Ammonium Tungsten Bronze Nanoribbon for

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Ultrathin Tellurium Oxide/Ammonium Tungsten Bronze Nanoribbon for Multi-Modality Imaging and Second Near-Infrared Region Photothermal Therapy Yaru Cheng, Fan Yang, Guolei Xiang, Kai Zhang, Yu Cao, Dongdong Wang, HaiFeng Dong, and Xueji Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04618 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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Ultrathin Tellurium Oxide/Ammonium Tungsten Bronze Nanoribbon for Multi-Modality Imaging and Second NearInfrared Region Photothermal Therapy Yaru Cheng, † Fan Yang, † Guolei Xiang, ‡ Kai Zhang, † Yu Cao, † Dongdong Wang, † Haifeng Dong, *, † and Xueji Zhang, *, † †Beijing

Advanced Innovation Center for Materials Genome Engineering, University of

Science & Technology Beijing, 30 Xueyuan Road, Beijing 100083 (P.R. China) †Research

Center for Bioengineering and Sensing Technology, University of Science &

Technology Beijing, 30 Xueyuan Road, Beijing 100083 (P.R. China) State Key Laboratory of Chemical Resource Engineering, School of Science, Beijing

‡

University of Chemical Technology, 15 Beisanhuan Road, Beijing 100029, P.R. China. *Haifeng *Xueji

Dong. Phone: +86 10 82375840. E-mail: [email protected]

Zhang. Phone/fax: +86 10 82376993. E-mail: [email protected]

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ABSTRACT: Developing nano-photothermal agents (PTAs) with satisfied photothermal conversion efficiency (PTCE) in the second NIR window (1000-1350 nm, NIR II) holds great promise for enhanced photothermal therapy (PTT) effect. Herein, we develop a NIRII PTA with advanced PTCE, based on a new two-dimensional (2D) ultrathin tellurium oxide/ammonium tungsten bronze (TeO2/(NH4)xWO3) nanoribbons (TONW NRs). The doped ammonia ions mediated-free electrons injection into the LUMO band of WO3 combined with the electronic transitions between W6+ ions and the lone pair of electrons in Te atoms achieve excellent NIR absorption of TONW NRs resulted from localized surface plasmon resonance (LSPR). The polyethylene glycol functionalized TONW NRs (PEG-TONW NRs) exhibit good stability and biocompatibility, displaying a PTCE high to 43.6%, surpassing many previous nanoPTAs active in the NIR II region, leading to remarkable tumor ablation ability both in vitro and in vivo. Meanwhile, advanced X-ray computed tomography (CT) and photoacoustic (PA) imaging capability of PEG-TONW NRs were also realized. Given the admirable photothermal effect in NIR II region, good biocompatibility and advanced CT/PA imaging diagnosis capability, the novel PEG-TONW NRs is promising in future personalized medicine application.

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KEYWORDS: ammonium tungsten bronze, tellurium oxide, photothermal therapy, the second NIR window, two-dimensional nanomaterials, multimodal imaging

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Photothermal therapy (PTT) can ablate tumors through the local heat produced by the light absorber under NIR radiation. This minimally invasive means by hyperthermia is a potential alternative to traditional clinical cancer treatment methods.1 Many kinds of photothermal transducing agents (PTAs) were exploited to enhance the photothermal conversion efficiency (PTCE) for enhanced tumor ablation efficiency.2-6 Owing to the intriguing physical and optical properties, ultrathin two-dimensional (2D) nanomaterials offer new possibilities for achieving efficient PTAs possessing high extinction coefficients and high PTCE.7,8 Many 2D nanomaterials, such as graphene and its derivatives,9 transition metal dichalcogenide (TMD),10,11 transition metal carbides and nitrides (MXenes)12, black phosphorus nanosheets13,14 and Pd nanosheets15,16, have been reported as efficient PTAs. However, most reported 2D nanomaterials as PTAs can only work in the NIR-I region (650-950 nm). Compared to the NIR I region, the NIR II region (1000-1350 nm) exhibits a bigger laser maximum permissible exposure (MPE), and provides a larger tissue penetration depth owing to the lower photon scattering and reduced tissue background.17 Previous reports have demonstrated that WO3-x nanostructures showed strong NIR absorption resulted from localized surface plasmon resonance (LSPR) of free electrons for PTT.18 However, little attention has been devoted to the PTT treatment in the NIR II region due to insufficient PTCE. Doping heterogeneous atoms to construct MxWO3 is efficient approach to improve NIR absorption. The [WO6] octahedra in tungsten bronzes are connected by sharing the points, and the extracellular cations 'M' are embedded in one of the two channels along the c direction.19 Hydrogen and small alkali metal ions like Li+ can occupy the narrower triangular channels, while large cations like NH4+ can occupy the hexagonal channels.20 The oxygen deficiency or third element insertion21 will inject free electrons into the LUMO band of WO3, resulting in conductive reduction tungsten oxides (WO3-x, WV2xWVI1-2xO3-x) or tungsten bronzes (MxWO3, M = Tl, Rb, Cs, K, Na, NH4, et al).22 On the other hand, WO3-x nanocomposite with high NIR absorption are also explored. For example, WO3-TeO2 with an enhanced absorption band resulted 3 ACS Paragon Plus Environment

