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Oxygen-Deficient Black Titania for Synergistic/Enhanced Sonodynamic and Photo-Induced Cancer Therapy at Near Infrared-II Biowindow Xiaoxia Han, Ju Huang, Xiangxiang Jing, Dayan Yang, Han Lin, Zhigang Wang, Pan Li, and Yu Chen ACS Nano, Just Accepted Manuscript • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Oxygen-Deficient Black Titania for Synergistic/Enhanced Sonodynamic and Photo-Induced Cancer Therapy at Near Infrared-II Biowindow Xiaoxia Han1, Ju Huang1, Xiangxiang Jing2*, Dayan Yang2, Han Lin3, Zhigang Wang1, Pan Li1* and Yu Chen3* 1

Chongqing Key Laboratory of Ultrasound Molecular Imaging, Ultrasound Department of the

Second Affiliated Hospital of Chongqing Medical University. Chongqing, 400010, P. R. China. 2

Department of Ultrasound, Hainan General Hospital, Haikou, 570311, P. R. China.

3

State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China.

ABSTRACT The conventional inorganic semiconductors are not suitable for in vivo therapeutic nanomedicine because of lacking adequate and safe irradiation source to activate them. This work report, on the rational design of titania (TiO2)-based semiconductors for enhanced and synergistic sono-/photo-induced tumor eradication by creating an oxygen-deficient TiO2-x layer onto the surface of TiO2 nanocrystals, which can create a crystalline-disordered core/shell structure (TiO2@TiO2-x) with black color. By learning the lessons from traditional photocatalysis, such an oxygen-deficient TiO2-x layer with abundant oxygen defects facilitates and enhances the separation of electrons (e-) and holes (h+) from the energy-band structure 1

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upon the external ultrasound irradiation, which can significantly improve the efficacy of sono-triggered sonocatalytic tumor therapy. Such an oxygen-deficient TiO2-x layer can also endow black titania nanoparticles with high photothermal-conversion efficiency (39.8%) at NIR-II bio-window (1064 nm) for enhanced photothermal hyperthermia. Both in vitro cell-level and systematic in vivo tumor-bearing mice xenograft evaluations have demonstrated the high synergistic efficacy of combined and enhanced sonodynamic therapy and photothermal ablation as assisted by oxygen-deficient black titania, which has achieved the complete tumor eradication with high therapeutic biosafety and without obvious reoccurrence. This work not only provides the paradigm of high therapeutic efficacy of combined sono-/photo-induced tumor-treatment protocol, but also significantly broadens the nanomedical applications of semiconductor-based nanoplatforms by rational design of their nanostructures and control of their physiochemical properties. KEYWORDS. black titania, sonodynamic therapy, photothermal therapy, nanomedicine, cancer

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The fast development of theranostic nanomedicine has provided versatile therapeutic modalities for cancer treatment, among which the efficient tumor eradication by external non-invasive energy triggers has been considered as one of the most promising strategies because of its high tumor specificity, controllability, desirable therapeutic efficacy and mitigated damages to normal organs/tissues.1-9 On one hand, ultrasound (US) is one of the mostly explored physical triggers based on its features of non-invasiveness, high tissue-penetrating depth and low cost.10-12 Especially, US-triggered sonodynamic therapy (SDT) has been demonstrated to be effective in the production of reaction oxygen species (ROS) for killing cancer cells, which is realized by triggering the elaborately designed nanosonosensitizers.13,14 On the other hand, light, especially near infrared (NIR) light, has been extensively adopted for photo-therapies, such as photothermal therapy (PTT) and photodynamic therapy (PDT).15-18 Although traditional NIR-I bio-window (750 nm - 1000 nm) has improved the penetration depth of light for photo-therapy, it is still not satisfactory for efficient photo-induced tumor therapy. Recent progress on photo-therapy has been focused on the NIR-II bio-window (1000 nm - 1350 nm) because such a long wavelength range can achieve the improved penetration depth and larger maximum permissible exposure (MPR).19,20 Diverse nanoplatforms have been constructed as the nanosonosensitizers for enhancing the SDT effect. Most of these nanosystems were based on the loading of organic sonosensitizer molecules into nanoparticles.14, 21-24 Especially, inorganic titanium oxide (TiO2) nanoparticles have also been explored for SDT.25-28 The sonosensitizing mechanism is based on the US-triggered separation of electrons (e-) and holes (h+) from the energy-band structure 3


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of TiO2 because of its semiconductor nature.27 In addition, diverse organic or inorganic nanoplatforms have been explored for photo-therapy, especially for photothermal hyperthermia. These nanosystems include the typical Au, Prussian, CuS2-x and other two-dimensional (2D) nanosheets such as graphene, transition-metal dichalcogenides, black phosphorus and MXenes.29-33 However, there is still no adequate nanoplatforms with the fast response to both US and light irradiations for concurrent and synergistic US therapy (e.g., SDT) and photo-therapy (e.g., PTT). Bearing the US response of TiO2 nanoparticles for SDT, we herein report, on the construction of a TiO2-based nanoplatform for simultaneous SDT and PTT in NIR-II bio-window simply by creating an oxygen-deficient TiO2-x layer on the surface to produce black core/shell-structured TiO2@TiO2-x nanoplatforms according to an aluminum (Al) reduction procedure. Such an oxygen-deficient TiO2-x layer with abundant defects facilitates and enhances the separation of electrons (e-) and holes (h+) from the energy-band structure upon the US irradiation, causing the high quantum yield of ROS for cancer therapy. This design idea originates from conventional photocatalysis where the black TiO2-x (designated as B-TiO2-x) with oxygen defects in the crystalline structure has been demonstrated to improve the separation efficacy of electrons and holes and enhance photocatalytic efficacy subsequently.34-41 Therefore, US-triggered SDT effect as assisted by black TiO2-x would be significantly enhanced compared to traditional white TiO2 nanoparticles. Importantly, these B-TiO2-x nanosystems are featured with response to NIR irradiation in NIR-II bio-window, which could assist the photo-induced ablation and hyperthermia. This B-TiO2-x-enhanced synergistic SDT and PTT has been demonstrated to completely eradicate the tumor on breast 4


