Two-Dimensional Graphene Augments Nanosonosensitized

Subscriber access provided by PEPPERDINE UNIV

Article

Two-Dimensional Graphene Augments Nanosonosensitized Sonocatalytic Tumor Eradication Chen Dai, Shengjian Zhang, Zhuang Liu, Rong Wu, and Yu Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05215 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Two-Dimensional Graphene Augments Nanosonosensitized Sonocatalytic Tumor Eradication Chen Dai,1,3 Shengjian Zhang,2* Zhuang Liu,2 Rong Wu1* and Yu Chen4*

1

Department of Ultrasound in Medicine, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China. E-mail: [email protected]

2

Department of Radiology, Fudan University Shanghai Cancer Center, Shanghai, 200032, P. R. China. E-mail: [email protected] 3

Department of Ultrasound, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.

4

State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China. E-mail: [email protected]

ACS Paragon Plus Environment

1

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 49

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 electrons (e-) and holes (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 tumor. By further MnOx functionalization, these 2D composite nanosonosensitizers achieved tumor microenvironmentsensitive (mild acidity) T1-weighted magnetic resonance imaging of tumor for therapeutic guidance and monitoring. The high photothermal-conversion capability of graphene also synergistically enhanced the SDT efficiency, achieving the complete eradication of 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 non-invasive and safe therapeutic modality for tumor eradication by the nanosonosensitized sonocatalytic process. Keywords: sonodynamic therapy, TiO2 sonosensitizer, graphene, synergistic therapy, nanomedicine


2

Page 3 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

The ideal therapeutic modality for cancer aims to trigger the tumor-specific treatment,1-4 which means that the therapeutic toxicity is only generated at the tumor site while the normal tissue/organ should not be damaged. On this ground, 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 clinic but the severe side effects and damage to normal organs/tissues usually cause the undesirable therapeutic outcome.14 Photodynamic therapy (PDT, laser as outer source) activates photosensitizers to generate toxic singlet oxygen (1O2) for inducing the tumor-cell death, but the low tissue-penetrating depth of light hinders the treatment of deep-seated tumor.15-17 Ultrasound (US), as a mechanical wave in physics, has been extensively explored for diagnostic imaging in clinic.18, 19 It can also exert the therapeutic function such as high intensityfocused 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 as “sonodynamic therapy” (SDT).23, 24 SDT shows its high application potential in clinic based on the US features such as its non-invasiveness 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, etc.) 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


3

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 49

sonosensitizer delivery into tumor.28 As compared to traditional organic sonosensitizers, the physiochemical property of inorganic nanomaterials makes them the 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.2931

The low quantum yield of ROS generation, however, hinders their further separation of

electron (e-) and hole (h+) pairs from energy band. The sonosensitizing effect is therefore low, unfortunately causing the insufficient SDT efficiency. It is still highly challenging to prevent the recombination of US-triggered electron and hole pairs from TiO2 nanosonosensitizer and further augment its SDT efficiency.29 In this work, we report on the integration of two-dimensional (2D) reduced graphene oxide (GR) nanosheets with TiO2 nanosonosensitizer for enhancing the SDT outcome against cancer by taking the advantage of 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 MnOx/TiO2-GR) for diagnostic-imaging guidance and monitoring. These 2D MnOx/TiO2-GR nanocomposites exhibit the features for imaging-guided synergistic SDT on combating cancer. First, the presence of GR can effectively separate the electrons (e-) and holes (h+) pairs as generated by the US irradiation-induced cavitation effect based on the high electroconductivity of GR nanosheets. Second, the high photothermal-conversion capability of GR is capable of synergistically enhancing the SDT efficiency by photothermal therapy (PTT). Third, the integrated MnOx NPs on the surface of 2D MnOx/TiO2-GR nanocomposites act


4

Page 5 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

as the pH-responsive contrast agents (CAs) for T1-weighted magnetic resonance imaging (MRI), potentially providing the guidance for the synergistic PTT-enhanced 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 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 electrons (e-) and holes (h+) pairs have been demonstrated as the main contributor for the synergistic effect between GO and TiO2. It is considered that SDT is generally activated by 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 oxygens (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 onto the surface of GO by a simple but facile hydrothermal methodology (Fig. 1a). To be specific, ultrathin GO nanosheets were initially fabricated by the exfoliation of multi-layer GO for 12 h via sonication, which were further dispersed into the co-solvents of water and ethanol. TiO2 NPs could be directly grown onto GO’s surface by the hydrothermal treatment of the co-solvent solution of TiO2 NPs and as-


5

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 49

exfoliated GO suspension. 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 MnOx/TiO2-GR composites was finally modified with biocompatible polyvinylpyrrolidone (PVP) to improve the colloidal stability of composite nanosheets in physiological condition (designated as MnOx/TiO2-GRPVP). These composite nanosheets could freely transport within the blood vessel and accumulate into the tumor tissue via the typical enhanced permeability and retention (EPR) effect (Fig. 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 the enhanced synergistic efficiency for concurrent PTT and SDT against tumor growth, and even complete tumor eradication. Ultrathin GO nanosheets were fabricated with the average planar size of nearly 230.0 nm after ultrasonic exfoliation of multilayer-structured and large sheet-sized GO for 12 h (Fig. 2a, Fig. S1). The nanoscale size of as-synthesized GO nanosheets can guarantee their facile transport within the blood vessel and further systematic in vivo theranostic application. After hydrothermal reaction between TiO2 NPs and GO nanosheets in the co-solvent of ethanol and water, TiO2 NPs were firmly attached onto the surface of reduced GO nanosheets (GR). TEM and SEM images (Fig. 2b-c, Fig. S2) evidenced the formation of TiO2-GR composites where many decorated TiO2 NPs were clearly observed on the GR’s surface. High resolution TEM image (HRTEM) and selected area electron diffraction (SAED) patterns of TiO2-GR (inset of Fig. S2c) demonstrate that the attached TiO2 NPs are highly crystallized, which is favorable for the


