Accepted Manuscript

Graphene oxide / MnWO4 nanocomposite for magnetic resonance / photoacoustic

dual-model imaging and tumor photothermo-chemotherapy

Xu Chang, Yixue Zhang, Puqun Xu, Mengqing Zhang, Huixia Wu, Shiping Yang

PII: S0008-6223(18)30708-5
DOI: 10.1016/j.carbon.2018.07.058
Reference: CARBON 13339

To appear in: Carbon

Received Date: 30 April 2018

Revised Date: 24 July 2018
Accepted Date: 25 July 2018

Please cite this article as: X. Chang, Y. Zhang, P. Xu, M. Zhang, H. Wu, S. Yang, Graphene oxide /
MnWO4 nanocomposite for magnetic resonance / photoacoustic dual-model imaging and tumor
photothermo-chemotherapy, Carbon (2018), doi: 10.1016/j.carbon.2018.07.058.

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 ACCEPTED MANUSCRIPT

Graphical abstract

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Graphene oxide / MnWO4 nanocomposite for magnetic resonance /

photoacoustic dual-model imaging and tumor photothermo-chemotherapy

Xu Chang, Yixue Zhang, Puqun Xu, Mengqing Zhang, Huixia Wu*, Shiping Yang

The Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key

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Laboratory of the Rare Earth Functional Materials, and Shanghai Municipal

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Education Committee Key Laboratory of Molecular Imaging Probes and Sensors,

Shanghai Normal University, Shanghai 200234, China.

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* Corresponding author.
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E-mail address: wuhuixia@shnu.edu.cn (H.-X. Wu)

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Abstract

A multifunctional theranostic nanoplatform based on nanosized graphene oxide

(GO) and MnWO4 has been developed by in situ growth of MnWO4 nanoparticles

onto GO surfaces in a hyperthermia polyol medium containing polyethylene glycol

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(PEG). In comparison with GO and MnWO4/PEG, the near infrared (NIR) absorbance

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of the produced GO/MnWO4/PEG nanocomposite is significantly improved, resulting

in an enhanced photothermal conversion capability and a good photoacoustic (PA)

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imaging performance. It is worth to note that the longitudinal relaxivity r1 of

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GO/MnWO4/PEG reaches impressively 11.34 mM-1 s-1 in a 0.5 T magnetic field,
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which is significantly higher than ordinary Mn(II)-based T1 agents. In vivo magnetic

resonance and PA imaging demonstrate that GO/MnWO4/PEG could serve as an

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efficient bimodal contrast agent to guide the cancer treatment. GO/MnWO4/PEG

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shows a high loading capacity for anticancer drug doxorubicin hydrochloride (DOX)
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by noncovalent forces, and the drug release could be triggered by lower pH value and

external NIR light. Effective synergistic photothermo-chemotherapy has been

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performed in xenograft 4T1 tumor models after intravenous administration of

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GO/MnWO4/PEG/DOX. No obvious toxicity and side effect have been observed

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during in vivo treatment. These positive results suggest the promising application of

GO/MnWO4/PEG as a versatile nanoplatform for multimodal imaging-guided cancer

synergistic therapy.

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1. Introduction

As an emerging technique for cancer treatment, photothermal therapy (PTT) has

attracted increasing attention in recent years. PTT can specifically cause thermal

ablation of solid tumors with the help of near infrared (NIR) photothermal agents

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[1,2]. Compared with traditional treatment methods (such as surgery, radiotherapy,

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and chemotherapy), PTT provides patients with a more comfortable and acceptable

way to fight against cancer, because of its unique benefits including high selectivity,

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slight invasiveness, and minimal side effects [3]. With high optical absorbance in NIR

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region, photothermal agents can convert the absorbed light energy into heat to induce
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hyperthermia, which has been proved to be of higher toxicity to cancer cells in

comparison with normal cells [4,5]. To date, various photothermal agents have been
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reported to exhibit high light-to-heat conversion efficiency and good PTT efficacy
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both in vitro and in vivo. Among them, gold nanostructures [6-7], carbon
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nanomaterials [8-10], and organic nanoparticles (NPs) [11,12] have been extensively

studied. However, cancer treatment using only PTT is often hard to completely ablate
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tumors due to inhomogeneous heat distribution and might result in inevitable tumor
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recurrence [13]. For improving the treatment efficiency, an effective solution is to

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combine PTT with other therapeutic strategies by utilizing the merits of different

modalities. Among various combination therapies, photothermo-chemotherapy has

been considered as a promising strategy, which uses an integrated system capable of

delivering both photothermal and chemotherapeutic agents to the targeted tumor site

[14-16]. In this approach, the heat produced by photothermal agents can promote the

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drug release and enhance the drug intake of cancer cells, thus bringing in synergistic

therapeutic effect. As a single layer of graphite with ultrahigh surface area and good

biocompatibility, graphene oxide (GO) has been considered as a promising carrier for

loading drugs containing aromatic rings through π–π stacking and hydrophobic

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interaction [17]. Moreover, the typical structures of sp2 carbon atoms enable GO

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(especially reduced GO) to absorb NIR light and produce thermal energy for PTT [18].

Therefore, GO is able to serve as a favorable candidate for fabricating versatile

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platforms for efficient photothermo-chemotherapy.

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Nowadays, imaging-guided therapy strategy has become more and more important
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for precise cancer treatment, because it could identify the location and size of tumors,

monitor the accumulation of therapeutic agents at tumor site, and evaluate the
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treatment efficiency [19,20]. Up to now, the rapid development of tumor imaging

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technology still cannot guarantee to provide accurate and comprehensive diagnostic

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information only by single-modality imaging [21]. Hence, multimodal imaging

strategies has drawn much attention, as they may achieve complementary advantages
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of different imaging modalities and circumvent the shortcomings of each imaging

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technique [22,23]. As one of the most reliable clinical diagnostic tools, magnetic
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resonance imaging (MRI) has merits of good spatial resolution, high penetration depth,

and excellent soft tissue contrast [24]. Among the MRI contrast agents that have been

developed, T1 agents (such as Gd(III)-based [25] or Mn(II)-based [26] agents) which

induce positive contrast are superior to T2 agents with negative contrast, because

T1-weighted imaging can provide more accurate information of the target tissues due

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to less artifacts and higher signal intensity [27]. On the other hand, photoacoustic

imaging (PAI) is an emerging biomedical imaging technology that integrates the high

contrast of optical imaging with the good resolution of ultrasound imaging. PAI has

exhibited great superiority for the visualization of tissue structures and functions at

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the molecular level [28]. And moreover, PAI is proposed as the best guidance for PTT

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by using the same photosensitizer with strong absorption in NIR region [29]. Despite

the above-mentioned outstanding advantages, both imaging modalities have their own

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limitations. For example, MRI is suffering from low sensitivity [30], while PAI is

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limited by the insufficient penetration depth of light [31]. Therefore, the combination
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of MRI and PAI may offer more beneficial information for precisely guiding cancer

therapies [32,33].
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In this work, MnWO4 NPs were in situ grown on GO surfaces in a hyperthermia

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polyol medium containing polyethylene glycol (PEG) to produce the hydrophilic

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GO/MnWO4/PEG nanocomposite. MnWO4 NPs can act as an effective T1-weighted

MRI contrast agent with little in vivo toxicity [34], and importantly, after in situ
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growth of MnWO4 NPs on GO, the resultant GO/MnWO4/PEG shows a much higher
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longitudinal relaxivity r1 than bare MnWO4/PEG NPs. Reductive environment in the

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preparation process makes GO nanosheets partially reduced, and moreover, MnWO4

NPs also show photothermal property. These favorable factors endowed

GO/MnWO4/PEG with enhanced NIR absorption, outstanding PAI capability and

good photothermal property. Therapeutic agent doxorubicin hydrochloride (DOX)

was then loaded on GO/MnWO4/PEG via π-π interactions and electrostatic forces.