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cancer 4T1 nude-mice xenograft. The in vivo high therapeutic biosafety of these B-TiO2-x nanoparticles was also evaluated in this work to guarantee their further clinical translation. RESULTS AND DISCUSSION Core/shell-structured

B-TiO2-x

nanoparticles

were

fabricated

by

a

facile

aluminum-reduction strategy according to previous report (Figure 1a).34,35 To facilitate the biomedical application and improve the dispersity in aqueous solution, the surface of B-TiO2-x nanoparticles was modified with NH2-PEG2000 molecules by the sonication process for 2 h. PEGylation was simply achieved by the coordination interaction between N atom of PEG molecules and Ti atom of B-TiO2-x nanoparticles. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) were used to identify the microstructure and morphology of B-TiO2-x nanoparticles. It has been found that B-TiO2-x nanoparticles exhibit the typical core/shell structure with an obvious oxygen-deficient amorphous shell on the surface and a single-crystalline core (Figure 1b). Selected area electron diffraction (SAED) pattern further demonstrates the single-crystalline nature of B-TiO2-x nanoparticles (inset of Figure 1b). The low-magnification TEM image shows the relatively high dispersity of B-TiO2-x nanoparticles after the aluminum reduction (Figure 1c), guaranteeing the possible biomedical applications where the small particle size is typically required. The X-ray energy dispersive spectrometer (EDS) analysis (Figure 1d) and electron energy loss spectrum (EELS) (Figure 1e) prove that the main composition of B-TiO2-x nanoparticles are titanium (Ti) and oxygen (O) elements, which have been demonstrated to be highly biocompatible for in vivo biomedical applications.42-44 5


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X-ray diffraction (XRD) was used to characterize the crystalline structure of TiO2 nanoparticles before and after aluminum reduction, which shows that the crystalline degree was reduced after the reduction because of the formation of oxygen-deficient amorphous TiO2-x layer (Figure 1f). Both TiO2 and B-TiO2-x nanoparticles show the major characteristic diffraction peaks of anatase TiO2 at 25.4° (101), 48.3° (200) and 55.2° (211), indicating that the crystalline structure of B-TiO2-x was remained after aluminum reduction. Raman spectrum of TiO2 nanoparticles shows the typical Raman-active modes at 144.0, 199.3, 397.4, 516.1 and 639.4 cm-1. Comparatively, the B-TiO2-x nanoparticles exhibit new bands at 246.9 and 352.9 cm-1 in Raman spectrum (Figure 1g), indicating the structural changes after aluminum reduction due to the formation of disordered shell layer and the layer could break down the Raman selection rule to activate zone-edge and Raman-forbidden modes.38 X-ray photoelectron spectroscopy (XPS) was used to investigate the element-valence status of TiO2 before and after aluminum reduction. The survey signals (Figure 1h and 1i) of Ti and O elements in XPS are similar, and there is no Al element signal indicating no residual Al during reduction. The Ti 2p XPS spectra for both TiO2 and B-TiO2-x nanoparticles are almost identical (Figure 1j and 1k), which indicates that Ti elements have a semblable bonding with environment. The O and C element status was analyzed (Figure S1) and the typical Ti-O binding energy is changed from 529.80 eV to 529.20 eV, indicating the existence of oxygen vacancies.34,35 After the surface PEGylation of B-TiO2-x nanoparticles (designated as B-TiO2-x-PEG) to enhance the stability and dispersity in aqueous solution and facilitate the further biomedical evaluation, the Fourier transform infrared (FTIR) spectroscopy shows one characteristic peak 6


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around 1500 cm-1 of B-TiO2-x-PEG, which is indexed to Ti-N-O groups of NH2-PEG2000, indicating the successful surface PEGylation (Figure S2).45 Dynamic light scattering (DLS) (Figure S3) measurement of PEGylated B-TiO2-x shows an increase of average hydrodynamic particle size from 109.4 nm to 141.8 nm after the surface PEGylation. The changes of zeta potential (Figure S4) before and after PEGylation further confirm the stepwise formation of B-TiO2-x-PEG (from -30.9 mV to -6.5 mV). Importantly, the PEGylated B-TiO2-x could be easily dispersed into physiological solutions (Figure S5) such as Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum, saline, simulated body fluid (SBF) and phosphate buffer saline (PBS) with the desirable colloidal stability, guaranteeing the further biomedical use. To evaluate the sonocatalytic effect of B-TiO2-x nanoparticles, 1,3-diphenylisobenzofuran (DPBF), a commonly used molecular probe, was used to characterize the in vitro ROS-generating efficacy by US-activated B-TiO2-x nanosonosensitizers, such as superoxide anion (O2-) and singlet oxygen (1O2).27,28 The produced ROS could react with DPBF, causing the decrease of characteristic absorption at the wavelength of 410 nm in UV-vis spectrum. It has been found that the absorption of DPBF solution dramatically decreased containing B-TiO2-x as compared with white TiO2 at the same concentration under US irradiation at the power intensity of 1.5 W cm-2 for 120 s irradiation (Figure 2a and 2b). Furthermore, the absorption intensity of DPBF at 410 nm in different time points were measured to calculate the proportion of DPBF decrease (Figure 2c). It has been found that the ROS-generation efficacy of US-triggered B-TiO2-x is much higher than that of white TiO2, demonstrating that the presence of oxygen-deficient amorphous shell in B-TiO2-x could significantly improve the 7