6

Page 7 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 were shown in Fig. 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 attributing 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°. Fig. S3b showed the FTIR spectrum of GO, TiO2 and TiO2-GR from the range of 400 cm-1 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 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 (Fig. 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 insitu grown onto the surface of TiO2-GR nanocomposites (designated as MnOx/TiO2-GR) based on the redox reaction of post-introduced oxidative MnO4- and reducing TiO2-GR surface. Highly uniform and dispersive MnOx species could be found on the surface of TiO2-GR nanocomposites


7

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 49

(Fig. 2f-g, Fig. S4), which also exhibited the sheet-like topology firmly attached on the surface of 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 GR nanosheets (Fig. 3a). The X-ray energy dispersive spectroscopy (EDS, Fig. 3b) and corresponding electron energy loss spectrum (EELS) of MnOx/TiO2-GR nanocomposites (Fig. 3c) showed the co-existence of Ti, O and Mn elements, demonstrating the successful construction of TiO2-GR via hydrothermal treatment and further MnOx grafting onto the 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 condition and prolonging their in vivo blood circulation, their surface was grafted with the biocompatible polyvinyl pyrrolidone (PVP) (designated as MnOx/TiO2-GR-PVP). After PVP coating, one characteristic peak around 1600 cm−1 in FTIR spectrum occurred (Fig. S5), which was indexed to the carbonyl groups of PVP, further indicating the successful PVP modification. The average particle size of MnOx/TiO2-GR-PVP nanocomposites measured by dynamic light scattering (DLS) was determined to be around 260.0 nm, and the further surface PVP modification showed only slight increase on the particle size (Fig. 3d). The series changes on Zeta potential of each grafting step further indicate the stepwise formation of MnOx/TiO2-GR-PVP nanocomposites (Fig S6). Especially, these surface-modified MnOx/TiO2-GR-PVP nanocomposites are featured with 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 (Fig. 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


8

Page 9 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

presence of TiO2 and MnOx on the surface of GR nanosheets (Fig. S8). For as-prepared MnOx/TiO2-GR, 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 (Fig. 3f), respectively. An additional shoulder peak centered at 282.6 eV was observed, which was attributed to the formation of the 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 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 eV and 466.0 eV, another two peaks around 456.7 and 462.5 eV were clearly identified (Fig. 3g) because of the formation of C-Ti bond in the MnOx/TiO2-GR nanocomposites. The obvious Mn signal in XPS demonstrated the integration of MnOx on the surface of TiO2-GR (Fig. S8d), The fitted peaks around 642.4 eV 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.

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 able 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 the electron-hole pair recombination due to its excellent electroconductivity with 2D planar πconjugation structure. Subsequently, the lifetime of charge carriers is prolonged, resulting in the


9

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 49

formation of the larger amount of radical species with strong oxidation capability (Fig. 4a), such as, singlet oxygen (1O2), superoxide radical (O2-) and hydroxyl radical (•OH) species. The 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 MnOx/TiO2-GR nanocomposite was obviously smaller as compared with TiO2 NPs, demonstrating the decreased charge-transfer resistance within MnOx/TiO2-GR nanocomposites. Therefore, the MnOx/TiO2-GR nanocomposite is more effective in 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 UV-vis spectrum. As shown in Fig. 4c, the absorbance intensity of DPBF sharply decreased in the presence TiO2-GR than 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 manner of TiO2-GR for ROS generation. 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 (Fig 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 (Fig. 4d), demonstrating


10

Page 11 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

that the presence of GO could efficiently enhanced the ROS generation capability during the SDT process. 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-1oxyl, which then induces a characteristic 1:1:1 triplet signal in ESR spectrum. As shown in Fig. S10a, both TiO2 and TiO2-GR produced 1O2 under the same US irradiation (1.0 MHz, 1.5 W cm2

, 50% duty cycle, 60 s), and the signal intensity of TiO2-GR-induced 1O2 generation was

obviously higher than that of pure TiO2 NPs, indicating more amounts of 1O2 production by USexcited TiO2-GR. These results were in accordance with the measurement of DPBF absorption spectra (Fig. 4c and 4d). 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 exposure 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 TiO2GR was also higher than that of DMPO-•OH adduct as induced by TiO2 NPs (Fig. S10b). These results demonstrated that TiO2-GR could serve as desirable nanosonosensitizer for the USactivated generation of toxic ROS with higher efficiency as compared to bare TiO2 NPs, guaranteeing further enhanced SDT for in vivo tumor therapy. 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 (PTT).32, 51, 52 MnOx/TiO2-GR with different weight ratios of GR (9%, 23% and 33%)


11

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 49

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 o

C (2 W cm-2, 5 min) at the Ti concentration of 118 ppm (Fig. 11a), 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 (Fig. 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 (Fig. 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 75ppm) was then tested and the extinction coefficient at 808 nm was measured to be 10.1 Lg-1 cm-1 (Fig. 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% (Fig. 4g and 4h), high enough for efficient photothermal ablation of tumor. The photothermal performance of the MnOx/TiO2-GR composite nanosheets did not show obvious deterioration during five laser on/off recycles, demonstrating the high potential of MnOx/TiO2-GR composite nanosheets as a durable photothermal agent for PTT-based hyperthermia (Fig. 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 bio-application by using 4T1 murine breast cancer cells as a model cell. 4T1 cells were firstly incubated with


