full papers Cancer Theranostics
Multifunctional RbxWO3 Nanorods for Simultaneous Combined Chemo-photothermal Therapy and Photoacoustic/CT Imaging Gan Tian, Xiao Zhang, Xiaopeng Zheng, Wenyan Yin, Longfei Ruan, Xiaodong Liu, Liangjun Zhou, Liang Yan, Shoujian Li, Zhanjun Gu,* and Yuliang Zhao*
Light-triggered drug delivery based on near-infrared (NIR)-mediated photothermal nanocarriers has received tremendous attention for the construction of cooperative therapeutic systems in nanomedicine. Herein, a new paradigm of light-responsive drug carrier that doubles as a photothermal agent is reported based on the NIR lightabsorber, RbxWO3 (rubidium tungsten bronze, Rb-TB) nanorods. With doxorubicin (DOX) payload, the DOX-loaded Rb-TB composite (Rb-TB-DOX) simultaneously provides a burst-like drug release and intense heating effect upon 808-nm NIR light exposure. MTT assays show the photothermally enhanced antitumor activity of Rb-TB-DOX to the MCF-7 cancer cells. Most remarkably, Rb-TB-DOX combined with NIR irradiation also shows dramatically enhanced chemotherapeutic effect to DOX-resistant MCF-7 cells compared with free DOX, demonstrating the enhanced efficacy of combinational chemo-photothermal therapy for potentially overcoming drug resistance in cancer chemotherapy. Furthermore, in vivo study of combined chemo-photothermal therapy is also conducted and realized on pancreatic (Pance-1) tumor-bearing nude mice. Apart from its promise for cancer therapy, the as-prepared Rb-TB can also be employed as a new dual-modal contrast agent for photoacoustic tomography and (PAT) X-ray computed tomography (CT) imaging because of its high NIR optical absorption capability and strong X-ray attenuation ability, respectively. The results presented in the current study suggest promise of the multifunctional RbxWO3 nanorods for applications in cancer theranostics.
Dr. G. Tian, Prof. S. Li, Prof. Y. Zhao College of Chemistry Sichuan University Chengdu 610064, P. R. China Dr. G. Tian, Dr. X. Zhang, Dr. X. Zheng, Prof. W. Yin, Dr. L. Ruan, Dr. X. Liu, Dr. L. Zhou, Dr. L. Yan, Prof. Z. Gu, Prof. Y. Zhao Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics Chinese Academy of Sciences Beijing 100049, P. R. China E-mail: [email protected]; [email protected]
Dr. X. Zhang, Prof. Y. Zhao Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanosciences and Technology of China Beijing, 100190, P. R. China Dr. X. Zheng, Dr. L. Zhou College of Materials Science and Opto-electronic Technology Graduate University of Chinese Academy of Sciences Beijing, 100049, P. R. China
DOI: 10.1002/smll.201401237
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1. Introduction There is growing interest in combating cancer with nanoparticle-based therapeutics since nanocarriers-mediated drug delivery can overcome the intrinsic defects originating from the conventional therapeutic drugs, including poor watersolubility, limited stability, short circulation period and low selectivity.[1] Moreover, the development of external stimulus-sensitive drug delivery nanocarriers for cancer therapy has received tremendous attention in recent years, as these systems can differentially increase the drug accumulation at targeted cancer cells/tissues, drastically decrease the systemic toxicity, and potentially avoid under- or over-dosing.[2] Among them, light-responsible vehicles, especially those plasmonic nanomaterials that can convert near-infrared (NIR) light into heat energy and then serve as photothermal agents for localized hyperthermia cancer therapy are preferentially attractive because of the on-demand drug release at the desired sites in time with controllable therapeutic outcome due to the high spatial-temporal resolution of light stimuli,[3] the non-invasive nature of NIR laser-induced photothermal therapy (PTT) benefited from the high tissue-penetration ability and minimal photo-damage of NIR light,[4] and most importantly, the expected synergistic antitumor efficacy ascribed to the combination of PTT and chemotherapy.[5] Therefore, exploration on NIR light-responsive photothermal agents potentially engineered as drug vehicles to achieve enhanced anti-cancer effect is highly desired and important. Transition-metal oxide nanostructures with tunable localized surface plasmon resonance (LSPR) in the NIR region arising from the unique character of their outer-d valence electrons are of particular interest.[6] Recently, oxygen-deficient tungsten oxide (WO2.83) nanorods were certified to be capable to support a strong LSPR ranging from the red edge of the visible to the NIR region of light originating from the intervalence charge-transfer transition between W6+ and W5+.[7] Chen et al. developed the first tungsten oxide-based PTT agent, W18O49 nanowires, for cancer treatment and demonstrated that tumor suppression observed upon 980-nm NIR light irradiation of cancer cells with internalized PEG-coated W18O49 nanowires was solely attributed to the photothermally-induced hyperthermia.[8] Zhou et al. engineered the WO2.9 nanorods as a promising theranostic agent for simultaneous CT imaging and 980-nm NIR photothermal therapy of tumors in vivo.[9] In addition, metal tungsten oxide materials, such as tungsten bronzes (MxWO3, M = Cs, K, Na, and NH4; 0 < x < 1/3), which are constructed by the intercalation of small cationic ions into the WO3 framework,[10] possess similar LSPR properties with tungsten oxide and have also been emerged as a novel PTT agent for efficient photothermal ablation of human cancer cells in vitro under 980-nm NIR irradiation.[10d] However, despite those few pioneer work, the possibility of using either tungsten oxides or tungsten bronzes as drug delivery systems for synergistic thermo-chemotherapy has not yet been demonstrated to our best knowledge. In addition, 980-nm NIR laser is preferentially chosen as the light source in theses pioneer reports to heighten the photothermal efficacy in consideration of the increased small 2014, 10, No. 20, 4160–4170
optical absorbance at longer NIR wavelength of tungsten oxides based nanomaterials.[8,10d] However, the 980-nm light could be easily adsorbed by the water-rich biological tissues, which will result in limited penetration ability. Furthermore, the absorbed light energy would quickly transform into heat energy, inducing irreversible overheating effect, and thus causing significant cell death and tissue damage.[11] Alternatively, 808-nm laser with wavelength within the “biological window” has low absorption coefficient to water molecules and thus could greatly minimize the laser-induced heating effect and significantly improve the penetration depth in biotissue. Hence, using 808-nm light to trigger the generation of localized heat is much more safe and desirable.[12] Here, PVP-coated multifunctional rubidium tungsten bronze (RbxWO3) nanorods were engineered as a smart 808-nm NIR light-responsive nanocarries for synergistic chemo-photothermal therapy and yet as a new dual-modal probe for photoacoustic tomography (PAT)/X-ray computed tomography (CT) imaging (Scheme 1). Doxorubicin (DOX) was attached to the nanorods as the model drug through physical adsorption and could be on-demand released by the NIR light stimuli. In order to evaluate the therapeutic efficacy of the light-enhanced drug delivery system, a couple of normal MCF-7 and DOX-resistant MCF-7 (MCF-7-DOXR) cancer cells were chosen for in vitro cell killing experiments. As expected, synergistic effect was achieved for both of the two tested cancer cells. Moreover, the combination of chemotherapy and photothermal therapy with fascinating synergistic effect in vivo was also demonstrated for Pance-1 tumor-bearing nude mice. Due to the intrinsic high NIR optical absorbance of RbxWO3 and high X-ray absorption coefficient (4.438 cm2/kg at 100 keV) of tungsten (W),[9] in vivo PAT and CT imaging of MCF-7 tumor-bearing mice were also demonstrated. Therefore, this work demonstrates the potential of the tungsten bronze-based light-absorber as a new multifunctional platform for cancer imaging and therapy, and encourages further in-depth exploration of other plasmonic transition-metal oxides for theranostic nanomedicine.
