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10.1039/C7NR08747K.

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Paper

Nano-graphene oxide-manganese dioxide nanocomposites for

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overcoming tumor hypoxia and enhancing cancer radioisotope

Received 00th January 20xx,
Accepted 00th January 20xx
therapy
Yugui Taoa, Longlong Zhua,b, Yunayuan Zhaod, Xuan Yib,c, Longbao Zhua, Fei Gea, Xiaozhou Moud*,

Nanoscale Accepted Manuscript

DOI: 10.1039/x0xx00000x
Lei chenb,c*,Liang Sunb, Kai Yangb*
www.rsc.org/

While radiotherapy (RT) is commonly used in clinic for cancer treatment, the therapeutic efficiency is not satisfactory
owing to the existence of the hypoxia tumor microenvironment which seriously affect the efficiency of RT. Herein, we
design polyethylene glycol (PEG) modified reduced nano-graphene oxide-manganese dioxide (rGO-MnO2-PEG)
nanocomposites to trigger oxygen generation from H2O2 to reduce the tumor hypoxic microenvironments. We use
radioisotope, 131I labeled rGO-MnO2-PEG as therapeutic agents for in vivo tumor radioisotope therapy (RIT), achieving
excellent tumor killing and further enhancing the therapeutic efficiency of RIT. More importantly, dissolution of MnO2 in
acidic condition and the redox process during catalytic pathway of H2O2 decomposition in cellular microenvironment direct
to the production of enormous amount of Mn2+ which has been used as contrast agents for magnetic resonance imaging
(MRI). Our proposed work provides a strategy to trigger oxygen formation via internal stimulus to enhance imaging guided
RIT efficiency.

In our previous work, it was found that mild heating could

Introduction
Hypoxia, severe oxygen deficiency, is widespread in the solid
tumors and plays an important role in tumor invasion and
metastasis.1-3 It is also known that tumor hypoxia is closely
related with the advanced stages of malignancy.4-6 Owing to the
limited oxygen supply, hypoxic tumor often exhibits the
resistance to the conventional therapies such as chemotherapy
and radiotherapy7, inducing the unsatisfactory or failure of
treatments.8-14 While radiotherapy including RT and internal
radioisotope therapy (RIT) 15 have been widely applied in
clinical cancer treatment, the therapeutic efficiency is not
satisfactory owing to the existence of the tumor hypoxic
microenvironment.16-19 As a result, the existence of hypoxia
tends to promote the repair of DNA damage and reduce the
radiosensitivity of cancer cells.20 To date, various strategies
including external stimuli and internal stimuli have been
applied to overcome the tumor hypoxic microenvironments.21-31
enhance bloodstream to the tumor sites and increase the oxygen
supply to tumor, reducing the hypoxic microenvironment.32, 33
Besides external stimuli such as light irradiation, internal
stimuli such as the catalase loaded by nanoparticles or
manganese dioxide (MnO2) nanoparticles could also be used to
trigger the formation of oxygen from hydrogen peroxide
(H2O2)34, lead to overcome the hypoxia and enhance the
therapeutic efficiency of RT.22, 30 Therefore, the generation of
oxygen in the tumor sites may be considered as a more
effective pathway to reduce the hypoxia microenvironment.
Graphene, as typical two-dimensional nanomaterial, has
been used in different fields.35, 36 Owing to its intrinsic
chemical-physical properties, nano-graphene and graphene
based nanocomposites are widely used for biomedicine.37-41 For
example, nano-graphene has been used as an excellent nano-
carrier to deliver anticancer drug or gene due to the high
surface area.42, 44 Utilizing its high near-infrared (NIR)
absorbance, nano-graphene and its derivatives have been acted
as thermal agent for cancer ablation under light exposure.45
Numbers of papers have reported that nano-graphene and its
derivatives could be used as contrast agents for cancer bio-
imaging.46-49 Different radioisotopes such as 64 Cu, 66Ga, 125I,
and 131I have also been used to trace the in vivo biodistribution
of nano-graphene.50-52 In our recent work, radioisotope 131I that
could emit the β and γ rays, was selected to label reduced nano-
graphene oxides functionalized with polyethylene glycol
(RGO-PEG) for radiotherapy of cancer.50 Besides, we also used
the thermal effect of RGO-PEG under 808 nm laser irradiation
to ablate the tumors. It was found that tumors received 808 nm