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US-triggered separation efficacy of electrons (e-) and holes (h+) and enhance the ROS generation subsequently, i.e., B-TiO2-x shows the enhanced sonocatalytic efficacy upon external US irradiation as compared to traditional white TiO2 nanoparticles. Importantly, the presence of oxygen-deficient shell in B-TiO2-x endows these black nanoparticles with high photothermal-conversion efficacy in a broad range of light wavelength. UV-vis spectra of B-TiO2-x aqueous solution at elevated concentrations (6.25, 12.5, 25, 50 and 100 ppm) show that these nanoagents have a high light absorption in NIR range, even in the second bio-window (NIR-II). It is well known that the light in the second bio-window has much higher tissue-penetrating capability as compared to that in the first bio-window, but the photothermal nanoagents for photothermal hyperthermia at NIR-II have been rarely developed. On this ground, this work provides the paradigm that B-TiO2-x-based semiconductors could act as the efficient photothermal-conversion nanoagents for PTT in the NIR-II bio-window (1064 nm in this work). The extinction coefficient of B-TiO2-x at 1064 nm was calculated to be 5.54 Lg-1cm-1 (Figure 2d), which is higher than that of traditional graphene oxide in NIR-I bio-window (808 nm, 3.6 Lg-1cm-1) for PTT.46 The photothermal performance of B-TiO2-x aqueous solution was further evaluated at different concentrations (0, 25, 50, 100, 200 and 400 ppm) and varied laser power densities of 1.0 W cm-2 (Figure S6) and 1.5 W cm-2 (Figure 2e), respectively. It has been found that the temperature could research as high as 62.8℃ at the power density of 1.5 W cm-2 (400 ppm, Figure 2e), which is sufficiently high to kill the cancer cells by the hyperthermia. The laser power-dependent photothermal effect (0.5, 0.75, 8


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1.0, 1.25 and 1.5 W cm-2) was also demonstrated (Figure 2f). Especially, the photothermal-conversion efficiency (η) of B-TiO2-x was calculated to be as high as 39.8% (Figure 2g and h), which is much higher than traditional photothermal nanoagents, such as Au nanorods (21%),47 Cu2-x-Se NCs (22%)48 and Au nanovesicles (37%)49 in the NIR-I bio-window. Importantly, there was no obvious deterioration during five laser on/off cycles for the photothermal performance of B-TiO2-x nanoparticles, showing the high photothermal stability of B-TiO2-x nanoagents for photothermal hyperthermia (Figure 2i). Furthermore, the photothermal performance of B-TiO2-x nanoparticles was evaluated in NIR-I biowindow (λ = 808nm, Figure S7), which exhibited that these B-TiO2-x nanoparticles also possessed the photothermal-conversion performance in NIR-I biowindow. Especially, the photothermal performance of B-TiO2-x had no obvious change before and after surface PEGylation in both NIR-I (λ = 808nm, Figure S8) and NIR-II biowindows (λ=1064nm, Figure S9). These B-TiO2-x-PEG nanoparticles could efficiently enter the cancer cells via the typical endocytosis process (Figure 3a).50,51 The US-triggered separation of electrons (e-) and holes (h+) from B-TiO2-x-PEG enables the generation of ROS for killing the cancer cells. Especially, these B-TiO2-x-PEG nanoparticles could act as the photothermal nanoagents for hyperthermia in the NIR-II bio-window. The cytotoxicity of B-TiO2-x-PEG nanoparticles were evaluated by the typical cell-counting kit 8 (CCK-8) protocol. These B-TiO2-x-PEG nanoparticles show no obvious cytotoxicity even at the concentration of as high as 600 ppm (Figure 3b) for different incubation durations (12, 24 and 48 h). The standard CCK-8 assay was initially conducted to investigate the in vitro cytotoxicity of B-TiO2-x-PEG exposed to 1064 nm laser, B-TiO2-x-PEG irradiated by US activation and 9


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B-TiO2-x-PEG combined with US and 1064 nm laser irradiations. After 1064 nm laser irradiation (1.5 W cm-2, 5 min), the cancer-cell viability incubated with B-TiO2-x-PEG as PTT agent for 24 h was decreased with the cell-viability rate of 64.5% (Figure 3c), which was also power intensity-dependent, concentration-dependent and irradiation duration-dependent (Figure S10). When further US irradiation (1.0 MHz, 1.5 W cm-2, 50% duty cycle, 5 min) was conducted, the cell-viability rate dramatically decreased to 16.5%, which was much higher than that of B-TiO2-x-PEG combined US only (34.2%). The cell-killing effect of B-TiO2-x-PEG

as

nanosonosensitiers

was

also

power

intensity-dependent,

concentration-dependent and irradiation duration-dependent (Figure S11). Comparatively, the control group, B-TiO2-x-PEG only group, laser only group and US only group show no significant cell damage and cell-viability decrease. Such a synergistic SDT/PTT effect was also power intensity-dependent (1.0 Wcm-2, Figure S12). Furthermore, the intracellular uptake of B-TiO2-x-PEG was investigated. Fluorescein isothiocyanate (FITC) was used to label B-TiO2-x-PEG. Confocal laser scanning microscopy (CLSM) and flow cytometry were used to investigate the intracellular uptake of B-TiO2-x-PEG at prolonged incubation durations (0, 1, 2 and 4 h). After 4 h co-incubation with 4T1 cells, the green fluorescence originated from FITC-labeled B-TiO2-x-PEG could be directly observed in the cytoplasm of 4T1 cancer cells (Figure S13). An obvious increase of fluorescence intensity of FITC-labeled B-TiO2-x-PEG was measured from 0% to 89.2% by flow cytometry (Figure S14). Especially, the synergistic effect of SDT and PTT was also confirmed by flow-cytometry apoptosis assay based on the typical AnnexinV-FITC and PI co-staining protocol (Figure 3d). Furthermore, after various treatments, the cell-killing effect 10