2. Results and Discussion Rubidium tungsten bronze (Rb-TB) nanorods were prepared by the hydrothermal treatment of WO3 precursor and Rb2SO4 in an mixture solution containing ethylene glycol (EG) and polyvinyl pyrrolidone (PVP) at 180 °C for 16 h.[10a] TEM image in Figure 1a shows that the Rb-TB nanocrystals are predominantly rod-like in shape, and the particle size of nanorods is found to be 20–40 nm in length and about 5 nm in diameter (Figure S1 in the Supporting Information). XRD patterns presented in Figure 1b reveal that all the reflections could be well indexed to the hexagonal Rb0.27WO3 (JCPDS No. 73–1549) and no impurities are detected. X-ray photoelectron spectroscopy (XPS) analysis (Figure 1c) reveals that the elements O (1s, 532.1 eV), N (1s, 399.3 eV), C (1s, 284.7 eV), Rb (3d, 109.7 eV) and W (4f, 35.32 eV) could be determined and the atomic ratio of Rb/W is calculated to be 0.24, which is very close to the value from the XRD results. In addition, the oxidation state of tungsten could also be
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Scheme 1. Illustration of the multifunctional RbxWO3 nanorods for NIR light-triggered synergetic chemo-photothermal therapy and dual-modal PAT/CT imaging.
distinguished by the XPS analysis. Figure 1d shows the W4f XPS spectra of the as-obtained Rb0.27WO3 nanorods. The W4f core-level spectrum can be well fitted into two spin-orbit doublets, corresponding to two different oxidation states of W atoms. Two peaks centered at 37.52 and 35.42 eV correspond to W6+, while the other two peaks with lower binding energy at 36.37 and 34.72 eV belong to W5+.[10a,13] The existence of W5+ should be due to the fact that a portion of the W6+ ions are reduced by PVP and ethylene glycol under hydrothermal condition. Figure 1e exhibits the FT-IR spectrum of the as-prepared samples. The peak at 1639 cm−1 should be related to a strong C=O adsorption from the lactam group in the side chains of the PVP.[4b,14] The bands at 1430 and 1284 cm−1 are corresponding to the pyrollidone ring and the N→HO complex vibrations, respectively.[14a] Moreover, the broad band centered at 1104 cm−1 corresponds to C–N stretching vibration.[14b] Based on the above results, the successful coating of PVP ligands on the surface of Rb-TB could be confirmed. In addition, the amount of PVP on nanoparticles was determined to be 6.2% by thermogravimetric analysis (TGA, Figure S2, Supporting Information). As a result, the PVP-coated Rb0.27WO3 nanorods are hydrophilic and can be readily dispersed in water, showing a dark blue color (Figure 1f inset). The optical absorption spectrum of their aqueous dispersion was investigated using UV–vis– NIR spectroscopy (Figure 1f). A very large absorption tail presented in the visible and near infrared (NIR) regions can be clearly observed, which can be explained by the free electrons induced SPR effect and/or oxygen-deficiency induced small polarons absorption.[7–9] In addition, the UV–vis–NIR reflectance diffuse spectrum (Figure S3, Supporting Information) confirms the enhanced photo-absorption ability as the wavelength increases in the NIR region.
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The strong photo-absorption in NIR region promotes us to investigate their photo-thermal effect property under NIR laser irradiation. As mentioned above, previous reports always used the 980-nm laser other than 808-nm laser as the light source to investigate the photothermal behavior of tungsten oxide based plasmonic nanomaterials in consideration of the gradually increased optical absorbance at longer wavelength region. However, 980-nm light with water absorption coefficient at 0.48 cm−1 would be significantly attenuated to cause limited penetration depth and irreversible overheating effect in bio-tissue, while this vital side-effect could be largely alleviated when using 808-nm laser with water adsorption coefficient at 0.02 cm−1.[11b,12b] As seen in the Figure 2a, the temperature of the water irradiated by 980-nm laser increased from 25 °C to 37.35 °C, a rise of 12.35 °C, while the one under 808-nm irradiation was heated to a temperature at 28.31 °C with a rise of only 3.31 °C. In order to demonstrate the weak water absorption of 808-nm NIR light, we measured the energy loss of 808-nm and 980-nm laser after they pass through water columns with different thickness. As clearly presented in the Figure 2b, after penetrating the same thickness of water, the fall of laser intensities of 808-nm were much smaller than that of 980-nm laser. Meanwhile, the temperature elevation (Figure 2c) and fluctuation (Figure 2d) of the Rb0.27WO3 dispersion (≈0.25 mg/mL) induced by 808-nm or 980-nm irradiation after passing through a certain thickness of water accordingly showed that temperature rising induced by 980-nm irradiation decreased much more rapidly along with the increased thickness of the water block compared with that of 808-nm irradiation, demonstrating that 808-nm light is much more resistant to water absorption and more reliable for water-rich deep-tissue application.
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Figure 1. Materials characterization. a) TEM image and b) wide-angle XRD pattern of the as-prepared RbxWO3 nanorods. Bottom in (b): the standard pattern of pure hexagonal Rb0.27WO3 (JCPDS No: 73–1549). c) Full range and d) W4f core-level XPS spectrum of the Rb0.27WO3 nanorods. e) FT-IR spectra of the as-prepared PVP-coated samples, inset: chemical structure of PVP molecule. f) UV–vis–NIR absorption spectrum of an aqueous dispersion containing 1 mg/mL Rb0.27WO3. Inset: Photograph of the corresponding aqueous dispersion.
Then, we systematically investigated the photothermal behavior and conversion efficiency of Rb0.27WO3 under 808-nm laser irradiation with a power density at 1.0 W/cm2. Temperature trends of this material dispersed within water as shown in Figure 3a declare that Rb0.27WO3 nanorods can efficiently convert the 808-nm laser energy into heat energy and the photothermal heating effect is positively correlated with concentration. Figure 3b shows the temperature changes (ΔT) in 10 min irradiation rise rapidly with the concentration increasing to 0.25 mg/mL and then slowly go up upon increasing the concentration to 1.0 mg/mL. This trend could be explained by the faster heat loss at higher temperature.[3b,4c] The conversion efficiency (η) is an important character for photothermal materials. As far as we know, no relative data about theη value of the tungsten oxides or tungsten bronzes has been reported.[15] Here, the η value of the Rb0.27WO3 NPs was measured by a modified method and calculated as follows:[3c] small 2014, 10, No. 20, 4160–4170
η=
hS (Tmax − Tsurr ) − QDis I (1 − 10 − A808 )
(1)
θ=
T − Tsurr Tmax − Tsurr
(2)
t = −τ s ln θ
τs =
mC p hS
(3) (4)
Where h is the heat transfer coefficient, S is the surface area of the container, Tmax is the maximum steady-state temperature, Tsurr is the ambient temperature of the surroundings, T is the solution temperature, QDis is the heat associated with the light absorbance of the solvent, I is the laser power, A808 is the absorbance of Rb0.27WO3 solution at 808 nm, τs is the system time constant, m and Cp are the mass and heat capacity of water, respectively.
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Figure 2. Comparative study of 980-nm and 808-nm NIR laser. a) Temperature elevation of water (1 mL) under 808-nm and 980-nm laser irradiation, respectively. b) Power attenuation trend of 808-nm and 980-nm lasers after they passed a water column with different thickness. c) Temperature elevation and d) temperature change of Rb0.27WO3 aqueous suspensions (≈0.25 mg/mL) irradiated by 808-nm and 980-nm irradiation with the same power density at 1.0 W/cm2 when the laser were shielded by water column with different thickness. Error bars are calculated based on triplicated samples.