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The resulting GO/MnWO4/PEG/DOX formulation exhibited both pH and NIR light

responsive drug release behaviors. Finally, an 808 nm laser with a low power density

(0.6 W/cm2) was involved for in vivo treatment of 4T1 xenograft tumors after tail vein

injection of GO/MnWO4/PEG/DOX, and the effect of combined

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photothermo-chemotherapy was evaluated. The excellent dual-modal imaging

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contrast performance and combination therapy efficacy demonstrate the great

potential of GO/MnWO4/PEG/DOX as an appealing theranostic agent for efficient

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cancer diagnosis and treatment (Fig. 1).

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Fig. 1. Schematic diagram of GO/MnWO4/PEG nanocomposite for MRI/PAI and

photothermo-chemotherapy.

2. Experimental

2.1. Materials
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PEG (HOCH2(CH2OCH2)nCH2OH, Mw = 1500), natural graphite powder,

KMnO4, absolute ethyl alcohol, and 30% H2O2 were purchased from Sinopharm

Chemical Reagent Co., Ltd. Mn(NO3)2·4H2O, NaWO4·2H2O, 6-aminocaproic acid

(ACA), and triethylene glycol (TEG) were obtained from Shanghai Energy Chemical

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Co., Ltd. DOX was purchased from Alfa Aesar. Ultrapure water (resistivity > 18 MΩ

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cm) was used in all experiments. GO was prepared by a modified Hummers’ method

which used natural graphite powder as a start material.

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2.2. Characterization

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Transmission electron microscopy (TEM) images were obtained on a JEOL
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JEM-2100 high-resolution transmission electron microscope. Morphological analysis

was performed with a NT-MDT NETGRA atomic force microscopy instrument

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(AFM). Scanning electron microscopy (SEM) images were acquired with a Hitachi
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S-4800 scanning electron microscope. Fourier transform infrared (FT-IR) spectra

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were recorded on a Nicolet Avatar 370 FT-IR spectrophotometer with KBr pellets.

X-ray diffraction (XRD) patterns were taken using a Rigaku DMAX 2000 X-ray
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diffractometer. Surface Zeta potential and hydrated particle size were measured using
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a Malvern Zetasizer Nano ZS model ZEN3690. Visible-NIR (Vis-NIR) absorption

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measurements were carried out using a BeckMan coulter DU 730 spectrophotometer.

Thermogravimetric analysis (TGA) was carried out on a Rigaku DMAX 2000 thermal

analyzer in an air atmosphere at a heating rate of 10 °C/min. X-ray photoelectron

spectroscopy (XPS) spectra were measured on an RBD-upgraded PHI-5000C ESCA

system (Perkin Elmer) with Al/Kα radiation ( hν = 1486.6 eV).

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2.3. Synthesis of GO nanosheets

Natural graphite powder (1.0 g) and NaNO3 (0.5 g) were mixed with sulfuric

acid (98%, 25 mL), and the mixture was stirred under ice bath for 30 min. After that,

KMnO4 (5 g) was slowly added into the solution with extreme caution. After 3 days of

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stirring at ~ 4 oC in an ice bath, the mixture was first heated to 40 oC for 30 min, and

then to 80 oC for another 30 min. The final temperature was kept as 95 oC for 2 h.

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After heating process, H2O2 (30%, 30 mL) and water (80 mL) were added into the

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mixture, followed by natural cooling to room temperature. The solid was collected by

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centrifugation, washed first with 5% HCl solution and then with water, dispersed in
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water and exposed to sonication treatment for 3 h. Finally, the produced GO was

transferred into a dialysis bag to remove the adsorbed sulfate ions.

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2.4. Synthesis of GO/MnWO4/PEG

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The as-prepared GO (70 mg) was fully dispersed in TEG (30 mL), and
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Mn(NO3)2·4H2O (50 mg) was dissolved in another 30 mL of TEG. These two

solutions were mixed slowly and the pH value of the mixture was regulated to values
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of 5-6, followed by vigorous stirring. Na2WO4·2H2O (66 mg) dissolved in water (6

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mL) was then added to the above mixture at the speed of 0.1 mL/min. After stirring
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for an hour, the aqueous solution (2 mL) of ACA (113 mg) was introduced into the

reaction solution, which was then heated to 220 oC and allowed to react at this

temperature for 2 h. After that, PEG (500 mg) dissolved in TEG (10 mL) was added

and the solution temperature was kept at 220 oC for another 1 h before a naturally

cooling process. Magnetic stirring was used throughout the reaction. The solid was

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collected by centrifugation, washed alternately with water and ethanol for five times.

A small amount of free GO nanosheets and MnWO4 NPs that may be present can be

removed by controlling the centrifugation speed and time, because free GO

nanosheets and MnWO4 NPs are much lighter in weight in comparison with

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GO/MnWO4/PEG. The final product GO/MnWO4/PEG was fully dispersed in water

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by sonication for further use.

MnWO4/PEG was synthesized by the similar experimental procedure in the

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absence of GO nanosheets. GO/PEG was prepared under similar conditions without

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addition of Mn(NO3)2·4H2O and Na2WO4·2H2O.
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2.5. Photothermal experiments in solution

The GO/MnWO4/PEG nanocomposite suspended in water was diluted to

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different concentrations (25, 50, 75, 100, 150, and 200 µg/mL). Each sample was
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transferred to a quartz cuvette (1 cm × 1 cm) and illuminated at a laser power density

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of 0.6 W/cm2 for 15 minutes using an 808-nm NIR laser. The laser power was

detected by a power densimeter. The temperature was recorded on an infrared thermal

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camera system. Both original GO nanosheets and MnWO4/PEG NPs were also
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studied as contrast with the NIR laser (808 nm) power density fixed at 0.6 W/cm2.
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Photothermal stability of GO/MnWO4/PEG nanocomposite (150 µg/mL) was also

evaluated using the same NIR laser source (0.6 W/cm2).

2.6. Drug loading and release

DOX (5.0 mg) was added into the GO/MnWO4/PEG suspension (5.0 mL, 1.0

mg/mL). The above solution was allowed to incubate in the shaking bed at room

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temperature overnight. After that, GO/MnWO4/PEG/DOX was collected by

centrifugation and washed repeatedly with water until the supernatant became

colorless. The supernatant and washed solutions were then collected for measuring

absorption at 490 nm. The amount of unloaded DOX was calculated and subtracted

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from the overall DOX to quantify the actual amount of loaded DOX.