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was directly observed by CLSM where the live and dead cells were stained by calcein-AM (green) and PI (red), respectively. A host of dead cells were observed in B-TiO2-x-PEG combined with US and laser group, indicating that the cell apoptosis and death extensively occurred for synergistic SDT and PTT (Figure 3e). To directly evaluate the in vitro ROS production in 4T1 cancer cells which were co-incubated with B-TiO2-x-PEG nanoparticles for 4 h after US irradiation (1.0 MHz, 1.5 W cm-2, 50% duty cycle, 5 min), 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to demonstrate the generation of ROS due to the high fluorescence of 2′,7′-dichlorofluorescein (DCF), which was derived from DCFH-DA in the presence of ROS. The presence of obvious green fluorescence representing DCF was clearly observed in the group of B-TiO2-x-PEG combined with US irradiation (Figure 3f). Especially, the combined US and laser treatment further enhanced the ROS generation, indicating that the photothermal-induced temperature elevation facilitates the sonocatalytic process where the separation of electrons and holes could be enhanced, which has been demonstrated in photothermal-accelerated photocatalytic process based on TiO2 semiconductor photocatalysts.52,53 In addition, the ROS production was further confirmed by flow cytometry where the fluorescent-intensity trend was in consistent with the CLSM observation (Figure 3g). The high in vitro synergistic effect of SDT and PTT indicates the potential in vivo high tumor-suppression efficacy as assisted by B-TiO2-x-PEG acting as both nanosonosensitizer and photothermal-conversion nanoagent. Therefore, 4T1 breast tumor xenograft was established on nude mice to evaluate the in vivo synergistic effect. Initially, the circulation of B-TiO2-x-PEG nanoparticles in bloodstream was investigated and the circulation half-time was 11


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calculated to be 0.95 h (Figure 4a). The eliminating rate for the first 2 h (0.70 ppm/h) is 35 times higher (Figure 4b) than that of B-TiO2-x-PEG in the next 22 h (0.02 ppm/h). Importantly, the intravenously administrated B-TiO2-x-PEG nanoparticles could efficiently accumulate into tumor tissue with the accumulation efficiency of 1.3% in 4 h and 4.4% in 24 h (Figure 4c), which was attributed to the typical EPR effect for the passively targeted tumor accumulation of B-TiO2-x-PEG nanoparticles.54,55 To evaluate the enhanced and synergistic efficacy of SDT (the generation of ROS) and PTT (photothermal hyperthermia, Figure 4d), thirty-five 4T1 tumor-bearing mice were randomly separated into seven groups (n = 5 per group), including (a) control group (treated with intravenously injected saline), (b) B-TiO2-x-PEG group (only intravenously injected with B-TiO2-x-PEG nanoparticles), (c) laser group (only irradiated by 1064 nm laser), (d) US group (only exposed to US), (e) B-TiO2-x-PEG combined with laser group (treated with 1064 nm laser after intravenous injection of B-TiO2-x-PEG nanoparticles), (f) B-TiO2-x-PEG + US group (treated with US after intravenous injection of B-TiO2-x-PEG nanoparticles), (g) B-TiO2-x-PEG combined with laser and US group (treated with both laser and US after intravenous injection of B-TiO2-x-PEG). The above-mentioned protocol was conducted after intravenous injection of B-TiO2-x-PEG at the dose of 15 mg/kg for 24 h. The tumor volume and body weight were measured every two days after the treatments. Furthermore, the US irradiation (1 MHz, 1.5 W cm-2, 50% duty cycle, 5 min) was repeated on the third and the fifth day during the therapeutic process (Figure 4e). The efficient accumulation of B-TiO2-x-PEG into tumor could quickly elevate the tumor 12


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temperature upon laser irradiation in NIR-II bio-window (1064 nm). The tumor temperature increased from 30.3℃ to 53.4℃ after exposure to 1064 nm laser at a power density of 1.5 W cm-2 for 10 min (Figure 4f and g), which was sufficiently high to ablate the tumor tissue. In contrast, the tumor temperature in laser only group rose by 4℃. No obvious changes were monitored in body weight among these treatment groups (Figure 5a), indicating the relatively high therapeutic biosafety. Both photothermal hyperthermia and sonodynamic process as assisted by B-TiO2-x-PEG nanoparticles could efficiently suppress the tumor growth (Figure 5b and c), but the obvious tumor reoccurrence occurred after the therapy. Comparatively, the combined photothermal ablation and US irradiation in the therapeutic (g) group completely eradicated the tumor without the reoccurrence in the following monitoring duration, demonstrating the high synergistic efficacy of combined PTT and SDT. Especially, the tumor-inhibition rate results show the 100% inhibition rate in group (g), which is significantly higher than group (e) (54.2%) and group (f) (74.6%). The survival rate in group (g) is also highest among these groups over the monitoring duration of fifty days (Figure 5c). Comparatively, all 4T1 tumor-bearing mice treated with only saline, B-TiO2-x-PEG only, laser only and US only show the rapid tumor growth and low tumor-inhibition rate (Figure 5d and e). Hematoxylin and eosin (H&E), TdT-mediated dUTP nick-end labeling (TUNEL) and Ki-67 antibody staining of tumor tissues were conducted to investigate the mechanism of synergistic SDT/PTT after different treatments. There is no obvious change of cell status in control group, B-TiO2-x-PEG group, laser group and US group. Comparatively, H&E and TUNEL results show a plenty of dead cells in the tumor sections in the groups of (e), (f) and 13