Figure 3. Photothermal properties investigation. a) The temperature elevation of aqueous dispersions of Rb0.27WO3 with different concentrations as a function of irradiation time of an 808 nm CW laser with power density (1.0 W/cm2). b) Plot of temperature change (ΔT) over a period of 10 min irradiation versus Rb0.27WO3 concentration. Inset: IR thermal images of water and Rb0.27WO3 solution (1.0 mg/mL) after laser irradiation at 1.0 W/cm2 for 10 min. Error bars are calculated based on triplicated samples. c) The photothermal response of the Rb0.27WO3 aqueous solution (≈1.0 mg/mL) under an 808-nm laser irradiation (1 W/cm2) for 720 s and then the laser was turned off. d) Linear time data from the cooling period (after 720 s) versus negative natural logarithm of driving force temperature (−lnθ) for time constant (τs) determination.
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We collected the temperature rising (laser on) and cooling (laser off) curves of the Rb0.27WO3 dispersion with concentration at 1.0 mg/mL (Figure 3c). From the temperature cooling curve, we could calculate the sample system time constant τs (Figure 3d) based on Equation 2,3 and then obtain the hS value from Equation 4. Finally, according to Equation 1, the η value of was determined to be 17.8%. In addition, our Rb0.27WO3 samples exhibit good photostability towards 808-nm laser since no significant difference in optical absorbance or color of the tested Rb0.27WO3 suspension has been obtained even after 808-nm laser exposure for 3 h (Figure S4, Supporting Information), indicating they are promising as an ideal photothermal converter in cancer therapy. Nanomaterials with versatile surface chemistry and large surface area have been widely employed as nanocarriers for therapeutic drug delivery. The as-prepared Rb0.27WO3 nanorods were coated with a PVP layer with weight ratio up to 6.2%, which may provide adsorption sites for drug molecules intercalation through physical interactions. To test our hypothesis, DOX was loaded as a guest molecule by soaking PVP-coated Rb0.27WO3 nanorods with DOX aqueous solutions overnight. Reddish precipitate and nearly colorless supernatant were observed after the mixture was centrifuged (Figure 4a inset), demonstrating the successful loading of DOX onto Rb-TB nanorods. Based on UV–vis absorption measurements, the loading capacity for DOX molecules stored on the Rb-TB is 0.326 mmol/g (Figure 4a). The in vitro DOX release behaviors of Rb-TB-DOX were performed in PBS medium at different pH values. In the absence of laser irradiation, only 14.65% and 21.65% DOX are released within 7 h at the pH 7.4 and pH 5.0 under ambient temperature, respectively. However, burst release was observed upon 808-nm laser irradiation, and the rate subsequently slowed when the laser was shut off. After initial irradiation for 15 min (1.0 W/cm2), the DOX release was significantly increased from 8.50% to 16.77% at pH 7.4, and from 12.07% to 28.83% at pH 5.0. The cumulative release of DOX reached up to 40.86% and 63.31% within 7 h at pH 7.4 and pH 5.0, respectively, which are much higher than their counterparts without irradiation (Figure 4b). The stepwise triggered rapid DOX release under NIR irradiation should be ascribed to the photothermal effect of Rb-TB nanorods since increasing local temperature would dissociate the interactions between DOX and PVP and thus more DOX molecules are detached (Figure S5, Supporting Information) from the nanocarriers.[5e] In addition, the release of DOX exhibited a pH-responsive pattern, where a higher cumulative DOX release is achieved in relation to a lower pH value. The phenomenon can be attributed to the reduction of the hydrophobic-hydrophobic interaction between DOX and PVP polymer, where DOX molecule becomes more hydrophilic at lower pH values, weakening its binding to the long hydrophobic alkyl chain of PVP polymer. Such a pH-dependent release hallmark is important for cancer therapy because of the lower pH value in tumor microenvironment compared with the normal tissue.[16] To further illustrate the potential of light-responsible Rb-TB nanocarries in the NIR-stimulus synergistic chemo/ small 2014, 10, No. 20, 4160–4170
photothermal-therapy field, a couple of cancer cells, MCF-7 and DOX-resistant MCF-7 (MCF-7-DOXR) cells were tested. MTT assay showed that Rb-TB nanocarrier was nontoxic to both MCF-7 and MCF-7-DOXR cells up to a tested concentration of 800 µg/mL (Figure S6, Supporting Information). First, we incubated MCF-7 cells with Rb-TB and Rb-TBDOX and evaluated the cytotoxicity with and without laser irradiation. As expected, the viability of MCF-7 cells treated with Rb-TB-DOX dramatically decreased upon 808-nm laser irradiation with 1.0 W/cm2 for 5 min, the dose of which has been proved to be safe for living cells (Figure S7, Supporting Information). Compared with the control groups without laser irradiation or no DOX loading, those treated with Rb-TB-DOX under irradiation held higher potency against MCF-7 cells at each concentration (Figure 4c). For instance, at the equivalent Rb-TB concentration of 25 µg/mL, 18.5% and 32.5% of the cells were killed in the case of being treated with bare Rb-TB with NIR irradiation and Rb-TB-DOX without NIR irradiation, respectively. In comparison, 78.2% of the cells were killed in the presence of Rb-TB-DOX with 5 min NIR irradiation. The cell-killing efficacy by Rb-TBDOX under NIR irradiation was even higher than the sum of chemotherapy by Rb-TB-DOX and photothermal therapy by DOX-free Rb-TB, indicating the synergy was achieved. This synergistic effect was probably ascribed to the increased drug release inside cells upon NIR irradiation and the enhanced cytotoxicity of DOX at elevated temperatures.[5b,5d] The occurrence of multi-drug resistance (MDR) is a major impediment to the success of cancer chemotherapy. In the following, we assessed the anti-tumor efficacy of Rb-TB-DOX in MCF-7-DOXR cells treatment. From Figure 4d, we could find that MCF-7-DOXR cells had strong drug resistance since free DOX induced no significant inhibition even at a DOX dosage of 50 µm. Meanwhile, Rb-TB-DOX without laser irradiation showed the comparable inhibiting ability with free DOX. Interestingly, potent inhibition of MCF-7-DOXR cells was obtained when cells were treated with Rb-TBDOX under 5 min laser irradiation (1.0 W/cm2), suggesting these photo-responsive Rb-TB-DOX composites are able to overcome MDR mechanism, likely due to a combination of the decreased chemoresistance sensitiveness contributed by thermal effect[17] and the light-triggered boosted drug release since the fast intracellular drug release feature is able to reverse MDR mechanisms, such as drug efflux.[18] Moreover, we also performed MCF-7 or MCF-7-DOXR nucleus staining experiments using Hochest 33342 to visually illuminate the synergy effect, where brighter ones are dead cells. As seen in the Figure 4e, when treated with Rb-TB-DOX as well as laser irradiation, much more number of MCF-7 cells were found to be brighter compared to Rb-TB without laser or RbTB-DOX treated case at equivalent Rb-TB concentration (25 µg/mL). Similar results were obtained for MCF-7DOXR nucleus staining with equivalent DOX concentration (12.5 µm), larger amount of cells treated with Rb-TB-DOX under NIR exposure were bright than that of free DOX or Rb-TB-DOX treated case (Figure 4f). Encouraged by the apparent synergistic effect in vitro, we then performed a pilot chemo-photothermal therapy study in vivo. Five groups of Pance-1 tumor-bearing nude mice with
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Figure 4. Controlled drug loading/releasing behavior and combined photothermal-chemotherapy treatment. a) Quantification of drug loading capacity at different DOX concentrations. Inset: photographs showing the reddish precipitate and nearly colorless supernatant were observed after the mixture of Rb-TB and DOX (100 µm) was centrifuged. b) Release kinetics of DOX from Rb-TB in PBS medium at pH values of 7.4 and 5.0 with or without laser irradiation at 808 nm (1.0 W/cm2). c) Viabilities of normal MCF-7 cells incubated with different concentrations of testing materials. d) Fluorescent images of normal MCF-7 nucleus stained with Hoechst 33342. The tested materials are of equivalent Rb-TB concentrations (25 µg/ mL). Brighter ones are dead cells. e) Dependence of cell viabilities on DOX concentrations for MCF-7-DOXR. f) Hoechst 33342 staining MCF-7-DOXR nucleus. The tested materials are of equivalent DOX concentration at 12.5 µm. 808-nm laser irradiation parameters: 1.0 W/cm2, 5 min. Error bars are calculated based on triplicated samples. All scale bars in fluorescent images are 100 µm.