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For drug release study, phosphate buffer saline (PBS) solutions (2.0 mL, pH 7.4

and 5.2) of GO/MnWO4/PEG/DOX (5 mg) were sealed in dialysis bags (Mw cutoff =

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3500) and immersed in PBS buffer with the same pH value (8.0 mL, in glass

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reservoirs) at 37 oC under a shaking speed of 110 rpm min-1. At a given time point, 3.0
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mL of the solution outside the dialysis bag was taken out to determine the

concentration of released DOX using the UV–vis spectrophotometer (490 nm), and
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then returned to the original solution. Each experiment was repeated three times. For
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the study of the drug release induced by photothermal effect, the glass reservoirs were
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irradiated by an 808 nm laser for 10 min (0.6 W/cm2) at 1, 2, 4, and 6 h, and the

concentration of released DOX before and after laser irradiation was measured.
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2.7. Cell culture

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Human umbilical vein endothelial cells (HUVECs) were cultured in Roswell

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Park Memorial Institute 1640 medium, while the 4T1 cells were cultured in

Dulbecco’s Modified Eagle Medium (DMEM) with high glucose. Both mediums

contained 10% fetal bovine serum and 1% penicillin-streptomycin solution. These

two cell lines were incubated at 37 oC in a humidified atmosphere of 5% CO2. The

ethylene diamine tetraacetic acid solution containing 0.25% trypsin was used during

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subculture.

2.8. Cytotoxicity of GO/MnWO4/PEG and GO/MnWO4/PEG/DOX

The cell cytotoxicity test of GO/MnWO4/PEG was performed with both HUVEC

and 4T1 cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) assays. Cells were cultured in 96-well plates at the density of 5 × 103

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cells per well for 24 h before treated with 100 µL of as-prepared GO/MnWO4/PEG

samples (0, 25, 50, 100, 150, and 200 µg/mL). Cells were allowed to be incubated

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with the samples for 12 or 24 h followed by phosphate buffered saline (PBS) washing.

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MTT (5 mg/mL) dissolved in PBS was diluted by the medium in a ratio of 1:10 and
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added to the cells at the amount of 100 µL per well. After 4 h of incubation, dimethyl

sulfoxide was added and the absorbance at 490 nm was measured on a microplate
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reader (Multiskan MK3, USA).

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For cytotoxicity of GO/MnWO4/PEG/DOX, the nanocomposite was diluted to

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various drug concentrations (0, 2, 4, 8, 16, and 32 µg/mL) by DMEM. MTT assays

were carried out to measure the viabilities of 4T1 cells after incubation with the
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nanocomposite for 6 and 12 h.

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2.9. PTT and chemotherapy in vitro

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In PTT experiments of GO/MnWO4/PEG, 4T1 cells seeded in 96-well plates

were cultured for 6 to 12 h until cells covered 80% area of each well.

GO/MnWO4/PEG of different concentrations (0, 25, 50, 100, 150, and 200 µg/mL)

were added into the 96-well plates before 6 h of incubation. The cells were then

washed by PBS to remove the excess material and fresh medium was added. After

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exposure to the NIR laser (808 nm, 0.6 W/cm2) for 10 minutes, the cells were

incubated for another 1 h, followed by MTT assays.

As for combination therapy, GO/MnWO4/PEG/DOX was diluted by medium to

50 µg/mL (containing 2 µg/mL of DOX) and 100 µg/mL (containing 5 µg/mL of DOX)

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in concentration. 4T1 cells were incubated in 96-well plates and

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GO/MnWO4/PEG/DOX was added after 4T1 cells reached experiment conditions.

The incubation time is 6 h. Before MTT assays, 4T1 cells were exposed to NIR laser

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(808 nm, 0.6 W/cm2, and 10 min) for photothermo-chemotherapy treatment.

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2.10. Animals
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Female athymic nude mice (4-5 weeks) were used in our experiments. 4T1 tumor

models were established by subcutaneously injection of 4T1 tumor cells (5 × 104 cells
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in 100 µL of PBS) into the right flank of the mice. 3 to 7 days after injection, when
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tumor volumes reached about 100 mm3, 4T1 tumor models were already established
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for further use.

2.11. MRI in vitro and in vivo

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The relaxivity and MRI images of GO/MnWO4/PEG serial dilutions were

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acquired from a 0.5 T Niumag Imaging and Analyzing system NMI20-Analyst. The T
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values and MRI images of these solutions were measured with a conventional

spin-echo sequence sequentially (T1: repetition time (TR) = 500 ms, echo time (TE) =

200 ms; T2: TR = 4000 ms, TE = 100 ms). The concentrations of Mn element in the

samples were obtained by the inductively coupled plasma-atomic emission

spectroscopy (ICP-AES) and then fitted with 1/T values to calculate the r1 and r2

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relaxivities.

In vivo MRI was performed on a 3.0 T MRI system (3 T Siemens Magnetom

Trio). Animals were anesthetized with chloral hydrate and imaged as contrast. Then

PBS solution of GO/MnWO4/PEG (200 µL, 8 mg/mL) was injected intravenously into

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the animals. Thereafter, T1-weighted MRI images were recorded at 2 and 4 h post

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injection. The sequence parameters were TR 200 ms, TE 11 ms, acquisition matrix

320 × 100, flip angle 90o, slice thickness 1.5 mm, and field of view 80 × 60 mm.

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2.12. PAI in vitro and in vivo

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For PAI in vitro, 200 µL of GO/MnWO4/PEG solutions with different
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concentrations (0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 µg/mL) were sealed

into disposable pipette tips and then imaged by a PAI system for small animals
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(MOST inVision128, iThera Medical). The PAI signals at 808 nm of the samples were
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measured and fitted with the concentration to obtain the curve.

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PAI in vivo was performed with the same PAI system. Mice were anesthetized

with isoflurane flow and imaged as control. The PBS solution of GO/MnWO4/PEG
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(200 µL, 3 mg/mL) was then injected intravenously into the mice. The photoacoustic
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images of these mice were obtained at 2, 4, 6, 8, and 24 h after the injection.

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Thereafter, images were reconstructed using the small animal photoacoustic MSOT

system and signals of tumor sections were acquired.

2.13. Combination therapy in vivo

Tumor bearing mice were divided randomly into six groups (4 mice per group):

PBS injection treatment as control group, PBS injection plus laser irradiation (laser

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only group), free DOX injection only, GO/MnWO4/PEG plus laser irradiation,

GO/MnWO4/PEG/DOX only and GO/MnWO4/PEG/DOX plus laser irradiation.

GO/MnWO4/PEG (200 µL, 2 mg/mL), GO/MnWO4/PEG/DOX (200 µL, 3 mg/mL,

DOX 1 mg/mL), and free DOX (200 µL, 1 mg/mL) were injected into the 4T1

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tumor-bearing mice via tail vein. At the time points of 6 and 24 h after injection,

tumor region was irradiated for 10 minutes using an 808-nm laser (0.6 W/cm2).

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Thereafter, the body weight and tumor volumes of each group were monitored every

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two days for 12 or 16 days.

