibit negligible toxicity to several 4259

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Figure 4. Magnetic resonance angiographic images of normal SD rats immediately after injection of PEG-NaxGdWO3 nanorods (a) or Magnevist (b) at a rather low concentration of 2.5 mg Gd3+/kg. (c) In vivo T1-weighted MR images of 4T1-tumor-bearing mice before and after I.T. injection of PEG-NaxGdWO3 nanorods. (d) In vivo MRI signal enhancement of tumors obtained from mice before and 1 h after I.V. injection or immediate I.T. injection of PEG-NaxGdWO3 nanorods.

Figure 5. (a) In vivo photothermal images of mice before and after I.T. injection with PEG-NaxWGdO3 nanorods under continuous 980 nm laser irradiation for different durations. (b) In vivo temperature of tumors obtained from mice before and 1 h after I.V. injection and immediately after I.T. injection of PEG-NaxGdWO3 nanorods under continuous 980 nm laser irradiation for 5 min (n = 4, mean ± SD, *p < 0.05, **p < 0.01, versus control). (c) Tumor growth profiles of 4T1 tumors after different treatments (for each group, n = 6, mean ± SD, ***p < 0.001, versus control). (d) Representative photos of 4T1-tumor-bearing mice on day 14 after different treatments (left) and H&E staining of tumor sections after various treatments (right). Scale bar: 50 μm.

rods as a PTT agent for in vitro cancer cell ablation underwent 980 nm laser irradiation. As shown in Figure S13, the relative viabilities of the cells decrease markedly at an elevated power

types of cells, which is consistent with previous literature that PEGylation can significantly enhance the biocompatibility of nanomaterials (Figure S12).48,49 Next, PEG-NaxGdWO3 nano4260

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observed in all groups (Figure S18), indicating insignificant in vivo side effects of the nanorods. The H&E-stained images of different organs (heart, liver, spleen, lung, and kidney) showed no noticeable organ damage or inflammatory lesions in 30 days (Figure S19), suggesting the low toxicity of PEG-NaxGdWO3 nanorods.

density of the incident laser or increased concentrations. In contrast, cells not incubated with the nanorods remain alive even after laser exposure at the highest power density. These cells were then stained by trypan blue to differentiate dead cells from live ones (Figure S14), the result of which was consistent with MTT results. One serious shortcoming of clinical Gd3+ chelates is their relatively low proton relaxation efficiency, which means that high doses are necessary (mM for Gd3+ chelates).10,16 The high relaxivity of PEG-NaxGdWO3 nanorods greatly favors overcoming the above obstacle by achieving enhanced imaging performance at lowered doses. We first explored their application in magnetic resonance angiography (MRA) imaging because MRA plays an important role in clinics for detecting atherosclerotic plaque, thrombosis, and myocardial infarction.50−52 As shown in Figure 4a, even at a rather low injection dose (2.5 mg Gd3+/kg), an excellent blood pool MR image is obtained after the injection of PEG-NaxGdWO3 nanorods, with the jugular vein and carotid artery being clearly delineated. The resolution of the image is much higher than that of the Magnevist-injected group, which fails to deliver clear images to reveal any vessels (Figure 4b). The in vivo half-life and biodistribution of PEG-NaxGdWO3 nanorods were also measured. As shown in Figure S16a, the blood circulation half-life of PEG-NaxGdWO3 was calculated to be 0.82 h. The biodistribution of PEG-NaxGdWO3 in main organs and tumor was measured, which showed that 2% of injected nanoprobes were accumulated in the tumor through the enhanced permeability and retention (EPR) effect after 1 h injection (Figure S16b). Similar to other nanoparticles, most of the PEGNaxGdWO3 nanorods would be captured by reticuloendothelial system (RES) such as liver and spleen and would be cleared via the hepatobiliary route within a long time period. Escaping from RES organs with enhanced tumor uptake and rapid renal clearance can be realized by decreasing the size of nanomaterials and protecting them with a natural biomolecule such as glutathione.53,54 Then, in vivo tumor MR imaging was also performed by administering the PEG-NaxGdWO3 nanorods via an intravenous (I.V.) or intratumoral (I.T.) injection manner into the 4T1-tumor-bearing mice. Importantly, the MR images demonstrate the dramatic brightening effect in the tumor area at a low Gd3+ dose (Figure 4c,d), revealing the highperformance of T1-weighted MR imaging of PEG-NaxGdWO3 nanorods. In Vivo Therapeutic Efficacy Evaluation for Tumors. The strong photothermal conversion effect of the nanorods encouraged us to apply it for in vivo tumor photothermal therapy. As shown in Figure 5a, the temperature of the tumor area (I.T. injection) rapidly increases to above 45 °C in 30 s and to 57 °C in 5 min under laser irradiation, while the control group shows a slight temperature increase to about 36 °C. Figure 5b shows that both administration manners of I.V. and I.T. injections result in increased surface temperatures of the tumors. With 980 nm laser irradiation, the tumor growth is substantially inhibited by I.V. administration and is completely eliminated by I.T. administration without later recurrence during a prolonged period up to 95 days, whereas the control groups show rapid tumor growth (Figure 5c). Meanwhile, the above tumor growth results were further confirmed by examining the histological changes of tumor tissues by the hematoxylin and eosin (H&E) staining, where treatment groups showed the most tumor damage (Figure 5d). In 16 days, neither abnormal behavior nor significant weight loss was