3 mice per group were used in our experiment. Each group of mice were intratumorally injected with 20 µL of saline, free DOX, Rb-TB or Rb-TB-DOX ([DOX] = 0.5 mg/kg) with or without 808-nm NIR irradiation (1 W/cm2) for 10 min. As seen from the Figure 5a,b, the temperature of tumor injected with Rb-TB or Rb-TB-DOX could increase up to 50 °C, with a raise of ≈17.5 °C, while the control group treated with saline
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showed insignificant change (ΔT = ≈5 °C), indicating that the injected Rb-TB or Rb-TB-DOX could induce sufficient hyperthermia to kill cancer cells under 808-nm NIR irradiation. The tumor volume measured for each group is plotted as a function of time intervals in Figure 5c. The control group treated only with saline exhibited rapidly increasing tumor volumes. Compared with the control group, there was only
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Figure 5. In vivo combined chemo-photothermal therapy study. a) IR thermal images of Pance-1 tumor-bearing mice collected by an IR camera at different time intervals during 808-nm laser irradiation (1.0 W/cm2 for 10 min). b) Temperature change of tumors monitored by the IR thermal camera during laser irradiation. c) Tumor volume growth curves of mice after different treatments. d) Photograph of tumors after excision. e) Tumor weights of each group. Error bars are calculated based on triplicated samples.
a slight inhibition of tumor growth treated with free DOX, suggesting that free DOX at this low dose was not sufficient to inhibit the tumor growth. The tumors treated with RbTB-DOX without NIR laser irradiation showed enhanced tumor regression compared with free DOX-treated group, mainly due to the longer retention of Rb-TB-DOX inside the tumor after local administration.[5e] For the mice treated with Rb-TB under NIR light, noticeable tumor inhibition in the preliminary stage could be certified due to the laser induced hyperthermia. Unfortunately, the tumor growth in this group became uncontrollable later. In marked contrast, the tumor growth in the group treated with Rb-TB-DOX under NIR irradiation was dramatically inhibited as a result of combined chemotherapy and photothermal therapy. Figure 5d,e represented the photographs and mean weight of the excised tumors of each group, which could well coincide with the final results as indicated in Figure 5c. These results demonstrated the in vivo synergistic effect of combining photothermal therapy and chemotherapy in our drug delivery system. Ideal nanoparticle systems in nanomedicine should possess multimodality in both imaging and therapy. Recently, small 2014, 10, No. 20, 4160–4170
LSPR in nanomaterials has been utilized for photoacoustic tomography (PAT), an emerging noninvasive and nonionizing biomedical imaging modality that is of great current interest.[19] PAT is developed based on the photoacoustic effect of light-absorbers[19e] and thus Rb-TB with high NIR absorbance appeared to be a desired contrast agent for PAT imaging. As expected, the photoacoustic signal of Rb-TB increased upon increasing the concentration (Figure 6a) and correlated highly with Rb-TB concentrations (Figure 6b), indicating that Rb-TB should be a promising candidate for PAT imaging. Next, MCF-7 tumor-bearing nude mice were intratumorally injected with Rb-TB (3 mg/mL, 20 µL) and imaged under the PAT imaging system. No obvious signal could be found by PAT imaging prior to the Rb-TB injection (Figure 6c), while strong photoacoustic signals in the tumor of Rb-TB injected mice were observed (Figure 6d), confirming the good contrast enhancement property of Rb-TB for PAT imaging. In addition, considering the high atomic number and X-ray absorption coefficient of the tungsten (W) element, the Rb-TB could be used as a contrast agent in CT imaging, which is one of the most commonly used imaging
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Figure 6. Dual-modal PAT/CT imaging in vivo. a) PAT phantom images of Rb-TB aqueous solutions with different concentrations. b) Plot of photoacoustic signal versus Rb-TB concentrations. c,d) In vivo PAT images of MCF-7 tumor-bearing mice before and after intratumoral injection of Rb-TB (3 mg/mL, 20 µL), all scale bars are 3 mm. Tumor sites are marked with white dashed circles. e) CT images of Rb-TB and Ultravist 300 dispersed in agarose gel with different concentrations. f) HU values of Rb-TB and Ultravist 300 as the function of the sample concentrations. g) In vivo 3D CT images of mice after intratumoral injection of Rb-TB (3 mg/mL, 20 µL). Tumor sites are marked with red dashed circles.
tools for medical diagnosis and can afford high-resolution 3D structure details of tissues.[9,19f] The combination of PAT with CT imaging could offer whole-body imaging with high spatial resolution without imaging depth limitation and thus allow better understanding of the subtle changes in tumor micro-structures.[19f] Figure 6e,f presented the CT phantom images and Hounsfield units (HU) values of different concentrations of our prepared Rb-TB and a conventional iodine-based CT contrast agent named Ultravist 300 for comparison. As the sample concentration increased, the positive contrast enhancement signals were observed, and the signals from Rb-TB were much stronger than that of the commercial agent at each equivalent concentration. Correspondingly,
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the slope of HU values for Rb-TB was about 38.66 HU L/g, much higher than that of Ultravist 300 (13.25 HU L/g), indicating the good contrast efficacy of Rb-TB for CT imaging. We next tested the feasibility to use Rb-TB as a CT contrast agent for in vivo tumor imaging. Rb-TB dispersed in physiological saline were intratumorally injected into the MCF-7 tumor model in a nude mouse (3 mg/mL, 20 µL) and the tumor signal was clearly seen immediately after injection (Figure 6g), revealing that the developed Rb-TB were able to be used as an efficient contrast agent for tumor CT imaging.[20] Therefore, the as-prepared PVP-coated Rb-TB NRs could be potentially served as a dual-modal PTA/CT probe for deep-tissue imaging.
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3. Conclusion In summary, for the first time, NIR resonant tungsten bronze nanomaterials, such as Rb0.27WO3 nanorods used in this work, could be proposed as a new class of the light-responsible nanocarries for therapeutic drug delivery. The Rb-TB nanorods have satisfactory photo-thermal conversion efficacy at 17.8%. Utilizing DOX loaded Rb-TB, the combination of photothermal therapy and chemotherapy is achieved in vitro. Upon application to 808-nm NIR light, the photothermal effect of the Rb-TB led to a rapid rise in the local temperature and thus resulted in the accelerated release of the toxic DOX as well as the enhanced toxicity of DOX, exhibiting obviously synergistic therapy for normal MCF-7 and DOX-resistant MCF-7 cells in vitro. Moreover, the in vivo synergistic effect of combining photothermal therapy and chemotherapy in our drug delivery system was also demonstrated for Pance-1 tumor-bearing nude mice. In addition, Rb-TB with high NIR optical absorbance and X-ray absorption coefficient could serve as a promising dual-modal contrast agent for in vivo PAT/CT imaging with high spatial resolution. Our results not only promise the use of LSPRtunable tungsten oxide for imaging-guided combined cancer therapies but also encourage further exploration of other light-absorbing tungsten based nanomaterials for biomedical application.
4. Experimental Section All animal experiments were carried out under the standard protocols approved by the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety (Institute of High Energy Physics, CAS). Further experimental details can be found in the Supporting Information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements G.T. and X.Z. contributed equally to this work. This work was supported by National Basic Research Programs of China (973 program, No. 2012CB932504, 2011CB933403 and 2013CB933704), and National Natural Science Foundation of China (No. 21177128, 21171122 and 21101158).