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For ex vivo analysis, tumors of control group (PBS injection only),
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GO/MnWO4/PEG plus laser irradiation group, GO/MnWO4/PEG/DOX only group

and GO/MnWO4/PEG/DOX plus laser irradiation group were removed for

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Hematoxylin-Eosin (H&E) and terminal deoxynucleotidyl transferase-mediated

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deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. The major

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organs (heart, liver, spleen, lung, and kidneys) were also harvested for histological

analysis.
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3. Results and discussion

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3.1. Characterization of GO/MnWO4/PEG

The original GO nanosheets were prepared from sonication treatment of

oxidized graphite obtained by a modified Hummers' method. The as-made GO

nanosheets are well dispersed in water and have a mean size of approximate 130 nm,

as indicated by the TEM image (Fig. 2a) and size distribution (Fig. 2b). FT-IR

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spectrum of GO is shown in Fig. 2c. GO nanosheets exhibit absorption bands at 1720

cm-1 and 1060 cm-1 to 1300 cm-1, characteristic of C=O and C-O functional groups,

respectively [35].

To prepare GO/MnWO4/PEG nanocomposite, a thermochemical method was

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employed, in which ACA was used as a capping agent to control the size of MnWO4

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NPs and PEG was added to improve the biocompatibility of the resultant

nanocomposite. TEM imaging of GO/MnWO4/PEG was first performed to confirm

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successful in situ growth of MnWO4 NPs on GO surfaces (Fig. 2d). The MnWO4 NPs

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with a length diameter of 11.7 ± 2.2 nm were firmly and uniformly attached to the GO
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sheets and the size of GO carriers are barely changed compared with the original

nanosheets, suggesting no obvious aggregation occurred during the preparation of the

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nanocomposite. As shown in the XRD pattern of GO/MnWO4/PEG (Fig. 2e), the

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diffraction peaks are verified to fit the standard monoclinic phase of MnWO4 (JCPDS
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no. 80-152) [36]. This confirmed that the deposited NPs on GO were MnWO4 NPs.

Fig. 2f is the high-resolution TEM (HRTEM) image of NPs. The crystal lattice is clear
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and the interplanar spacing is about 0.297 nm, which is assigned to the (111) lattice
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plane of monoclinic MnWO4 [36]. The surface topography of GO/MnWO4/PEG

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nanocomposite was further investigated by AFM, which clearly revealed that MnWO4

NPs were deposited on GO (Fig. 2g). The height profile analysis shows that the mean

thickness of GO/MnWO4/PEG is ~16.2 nm, while the thickness is only ~3.4 nm at the

edges of GO sheets where GO is not covered by the NPs (Fig. S1). SEM image (Fig.

S2) also demonstrates the successful deposition of MnWO4 NPs on GO surfaces.

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The zeta potential of initial GO nanosheets is about -41.8 mV, which is

conducive to the adsorption of Mn2+ ions for subsequent growth of MnWO4 NPs on

the surface of GO. After deposition of MnWO4 NPs, the zeta potential of

GO/MnWO4/PEG rises up to around -26.2 mV (Fig. S3). In the FT-IR spectrum of

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GO/MnWO4/PEG (Fig. 2c), several new absorbance peaks appear at 2922, 2858,

2822, and 1120 cm-1, due to the presence of ACA and PEG in the nanocomposite

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[37,38]. The weight ratio of GO to MnWO4 was estimated by TGA (Fig. 2h) of

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GO/MnWO4/PEG, MnWO4/PEG, and GO/PEG. For GO/PEG, when the temperature

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was increased to ~665 oC, the adsorbed organic molecules and GO nanosheets were
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completely burned out, leaving only a small amount of residue (~1.5%).

MnWO4/PEG exhibited about 27.5% of weight loss before 360 oC due to the removal
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of surface absorbed water and the decomposition of ACA and PEG, and no obvious
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weight loss was observed when the temperature continued to rise, indicative of a good
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thermal stability of MnWO4 NPs. In the TGA curve of GO/MnWO4/PEG, the total

weight loss percentage is 40.4%, indicating that the weight percentage of MnWO4 in
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the nanocomposite is ~59.6%. By comparing the weight loss curve of

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GO/MnWO4/PEG with that of MnWO4/PEG and GO/PEG, the weight percentage of

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GO in GO/MnWO4/PEG is estimated to be about 13.0%. The chemical state of the

main elements in GO/MnWO4/PEG was identified by XPS analysis (Fig. 2i, Fig. S4).

The survey XPS spectrum of GO/MnWO4/PEG demonstrates the presence of Mn, W,

C, O, and N elements, and the Mn : W molar ratio was close to 1 : 1. In the Mn 2p

XPS spectrum (Fig. S4a), two peaks are observed at 640.5 eV (Mn 2p3/2) and 653.0

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eV (Mn 2p1/2), and an additional satellite peak is also found at 646.0 eV, revealing the

oxidation state of Mn2+ [39]. The deconvoluted XPS spectra for W 4f (Fig. S4b) show

two peaks at 35.1 eV (W 4f7/2) and 37.2 eV (W 4f5/2) with a 2.1 eV spin–orbit

coupling, indicating the presence of W in the +6 oxidation state [39]. The XPS spectra

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of O 1s (Fig. S4c) can be deconvoluted into four peaks at 530.1, 531.5, 532.8 and

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534.0 eV, which can be assigned to Mn–O–W (MnWO4), C–O–C (PEG), -COOH

(ACA, GO) / -COH (PEG, GO) groups, and surface hydration, respectively [40,41].

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The XPS spectra of C 1s (Fig. S4b) consist of four XPS peaks at 284.8, 285.4, 286.3,

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and 288.1 eV arising from Csp2 (GO) / C–C (ACA, PEG), C–N (ACA) / C–OH
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(PEG), C–O (PEG, GO), and C=O (ACA, GO) groups, respectively [40,42,43]. The

above characterizations demonstrate the successful preparation of GO/MnWO4/PEG

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nanocomposites and the presence of PEG in the nanocomposite. MnWO4 NPs are
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mainly non-covalently deposited on GO surface, and some Mn2+ ions of MnWO4 NPs
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may coordinate with oxygen-containing groups on GO surface. PEG may covalent

link to MnWO4 NPs which grow on the surface of GO by the coordination between
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oxygen atoms of PEG and Mn or W ions on MnWO4 surface. PEG may also
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non-covalently link to GO by hydrophobic interaction between the –CH2–CH2– group

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and GO surface or by hydrogen bonding interactions between PEG and residual

oxygen-containing functional groups on GO.

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D
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Fig. 2. Characterization of GO and GO/MnWO4/PEG. (a) TEM image of GO

nanosheets. Scale bar = 200 nm. (b) Size distribution of GO nanosheets. (c) FT-IR
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spectra of GO, PEG, and GO/MnWO4/PEG. (d) TEM image of GO/MnWO4/PEG. (e)
C

XRD patterns of GO/MnWO4/PEG. (f) HR-TEM image of GO/MnWO4/PEG. (g)

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AFM image of GO/MnWO4/PEG. (h) TGA weight loss curves of MnWO4/PEG,

GO/MnWO4/PEG, and GO/PEG. (i) The survey XPS spectra of GO/MnWO4/PEG.