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from the electronic transitions between W6+ ions and the lone pair of electrons on Te atoms has been reported.23 Thus, we speculated that fabricating of WO3-x nanocomposite with heterogeneous atoms doping would hold great potential for improved PTCE. As proof-of-concept, the TeO2/(NH4)xWO3 nanoribbons was synthesized that inspired by the enhanced localized LSPR mechanism of the WO3-x nanostructures. The doped ammonia ions enable to free electrons injection into the LUMO band of WO3 for the (NH4)xWO3 nanostructures, leading to enhanced NIR absorption resulted from LSPR of free electrons. Meanwhile, the electronic transitions between W6+ ions and the lone pair of electrons on Te atoms resulted from the introduction of tellurium oxide would further improve the NIR absorption of ammonium tungsten bronze.23 As we known, the [WO6] octahedra in tungsten bronzes are connected by sharing the points, and the extraneous cations 'M' are embedded in one of the two channels along the c direction. During the growth process of TeO2/(NH4)xWO3, the large NH4+ cations can occupy the hexagonal channels to form (NH4)xWO3.20 So, it is presumably that Te atoms would occupy the triangular channels to further produce TeO2/(NH4)xWO3 nanoribbons (Figure S2).

Scheme 1. Schematic illustration of the applications of PEG-TONW NRs in CT imaging, PA imaging, thermal imaging and PTT of cancer.

Herein, for the first time, we report a new two-dimensional (2D) ultrathin tellurium 4 ACS Paragon Plus Environment

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oxide/ammonium tungsten bronze (TeO2/(NH4)xWO3) nanoribbons (TONW NRs) as a NIR-II PTA with advanced PTCE. The enhanced mechanism of PTCE was appropriately explained. As proof-of-concept, the TeO2/(NH4)xWO3 nanoribbons was synthesized that inspired by the enhanced localized surface plasmon resonance (LSPR) mechanism of the WO3-x nanostructures. The doped ammonia ions enable to free electrons injection into the LUMO band of WO3 for the (NH4)xWO3 nanostructures, leading to enhanced NIR absorption resulted from LSPR of free electrons.22 Meanwhile, the electronic transitions between W6+ ions and the lone pair of electrons on Te atoms resulted from the introduction of tellurium oxide would further improve the NIR absorption of ammonium tungsten bronze.23 The as-synthesized TONW NRs exhibited broad and strong photoabsorption in both NIR II and I window with advanced PTCE. Specifically, the PTCE of nanocomposite in the NIR II window was comparable to most previous reports such as Au−Cu9S5 nanoparticles (37%), Cu3BiS3 nanorods (40.7%), Au@Cu2−xS Core–Shell nanocrystals (43.2%).17 After coating with DSPE-PEG