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Received: May 6, 2014 Revised: June 3, 2014 Published online: June 30, 2014
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Multifunctional RbxWO3 Nanorods for Simultaneous Combined Chemo-photothermal Therapy and Photoacoustic/CT Imaging Gan Tian, Xiao Zhang, Xiaopeng Zheng, Wenyan Yin, Longfei Ruan, Xiaodong Liu, Liangjun Zhou, Liang Yan, Shoujian Li, Zhanjun Gu,* and Yuliang Zhao*
Light-triggered drug delivery based on near-infrared (NIR)-mediated photothermal nanocarriers has received tremendous attention for the construction of cooperative therapeutic systems in nanomedicine. Herein, a new paradigm of light-responsive drug carrier that doubles as a photothermal agent is reported based on the NIR lightabsorber, RbxWO3 (rubidium tungsten bronze, Rb-TB) nanorods. With doxorubicin (DOX) payload, the DOX-loaded Rb-TB composite (Rb-TB-DOX) simultaneously provides a burst-like drug release and intense heating effect upon 808-nm NIR light exposure. MTT assays show the photothermally enhanced antitumor activity of Rb-TB-DOX to the MCF-7 cancer cells. Most remarkably, Rb-TB-DOX combined with NIR irradiation also shows dramatically enhanced chemotherapeutic effect to DOX-resistant MCF-7 cells compared with free DOX, demonstrating the enhanced efficacy of combinational chemo-photothermal therapy for potentially overcoming drug resistance in cancer chemotherapy. Furthermore, in vivo study of combined chemo-photothermal therapy is also conducted and realized on pancreatic (Pance-1) tumor-bearing nude mice. Apart from its promise for cancer therapy, the as-prepared Rb-TB can also be employed as a new dual-modal contrast agent for photoacoustic tomography and (PAT) X-ray computed tomography (CT) imaging because of its high NIR optical absorption capability and strong X-ray attenuation ability, respectively. The results presented in the current study suggest promise of the multifunctional RbxWO3 nanorods for applications in cancer theranostics.
Dr. G. Tian, Prof. S. Li, Prof. Y. Zhao College of Chemistry Sichuan University Chengdu 610064, P. R. China Dr. G. Tian, Dr. X. Zhang, Dr. X. Zheng, Prof. W. Yin, Dr. L. Ruan, Dr. X. Liu, Dr. L. Zhou, Dr. L. Yan, Prof. Z. Gu, Prof. Y. Zhao Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics Chinese Academy of Sciences Beijing 100049, P. R. China E-mail: [email protected]; [email protected]
Dr. X. Zhang, Prof. Y. Zhao Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanosciences and Technology of China Beijing, 100190, P. R. China Dr. X. Zheng, Dr. L. Zhou College of Materials Science and Opto-electronic Technology Graduate University of Chinese Academy of Sciences Beijing, 100049, P. R. China
DOI: 10.1002/smll.201401237
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1. Introduction There is growing interest in combating cancer with nanoparticle-based therapeutics since nanocarriers-mediated drug delivery can overcome the intrinsic defects originating from the conventional therapeutic drugs, including poor watersolubility, limited stability, short circulation period and low selectivity.[1] Moreover, the development of external stimulus-sensitive drug delivery nanocarriers for cancer therapy has received tremendous attention in recent years, as these systems can differentially increase the drug accumulation at targeted cancer cells/tissues, drastically decrease the systemic toxicity, and potentially avoid under- or over-dosing.[2] Among them, light-responsible vehicles, especially those plasmonic nanomaterials that can convert near-infrared (NIR) light into heat energy and then serve as photothermal agents for localized hyperthermia cancer therapy are preferentially attractive because of the on-demand drug release at the desired sites in time with controllable therapeutic outcome due to the high spatial-temporal resolution of light stimuli,[3] the non-invasive nature of NIR laser-induced photothermal therapy (PTT) benefited from the high tissue-penetration ability and minimal photo-damage of NIR light,[4] and most importantly, the expected synergistic antitumor efficacy ascribed to the combination of PTT and chemotherapy.[5] Therefore, exploration on NIR light-responsive photothermal agents potentially engineered as drug vehicles to achieve enhanced anti-cancer effect is highly desired and important. Transition-metal oxide nanostructures with tunable localized surface plasmon resonance (LSPR) in the NIR region arising from the unique character of their outer-d valence electrons are of particular interest.[6] Recently, oxygen-deficient tungsten oxide (WO2.83) nanorods were certified to be capable to support a strong LSPR ranging from the red edge of the visible to the NIR region of light originating from the intervalence charge-transfer transition between W6+ and W5+.[7] Chen et al. developed the first tungsten oxide-based PTT agent, W18O49 nanowires, for cancer treatment and demonstrated that tumor suppression observed upon 980-nm NIR light irradiation of cancer cells with internalized PEG-coated W18O49 nanowires was solely attributed to the photothermally-induced hyperthermia.[8] Zhou et al. engineered the WO2.9 nanorods as a promising theranostic agent for simultaneous CT imaging and 980-nm NIR photothermal therapy of tumors in vivo.[9] In addition, metal tungsten oxide materials, such as tungsten bronzes (MxWO3, M = Cs, K, Na, and NH4; 0 < x < 1/3), which are constructed by the intercalation of small cationic ions into the WO3 framework,[10] possess similar LSPR properties with tungsten oxide and have also been emerged as a novel PTT agent for efficient photothermal ablation of human cancer cells in vitro under 980-nm NIR irradiation.[10d] However, despite those few pioneer work, the possibility of using either tungsten oxides or tungsten bronzes as drug delivery systems for synergistic thermo-chemotherapy has not yet been demonstrated to our best knowledge. In addition, 980-nm NIR laser is preferentially chosen as the light source in theses pioneer reports to heighten the photothermal efficacy in consideration of the increased small 2014, 10, No. 20, 4160–4170
optical absorbance at longer NIR wavelength of tungsten oxides based nanomaterials.[8,10d] However, the 980-nm light could be easily adsorbed by the water-rich biological tissues, which will result in limited penetration ability. Furthermore, the absorbed light energy would quickly transform into heat energy, inducing irreversible overheating effect, and thus causing significant cell death and tissue damage.[11] Alternatively, 808-nm laser with wavelength within the “biological window” has low absorption coefficient to water molecules and thus could greatly minimize the laser-induced heating effect and significantly improve the penetration depth in biotissue. Hence, using 808-nm light to trigger the generation of localized heat is much more safe and desirable.[12] Here, PVP-coated multifunctional rubidium tungsten bronze (RbxWO3) nanorods were engineered as a smart 808-nm NIR light-responsive nanocarries for synergistic chemo-photothermal therapy and yet as a new dual-modal probe for photoacoustic tomography (PAT)/X-ray computed tomography (CT) imaging (Scheme 1). Doxorubicin (DOX) was attached to the nanorods as the model drug through physical adsorption and could be on-demand released by the NIR light stimuli. In order to evaluate the therapeutic efficacy of the light-enhanced drug delivery system, a couple of normal MCF-7 and DOX-resistant MCF-7 (MCF-7-DOXR) cancer cells were chosen for in vitro cell killing experiments. As expected, synergistic effect was achieved for both of the two tested cancer cells. Moreover, the combination of chemotherapy and photothermal therapy with fascinating synergistic effect in vivo was also demonstrated for Pance-1 tumor-bearing nude mice. Due to the intrinsic high NIR optical absorbance of RbxWO3 and high X-ray absorption coefficient (4.438 cm2/kg at 100 keV) of tungsten (W),[9] in vivo PAT and CT imaging of MCF-7 tumor-bearing mice were also demonstrated. Therefore, this work demonstrates the potential of the tungsten bronze-based light-absorber as a new multifunctional platform for cancer imaging and therapy, and encourages further in-depth exploration of other plasmonic transition-metal oxides for theranostic nanomedicine.