3.2. Photothermal capabilities in vitro

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It is clear that the photothermal capability is of vital importance for a

photothermal agent. Before investigating the photothermal behavior of

GO/MnWO4/PEG, the UV-vis-NIR absorptions of the nanocomposite with various

concentrations were measured. GO/MnWO4/PEG has a strong absorption in near UV

PT
region mainly due to charge-transfer transition between oxygen 2p orbits and empty d

RI
orbits of the central W ion in MnWO4 [36]. Hence, only the absorptions in the

wavelength range from 500 to 1000 nm are presented in Fig. 3a. The absorption

SC
spectra from 190 to 1000 nm for GO/MnWO4/PEG with a low concentration (5

U
µg/mL) is shown in Fig. S5. GO/MnWO4/PEG showed significant absorbance in the
AN
NIR region (Fig. 3a) and the absorbance rose with the increase of material

concentration. When exposed to an 808 nm laser with a power density of 0.6 W/cm2,
M

GO/MnWO4/PEG exhibited significant temperature increase with a

D

concentration/time-dependent manner, while pure water showed little change. The

TE

high NIR absorbance of GO/MnWO4/PEG resulted in a temperature increase of 26.5

o
C for GO/MnWO4/PEG solution (200 µg/mL) under the NIR laser irradiation for 10
EP

min (Fig. 3b).

C
AC

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Fig. 3. Photothermal studies of GO/MnWO4/PEG in aqueous solution. (a) Vis-NIR

D

absorptions of GO/MnWO4/PEG solutions with various concentrations. (b)

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Temperature increase curves of GO/MnWO4/PEG solutions exposed to an 808 nm

EP

laser with a power density of 0.6 W/cm2 for 15 min. (c) Vis-NIR absorbance of GO

nanosheets and MnWO4/PEG (200 µg/mL). (d) Temperature increase curves of GO

C
AC

and MnWO4/PEG solutions (200 µg/mL) under the same irradiation conditions.

To find out reasons for enhanced photothermal performance of GO/MnWO4/PEG,

we measured the absorption spectrum and photothermal properties of GO nanosheets

and MnWO4/PEG NPs (Fig. 3c and d). The absorption spectra of these two samples

from 190 to 1000 nm can be found in Fig. S5. The photothermal heating curve of GO

aqueous solution was studied under the irradiation of an 808 nm laser with power
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intensity of 0.6 W/cm2 for 10 min. As a result of poor absorbance in NIR region, a

temperature increase of only about 7 oC was caused by GO with a concentration of

200 µg/mL. This is much lower than the temperature change that GO/MnWO4/PEG

can reach. At the same concentration (200 µg/mL), MnWO4/PEG exhibited a slightly

PT
higher NIR absorption than GO, and it caused a temperature rise of about 9 oC under

RI
the same irradiation conditions, still significantly lower than that induced by

GO/MnWO4/PEG (200 µg/mL). Therefore, neither GO nanosheets nor MnWO4/PEG

SC
NPs could be responsible for the high temperature increase of GO/MnWO4/PEG

U
under NIR laser irradiation. As a matter of fact, the preparation of GO/MnWO4/PEG
AN
was carried out using TEG as solvent at a high reaction temperature (220 oC) in a

nitrogen atmosphere. The reductive environment resulted in partially deoxidation of

M

the GO carriers, as indicated by the color change of the mixture during reaction
D

(turning black) and the significant enhancement of NIR absorbance for

TE

GO/MnWO4/PEG. In the UV-vis spectrum of GO (Fig. S5), an absorption band is

found at ~225 nm, corresponding to the π–π∗ transition of the C=C bond of GO. A
EP

redshift of this peak will be observed when GO is reduced in the reductive

C

environment, due to the restoration of the conjugated system. The as-prepared

AC

GO/MnWO4/PEG exhibits an absorption band at ~250 nm, which appears at ~268 nm

for the reduced GO prepared by using hydrazine or vitamin C as reducing agent [42],

so we believe that GO nanosheets in GO/MnWO4/PEG is partially reduced. This is

favorable for the hydrophilicity of the material. Therefore, the significant

enhancement of photothermal heat generation by GO/MnWO4/PEG was mainly due

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to deoxidation of GO during the growth of MnWO4 NPs under reductive environment

[44].

For a photothermal agent, the heat stability and light conversion efficiency play

key roles in its functions. Herein, the photothermal stability of GO/MnWO4/PEG was

PT
evaluated by exposing an aqueous solution of the material (150 µg/mL) to NIR laser

RI
irradiation for several laser on/off cycles. During eight cycles of heating and cooling,

the temperature variation curves remained basically the same with repetitive heating

SC
and cooling (Fig. S6). Moreover, GO/MnWO4/PEG was able to maintain its colloidal

U
stability in aqueous solutions after 8 cycles of monitoring, and the Vis-NIR absorption
AN
of the solution barely changed before and after 8 cycles of laser on/off (Fig. S7).

These results indicate that GO/MnWO4/PEG is a stable photothermal material. In

M

contrast to GO/MnWO4/PEG, some photothermal agents such as gold nanorods [45]

D

and organic photosensitizers (e.g., indocyanine green) [46] showed poor stability
TE

under NIR laser treatment. Additionally, the photothermal-conversion efficiency (η)

of GO/MnWO4/PEG was measured using the reported method [47] (Fig. S8). The
EP

temperature change of a GO/MnWO4/PEG solution versus time was recorded under

C

continuous irradiation (808 nm) until a constant temperature was observed and then
AC

natural cooldown to room temperature by turning off the laser. By plotting cooling

time versus negative natural logarithm of the temperature driving force obtained from

the cooling stage, the photothermal efficiency of GO/MnWO4/PEG was calculated to

be 30.6%, being higher than those of Cu2-xSe NPs (22.0%) [48] and iron/iron oxide

core/shell NPs (~20%) [49], but lower than those of biodegradable Au nanovesicles

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(37%) [50] and cysteine-coated CuS NPs (38.0%) [51]. Hence, according to the above

photothermal experiments, GO/MnWO4/PEG was demonstrated to be an ideal

photothermal agent holding high photothermal conversion capability and good

photostability.

