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Multifunctional Bi2WO6 Nanoparticles for CT-Guided Photothermal and Oxygen-Free Photodynamic Therapy Chao Zhang, Jing Ren, Jisong Hua, Luyao Xia, Jian He, Da Huo, and Yong Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16000 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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ACS Applied Materials & Interfaces

Multifunctional Bi2WO6 Nanoparticles for CT-Guided Photothermal and Oxygen-Free Photodynamic Therapy Chao Zhang,†,‡ Jing Ren,§ Jisong Hua,† Luyao Xia,† Jian He,*, ‡ Da Huo,*,† and Yong Hu*,† †

Collaborative Innovation Center of Chemistry for Life Sciences, College of Engineering and Applied Sciences, Nanjing University, Jiangsu, 210093, China. ‡

Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing, 210008, China. §

The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, China.

ABSTRACT: The consumption of oxygen in photodynamic therapy (PDT) significantly exacerbates the degree of hypoxia in tumors, which not only impedes the therapeutic effect of PDT but also drives local tumor recurrence. To relieve the PDT-induced hypoxia and improve the therapeutic outcome of PDT in cancer treatment, herein we reported a class of Bi2WO6 nanoparticles (NPs) as a robust multifunctional platform, which integrates the abilities for contrastenhanced computed tomography (CT) imaging, photothermal therapy (PTT) and PDT in an oxygen-free manner. The as-obtained Bi2WO6 NPs with a mean diameter of 5.2 nm are stable in PBS and an in vivo microenvironmentmimicking buffer. The location of the solid tumor could be accurately positioned using Bi2WO6-enhanced CT with higher spatial resolution. After being irradiated with an 808-nm laser, these Bi2WO6 NPs could realize CT-guided local photothermal ablation of the tumor. Meanwhile, •OH radicals were generated simultaneously from the treatment without consuming an oxygen molecule, which enabled these Bi2WO6 NPs to exert photodynamic killing effect in an oxygen-free manner during cancer therapy. Remarkable tumor suppression was observed in mice bearing the HeLa xenograft, supporting the promising application of these multifunctional Bi2WO6 NPs in the combat against cancers through synergistic photothermal and oxygen-free PDT treatment.

KEYWORDS: photodynamic therapy, photothermal therapy, nanoparticles, computed tomography, hypoxia

1. INTRODUCTION Hypoxia is generally characterized by an insufficient oxygen supply and has been shown to exist in most solid tumors due to the rapid proliferation of the tumor cells and the incomplete development of vasculature.1,2 Hypoxia not only induces the development and metastasis of the tumor3-5 but also severely reduces the sensitivity of the tumor tissue to conventional chemotherapy, radiotherapy and photodynamic therapy (PDT),6-9 leading to a major cause of cancer treatment failure. Photodynamic therapy (PDT) has been proven to be one of the most promising clinical cancer treatments and has drawn much attention because it is noninvasive and highly se-

lective.10-15 In PDT, reactive oxygen species (ROS) can be generated from a PDT agent illuminated by light to kill the cancer cells. However, most conventional PDT agents, such as chlorine e6 (Ce6) and indocyanine green (ICG), can only generate singlet oxygen (1O2) by reacting with the local O2, which exacerbates the lack of oxygen and serious degree of hypoxia inside a tumor, making the tumor cells less sensitive to the PDT treatment and enhancing their survival against PDT.16-18 Moreover, the tumor cells that survive the PDT treatment are more resistant and malignant, and they proliferate throughout the entire solid tumor and contribute to rapid local recurrence.19 Therefore, oxygen-loaded carriers are developed to supply gaseous oxygen to a solid tumor to enrich the

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intratumoral oxygen level and potentiate the PDT effect.20-23 However, the system capable of loading and delivering O2 is very complicated and costly. Most importantly, the high interstitial pressure in the hypoxic region greatly hinders the penetration of the oxygen gas, thus restricting the PDT effect.22, 24 To address this limitation, PDT systems that can endogenously generate molecular oxygen are reported. O2 can be obtained by the decomposition of H2O2 upon manganese dioxide nanoparticles25 or from water splitting inside the tumor,26 and ROS can be produced inside the tumor from conventional PDT agents illuminated by light without the addition of external O2. Inspired by these works and the principle of PDT, we think that novel agents that can directly split the water to ROS will also exert a PDT effect. Meanwhile, the success of PDT against the tumor also relies on the accurate location of the tumor for light irradiation, which can maximize the light coverage on the tumor while minimizing the damage to surrounding normal tissues. To this end, PDT agents, integrating ROS generation and imaging ability, are highly desired in PDT treatment.27-34 Recently, Wang et al. reported that a monolayer of Bi2WO6 had a highly active surface for photocatalytic oxidation reactions, which could oxidize H2O (or OH-) to •OH upon laser irradiation ranging from UV to near infrared (NIR).35 Thus, we envisioned that Bi2WO6 can produce ROS without the consumption of molecular oxygen and thus open a new avenue for oxygen-free PDT. However, the monolayer Bi2WO6 is large in size and has poor water solubility, which makes is difficult to directly translate into in vivo practical applications. To circumvent these hurdles, in this study, we reported the synthesis of ultrafine Bi2WO6 NPs (~5 nm) that were functionalized with carboxylic acid groups and exhibited excellent water dispersion. These Bi2WO6 NPs could efficiently generate •OH under laser irradiation in aqueous solution without the presence of oxygen and thus kill the cancer cells and destroy the solid tumor effectively. Importantly, it is known that metals with a high atomic number (Z > 50) have strong X-ray attenuating potency, and therefore, they can be used as enhanced computed tomography (CT) contrast agents.36,37 As expected, since they contain W (Z=74) and Bi (Z=83) with high atomic numbers, these Bi2WO6 NPs also showed an excellent CT imaging capability, featuring CT imaging guidance in PDT. Furthermore, because of their strong localized surface plasmon resonance (LSPR), Bi2WO6

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NPs have the promise of inducing hyperthermia and greatly contributing to tumor elimination. The in vitro antitumor results revealed that Bi2WO6 NPs have both photothermal therapy (PTT) and PDT functions to destroy cancer cells. The in vivo antitumor results clearly demonstrated that these Bi2WO6 NPs can efficiently erase the solid tumor tissue through CT-guided PTT and PDT without the aid of oxygen. To the best of our knowledge, this is the first attempt to develop multifunctional NPs integrating the abilities for CT imaging, PTT and oxygen-free PDT. We believe that this novel system will play an active role in future clinical practice by overcoming the PDT-induced hypoxia in cancer treatment. 2. EXPERIMENTAL SECTION 2.1 Materials. Na2WO4•2H2O, Bi(NO3)3• 5H2O, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cobalt(II) chloride hexahydrate (CoCl2•6H2O, Bioreagent), and 3,4-dihydro-2,2dimethyl-2H-pyrrole 1-oxide (DMPO) were all bought from Sigma-Aldrich (St. Louis, MO). Calcein-AM, Ethidium Homodimer-1 (EthD-1), 2’,7’dichlorodihydrofluoresceindiacetate (H2DCFDA), phosphate buffered saline (PBS, pH~7.2), 2,2,6,6tetramethylpiperidine (TEMP), N-acetylcysteine (NAC), Singlet Oxygen Sensor Green (Thermo Fisher Scientific) and Tempo-9-AC (Synchem UG & Co. KG, Germany) were all obtained from Invitrogen (Thermo Fisher Scientific, Carlsbad, CA). Oleic acid, potassium permanganate (KMnO4), ammonia water (NH3•H2O), and nitric acid (65%-68%, wt%) were all purchased from J&K SCIENCE (Beijing, China). The human cervical carcinoma cell line (HeLa) and human umbilical vein endothelial cells (HUVECs) were both purchased from the Shanghai Institute of Cell Biology (Shanghai, China). 2.2 Synthesis of Bi2WO6 NPs. Bi2WO6 NPs were synthesized as indicated in Scheme S1. To prepare Bi2WO6 NPs, 0.2 mmol of Bi(NO3)3•5H2O was dissolved in 5 mL of nitric acid. After 10 min, 45 mL of distilled water containing 0.1 mmol Na2WO4•2H2O was added to the solution. The oil bath was heated to 100 °C and the reaction was allowed to proceed react for 50 min at this temperature. Afterwards, 0.2 mL of NH3•H2O and 0.1 mL of oleic acid were added, and the temperature of oil bath was raised to 170 °C and then reacted for another 50 min. After that, the resultant yellow precipitate was harvested via centrifugation, washed thoroughly with