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(g). Especially, the group (g) with combined laser and US irradiation based on B-TiO2-x-PEG exhibits much higher cell necrosis in the tumor tissues (Figure 5f) as compare to other therapeutic groups. Ki-67 antibody staining was commonly used to test proliferative status of cancer cells, and the result is in accordance with H&E and TUNEL results, confirming the synergistic effects induced by combined SDT and PTT assisted by B-TiO2-x-PEG as both nanosonosensitizer and photothermal nanoagent. Furthermore, H&E staining of the major organs (heart, liver, spleen, lung and kidney) was conducted to evaluate the therapeutic safety after varied treatments. No obvious pathological changes of tissues are observed in therapeutic groups (Figure S15), indicating the relatively high therapeutic biosafety. Additionally, the excretion process in urine and faeces show that almost 55.4% of Ti content was excreted out of the body after intravenous injection in 48 h, indicating the easy excretion of these B-TiO2-x-PEG out of the body after the synergistic SDT/PTT treatment (Figure 5g). CONCLUSIONS In summary, this work reports on the creation of an oxygen-deficient TiO2-x layer onto the surface of TiO2 nanocrystals (B-TiO2-x) to enhance their sonocatalytic efficacy for improved tumor sonodynamic therapy. Such an oxygen-deficient TiO2-x layer with abundant defects facilitates the separation of electrons (e-) and holes (h+) from the energy-band structure of B-TiO2-x upon the US irradiation. Especially, this core/shell structure of B-TiO2-x can endow these B-TiO2-x nanoparticles with high photothermal-conversion efficiency (39.8%) in NIR-II bio-window. Both in vitro cell-level and systematic in vivo tumor-bearing mice 14


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xenograft evaluations have demonstrated the high synergistic efficacy of combined and enhanced SDT and PTT, which has achieved the complete tumor eradication without obvious reoccurrence. The high therapeutic biosafety and easy excretion of these B-TiO2 nanoparticles have also been evaluated and demonstrated to guarantee their further clinical translation. Therefore, this work not only provides the demonstration of high therapeutic efficacy of combined sono-/photo-induced tumor-treatment protocol (SDT and PTT), but also significantly extends the biomedical applications of TiO2-based nanoplatforms by rational design of their nanostructures and control of their physiochemical properties.

EXPERIMENTAL SECTION Synthesis of B-TiO2-x and B-TiO2-x-PEG. B-TiO2-x nanoparticles were fabricated by an aluminum (Al) reduction procedure according to previous report.34,35 To improve the dispersity and stability of B-TiO2-x in the physiological environment, NH2-PEG2000 (JenKem Technology (Beijing) Co., Ltd.) was used to modify the surface of B-TiO2-x nanoparticles. Typically, B-TiO2-x (10 mg) and NH2-PEG2000 (100 mg) were mixed together and sonicated by an ultrasonic cell crusher (Bilon-1000Y Shanghai Bilon Instrument Manufacturing Co., Ltd) for 90 min in an ice bath. The resulting B-TiO2-x-PEG2000 was collected by centrifugation and further washed for several times with deionized water, followed by re-dispersing in water for further use. Characterization. JEM-2100F transmission electron microscope was used to acquire the TEM image, HRTEM image and corresponding EDS and EELS spectra. X-ray diffraction (XRD) patterns were obtained on a Rigaku D/MAX-2200 PX XRD system, and the 15


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parameters were set as Cu Kα, 40 mA and 40 kV. Confocal microscope Raman spectrometer system (in Via Qontor, Renishaw) was operated to record Raman spectra. X-ray photoelectron spectroscopy (XPS) was conducted on ESCAlab250 Thermal Scientific. The particle-size distribution and zeta potential measurements were conducted on a Zetasizer system (Nano ZS90, Malvern Instrument Ltd.). UV-3600 Shimadzu UV-vis-NIR spectrometer was used to record UV-vis-NIR absorption spectra. A 1064 nm multimode pump laser (Shanghai Connect Fiber Optics Co. Ltd) was used as the irradiation source for photothermal hyperthermia. An Agilent 725 Inductively coupled plasma-optical emission spectrometry (Agilent Technologies) was operated to confirm the quantitative analysis of the contents of nanoparticles. Confocal laser scanning microscopy images were recorded by FV1000 (Olympus Company, Japan). Intracellular uptake and cell apoptosis was obtained by BD LSRFortessa flow cytometry (Becton, Dickinson and Company, USA). Ultrasound irradiation for sonodynamic therapy was conducted by an Intelect Transport Ultrasound (Chattanooga Group, USA). In Vitro ROS Generation from B-TiO2-x by Ultrasound Activation. As a typically used molecular

probe

for

detecting

in

vitro

singlet

oxygen

(1O2)

production,

1,3-diphenylisobenzofuran (DPBF, 2 mL) dissolved in DMF was added into TiO2 or B-TiO2-x aqueous solution (1 mL, Ti concentration: 500 ppm). Then, the mixture was irradiated by ultrasound (1 MHz, 50% duty cycle, 1.5 W·cm-2) for prolonged durations in the dark. The changes of DPBF was characterized by recording the absorption intensity in UV-vis-NIR spectrum. In Vitro Photothermal Effect of B-TiO2-x and B-TiO2-x-PEG by Laser Irradiation in 16