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3.3. MRI and PAI in vitro

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GO is a desirable platform for loading inorganic NPs with theranosis functions to

construct promising multifunctional nanomaterials. With successful growth of

SC
MnWO4 NPs, GO/MnWO4/PEG nanocomposite exhibits excellent capability to serve

U
as MRI contrast agents. The relaxivity of the material were measured at 0.5 T both in
AN
T1 and T2 modes (Fig. 4a). The longitudinal relaxivity r1 was calculated to be 11.34

mM-1 s-1, and the transverse relaxivity r2 was 24.06 mM-1 s-1. In contrast, the r1 value
M

of MnWO4/PEG is only 1.07 mM-1 s-1 (Fig. S9). Moreover, such an r1 value of
D

GO/MnWO4/PEG is also several times larger than normal Mn-based materials

TE

reported in the literature. For instance, glycine-capped MnWO4 nanorods were

reported and the relaxivity value r1 was tested to be 0.88 mM-1 s-1 [34], while the r1
EP

value of Mn-PPC materials was reported to be 3.7 mM-1 s-1 [52]. In our work, the
C

loading of several MnWO4 NPs on one GO nanosheet will lead to the local
AC

concentration increase of surface Mn2+ and the rotation speed decrease of Mn-based

NPs, favorable for reducing the relaxation time of water. Moreover, the large surface

to volume ratio of GO/MnWO4/PEG will also enhance the synergistic effect of Mn2+

ions and speed up the r1 relaxation process [53-55]. The high r1 value of

GO/MnWO4/PEG also suggests that the contact of surface Mn2+ ions with water is

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rarely hindered after the NPs were deposited on GO. The r2/r1 ratio was about 2:1,

which indicated that GO/MnWO4/PEG was more favorable to serve as a T1-weighted

contrast agent. Fig. 4b shows the T1-weighted images of GO/MnWO4/PEG solutions

at 0.5 T. Similar with relaxivity curves in Fig. 4a, the MRI images exhibited a Mn2+

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concentration dependent brightening manner, further confirming the excellent

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positive-enhanced effect of GO/MnWO4/PEG solutions in MRI.

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M
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Fig. 4. MRI and PAI of GO/MnWO4/PEG in aqueous solutions. (a) Plot of 1/T1 and
EP

1/T2 versus concentration of Mn. (b) T1-wighted MRI images of GO/MnWO4/PEG at

C

different Mn concentrations. (c) Plot of PAI signal intensity versus concentration of

AC

GO/MnWO4/PEG. (d) PAI images of GO/MnWO4/PEG at different concentrations.

PAI is an emerging noninvasive imaging technology based on the photoacoustic

effect of NIR laser absorbers. PAI has attracted extensive interest because of its

reasonable penetration depth and good spatial resolution [28]. In addition to MRI, PAI

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was supposed to be another imaging mode of GO/MnWO4/PEG due to the strong

absorption in NIR region and high photothermal transduction capability. To

investigate the potential of GO/MnWO4/PEG as a PAI contrast agent, we examined

the PAI signals generated by the solutions of the nanocomposite (from 0 to 200 µg/mL)

PT
using a PAI system. As indicated in Fig. 4c and d, the PAI signal of GO/MnWO4/PEG

RI
was found to rise up as the concentration increased. Moreover, the PAI signals

produced by GO/MnWO4/PEG seems like to be linearly dependent on the material

SC
concentration, especially at low material concentration (Fig. 4c), suggesting the high

U
photoacoustic contrast potential of this material. The above in vitro MRI and PAI
AN
results demonstrate that the GO/MnWO4/PEG nanocomposite has promising potential

to serve as an appealing MRI/PAI dual-modal contrast agent.

M

3.4. Drug loading and release of GO/MnWO4/PEG

D

Besides a PTT agent, the GO/MnWO4/PEG nanocomposite can also act as a

TE

vehicle for drug delivery. The large specific surface area, extended π–π conjugated

system of GO, and negative surface potential endow GO/MnWO4/PEG with a high
EP

capacity for aromatic drug loading via π–π stacking and electrostatic interactions [17].
C

Herein, a popular anticancer drug DOX was used as a model chemotherapeutic agent
AC

to evaluate the drug loading and release behavior of GO/MnWO4/PEG. The drug

loading capacity of the nanocomposite, which was determined using the absorption of

DOX at 490 nm, increases with the increase of initial drug concentration. The

saturated loading capacity of DOX was determined to be about 550 mg/g (Fig. S10).

Although some GO surface has been occupied by MnWO4 NPs, the nanocomposite

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still showed a high drug loading capacity. The initial Zeta potential of

GO/MnWO4/PEG was –26.2 mV, and it increased to –5 mV after adsorption of DOX

(550 mg/g). Although the absolute value of the Zeta potential is relatively small,

GO/MnWO4/PEG/DOX still shows good colloidal stability in PBS buffer, DMEM

PT
medium, and FBS (Fig. S11). This is mainly due to PEG modification, good

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hydrophilicity and small size of GO/MnWO4/PEG.

As early reported, the micro-environment of tumor tissues tended to be slightly

SC
acid compared with normal tissues [56]. In view of this, we monitored the drug

U
release of the GO/MnWO4/PEG/DOX nanosystem at pH 5.2 and 7.4, respectively. As
AN
shown in Fig. 5a, the release of DOX in slightly acidic environment (28.1%) is

considerably larger than that in the weak alkaline solutions (12.4%). This
M

demonstrated that little drug leakage would be caused during the transport of
D

GO/MnWO4/PEG/DOX under normal physiological conditions. In addition to pH,

TE

laser treatment was also found to enhance the DOX release (Fig. 5b). The drug loaded

nano-carriers under laser illumination at pH 5.2 experienced the most drastic drug
EP

leakage (35.7%) within 370 min. Besides, both two laser treatment groups undertook
C

an increasing trend compared with non-laser groups. Among these four groups, the pH
AC

= 7.4 group without laser illumination showed the least drug release. All these results

demonstrate the superior drug loading and releasing abilities of GO/MnWO4/PEG

nanocomposite and suggest the potential use of the material as drug delivery vehicles.

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PT
RI
Fig. 5. Drug release of GO/MnWO4/PEG. (a) durg release curves of

SC
GO/MnWO4/PEG/DOX in PBS solutions with different pH values (pH 5.2 and 7.4).

U
(b) drug release curves of GO/MnWO4/PEG/DOX in different pH solutions with and
AN
without laser irradiation (808 nm, 0.6 W/cm2).
M

3.5. Cytotoxicity, PTT and chemotherapy in vitro

D

Safety and toxicity of the nanocarriers are major concerns when they were used
TE

for biomedical applications. We accessed the in vitro toxicity of GO/MnWO4/PEG in

4T1 and HUVEC cell lines utilizing a standard MTT assay. As shown in Fig. 6a,
EP

pretty low cytotoxicity of GO/MnWO4/PEG was observed after incubation with 4T1
C

cancer cells. Similarly, no significant toxicity was found in HUVECs incubated with
AC

the material (Fig. 6b). Even at a large concentration of 200 µg/mL, more than 90% of

4T1 cancer cells and 80% of HUVECs were still viable after 12 and 24 h of

incubation. This suggests the good bio-compatibility of GO/MnWO4/PEG in vitro.

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PT
RI
Fig. 6. Cytotoxicity, PTT, and chemotherapy in vitro. (a) Cell viabilities of 4T1 cells

SC
after incubated with GO/MnWO4/PEG at concentrations of 0, 25, 50, 100, 150, 200

U
µg/mL for 12 and 24 h. (b) Cell viabilities of HUVECs after incubated with different
AN
concentration of the material for 12 and 24 h. (c) Relative survival rate of 4T1 cells

after incubation with different concentrations of GO/MnWO4/PEG for 6 h and then

M

exposed to an NIR laser. (d) Relative survival rate of 4T1 cells after incubation with
D

GO/MnWO4/PEG/DOX at DOX concentrations of 0, 2, 4, 8, 16, and 32 µg/mL for 6

TE

and 12 h. (e) Cell viabilities of 4T1 cells after incubated with GO/MnWO4/PEG, DOX,

and GO/MnWO4/PEG/DOX for 6 h, and then treated with or without laser irradiation.
EP

***p < 0.001. The laser used above is 808 nm in wavelength, 0.6 W/cm2 in power
C

density, and 10 min in exposure time.