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ethyl alcohol and PBS in triplicate to remove the residue. The yellow precipitate was then mixed with 50 mL of 10 mg/mL KMnO4 solution and reacted under ultrasonication for 6 h to obtain carboxylic acidfunctionalized Bi2WO6 NPs. The resultant carboxylic acid-functionalized Bi2WO6 NPs (abbreviated as Bi2WO6 NPs) were obtained by thrice centrifugation and washing with PBS. The purified carboxylic acidfunctionalized Bi2WO6 NPs were freeze-dried and stored at 4 °C before use. 2.3 Characterization. The morphology of the Bi2WO6 NPs was observed by transmission electron microscopy (JEOL, TEM-2100, TEM-100). Element of W and Bi were detected by a Sectional Energy Dispersive Spectral (EDS) analysis with corresponding color mapping. The hybrid bonding state of the elements W and Bi was tested with the help of X-ray photoelectron spectroscopy (XPS, ThermoFisher KAlfa). The UV-vis absorption spectra of the Bi2WO6 NPs were recorded on a spectrophotometer (UV3100, Shimadzu, Japan). The X-ray diffraction (XRD) analyses was performed on a Hitachi X-ray diffractometer (λ=1.54056 Å, 40 kV, 200 mA). The size distribution was tested using a dynamic light scattering (DLS) technology (BI-9000AT, Brookhaven, USA). The results were carried out in triplicate. The tungsten concentrations were measured with an inductively coupled plasma-mass spectrometer (ICP-MS, NexION 300 D, Perkin-Elmer Corporation, USA). The data were presented as an average of three measurements. An 808-nm diode laser (LEO photonics Co. Ltd) was used to study the photothermal effect and photodynamic effect. The change of temperature of NPs exposed to 808-nm laser was investigated in a tin foil-capped quartz cell to prevent the evaporation of water. The photothermal conversion efficiency (η) was calculated as following equations38 (c: specific heat capacity of Bi2WO6 NP solution, 4.2×103 J/(kg·K); m: mass of Bi2WO6 NP solution, 3.83×10-3 kg; ∆t: temperature increase of Bi2WO6 NP solution; w: the laser power, 1W; t: the laser irradiation time, 600s).

η (%) = 100%

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× 100% =

ସ.ଶ×ଵ଴య ×ଷ.଼ଷ×ଵ଴షయ ∆௧ ଵ×଺଴଴

×

2.4 In Vitro Cytotoxicity Assay (MTT Assay). The cytotoxicity of Bi2WO6 NPs was analyzed with the aid of an MTT test. Briefly, HeLa cells and HUVECs were seeded in 96-well plates (2 × 103 cells/ well) and incubated overnight. After that, the cells were washed and incubated with 200 µL of fresh culture medium containing various concentrations of Bi2WO6 NPs. A 20 µL of MTT (5 mg/mL) was added into each well at 24 or 48 h

later, respectively. After another 4 h of incubation, the medium containing the MTT was removed, and the formazan crystals were dissolved after the addition of 150 µL of DMSO for 0.5 h. The absorbance of each well at 490 nm was determined with an iMark Enzyme mark instrument (BIO-RAD Inc., USA). The results were obtained from the average of thrice test. 2.5. Biodistribution of Bi2WO6 NPs in vivo. 0.1 mL of Bi2WO6 NPs in a concentration of 1 mg tungsten/mL saline were injected intratumorally and intravenously respectively into the HeLa-xenograft-bearing mice, and the mice were sacrificed (for each time point n = 5) 0.5, 1, 3, 8, 12 and 24 h after injection. The tumor, heart, liver, spleen, lung and kidney were excised and weighed. Then, the collected organs were completely lysed in aqua regia, and quantified by an inductively-coupled plasma mass spectrometer (ICP-MS, NexION 300 D, Perkin-Elmer Corporation, USA). 2.6 In Vitro and in Vivo CT Imaging. For in vitro CT imaging, suspensions of Bi2WO6 NPs (in terms of the concentrations of tungsten, 6.25, 12.5, 25, 50, and 100 mM, respectively) and suspensions of the clinical iodine contrast agent iodixanol (in terms of the concentrations of iodine, 6.25, 12.5, 25, 50, and 100 mM, respectively) were added to 48-well plates. The CT imaging was performing on a clinical CT Gemstone spectral 64-Detector CT (Discovery CT 750HD, GE Amersham Healthcare System, Milwaukee, WI) with X-ray anode voltage of 70 kV and a current of 400 µA. For in vivo CT imaging, HeLa-tumor-bearing mice were injected intratumorally with 40 µL of Bi2WO6 NPs (the concentration of tungsten was 50 mM) and 40 µL of iodixanol (the concentration of iodine was 100 mM). The analysis was carried out at 3 h after injection, and the instrument and the parameters were the same as mentioned in the in vitro assay. 2.7 In Vivo Infrared Thermal Imaging of Mice. HeLa-tumor-bearing mice that received an intratumoral injection of 100 µL of PBS or Bi2WO6 NPs (1 mg/mL) were irradiated by an 808-nm laser 8 h after injection. The laser device had a 200 µm diameter fiber, and the beam diameter could be expanded to 11.4 mm with the help of an optical lens, so that the entire tumor area could be exposed. The output power density was 1 W/cm2 and the irradiation time was 20 min. The change in temperature caused by the irradiation was determined



using an infrared thermographic camera (ThermaCAM SC3000, FLIR, USA). 2.8 Detection of ROS. The capability of Bi2WO6 to sensitize ROS formation upon photoexcitation was investigated using the DMPO and TEMP spin-trapping electron paramagnetic resonance (EPR) technique. Briefly, solutions containing Bi2WO6 NPs (1 mg/mL) and DMPO (25 mM) or TEMP (25 mM) were mixed and transferred into a sealed glass capillary tube of 0.9-1.1 mm diameter, followed by irradiation with the 808-nm laser for 5 min. The capillary tube was inserted into the resonance cavity. The EPR spectrum was recorded at room temperature with a Bruker EMXplus-10/12 spectrometer (Bruker, Germany) operating at 9.85 GHz with a variable temperature control unit (Bruker ER4141 VTI). Bi2WO6 NPs alone, DMPO alone and TEMP alone were also prepared and tested in the same manner. All analyses were performed in triplicate. ROS generated in HeLa cells that had taken up Bi2WO6 NPs after irradiation with NIR was evaluated with a H2DCFDA probe. The HeLa cell lines were seeded into a 6-well plate (1 × 104 cells/well) and incubated for 24 h at 37 °C. Then, CoCl2·6H2O was employed to stabilize the expression of hypoxia inducible factor-1α (HIF-1α) to create a hypoxia microenvironment. The HeLa cells were subsequently incubated with 200 µL Bi2WO6 NPs (200 µg/mL) for 24 h at 37 °C. Positive control cells were prepared with 200 µL of 50 mM H2O2 for 30 min at 37 °C, followed by incubation with 50 µL of H2DCFDA (10 mM in DMSO) for another 1 h at room temperature, then washed with PBS twice and exposed to the 808-nm laser (1 W/cm2) for 5 min. The ROS were immediately determined with confocal laser scanning microscopy with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. ROS generated in HeLa cells was also obtained using a flow cytometer (Cytomics FC 500 MCL, Beckman Coulter). To investigate the types of ROS, Singlet Oxygen Sensor Green (Thermo Fisher Scientific) and Tempo-9-AC (Synchem UG & Co. KG, Germany) were used to detect the level of singlet oxygen and hydroxyl radicals, respectively. The operation was performed under identical conditions and the results were obtained using the same flow cytometer. 2.9 Intracellular ROS/Hypoxia Detection. An oxidative stress/hypoxia detection kit (Enzo Life Sciences) was employed to investigate intracellular ROS/hypoxia