(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(polyethylene glycol)-2000]) via van der Waals forces and hydrophobic interactions,5 the PEGylated TONW NRs with pretty stable physiological dispersibility and good biocompatibility were obtained for further biomedical applications. It achieved excellent tumor ablation in both in vitro and in vivo upon irradiation with a 1064 nm laser due to the admirable PTCE. The good X-ray computed tomography (CT) and photoacoustic (PA) imaging capability of TONW NRs was also demonstrated (Scheme 1). It not only provided comparable PTCE to recently reported NIR-II counterparts such as cyclo(Arg-Gly-Asp-DPhe-Lys(mpa)) decorated polymer nanoparticles nanoparticles (30.1%) and 2D Nb2C nanosheets (45.2%),24,25 but also possessed multimodal tumor imaging capability. Meanwhile, no complicated synthesized process was involved in comparison with some organic conjugated polymer (TBDOPV-DT) PTAs.26 The TONW NRs with advanced PTCE in the NIR II window and good multimodal tumor imaging capability contributes to rational design new multifunctional PTA nanocomposite active in the NIR II window, 5 ACS Paragon Plus Environment

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holds great promise for improved photothermal therapy (PTT) effect and further personalized medicine application.

Figure 1. Characterization of TONW NRs: a) SEM image, b) TEM and HRTEM image (inset of b) of TONW NRs. c) AFM image of TONW NRs and the AFM-measured thickness of TONW NRs (inset of d). d) XRD pattern and e) TEM-EDX mapping of TONW NRs.

The TONW NRs were prepared by adopting a liquid-liquid interface-mediated method using Te powder and paratungstate ((NH4)10[H2W12O42]4H2O) power as precursors (Figure S1), and the detailed information was provided in the Supporting Information. In the first stage, the Te powder and ((NH4)10[H2W12O42]4H2O) power were evenly distributed at the oil/water interface after intensive stirring. With the help of heating in the second stage, the Te and (NH4)10[H2W12O42]4H2O gradually reacted with each other to form TeO2/(NH4)xWO3 nanoribbons. Scanning electron microscopy (SEM) (Figure 1a) and the transmission electron microscopy (TEM) (Figure 1b) analysis show the uniform ribbon structure of the TONW NRs. It displayed a narrow size distribution with a length of about 210 nm and a width of about 21 nm (Figure S3) from the TEM analysis (Figure 1b). The HRTEM result showed the nanoribbons are crystalline, and grow along the (002) direction as indicated by the lattice fringe of 6 ACS Paragon Plus Environment

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0.378 nm (inset of Figure 1b). The thickness of the as-prepared TONW NRs measured by atomic force microscopy (AFM) was about 6 nm (Figures 1c), which proved the as-prepared TONW NRs was a kind of ultrathin 2D nanosheets. These results demonstrated the unique nanoribbon structure of the TONW NRs, and the high aspect ratio (AR: length/width) facilitated the uptake of nanomaterials by cells for further biomedical application.27 X-ray diffraction (XRD) was employed to characterize the phase composition of the TONW NRs (Figure 1d). The nanoribbons are mainly hexagonal (NH4)xWO3, matching the JCPDS No. 42-0452. The number of sheets of the nanoribbons was estimated to 8 in term of the main hexagonal (NH4)xWO3 cell parameters. Meanwhile, the XRD analysis indicated the sample also contained a small amount of (TeO2)0.95(WO3)0.05 phase (JCPDS No. 52-0796), indicating the TeO2 was grown along WO3. The energy dispersive X-ray fluorescence spectroscopy (EDX) mapping of elements derived from the TEM showed obvious O, W and Te elements (Figure 1e). The distributions of different elements were largely co-localized for TONW NRs, which suggested TeO2 was homogeneously grew on the as-prepared TONW NRs. The energy dispersive spectroscopy (EDS) analysis of TONW NRs further proved the good co-localization of different elements in the sample (Figure S4).

Figure 2. Survey XPS spectrum of a) TONW NRs and corresponding XPS spectra of b) W 4f and c)

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Te 3d in TONW NRs. d) Survey XPS spectrum of (NH4)xWO3 NRs.