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NIR-I and NIR-II Bio-window. An infrared thermal image recorder (FLIR TM A325SC camera) was used to characterize the photothermal performance of B-TiO2-x and B-TiO2-x-PEG by recording the temperature changes during laser irradiation in NIR-I bio-window (808 nm) and NIR-II bio-window (1064 nm). B-TiO2-x and B-TiO2-x-PEG dispersed in deionized water at different Ti concentrations (0, 25, 50, 100, 200 and 400 ppm) was exposed to 808 nm and 1064 nm pump laser irradiation at the laser-power density of 1.0 W·cm-2 and 1.5 W·cm-2. In addition, the temperature increase of B-TiO2-x and B-TiO2-x-PEG aqueous solution at the Ti concentration of 400 ppm as irradiated by a 1064 nm laser at different power intensities (0.5, 0.75, 1.0, 1.25 and 1.5 W·cm-2) was tested. Cell Culture and In Vitro Cytotoxicity Assay. 4T1 murine breast cancer cells were used for the following in vitro and in vivo evaluations. These cells were purchased from Shanghai Institute of Cells, Chinese Academy of Sciences and cultured in a humidified incubator in the setting of partial pressure of 5% CO2 at 37℃ in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, GIBCO, Invitrogen), which was supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). 4T1 cells were generally seeded in cell culture flask (Corning, USA) for 12 h to adhere. Then, they were harvested by 0.25% trypsin-EDTA solution (GIBCO, Invitrogen) for 2 min for the following experiments. To evaluate in vitro cytotoxicity of B-TiO2-x-PEG, a standard CCK-8 viability assay (Sihai Bio-Tech Co., Ltd, Shanghai) was conducted. Varied Ti concentrations of B-TiO2-x-PEG (0, 25, 50, 100, 200 and 400 ppm) was co-incubated with 4T1 breast cancer cells pre-seeded in 96-well plants for 12, 24 and 48 h, then CCK-8 diluted by DMEM at a ratio of 1:10 was added into plates to test the cell viabilities at a wavelength of 450 nm after 17


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60 min on a microplate reader. In Vitro Synergistic Sonocatalytic and Photothermal Ablation of Cancer Cells Assisted by B-TiO2-x-PEG. A standard CCK-8 protocol was used to evaluate the synergistic sonocatalytic and photothermal performance of B-TiO2-x-PEG for killing cancer cells. 4T1 cells were seeded in 96-well plates in DMEM containing 10% FBS for 12 h to adhere the plates at the density of 1×104 per well, and B-TiO2-x-PEG (Ti concentration: 100 ppm) was then added for co-incubation. To evaluate the killing effect of synergistic therapy, the wells were divided into seven groups, including control group, B-TiO2-x-PEG only group, NIR-I laser only group, US only group, B-TiO2-x-PEG combined laser group, B-TiO2-x-PEG combined US group and B-TiO2-x-PEG co-combined US and laser group. The cell viabilities in each group were determined by comparing with control group (set as 100%). The laser-power intensity was set as 1.0 W·cm-2 and 1.5 W·cm-2, and the US irradiation parameters were set as 1.0 MHz, 50 % duty cycle, 1.0 W·cm-2 and 1.5 W·cm-2. Intracellular Endocytosis Analysis. CLSM was used to observe the endocytosis process of B-TiO2-x-PEG into cancer cells. 4T1 cells were seeded into CLSM-specific culture dishes (1 × 105 per dish) in humidified incubator at 37℃ for 12 h, then the media was replaced by FITC-labelled B-TiO2-x-PEG dispersed in DMEM containing 10 % FBS (1mL,100 ppm). After various co-incubating periods (0, 1, 2 and 4 h), DAPI (100 μ L, Beyotime Biotechnology) diluted by methyl alcohol (1:10) was used to stain cell nuclei for 20 min and then washed by PBS for CLSM observation. For flow-cytometry test, 4T1 cells were planted into 6-well plates at a density of 3 × 105 for 12 h. Then, the culture media was replaced with FITC-labeled B-TiO2-x-PEG (1mL, 100 18


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ppm in DMEM) and co-incubated for 0, 1, 2 and 4 h. The cancer cells were collected by trypsin to a specific test tube, and flow cytometry analysis was used to determine the fluorescence intensity of FITC-labeled B-TiO2-x-PEG in cancer cells. In Vitro Synergistic SDT/PTT Efficacy as Determined by CLSM and Flow Cytometry. 4T1 breast cancer cells were seeded into CLSM-specific dishes and cultured in DMEM supplemented with 10% FBS overnight, and then co-incubated with B-TiO2-x-PEG for 4 h. The dishes were treated with different patterns, including control group, B-TiO2-x-PEG group, NIR-I laser group, US group, B-TiO2-x-PEG + laser group, B-TiO2-x-PEG + US group and B-TiO2-x-PEG + laser/US group. The NIR-II laser was set at the power density of 1.5 W·cm-2. The parameters of US were set as 1 MHz, 50% duty cycle and 1.5 W·cm-2. The Calcein-AM (15 μL) and PI (15 μL) (Donjindo Molecular Technologies, Inc) dispersed in PBS (7.5 mL) were used to replace the cell culture media and stained live (green) and dead (red) cells after varied treatments. After 15 min staining, the cells were washed by PBS for three times and observed by CLSM. For apoptosis analysis by flow cytometry, 4T1 cells were plated in 6-well plate for 12 h for obtaining a high cell attachment rate and B-TiO2-x-PEG (100 ppm, 3mL) was then added into wells and subsequently incubated for another 4 h. The adherent cells were treated by different protocols as mentioned above, and the cells were collected via Trypsin-EDTA solution after the treatments. After 2 min of cell dissociation, the cells were collected by centrifugation and then added with PBS into two batches for other two centrifugations. Then, 500 µL binding buffer was added to re-disperse the cancer cells. Annexin V (5 μL) and FITC (5 μL) (Dojindo Laboratorise, Co., Ltd) were used to stain the live and dead cells for 20 min 19


