Two-Dimensional Graphene Augments Nanosonosensitized

Two-Dimensional Graphene Augments Nanosonosensitized Sonocatalytic Tumor Eradication Chen Dai,†,§ Shengjian Zhang,*,‡ Zhuang Liu,‡ Rong Wu,*,† and Yu Chen*,∥ †

Department of Ultrasound in Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, People’s Republic of China ‡ Department of Radiology, Fudan University Shanghai Cancer Center, Shanghai 200032, People’s Republic of China § Department of Ultrasound, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, People’s Republic of China ∥ State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China S Supporting Information *

ABSTRACT: Ultrasound (US) can activate sonosensitizers for sonodynamic therapy (SDT), but the low activation efficiency and therapeutic outcome significantly hinder its further clinical translation. Inspired by the principles of semiconductor physics and photocatalysis chemistry, we herein report on augmenting the sonocatalytic efficiency of semiconductor TiO2-based nanosonosensitizers for highly efficient SDT by the integration of two-dimensional (2D) ultrathin graphene with TiO2 nanosonosensitizers. The high electroconductivity of graphene facilitates the separation of the electron (e−) and hole (h+) pairs from the energy band of TiO2 and avoids their recombination upon external US irradiation; thus it significantly augments the therapeutic efficiency of TiO2 nanosonosensitizers for SDT against tumors. By further MnOx functionalization, these 2D composite nanosonosensitizers achieved tumor microenvironment-sensitive (mild acidity) T1-weighted magnetic resonance imaging of tumors for therapeutic guidance and monitoring. The high photothermal-conversion capability of graphene also synergistically enhanced the SDT efficiency, achieving the complete eradication of a tumor without reoccurrence. This work provides a paradigm for augmenting semiconductor TiO2-based sonocatalytic therapeutic nanomedicine by learning the physiochemical principles from traditional photocatalysis, which also demonstrates a highly efficient noninvasive and safe therapeutic modality for tumor eradication by the nanosonosensitized sonocatalytic process. KEYWORDS: sonodynamic therapy, TiO2 sonosensitizer, graphene, synergistic therapy, nanomedicine

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the severe side effects and damage to normal organs/tissues usually cause an undesirable therapeutic outcome.14 Photodynamic therapy (PDT, laser as outer source) activates photosensitizers to generate toxic singlet oxygen (1O2) for inducing tumor-cell death, but the low tissue-penetrating depth of light hinders the treatment of deep-seated tumors.15−17 Ultrasound (US), as a mechanical wave in physics, has been extensively explored for diagnostic imaging in the clinic.18,19 It can also exert a therapeutic function such as high-intensity-

he ideal therapeutic modality for cancer aims to trigger the tumor-specific treatment,1−4 which means that the therapeutic toxicity is generated only at the tumor site while the normal tissue/organ should not be damaged. On these grounds, various tumor microenvironment (TME)responsive treatment protocols (e.g., pH, redox, hypoxia, and immune response) have been proposed to achieve this tumor specificity.5−9 However, this endogenous TME response is substantially influenced by tumor type/stage, individual patient difference, and thereby low sensitivity/specificity.10−13 Comparatively, external triggers can precisely focus the outer energy sources on the tumor position with high controllability and specificity. Radiation therapy (X-ray or Cerenkov radiation as outer source) has been extensively explored in the clinic, but © 2017 American Chemical Society

Received: July 23, 2017 Accepted: August 22, 2017 Published: August 22, 2017 9467

DOI: 10.1021/acsnano.7b05215 ACS Nano 2017, 11, 9467−9480

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Figure 1. Schematic illustration of the synthetic procedure of MnOx/TiO2-GR-PVP and MR imaging-guided synergistic SDT/PTT against cancer. (a) Scheme of the synthetic procedure for MnOx/TiO2-GR-PVP nanocomposites, including exfoliation of GO, hydrothermal treatment for the integration of GO with TiO2, in situ redox reaction between TiO2-GR and postintroduced KMnO4, and surface PVP modification. (b) Schematic illustration of theranostic functions of MnOx/TiO2-GR-PVP nanocomposites, including free transport with the blood vessels after intravenous injection, TME-responsive MRI guidance prior to cancer therapy, and synergistic SDT/PTT against cancer.

focused US for tumor ablation.20−22 As compared to traditional PDT, US can activate sonosensitizers to generate toxic reactive oxygen species (ROS) for therapeutics, termed “sonodynamic therapy” (SDT).23,24 SDT shows its high application potential in the clinic based on US features such as its noninvasiveness and high tissue-penetrating ability. However, the deficiency of sonosensitizers substantially hinders its extensive clinical translation. Traditional organic sonosensitizers (e.g., photofrin, ATX-70, chlorophyll derivative) suffer from low stability and fast excretion out of the body.25−27 We recently improved the delivery efficiency of organic sonosensitizers based on nanomedicine where well-defined mesopores were used for sonosensitizer delivery into a tumor.28 As compared to traditional organic sonosensitizers, the physiochemical property of inorganic nanomaterials makes them excellent candidates as sonosensitizers. The most representative paradigm of inorganic nanosonosensitizers is semiconductor titanium dioxide (TiO2) nanoparticles (NPs), which can generate ROS such as singlet oxygen (1O2), hydroxyl radical (•OH), and superoxide radical (O2−) upon US activation.29−31 The low quantum yield of ROS generation, however, hinders their further separation of electron (e−) and hole (h+) pairs from the energy band. The sonosensitizing effect is therefore low, unfortunately causing insufficient SDT efficiency. It is still highly challenging to prevent the recombination of US-triggered electron and hole pairs from a TiO2 nanosonosensitizer and to further augment its SDT efficiency.29

In this work, we report on the integration of two-dimensional (2D) reduced graphene oxide (GR) nanosheets with a TiO2 nanosonosensitizer for enhancing the SDT outcome against cancer by taking advantage of the high electroconductivity, ultrathin planar nanostructure, abundant surface chemistry, and high photothermal-conversion capability of GR nanosheets. TiO2 NPs have been directly grown on the surface of GR nanosheets, followed by in situ surface growth of MnOx NPs (designated as MnO x/TiO2-GR) for diagnostic-imaging guidance and monitoring. These 2D MnOx/TiO2-GR nanocomposites exhibit features for imaging-guided synergistic SDT to combat cancer. First, the presence of GR can effectively separate the electron (e−) and hole (h+) pairs as generated by the US irradiation-induced cavitation effect based on the high electroconductivity of the GR nanosheets. Second, the high photothermal-conversion capability of GR is capable of synergistically enhancing the SDT efficiency of photothermal therapy (PTT). Third, the integrated MnOx NPs on the surface of 2D MnOx/TiO2-GR nanocomposites act as pH-responsive contrast agents for T1-weighted magnetic resonance imaging (MRI), potentially providing the guidance for synergistic PTTenhanced SDT.

RESULTS AND DISCUSSION Design, Synthesis, and Characterization of 2D MnOx/ TiO2-GR Nanocomposites. GO, as a class of 2D carbon nanomaterials with its intrinsic physiochemical property, has 9468


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Figure 2. Characterization of TiO2-GR and MnOx/TiO2-GR nanocomposites. (a) TEM image of highly dispersed GO nanosheets. (b) TEM and (c) SEM images of TiO2-GR nanocomposites. (d) High-resolution TEM (HRTEM) image of TiO2-GR. (e) 3D schematic illustration of the growth of TiO2 and MnOx on the GR’s surface. (f) TEM and (g) SEM images of MnOx/TiO2-GR nanocomposites.