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induced by ICG, W18O49 NPs, and Bi2WO6 NPs. HeLa cells were seeded in a confocal petri-dish for 24 h, then 100 µL of ICG, W18O49 NPs, or Bi2WO6 NPs (1 mg tungsten/mL) were added to the cells and incubated for 2 h in the dark. A hypoxia/oxidative stress detection mixture was sequentially added into cells following the manufacturer’s instructions. After an another 30 min incubation, the HeLa cells were washed with PBS and irradiated with the 808-nm laser (1 W/cm2) for 10 min. After-irradiation confocal fluorescence imaging was done with a confocal laser scanning microscope. The ROS signal was monitored at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. A hypoxia signal was detected at an excitation wavelength of 488 nm and an emission wavelength of 590 nm. 2.10 Evaluation of in Vitro PTT, PDT and PTT/PDT with the MTT Assay. The MTT assay was employed to detect the relative cell viability. HeLa cells were incubated in a 6-well plate for 24 h at 37 °C to assess PTT, PDT and PTT/PDT at the cellular level. For PTT, HeLa cells that had taken up Bi2WO6 NPs were treated with 30 µL of 20 µM NAC to quench the ROS for 24 h, 39 followed by a 5 min irradiation with the 808nm laser (1 W/cm2). For PDT, HeLa cells that had taken up Bi2WO6 were subjected to a 5 min irradiation with the 808-nm laser on an ice box (to ensure that the temperature of HeLa cells below 10 °C (1 W/cm2). For PTT/PDT, HeLa cells that had taken up Bi2WO6 were exposed to the 808-nm laser (1 W/cm2) for 5 min. After irradiation, 20 µL of MTT (5 mg/mL in PBS) was added to each well for all groups. After another 4 h incubation, the MTT-containing medium was removed, and 150 µL of DMSO was added to dissolve the formazan crystals at room temperature for 30 min. The absorbance of each well at 490 nm was determined with an iMark Enzyme mark instrument (BIO-RAD Inc., USA). 2.11 In Vitro Fluorescence-Visual Phototherapy. To assess PTT, PDT and PTT/PDT at the cellular level, HeLa cells were incubated in the confocal dish for 24 h at 37 °C. For PTT, HeLa cells that had taken up Bi2WO6 NPs were treated with 30 µL of 20 µM NAC for 24 h to quench ROS, followed by 5 min of irradiation with the 808-nm laser (1 W/cm2). For PDT experiment, HeLa cells that had taken up Bi2WO6 NPs were irradiated for 5 min with the 808-nm laser on an ice box (1 W/cm2). For PTT/PDT experiment, HeLa cells that had taken up Bi2WO6 were irradiated with an 808-nm laser at a power density of 1 W/cm2 for 5 min. After irradiation, all


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groups of cells were washed with PBS and stained with Calcein-AM or EthD-1. The cells were directly imaged with laser scanning confocal microscopy (LSCM) without formalin fixation. 2.12 In Vivo Antitumor Effect. Nude mice were subcutaneously injected with HeLa cells (1 × 107 cells per mouse) at the right armpits to acquire HeLa-tumor- bearing nude mice. When the tumor volume reached ~500 mm3, the injected mice were randomly divided into four groups with eight mice in each group: a saline group, an ICG group, a W18O49 NPs group, and a Bi2WO6 NPs group. All groups (1 mg tungsten/mL saline) were injected intratumorally at a dose of 100 µL and then irradiated with an 808-nm laser (1 W/cm2) for 8 min at 8 h after the injection. The tumor volume and the survival of each mouse were monitored and recorded. After 13 days, the mice were sacrificed, and the tumors were collected, weighed, washed thrice with PBS and fixed in a 10% neutral buffered formalin solution. Hematoxylin and eosin (H&E) staining, TUNEL apoptosis staining, and hypoxia-inducible factor (HIF)-1α staining of the tumors were prepared by Nanjing Pharmaceutical Company and observed via fluorescence microscopy (IX71, Olympus). 2.13 In Vivo Micro-PET. To assess the hypoxic condition in HeLa-tumor-bearing nude mice post different treatments, Saline suspension (100 µL) containing 18FMISO (75 µCi/mouse) was intravenously injected into the mice. Micro-PET scans were carried out on Inveon PET/CT system (Siemens, Malvern, PA) 1 h post injection. All the PET scanners were cross-calibrated periodically. All the data was processed and reconstructed on the workstation. 2.14 Pathology Analysis. We performed a time course of histological changes in the mice organs (heart, liver, spleen, lung, and kidney) at 48 h post PTT/PDT. The organs collected from the mice were embedded in paraffin after immobilization in 4% paraformaldehyde at 4 °C for 4 h, and then the sections were stained with hematoxylin and eosin (H&E) and observed using a light microscope, and representative images were taken. 2.15 Hematology and Biochemical Assay. HeLatumor-bearing nude mice were treated with PTT/PDT for a period of 1, 7, 14 or 21 days. We used a sodium EDTA anticoagulant tube to collect the blood for hematology. Lymphocytes (LYM), neutrophils (NEU), and monocytes (MON) were tested to assess the immune

response, while the cytotoxicity of PTT/PDT was confirmed by a behavioral approach system (BAS), eosinophils (EOS), and red blood cell (RBC) levels. Blood serum was collected from the separated gel for biochemical analysis. The alanine aminotransferase (ALT) level, the total bilirubin level (TBIL), and total protein (TP) of the collected serum were measured to evaluate liver function. The production of platelets (PLT) was used to assess the spleen function. Kidney function was evaluated by the levels of creatinine (CRE) and blood urea nitrogen (BUN). 2.16 Statistical Analysis. Triplicate data were analyzed via Student’s t-test using the GraphPad Prism (version 7.0) software at a significance level of p < 0.05. Asterisks are used to indicate the significant differences. 3. RESULTS AND DISCUSSION 3.1 Characterization of Bi2WO6 NPs. Bi2WO6 NPs were fabricated and analyzed. As indicated in Figure 1A, Bi2WO6 NPs show a homogeneously dispersed spherical morphology with a mean diameter of 5.2 nm and an interplanar d-spacing equal to 0.192 nm, which matched with the {202} lattice of the Bi2WO6 NPs.40 Figure 1B shows the size distribution of the Bi2WO6 NPs, and approximately 170 of the 200 selected NPs were spheres with a size ranging from 4-6 nm. The mean diameter obtained from the DLS measurement was approximately 15 nm (Figure 1C), which is larger than that found in the TEM images, partly due to the hydration of the carboxylic acid groups. Bi2WO6 NPs exhibited a zeta potential of -36.2 ± 2.4 mV due to the presence of carboxylic groups on their surface. These Bi2WO6 NPs showed remarkable stability without significant size changes in PBS, acidic PBS (pH~5.0, mimicking the acidic microenvironment of lysosome), and fetal bovine serum buffer for 7 days (Figure 1D), which ensured their feasibility for use in in vivo applications. To further verify the chemical structure of these NPs, they were then investigated by energy dispersive X-ray spectroscopy (EDS). EDS mapping results (Figure 1E and F) clearly indicated the existence of W and Bi elements in the Bi2WO6 NPs, and the atomic ratio of Bi to W was determined by ICPMS analysis to be 2:1 as indicated by Figure S1, consistent with that of the theoretic value of 2:1. Peaks in XPS analysis corresponding to tungsten displayed the mixed bonding state (Figure 2A, W5+ and W6+, respectively) of the elemental tungsten, and peaks corresponding to bismuth also indicated the hybrid bonding state



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Figure 1. (A) TEM of Bi2WO6 NPs (inset: high resolution TEM image of an individual Bi2WO6 nanoparticle). (B) Size distribution of the obtained Bi2WO6 NPs. (C) DLS of the Bi2WO6 NPs. (D) Stability investigation of the Bi2WO6 NPs diluted with PBS, acidic PBS (pH~5.0) or fetal bovine serum buffer and stored at room temperature for a week via the hydrodynamic diameter. (E, F) EDS mapping images of W (E) and Bi (F) in the Bi2WO6 NPs, respectively. (Figure 2B, Bi2+ and Bi3+, respectively) of bismuth in the Bi2WO6 NPs.40 The X-ray diffraction (XRD) results (Figure 2C) confirmed that these as prepared NPs were Bi2WO6 NPs and all of the peaks completely correspond to the monoclinic phase.40 It was reported that the Bi2WO6 film showed a high absorption capacity in the near-infrared (NIR) region, which was the precondition for its use in the PTT and PDT.40 Therefore, the absorbance of Bi2WO6 NPs was determined using a UV-vis-NIR spectrum. As shown in Figure 2D, although, a profile with a slight decrease in the absorption intensity as a function of wavelength up to the NIR region was observed, Bi2WO6 NPs still had a remarkable absorption intensity at 808-nm in the UV-

vis-NIR spectrum. Given the wide and strong light absorption of Bi2WO6 NPs, the laser-induced heat generation of aqueous suspensions of Bi2WO6 NPs was assessed. One milliliter of aqueous solution containing Bi2WO6 NPs with various concentrations (0, 0.0625, 0.125, 0.25, and 0.5 mg/mL) was placed in a quartz cell and exposed to the irradiation of an 808-nm laser, and the laser power was set at 1 W/cm2, as widely accepted.41,42 The increase in temperature was tracked using a thermographic camera. Figure 2E shows that the temperature of all Bi2WO6 dispersions rose rapidly at the beginning and then began to level off 5 min after the irradiation. The concentration-dependent temperature elevation indicated that Bi2WO6 NPs could rapidly and