2. Results and Discussion Rubidium tungsten bronze (Rb-TB) nanorods were prepared by the hydrothermal treatment of WO3 precursor and Rb2SO4 in an mixture solution containing ethylene glycol (EG) and polyvinyl pyrrolidone (PVP) at 180 °C for 16 h.[10a] TEM image in Figure 1a shows that the Rb-TB nanocrystals are predominantly rod-like in shape, and the particle size of nanorods is found to be 20–40 nm in length and about 5 nm in diameter (Figure S1 in the Supporting Information). XRD patterns presented in Figure 1b reveal that all the reflections could be well indexed to the hexagonal Rb0.27WO3 (JCPDS No. 73–1549) and no impurities are detected. X-ray photoelectron spectroscopy (XPS) analysis (Figure 1c) reveals that the elements O (1s, 532.1 eV), N (1s, 399.3 eV), C (1s, 284.7 eV), Rb (3d, 109.7 eV) and W (4f, 35.32 eV) could be determined and the atomic ratio of Rb/W is calculated to be 0.24, which is very close to the value from the XRD results. In addition, the oxidation state of tungsten could also be
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Scheme 1. Illustration of the multifunctional RbxWO3 nanorods for NIR light-triggered synergetic chemo-photothermal therapy and dual-modal PAT/CT imaging.
distinguished by the XPS analysis. Figure 1d shows the W4f XPS spectra of the as-obtained Rb0.27WO3 nanorods. The W4f core-level spectrum can be well fitted into two spin-orbit doublets, corresponding to two different oxidation states of W atoms. Two peaks centered at 37.52 and 35.42 eV correspond to W6+, while the other two peaks with lower binding energy at 36.37 and 34.72 eV belong to W5+.[10a,13] The existence of W5+ should be due to the fact that a portion of the W6+ ions are reduced by PVP and ethylene glycol under hydrothermal condition. Figure 1e exhibits the FT-IR spectrum of the as-prepared samples. The peak at 1639 cm−1 should be related to a strong C=O adsorption from the lactam group in the side chains of the PVP.[4b,14] The bands at 1430 and 1284 cm−1 are corresponding to the pyrollidone ring and the N→HO complex vibrations, respectively.[14a] Moreover, the broad band centered at 1104 cm−1 corresponds to C–N stretching vibration.[14b] Based on the above results, the successful coating of PVP ligands on the surface of Rb-TB could be confirmed. In addition, the amount of PVP on nanoparticles was determined to be 6.2% by thermogravimetric analysis (TGA, Figure S2, Supporting Information). As a result, the PVP-coated Rb0.27WO3 nanorods are hydrophilic and can be readily dispersed in water, showing a dark blue color (Figure 1f inset). The optical absorption spectrum of their aqueous dispersion was investigated using UV–vis– NIR spectroscopy (Figure 1f). A very large absorption tail presented in the visible and near infrared (NIR) regions can be clearly observed, which can be explained by the free electrons induced SPR effect and/or oxygen-deficiency induced small polarons absorption.[7–9] In addition, the UV–vis–NIR reflectance diffuse spectrum (Figure S3, Supporting Information) confirms the enhanced photo-absorption ability as the wavelength increases in the NIR region.
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The strong photo-absorption in NIR region promotes us to investigate their photo-thermal effect property under NIR laser irradiation. As mentioned above, previous reports always used the 980-nm laser other than 808-nm laser as the light source to investigate the photothermal behavior of tungsten oxide based plasmonic nanomaterials in consideration of the gradually increased optical absorbance at longer wavelength region. However, 980-nm light with water absorption coefficient at 0.48 cm−1 would be significantly attenuated to cause limited penetration depth and irreversible overheating effect in bio-tissue, while this vital side-effect could be largely alleviated when using 808-nm laser with water adsorption coefficient at 0.02 cm−1.[11b,12b] As seen in the Figure 2a, the temperature of the water irradiated by 980-nm laser increased from 25 °C to 37.35 °C, a rise of 12.35 °C, while the one under 808-nm irradiation was heated to a temperature at 28.31 °C with a rise of only 3.31 °C. In order to demonstrate the weak water absorption of 808-nm NIR light, we measured the energy loss of 808-nm and 980-nm laser after they pass through water columns with different thickness. As clearly presented in the Figure 2b, after penetrating the same thickness of water, the fall of laser intensities of 808-nm were much smaller than that of 980-nm laser. Meanwhile, the temperature elevation (Figure 2c) and fluctuation (Figure 2d) of the Rb0.27WO3 dispersion (≈0.25 mg/mL) induced by 808-nm or 980-nm irradiation after passing through a certain thickness of water accordingly showed that temperature rising induced by 980-nm irradiation decreased much more rapidly along with the increased thickness of the water block compared with that of 808-nm irradiation, demonstrating that 808-nm light is much more resistant to water absorption and more reliable for water-rich deep-tissue application.
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Figure 1. Materials characterization. a) TEM image and b) wide-angle XRD pattern of the as-prepared RbxWO3 nanorods. Bottom in (b): the standard pattern of pure hexagonal Rb0.27WO3 (JCPDS No: 73–1549). c) Full range and d) W4f core-level XPS spectrum of the Rb0.27WO3 nanorods. e) FT-IR spectra of the as-prepared PVP-coated samples, inset: chemical structure of PVP molecule. f) UV–vis–NIR absorption spectrum of an aqueous dispersion containing 1 mg/mL Rb0.27WO3. Inset: Photograph of the corresponding aqueous dispersion.
Then, we systematically investigated the photothermal behavior and conversion efficiency of Rb0.27WO3 under 808-nm laser irradiation with a power density at 1.0 W/cm2. Temperature trends of this material dispersed within water as shown in Figure 3a declare that Rb0.27WO3 nanorods can efficiently convert the 808-nm laser energy into heat energy and the photothermal heating effect is positively correlated with concentration. Figure 3b shows the temperature changes (ΔT) in 10 min irradiation rise rapidly with the concentration increasing to 0.25 mg/mL and then slowly go up upon increasing the concentration to 1.0 mg/mL. This trend could be explained by the faster heat loss at higher temperature.[3b,4c] The conversion efficiency (η) is an important character for photothermal materials. As far as we know, no relative data about theη value of the tungsten oxides or tungsten bronzes has been reported.[15] Here, the η value of the Rb0.27WO3 NPs was measured by a modified method and calculated as follows:[3c] small 2014, 10, No. 20, 4160–4170
η=
hS (Tmax − Tsurr ) − QDis I (1 − 10 − A808 )
(1)
θ=
T − Tsurr Tmax − Tsurr
(2)
t = −τ s ln θ
τs =
mC p hS
(3) (4)
Where h is the heat transfer coefficient, S is the surface area of the container, Tmax is the maximum steady-state temperature, Tsurr is the ambient temperature of the surroundings, T is the solution temperature, QDis is the heat associated with the light absorbance of the solvent, I is the laser power, A808 is the absorbance of Rb0.27WO3 solution at 808 nm, τs is the system time constant, m and Cp are the mass and heat capacity of water, respectively.
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Figure 2. Comparative study of 980-nm and 808-nm NIR laser. a) Temperature elevation of water (1 mL) under 808-nm and 980-nm laser irradiation, respectively. b) Power attenuation trend of 808-nm and 980-nm lasers after they passed a water column with different thickness. c) Temperature elevation and d) temperature change of Rb0.27WO3 aqueous suspensions (≈0.25 mg/mL) irradiated by 808-nm and 980-nm irradiation with the same power density at 1.0 W/cm2 when the laser were shielded by water column with different thickness. Error bars are calculated based on triplicated samples.