12

Page 13 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 prolonged incubation time of 48 h, no obvious cytotoxicity of MnOx/TiO2-GR-PVP nanocomposites was observed (Fig. 5a). The nanoscale size of MnOx/TiO2-GR-PVP nanocomposites guarantees their easy endocytosis into cancer cells, which are mainly distributed within the cytoplasm of cells. After sequential US and laser irradiations, MnOx/TiO2-GR-PVP nanocomposites trigger the intracellular ROS generation and temperature elevation to kill the cancer cells (Fig. 5b). The intracellular uptake of MnOx/TiO2GR-PVP at different durations (0, 1, 2, 4 and 8 h) was further tested. Most of fluorescein isothiocyanate (FITC)-labeled MnOx/TiO2-GR-PVP nanocomposites were found in the cytoplasm of 4T1 cells after 4 h co-incubation, where the green fluorescence originated from FITC and the blue fluorescence represented cell nucleus as stained by DAPI (Fig. 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-dihydro-fluorescien diacetate (DCFH-DA) was used to test the ROS generation by monitoring the green fluorescence in confocal laser scanning microscope (CLSM) imaging, attributing to highly fluorescent 2,7dichlorofluorescein (DCF) transformed from DCFH-DA in the presence of ROS. As expected, a strong green fluorescence of MnOx/TiO2-GR-PVP + US group was observed in 4T1 cancer cells by CLSM, which is much brighter than that of TiO2-PVP + US group, while the control and USonly group exhibited a negligible green fluorescence, demonstrating the large production of ROS during the sonocatalytic process assisted by MnOx/TiO2-GR-PVP (Fig. S13). To compare the in vitro cytotoxicity of TiO2-PEG and MnOx/TiO2-GR-PVP under US activation, MnOx/TiO2-GR-PVP under 808 nm-laser irradiation and MnOx/TiO2-GR-PVP under sequential US/808nm-laser irradiation, the viability of cells was determined by the typical CCK-


13

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 49

8 assay. As shown in Fig. 8b, the cells were substantially killed by US irradiation (1.0 MHz, 1.0 W cm-2, 50% duty cycle, 3 min) after the co-incubation 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 durationdependent (Fig S14). The inhibition rate of cancer cells by MnOx/TiO2-GR-PVP under US irradiation could reach nearly 56%, much higher as compared to TiO2-PEG (22%) at the same Ti concentration (120 ppm) under the same US-irradiation condition. The significantly improved sono-toxicity of MnOx/TiO2-GR-PVP was attributed to generation of more 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, MnOx/TiO2-GR-PVP only group and laser-only group showed the negligible effect on the survival of 4T1 cells. The cell apoptosis after different treatment was further confirmed by CLSM observation, where the live and dead cells were stained by calcein-AM (green) and PI (red), respectively (Fig. 5e). Large amounts of dead cancer cells presenting red fluorescence were clearly distinguished in the group of MnOx/TiO2-GR-PVP under sequential US/808nm-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 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 the mild acidic condition to release the paramagnetic Mn2+ (Fig. 6a). To evaluate


14

Page 15 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

the contrast-enhanced MRI capability of MnOx/TiO2-GR-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 condition (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 at acidic solution (Fig. 6b and c). The r1 relaxivity of initial MnOx/TiO2-GRPVP was measured to be only 0.06 mM-1 s-1. Importantly, this relaxivity reached 1.61 and 5.77 mM-1s-1 at the acidic pHs of 6.0 and 5.0, respectively, significantly higher than their original r1 values at neutral condition (nearly 96-fold increase). This enhanced T1-weighted MRI capability under acidic condition was due to the released paramagnetic Mn2+ ions, which can get the 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 T2weighted MRI where substantially enhanced negative T2 MRI signals were observed under acidic condition (Fig. 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 tumor-bearing mice were recorded at the given time intervals. As shown in Fig. 6d, a remarkable signal enhancement and brightening effect of MRI signals at the tumor site were observed, attributing 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 mild acidic microenvironment of tumor tissue. This MRI-signal enhancement in tumor site was further demonstrated by


15

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 49

quantitatively positive MRI signal intensities (Fig. 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 by MnOx/TiO2-GRPVP. 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 organs and tumor was investigated after intravenous injection for 4 h and 24 h using nude 4T1 tumor-bearing mice model. Nearly 10% of Mn ion had accumulated into tumor sites via the EPR effect of MnOx/TiO2-GR-PVP nanocomposites (Fig. 7a). The circulation of MnOx/TiO2-GR-PVP nanocomposites in bloodstream was investigated and the blood-circulation half-time of MnOx/TiO2-GR-PVP was calculated to be 0.80 h (Fig. 7b). The eliminating rate constants of MnOx/TiO2-GR-PVP were calculated to be -0.014 µg ml-1h-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 (Fig. 7c). Forty tumor-bearing mice were then randomly separated into eight groups (n = 5 per group) for therapeutic evaluation, including (a) control group (only treated with saline), (b) MnOx/TiO2GR-PVP group (only intravenously injected with MnOx/TiO2-GR-PVP nanocomposites), (c) laser group (only exposed to 808nm 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-