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Figure 2. (A) W4f core-level XPS spectra of Bi2WO6. (B) Bi4f core-level XPS spectra of Bi2WO6. (C) XRD of the Bi2WO6 NPs. (D) UV-vis spectra of the Bi2WO6 NPs (1 mg/mL dissolved in PBS). (E) The increase of temperature at different concentrations of Bi2WO6 NPs irradiated by an 808-nm laser (1 W/cm2). (0.0625 mg/mL NPs: 13.175 µg W and 29.95 µg Bi; 0.125 mg/mL NPs: 26.35 µg W and 59.9 µg Bi; 0.25 mg/mL NPs: 52.7 µg W and 119.8 µg Bi; 0.5 mg/mL NPs: 105.4 µg W and 239.6 µg Bi). (F) The EPR spectra of Bi2WO6, DMPO, and Bi2WO6 + DMPO exposed to an 808-nm laser (1 W/cm2). efficiently convert the laser energy into local hyperthermia under 808-nm irradiation. As indicated in Figure S2, the conversion efficiency was calculated to be around 68%, and this value did not experience remarkable reduction over repeated laser irradiation, confirming the stability of Bi2WO6 as photothermal agents. The Bi2WO6 monolayer film had a highly active surface, which was highly effective for photocatalytic oxidation reactions to convert H2O (OH-) to •OH without the presence of oxygen molecules.35 To clarify whether these Bi2WO6 NPs could also oxidize H2O (OH-) to •OH, we employed the DMPO spin-trapping EPR technique (Figure 2F). The •OH radical signal was very strong from Bi2WO6 NPs after 5 min 808-nm laser illumination, which depicted the superior activity of photocatalytic oxidation reactions. Besides, we have also analyzed the induction of singlet oxygen upon laser irradiation by using TEMP spin-trapping EPR technique. As shown in Figure S3, we observed negligible generation of singlet oxygen upon laser irradiation. The EPR results indicated that Bi2WO6 NPs could generate •OH radical

and act as light-induced PDT agents for killing cancer cells and destroying solid tumors after laser irradiation. 3.2 In Vitro CT Study and Cytotoxicity Assay. To successfully detect the tumor site and monitor the development of tumors, imaging modalities, such as MRI, CT or optical imaging technologies have been applied in cancer therapy. According to our previous report, tungsten oxide nanoparticles have been successfully applied as CT-guided PTT agents against breast malignancy.43 Therefore, the potential of these Bi2WO6 NPs for use as CT contrast agents was evaluated. Figure 3A shows the in vitro CT images obtained from Bi2WO6 NPs and the most used clinical contrast agent (iodixanol). Clearly, Bi2WO6 NPs showed brighter images than iodixanol at equivalent concentrations, revealing their superior potential for CT enhancement. As displayed in Figure 3B, both Bi2WO6 NPs and iodixanol revealed a concentration-dependent CT enhancement, but the Hounsfield unit (HU) values of Bi2WO6 NPs were much higher than that of iodixanol at the same concentration. These data also suggested that Bi2WO6 NPs could be used as CT



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Figure 3. (A) CT image of Bi2WO6 NPs and iodixanol. (B) In vitro X-ray attenuation assay of Bi2WO6 NPs and iodixanol. (C) In vitro viability of HeLa cells incubated with Bi2WO6 NPs without irradiation. (D) In vitro viability of HUVECs incubated with Bi2WO6 NPs without irradiation. The results are represented as the mean ± SD (n = 3). contrast agents to visualize the tumor tissue and guide the cancer therapy. Prior to the in vivo CT imaging test, the cytotoxicity of these Bi2WO6 NPs was evaluated. One cancer cell line (HeLa cells) and one normal cell line (HUVECs) were selected to perform the cytotoxicity test by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Figure 3C and D). After these cells were incubated with Bi2WO6 NPs at various concentrations ranging from 0.1-1000 µg/mL for 24 h, negligible cytotoxic effects on cell viability were observed both for the HeLa cells and HUVECs. After 48 h, a slight reduction in cell viability (17.5% for HeLa cells, 18.2% for HUVECs) was observed at the highest tungsten concentration (1000 µg/mL), which indicated that Bi2WO6 NPs had low cytotoxicity and were suitable for in vivo applications. 3.3 In Vivo CT Imaging and Infrared (IR) Thermal Imaging. To evaluate the potential capacity of Bi2WO6 for CT enhancement, HeLa-tumor-bearing nude mice were treated with an intratumoral injection of 40 µL of Bi2WO6 NPs (a tungsten concentration of 50 mM) or 40

µL of iodixanol (an iodine concentration of 100 mM). Before the CT Imaging, the in vivo distribution profiles of Bi2WO6 NPs were analyzed in HeLa-xenograftbearing mice at 0.5, 1, 3, 8, 12 and 24 h post an intratumoral (i.t., Figure S4) or intravenous injection (i.v., Figure S5), respectively, (Dosage: 0.1 mL of Bi2WO6 NPs at a concentration of 1 mg tungsten/mL saline). We found that only a small proportion of the i.v. administrated-Bi2WO6 NPs enriched in the tumor tissue through the EPR effect, owing to the rapid renal clearance. By contrast, the clearance of Bi2WO6 NPs was fairly slower over time via i.t. injection. Though the amount of Bi2WO6 NPs gradually reduced over time, we noticed that an appropriate time window should be set between the injection and collection of images, as the contrast agents required time to realize a roughly even distribution in solid tumor. The CT images of tumor tissue were collected at 3 hours post the injection. Clearly, mice injected with Bi2WO6 NPs showed an enhanced signal as indicated by a yellow arrow in Figure 4A, and the HU value dramatically increased from 256.5 ± 37.2 prior to


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Figure 4. In vivo CT and infrared thermal imaging. (A) CT images of mice before and after intratumoral injection with Bi2WO6 NPs (tungsten concentration, 50 mM, 40 µL) and iodixanol (iodine concentration, 100 mM, 40 µL), respectively. The tumor is clearly indicated with the yellow arrow. (B) IR thermal imaging of HeLa-tumor-bearing mice intratumorally injected with PBS or Bi2WO6 NPs after irradiation of 808-nm (1 W/cm2) for 20 min. (C) Temperature profiles of (B). injection to a value of 624.6 ± 45.8. In contrast, no significant tumor signals were seen in the CT images of mice injected with iodixanol (Figure 4A), and the HU value slightly increased from 264.3 ± 38.1 to 304.7 ± 40.3, partially due to the poor retention of the small molecular iodine contrast agent at the tumor site.44 These results demonstrated that although Bi2WO6 NPs have a small size, they still can be retained inside the tumor for a long time and are promising to serve as CT contrast agents to determine the position of the tumor. To investigate the photothermal conversion properties of Bi2WO6 NPs, the in vivo photothermal effect of Bi2WO6 NPs on tumor tissue was investigated. Considering that the hypoxic zone is in the center of the tumor, the NPs administrated via intravenous injection will

hardly penetrate into the center of the tumor tissue due to the high intratumoral pressure, thus leading to limited accumulation in the hypoxic region.45,46 Therefore, in this work, Bi2WO6 NPs were i.t. injected into the center of each tumor to precisely deliver these NPs into the hypoxic zone. Additionally, due to their ultra-small size, these Bi2WO6 NPs can feasibly diffuse to the entire tumor tissue from the original site, which can strengthen the therapeutic effect.47 Tumor-bearing mice were treated with an i.t. injection of Bi2WO6 NPs, and the tumor region was exposed to NIR radiation (1 W/cm2) for 20 min at 8 h post injection. An infrared thermal camera was employed to monitor the local temperature of the tumor region, as shown in Figure 4B. Clearly, the temperature of the PBS group did not change greatly after the laser irradiation. However, the thermal signal of tu-