enhanced permeability and retention (EPR) effect (Figure 1b).9,50 The integrated MnOx NPs on the surface provide the TME-responsive MRI guidance for the subsequent therapeutic treatment.51−54 Upon external triggering by both laser and US, these MnOx/TiO2-GR-PVP composite nanosheets induce an enhanced synergistic efficiency for concurrent PTT and SDT against tumor growth and even complete tumor eradication. Ultrathin GO nanosheets were fabricated with an average planar size of nearly 230.0 nm after ultrasonic exfoliation of multilayer-structured and large-sheet-sized GO for 12 h (Figure 2a, Figure S1). The nanoscale size of as-synthesized GO nanosheets can guarantee their facile transport within the blood vessels and further systematic in vivo theranostic application. After hydrothermal reaction between TiO2 NPs and GO nanosheets in the cosolvent of ethanol and water, TiO2 NPs were firmly attached onto the surface of reduced GO nanosheets (GR). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images (Figure 2b,c, Figure S2) evidenced the formation of TiO2-GR composites where many decorated TiO2 NPs were clearly observed on the GR’s surface. A high-resolution TEM image (HRTEM) and selected area electron diffraction (SAED) patterns of TiO2-GR (inset of Figure S2c) demonstrate that the attached TiO2 NPs are highly crystallized, which is favorable for the sonocatalytic process to generate electrons (e−) and holes (h+) from the energy band of TiO2 and avoid their recombination. X-ray diffraction (XRD) patterns of TiO2 and as-obtained TiO2-GR nanocomposites are shown in Figure S3a. Both patterns exhibit characteristic diffraction peaks of 25.4°, 37.9°, 48.3°, 54.0°, 55.2°, 62.9°, 69.0°, 70.4°, and 75.3°, which were assigned to (101), (004), (200), (105), (211), (204), (116), (220), and (215) crystal planes of anatase TiO2, respectively. In addition, characteristic diffraction peaks at 27.5°, 36.2°, and 41.3° were indexed to the (110), (101), and (111) faces of rutile TiO2, respectively. No diffraction peaks for carbon species were discovered in the nanocomposites, probably attributed to the relatively low diffraction intensity of reduced graphene and the overlap of diffraction peaks between graphene and TiO2 from the angle range of 20° to 30°. Figure S3b showed the

been extensively explored for diverse applications such as theranostic nanomedicine.32−38 Especially, the previous results have demonstrated that TiO2−graphene nanocomposites showed improved photocatalytic performance as compared to single TiO2 NPs.39−44 The enhanced separation and reduced combination of light-triggered electron (e−) and hole (h+) pairs have been demonstrated as the main contributor to the synergistic effect between GO and TiO2. It is considered that SDT is generally activated by a US-triggered cavitation effect, which can cause the sonoluminesence emission to activate TiO2 sonosensitizers and generate electrons (e−) and holes (h+) subsequently.45−49 The produced electrons and holes further react with surrounding water (H2O) and oxygen (O2) molecules to generate toxic ROS with therapeutic functionality. This process is similar to photocatalysis and therefore is comparatively defined as “sonocatalysis”. Similar to the principle of graphene-enhanced photocatalytic performance, the presence of GO is therefore also expected to be effective in enhancing the sonocatalytic efficiency. To verify this assumption, TiO2 NPs were in situ grown on the surface of GO by a simple but facile hydrothermal methodology (Figure 1a). To be specific, ultrathin GO nanosheets were initially fabricated by the exfoliation of multilayer GO for 12 h via sonication, which were further dispersed into the cosolvents of water and ethanol. TiO2 NPs could be directly grown onto GO’s surface by the hydrothermal treatment of the cosolvent solution of TiO2 NPs and asexfoliated GO suspension. The GO component was concurrently reduced during the hydrothermal process (reduced GO, designated as TiO2-GR). To render the hydrophilic surface of TiO2−GR composite nanosheets, they were further oxidized by strongly oxidative KMnO4, resulting in the in situ growth of MnOx NPs (MnOx/TiO2-GR). The surface of MnO x /TiO 2 -GR composites was finally modified with biocompatible polyvinylpyrrolidone (PVP) to improve the colloidal stability of composite nanosheets in physiological conditions (designated as MnOx/TiO2-GR-PVP). These composite nanosheets could freely transport within the blood vessels and accumulate into the tumor tissue via the typical 9469


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Figure 3. Structure and composition characterization of MnOx/TiO2-GR nanocomposites. (a) SEM image of elemental mappings of MnOx/ TiO2-GR nanocomposites, including C, O, Ti, and Mn elements. (b) X-ray energy dispersive spectrum (EDS) of MnOx/TiO2-GR nanocomposites. (c) Electron energy loss spectra (EELS) of MnOx/TiO2-GR nanocomposites. (d) Dynamic light scattering (DLS) size distribution profiles of MnOx/TiO2-GR and MnOx/TiO2-GR-PVP nanosheets dispersed in aqueous solution. (e) 3D schematic illustration of formation of a Ti−C bond between GO and TiO2. (f) X-ray photoelectron spectroscopy (XPS) of MnOx/TiO2-GR in the C 1s region. (g) XPS spectrum of MnOx/TiO2-GR in the Ti 2p region.

surface of TiO2-GR. The weight ratio of Mn:Ti:GO was determined to be 5.5:33:28. For enhancing the stability of MnOx/TiO2-GR nanocomposites in physiological conditions and prolonging their in vivo blood circulation, their surface was grafted with the biocompatible PVP (designated as MnOx/TiO2-GR-PVP). After PVP coating, one characteristic peak around 1600 cm−1 in the FTIR spectrum occurred (Figure S5), which was indexed to the carbonyl groups of PVP, further indicating the successful PVP modification. The average particle size of MnOx/TiO2GR-PVP nanocomposites measured by dynamic light scattering (DLS) was determined to be around 260.0 nm, and further surface PVP modification showed only a slight increase in the particle size (Figure 3d). The series changes on zeta potential of each grafting step further indicate the stepwise formation of MnOx/TiO2-GR-PVP nanocomposites (Figure S6). Especially, these surface-modified MnOx/TiO2-GR-PVP nanocomposites feature high colloidal stability in physiological solutions, such as Dulbecco’s modified Eagle’s medium (DMEM), simulated body fluid (SBF), saline, and phosphate buffer saline (PBS), which can guarantee their further in vivo intravenous administration (Figure S7). X-ray photoelectron spectroscopy (XPS) was used to analyze the chemical state of elements in MnOx/TiO2-GR nanocomposites. The obvious Ti and Mn signals in XPS demonstrate the presence of TiO2 and MnOx on the surface of GR nanosheets (Figure S8). For as-prepared MnOx/TiO2GR, the peaks around 284.0, 285.1, and 286.3 eV of C 1s were indexed to the C−C, C−O, and CO bonds (Figure 3f), respectively. An additional shoulder peak centered at 282.6 eV was observed, which was attributed to the formation of the

FTIR spectrum of GO, TiO2, and TiO2-GR from the range of 400 to 4000 cm−1. For TiO2-GR, the absorption peak below 1000 cm−1 in the FTIR spectrum is the same as that of TiO2, where the low-frequency absorption peak around 685 cm−1 was assigned to Ti−O−Ti stretching, indicating the successful formation of TiO2-GR nanocomposites via the hydrothermal process. After grafting TiO2 NPs onto GO, the Raman spectrum of TiO2-GR changed obviously as compared to that of GO. The characteristic peaks around 1360, 1580, and 2680 cm−1 were indexed to GO (Figure S3c). The supplementary peaks observed in the spectrum of TiO2-GR around 144, 399, 513, and 639 cm−1 were attributed to TiO2 NPs, further confirming the formation of TiO2-GR nanocomposites. To achieve the diagnostic imaging-guided therapy (theranostics), MnOx NPs were further in situ grown onto the surface of TiO2-GR nanocomposites (designated as MnOx/TiO2-GR) based on the redox reaction of postintroduced oxidative MnO4− and reducing TiO2-GR surface. Highly uniform and dispersive MnOx species could be found on the surface of TiO2GR nanocomposites (Figure 2f,g, Figure S4), which also exhibited a sheet-like topology firmly attached on the surface of the nanocomposites. The element distribution mappings of C, Ti, O, and Mn elements exhibited the high uniformity of TiO2 and MnOx distribution on the surface of the GR nanosheets (Figure 3a). The X-ray energy dispersive spectroscopy (EDS, Figure 3b) and corresponding electron energy loss spectrum (EELS) of MnOx/TiO2-GR nanocomposites (Figure 3c) showed the coexistence of Ti, O, and Mn elements, demonstrating the successful construction of TiO2-GR via hydrothermal treatment and further MnOx grafting onto the 9470


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Figure 4. In vitro sonocatalytic and photothermal performance of MnOx/TiO2-GR nanocomposites. (a) Schematic illustration of the principle for GR-enhanced SDT employing TiO2 NPs as the sonosensitizers. (b) Nyquist plots of TiO2 and MnOx/TiO2-GR obtained from electrochemical impedance spectrum (EIS) measurements. EIS changes of TiO2 in the presence of a 2.5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe in 0.1 M KCl aqueous solution. (c) UV−vis absorption spectra of the 1,3-diphenylisobenzofuran (DPBF) in the presence of TiO2 and MnOx/TiO2-GR upon exposure to US irradiation for prolonged durations. (d) Decay curves of the relative absorption of DPBF at 410 nm with different irradiation durations in the presence of MnOx/TiO2-GR as compared with TiO2 NPs. (e) UV− vis spectra of MnOx/TiO2-GR nanocomposites dispersed in aqueous solution at elevated concentrations (4.69, 9.38, 18.8, 37.5, and 75.0 ppm). Inset: Normalized absorbance intensity at λ = 808 nm divided by the characteristic length of the cell (A/L) at varied concentrations. (f) Photothermal-heating curves of MnOx/TiO2-GR nanocomposites at elevated concentrations under 808 nm laser irradiation (2.0 W cm−2). (g) Photothermal effect of an aqueous dispersion of MnOx/TiO2-GR under irradiation by a NIR laser (808 nm, 2.0 W cm−2). (h) The time constant for heat transfer from the system was determined to be 146.71 s by applying the linear time data from the cooling period versus the negative natural logarithm of the driving force temperature, which originated from the cooling stage. (i) Recycling heating profiles of a MnOx/TiO2-GR composite nanosheet-dispersed suspension using an 808 nm laser (2 W cm−2) for five laser on/off cycles.