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according to the instructions. Finally, the flow cytometer was operated to detect cell apoptosis after treatments. In Vitro ROS Generation as Observed by CLSM and Flow Cytometry. 4T1 cells were planted into CLSM-specific culture dishes at the density of 1×105 cells per dish for 12 h, and co-incubated with B-TiO2-x-PEG at 37℃ for 4 h. After incubation, the media was removed and cells were gently washed by PBS for three times. DCFH-DA (Beyotime Biotechnology, Shanghai) in DMEM was added to dishes for 1 h. Then, the cells were treated by US radiation (1 MHz, 50% duty cycle, 1.5 W·cm-2) for 5 min. The cells were then observed by CLSM, where the cells were stained in green because of the ROS generation by sonocatalytic process. For measuring the ROS production by flow cytometry, 4T1 cells were seeded into 6-well plates at the density of 3×105 cells per cell. After 12 h, B-TiO2-x-PEG was added into the plates to co-incubate with 4T1 cells for 4 h, which was then washed by PBS for three times. DMEM containing DCFH-DA was added for further 1 h co-incubation. After different treatments (US irradiation for 5 min), the cells were collected by Trypsin-EDTA solution and centrifugation for detecting the intracellular fluorescence intensity of DCF. Pharmacokinetics, Biodistribution and Metabolism Evaluation of B-TiO2-x-PEG. All animal procedures were managed in agreement with the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of Chongqing Medical University. For establishing the animal models, 4T1 cells (1×106 cells, 100 μL PBS) were subcutaneously injected into right back of 6-week old female nude mice. For pharmacokinetic analysis, 15 μL of blood of 4T1-bearing mice (n = 5) were collected after tail vein injection of B-TiO2-x-PEG 20


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at different durations (2 min, 8 min, 10 min, 15 min, 30 min, 1 h, 4 h, 6 h, 12 h and 24 h). The Ti concentration in blood was measured by ICP-OES. Biodistribution of B-TiO2-x-PEG in tumor tissues and major organs were determined in 4T1 breast tumor bearing mice (n = 9). 4T1 female tumor bearing mice were divided into 3 groups randomly and intravenously injected with B-TiO2-x-PEG in saline at the dosage of 15 mg·mL-1. The mice were sacrificed in pre-planned time intervals (4, 24 and 48 h) and dissected to collect the main organs and tumors. These sections were weighed, homogenized and dissolved in aqua regia. The Ti content in these organs and tumor tissue was measured by ICP-OES and the distribution was calculated by the original dose of per gram of tissue. Metabolism of B-TiO2-x-PEG was investigated in 4T1 tumor-bearing mice (n = 3), B-TiO2-x-PEG in saline (15 mg·kg-1 per mouse) were intravenous injected into mice. The faeces and urine were collected at pre-planned time periods (2, 6, 12, 24 and 48 h). Finally, the Ti content in faeces and urine was analyzed by ICP-OES. In Vivo Synergistic SDT and PTT as Assisted by B-TiO2-x-PEG. Thirty-five female 4T1 tumor-bearing mice were fed at Laboratory Animal Center, Chongqing Medical University. 4T1 cells (1×106 cells per mouse) were suspended in 100 μL PBS and injected into right back of mice to establish animal tumor xenograft. After the tumors grew up to nearly 50 mm3, the mice were divided into seven groups (n = 5) as following: (a) control group (treated with saline), (b) B-TiO2-x-PEG only group (treated with B-TiO2-x-PEG injected via tail vein), (c) laser only group (treated with 1064 nm laser), (d) US only group (treated with US irradiation), (e) B-TiO2-x-PEG + laser group (injected with B-TiO2-x-PEG followed by 1064 nm laser 21


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irradiation), (f) B-TiO2-x-PEG + US group (injected with B-TiO2-x-PEG followed by US irradiation), (g) B-TiO2-x-PEG + laser + US group (injected with B-TiO2-x-PEG followed by laser and US irradiation). The injection dose of B-TiO2-x-PEG was 15 mg·mL-1. After 4 h of intravenous injection, the 1064 nm laser (1.5 W·cm-2, 10 min) and US (1 MHz, 50 % duty cycle, 1.5 W·cm-2, 5 min) were operated to execute the above therapeutic schedule. The body weight and length and width of tumor were measured by digital scale and caliper every two days, respectively. The tumor volume was calculated by following formula: Tumor volume (mm3) = a × b2/2. where a and b mean the maximum length (mm) and minimum width (mm) of tumor, respectively. The tumors were collected and sliced for further being stained by Hematoxylin and eosin (H&E) for observing the structure and status of cells, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) for detecting DNA fragmentation during apoptosis and Ki-67 antibody staining for determining the growth fraction of cells after 24 h of different treatments. According to the standard animal protocol, mice with tumors larger than 1000 mm3 should be euthanized. Statistical Analysis. The data were shown as mean ± standard deviation (SD) and the significance between two groups of the data in this work was analyzed based on Student’s two-tailed t test (*p < 0.05, **p < 0.01, ***p < 0.001).

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Figure 1. Composition and structure characterization of B-TiO2-x. (a) The scheme of the fabrication of B-TiO2-x by aluminum reduction. (b) High-resolution TEM image of B-TiO2-x. Inset: SAED pattern of B-TiO2-x. (c) Low-magnification TEM image of B-TiO2-x. (d) X-ray energy dispersive spectrum (EDS) and (e) electron energy loss spectrum (EELS) of B-TiO2-x. (f) X-ray diffraction (XRD) patterns and (g) Raman spectra of TiO2 and B-TiO2-x. (h) X-ray photoelectron spectroscopy (XPS) spectra of TiO2 and B-TiO2-x and (i) corresponding element contents. Fitted Ti 2p XPS spectra of (j) TiO2 and (k) B-TiO2-x. Inset: Element-content distribution in TiO2 and B-TiO2-x.