Furthermore, elemental composition of TONW NRs was examined using XPS (Xray photoelectron spectroscopy). The TONW NRs exhibited characteristic peaks assigned to W, O, N and Te, and Si peak from the wafer as well as weak C peak from the residual organic solvent were also observed (Figure 2a). By fitting the XPS spectra in Figure 2b, the two strong binding energy peaks at 35.46 eV (W6+ 4f7/2) and 37.78 eV (W6+ 4f5/2) was corresponded to W6+, and the other two peaks located at around 34.48 eV (W5+ 4f7/2) and 36.02 eV (W5+ 4f5/2) were assigned to W5+.28 Moreover, in the Te 3d XPS spectra (Figure 2c), the two strong peaks at around 586.82 eV (Te 3d5/2) and 576.45 eV (Te 3d3/2) were ascribed to Te-O bond, and the other two weak peaks at 583.89 eV (Te 3d5/2) and 573.42 eV (Te 3d3/2) were assigned to Te-W bond.29 In order to prove the enhancement of the photothermal effect of TONW NRs by the introduction of TeO2, we also synthesized (NH4)xWO3 NRs.28 The Figure S5 showed the size and morphology of (NH4)xWO3 NRs were similar with TONW NRs, and the survey XPS spectrum of (NH4)xWO3 NRs in Figure 2d proved the absence of TeO2.29

Figure 3. Synthesis and characterization of PEG-TONW NRs: a) Synthesis process of the PEG-TONW NRs. b) TEM images of PEG-TONW NRs and TONW NRs (inset of b). c) TG plot of the TONW NRs and PEG-TONW NRs record in argon. d) FTIR spectrum of TONW NRs and PEG-TONW NRs. e) Zeta potential of TONW NRs, DSPE-PEG and PEG-TONW NRs.

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To increase the solubility and biocompatibility of TONW NRs, DSPE-PEG was wrapped around the TONW NRs to form PEG-TONW NRs by hydrophobic interactions and van der Waals forces (Figure 3a).11 After coating with DSPE-PEG, the PEG-TONW NRs showed excellent dispersibility in water, PBS (pH 7.4, 10 mM) and DMEM medium (Figure S6). The dynamic light scattering (DLS) and UV-Vis absorption indicated negligible change in aqueous diameter and NIR absorb ability of DSPE-PEG for the PEG-TONW NRs in PBS (pH 7.4, 10 mM), DMEM medium (at room temperature) and serum (37 ℃) compared to that in water, indicating the hydrophilic PEG chain extending into the aqueous phase while solubilizing and stabilizing the TONW NRs in physiological condition (Figure S7). The TEM image of PEG-TONW NPs shown in Figure 3b exhibited an obvious layer of PEG film wrapped evenly around the surface of TONW NRs, and the thickness of the film was between 5 nm to 10 nm. The DLS results suggested the significant aggregation of TONW, and good dispersity of PEG-TONW was also observed after PEG functionalization, which exhibited a mean hydrodynamic size of 200 nm, in agreement with the TEM analysis (Figure S8). The coating amount of DSPE-PEG was further investigated using thermogravimetry (TG) measurement in a nitrogen atmosphere at a heating rate of 10 ℃ min-1 (Figure 3c). The first weight loss stage before 240 ℃ was resulted from the desorption of surface and structural water. The weight loss from 240 ℃ to 413 ℃ and 413-435 ℃ was assigned to the PEG burning and sublimation of NH3 as a result of decomposition of (NH4)xWO3 NRs.18,30 The lose-on-ignition (LOI) of TONW NRs and PEG-TONW NRs were 25.4% and 48.2% within the selected temperature range from 30-600 ℃. Therefore, the coating amount of DSPE-PEG could be calculated to be 22.8% by the discrepancy of normalized LOIs between TONW NRs and PEG-TONW NRs.25 Furthermore, Fourier-transform infrared spectroscopy (FT-IR) and zeta potential were applied to verify the PEGylation process. The FT-IR spectrum of PEG-TONW NRs exhibited the characteristic absorption bands of DSPE-PEG located at around 2900 cm-1 attributed to the C–H vibration and 1650–1660 cm-1 assigned to C=O 9 ACS Paragon Plus Environment

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stretching vibrations (Figure 3d).5 Zeta potential analysis revealed the coating of negative DSPE-PEG polymer on the surface of TONW NRs induced the change of positive TONW NRs to negatively charged PEG-TONW NRs (Figure 3e). These results revealed that DSPE-PEG was successfully coated on the surrounding of TONW NRs, and the resulted PEG-TONW NRs showed excellent dispersion and stability in water and phosphate-buffered saline (PBS; pH 7.4, 10 mM) as well as cell media without detectable agglomeration.