In Vitro ROS Generation upon US Activation and Photothermal Performance. The catalytic performance of TiO2 can be substantially improved by the integration of TiO2 with various types of carbon-based materials.39−44 The key role of carbon in the TiO2-GR nanocomposites is to trap and transfer excited electrons. The excited electrons of TiO2 can transfer from the conduction band to GR by a percolation mechanism. The presence of GR can effectively inhibit electron−hole pair recombination due to its excellent electroconductivity with 2D planar π-conjugation structure. Subsequently, the lifetime of charge carriers is prolonged, resulting in the formation of a larger amount of radical species with strong oxidation capability (Figure 4a), such as singlet oxygen (1O2), superoxide radical (O2−), and hydroxyl radical (•OH) species. A typical electrochemical impedance spectrum (EIS) was used to describe the interface and conductive properties of composite nanosheets. By the integration with GR, the semicircle in the Nyquist plots of the MnOx/TiO2-GR nanocomposite was obviously smaller as compared with TiO2

chemical C−Ti bond during the hydrothermal treatment. The area percentages of C−O and CO in MnOx/TiO2-GR nanocomposites increased significantly because of the oxidation by KMnO4, resulting in the decline of the consumed C−C bond. The formation of a C−Ti bond could also be observed and demonstrated by the analysis of the XPS in the Ti 2p region of MnOx/TiO2-GR nanocomposites. In addition to the two characteristic peaks of TiO2 at 460.0 and 466.0 eV, another two peaks around 456.7 and 462.5 eV were clearly identified (Figure 3g) because of the formation of a C−Ti bond in the MnOx/TiO2-GR nanocomposites. The obvious Mn signal in the XPS demonstrated the integration of MnOx on the surface of TiO2-GR (Figure S8d). The fitted peaks around 642.4 and 655.1 eV were assigned to Mn 2p3/2 and Mn 2p1/2, respectively. The relative contents of bivalent, trivalent, and quadrivalent Mn in MnOx/TiO2-GR nanocomposites were determined to be around 17.7%, 22.9%, and 39.6%, respectively. 9471


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Figure 5. In vitro SDT/PTT-based synergistic therapy of cancer. (a) Relative viabilities of 4T1 cells after incubation with various concentrations of MnOx/TiO2-GR-PVP nanocomposites for 24 and 48 h. Error bars were based on the standard deviations (SD) of five parallel samples. (b) Schematic illustration of MnOx/TiO2-GR-PVP nanocomposites as SDT/PTT synergistic agents for cancer cell therapy, including efficient uptake into cancer cells, the intracellular distribution within the cytoplasm of cells, and simultaneous SDT/PTT for killing the cancer cells. (c) CLSM images of 4T1 cells incubated with FITC-labeled MnOx/TiO2-GR-PVP for varied durations of 0, 1, 2, 4, and 8 h. All the scale bars are 20 μm. (d) Relative viabilities of 4T1 cells after different treatments, including control (without any treatment), MnOx/ TiO2-GR-PVP only, laser only, US only, TiO2-PEG combined with US irradiation, MnOx/TiO2-GR-PVP combined with US irradiation, MnOx/TiO2-GR-PVP combined with NIR laser irradiation, and MnOx/TiO2-GR-PVP combined with US/laser coirradiations. Error bars were based on the SD of five parallel samples (**p values < 0.01.). (e) CLSM images of 4T1 cells stained by calcein AM and PI after different treatments.

The electron spin resonance (ESR) technique was employed to detect the ROS generation by TiO2 and TiO2-GR under US activation. 2,2,6,6-Tetramethylpiperidine (TEMP), serving as a spin-trapping agent, could selectively react with 1O2 to yield 2,2,6,6-tetramethylpiperidine-1-oxyl, which then induces a characteristic 1:1:1 triplet signal in the ESR spectrum. As shown in Figure S10a, both TiO2 and TiO2-GR produced 1O2 under the same US irradiation (1.0 MHz, 1.5 W cm−2, 50% duty cycle, 60 s), and the signal intensity of TiO2-GR-induced 1 O2 generation was obviously higher than that of pure TiO2 NPs, indicating more amounts of 1O2 production by US-excited TiO2-GR. These results were in accordance with the measurement of DPBF absorption spectra (Figure 4c and d). Additionally, ESR spectroscopy was further used to monitor the generation of hydroxyl radicals (•OH) by employing 5,5′dimethylpyrroline-1-oxide (DMPO) as spin trap agent. Hydroxyl radicals were demonstrated to be produced when TiO2 and TiO2-GR were exposed to US irradiation (1.0 MHz, 1.5 W cm−2, 50% duty cycle, 60 s), which generated DMPO-•OH adducts in the presence of the reagent DMPO with the characteristic ESR signal intensity of 1:2:2:1. The intensity of the ESR signal of the DMPO-•OH adduct induced by TiO2-GR was also higher than that of the DMPO-•OH adduct as induced by TiO2 NPs (Figure S10b). These results demonstrated that TiO2-GR could serve as a desirable nanosonosensitizer for the US-activated generation of toxic ROS with higher efficiency as compared to bare TiO2 NPs, guaranteeing further enhanced SDT for in vivo tumor therapy.

NPs, demonstrating the decreased charge-transfer resistance within the MnOx/TiO2-GR nanocomposites. Therefore, the MnOx/TiO2-GR nanocomposite is more effective at shuttling charges and the suppression of charge recombination, and consequently an enhanced catalytic activity by external triggering can be achieved. To test the in vitro ROS generation capability of MnOx/ TiO2-GR, 1,3-diphenylisobenzofuran (DPBF) was used as a typical molecular probe to test the generation level of singlet oxygen (1O2) or superoxide anion (O2−). The 1O2 and/or O2− generation can react with DPBF, leading to the decline of its intensity of the characteristic absorption at the wavelength of 410 nm in the UV−vis spectrum. As shown in Figure 4c, the absorbance intensity of DPBF sharply decreased in the presence TiO2-GR compared with that of TiO2 at the same Ti concentration under US activation at 1.5 W cm−2 for 60 s, which also indicated a time-dependent ROS generation for TiO2-GR. We also tested the ROS generation capability of TiO2-GR under different US power densities of 0.5, 1.0, and 1.5 W cm−2, showing the US power-dependent ROS generation by MnOx/TiO2-GR (Figure S9). The decrease of the absorption intensity of DPBF at 410 nm upon US irradiation in the presence of TiO2-GR and TiO2 was measured respectively as a function of time to further assess the ROS generation efficiency. It is important to find that the ROS production efficiency of TiO2-GR was significantly higher as compared to that of single TiO2 (Figure 4d), demonstrating that the presence of GO could efficiently enhanced the ROS generation capability during the SDT process. 9472


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Figure 6. Contrast-enhanced pH-responsive MR imaging of tumor cells by MnOx/TiO2-GR-PVP nanocomposites both in vitro and in vivo. (a) Schematic illustration of the broken Mn−O bond from MnOx/TiO2-GR-PVP nanocomposites under the mildly acidic TME for contrastenhanced T1-weighted MR imaging. (b) 1/T1 vs Mn concentration for MnOx/TiO2-GR-PVP nanocomposites in buffer solution at different pH values after soaking for 4 h. (c) In vitro T1-weighted MR imaging of MnOx/TiO2-GR-PVP nanosheets in buffer solution at varied pH values after soaking for 4 h. (d) 1/T2 vs Mn concentration for MnOx/TiO2-GR-PVP nanocomposites in buffer solution at different pH values after soaking for 4 h. (e) In vitro T2-weighted MR imaging of MnOx/TiO2-GR-PVP nanosheets in buffer solution at varied pH values after soaking for 4 h. (f) T1-weighted imaging and (g) corresponding MRI-signal intensity of 4T1 tumor-bearing mice after intravenous administration of MnOx/TiO2-GR-PVP nanosheets at different time intervals.