mors of mice treated with Bi2WO6 NPs continually increased upon laser irradiation, which implied that the Bi2WO6 NPs had a superb photothermal conversion ability. Figure 4C shows the quantitative temperature changes of the tumors as a function of the irradiation time. The temperature rose as the irradiation time increased for both the PBS and Bi2WO6 NP groups. The average temperature of the tumor region only rose slightly in the PBS group and reached 34.5 °C at 20 min post irradiation, indicating that PBS alone did not demonstrate NIR irradiation-induced hyperthermia. On the other hand, the average temperature of the tumor region in the Bi2WO6 NPs group increased significantly and reached 47 °C at 20 min after irradiation, indicating that Bi2WO6 NPs were able to serve as a promising PTT agent for cancer treatment. 3.4 ROS Production of Bi2WO6 NPs. According to our above analysis, ROS, such •OH, were generated by Bi2WO6 NPs during the laser irradiation. To clarify whether Bi2WO6 NPs can generate ROS in a hypoxia microenvironment under NIR irradiation, CoCl2·6H2O was employed to stabilize the expression of hypoxia inducible factor-1α (HIF-1α) to create a hypoxia microenvironment. Next, the HeLa cells were incubated with Bi2WO6 NPs under identical conditions as mentioned above for 24 h and exposed to an 808-nm laser (1 W/cm2) for 5 min or 30 min. HeLa cells treated with H2O2 were used as the positive control. ROS generated in HeLa cells was evaluated by staining cells with H2DCFDA, which is a standard probe for ROS detection, followed by confocal laser scanning microscopy (CLSM) observation and flow-cytometry measurements (Figure 5). A strong green fluorescence representing ROS was detected by CLSM in HeLa cells treated with either 50 mM H2O2 or Bi2WO6 NPs combined with NIR radiation, and the intensity of the green fluorescence signal significantly increased with the extension of the irradiation time (Figure 5A). In contrast, no green color was detected in the negative controls, e.g., cells treated by laser irradiation alone or by the Bi2WO6 NPs without irradiation. Quantification of the ROS intensity was achieved by flow-cytometry (Figure 5B and C). The intracellular fluorescence intensity inside cells treated by Bi2WO6 NPs combined with laser irradiation was higher than that of the cells treated by laser irradiation alone or by Bi2WO6 NPs without irradiation. The fluorescence intensity at 30 min post irradiation was approximately 1.75 times higher than that at 5 min post irradiation. To-

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gether, all the results suggested that ROS can be selectively generated in the cells by the uptake of Bi2WO6 NPs plus NIR radiation. This observation was further verified by in vitro flow-cytometry analysis (Figure S6). HeLa cells were incubated with Bi2WO6 (1 mg/mL) for 6 h, followed by vigorous washing with warm PBS to remove non-internalized nanoparticles. Afterwards, the cells were subjected to laser irradiation (1 W/cm2, 5 min), and stained with Singlet Oxygen Sensor Green and Tempo-9-AC respectively to analyze the level of singlet oxygen and hydroxyl radicals. The enhancement of expression was calculated as the ratio of [ROS]post/[ROS]pre (post: Post laser irradiation, denoted by PDT; pre: Before laser irradiation, denoted by control). The mass production of hydroxyl radicals is in consistent with the results of EPR study. As the level of singlet oxygen only increased slightly (1.13-fold), its contribution to cell killing is speculated to be limited, especially when comparing with that of hydroxyl radicals (87.5-fold). Collectively, these findings confirm that the Bi2WO6 could mediate PDT, in which hydroxyl radicals plays dominant role, a manner largely independent of oxygen availability. Additionally, to clarify the characteristics and advantages of the PDT effect by Bi2WO6 NPs, we also investigated light-triggered intracellular ROS generation and the induction of a hypoxic environment by staining with the oxidative stress/hypoxia detection kit and analyzed the cells with CLSM in three different groups (Bi2WO6 NPs, ICG and W18O49 NPs synthesized by our previous method43). It was reported that photogenerated electrons of oxygen-deficient tungsten oxides such as W18O49 could react with the absorbed O2 to generate reactive oxygen species (ROS, including singlet oxygen and ·O2−).27-29 Here, ICG and W18O49 NPs will exert a photodynamic killing effect in an oxygen-dependent manner. Untreated cells were used as the negative control. As shown in Figure 6, the green fluorescence representing ROS could be observed in all of the cells treated by Bi2WO6 NPs, ICG and W18O49 NPs, respectively (all of them followed by laser irradiation). However, red fluorescence indicated that the presence of hypoxia was only detected in the ICG and W18O49 NP groups, demonstrating that ICG and W18O49 NPs will induce hypoxia post PDT, while Bi2WO6 NPs generate ROS under laser irradiation without the induction of hypoxia.


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Figure 5. (A) CLSM images of ROS generation in HeLa cells that received different treatments as displayed. The H2DCFDA probe was used as a ROS indicator. Scale bar: 50 µm. (B) Flow cytometry analysis of ROS generation inside the cells after incubation with Bi2WO6 NPs for 2 h. (C) The mean fluorescence intensity (MFI) of ROS generated in each group as displayed, and untreated HeLa cells were used as the control.

Figure 6. Confocal fluorescence images of HeLa cells subjected to different treatments, and ROS/hypoxia detection probes were used as indicators. Scale bar: 50 µm. 3.5 In Vitro Antitumor Effect. The negligible cytotoxic effect rendered by Bi2WO6 NPs without an 808-nm laser was indicated by the MTT assay, and thus, we next assessed the PTT, PDT, and PTT/PDT effects on the treatment of cancer at the cell level. Before the investigation of combined NIR-light-triggered PTT and PDT, we first performed a pure PTT treatment on the cancer cells. Cell viability under 808-nm light irradiation (1 W/cm2) was measured with Bi2WO6-internalized HeLa cells, which were pre-incubated with NAC to quench ROS to eliminate the PDT effect and ensure that only

the PTT effect was assessed.39 After irradiation, the cellular inhibition rate of PTT alone was kind of low (less than 10%, Figure 7A). However, to avoid the interference of PTT effects, HeLa cells that had taken up Bi2WO6 NPs were irradiated with an 808-nm laser (1 W/cm2) on an ice box to ensure that the temperature of the irradiated area never exceeded 10 °C, and NIR-lightinduced photothermal ablation was almost entirely suppressed.29 After this procedure, the cell inhibition rate was 47.5%, which was much larger than that of the PTT,



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Figure 7. (A) Relative HeLa cell viabilities after different treatments (PTT alone, PDT alone and the combination of PTT and PDT) with Bi2WO6 (200 µg/mL) after 24 h of incubation (irradiation time: 5 min). (B) Fluorescence images of HeLa cells incubated with Bi2WO6 NPs (200 µg/mL) assisted with the irradiation of an 808-nm laser to examine the in vitro PTT, PDT, and PTT/PDT effects of Bi2WO6 NPs against HeLa cells. Calcein AM (green fluorescence representing live cells) and EthD-1 (red fluorescence representing dead cells) were used as indicators. Scale bar: 50 µm. fully demonstrating the excellent efficacy of the PDT effect by these Bi2WO6 NPs (Figure 7A). The combined treatment of PTT and PDT caused more than 87% cell death, which was considerably larger than the sum of the outputs of PTT and PDT alone. Taken together, these results clearly reveal that the mechanism of the photodestruction of cancer cells by Bi2WO6 NPs is comprised of both PTT and PDT abilities, and the combined PTT/PDT treatment provides a superior synergistic antitumor effect. To further demonstrate that combined PTT/PDT treatment is superior to PTT or PDT alone, we conducted a live/dead cell double staining experiment. Figure 7B shows no red color in the HeLa cells exposed to laser irradiation alone or incubated with Bi2WO6 NPs

alone. Besides, only a slight red color was observed in HeLa cells subjected to PTT or PDT alone after being incubated with Bi2WO6 NPs, indicating that these treatments did not show obvious damage against HeLa cells. When the cells were subjected to the combined PTT/PDT treatment, a large amount of red fluorescence representing dead cells was seen, confirming the superior phototherapeutic effect of Bi2WO6 NPs against HeLa cells after laser irradiation. These results further proved that Bi2WO6 NPs can be used for the treatment of cancer via PTT/PDT.


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Figure 8. (A) Tumor volumes of HeLa-tumor-bearing mice that received different treatments as displayed. The data are expressed as the mean ± SD (n = 8). ** p < 0.01, *** p < 0.001. (B) Number of tumor-free mice after treatment during the observation. (C) Survival curves of HeLa-tumor-bearing mice that received different treatments as displayed. (D) Body weight of HeLa-tumor-bearing mice that received different treatments as indicated. 3.6 In Vivo Antitumor Effect. Subsequently, the in vivo therapeutic effect of Bi2WO6 NP-assisted PTT/PDT on a HeLa-tumor-bearing mouse model was evaluated. When the tumor size reached approximately 500 mm3, the tumor-bearing nude mice were randomly divided into the following four groups with eight mice per group: saline group, ICG group, W18O49 group, and Bi2WO6 group. All groups (1 mg tungsten/mL saline) were injected intratumorally at a dose of 100 µL and then irradiated with an 808-nm laser (1 W/cm2) for 8 min at 8 h after the injection. The tumor in the saline group grew rapidly, and the tumor volume reached approximately 7.5-fold (day 13) that of day 1 (Figure 8A). For the ICG group and W18O49 group, during the first 5 days, the tumor size became smaller at first, and then it relapsed over time. These results showed that ICG and W18O49 NPs could initially suppress the tumor out-

growth after laser irradiation to some extent. However, the tumoral inhibition was temporary, and the tumor would likely recur later. Eventually, the tumor volume increased by approximately 4-fold (ICG group) and 3.4fold (W18O49 group) on day 13 compared to that of day 1, respectively. Interestingly, only the Bi2WO6 NPtreated group caused the continuous abatement of the tumor volume, and the tumor volume reached to 0.4-fold on day 13 compared to day 1, showing a significant therapeutic effect for the elimination of the solid tumor. Although ICG, W18O49 NPs, and Bi2WO6 NPs all exhibited anti-tumor effects, tumor growth only recurred in the ICG and W18O49 groups. Taking the observations of Figure 6 together, it is reasonable to propose that Bi2WO6 NPs induce photodynamic killing in a hypoxiafree manner different from that of the W18O49 and ICG groups.