AC

Before evaluating the efficacy of combination therapy, we carried out parallel

experiments to select proper laser power density. For choice of laser power density,

4T1 cells were incubated with the nanomaterial (200 µg/mL) and then exposed to the

NIR laser with different densities (0.2, 0.4, and 0.6 W/cm2). After that, the cell

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viabilities were tested by MTT assays (Fig. S12). The cell inhibition rate increases

significantly as the power density increased, and less than 20% of cells were found

alive when exposed to a laser at 0.6 W/cm2. Next, 4T1 cells were incubated with

different concentrations of GO/MnWO4/PEG and then suffered the laser irradiation at

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0.6 W/cm2 (Fig. 6c). The cell viabilities decreased with the increase of nanomaterial

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concentration. At a material concentration of 100 µg/mL, about 49% of the cancer

cells were killed after laser irradiation. As for drug dose, GO/MnWO4/PEG/DOX with

SC
0, 2, 4, 8, 16, and 32 µg/mL of DOX was added and incubated with 4T1 cells for 6

U
and 12 h (Fig. 6d). When DOX concentration reached 4 µg/mL, the cell viability after
AN
6 and 12 h of co-incubation dropped to about 66% and 28%, respectively, indicating a

significant anti-cancer activity at this drug concentration. In the subsequent research,

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the drug concentration was controlled as 2 and 5 µg/mL for GO/MnWO4/PEG/DOX

D

solutions.
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For combination treatment, 4T1 cells were incubated with different

concentrations of GO/MnWO4/PEG, DOX, and GO/MnWO4/PEG/DOX for 6 h, and

EP

then treated with or without a NIR laser (808 nm, 0.6 W/cm2, 10 min). As shown in
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Fig. 6e, GO/MnWO4/PEG treatment only or laser irradiation only caused negligible
AC

cell death, while GO/MnWO4/PEG treated cells under laser exposure exhibited

notably increased death rate with the increase of material concentration. As the drug

concentration increased (laser off), it was found that the cancer cell killing efficiency

of GO/MnWO4/PEG/DOX was significantly improved, and meanwhile, the

cytotoxicity of the DOX itself was not affected by NIR laser exposure. In contrast to

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the groups of GO/MnWO4/PEG + laser and GO/MnWO4/PEG/DOX, the cells treated

with both GO/MnWO4/PEG/DOX and laser irradiation showed remarkably reduced

viabilities. The cells incubated with 100 µg/mL of GO/MnWO4/PEG/DOX

(containing 5 µg/mL of DOX) and then exposed to laser irradiation showed the

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highest mortality rate (about 90%) in comparison with GO/MnWO4/PEG/DOX or

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GO/MnWO4/PEG + laser groups with similar conditions (P < 0.001). This indicated

the excellent performance of GO/MnWO4/PEG/DOX in combination therapy of PTT

SC
and chemotherapy. The efficiency of combination therapy is much higher than that of

U
PTT by GO/MnWO4/PEG and that of chemotherapy by GO/MnWO4/PEG/DOX,
AN
indicative of significant synergistic effect. This may be attributed to two main reasons.

On one hand, GO/MnWO4/PEG is an effective NIR photothermal agent that can

M

generate heat upon NIR laser irradiation to kill the cancer cells. On the other hand, the
D

elevated temperature could induce increased cellular uptake of

TE

GO/MnWO4/PEG/DOX and meanwhile accelerate the release of DOX to enhance the

drug toxicity [14,57].

EP

3.6. MRI and PAI of GO/MnWO4/PEG in vivo

C

Since GO/MnWO4/PEG showed excellent MRI contrast performance in aqueous

AC

solutions, it is necessary to further assess MRI of tumor xenografts using the material.

4T1 tumor models were established, and when the tumor reached a proper size, the

mice were anesthetized and imaged with a 3T MRI scanner as control. A PBS solution

of GO/MnWO4/PEG (200 µL, 8 mg/mL) serving as the contrast agent was then

intravenously injected into the mice for T1-weighted tumor MRI. Fig. 7a reveals that

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the tumor region appeared brighter in a time-dependent manner after injection. At 4 h

post-injection, the contrast enhancement of tumor region was clear, whereas the

muscle tissue of the mice showed no significant changes. It was also observed from

Fig. 7b that the quantitative signal values at tumor regions significantly increased

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from 87.5 ± 0.4 to 124.9 ± 4.2 at 4 h post-injection (P < 0.001). Furthermore, the ratio

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of signal values between tumor and muscle raised from 1.2 : 1 (pre-injection) to 1.6 :

1 (4 h post-injection), owing to the good T1-weighted contrast enhancement of

SC
GO/MnWO4/PEG. Based on the enhanced permeation and retention (EPR) effect,

U
GO/MnWO4/PEG successfully accumulated in the tumor regions and resulted in
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distinct positive contrast-enhanced MRI [27].
M
D
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C EP
AC

Fig. 7. MRI and PAI in vivo. (a) T1-weighted longitudinal section MRI images of a

4T1-tumor bearing mouse before and at different time (2 and 4 h) after intravenous

injection with GO/MnWO4/PEG. Dotted red circles refer to the tumor region. (b) Grey

values of tumor and muscles collected from the small animal MRI software. (c) PAI

of a 4T1-tumor bearing mouse injected with GO/MnWO4/PEG at different time.

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Dotted red circles refer to the tumor sites. Color bar of the intensity is from low (blue)

to high (red). (d) Mean photoacoustic intensity of the tumor sites calculated from the

software of PAI machine. ***p < 0.001. NS: No significant difference.

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On the other hand, GO/MnWO4/PEG has been demonstrated above to generate

RI
strong photoacoustic signal in vitro. We then performed in vivo PAI research using the

mouse 4T1 tumor model with a small animal PAI system. 4T1 tumor-bearing mice

SC
were scanned 2, 4, 6, 8, and 24 h after tail-vein injection of GO/MnWO4/PEG (200

U
µL, 3 mg/mL). Control images were recorded pre-injection and isoflurane was
AN
utilized during the test. As shown in Fig. 7c and d, significantly increased

photoacoustic signal was observed in the tumor sites at 2, 4, 6 h post-injection,

M

compared to the signal level of pre-injection. This is mainly attributed to the strong
D

NIR absorption of GO/MnWO4/PEG nanocomposite, which was enriched in the

TE

tumor site due to the EPR effect. At 6 h after injection, maximum photoacoustic signal

was observed at tumor region, and then the photoacoustic signal started to drop. This
EP

suggests that the enrichment of GO/MnWO4/PEG at tumor site reached the maximum
C

extent and then gradually decreased due to the clearance of the material in vivo. The
AC

photoacoustic signal still maintained to be 1.4 times that of pre-injection at 24 h

post-injection. The positive results demonstrate the good contrast enhancement effect

of GO/MnWO4/PEG for PAI in vivo. Therefore, GO/MnWO4/PEG nanocomposite

could act as a promising dual-modal MRI/PAI contrast agent to provide more accurate

information for guiding cancer therapy.