photothermal ablation of a tumor. The photothermal performance of the MnOx/TiO2-GR composite nanosheets did not show obvious deterioration during five laser on/off cycles, demonstrating the high potential of MnOx/TiO2-GR composite nanosheets as a durable photothermal agent for PTT-based hyperthermia (Figure 4i). In Vitro Synergistic SDT and PTT against Tumor Cells by MnOx/TiO2-GR-PVP Nanocomposites. The standard cell-counting kit 8 (CCK-8) assay was initially conducted to determine the intrinsic toxicity of MnOx/TiO2-GR-PVP prior to its in vivo bioapplication by using 4T1 murine breast cancer cells as a model cell. 4T1 cells were first incubated with MnOx/ TiO2-GR-PVP at varied concentrations (12, 25, 50, 100, and 200 ppm) for 24 and 48 h. Even at a high concentration up to 200 ppm with a prolonged incubation time of 48 h, no obvious cytotoxicity of MnOx/TiO2-GR-PVP nanocomposites was observed (Figure 5a). The nanoscale size of MnOx/TiO2-GRPVP nanocomposites guarantees their easy endocytosis into cancer cells, which are mainly distributed within the cytoplasm of the cells. After sequential US and laser irradiations, MnOx/ TiO2-GR-PVP nanocomposites trigger intracellular ROS generation and temperature elevation to kill the cancer cells (Figure 5b). The intracellular uptake of MnOx/TiO2-GR-PVP at different durations (0, 1, 2, 4, and 8 h) was further tested. Most of the fluorescein isothiocyanate (FITC)-labeled MnOx/ TiO2-GR-PVP nanocomposites were found in the cytoplasm of 4T1 cells after 4 h of co-incubation, where the green

In addition to the synergistic capability of GR for enhancing the sonocatalytic efficiency, its intrinsic photothermal-conversion property provides the potential for simultaneous photothermal therapy.32,55,56 MnOx/TiO2-GR with different weight ratios of GR (9%, 23%, and 33%) could be easily fabricated via the change of the initial GR amount during the synthesis. With the elevated addition of GR to 33% in the MnOx/TiO2-GR, the temperature could reach as high as 57 °C (2 W cm−2, 5 min) at a Ti concentration of 118 ppm (Figure S11), which was sufficiently high to kill the cancer cells by hyperthermia. MnOx/TiO2-GR nanocomposites at elevated concentrations (75, 150, and 300 ppm) were also tested upon 808 nm laser irradiation at the power density of 2.0 W cm−2 (Figure 4f), showing their concentration-dependent photothermal profiles. The process on the integration of TiO2 and MnOx components could improve the photothermal performance of initial GO, which is attributed to the reduction of GO during the growth of TiO2 nanoparticles onto the GO’s surface (Figure S12). The UV−vis adsorption intensity of MnOx/ TiO2-GR over the length of the cell (A/L) at λ = 808 nm at elevated concentrations (C) (4.69, 9.38, 18.8, 37.5, and 75 ppm) was then tested, and the extinction coefficient at 808 nm was measured to be 10.1 Lg−1 cm−1 (Figure 4e, inset), which is much higher than that of GO (3.6 Lg1−cm−1).36 The photothermal-conversion efficiency (η), which reveals the performance in translating the light into heat, was measured to be 18.4% (Figure 4g and h), high enough for efficient 9473


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Figure 7. In vivo synergistic SDT/PTT for tumor eradication. (a) Biodistribution assay after the intravenous administration of MnOx/TiO2GR-PVP nanocomposites into tumor-bearing mice for 4 and 24 h (n = 3). (b) Blood circulation curve of intravenously injected MnOx/TiO2GR-PVP (n = 3). The half-time (T1/2) was calculated to be approximately 0.80 h. (c) Eliminating rate curve of intravenously injected MnOx/ TiO2-GR-PVP from the blood circulation curve according to the ln(concentration)−T relationship. (d) IR thermal images of 4T1 tumorbearing nude mice with or without receiving an intravenous injection of MnOx/TiO2-GR-PVP nanocomposites followed by 808 nm laser irradiation (2.0 W cm−2) at varied time intervals. (e) Elevated temperature at the tumor site of 4T1 tumor-bearing nude mice upon 808 nm laser irradiation for 600 s. (f) In vivo therapeutic protocol of PTT and/or SDT on mice tumor xenograft. (g) Time-dependent tumor-growth and (h) time-dependent body-weight curves of nude mice in eight experimental groups after different treatments, including control group, MnOx/TiO2-GR-PVP group, laser group, US group, TiO2-PEG + US group, MnOx/TiO2-GR-PVP + US group, MnOx/TiO2-GR-PVP + laser group, and MnOx/TiO2-GR-PVP + US/laser group (*p < 0.05, **p < 0.01, ***p < 0.001). (j) Digital images of tumors from each group at the end of various treatments. (f) H&E staining, TUNEL staining, and Antigen Ki-67 immunofluorescence staining in tumor tissues from each group after different treatments (I−VIII represent control group, MnOx/TiO2-GR-PVP group, laser group, US group, TiO2-PEG + US group, MnOx/TiO2-GR-PVP + US group, MnOx/TiO2-GR-PVP + laser group, and MnOx/TiO2-GR-PVP + US/laser group, respectively). All the scale bars are 50 μm.

fluorescence originated from FITC and the blue fluorescence represented the cell nucleus as stained by DAPI (Figure 5c). To verify the sonodynamic effect induced by US activation (1.0 MHz, 1.0 W cm−2, 50% duty cycle) in the presence of MnOx/TiO2-GR-PVP, 2,7-dichloro-dihydrofluorescien diacetate (DCFH-DA) was used to test the ROS generation by monitoring the green fluorescence in confocal laser scanning microscope (CLSM) imaging, attributed to highly fluorescent 2,7-dichlorofluorescein (DCF) transformed from DCFH-DA in the presence of ROS. As expected, a strong green fluorescence of the MnOx/TiO2-GR-PVP + US group was observed in 4T1 cancer cells by CLSM, which is much brighter than that of the TiO2-PVP + US group, while the control and US-only group exhibited a negligible green fluorescence, demonstrating the large production of ROS during the sonocatalytic process assisted by MnOx/TiO2-GR-PVP (Figure S13). To compare the in vitro cytotoxicity of TiO2-PEG and MnOx/TiO2-GR-PVP under US activation, MnOx/TiO2-GRPVP under 808 nm laser irradiation and MnOx/TiO2-GR-PVP under sequential US/808 nm laser irradiation, the viability of

cells was determined by a typical CCK-8 assay. As shown in Figure 5d, the cells were substantially killed by US irradiation (1.0 MHz, 1.0 W cm−2, 50% duty cycle, 3 min) after coincubation with MnOx/TiO2-GR-PVP nanocomposites as the SDT agents followed by US irradiation, which was also US power density-dependent, MnOx/TiO2-GR-PVP concentration-dependent, and irradiation duration-dependent (Figure S14). The inhibition rate of cancer cells by MnOx/TiO2-GRPVP under US irradiation could reach nearly 56%, much higher compared to TiO2-PEG (22%) at the same Ti concentration (120 ppm) under the same US-irradiation conditions. The significantly improved sonotoxicity of MnOx/TiO2-GR-PVP was attributed to the generation of greater amounts of ROS because of GR-assisted separation of electrons (e−) and holes (h+) under US activation. When NIR laser irradiation was further employed (2.0 W cm−2, 5 min), the inhibition rate increased up to 81%, which was also higher than that of MnOx/ TiO2-GR-PVP treated with laser irradiation only (74%). Comparatively, the control group, the MnOx/TiO2-GR-PVPonly group, and the laser-only group showed a negligible effect 9474