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Figure 9. (A) Histological observation of the tumor tissues after different treatments. The tumor sections were stained with hematoxylin and eosin (H&E), (red arrow: necrosis; blue arrow: apoptosis). (B) Detection of cell apoptosis in the tumor tissues after the different treatments was detected with immunofluorescence. The tumor sections were stained with deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and then were observed with an optical microscope. (C) Hypoxia-inducible factor (HIF)-1α staining of the tumor slides from HeLa tumor-bearing mice after different treatments. Scale bars: 50 µm It should be noted that the tumor growth curves were completely based on the mice that still had tumors on day 13. However, 0, 3, 4, and 5 mice showed the complete disappearance of the tumor in the saline, ICG, W18O49, and Bi2WO6 groups, respectively (Figure 8B). The survival curves for the mice in Figure 8C show that the life spans in the saline group, ICG group and W18O49 NPs group are no longer than 32 days, 44 days, and 56 days, respectively. On the other hand, Bi2WO6 group had the longest life span; only three mice died at the end. The body weight of the mice was also determined, and no obvious changes in the body weight of the mice were observed during the course of the study for all groups (Figure 8D), implying that Bi2WO6 NPs displayed no obvious acute toxicity. The therapeutic efficacy in all groups was further assessed by hematoxylin and eosin (H&E) staining and immunofluorescence. The tumor tissue in the ICG group, W18O49 group, and Bi2WO6

group all indicated a large degree of necrosis (red arrow in Figure 9A) and apoptosis (blue arrow in Figure 9A and the nuclei were stained with cyan in Figure 9B). However, the Bi2WO6 NPs group had the greatest degree of necrosis and apoptosis (Figure 9A and B). Altogether, from Figure 8, Figure 9A and Figure 9B, respectively, we can conclude that the Bi2WO6 NPs are the best agents to induce tumor necrosis and apoptosis, and they possess a superior effect on hindering tumor growth and prolonging the life span of mice to that of ICG or W18O49 NPs. To examine the mechanism of the superior therapeutic efficacy of Bi2WO6 NPs during the PTT/PDT procedure, the degree of hypoxia inside the tumor was measured via hypoxia-inducible factor (HIF)1α staining.48 As shown in Figure 9C, the tumor tissues in the ICG group and in the W18O49 group expressed (HIF)-1α to a greater degree (the nuclei were stained by dark brown) than the control group and the Bi2WO6


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Figure 10. (A) PET images of mice after different treatments (Day 21, I: saline; II: ICG + laser; III: W18O49 + laser; IV: Bi2WO6 + laser). Tumor lesions are highlighted with white circles. Right panels show the images of the tumor in the transversal view. (B) Standardized uptake values (SUV) and (C) normalized values (to the weight) in solid tumor regions obtained from PET images taken at 1 h after the injection of 18F-MISO. *** p < 0.001. group (numerous nuclei remained blue). The high degree

been validated as the gold standard for tracking hypoxia

of hypoxia reduced the sensitivity of the tumor tissue to

due to its high sensitivity despite its low spatial resolu-

PDT treatments and caused a compromised anti-tumor

tion. The residual hypoxic zone inside the tumor after

effect. This result also demonstrated that ICG and

different treatments was verified by the PET modality.

W18O49 NPs exacerbated hypoxia in the tumor and

As indicated in Figure 10A, the tumor volumes in the

caused the recurrence of the tumor, while Bi2WO6 NPs

ICG group, W18O49 group, and Bi2WO6 group all re-

did not worsen hypoxia in the tumor and effectively

gressed 21 day post-treatment compared to those of the

eliminated the tumor during the PDT procedure.

saline group (~100 mm3 for all group prior to the treatment, ~2900 mm3, ~320 mm3, ~240 mm3, and ~75 mm3

3.7 In Vivo Micro-PET. Different nanomedicines exert distinct effects on the tumor microenvironment, and here we investigate the hypoxia condition after four different therapies (I: saline; II: ICG + laser; III: W18O49 + laser; and IV: Bi2WO6 + laser) to monitor their long-term therapeutic effects. Positron emission tomography (PET) imaging with

18

F fluoromisonidazole (FMISO) has long

for saline, ICG + laser, W18O49 + laser, Bi2WO6 + laser groups, respectively, 21 days post-treatment). However, the hypoxia in the four different groups displayed totally different degrees. Almost no positively hypoxic zone was observed inside the tumor after the combination treatment of PTT and PDT rendered by the Bi2WO6 NPs (Figure 10A, IV, white circle). This region had lowest



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Figure 11. (A) The cytotoxicity of the combination treatment of PTT and PDT rendered by Bi2WO6 NPs was detected by the H&E staining of the mice organs (heart, liver, spleen, lung and kidney). (B) Serum bio-chemical study and hematology assay of lymphocytes (LYM), monocytes (MON), neutrophils (NEU), red blood cells (RBCs), eosinophils (EOS), the behavioral approach system (BAS), total bilirubin level (TBIL), creatinine (CRE), total protein (TP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and platelet (PLT) levels at 1, 7, 14, and 21 days after PTT/PDT treatment. PET signal and smallest amount of 18F-MISO (SUV: mean: 0.148 ± 0.022; max: 0.2147 ± 0.043, p < 0.001; uptake: 0.97% ID/g tumor, Figure 10B, C). In contrast, the ICG group (SUV: mean: 0.8422 ± 0.1132; uptake: 4.48% ID/g tumor) and W18O49 group (SUV: mean: 0.8262 ± 0.1098; uptake: 4.67% ID/g tumor) both exac-

erbate the degree of hypoxia, again demonstrating that ICG and W18O49 NPs will induce hypoxia posttreatment, while Bi2WO6 NPs generate ROS under laser irradiation without the induction of hypoxia and thus open a new avenue for oxygen-free PDT.


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3.7 Pathological Analysis and Hematology Assay. A histological analysis and H&E staining of the mice organs (heart, liver, spleen, lung, and kidney) harvested from the tumor-bearing nude mice after the Bi2WO6 NPassisted PTT/PDT treatment were performed to evaluate the in vivo cytotoxicity. Figure 11A shows representative H&E stained pictures of these organs at different times. Compared to images from normal mice, no apparent necrosis, hydropic degeneration, pulmonary fibrosis, histological abnormalities and inflammation lesions were seen in any of the organs from the mice treated with the Bi2WO6 NP-assisted PTT/PDT treatment, which provides solid evidence of the good biocompatibility of these Bi2WO6 NPs in vivo. A hematology study was carried out to estimate the potential cytotoxicity in the body after the Bi2WO6-assisted PTT/PDT treatment because hepatotoxicity is concerned with the in vivo application of nanomaterials. Figure 11B shows that the counts of red blood cells (RBC), eosinophils (EOS), and the behavioral approach system (BAS) level with respect to the hemopoietic and aerobic capacity were similar to those in normal mice. Peripheral blood lymphocytes (LYM), neutrophils (NEU) and monocytes (MON) were also tested to evaluate the immune response (Figure 11B). It was observed that the Bi2WO6 NP-assisted PTT/PDT combined treatment enhanced LYM proliferation and slightly activated NEU. The levels of both cell types gradually recovered by 21 days after treatment. The activation of LYM and NEU could be attributed to a tumoricidal immune response induced by the PTT/PDT combined treatment. The serum levels of alanine aminotransferase (ALT), total bilirubin (TBIL), and total protein (TP) were used to monitor the liver function, while the serum levels of the blood urea nitrogen (BUN), platelets (PLT), and creatinine (CRE) were used to monitor the kidney and spleen function (Figure 11B). Little change in the statistical significance was seen over time in TP, BUN, CRE and PLT after the PTT/PDT combined treatment, which indicated that the Bi2WO6 NP-assisted PTT/PDT combined treatment did not break down the metabolic system. 4. CONCLUSION In summary, we synthesized multifunctional Bi2WO6 NPs that combined the CT imaging ability, PTT and oxygen-free PDT functions. These NPs showed low in