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Figure 2. In vitro sonodynamic and photothermal effect of B-TiO2-x. UV-vis spectra of (a) TiO2 and (b) B-TiO2-x solution containing DPBF and further exposure to US irradiation (power density: 1.5 W cm-2; Duty cycle: 50%) for prolonged duration (0, 1, 2, 3 and 4 min). (c) Relative absorption of DPBF after co-incubation with TiO2 and B-TiO2-x followed by US irradiation under different conditions. (d) UV-vis spectra of B-TiO2-x dispersed in aqueous solution at different concentrations (6.25, 12.5, 25, 50 and 100 ppm). Inset: Mass extinction coefficient of B-TiO2-x at 1064 nm. Normalized absorbance intensity at λ = 1064 nm divided by the characteristic length of cell (A/L). (e) Elevated concentrations (0, 25, 50, 100, 200 and 400 ppm) of B-TiO2-x and TiO2 irradiated by NIR-II (1064 nm) at the power intensity of 1.5 W cm-2. (f) Photothermal-heating curves of B-TiO2-x dispersed in aqueous solution irradiated under varied power densities (0.5, 0.75, 1.0, 1.25 and 1.5 W cm-2). (g) Photothermal performance of the aqueous dispersion of B-TiO2-x under the NIR irradiation by 1064 nm laser at the power intensity of 1.5 W cm-2 for periods, and the laser was cut off when the temperature tended to be stable. (h) Time constant for heat transfer calculated from the cooling period. (i) Heating curve of B-TiO2-x dispersed in water for five cycles at the power intensity of 1.5 W cm-2 under the irradiation by 1064 nm laser.

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Figure 3. In vitro SDT/PTT-based synergistic cancer therapy. (a) Schematic illustration of B-TiO2-x-PEG for enhanced/synergistic sonodynamic and photothermal therapy of cancer cells. (b) Relative cell viability of 4T1 cells after incubation with B-TiO2-x-PEG at elevated concentrations (0, 37.5,75, 150, 300 and 600 ppm) for 4 h, 24 h and 48 h. (c) Relative cell viability of 4T1 cells after different treatments, including control (without treatment), B-TiO2-x-PEG only, Laser only, US only, B-TiO2-x-PEG combined with laser irradiation, B-TiO2-x-PEG combined with US irradiation and B-TiO2-x-PEG combined with Laser/US co-irradiation. (***P < 0.001). (d) Flow-cytometry apoptosis assay of 4T1 cells after the incubation with B-TiO2-x-PEG under different treatments followed by staining with Annexin-FITC and PI. (e) CLSM images of 4T1 cells after different treatments, which were stained by PI (red fluorescence) and calcein-AM (green fluorescence). The scale bar is 40 µm. (f) CLSM images of cells stained with DCFH-DA after different treatments. The scale bar is 20 µm. (g) Flow cytometry of ROS production in cancer cells as stained with DCFH-DA after different treatments. 25


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Figure 4. In vivo pharmacokinetic evaluation, biodistribution analysis and photothermal-performance assessment. (a) The blood-circulation curve of intravenously administrated B-TiO2-x-PEG. (b) The eliminating rate curve originated from the blood-circulation curve. (c) The biodistribution of Ti (% ID of Ti per gram of tissues) in main organs and tumor after intravenous injection of B-TiO2-x-PEG for 2, 24 and 48 h. (d) Schematic illustration of synergistic SDT and PTT as assisted by B-TiO2-x-PEG for tumor eradication. (e) In vivo therapeutic protocol of separate and combined SDT and PTT. (f) Temperature elevation of tumor region in 4T1 tumor-bearing mice under irradiation of 1064 nm laser at the laser density of 1.5 W cm-2 for 600 s with or without the assistance of intravenously administrated B-TiO2-x-PEG, and (g) corresponding IR images of 4T1 tumor-bearing mice under irradiation at varied time intervals (0, 2, 4, 6, 8 and 10 min).

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Figure 5. In vivo synergistic SDT/PTT on tumor suppression at NIR-II Bio-window. (a) Time-dependent body-weight curves and (b) time-dependent tumor-volume curves of 4T1 tumor-bearing mice in groups of control group, B-TiO2-x-PEG only group, laser only group, US only group and B-TiO2-x-PEG combined with laser group, B-TiO2-x-PEG combined with US group and B-TiO2-x-PEG combined with laser and US group. (c) Digital photos of 4T1 tumor-bearing mice and their tumor regions after varied treatments at the 15th day. (d) Tumor-inhibition rate and (e) survival curves of 4T1 tumor-bearing mice after different treatments. (f) H&E staining, TUNEL staining and Antigen Ki-67 immunohistochemistry staining in tumor region of each group after the treatments. Scale bar: 50 µm. (g) Accumulated Ti excretion out of the body after the intravenous injection of B-TiO2-x-PEG for different periods (2, 6, 12, 24 and 48 h).

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ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available from the ACS Publications website at DOI: (will be filled in by the editorial staff). Supplementary experimental details and calculation of photothermal-conversion efficiency, additional data for characterization of black-titania nanoparticles, in vitro photothermal performance, their therapeutic outcome and in vivo biocompatibility.

AUTHOR INFORMATION Corresponding Author *[email protected]; *[email protected]; *[email protected].

The authors declare no competing financial interest.

ACKNOWLEDGMENT We greatly acknowledge the financial support from the National Key R&D Program of China (Grant No. 2016YFA0203700), National Natural Science Foundation of China (Grant No. 51722211, 51672303, 81760317, 31630026 and 81771847) and Young Elite Scientist Sponsorship Program by CAST (Grant No. 2015QNRC001). We thank Dr. Tianquan Lin from SICCAS for providing the assistance on the fabrication of black TiO2-x nanoparticles.

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