CONCLUSION In summary, we present here the paradigm of oxygen-vacancyenhanced relaxivity of a Gd3+-doped tungsten bronze (PEGNaxGdWO3) nanorod, which exhibits high-performance MR imaging with remarkable r1 value up to 80 mM−1 s−1 at 0.7 T. The r1 value at clinical field strength of 3.0 T reaches 32.1 mM−1 s−1, which is much higher than those of previously reported nanosized Gd3+-based CAs. Excellent blood pool MR images have been achieved at rather low doses using this highperformance CA. The oxygen vacancies are deemed to be responsible for both the high MR relaxivity as well as the outstanding PTT performance. Both in vitro and in vivo results show that PEG-NaxGdWO3 nanorods can be used as an efficient theranostic platform for MRI-guided photothermal therapy by simply regulating the oxygen vacancy. As a proof-ofconcept, such a reported proton relaxation influenced by oxygen vacancy will offer possibilities for developing MRI biosensors, molecular MR imaging (e.g., detecting H2O2), and so on. METHODS Materials. GdCl3·6H2O and 1-octadecene (90%) were purchased from Sigma-Aldrich. Oleic acid and cyclohexane (C6H12) were obtained from Adamas-beta Company. The ammonia (NH3·H2O) and sodium tungstate dehydrate (Na2WO4·2H2O) were acquired from Sinopharm Chemical Reagent Co., Ltd. MeO-PEG2k-SH were obtained from Jenkem Co., Ltd. All reagents were of analytical grade and used without any purification. Synthesis of PEG-NaxGdWO3 Nanorods. In a typical synthesis of NaxGdWO3 nanorods, 0.2 mmol of GdCl3·6H2O was dissolved in 2 mL of deionized water and placed into a 100 mL flask; 10 mL of oleic acid and 30 mL of 1-octadecene were put into the flask and mixed for 1 h at room temperature. Then the mixture was heated to 120 °C to get remove water under an argon atmosphere and maintained at 160 °C for 1 h. After being cooled back to room temperature, the mixture was added with 5 mL of ammonia solution dissolving 2 mmol Na2WO4·2H2O. The mixture was then stirred at room temperature for 3 h. After the evaporation of ammonia at 80 °C, the solution was heated to 280 °C and maintained for 1 h, then cooled to room temperature. The product was centrifuged to disperse in 10 mL of cyclohexane. After being washed with 5 mL of ethanol, the mixed solution was centrifuged and the NaxGdWO3 nanorods were dispersed 10 mL of DMSO. The PEG-NaxGdWO3 nanorods with good biocompatibility were prepared by strong thiol−metal interactions. Briefly, the as-synthesized NaxGdWO3 nanorods were redispersed in 10 mL of deionized water, followed by adding 10 mL of MeO-PEG2kSH (300 mg) and stirring for 24 h at room temperature. Excess PEG was removed by centrifugation (20 000 rpm, 15 min). Stability Assessment. The stability of PEG-NaxGdWO3 nanorods was examined under acid/alkali and salt conditions to ensure their stability for in vivo applications. PEG-NaxGdWO3 nanorods were incubated in 50 mM HEPES buffer at pH 5, 7, and 9 for 24 h or in carbonate (200 mM Na2CO3 in water) for 1, 3, 7, and 14 days. To further simulate the physiological environment, normal human serum was used to disperse the PEG-NaxGdWO3 nanorods, and their stability was measured in 2 weeks. ESR Measurement of Oxygen Vacancy. The PEG-NaxGdWO3 nanorods before (1#) and after oxidation by H2O2 for 0.5 h (2#) or 3 h (4#) were dried in a lyophilizer to obtain powders. All samples 4261