16

Page 17 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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), (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/TiO2GR-PVP nanocomposites (20 mg kg-1) for 4 h, the mice were treated by aforementioned therapeutic protocols, and the tumor-growth status was carefully monitored. Fig. 7d and 7e show that the tumor temperature in the group (g) treated with MnOx/TiO2-GRPVP and 808 nm laser irradiation increased significantly from 25oC to 60oC at a laser power density of 2 W cm-2 for ten minutes, which is sufficient for causing the tumor-cell death. In contrast, the tumor temperature in group (c) treated with laser only increased by ~3 oC. The US irradiation for group (d), (e), (f) and (h) was repeated on the third and fifth day (Fig. 7f). The tumor volumes of eight groups were recorded every 2 days using a digital caliper (Fig. 7g), and the digital photos of tumor sites were taken every 2 days during two weeks after varied treatments (Fig. S15). The body weight of mice in each group shows no obvious change during two weeks (Fig. 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 MnOx/TiO2GR-PVP + US group reaches up to nearly 78%, significantly higher than that of TiO2-PEG + US group (32.2%) and U.S group (22.9%). Though 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


17

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 49

reoccurrence during two weeks (Fig. 7i), demonstrating the high synergistic effect of combinatorial SDT/PTT therapy in comparison to single-modality SDT or PTT therapy. 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 collected from 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 group (f), (g) and (h). The MnOx/TiO2-GR-PVP combined with SDT/PTT exhibit higher necrosis of tumor cells as compared to either MnOx/TiO2-GR-PVP + US group or MnOx/TiO2-GR-PVP + laser group. There is much less cell necrosis on the examined tumor sections of US group and TiO2-PEG group. No obvious change of cell status was observed in the mice groups of the control, NIR laser only and MnOx/TiO2GR-PVP only. 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 photothermalconversion nanoagents (Fig. 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-GR-PVP at elevated doses


18

Page 19 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(control, 5 mg kg-1, 10 mg kg-1 and 20 mg kg-1). These mice were sacrificed after one-month feeding for blood collection and organ harvest. The complete blood panel test and biochemical analysis were then conducted and their major organs, including heart, liver, lung, spleen and kidney, were collected for histological characterization. No behavior abnormality of experimental mice was observed, and no obvious body-weight loss of mice were recorded (Fig. S16). The blood indexes were then tested, including the white blood cells (WBC), red blood cells (RBC), platelets (PLT), lymphocytes (LYM), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), haematocrit (HCT) and related kidney and liver function indexes (Fig. S17). Compared with the control group, all the indexes of the three MnOx/TiO2-GR-PVP-treated groups show no significant differences as compared to control group without any treatment. No obvious signals of organ damage and inflammatory lesion were discovered from H&E-stained organ slices (heart, liver, spleen, kidney and lung) (Fig. S18), suggesting the high histocompatibility of the as-designed 2D MnOx/TiO2-GR-PVP theranostic nanoagents.

Conclusions In summary, this work report on the integration of 2D graphene with TiO2 nanosonosensitizers (MnOx/TiO2-GR-PVP nanocomposites) for augmenting the sonocatalytic therapeutic efficiency against tumor 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 MnOx component has achieved the tumor microenvironmentsensitive (mild acidity) T1-weighted MR imaging of tumor, providing the potential for


19

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 49

therapeutic guidance and monitoring. Importantly, the designed MnOx/TiO2-GR-PVP nanocomposites substantially suppressed the 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/TiO2-GR-PVP nanosonosensitizers, achieving the complete eradication of tumor without reoccurrence.

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 co-solvent of deionized water (20 mL) and ethanol (10 mL), followed by ultrasonic treatment for 12 h. Then, 50 mg of TiO2 NPs were 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 for several times.

Synthesis of MnOx/TiO2-GR nanocomposites. MnOx/TiO2-GR nanocomposites were obtained by a simple redox reaction on the surface of graphene to in-situ grow MnOx components. Typically, 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 was lasted for another 12 h at room temperature. The resulting MnOx/TiO2-GR was collected by centrifugation, and further washed with deionized water for several times.


20

Page 21 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Surface PVP modification of MnOx/TiO2-GR nanocomposites (MnOx/TiO2-GR-PVP). To improve the stability of MnOx/TiO2-GR nanocomposites in physiological environment, MnOx/TiO2-GR (90 mg) and polyvinyl pyrrolidone (PVP) (Sigma-Aldrich, 300 mg) were added into a wide-necked bottle (100 mL in volume), which was then magnetically stirred at the 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 for several times to remove un-attached PVP.

Characterization. Transmission electron microscopy (TEM) images and corresponding X-ray energy dispersive spectroscopy (EDS) spectrum were acquired on a JEM-2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) images and element mapping were obtained on a field-emission Magellan 400 microscope (FEI Company). UV-visNIR absorption spectra were recorded by UV-3600 Shimadzu UV-vis-NIR spectrometer with QS-grade quartz cuvettes at room temperature. X-ray photoelectron spectroscopy (XPS) spectrum was recorded by ESCAlab250 (Thermal Scientific). Size and Zeta potential measurements were conducted on Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). The confocal laser scanning microscopy (CLSM) images were measured in FV1000 (Olympus Company, Japan). The MnOx/TiO2-GR concentration was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies, US). The electron spin resonance (ESR) characterization was performed on Bruker EMX Electron Paramagnetic Resonance (EPR) 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.