vitro cytotoxicity and better CT contrast ability than that of the commercial CT contrast agents. Upon the irradiation with an 808-nm laser, these NPs can not only photothermally kill the tumor cells but also generate •OH radicals without the consumption of oxygen to perform the PDT function, which greatly suppresses the PDTinduced hypoxia as generated by the conventional PDT. Furthermore, the superior in vivo antitumor results clearly demonstrated that the synergistic effect of PTT and oxygen-free PDT arising from these Bi2WO6 NPs will play a vital role in future clinical practice by overcoming PDT-induced hypoxia in cancer treatment.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * Email: [email protected]; * Email: [email protected] * Email: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2017YFA0205400), National Natural Science Foundation of China (No. 21474047, 51773089, 81671751, 81371516), the Industry-University-Research Collaboration project of Jiangsu Province (BY2015041-01), the Key Project and Outstanding Youth supported by Medical Science and Technology Development Foundation Nanjing Department of Health (ID: YKK15067), and the Jiangsu province key medical young talents, "13th Five-Year" health promotion project of Jiangsu province (JH.2016-2020) REFERENCES (1) Goel, S.; Duda, D. G.; Xu, L.; Munn, L. L.; Boucher, Y.; Fukumura, D, Jain R. K. Normalization of the Vasculature for Treatment of Cancer and Other Diseases. Physiol. Rev. 2011, 91, 1071-1121. (2) Mankoff, D. A.; Dunnwald, L. K.; Partridge, S. C.; Specht, J. M. Blood Flow-Metabolism Mismatch: Good

17 ACS Paragon Plus Environment


for the Tumor, Bad for the Patient. Clin. Cancer Res. 2009, 15, 5294-5296. (3) Masuda, S.; Belmonte J. C. I. The Microenvironment and Resistance to Personalized Cancer Therapy. Nat. Rev. Clin. Oncol. 2012, 10, 79. (4) Bristow, R. G.; Hill, R. P. Hypoxia and Metabolism: Hypoxia, DNA Repair and Genetic Instability. Nat. Rev. Cancer 2008, 8, 180-192. (5) Vaupel, P.; Mayer, A. Hypoxia in Cancer: Significance and Impact on Clinical Outcome. Cancer Metastasis Rev. 2007, 26, 225-239. (6) Semenza, G. L. The Hypoxic Tumor Microenvironment: a Driving Force for Breast Cancer Progression. Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863, 382391. (7) Brown, J. M.; Giaccia, A. J. The Unique Physiology of Solid Tumors: Opportunities (and Problems) for Cancer Therapy. Cancer Res. 1998, 58, 1408-1416. (8) Swartz, M. A.; Iida, N.; Roberts, E. W.; Sangaletti, S.; Wong, M. H.; Yull, F. E.; Coussens, L. M.; DeClerck, Y. A. Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy. Cancer Res. 2012, 72, 24732480. (9) Barker, H. E.; Paget, J. T. E.; Khan, A. A.; Harrington, K. J. The Tumor Microenvironment after Radiotherapy: Mechanisms of Resistance and Recurrence. Nat. Rev. Cancer 2015, 15, 409-425. (10) Yuan, Y.; Zhang, C.-J.; Kwok, R. T. K.; Xu, S.; Zhang, R.; Wu, J.; Tang B.-Z.; Liu, B. Light-Up Probe for Targeted and Activatable Photodynamic Therapy with Real-Time in Situ Reporting of Sensitizer Activation and Therapeutic Responses. Adv. Funct. Mater. 2015, 25, 6586-6595. (11) Kamkaew, A.; Cheng, L.; Goel, S.; Valdovinos, H. F.; Barnhart, T. E.; Liu, Z.; Cai, W.-B. Cerenkov Radiation Induced Photodynamic Therapy Using Chlorin e6-Loaded Hollow Mesoporous Silica Nanoparticles. ACS Appl. Mater. Interfaces 2016, 8, 26630-26637. (12) Cheng, L.; Kamkaew, A.; Sun, H.; Jiang, D.; Valdovinos, H. F.; Gong, H.; England, C. G.; Goel, S.; Barnhart, T. E.; Cai, W.-B. Dual-Modality Positron Emission Tomography/Optical Image-Guided Photodynamic Cancer Therapy with Chlorin e6-Containing Nanomicelles. ACS Nano 2016, 10, 7721-7730. (13) Nakki, S.; Martinez, J. O.; Evangelopoulos, M.; Xu, W.-J.; Lehto, V. P.; Tasciotti, E. Chlorin e6 Functionalized Theranostic Multistage Nanovectors Trans-

Page 18 of 21

ported by Stem Cells for Effective Photodynamic Therapy. ACS Appl. Mater. Interfaces 2013, 25, 3055-3061. (14) Yu, M.; Guo, F.; Wang, J.-P.; Tan, F.-P.; Li, N. Photosensitizer-Loaded pH-Responsive Hollow Gold Nanospheres for Single Light-Induced Photothermal/Photodynamic Therapy. ACS Appl. Mater. Interfaces DOI: 10.1021/acsami.7b05766 (15) Zhao, Z.-X.; Shi, S.-G.; Huang, Y.-Z.; Tang, S.H.; Chen, X.-L. Simultaneous Photodynamic and Photothermal Therapy Using Photosensitizer-Functionalized Pd Nanosheets by Single Continuous Wave Laser. ACS Appl. Mater. Interfaces 2014, 6, 8878-8885. (16) Qian, C.-G.; Yu, J.-C.; Chen, Y.-L.; Hu, Q.-Y.; Xiao, X.-Z.; Sun, W.-J.; Wang, C.; Feng, P.-J.; Shen, Q.-D.; Gu, Z. Light-Activated Hypoxia-Responsive Nanocarriers for Enhanced Anticancer Therapy. Adv. Mater. 2016, 28, 3313-3320. (17) Qian, C.-G.; Feng, P.-J.; Yu, J.-C. Chen, Y.-L.; Hu, Q.-Y.; Sun, W.-J.; Xiao, X.-Z.; Hu, X.-L.; Bellotti, A.; Shen, Q.-D.; Gu, Z. Anaerobe-Inspired Anticancer Nanovesicles. Angew. Chem., Int. Ed. 2017, 56, 25882593. (18) Feng, L. Z.; Cheng, L.; Dong, Z.-L.; Tao, D.-L.; Barnhart, T. E. Cai, W.-B. Chen, M.-W, Liu, Z. Theranostic Liposomes with Hypoxia-Activated Prodrug to Efectively Destruct Hypoxic Tumors PostPhotodynamic Therapy. ACS Nano 2017, 11, 927-937. (19) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. (20) Song, X.; Feng, L.; Liang, C.; Yang, K.; Liu, Z. Ultrasound Triggered Tumor Oxygenation with OxygenShuttle Nanoperfluorocarbon to Overcome HypoxiaAssociated Resistance in Cancer Therapies. Nano Lett. 2016, 16, 6145-6153. (21) Luo, Z.; Zheng, M.; Zhao, P.; Chen, Z.; Siu, F.; Gong, P.; Gao, G.; Sheng, Z.; Zheng, C.; Ma, Y.; Cai, L.; Self-Monitoring Artificial Red Cells with Sufficient Oxygen Supply for Enhanced Photodynamic Therapy. Sci. Rep. 2016, 6, 23393-23403. (22) Cheng, Y.; Cheng, H.; Jiang, C.; Qiu, X.; Wang, K.; Huan, W.; Yuan, A.; Wu, J.; Hu, Y. Perfluorocarbon Nanoparticles Enhance Reactive Oxygen Levels and Tumor Growth Inhibition in Photodynamic Therapy. Nat. Commun. 2015, 6, 8785-8792. (23) Zhu, W.-W.; Dong, Z.-L.; Fu, T.-T.; Liu, J.-J.; Chen, Q.; Li, Y.-G.; Zhu, R.; Xu, L.-G.; Liu, Z. Modulation of Hypoxia in Solid Tumor Microenvironment with