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ACS Nano weighing 200 mg were used for the ESR detection, and the EPR spectra were measured at room temperature in perpendicular mode on a Bruker EMX-8/2.7 spectrometer and recorded with the following settings: center field = 3515.000 GHz, sweep width = 150.000 GHz, microwave frequency = 9.876 GHz, microwave power = 6.385 mW, modulation frequency = 100.00 kHz, and modulation amplitude = 1.00 G. Photothermal Evaluations of PEG-NaxGdWO3 Nanorods. An aqueous suspension containing PEG-NaxGdWO3 nanorods at different concentrations was poured into a quartz cuvette. The cuvette was illuminated by a 980 nm laser with power density 0.7 W/cm2 for 5 min. The increase of temperature was monitored by a digital thermocouple device. Leakage Studies of Gd3+ from the Matrix. To check the leakage of the Gd3+ from the nanorods, the dialysis of solutions containing nanorods was performed. After being stirred for 7 days, the obtained filtrates were incubated with Arsenazo III (0.05 mM) to monitor any Gd3+ presence by detecting the absorption band of Gd3+−arsenazo complex at 658 nm. The GdCl3 solution and pure water were used as positive and negative controls, respectively. Meanwhile, the filtrates were further measured by inductively coupled plasma optical emission spectrometry. Relaxivity Measurement. The MR imaging experiments of PEGNaxGdWO3 nanorods in water solutions were performed on a 3.0 T clinical MRI scanner (GE Signa 3.0 T), and the pulse sequence used was a T1-weighted FSE-XL/90 sequence: TR/TE = 1000, 2000, 3000, 4000/7.9 ms; FOV = 18 cm; matrix = 128 × 128; NEX = 2; slice thickness = 2 mm; space = 0.5 mm; FOV = 18 cm; and coil= QUADKNEE. The images were analyzed at the workstation provided by GE Healthcare.The Gd3+concentrations of all samples were measured with coupled plasma optical emission spectrometry. The r1 value was calculated as the slope of curves of longitudinal relaxation rate (1/T1) versus Gd3+ concentration. Cell Culture and Cytotoxicity Assessment. Murine breast carcinoma tumor 4T1 cells, murine macrophage cells (RAW 264.3), and brain capillary endothelial cells were cultured at 37 °C and with 5% CO2 in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. The above metioned cells were seeded into a 96-well cell culture plate at 106/well and then incubated for 24 h. Cell culture media of PEG-NaxGdWO3 nanorods with different concentrations (0.006, 0.013, 0.025, 0.05, 0.1, and 0.2 mg/mL) were put into the wells. The cells were then incubated for 24 h at 37 °C under 5% CO2, and the cell viability was detected using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Photothermal Ablation of 4T1 Cells in Vitro. The 4T1 cells were seeded into a 96-well plate at a density of 104/well and then incubated at 37 °C under 5% CO2 for 24 h prior to treatment. The PEG-NaxGdWO3 nanorods dispersed in RPMI 1640 with different concentrations (0.05, 0.1, and 0.2 mg/mL) were added to the wells and incubated for 4 h. The cells were irradiated for 5 min using a 980 nm laser with a power density of 0.7 and 1.5 W/cm2. The cell viability was calculated using a typical MTT assay. Trypan Blue Stain. Phosphate-buffered saline (PBS) of the solution of PEG-NaxGdWO3 nanorods with the concentration of 0.1 mg/mL was added to a cell culture plate containing 4T1 cells for 4 h incubation. Next, an adherent cell solution was exposed to a 980 nm laser with a power density of 1.5 W/cm2 for 5 min. After that, 4T1 cells were stained with 0.4% trypan blue solution for 15 min. Then, the cells were washed with PBS three times. Cell morphology of the adherent cells was observed by an inverted optical microscope (Olympus, IX71, Japan). Cells stained in blue were counted as dead cells. Animal Experiments. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee. All the Balb/c and Kunming mice with an average weight of 20 g were purchased from Laboratory Animal Centre, Shanghai Medical College of Fudan University, China. 4T1 cells (106 in 100 μL of PBS) were subcutaneously injected into the right front leg region of the Balb/c mice to establish a 4T1 tumor model. Healthy male Sprague−Dawley