21

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 49

Electrochemical Impedance Spectra (EIS) measurement. The EIS measurement was carried out on a CH Instruments 760E electrochemical workstation by using three-electrode cells. A glassy carbon electrode (GCE) coated with samples, an Ag/AgCl electrode and a Pt foil were employed as the working electrode, reference electrode and counter electrode, respectively, which was performed 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. 2.5 µL of sample (2500 ppm) dispersed in deionized water and absolute ethanol solution (v : v = 1:1) was pipetted onto a 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 (ESR) spectra test. TEMP (Dojindo Molecular Technologies, Inc) was used to test the generation of 1O2 by TiO2 and TiO2-GR. Typically, TiO2 and TiO2-GR (Ti： 100 µg mL-1) solutions were exposed to U.S 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 electron spin resonance (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 exposed to U.S 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 the


22

Page 23 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

either TiO2 or MnOx/TiO2-GR (Ti: 500 ppm) aqueous solution was added to 2 mL of DPBF (60 µM) solution which was dissolved in DMF. Then, the mixture was exposed to US irradiation (1.0 MHz, 1.5 W cm-2, 50% duty cycle) in dark. The intensity of DPBF was measured using UV-Vis spectrometer.

Photothermal performance of MnOx/TiO2-GR nanocomposites. 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). Photothermal performance of MnOx/TiO2-GR with different GR ratio 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

(1)

where A represents the absorbance at a wavelength λ, ε represents the extinction coefficient, L represents 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 Lg-1 cm-1) of each linear fit against wavelength. The 808 nm laser extinction coefficient (ε) of MnOx/TiO2-GR can be calculated to be 10.1 Lg-1 cm-1. b. Calculation of the Photothermal Conversion Efficiency


23

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 49

According to previous report, the total energy balance for the whole system is ∑ ,

= / + −

(2)

where m and Cp are the mass and heat capacity of solvent (water) and T is the solution temperature, / is the photothermal energy inputted by MnOx/TiO2-GR nanocomposites. is the baseline energy inputted by the sample cell and is heat conduction away from the system surface by air. The 808nm laser induced source term. / , is heat dissipated by electron-phonon relaxation of plasmons on the MnOx/TiO2-GR surface induced by 808 nm laser irradiation of MnOx/TiO2-GR nanocomposites at a resonant wavelength λ / = 1 − 10

!"!

#$

(3)

where I represents incident laser power (in unit of mW), %&'& is the absorbance of the MnOx/TiO2-GR at wavelength of 808 nm, and $ represents the photothermal transduction efficiency. In addition, is heat dissipated from light absorbed by the sample cell itself, and it was calculated independently to be = 5.4 × 10, # (in mW) using a sample cell containing pure water without MnOx/TiO2-GR. , represents nearly proportional to the linear thermal driving force in this system, with a heat-transfer coefficient, ℎ, as the proportionality constant = ℎ./ − / #

(4)

where S is the surface area of the container, and / is ambient surrounding temperature. In order to acquire the hS, a dimensionless driving force temperature, θ, is introduced, using the maximum system temperature, /012 3=

456

78 456

(5)


24

Page 25 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

and a sample system time constant 9 9 =

∑: 0: ;

(6)

which is substituted into equation (2) and rearranged to yield ?

@ DEF4 /G:4 HIJ KDL:B =>E8 456 # B

=A C

− 3M

(7)

While turning off the laser source, / + = 0, reducing equation (7) to N = −9 OP 3

(8)

Therefore, 9 was obtained to be 146.71 from the data in Fig. 4h. In addition, the m is 0.1 g and the C is 4.2 J g-1. According to equation (6), the hS is calculated to be 2.66 mW °C-1. During laser irradiation, / + is finite, and the heat output # is increased along with the increase of the temperature according to the equation (4), the system temperature will rise to a maximum value when external heat flux equals to heat input / + = ℎ./ 12 − / #

(9)

where / 12 is the equilibrium temperature. The 808 nm laser photothermal conversion efficiency (η) can be calculated by substituting equation (3) for / into equation (9) and rearranging to obtain $ =

=>E8 456 #DL:B QR@@'HS!"! T

(10)

where the / 12 − / # was 36.51 °C according to Fig. 4g, I is 560 mW, was measured independently to be 0.1728 mW, %&'& is the absorbance (3.0) of MnOx/TiO2-GR at 808 nm (Fig. 4e). Substituting these values into equation (10), the 808 nm laser photothermal conversion efficiency (η) of MnOx/TiO2-GR can be calculated to be 18.4%.


25

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 49

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 Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, GIBCO, Invitrogen) media and supplemented with 10% fetal bovine serum (FBS), 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) were added into the well and co-incubated for another 24 h 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 the wavelength 405 nm after 60 min.

In vitro synergistic SDT and PTT against cancer cells. For assessing the synergistic SDT and PTT for killing the cancer cell by 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-GR-PVP + laser, MnOx/TiO2-GR-PVP+ US/laser), 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 50% duty cycle.

Intracellular endocytosis observed by CLSM observation. 4T1 cancer cells were seeded in confocal laser scanning microscope (CLSM)-specific culture dishes (35 mm × 10 mm, Corning Inc., New York) and incubated with MnOx/TiO2-GR-PVP nanocomposites at 37 oC in a humidified 5% CO2 for 24 h. Then, the culture media was replaced with FITC-labeled


26

Page 27 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

MnOx/TiO2-GR-PVP 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 for 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 CLSMspecific culture dishes and incubated with MnOx/TiO2-GR-PVP nanocomposites at 37 oC in a humidified 5% CO2 for 24 h. The cells were then treated by US irradiation (1.0 MHz, 1.0 W cm2

, 50% duty cycle) for 5 min. Subsequently, the cell culture medium was replaced with 2, 7-

dichloro-dihydro-fluorescien diacetate (DCFH-DA, Beyotime Biotech-nology) solution and incubated for another 30 min. The cells were finally washed gently with PBS for three times and observed by CLSM.