Figure 4. a) UV–vis absorption spectra of PEG-TONW NRs with different concentration. b) Photothermal heating curves of deionized water and PEG-TONW NRs aqueous solutions at different concentrations under NIR laser (1064 nm,1 W cm-2) and the corresponding temperature change c). (d) Photothermal effect of an aqueous solutions of TONW NRs (100 μg mL-1) under 1064 nm laser irradiation (1. W cm−2). (e) The time constant for heat transfer was tested by using the linear time data from the cooling period versus negative natural logarithm of driving force temperature, which was obtained from the cooling stage. f) Photothermal conversion stability PEG-TONW NRs (100 µg mL-1). The laser was turned on/turned for four cycles (1064 nm, 1 W cm-2). The inset shows photos of PEGTONW NRs solution before and after laser irradiation for 40 h. g) Infrared thermal pictures and h) corresponding temperature change of tumor-bearing mice exposed to NIR laser (1064 nm, 1 W cm-2) for 6 min after intravenously injected with PBS (control) and PEG-TONW NRs (20 mg kg-1) for 4 h.

As shown in Figure 4a, PEG-TONW NRs presented a broad absorption range from ultraviolet (UV) to NIR arising from the LSPR. The absorbance at 1064 nm increased 10 ACS Paragon Plus Environment

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gradually with the concentration of PEG-TONW NRs. Given the bigger laser MPE a larger tissue penetration depth of the NIR II region (1000-1350 nm) window compared to the NIR I region window, a 1064 nm laser was employed for the following experiments.1,25 As shown in Figure 4b, the temperature of the PEGTONW NRs aqueous solutions with different concentrations raised quickly with the time of irradiation. Meanwhile, both the heating rates and the ending temperatures increased with the concentrations of PEG-TONW NRs. After exposing to the laser for 10 min, the temperature of PEG-TONW NRs aqueous dispersions (100 µg mL-1) increased by 35.1 °C (Figure 4c), much higher than that of water increased by about 10.4 °C. It is worth noting that even at low concentrations (25 µg mL-1), the temperature was still increased by 23.6°C, and the rapid heating would trigger ablation of tumor cells.17 As showed in Figure S9a, (NH4)xWO3 NRs had lighter color and lower UV–vis absorption spectra of TONW NRs when the concentration was the same. When compared to (NH4)xWO3 NRs without TeO2 introduction (Figure 4b and Figure S9b), the PEG-TONW NRs presented superb photothermal performance, which was speculated that the electronic transitions between W6+ ions and the lone pair of electrons on Te atom would enhance the absorption and thereby improve the PTCE of (NH4)xWO3 NRs. The above experimental results proved PEGTONW NRs could effectively convert NIR energy into thermal energy, while the thermal effect caused by laser irradiation alone is very small. The PTCE (η) was calculated using the previously reported method.5

where h is the heat transfer coefficient, A is the surface area of the container, Tmax is the maximum temperature of system, Ts is the temperature of surrounding, I is the power density of the NIR laser, A1064 is the absorbance of PEG-TONW NRs at the wavelength of 1064 nm, and Q0 is the heat associated with the light absorption of the solvent. The η value of PEG-TONW NRs was determined to be 43.6% according to aforementioned equation (Figure 4d and e), which was not only comparable to the 11 ACS Paragon Plus Environment