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organs and tumor was investigated after intravenous injection for 4 and 24 h using a nude 4T1 tumor-bearing mice model. Nearly 10% of Mn ions had accumulated in the tumor sites via the EPR effect of MnOx/TiO2-GR-PVP nanocomposites (Figure 7a). The circulation of MnOx/TiO2-GR-PVP nanocomposites in the bloodstream was investigated, and the bloodcirculation half-time of MnOx/TiO2-GR-PVP was calculated to be 0.80 h (Figure 7b). The eliminating rate constants of MnOx/ TiO2-GR-PVP were calculated to be −0.014 μg mL−1 h−1 after 2 h of intravenous injection, nearly 37 times higher as compared to that of MnOx/TiO2-GR-PVP in the next 22 h (Figure 7c). Forty tumor-bearing mice were then randomly separated into eight groups (n = 5 per group) for therapeutic evaluation, including (a) control group (treated only with saline), (b) MnOx/TiO2-GR-PVP group (only intravenously injected with MnOx/TiO2-GR-PVP nanocomposites), (c) laser group (only exposed to 808 nm laser irradiation), (d) US group (only exposed to US irradiation), (e) TiO2-PEG + US group (intravenously injected with TiO2-PEG followed by US irradiation), (f) MnOx/TiO2-GR-PVP + US group (intravenously injected with MnOx/TiO2-GR-PVP followed by US irradiation), (g) MnOx/TiO2-GR-PVP + laser group (intravenously injected with MnOx/TiO2-GR-PVP followed by exposure to 808 nm laser irradiation), and (h) MnOx/TiO2GR-PVP + US + laser group (intravenously injected with MnOx/TiO2-GR-PVP followed by exposure to both US and laser irradiation). After the intravenous administration of MnOx/TiO2-GR-PVP nanocomposites (20 mg kg−1) for 4 h, the mice were treated by the aforementioned therapeutic protocols, and the tumor-growth status was carefully monitored. Figure 7d and e show that the tumor temperature in the group (g) treated with MnOx/TiO2-GR-PVP and 808 nm laser irradiation increased significantly from 25 °C to 60 °C at a laser power density of 2 W cm−2 for 10 min, which is sufficient to cause tumor-cell death. In contrast, the tumor temperature in group (c) treated with laser only increased by ∼3 °C. The US irradiation for groups (d), (e), (f), and (h) was repeated on the third and fifth day (Figure 7f). The tumor volumes of eight groups were recorded every 2 days using a digital caliper (Figure 7g), and digital photos of tumor sites were taken every 2 days during 2 weeks after the varied treatments (Figure S15). The body weight of mice in each group showed no obvious change during the 2 weeks (Figure 7h). The tumor growth in the group (f) treated with MnOx/TiO2-GR-PVP + US irradiation was obviously suppressed after US irradiation as compared to group (e), treated with TiO2-PEG + US irradiation, indicating that the presence of GR significantly augmented the sonosensitization effect of TiO2-based sonosensitizers. The tumor-inhibition rate of the MnOx/TiO2-GRPVP + US group reached nearly 78%, significantly higher than that of the TiO2-PEG + US group (32.2%) and US group (22.9%). Although the tumor volume in group (g) (mice treated with MnOx/TiO2-GR-PVP and 808 nm laser) was suppressed in the first 6 days, the tumors reoccurred again at the original sites. When combining sonocatalytic therapy with PTT assisted by MnOx/TiO2-GR-PVP, the tumor tissues were completely eradicated without obvious reoccurrence during 2 weeks (Figure 7i), demonstrating the high synergistic effect of combinatorial SDT/PTT therapy in comparison to singlemodality SDT or PTT therapy.

on the survival of 4T1 cells. The cell apoptosis after different treatments was further confirmed by CLSM observation, where the live and dead cells were stained by calcein-AM (green) and PI (red), respectively (Figure 5e). Large amounts of dead cancer cells presenting red fluorescence were clearly distinguished in the MnOx/TiO2-GR-PVP group under sequential US/808 nm laser irradiation, further demonstrating the high killing effect of MnOx/TiO2-GR-PVP for synergistic sonocatalytic therapy and PTT. Tumor Microenvironment-Responsive MR Imaging of Cancer. The presence of the MnOx component within MnOx/ TiO2-GR-PVP endows the composite nanosheets with MR imaging capability. It is also sensitive to the mild pH variations of TME because of the low stability of MnOx under mild acidic conditions to release the paramagnetic Mn2+ (Figure 6a). To evaluate the contrast-enhanced MRI capability of MnOx/TiO2GR-PVP at different pH values, we measured their relaxivities under a 3.0 T clinical MRI scanner. An obvious concentrationdependent brightening effect was observed in phantom images of corresponding SBF solutions at both neutral and acidic conditions (pH = 7.4, 6.0, and 5.0). The acidic SBF was used to mimic the mild acidic microenvironment of tumor tissue. The positively enhanced T1 MRI signal was clearly observed in acidic solutions (Figure 6b and c). The r1 relaxivity of the initial MnOx/TiO2-GR-PVP was measured to be only 0.06 mM−1 s−1. Importantly, this relaxivity reached 1.61 and 5.77 mM−1 s−1 at the acidic pHs of 6.0 and 5.0, respectively, significantly higher than their original r1 values at neutral conditions (nearly 96-fold increase). This enhanced T1-weighted MRI capability under acidic conditions was due to the released paramagnetic Mn2+ ions, which can obtain maximized interaction chances and accessibility with the surrounding water molecules.51,57−59 Interestingly, such a pH-sensitive phenomenon also occurs at the imaging modality of T2-weighted MRI, where substantially enhanced negative T2 MRI signals were observed under acidic conditions (Figure 6d and e). 4T1 tumor-bearing mice xenograft was further established to assess the feasibility of MnOx/TiO2-GR-PVP nanocomposites for in vivo MR imaging. When the tumor size reached around 150 mm3, the mice were treated with intravenous administration of MnOx/TiO2-GR-PVP nanocomposites (dose: 20 mg mL−1, 100 μL). The T1-weighted MR images of tumorbearing mice were recorded at the given time intervals. As shown in Figure 6d, a remarkable signal enhancement and brightening effect of MRI signals at the tumor site were observed, attributed to both the efficient tumor accumulation of MnOx/TiO2-GR-PVP nanocomposites via the typical EPR effect and the Mn-releasing behavior as triggered by the mildly acidic microenvironment of the tumor tissue. This MRI-signal enhancement in the tumor site was further demonstrated by quantitatively positive MRI signal intensities (Figure 6e). Such a pH-responsive MR imaging capability with high tumor specificity facilitates the subsequent imaging guidance for synergistic SDT and PTT against cancer. In Vivo Synergistic Sonocatalytic and Photothermal Therapy of Tumor Cells by MnOx/TiO2-GR-PVP. Encouraged by the fascinating in vitro NIR laser/US-introduced synergistic effect, the in vivo synergistic sonocatalytic therapy and PTT assisted by MnOx/TiO2-GR nanocomposites were further systematically assessed by using intravenous administration of the MnOx/TiO2-GR nanocomposites into 4T1 breast tumor-bearing mice followed by different treatments. The biodistribution of MnOx/TiO2-GR-PVP in the main 9475


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achieved tumor-microenvironment-sensitive (mild acidity) T1weighted MR imaging of tumors, providing the potential for therapeutic guidance and monitoring. Importantly, the designed MnOx/TiO2-GR-PVP nanocomposites substantially suppressed tumor growth during the SDT process, and the corresponding inhibition rate was significantly higher as compared to single TiO2-based nanosonosensitizers. Especially, the high photothermal-conversion capability of graphene synergistically enhanced the SDT efficiency of MnOx/TiO2GR-PVP nanosonosensitizers, achieving the complete eradication of a tumor without reoccurrence.

In addition, the tumor-bearing mice treated only with saline, laser, or MnOx/TiO2-GR-PVP experienced a rapid growth of tumor volume, indirectly indicating the high therapeutic efficiency of SDT and PTT. To further understand the mechanism of synergistic therapy after various treatments, hematoxylin and eosin (H&E), TdT-mediated dUTP nick-end labeling (TUNEL), and Ki-67 antibody staining of tumor sections were performed for all groups of mice at 24 h after different treatments. H&E and TUNEL staining results show that large amounts of dead cells were observed in the tumor tissues of groups (f), (g), and (h). The MnOx/TiO2-GR-PVP combined with SDT/PTT exhibited higher necrosis of tumor cells as compared to either the MnOx/TiO2-GR-PVP + US group or the MnOx/TiO2-GR-PVP + laser group. There is much less cell necrosis on the examined tumor sections of the US group and TiO2-PEG group. No obvious change of cell status was observed in the mice of the control, NIR laser-only, and MnOx/TiO2-GR-PVP-only groups. Ki-67 antibody staining was carried out to test the proliferative activities of tumor cells. The results of each group were in accordance with H&E and TUNEL staining results, confirming the efficient synergistic effects induced by SDT and PTT in the presence of MnOx/ TiO2-GR-PVP nanocomposites as concurrent nanosonosensitizers and photothermal-conversion nanoagents (Figure 7j). Systematic in Vivo Biocompatibility Assay. Systematic in vivo toxicity and biocompatibility of MnOx/TiO2-GR-PVP nanocomposites were evaluated to guarantee their further clinical translation. Twenty healthy Kunming mice were randomly assigned into four groups (n = 5 in each group) followed by intravenous administration of MnOx/TiO2-GRPVP at elevated doses (control, 5 mg kg−1, 10 mg kg−1, and 20 mg kg−1). These mice were sacrificed after one month of feeding for blood collection and organ harvest. Complete blood panel tests and biochemical analyses were then conducted, and major organs, including the heart, liver, lung, spleen, and kidney, were collected for histological characterization. No behavior abnormality of the experimental mice was observed, and no obvious body-weight loss of mice was recorded (Figure S16). The blood indexes were then tested, including white blood cells (WBC), red blood cells (RBC), platelets (PLT), lymphocytes (LYM), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), hematocrit (HCT), and related kidney and liver function indexes (Figure S17). Compared with the control group, all the indexes of the three MnOx/TiO2-GR-PVP-treated groups showed no significant differences as compared to the control group without any treatment. No obvious signals of organ damage or inflammatory lesions were discovered from H&E-stained organ slices (heart, liver, spleen, kidney, and lung) (Figure S18), suggesting the high histocompatibility of the as-designed 2D MnOx/TiO2-GRPVP theranostic nanoagents.