In vitro synergistic SDT/PTT effect as observed by CLSM observation. 4T1 cancer cells were seeded in CLSM-specific culture dishes and incubated with MnOx/TiO2-GR-PVP nanocomposites at 37 oC in a humidified 5% CO2. After 4 h 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-GRPVP+ 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.


27

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 49

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 37oC 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 test. The in vitro MR imaging experiment was carried out on Signa HDXT 3.0 T equipment (GE Medical System, Fudan University Cancer Hospital). The parameters for T1-weighted Fast-recovery spin-echo (FR-FSE) 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 settled 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, which 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 two days. After 30 days feeding, the mice


28

Page 29 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

were sacrificed and their blood samples and major organs (heart, liver, spleen, lung and kidney) were collected to conduct the complete blood panel test and hematoxylin and eosin (H&E) staining.

In vivo synergetic therapeutic efficacy of concurrent sonocatalytic therapy and PTT assisted by MnOx/TiO2-GR-PVP nanocomposites. For the in vivo synergistic PTT/SDT treatment, these female Balb/c nude mice received 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 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 808nm 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/TiO2-GR-PVP + US group (intravenously injected with MnOx/TiO2-GR-PVP followed by exposure to US irradiation), (g) MnOx/TiO2-GR-PVP + US + laser group (intravenously injected with MnOx/TiO2-GR-PVP followed by exposure to US and laser irradiation). Each mouse in the groups of (d)-(g) was intravenously injected with MnOx/TiO2-GR-PVP in PBS at the 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 (IR) imaging instrument (FLIRTM 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 two days for 2


29

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 49

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 post injection for further H&E, TUNEL, Ki-67 antibody staining of tumor sections for further histopathologic study.

Statistical Analysis. The data herein were presented as mean ± s.d, 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).


30

Page 31 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1. Schematic illustration of the synthetic procedure of MnOx/TiO2-GR-PVP and MR imaging-guided synergistic SDT/PTT against cancer. (a) The 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


31

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 49

functions of MnOx/TiO2-GR-PVP nanocomposites, including free transport with the blood vessel after intravenous injection, TME-responsive MRI guidance prior to cancer therapy, and synergistic SDT/PTT against cancer.

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 onto the GR’s surface. (f) TEM and (g) SEM images of MnOx/TiO2-GR nanocomposites.


32

Page 33 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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-GRPVP nanosheets dispersed in aqueous solution. (e) 3D schematic illustration of formation of Ti-C bond between GO and TiO2. (f) X-ray photoelectron spectroscopy (XPS) of MnOx/TiO2-GR in C 1s region. (g) XPS spectrum of MnOx/TiO2-GR in Ti 2p region.


33

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 49

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 DPBF in the presence of TiO2 and MnOx/TiO2-GR upon exposure to US irradiation for prolonged durations. (d) Decay curves of relative absorption of DPBF at 410 nm with different irradiation durations in 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:


34

Page 35 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Normalized absorbance intensity at λ = 808 nm divided by the characteristic length of the cell (A/L) at varied concentrations. (f) The 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 aqueous dispersion of MnOx/TiO2-GR under irradiation by the NIR laser (808 nm, 2.0 W cm-2) (h) 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 negative natural logarithm of driving force temperature, which was originated from the cooling stage. (i) Recycling heating profiles of MnOx/TiO2-GR composite nanosheets-dispersed suspension using an 808 nm laser (2 W cm−2) for five laser on/off cycles.


35

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 49

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 h and 48 h. Error bars were based on the standard deviations (s. d.) of five parallel samples. (b) Schematic illustration of MnOx/TiO2-GR-PVP nanocomposites as the 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-


36

Page 37 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 co-irradiations. Error bars were based on the standard deviations (s. d.) of five parallel samples (P values: **p < 0.01.). (e) CLSM images of 4T1 cells stained by calcein AM and PI after different treatments.


37

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 49

Figure 6. Contrast-enhanced pH-responsive MR imaging of tumor 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 mild acidic TME for contrast-enhanced T1weighted 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/TiO2GR-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.


38

Page 39 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 7. In vivo synergistic SDT/PTT for tumor eradication. (a) Biodistribution assay after the intravenous administration of MnOx/TiO2-GR-PVP nanocomposites into tumor-bearing mice for 4 h and 24 h (n = 3). (b) The 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) The 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 intravenous injection of MnOx/TiO2-GR-PVP nanocomposites followed by 808 nm laser irradiation (2.0 W cm-2) at varied time intervals. (e) The 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


39

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 49

nude mice of eight experimental groups after different treatments, including control group, MnOx/TiO2-GR-PVP group, laser group, US group, TiO2-PEG + US group, MnOx/TiO2-GRPVP + US group, MnOx/TiO2-GR-PVP + laser group and MnOx/TiO2-GR-PVP + US/laser group (P values: *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/TiO2GR-PVP + US/laser group, respectively). All the scale bars are 50 µm.


40

Page 41 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

ASSOCIATED CONTENT Supporting Information Available: Additional figures, table, and results as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected]

(Y.

Chen),

[email protected]

(S.

Zhang),

[email protected] (R. Wu).