(SD) rats (mean age, 6 weeks; mean weight, 250 g) were purchased from Laboratory Animal Center, Shanghai Medical College of Fudan University. In Vivo Half-Life and Biodistribution. For blood circulation experiments, 15 μL of blood from female Kunming mice was collected at the varied time (2 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h) after intravenous injection of PEG-NaxGdWO3 nanorods (n = 4). The blood was dispersed into 1 mL of physiological saline containing heparin sodium injection. The concentration of W was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The in vivo blood half-life of PEG-NaxGdWO3 nanorods was calculated by a single-component pharmacokinetic model. The biodistribution of PEG-NaxGdWO3 in main organs and the tumor was measured at 1 h, 1 day, and 3 days after the intravenous injection of 4T1-tumor-bearing mice (n = 4). Dissected organs and tumor were weighed and homogenized. The determination of W concentrations in the dissected organs and tumor was tested using ICP-AES. In Vivo MR and Photothermal Imaging. In vivo MR imaging was conducted on a clinical MRI scanner (GE Signa 3.0 T). For MRA imaging of normal SD rats, a 19 mm intravenous catheter (24GX; WeihaiJie SwissMedical Products Co., Ltd., Shandong, China) was put in the femoral vein instead of the more readily available tail vein for CA administration. Time-resolved magnetic resonance imaging of contrast kinetics (TRICKS-MRA) was acquired in the coronal after the injection of PEG-NaxGdWO3 nanorods or Magnevist at low concentrations (2.5 mg Gd3+/kg). A precontrast phase was obtained and served as a mask prior to the injection of CAs. Then, scanning of subsequent phases was initiated simultaneously with the injection of CAs. Complex subtraction of the mask was performed automatically to maximally suppress the background signal. Scan parameters for TRICKS-MRA are listed as follows: TE = 2.2 ms; TR = 6.1 ms; slice thickness = 1.6 mm; flip angle = 20°; bandwidth = ±35.71 kHz; FOV = 10 × 6 cm; matrix = 192 × 128; and NEX = 1.0. Forty phases were acquired with a temporal resolution of 2 s. For in vivo tumor MR imaging, 4T1-tumor-bearing mice were intratumorally (I.T. 0.25 mg Gd3+/kg) or intravenously (I.V. 1.9 mg Gd3+/kg) injected with PEGNaxGdWO3 nanorods in saline. Regions of interest with the same area were drawn on MR images before and after injection. For the in vivo photothermal imaging, 4T1 tumors were I.T. or I.V. (1 h and 3 h postinjection) injected with 20 or 150 μL of PEG-NaxGdWO3 nanorod dispersion (3 mg/mL in saline) and irradiated with a 980 nm laser (1.5 W/cm2) for 5 min. Tumor temperature and thermal imaging were visualized and recorded using the FLIR A320 camera. In Vivo Photothermal Therapy. For in vivo photothermal therapy, 4T1-tumor-bearing mice were randomly divided into five treatment groups: the first group was intravenously injected with the saline solution (150 μL); the second group was only exposed to NIR laser with a power density of 1.5 W/cm2 for 5 min; the third group was I.T. injected with 20 μL of PEG-NaxGdWO3 nanorods (3 mg/mL) without NIR laser irradiation; the fourth group was exposed to 980 nm laser with an output power density of 1.5 W/cm2 (duration time = 5 min) at 1 h post-I.V. injection of 150 μL of PEG-NaxGdWO3 nanorod solution (3 mg/mL in saline); the fifth group was I.T. injected with 20 μL of PEG-NaxGdWO3 nanorod solution (3 mg/mL in saline) and treated with a 980 nm laser (1.5 W/cm2) for 5 min. The H&E staining was performed at 3 days after the corresponding treatment to compare the therapy efficiency of different treatment groups. The tumor volume after different treatments was recorded every 2 days for 15 days. Relative tumor volume (V/V0, where V0 represents the initial tumor volume, i.e., day 0), body weight, and tumor appearance were monitored. Histological Assessment. The in vivo biocompatibility of PEGNaxGdWO3 nanorods was evaluated using the standard H&E staining. Kunming mice were euthanized at 3 days and 30 days post-I.V. injection with 150 μL of PEG-NaxGdWO3 nanorod solution (3 mg/ mL in saline). Tissues were H&E-stained to monitor the histological changes in heart, liver, spleen, lung, and kidney of mice. The histological sections were observed under an optical microscope. 4262