Figure 3. Photothermal properties investigation. a) The temperature elevation of aqueous dispersions of Rb0.27WO3 with different concentrations as a function of irradiation time of an 808 nm CW laser with power density (1.0 W/cm2). b) Plot of temperature change (ΔT) over a period of 10 min irradiation versus Rb0.27WO3 concentration. Inset: IR thermal images of water and Rb0.27WO3 solution (1.0 mg/mL) after laser irradiation at 1.0 W/cm2 for 10 min. Error bars are calculated based on triplicated samples. c) The photothermal response of the Rb0.27WO3 aqueous solution (≈1.0 mg/mL) under an 808-nm laser irradiation (1 W/cm2) for 720 s and then the laser was turned off. d) Linear time data from the cooling period (after 720 s) versus negative natural logarithm of driving force temperature (−lnθ) for time constant (τs) determination.
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We collected the temperature rising (laser on) and cooling (laser off) curves of the Rb0.27WO3 dispersion with concentration at 1.0 mg/mL (Figure 3c). From the temperature cooling curve, we could calculate the sample system time constant τs (Figure 3d) based on Equation 2,3 and then obtain the hS value from Equation 4. Finally, according to Equation 1, the η value of was determined to be 17.8%. In addition, our Rb0.27WO3 samples exhibit good photostability towards 808-nm laser since no significant difference in optical absorbance or color of the tested Rb0.27WO3 suspension has been obtained even after 808-nm laser exposure for 3 h (Figure S4, Supporting Information), indicating they are promising as an ideal photothermal converter in cancer therapy. Nanomaterials with versatile surface chemistry and large surface area have been widely employed as nanocarriers for therapeutic drug delivery. The as-prepared Rb0.27WO3 nanorods were coated with a PVP layer with weight ratio up to 6.2%, which may provide adsorption sites for drug molecules intercalation through physical interactions. To test our hypothesis, DOX was loaded as a guest molecule by soaking PVP-coated Rb0.27WO3 nanorods with DOX aqueous solutions overnight. Reddish precipitate and nearly colorless supernatant were observed after the mixture was centrifuged (Figure 4a inset), demonstrating the successful loading of DOX onto Rb-TB nanorods. Based on UV–vis absorption measurements, the loading capacity for DOX molecules stored on the Rb-TB is 0.326 mmol/g (Figure 4a). The in vitro DOX release behaviors of Rb-TB-DOX were performed in PBS medium at different pH values. In the absence of laser irradiation, only 14.65% and 21.65% DOX are released within 7 h at the pH 7.4 and pH 5.0 under ambient temperature, respectively. However, burst release was observed upon 808-nm laser irradiation, and the rate subsequently slowed when the laser was shut off. After initial irradiation for 15 min (1.0 W/cm2), the DOX release was significantly increased from 8.50% to 16.77% at pH 7.4, and from 12.07% to 28.83% at pH 5.0. The cumulative release of DOX reached up to 40.86% and 63.31% within 7 h at pH 7.4 and pH 5.0, respectively, which are much higher than their counterparts without irradiation (Figure 4b). The stepwise triggered rapid DOX release under NIR irradiation should be ascribed to the photothermal effect of Rb-TB nanorods since increasing local temperature would dissociate the interactions between DOX and PVP and thus more DOX molecules are detached (Figure S5, Supporting Information) from the nanocarriers.[5e] In addition, the release of DOX exhibited a pH-responsive pattern, where a higher cumulative DOX release is achieved in relation to a lower pH value. The phenomenon can be attributed to the reduction of the hydrophobic-hydrophobic interaction between DOX and PVP polymer, where DOX molecule becomes more hydrophilic at lower pH values, weakening its binding to the long hydrophobic alkyl chain of PVP polymer. Such a pH-dependent release hallmark is important for cancer therapy because of the lower pH value in tumor microenvironment compared with the normal tissue.[16] To further illustrate the potential of light-responsible Rb-TB nanocarries in the NIR-stimulus synergistic chemo/ small 2014, 10, No. 20, 4160–4170
photothermal-therapy field, a couple of cancer cells, MCF-7 and DOX-resistant MCF-7 (MCF-7-DOXR) cells were tested. MTT assay showed that Rb-TB nanocarrier was nontoxic to both MCF-7 and MCF-7-DOXR cells up to a tested concentration of 800 µg/mL (Figure S6, Supporting Information). First, we incubated MCF-7 cells with Rb-TB and Rb-TBDOX and evaluated the cytotoxicity with and without laser irradiation. As expected, the viability of MCF-7 cells treated with Rb-TB-DOX dramatically decreased upon 808-nm laser irradiation with 1.0 W/cm2 for 5 min, the dose of which has been proved to be safe for living cells (Figure S7, Supporting Information). Compared with the control groups without laser irradiation or no DOX loading, those treated with Rb-TB-DOX under irradiation held higher potency against MCF-7 cells at each concentration (Figure 4c). For instance, at the equivalent Rb-TB concentration of 25 µg/mL, 18.5% and 32.5% of the cells were killed in the case of being treated with bare Rb-TB with NIR irradiation and Rb-TB-DOX without NIR irradiation, respectively. In comparison, 78.2% of the cells were killed in the presence of Rb-TB-DOX with 5 min NIR irradiation. The cell-killing efficacy by Rb-TBDOX under NIR irradiation was even higher than the sum of chemotherapy by Rb-TB-DOX and photothermal therapy by DOX-free Rb-TB, indicating the synergy was achieved. This synergistic effect was probably ascribed to the increased drug release inside cells upon NIR irradiation and the enhanced cytotoxicity of DOX at elevated temperatures.[5b,5d] The occurrence of multi-drug resistance (MDR) is a major impediment to the success of cancer chemotherapy. In the following, we assessed the anti-tumor efficacy of Rb-TB-DOX in MCF-7-DOXR cells treatment. From Figure 4d, we could find that MCF-7-DOXR cells had strong drug resistance since free DOX induced no significant inhibition even at a DOX dosage of 50 µm. Meanwhile, Rb-TB-DOX without laser irradiation showed the comparable inhibiting ability with free DOX. Interestingly, potent inhibition of MCF-7-DOXR cells was obtained when cells were treated with Rb-TBDOX under 5 min laser irradiation (1.0 W/cm2), suggesting these photo-responsive Rb-TB-DOX composites are able to overcome MDR mechanism, likely due to a combination of the decreased chemoresistance sensitiveness contributed by thermal effect[17] and the light-triggered boosted drug release since the fast intracellular drug release feature is able to reverse MDR mechanisms, such as drug efflux.[18] Moreover, we also performed MCF-7 or MCF-7-DOXR nucleus staining experiments using Hochest 33342 to visually illuminate the synergy effect, where brighter ones are dead cells. As seen in the Figure 4e, when treated with Rb-TB-DOX as well as laser irradiation, much more number of MCF-7 cells were found to be brighter compared to Rb-TB without laser or RbTB-DOX treated case at equivalent Rb-TB concentration (25 µg/mL). Similar results were obtained for MCF-7DOXR nucleus staining with equivalent DOX concentration (12.5 µm), larger amount of cells treated with Rb-TB-DOX under NIR exposure were bright than that of free DOX or Rb-TB-DOX treated case (Figure 4f). Encouraged by the apparent synergistic effect in vitro, we then performed a pilot chemo-photothermal therapy study in vivo. Five groups of Pance-1 tumor-bearing nude mice with
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Figure 4. Controlled drug loading/releasing behavior and combined photothermal-chemotherapy treatment. a) Quantification of drug loading capacity at different DOX concentrations. Inset: photographs showing the reddish precipitate and nearly colorless supernatant were observed after the mixture of Rb-TB and DOX (100 µm) was centrifuged. b) Release kinetics of DOX from Rb-TB in PBS medium at pH values of 7.4 and 5.0 with or without laser irradiation at 808 nm (1.0 W/cm2). c) Viabilities of normal MCF-7 cells incubated with different concentrations of testing materials. d) Fluorescent images of normal MCF-7 nucleus stained with Hoechst 33342. The tested materials are of equivalent Rb-TB concentrations (25 µg/ mL). Brighter ones are dead cells. e) Dependence of cell viabilities on DOX concentrations for MCF-7-DOXR. f) Hoechst 33342 staining MCF-7-DOXR nucleus. The tested materials are of equivalent DOX concentration at 12.5 µm. 808-nm laser irradiation parameters: 1.0 W/cm2, 5 min. Error bars are calculated based on triplicated samples. All scale bars in fluorescent images are 100 µm.