METHODS Synthesis of TiO2-GR Nanocomposites. TiO2-GR nanocomposites were prepared via a facile hydrothermal method. Typically, 25 mg of GO (Sigma-Aldrich) was immersed in a cosolvent of deionized water (20 mL) and ethanol (10 mL), followed by ultrasonic treatment for 12 h. Then, 50 mg of TiO2 NPs was added to the solution and stirred for another 3 h. The obtained homogeneous suspension was put into a 100 mL Teflon-sealed autoclave and maintained at 120 °C for 12 h. During this process, the reduction of GO to GR and the deposition of TiO2 onto the carbon substrate were simultaneously achieved. The resulting TiO2-GR nanocomposites were collected by centrifugation and washed by water several times. Synthesis of MnOx/TiO2-GR Nanocomposites. MnOx/TiO2GR nanocomposites were obtained by a simple redox reaction on the surface of graphene to in situ grow MnOx components. Typically, a KMnO4 (Sinopharm Chemical Regaent Co.) aqueous solution (15 mg, 10 mL) was added into the as-prepared TiO2-GR aqueous solution (10 mL) under magnetic stirring, which lasted for another 12 h at room temperature. The resulting MnOx/TiO2-GR was collected by centrifugation and further washed with deionized water several times. Surface PVP Modification of MnOx/TiO2-GR Nanocomposites (MnOx/TiO2-GR-PVP). To improve the stability of MnOx/TiO2GR nanocomposites in a physiological environment, MnOx/TiO2-GR (90 mg) and polyvinylpyrrolidone (Sigma-Aldrich, 300 mg) were added into a wide-necked bottle (100 mL in volume), which was then magnetically stirred at a rate of 500 rpm at 60 °C for 12 h. The resulting MnOx/TiO2-GR-PVP was collected by centrifugation and washed with deionized water several times to remove unattached PVP. Characterization. TEM images and corresponding X-ray EDS spectra were acquired on a JEM-2100F electron microscope operated at 200 kV. SEM images and element mapping were obtained on a fieldemission Magellan 400 microscope (FEI Company). UV−vis−NIR absorption spectra were recorded by a UV-3600 Shimadzu UV−vis− NIR spectrometer with QS-grade quartz cuvettes at room temperature. XPS spectrum was recorded by ESCAlab250 (Thermal Scientific). Size and zeta potential measurements were conducted on a Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). The CLSM images were measured in an FV1000 (Olympus Company, Japan). The MnOx/TiO2-GR concentration was determined by inductively coupled plasma atomic emission spectroscopy (Agilent Technologies, USA). The ESR characterization was performed on a Bruker EMX electron paramagnetic resonance spectrometer. Raman spectra were recorded on a DXR Raman microscope (Thermal Scientific, USA) with a 532 nm excitation length. A CHI 760E electrochemical workstation (CH Instruments) was used to measure the electronic properties of the samples. Electrochemical Impedance Spectra Measurement. The EIS measurement was carried out on a CH Instruments 760E electrochemical workstation by using three-electrode cells. A glassy carbon electrode coated with samples, an Ag/AgCl electrode, and a Pt foil were employed as the working electrode, reference electrode, and counter electrode, respectively, which were used in the presence of a 2.5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe in 0.1 M KCl solution. A 2.5 μL amount of sample (2500 ppm) dispersed in a deionized water and absolute ethanol solution (v/v = 1:1) was

CONCLUSIONS In summary, this work reports on the integration of 2D graphene with TiO2 nanosonosensitizers (MnOx/TiO2-GRPVP nanocomposites) for augmenting the sonocatalytic therapeutic efficiency against tumors by inspirations from the principles of semiconductor physics and photocatalysis chemistry, which is based on the high electroconductivity of graphene for easily separating the electrons (e−) and holes (h+) and avoiding their recombination upon external US activation. Furthermore, the integration of the MnOx component has 9476


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using a sample cell containing pure water without MnOx/TiO2-GR. Qout is nearly proportional to the linear thermal driving force in this system, with a heat-transfer coefficient, h, as the proportionality constant:

pipetted onto the glassy carbon electrode. The electrochemical impedance spectroscopy measurement was conducted in a frequency range of 0.01 Hz to 100 kHz with an amplitude of 5 mV at a fixed voltage of 0.2 V. Electron Spin Resonance Spectra Test. TEMP (Dojindo Molecular Technologies, Inc.) was used to test the generation of 1 O2 by TiO2 and TiO2-GR. Typically, TiO2 and TiO2-GR (Ti: 100 μg mL−1) solutions were exposed to US irradiation (1.0 MHz, 1.5 W cm−2, 50% duty cycle) for 60 s in the presence of TEMP (100 μM). The 1O2 signal was immediately detected by the ESR spectrometer. Additionally, the aqueous solution containing TiO2 and TiO2-GR (Ti: 100 μg mL−1) was mixed with DMPO (100 μM, Dojindo Molecular Technologies, Inc.), which was a spin trap agent for hydroxyl radicals (•OH), followed by exposure to US irradiation (1.0 MHz, 1.5 W cm−2, 50% duty cycle) for 60 s for ESR measurement. In Vitro ROS Generation of TiO2 and MnOx/TiO2-GR. DPBF was used to evaluate the in vitro singlet oxygen (1O2) generation induced by TiO2 and MnOx/TiO2-GR. Briefly, 1 mL of either TiO2 or a MnOx/TiO2-GR (Ti: 500 ppm) aqueous solution was added to 2 mL of a DPBF (60 μM) solution that was dissolved in DMF. Then, the mixture was exposed to US irradiation (1.0 MHz, 1.5 W cm−2, 50% duty cycle) in the dark. The intensity of DPBF was measured using a UV−vis spectrometer. Photothermal Performance of MnOx/TiO2-GR Nanocomposites. The photothermal performance of MnOx/TiO2-GR was studied by testing the temperature increase of the MnOx/TiO2-GR aqueous solution at different Ti concentrations (75, 150, and 300 ppm) under an 808 nm high-power multimode pump laser (Shanghai Connect Fiber Optics Company). The photothermal performance of MnOx/ TiO2-GR with different GR ratios was also tested. The temperature and thermal images of the irradiated aqueous dispersion were recorded on an infrared thermal imaging instrument (FLIR TM A325SC camera, USA). a. Calculation of the Extinction Coefficient. To test the NIR absorption capability of MnOx/TiO2-GR nanocomposites, the extinction coefficient ε(λ) of the MnOx/TiO2-GR nanocomposites is given, according to the Lambert−Beer law:

A(λ) = εLC

Q Out = hS(T − TOut)

where S is the surface area of the container and Tout is ambient surrounding temperature. In order to acquire the hS, a dimensionless driving force temperature, θ, is introduced, using the maximum system temperature, TMax:

θ=

τs =

i

x

2 ‐ GR

= I(1 − 10−A808)η

∑i miCp , i (6)

hS

⎤ dθ 1 ⎡ Q MnOx /TiO2 ‐ GR + Q Dis = ⎢ − θ⎥ ⎥⎦ dt τs ⎢⎣ hS(TMax − TOut)

(7)

While turning off the laser source, QMnOx/TiO2‑GR + QDis = 0, reducing eq 7 to t = − τs ln θ

(8)

Therefore, τs was obtained to be 146.71 from the data in Figure 4h. In addition, m is 0.1 g and C is 4.2 J g−1. According to eq 6, hS is calculated to be 2.66 mW °C−1. During laser irradiation, QMnOx/TiO2‑GR + QDis is finite, and the heat output (Qout) is increased along with the increase of the temperature according to eq 4. The system temperature will rise to a maximum value when the external heat flux equals the heat input: Q MnO /TiO x