Author contributions Y.C. designated 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 experiment and analyzed the data. S.Z. and Z.L. performed in vitro and in vivo experiment of MRI and analyzed the data. C.D. wrote the manuscript. All the authors contributed to the discussion during the whole project.

ACKNOWLEDGMENT We greatly acknowledge financial support from National Key R&D Program of China (Grant No. 2016YFA0203700), National Natural Science Foundation of China (Grant No. 81471673, 81671699 and 51672303), Natural Science Foundation of Shanghai (15ZR1407700), Shanghai Hospital Development Center (Grant No. SHDC12016233), Science and Technology


41

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 49

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).

REFERENCES 1.

Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C., Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37.

2.

Tibbitt, M. W.; Dahlman, J. E.; Langer, R., Emerging Frontiers in Drug Delivery. J. Am. Chem. Soc. 2016, 138, 704-717.

3.

Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C., Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016, 1, 16014.

4.

Cheng, Z. L.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A., Multifunctional Nanoparticles: Cost versus Benefit of Adding Targeting and Imaging Capabilities. Science 2012, 338, 903-910.

5.

Jain, R. K.; Stylianopoulos, T., Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Oncol. 2010, 7, 653-664.

6.

Park, J.; Wrzesinski, S. H.; Stern, E.; Look, M.; Criscione, J.; Ragheb, R.; Jay, S. M.; Demento, S. L.; Agawu, A.; Limon, P. L. et al., Combination Delivery of TGF-Beta Inhibitor and IL-2 by Nanoscale Liposomal Polymeric Gels Enhances Tumour Immunotherapy. Nat. Mater. 2012, 11, 895-905.

7.

Chauhan, V. P.; Jain, R. K., Strategies for Advancing Cancer Nanomedicine. Nat. Mater. 2013, 12, 958-962.


42

Page 43 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

8.

Bertrand, N.; Wu, J.; Xu, X. Y.; Kamaly, N.; Farokhzad, O. C., Cancer Nanotechnology: The Impact of Passive and Active Targeting in the Era of Modern Cancer Biology. Adv. Drug Deliv. Rev. 2014, 66, 2-25.

9.

Danhier, F.; Feron, O.; Preat, V., To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Control. Release 2010, 148, 135-146.

10. Albini, A.; Sporn, M. B., The Tumour Microenvironment as a Target for Chemoprevention. Nat. Rev. Cancer 2007, 7, 139-147. 11. Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K., Regulation of Transport Pathways in Tumor Vessels: Role of Tumor Type and Microenvironment. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4607-4612. 12. Liotta, L. A.; Kohn, E. C., The Microenvironment of the Tumour-Host Interface. Nature 2001, 411, 375-379. 13. Kessenbrock, K.; Plaks, V.; Werb, Z., Matrix Metalloproteinases: Regulators of the Tumor Microenvironment. Cell 2010, 141, 52-67. 14. Kotagiri, N.; Sudlow, G. P.; Akers, W. J.; Achilefu, S., Breaking the Depth Dependency of Phototherapy

with

Cerenkov

Radiation

and

Low-Radiance-Responsive

Nanophotosensitizers. Nat. Nanotechnol. 2015, 10, 370-379. 15. Dolmans, D.; Fukumura, D.; Jain, R. K., Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380-387. 16. Ge, J.; Lan, M.; Zhou, B.; Liu, W.; Guo, L.; Wang, H.; Jia, Q.; Niu, G.; Huang, X.; Zhou, H., A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat. Commun. 2014, 5, 4596.


43

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 49

17. Idris, N. M.; Gnanasammandhan, M. K.; Zhang, J.; Ho, P. C.; Mahendran, R.; Zhang, Y., In Vivo Photodynamic Therapy Using Upconversion Nanoparticles as Remote-Controlled Nanotransducers. Nat. Med. 2012, 18, 1580-1585. 18. Mitragotri, S., Healing Sound: The Use of Ultrasound in Drug Delivery and Other Therapeutic Applications. Nat. Rev. Drug Discov. 2005, 4, 255-260. 19. Tang, H.; Zheng, Y.; Chen, Y., Materials Chemistry of Nanoultrasonic Biomedicine. Adv. Mater. 2017, 29, 1604105. 20. Kennedy, J. E., High-Intensity Focused Ultrasound in the Treatment of Solid Tumours. Nat. Rev. Cancer 2005, 5, 321-327. 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. Healthc. 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.


44

Page 45 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 MRIGuided 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., ROS-Generating 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., Site-Specific 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.


45

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 49

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 NanoGraphene for Cancer Therapy. Adv. Drug Deliver Rev. 2016, 105, 228-241. 39. Perera, S. D.; Mariano, R. G.; Vu, K.; Nour, N.; Seitz, O.; Chabal, Y.; Balkus Jr, K. J., Hydrothermal Synthesis of Graphene-TiO2 Nanotube Composites with Enhanced Photocatalytic Activity. ACS Catal. 2012, 2, 949-956. 40. Williams, G.; Seger, B.; Kamat, P. V., TiO2-Graphene Nanocomposites. UV-Assisted Photocatalytic Reduction of Graphene Oxide. ACS Nano 2008, 2, 1487-1491. 41. Xiang, Q. J.; Yu, J. G.; Jaroniec, M., Graphene-Based Semiconductor Photocatalysts. Chem. Soc. Rev. 2012, 41, 782-796. 42. Wang, D. H.; Choi, D. W.; Li, J.; Yang, Z. G.; Nie, Z. M.; Kou, R.; Hu, D. H.; Wang, C. M.; Saraf, L. V.; Zhang, J. G. et al., Self-Assembled TiO2-Graphene Hybrid Nanostructures for Enhanced Li-Ion Insertion. ACS Nano 2009, 3, 907-914. 43. Sakthivel, S.; Kisch, H., Daylight Photocatalysis by Carbon-Modified Titanium Dioxide. Angew. Chem. Int. Ed. 2003, 42, 4908-4911. 44. Qiu, B.; Xing, M.; Zhang, J., Mesoporous TiO2 Nanocrystals Grown in Situ on Graphene