3 mice per group were used in our experiment. Each group of mice were intratumorally injected with 20 µL of saline, free DOX, Rb-TB or Rb-TB-DOX ([DOX] = 0.5 mg/kg) with or without 808-nm NIR irradiation (1 W/cm2) for 10 min. As seen from the Figure 5a,b, the temperature of tumor injected with Rb-TB or Rb-TB-DOX could increase up to 50 °C, with a raise of ≈17.5 °C, while the control group treated with saline
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showed insignificant change (ΔT = ≈5 °C), indicating that the injected Rb-TB or Rb-TB-DOX could induce sufficient hyperthermia to kill cancer cells under 808-nm NIR irradiation. The tumor volume measured for each group is plotted as a function of time intervals in Figure 5c. The control group treated only with saline exhibited rapidly increasing tumor volumes. Compared with the control group, there was only
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Figure 5. In vivo combined chemo-photothermal therapy study. a) IR thermal images of Pance-1 tumor-bearing mice collected by an IR camera at different time intervals during 808-nm laser irradiation (1.0 W/cm2 for 10 min). b) Temperature change of tumors monitored by the IR thermal camera during laser irradiation. c) Tumor volume growth curves of mice after different treatments. d) Photograph of tumors after excision. e) Tumor weights of each group. Error bars are calculated based on triplicated samples.
a slight inhibition of tumor growth treated with free DOX, suggesting that free DOX at this low dose was not sufficient to inhibit the tumor growth. The tumors treated with RbTB-DOX without NIR laser irradiation showed enhanced tumor regression compared with free DOX-treated group, mainly due to the longer retention of Rb-TB-DOX inside the tumor after local administration.[5e] For the mice treated with Rb-TB under NIR light, noticeable tumor inhibition in the preliminary stage could be certified due to the laser induced hyperthermia. Unfortunately, the tumor growth in this group became uncontrollable later. In marked contrast, the tumor growth in the group treated with Rb-TB-DOX under NIR irradiation was dramatically inhibited as a result of combined chemotherapy and photothermal therapy. Figure 5d,e represented the photographs and mean weight of the excised tumors of each group, which could well coincide with the final results as indicated in Figure 5c. These results demonstrated the in vivo synergistic effect of combining photothermal therapy and chemotherapy in our drug delivery system. Ideal nanoparticle systems in nanomedicine should possess multimodality in both imaging and therapy. Recently, small 2014, 10, No. 20, 4160–4170
LSPR in nanomaterials has been utilized for photoacoustic tomography (PAT), an emerging noninvasive and nonionizing biomedical imaging modality that is of great current interest.[19] PAT is developed based on the photoacoustic effect of light-absorbers[19e] and thus Rb-TB with high NIR absorbance appeared to be a desired contrast agent for PAT imaging. As expected, the photoacoustic signal of Rb-TB increased upon increasing the concentration (Figure 6a) and correlated highly with Rb-TB concentrations (Figure 6b), indicating that Rb-TB should be a promising candidate for PAT imaging. Next, MCF-7 tumor-bearing nude mice were intratumorally injected with Rb-TB (3 mg/mL, 20 µL) and imaged under the PAT imaging system. No obvious signal could be found by PAT imaging prior to the Rb-TB injection (Figure 6c), while strong photoacoustic signals in the tumor of Rb-TB injected mice were observed (Figure 6d), confirming the good contrast enhancement property of Rb-TB for PAT imaging. In addition, considering the high atomic number and X-ray absorption coefficient of the tungsten (W) element, the Rb-TB could be used as a contrast agent in CT imaging, which is one of the most commonly used imaging
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Figure 6. Dual-modal PAT/CT imaging in vivo. a) PAT phantom images of Rb-TB aqueous solutions with different concentrations. b) Plot of photoacoustic signal versus Rb-TB concentrations. c,d) In vivo PAT images of MCF-7 tumor-bearing mice before and after intratumoral injection of Rb-TB (3 mg/mL, 20 µL), all scale bars are 3 mm. Tumor sites are marked with white dashed circles. e) CT images of Rb-TB and Ultravist 300 dispersed in agarose gel with different concentrations. f) HU values of Rb-TB and Ultravist 300 as the function of the sample concentrations. g) In vivo 3D CT images of mice after intratumoral injection of Rb-TB (3 mg/mL, 20 µL). Tumor sites are marked with red dashed circles.
tools for medical diagnosis and can afford high-resolution 3D structure details of tissues.[9,19f] The combination of PAT with CT imaging could offer whole-body imaging with high spatial resolution without imaging depth limitation and thus allow better understanding of the subtle changes in tumor micro-structures.[19f] Figure 6e,f presented the CT phantom images and Hounsfield units (HU) values of different concentrations of our prepared Rb-TB and a conventional iodine-based CT contrast agent named Ultravist 300 for comparison. As the sample concentration increased, the positive contrast enhancement signals were observed, and the signals from Rb-TB were much stronger than that of the commercial agent at each equivalent concentration. Correspondingly,
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the slope of HU values for Rb-TB was about 38.66 HU L/g, much higher than that of Ultravist 300 (13.25 HU L/g), indicating the good contrast efficacy of Rb-TB for CT imaging. We next tested the feasibility to use Rb-TB as a CT contrast agent for in vivo tumor imaging. Rb-TB dispersed in physiological saline were intratumorally injected into the MCF-7 tumor model in a nude mouse (3 mg/mL, 20 µL) and the tumor signal was clearly seen immediately after injection (Figure 6g), revealing that the developed Rb-TB were able to be used as an efficient contrast agent for tumor CT imaging.[20] Therefore, the as-prepared PVP-coated Rb-TB NRs could be potentially served as a dual-modal PTA/CT probe for deep-tissue imaging.
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3. Conclusion In summary, for the first time, NIR resonant tungsten bronze nanomaterials, such as Rb0.27WO3 nanorods used in this work, could be proposed as a new class of the light-responsible nanocarries for therapeutic drug delivery. The Rb-TB nanorods have satisfactory photo-thermal conversion efficacy at 17.8%. Utilizing DOX loaded Rb-TB, the combination of photothermal therapy and chemotherapy is achieved in vitro. Upon application to 808-nm NIR light, the photothermal effect of the Rb-TB led to a rapid rise in the local temperature and thus resulted in the accelerated release of the toxic DOX as well as the enhanced toxicity of DOX, exhibiting obviously synergistic therapy for normal MCF-7 and DOX-resistant MCF-7 cells in vitro. Moreover, the in vivo synergistic effect of combining photothermal therapy and chemotherapy in our drug delivery system was also demonstrated for Pance-1 tumor-bearing nude mice. In addition, Rb-TB with high NIR optical absorbance and X-ray absorption coefficient could serve as a promising dual-modal contrast agent for in vivo PAT/CT imaging with high spatial resolution. Our results not only promise the use of LSPRtunable tungsten oxide for imaging-guided combined cancer therapies but also encourage further exploration of other light-absorbing tungsten based nanomaterials for biomedical application.
4. Experimental Section All animal experiments were carried out under the standard protocols approved by the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety (Institute of High Energy Physics, CAS). Further experimental details can be found in the Supporting Information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements G.T. and X.Z. contributed equally to this work. This work was supported by National Basic Research Programs of China (973 program, No. 2012CB932504, 2011CB933403 and 2013CB933704), and National Natural Science Foundation of China (No. 21177128, 21171122 and 21101158).
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Received: May 6, 2014 Revised: June 3, 2014 Published online: June 30, 2014
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