DOI: 10.1021/acsnano.7b01297 ACS Nano 2017, 11, 4256−4264

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ACS Nano Characterization. Transmission electron microscopy images and energy-dispersive X-ray analysis were performed on a JEOL 200CX microscope with an accelerating voltage of 200 kV. Powder X-ray diffraction patterns were performed on a Rigaku D/MAX-2250 V diffractometer with graphite-monochromatized Cu Kα radiation. Fourier transform infrared spectroscopy (FT-IR) spectra were conducted on a Nicolet Avatar 370 FT-IR spectrophotometer using KBr pellets. Dynamic light scattering measurement was performed on Nano-Zetasizer (Malvern Instruments Ltd.). Water proton relaxation rates of PEG-NaxGdWO3 nanorods were measured using a fast field cycling Stelar relaxometer. The concentration was determined by ICPAES (Agilent Technologies, USA).

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01297. EDX spectrum, XRD, FT-IR spectrum, ESR spectra, NMRD profiles, in vitro stability, in vitro cell viabilities, blood circulation and biodistribution, in vivo toxicity studies (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wenbo Bu: 0000-0001-6664-3453 Jianlin Shi: 0000-0001-8790-195X Author Contributions

D.N. and J.Z. contributed equally to this work. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51372260 and 51132009) and the Shanghai Excellent Academic Leaders Program (Grant No. 16XD1404000). We thank Jianan Liu, Yanyan Liu, Li Jiang, Heliang Yao, Jingwei Feng, and Linlin Zhang from Shanghai Institute of Ceramics, Chinese Academy of Sciences, for useful discussions. REFERENCES (1) Shin, T. H.; Choi, Y.; Kim, S.; Cheon, J. Recent Advances in Magnetic Nanoparticle-Based Multi-Modal Imaging. Chem. Soc. Rev. 2015, 44, 4501−4516. (2) Lee, N.; Hyeon, T. Designed Synthesis of Uniformly Sized Iron Oxide Nanoparticles for Efficient Magnetic Resonance Imaging Contrast Agents. Chem. Soc. Rev. 2012, 41, 2575−2589. (3) Bottrill, M.; Kwok, L.; Long, N. J. Lanthanides in Magnetic Resonance Imaging. Chem. Soc. Rev. 2006, 35, 557−571. (4) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats, R.; Jacobs, R. E.; Fraser, S. E.; Meade, T. J. In vivo Visualization of Gene Expression Using Magnetic Resonance Imaging. Nat. Biotechnol. 2000, 18, 321−325. (5) Holbrook, R. J.; Rammohan, N.; Rotz, M. W.; MacRenaris, K. W.; Preslar, A. T.; Meade, T. J. Gd(III)-Dithiolane Gold Nanoparticles for T1-Weighted Magnetic Resonance Imaging of the Pancreas. Nano Lett. 2016, 16, 3202−3209. (6) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99, 2293−2352. 4263

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