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current reported PTAs active in NIR I, such as Cu9S5 nanocrystals (25.7%),31 black phosphorus quantum dots (28.4%),32 Au nanorods (21%),33 Ti3C2 nanosheets (30.6%),12 MoS2 nanosheets (24.37%)10 and antimonene quantum dots (45.5%),5 but also was comparable to most reported PTAs active in NIR II, including Au−Cu9S5 nanoparticles (37%),34 Cu3BiS3 nanorods (40.7%)35, Au@Cu2−xS Core–Shell nanocrystals (43.2%),36 and so on (Table S1). In addition, the PTCE of PEG(NH4)xWO3 NRs was calculated to be 13.73%, which was much lower than that of PEG-TONW NRs (Figure S9c and d), and this result also proved the enhancement of photothermal ability of (NH4)xWO3 NRs by introducing of TeO2. The high η value of PEG-TONW NRs implied it was a promising PTAs. PEG-TONW NRs solution (100 µg mL-1) was exposed to a 1064 nm laser (1 W cm-2) for 10 min, and the 1064 nm laser on/off repetition was carried out four times to evaluate the photostability. The corresponding changeable temperature were observed (Figure 4f), which indicated the high photostability of PEG-TONW NRs. Moreover, after irradiation for 40 min, there was no significant change in the light blue color of PEG-TONW NRs solution (inset in Figure 4f). Thus, the outstanding photothermal performance and high photostability indicated the PEG-TONW NRs were suitable PATs for PTT treatments of tumors. Considering the outstanding photothermal performance of PEG-TONW NRs, the in vivo infrared thermal imaging capability in vivo was investigated. As a result, the temperature of tumor displayed obvious increase compared to that without PEGTONW NRs treatment (Figure 4g). The temperature increased with the irradiation time after injection of PEG-TONW NRs (Figure 4h), which was increased by 28 °C after irradiation for 6 min. The results demonstrated that PEG-TONW NRs could be used as good infrared photothermal imaging contrast agents to achieve real-time monitoring of thermodynamic in the PTT process. By using pork with different thickness (0, 2, 4, 6, 8 and 10 mm) as model biological tissues, the in vitro tissue penetration depth of NIR-I (808 nm) and NIR-II (1064 nm) was explored. The temperature changes of the PEG-TONW NRs aqueous solutions (100 μg mL-1) with 12 ACS Paragon Plus Environment

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above pock under the laser irradiation was detected by a thermal imager. As showed in Figure S10, when the thickness of tissue increased to 6 mm, the temperature of PEG-TONW NRs aqueous solutions (100 μg mL-1) exposed to a 1064 nm laser (1 W cm-2) still showed increases. While by using an 808 nm laser (1 W cm-2) as laser source, the temperature showed negligible change even the tissue thickness was increased to 4 mm, suggesting the much deeper tissue penetration in NIR-II windows than NIR-I windows. And the tissue penetration depth of NIR-II laser at 1064 nm was between 6 mm to 8 mm, which was 2-4 mm deeper than 808 nm laser.

Figure 5. a) Cell viability of MCF-7 and A549 cells after 24 h incubation with different concentrations of PEG-TONW NRs. b) Cell viability of MCF-7 and A549 cells treated with PBS (control), only PEGTONW NRs, only laser irradiation (1064 nm, 1 W cm-2, 5 min) and both PEG-TONW NRs and laser irradiation (1064 nm, 1 W cm-2, 5 min). c) Confocal images of calcein AM (green)/PI (red) co-stained MCF-7 cells after different treatments (scale bar = 200 μm). d) Apoptosis and necrosis analysis of various treatments. d1) control without any treatment; d2) treated with only NIR laser; d3) treated with only PEG-TONW and d4) treated with PEG-TONW and 1064 nm laser.

The cytotoxicity and in vitro PTT performance of PEG-TONW NRs toward MCF-7 cells and A549 cells were explored using the MTT assay. Even though the concentration was high to 200 µg mL-1, the PEG-TONW NRs did not induce significant cytotoxicity for cell lines after incubation for 24 h (Figure 5a). Next, the PTT efficiency in vitro by PEG-TONW NRs was examined by using MCF-7 and 13 ACS Paragon Plus Environment

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A549 cells as model. It indicated either only NIR or only PEG-TONW NRs would induce negligible cytotoxicity toward cell lines. On the contrary, the PEG-TONW NRs treated cells received NIR irradiation presented significant cytotoxicity, and more than 80% cells had been thermally ablated (Figure 5b). The calcein AM/PI twostained assay confirmed the MTT results, in which the live cells were stained by PI to red and dead cells were stained by AM to green. Most of the PEG-TONW NRs treated MCF-7 (Figure 5c) with NIR laser irradiation were killed (Figure S11), similar results were observed for the A-549 cancer cells. On the contrary, cells treated with laser or PEG-TONW NRs alone showed negligible death. Furthermore, flow cytometry with Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide(PI) fluorescence staining was conducted to further demonstrate the cell death, apoptosis and necrosis assay. As showed in the Figure 5d, the control and laser group did not show apparent cell death (