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3.7 Photothermo-chemotherapy in vivo

The exciting results of in vitro combination therapy encourage us to further

assess the in vivo therapeutic effect of combined PTT and chemotherapy in the 4T1

xenograft mice model. According to the in vivo PAI study, the gathering of

PT
GO/MnWO4/PEG in the tumor site reached the maximum at 6 h post injection, and at

RI
24 h, there was still a certain amount of material accumulation. The bio-distribution of

Mn element quantitatively analyzed by ICP-AES method (Fig. S13) also confirms the

SC
significant intra-tumor remaining of GO/MnWO4/PEG at 24 h due to the EPR effect.

U
To figure out suitable time points for PTT in vivo, we further conducted in vivo
AN
photothermal imaging study. 4T1 tumor bearing mice were intravenously injected

with the material (2 mg/mL, 200 µL) and followed by 808 nm laser irradiation (0.6
M

W/cm2) for 10 min at 6 and 24 h post-injection. An IR thermal mapping camera was

D

utilized to take photos and monitored the temperature change of the tumor sites. At 6
TE

h after the injection, the temperature of tumor sites rapidly increased by 27 oC under

laser irradiation for 5 min (Fig. 8a and b). Such heat increase was high enough to
EP

induce hyperthermia for damage tumors and trigger drug release as well [14]. By
C

contrast, only a small temperature increase was observed for the PBS-treated group.
AC

Notably, the tumor region still exhibited a large increase in temperature (17 oC) when

it was exposed to laser irradiation for 5 min at 24 h post-injection. This is due to the

light-to-heat conversion induced by the remaining GO/MnWO4/PEG nanocomposite

in the tumor, as indicated by the ICP results. The high temperature improvement

revealed the considerable passively tumor-targeted accumulation of GO/MnWO4/PEG

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nanocomposite and the excellent photothermal efficiency of the material in vivo [58].

In the following PTT studies, we performed laser treatment twice at 6 and 24 h

post-injection to get the best therapeutic efficiency.

PT
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M
D
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C EP
AC

Fig. 8. Photothermo-chemotherapy in vivo. (a) Photothermal images of 4T1 tumor

bearing-mouse intravenously injected with PBS (6 h post-injection, as control) and

GO/MnWO4/PEG (6 and 24 h post-injection) under laser irradiation (808 nm, 0.6

W/cm2). The injection dose of GO/MnWO4/PEG (200 µL) is 20 mg per kg body

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weight. (b) Time-dependent temperature change of tumor sites in (a). (c) Relative

tumor volume change during the treatments in six groups. * p < 0.05. NS: No

significant difference. (d) Typical photos of 4T1 tumor bearing-mice before (up) and

after (down) GO/MnWO4/PEG/DOX + laser treatment. (e) Representative H&E and

PT
TUNEL stained images of the dissected tumors after various treatments: group A

RI
(mice only injected with PBS, as control), group B (GO/MnWO4/PEG/DOX), group

C (GO/MnWO4/PEG + laser), and group D (GO/MnWO4/PEG/DOX + laser).

U SC
In combination therapy research, the mice were divided into six groups including
AN
the control group (PBS injected only), laser irradiation only group, free DOX group,

GO/MnWO4/PEG/DOX group, GO/MnWO4/PEG plus laser irradiation group (PTT

M

group), and GO/MnWO4/PEG/DOX plus laser irradiation group (PTT +

D

chemotherapy group). As shown in Fig. 8c (The change of actual tumor volume is

TE

presented in Fig. S14), the tumors of the control group and laser irradiation only

group show similarly rapid growth, revealing that laser irradiation itself could not
EP

inhibit the tumor growth. For Free DOX group, the tumor is only slightly inhibited
C

and continues to grow. The tumor volumes of GO/MnWO4/PEG/DOX group and PTT
AC

group are relatively smaller than control group due to chemo-toxicity or

photo-toxicity induced by material. However, only PTT or chemotherapy is incapable

to completely inhibit the growth of tumor. PTT group showed a better treatment effect

than GO/MnWO4/PEG/DOX group, while it suffered from tumor recurrence in 12th

to 16th days despite that the PTT effect of GO/MnWO4/PEG is much obvious (Fig.

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8c). By contrast, mice (PTT + chemotherapy) subjected to both drug-loaded material

and laser irradiation showed the best results of tumor suppression (*P<0.05). The

solid tumor basically disappeared, leaving only black scab in the original tumor site

(Fig. 8d). After second laser irradiation treatment, H&E and TUNEL staining of the

PT
tumor sections from the control group and three GO/MnWO4/PEG-involved groups

RI
were carried out to further assess the tumor damage (Fig. 8e). Compared with control

group, the tumor slices of GO/MnWO4/PEG/DOX group and PTT group shows partly

SC
cell pleomorphic nuclei and pyknosis due to the medication or laser treatment. In

U
contrast, infiltrating tumor cells with highly pleomorphic nuclei and significant dark
AN
brown in TUNEL staining (suggesting broken DNA chains) was observed in tumor

sections of combined therapy group, indicating the excellent effectiveness of

M

drug/laser combination therapy using GO/MnWO4/PEG/DOX.

D

To assess the in vivo toxicity of nanomaterials and the side effects of the
TE

therapies, the relative weight of each group was monitored during the treatments. The

mice in combined therapy group showed no apparent weight changes in the whole
EP

monitoring process (Fig. S15). After the treatments, the mice tissue slices of main
C

organs including heart, liver, spleen, lung, and kidney from the control group and
AC

GO/MnWO4/PEG-involved groups were stained by H&E for histological analysis. No

obvious cell damage or inflammation was observed in the tissue sections (Fig. S16).

Taken together, GO/MnWO4/PEG is a promising PTT agent and drug carrier for

synergistic photothermo-chemotherapy in vivo with low side effect.

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4. Conclusions

In summary, GO/MnWO4/PEG nanocomposite was prepared by in situ growth of

MnWO4 NPs onto GO surface in the presence of ACA and PEG. Owing to Mn-based

inorganic particles and the strong NIR absorption, GO/MnWO4/PEG exhibited

PT
excellent MRI/PAI capabilities and promising photothermal properties. In addition,

RI
GO/MnWO4/PEG is an efficient carrier for drug delivery, and the release of loaded

DOX could be triggered by lower pH value and external NIR light. Both in vitro and

SC
in vivo studies demonstrate that the combined photothermo-chemotherapy using

U
GO/MnWO4/PEG/DOX showed a better efficacy than either drug delivery or
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hyperthermia alone. Combination therapy induced by the intravenously injected

GO/MnWO4/PEG/DOX can completely ablate the 4T1 tumors, indicative of a good

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synergistic antitumor effect in vivo. Such multifunctional GO-based nanocomposite is

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undoubtedly a promising platform for multimodal imaging-guided synergistic

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combination therapy.
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Acknowledgements
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This work is supported by the Environmental Functional Materials Innovation Team

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of Ministry of Education (IRT_16R49), the National Natural Science Foundation of

China (Grant No. 21671135), and the International Joint Laboratory on Resource

Chemistry (IJLRC).

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