2 ‐ GR

+ Q Dis = hS(TMax − TOut)

(9)

where TMax is the equilibrium temperature. The 808 nm laser photothermal conversion efficiency (η) can be calculated by substituting eq 3 for QMnOx/TiO2‑GR into eq 9 and rearranging to obtain

η=

hS(TMax − TOut) − Q Dis I(1 − 10−A808)

(10)

where (TMax − TOut) was 36.51 °C according to Figure 4g, I is 560 mW, QDis was measured independently to be 0.1728 mW, and A808 is the absorbance (3.0) of MnOx/TiO2-GR at 808 nm (Figure 4e). Substituting these values into eq 10, the 808 nm laser photothermal conversion efficiency (η) of MnOx/TiO2-GR can be calculated to be 18.4%. In Vitro Cytotoxicity Assay. Murine breast cancer line 4T1 cells (noted as 4T1 cells, Shanghai Institute of Cells, Chinese Academy of Sciences) were cultured at 37 °C under 5% CO2 in DMEM (high glucose, GIBCO, Invitrogen) media and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator. 4T1 cells (1 × 104 cells/well) were cultured to adhere for 24 h. Then, MnOx/TiO2-GR-PVP at different concentrations (0, 12.5, 25, 50, 100, and 200 ppm) was added into the wells and co-incubated for another 24 and 48 h. Then, the standard CCK-8 assay (Shanghai Ruicheng Bio-Tech Co., LTD) was carried out to test the cell viabilities, which were measured on a microplate reader at a wavelength of 405 nm after 60 min. In Vitro Synergistic SDT and PTT against Cancer Cells. For assessing the synergistic SDT and PTT for killing cancer cells by the CCK-8 assay, 4T1 cells were cultured in DMEM containing 10% fetal bovine serum and seeded in 96-well plates at a density of 1 × 104 cells per well for 24 h. To test the cytotoxicity of each group under different treatments (including control, MnOx/TiO2-GR-PVP-only, laser-only, US-only, TiO2-PEG + US, MnOx/TiO2-GR-PVP + US, MnOx/TiO2-

(2)

where m and Cp are the mass and heat capacity of the solvent (water), T is the solution temperature, QMnOx/TiO2‑GR is the photothermal energy inputted by MnOx/TiO2-GR nanocomposites, QDis is the baseline energy inputted by the sample cell, and Qout is the heat conducted away from the system surface by air. The 808 nm laser-induced source term, QMnOx/TiO2‑GR, is heat dissipated by electron−phonon relaxation of plasmons on the MnOx/ TiO2-GR surface induced by 808 nm laser irradiation of MnOx/TiO2GR nanocomposites at a resonant wavelength λ:

Q MnO /TiO

(5)

which is substituted into eq 2 and rearranged to yield

(1)

dT = Q MnO /TiO ‐ GR + Q Dis − Q Out x 2 dt

T − TOut TMax − TOut

and a sample system time constant τs,

where A represents the absorbance at a wavelength λ, ε represents the extinction coefficient, L represents the path length (1 cm), and C represents the molar concentration of the nanocomposites (in g L−1). The extinction coefficient ε is obtained by plotting the slope (in L g−1 cm−1) of each linear fit against the wavelength. The 808 nm laser extinction coefficient (ε) of MnOx/TiO2-GR can be calculated to be 10.1 L g−1 cm−1. b. Calculation of the Photothermal Conversion Efficiency. According to a previous report, the total energy balance for the whole system is

∑ miCp, i

(4)

(3)

where I represents the incident laser power (in unit of mW), A808 is the absorbance of the MnOx/TiO2-GR at a wavelength of 808 nm, and η represents the photothermal transduction efficiency. In addition, QDis is heat dissipated from light absorbed by the sample cell itself, and it was calculated independently to be QDis = (5.4 × 10−4)I (in mW) 9477


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PVP Nanocomposites. For the in vivo synergistic PTT/SDT treatment, female Balb/c nude mice receiving a subcutaneous injection into the armpit with 4T1 cells (1 × 106 cell/site) suspended in 100 μL of PBS were selected as the animal tumor xenograft. After the tumors grew to a mean volume of around 70 mm3, the mice were randomly assigned into seven groups (n = 5 per group): (a) control group (only treated with saline), (b) MnOx/TiO2-GR-PVP group (only intravenously injected with MnOx/TiO2-GR-PVP nanocomposites), (c) laser group (only exposed to the 808 nm laser irradiation), (d) US group (only exposed to the US irradiation), (e) MnOx/TiO2-GR-PVP + laser group (intravenously injected with MnOx/TiO2-GR-PVP followed by exposure to 808 nm laser irradiation), (f) MnOx/TiO2GR-PVP + US group (intravenously injected with MnOx/TiO2-GRPVP followed by exposure to US irradiation), (g) MnOx/TiO2-GRPVP + US + laser group (intravenously injected with MnOx/TiO2-GRPVP followed by exposure to US and laser irradiation). Each mouse in the groups (d)−(g) was intravenously injected with MnOx/TiO2-GRPVP in PBS at a dose of 20 mg kg−1. US exposure (1.0 MHz, 1.0 W cm−2, 50% duty cycle, 5 min) and 808 nm laser irradiation (2.0 W cm−2, 10 min) were performed after 4 h of intravenous injection. An infrared thermal imaging instrument (FLIR A325SC camera, USA) was used for IR thermal imaging to record the temperature and thermal image at different time intervals. After varied treatments, the tumor length (L) and width (W) were measured by a caliper every 2 days for 2 weeks and calculated according to the following formula: Tumor volume (V) = L × W2/2. The tumor tissues were collected from mice in each group 24 h postinjection for further H&E, TUNEL, and Ki-67 antibody staining of tumor sections for further histopathologic study. Statistical Analysis. The data herein were presented as mean ± SD, and the significance of the data in this work is analyzed based on a Kruskal−Walls test (*p < 0.05, **p < 0.01, and ***p < 0.001).

GR-PVP + laser, MnOx/TiO2-GR-PVP + US/laser), the CCK-8 assay was performed to determine the cell viabilities compared with the untreated control cells. The US irradiation parameters are 1.0 MHz, 1.0 W cm−2, and a 50% duty cycle. Intracellular Endocytosis Observed by CLSM. 4T1 cancer cells were seeded in CLSM-specific culture dishes (35 mm × 10 mm, Corning Inc., New York, USA) and incubated with MnOx/TiO2-GRPVP nanocomposites at 37 °C in humidified 5% CO2 for 24 h. Then, the culture media was replaced with FITC-labeled MnOx/TiO2-GRPVP nanocomposites (1 mL, 100 μg/mL in DMEM), which were then cultured for 0, 1, 2, 4, and 8 h, respectively. DAPI (100 μL, Beyotime Biotech-nology) was added into the dish to stain the cell nuclei. The cells were washed with PBS three times after staining for 15 min and observed by CLSM. In Vitro ROS Generation As Observed by CLSM. 4T1 cancer cells were seeded into CLSM-specific culture dishes and incubated with MnOx/TiO2-GR-PVP nanocomposites at 37 °C in humidified 5% CO2 for 24 h. The cells were then treated by US irradiation (1.0 MHz, 1.0 W cm−2, 50% duty cycle) for 5 min. Subsequently, the cell culture medium was replaced with DCFH-DA (Beyotime Biotechnology) solution and incubated for another 30 min. The cells were finally washed gently with PBS three times and observed by CLSM. In Vitro Synergistic SDT/PTT Effect As Observed by CLSM. 4T1 cancer cells were seeded in CLSM-specific culture dishes and incubated with MnOx/TiO2-GR-PVP nanocomposites at 37 °C in humidified 5% CO2. After 4 h of incubation, the cells were then treated by different treatments, including control, MnOx/TiO2-GR-PVP-only, laser-only, US-only, TiO2-PEG + US, MnOx/TiO2-GR-PVP + US, MnOx/TiO2-GR-PVP + laser, and MnOx/TiO2-GR-PVP + US/laser. Then, the cell culture medium was removed followed by calcein-AM (100 μL) and PI solution (100 μL, Dojindo Molecular Technologies, Inc.) staining for another 15 min. Finally, the cells were observed by CLSM, where live cells were stained in green and dead cells in red, respectively. In Vitro and in Vivo MR Imaging. The T1-weighted signals of MnOx/TiO2-GR-PVP at different concentrations treated with different pH values (5.0, 6.0, and 7.4) were measured. The prepared MnOx/ TiO2-GR-PVP nanocomposites were dispersed into different buffer solutions (pH = 7.4, 6.0, and 5.0), which were further shaken at a speed of 120 rpm at 37 °C for 3 h. Then, the MnOx/TiO2-GR-PVP buffer solutions were diluted with corresponding xanthan gum buffer solution and transferred into 2 mL Eppendorf tubes for MRI testing. The in vitro MR imaging experiment was carried out on a Signa HDXT 3.0 T equipment (GE Medical System, Fudan University Cancer Hospital). The parameters for T1-weighted fast-recovery spin−echo sequence were adopted as follows: TR = 1000, 2000, 3000, and 4000, Slice = 3 mm, Space = 0.5 mm, Fov = 20, Phase fov = 0.8, Freq × Phase = 384 × 256, Nex = 2, ETL = 2. The in vivo T1-weighted MR images of tumor-bearing mice were obtained before and at determined time intervals after intravenous injection of MnOx/TiO2-GR-PVP PBS solutions at a dose of 20 mg kg−1. The tumor model was established by implanting 4T1 murine breast cancer cells (1 × 106) suspended in PBS into the tested mouse. The in vivo MRI experiment was performed after the tumor size reached nearly 150 mm3. The corresponding parameters for in vivo MR imaging were the same as the in vitro experiment. The animal procedures were in agreement with the guidelines for the Animal Care Ethics Commission of Shanghai Tenth People’s Hospital, School of Medicine of Tongji University. In Vivo Biocompatibility Assay. Twenty-four healthy female Kunming mice were randomly divided into four groups to evaluate the in vivo biocompatibility and received intravenous administration of different doses of MnOx/TiO2-GR-PVP (control, 5 mg kg−1, 10 mg kg−1, and 20 mg kg−1). The body weight of mice was recorded every 2 days. After 30 days of feeding, the mice were sacrificed and their blood samples and major organs (heart, liver, spleen, lung, and kidney) were collected to conduct complete blood panel tests and hematoxylin and eosin (H&E) staining. In Vivo Synergetic Therapeutic Efficacy of Concurrent Sonocatalytic Therapy and PTT Assisted by MnOx/TiO2-GR-