46

Page 47 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Aerogels for High Photocatalysis and Lithium-Ion Batteries. J. Am. Chem. Soc. 2014, 136, 5852-5855. 45. Saksena, T.; Nyborg, W., Sonoluminescence from Stable Cavitation. J. Chem. Phys. 1970, 53, 1722-1734. 46. Didenko, Y. T.; McNamara, W. B.; Suslick, K. S., Molecular Emission from Single-Bubble Sonoluminescence. Nature 2000, 407, 877-879. 47. Hilgenfeldt, S.; Grossmann, S.; Lohse, D., A Simple Explanation of Light Emission in Sonoluminescence. Nature 1999, 398, 402-405. 48. McNamara, W. B.; Didenko, Y. T.; Suslick, K. S., Sonoluminescence Temperatures During Multi-Bubble Cavitation. Nature 1999, 401, 772-775. 49. Barber, B. P.; Hiller, R. A.; Lofstedt, R.; Putterman, S. J.; Weninger, K. R., Defining the Unknowns of Sonoluminescence. Phys. Rep.-Rev. Sec. Phys. Lett. 1997, 281, 65-143. 50. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K., Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: A Review. J. Control. Release 2000, 65, 271-284. 51. Chen, Y.; Ye, D.; Wu, M.; Chen, H.; Zhang, L.; Shi, J.; Wang, L., Break-Up of TwoDimensional MnO2 Nanosheets Promotes Ultrasensitive pH-Triggered Theranostics of Cancer. Adv. Mater. 2014, 26, 7019-7026. 52. Schladt, T. D.; Shukoor, M. I.; Schneider, K.; Tahir, M. N.; Natalio, F.; Ament, I.; Becker, J.; Jochum, F. D.; Weber, S.; Kohler, O.; Theato, P.; Schreiber, L. M. et al., Au@MnO Nanoflowers: Hybrid Nanocomposites for Selective Dual Functionalization and Imaging. Angew. Chem. Int. Ed. 2010, 49, 3976-3980. 53. Na, H. B.; Lee, J. H.; An, K. J.; Park, Y. I.; Park, M.; Lee, I. S.; Nam, D. H.; Kim, S. T.;


47

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 49

Kim, S. H.; Kim, S. W. et al., Development of a T1 Contrast Agent for Magnetic Resonance Imaging Using MnO Nanoparticles. Angew. Chem. Int. Ed. 2007, 46, 5397-5401. 54. Kim, T.; Momin, E.; Choi, J.; Yuan, K.; Zaidi, H.; Kim, J.; Park, M.; Lee, N.; McMahon, M. T.; Quinones-Hinojosa, A. et al., Mesoporous Silica-Coated Hollow Manganese Oxide Nanoparticles as Positive T1 Contrast Agents for Labeling and MRI Tracking of AdiposeDerived Mesenchyrnal Stem Cells. J. Am. Chem. Soc. 2011, 133, 2955-2961. 55. Bitounis, D.; Ali-Boucetta, H.; Hong, B. H.; Min, D. H.; Kostarelos, K., Prospects and Challenges of Graphene in Biomedical Applications. Adv. Mater. 2013, 25, 2258-2268. 56. Yang, K.; Zhang, S. A.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. A., Graphene in Mice: Ultrahigh in Vivo Tumor Uptake and Efficient Photothermal Therapy. Nano Lett. 2010, 10, 3318-3323. 57. Bennewitz, M. F.; Lobo, T. L.; Nkansah, M. K.; Ulas, G.; Brudvig, G. W.; Shapiro, E. M., Biocompatible and pH-Sensitive PLGA Encapsulated MnO Nanocrystals for Molecular and Cellular MRI. ACS Nano 2011, 5, 3438-3446. 58. Fan, H.; Zhao, Z.; Yan, G.; Zhang, X.; Yang, C.; Meng, H.; Chen, Z.; Liu, H.; Tan, W., A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing. Angew. Chem. Int. Ed. 2015, 127, 4883-4887. 59. Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.; Zhang, X.; Tan, W., Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging via MnO2 Nanosheet-Aptamer Nanoprobe. J. Am. Chem. Soc. 2014, 136, 11220-11223.


48

Page 49 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

For Table of Contents Only

We herein report on augmenting the sonocatalytic efficiency of semiconductor TiO2-based nanosonosensitizers for efficient sonodynamic therapy (SDT) by the integration of twodimensional (2D) ultrathin graphene with TiO2 nanosonosensitizers. By further MnOx functionalization, these 2D composite nanosonosensitizers achieved tumor microenvironmentsensitive (mild acidity) T1-weighted magnetic resonance imaging of tumor for therapeutic guidance and monitoring. The high photothermal-conversion capability of graphene also synergistically enhanced the SDT efficiency, achieving the complete eradication of tumor without reoccurrence.


49

Two-Dimensional Graphene Augments Nanosonosensitized

Recommend Documents