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MnO2 Nanoparticles to Enhance Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 5490-5498. (24) Song, G.; Liang, C.; Yi, X.; Zhao, Q.; Cheng, L.; Yang, K.; Liu, Z. Perfluorocarbon-Loaded Hollow Bi2Se3 Nanoparticles for Timely Supply of Oxygen under Near-Infrared Light to Enhance the Radiotherapy of Cancer Adv. Mater. 2016, 28, 2716-2723. (25) Prasad, P.; Gordijo, C. R.; Abbasi, A. Z.; Maeda, A.; Ip, A.; Rauth, A. M.; DaCosta, R. S.; Wu, X.-Y. Multifunctional Albumin-MnO2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response. ACS Nano 2014, 8, 32023212. (26) Zheng, D.-W.; Li, B.; Li, C.-X.; Fan, J.-X.; Lei, Q.; Li, C.; Xu, Z.-S.; Zhang, X.-Z. Carbon-DotDecorated Carbon Nitride Nanoparticles for Enhanced Photodynamic Therapy against Hypoxic Tumor via Water Splitting. ACS Nano 2016, 10, 8715-8722. (27) Huang, Z.-F.; Zou, J.-J.; Pan, L.; Wang, S.; Zhang, X.; Wang, L. Synergetic Promotion on Photoactivity and Stability of W18O49/TiO2 Hybrid. Appl. Catal., B 2014, 147, 167-174. (28) Kalluru, P.; Vankayala, R.; Chiang, C. S.; Hwang, K. C. Photosensitization of Singlet Oxygen and In Vivo Photodynamic Therapeutic Effects Mediated by PEGylated W18O49 Nanowires Angew. Chem., Int. Ed. 2013, 52, 12332-12336. (29) Guo, W.; Guo, C.; Zheng, N.; Sun, T.; Liu, S. CsxWO3 Nanorods Coated with Polyelectrolyte Multilayers as a Multifunctional Nanomaterial for Bimodal Imaging-Guided Photothermal/Photodynamic Cancer Treatment. Adv. Mater. 2017, 29, 1-9. (30) Dong, K.; Liu, Z.; Liu, J.-H.; Huang, S.; Li, Z.H.; Yuan, Q.-H.; Ren, J.-S.; Qu, X.-G. Biocompatible and High-Performance Amino Acids-Capped MnWO4 Nanocasting as a Novel Non-Lanthanide Contrast Agent for X-Ray Computed Tomography and T1-Weighted Magnetic Resonance Imaging. Nanoscale 2014, 6, 22112217. (31) Liu, Z.; Liu, X.-J.; Ran, X.; Ju, E.-G.; Ren, J. S.; Qu, X. G. Single-Layer Tungsten Oxide as Intelligent Photo-Responsive Nanoagents for Permanent Male Sterilization. Biomaterials 2015, 69, 56-64. (32) Liu, Z.; Liu, J.-H.; Wang, R.; Du, Y.-D.; Ren, J.S.; Qu, X.-G. An Efficient Nano-Based Theranostic System for Multi-Modal Imaging-Guided Photothermal

Sterilization in Gastrointestinal Tract. Biomaterials 2015, 56, 206-218. (33) Du, J.-Z.; Du, X.-J.; Mao, C.-Q.; Wang, J. TailorMade Dual pH-Sensitive Polymer Doxorubicin Nanoparticles for Efficient Anticancer Drug Delivery. J. Am. Chem. Soc. 2011, 133, 17560-17563. (34) Du, J.-Z.; Sun, T.-M.; Song, W.-J.; Wu, J.; Wang, J. A Tumor-Acidity-Activated Charge-Conversional Nanogel as an Intelligent Vehicle for Promoted Tumoral-Cell Uptake and Drug Delivery. Angew. Chem. Int. Ed. 2010, 49, 3621-3626. (35) Zhou, Y.; Zhang, Y.; Lin, M.; Long, J.; Zhang, Z.; Lin, H.; Wu, J. C. S.; Wang, X. Monolayered Bi2WO6 Nanosheets Mimicking Heterojunction Interface with Open Surfaces for Photocatalysis. Nat. Commun. 2015, 6, 8340-8347. (36) Ai, K.; Liu, Y.; Liu, J.; Yuan, Q.; He, Y.; Lu, L. Large-Scale Synthesis of Bi2S3 Nanodots as a Contrast Agent for in Vivo X-Ray Computed Tomography Imaging. Adv. Mater. 2011, 23, 4886-4891. (37) Liu, J.; Han, J.; Kang, Z.; Golamaully, R.; Xu, N.; Li, H.; Han, X. In Vivo Near-Infrared Photothermal Therapy and Computed Tomography Imaging of Cancer Cells Using Novel Tungsten-Based Theranostic Probe. Nanoscale 2014, 6, 5770-5776. (38) Mu, J.-N.; Meng, X.-Y.; Chen, L.; Lu, Z.-B.; Mou, Q.-W.; Li, X.-X.; Wang, S.-Q.; Yue, H.-Y. Highly stable and biocompatible W18O49@PEG-PCL hybrid nanospheres combining CT imaging and cancer photothermal therapy. RSC Adv., 2017, 7, 10692-10699. (39) Wang, J.; Li, X.-M.; Bai, Z.; Chi, B.-X.; Wei, Y.; Chen, X. Curcumol Induces Cell Cycle Arrest in Colon Cancer Cells via Reactive Oxygen Species and Akt/ GSK3Β/Cyclin D1 Pathway. J. Ethnopharmacol. 2018, 210, 1–9. (40) Tian, J.; Sang, Y.; Yu, G.; Jiang, H.; Mu, X.; Liu, H. A Bi2WO6-Based Hybrid Photocatalyst with Broad Spectrum Photocatalytic Properties under UV, Visible, and Near-Infrared Irradiation. Adv. Mater. 2013, 25, 5075-5080. (41) Li, W.; Zhang, H.-B.; Guo, X.-M.; Wang, Z.-H.; Kong, F.-F.; Luo, L.-H.; Li, Q.-P. Zhu, C.-Q.; Yang, J.; Lou, Y.; Du, Y.-Z.; You, J. Gold NanospheresStabilized Indocyanine Green as a Synchronous Photodynamic-Photothermal Therapy Platform That Inhibits Tumor Growth and Metastasis. ACS Appl. Mater. Interfaces 2017, 9, 3354-3367.



Page 20 of 21

(42) Yu, Y.-N.; Zhang, Z.-P.; Wang, Y.; Zhu, H.; Li, F.-Z.; Shen, Y.-Y.; Guo, S.-R. A New NIR-Triggered Doxorubicin and Photosensitizer Indocyanine Green Codelivery System for Enhanced Multidrug Resistant Cancer Treatment through Simultaneous chemo/Photothermal/Photodynamic Therapy. Acta Biomater. 2017, DOI: 10.1016. (43) Huo, D.; He, J.; Li, H.; Huang, A.-J.; Zhao, H.Y.; Ding, Y.; Zhou, Z.-Y.; Hu, Y. X-Ray CT Guided Fault-Free Photothermal Ablation of Metastatic Lymphnodes with Ultrafine HER-2 Targeting W18O49 Nanoparticles. Biomaterials 2014, 35, 9155-9166. (44) Peng, C.; Zheng, L.-F.; Chen, Q.; Shen, Q.-M.; Guo, R.; Wang, H.; Cao, X.-Y.; Zhang, G.-X.; Shi, X.-Y. PEGylated Dendrimer-Entrapped Gold Nanoparticles for in Vivo Blood Pool and Tumor Imaging by Computed Tomography. Biomaterials 2012, 33, 1107-1119. (45) Black, K. C. L.; Wang, Y.; Luehmann, H. P.; Cai, X.; Xing, W.; Pang, B.; Zhao, Y.-F.; Cutler, C. S.; Wang, L. H. V.; Liu, Y.-J.; Xia, Y.-N. Radioactive 198 Au-Doped Nanostructures with Different Shapes for In Vivo Analyses of Their Biodistribution, Tumor Uptake, and Intratumoral Distribution. ACS Nano 2014, 8, 4385-4394. (46) Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popović, Z.; Jain, R.K.; Bawendi, M. G.; Fukumura, D. Multistage Nanoparticle Delivery System for Deep Penetration into Tumor Tissue. Proc. Natl. Acad. Sci. 2011, 108, 2426-2431. (47) Park, W.; Cho, S.; Huang, X.; Larson, A. C.; Kim, D. H. Branched Gold Nanoparticle Coating of Clostridium Novyi-NT Spores for CT-Guided Intratumoral Injection. Small 2017, 13, 1602722-1602727. (48) Chen, H.; Tian, J.; He, W.; Guo, Z. H2O2Activatable and O2-Evolving Nnanoparticles for Highly Efficient and Selective Photodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547.


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