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b05215. Additional figures and results as described in the text (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] (S. Zhang). *E-mail: [email protected] (R. Wu). *E-mail: [email protected] (Y. Chen). ORCID

Yu Chen: 0000-0002-8206-3325 Author Contributions

Y.C. originated the idea of the present work. Y.C. and R.W. supervised the project and commented on the project. C.D. synthesized and characterized the nanocatalysts, performed in vitro and in vivo experiments, and analyzed the data. S.Z. and Z.L. performed in vitro and in vivo MRI experiments and analyzed the data. C.D. wrote the manuscript. All the authors contributed to the discussion during the whole project. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We greatly acknowledge financial support from National Key R&D Program of China (Grant No. 2016YFA0203700), National Natural Science Foundation of China (Grant Nos. 81471673, 81671699, and 51672303), Natural Science Foundation of Shanghai (15ZR1407700), Shanghai Hospital 9478


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ACS Nano

(21) Liu, H.-L.; Hua, M.-Y.; Yang, H.-W.; Huang, C.-Y.; Chu, P.-C.; Wu, J.-S.; Tseng, I.-C.; Wang, J.-J.; Yen, T.-C.; Chen, P.-Y. Magnetic Resonance Monitoring of Focused Ultrasound/Magnetic Nanoparticle Targeting Delivery of Therapeutic Agents to the Brain. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15205−15210. (22) Chen, Y.; Chen, H.; Shi, J. Nanobiotechnology Promotes Noninvasive High-Intensity Focused Ultrasound Cancer Surgery. Adv. Healthcare Mater. 2015, 4, 158−165. (23) Qian, X.; Zheng, Y.; Chen, Y. Micro/Nanoparticle-Augmented Sonodynamic Therapy (SDT): Breaking the Depth Shallow of Photoactivation. Adv. Mater. 2016, 28, 8097−8129. (24) Rosenthal, I.; Sostaric, J. Z.; Riesz, P. Sonodynamic Therapy−A Review of the Synergistic Effects of Drugs and Ultrasound. Ultrason. Sonochem. 2004, 11, 349−363. (25) Yumita, N.; Iwase, Y.; Nishi, K.; Komatsu, H.; Takeda, K.; Onodera, K.; Fukai, T.; Ikeda, T.; Umemura, S.; Okudaira, K.; Momose, Y. Involvement of Reactive Oxygen Species in Sonodynamically Induced Apoptosis Using a Novel Porphyrin Derivative. Theranostics 2012, 2, 880−888. (26) McEwan, C.; Fowley, C.; Nomikou, N.; McCaughan, B.; McHale, A. P.; Callan, J. F. Polymeric Microbubbles as Delivery Vehicles for Sensitizers in Sonodynamic Therapy. Langmuir 2014, 30, 14926−14930. (27) Tachibana, K.; Feril, L. B.; Ikeda-Dantsuji, Y. Sonodynamic Therapy. Ultrasonics 2008, 48, 253−259. (28) Huang, P.; Qian, X.; Chen, Y.; Yu, L.; Lin, H.; Wang, L.; Zhu, Y.; Shi, J. Metalloporphyrin-Encapsulated Biodegradable Nanosystems for Highly Efficient MRI-Guided Sonodynamic Cancer Therapy. J. Am. Chem. Soc. 2017, 139, 1275−1284. (29) Deepagan, V. G.; You, D. G.; Um, W.; Ko, H.; Kwon, S.; Choi, K. Y.; Yi, G.-R.; Lee, J. Y.; Lee, D. S.; Kim, K.; Kwon, I. C.; Park, J. H. Long-Circulating Au-TiO2 Nanocomposite as a Sonosensitizer for ROS-Mediated Eradication of Cancer. Nano Lett. 2016, 16, 6257− 6264. (30) You, D. G.; Deepagan, V. G.; Um, W.; Jeon, S.; Son, S.; Chang, H.; Yoon, H. I.; Cho, Y. W.; Swierczewska, M.; Lee, S.; et al. ROSGenerating TiO2 Nanoparticles for Non-Invasive Sonodynamic Therapy of Cancer. Sci. Rep. 2016, 6, 23200. (31) Wang, X.; Wang, W.; Yu, L.; Tang, Y.; Cao, J.; Chen, Y. SiteSpecific Sonocatalytic Tumor Suppression by Chemically Engineered Single-Crystalline Mesoporous Titanium Dioxide Sonosensitizers. J. Mater. Chem. B 2017, 5, 4579−4586. (32) Chung, C.; Kim, Y.-K.; Shin, D.; Ryoo, S.-R.; Hong, B. H.; Min, D.-H. Biomedical Applications of Graphene and Graphene Oxide. Acc. Chem. Res. 2013, 46, 2211−2224. (33) Feng, L. Y.; Wu, L.; Qu, X. G. New Horizons for Diagnostics and Therapeutic Applications of Graphene and Graphene Oxide. Adv. Mater. 2013, 25, 168−186. (34) Mao, H. Y.; Laurent, S.; Chen, W.; Akhavan, O.; Imani, M.; Ashkarran, A. A.; Mahmoudi, M. Graphene: Promises, Facts, Opportunities, and Challenges in Nanomedicine. Chem. Rev. 2013, 113, 3407−3424. (35) Chen, Y.; Tan, C.; Zhang, H.; Wang, L. Two-Dimensional Graphene Analogues for Biomedical Applications. Chem. Soc. Rev. 2015, 44, 2681−2701. (36) Robinson, J. T.; Tabakman, S. M.; Liang, Y. Y.; Wang, H. L.; Casalongue, H. S.; Vinh, D.; Dai, H. J. Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. J. Am. Chem. Soc. 2011, 133, 6825−6831. (37) Yang, K.; Feng, L.; Shi, X.; Liu, Z. Nano-Graphene in Biomedicine: Theranostic Applications. Chem. Soc. Rev. 2013, 42, 530−547. (38) Yang, K.; Feng, L.; Liu, Z. Stimuli Responsive Drug Delivery Systems Based on Nano-Graphene for Cancer Therapy. Adv. Drug Delivery Rev. 2016, 105, 228−241. (39) Perera, S. D.; Mariano, R. G.; Vu, K.; Nour, N.; Seitz, O.; Chabal, Y.; Balkus, K. J., Jr Hydrothermal Synthesis of Graphene-TiO2 Nanotube Composites with Enhanced Photocatalytic Activity. ACS Catal. 2012, 2, 949−956.

Development Center (Grant No. SHDC12016233), Science and Technology Commission of Shanghai Municipality (Grant No. 17411967400), Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant No. 2013169), Young Elite Scientist Sponsorship Program by CAST (Grant No. 2015QNRC001), and Development Fund for Shanghai Talents (2015).

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