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laser irradiation and the γ rays from the 131I labeled RGO-PEG
were significantly inhibited compared to that of the control
groups. Therefore, we for the first time successfully designed
graphene-based nanoplatform for combination of photothermal
therapy and radiotherapy in cancer treatment. However, it has
not been yet reported that the use of nano-graphene for imaging
guided radiotherapy simultaneously with hypoxia overcoming.
In our work, we design PEG modified 131I labelled,
reduced nano-graphene oxide and manganese dioxide
nanocomposites (131I-rGO-MnO2-PEG) nanocomposites as
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therapeutic agents for overcoming hypoxic microenvironments
and enhancing radioisotope therapy (RIT) of cancer (Figure 1).
The rGO-MnO2-PEG nanocomposites significantly improve the
oxygen formation in the solid tumor sites in the presence of
H2O2, further overcoming tumor hypoxic microenvironments.
In vivo cancer treatment has also performed using 131I-rGO-
MnO2-PEG, achieving excellent therapeutic efficiency of
cancer. More importantly, MnO2 nanoparticles would
decompose in the presence of H2O2 and release manganese ions
(Mn2+) for magnetic resonance (MR) imaging, realizing
imaging guided cancer therapy. Our work provides a novel
strategy to trigger oxygen formation via internal stimulus,
further enhancing RIT efficiency.
Results and discussion
In this work, GO was produced by a modified Hammer’s
method according to our former protocol.53 rGO-MnO2
nanocomposites were synthesized from the GO and potassium
permanganate (KMnO4). The MnO2 nanoparticles were in situ
grown on the surface of GO through the redox reaction in the
presence of borane morpholine complex, obtaining rGO-MnO2
nanocomposites. X-ray powder diffraction (XRD) was
conducted to analyse the phase purity of rGO-MnO2
nanocomposites (Supporting information figure S1). All the
reflection patterns matched well with the formation of pure
phase of MnO2 corresponding to JCPDS Card No. 21-1272.
The weight ratio of rGO with MnO2 was evaluated to be 1:0.82
by the inductive coupling plasma (ICP) measurement of Mn
content.
Transmission electron microscopy (TEM) imaging of rGO-
MnO2 nanocomposites uncovered that MnO2 nanoparticles
were attached on the surface of rGO (Figure 2a). The X-ray
energy dispersive spectroscopy (EDS) and element mapping of
rGO-MnO2 nanocomposites also revealed the coexistence of C,
Mn and O elements (Figure 2b&c). In order to improve the
stability of the rGO-MnO2 in physiological solution, the as-
made rGO-MnO2 nanocomposites were modified with
biocompatible PEG functionalized-poly (maleic anhydride-alt-
1-octadecene) (C18-PMH-PEG) as reported in our previous
article.45 The UV–vis–NIR spectra of rGO-MnO2-PEG showed
the wide absorbance band of MnO2 in the range of 300-400 nm
(Supporting information figure S2). Moreover, the obtained
rGO-MnO2-PEG nanocomposites exhibited great stability in
various physiological solutions (Supporting information figure
S2). The surface modification of rGO-MnO2 nanocomposites
by PEG was successfully proved by Fourier transform infrared
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spectroscopy (FTIR) analysis (Supporting information Figure
S3). After PEGylation, the size of rGO-MnO2-PEG became
smaller compared to uncoated rGO-MnO2 as revealed from
TEM and atomic force microscope (AFM) imaging
measurements (Figure 2a&d). The sizes distribution of rGO-
MnO2 before and after PEGylation were measured to be 80 nm
and 24 nm, respectively by dynamic light scattering (DLS)
assay (Figure 2f). The results were also corroborated with the
TEM and AFM images.
MnO2 nanoparticles have been widely considered as agents
to trigger the oxygenation in acidic condition. The catalytic
pathway of MnO2 for the decomposition of H2O2 in acidic pH is
shown by the following equation:
MnO2 +2H+ → Mn2+ + H2O + 1/2O2↑
Nanoscale Accepted Manuscript
MnO2 + H2O2 + 2H+ → Mn2+ + 2H2O + O2↑
Next, we conducted in vitro O2 evolution study from H2O2
solutions using an oxygen probe (JPBJ-608 portable Dissolved
Oxygen Meters, Shanghai REX Instrument Factory) in presence
of rGO-MnO2-PEG nanocomposites. The significant amount of
oxygen generation of rGO-MnO2-PEG in H2O2 (100 µM)
solution was recorded compared to that of only H2O2 or rGO-
MnO2-PEG, demonstrating that MnO2 could act as a catalyst to
trigger oxygenation in the presence of H2O2 (Figure 3a). In
addition, during the decomposition of H2O2 under acidic
conditions (as shown in above equation), the released Mn2+
from MnO2 nanoparticles could also be used as T1-weighted
contrast media for magnetic resonance imaging (MRI). We next
studied the in vitro MR imaging of rGO-MnO2-PEG
nanocomposites with different concentrations of Mn2+ under pH
7.4 and 5.8, respectively. It was found that the relaxation rate
(r1 value) at pH 5.8 was evaluated to be 3.86 mM−1s−1, and
considerably greater than that of pH 7.4 (r1= 0.79 mM–1·S–1)
(Figure 3b). Meanwhile, in vitro T1-weigted MR imaging at
different concentrations of Mn2+ in rGO-MnO2-PEG under
various pH values was carried out as shown in Figure 3c. The
substantial enhanced in the whitening effects with increased
Mn2+ concentrations at pH 5.8 made the nanocomposites as
suitable for MR imaging. Comparatively, rGO-MnO2-PEG
nanocomposites exhibited almost no significant signal
variations at pH 7.4 (Figure 3c). Therefore, during
decomposition of MnO2 to Mn2+ in rGO-MnO2-PEG
nanocomposites by H2O2, could act as contrast material for
tumor MRI. In order to perform the RIT experiments,
radioisotope 131I was selected to radiolabel the rGO-MnO2-PEG
nanocomposites, obtaining 131
I-rGO-MnO2-PEG. After
successful radiolabelling, we investigated the radiolabelling
constancy of 131I-rGO-MnO2-PEG in mouse serum at 37 °C. It
was found that a minimum amount of 131I was detached from
the 131I-rGO-MnO2-PEG nanocomposites after 7 d of
incubation, suggesting the excellent attachment of 131I with
rGO-MnO2-PEG nanocomposites (Figure 3d).
In order to confirm the feasible behaviour of rGO-MnO2-
PEG towards the cells, bio-related experiments were conducted
including cellular uptake efficiency and cytotoxicity. The
potential cytotoxicity of rGO-MnO2-PEG nanocomposite was
studied by cell counting kit-8 (CCK8) assay (Figure 4a). rGO-
MnO2-PEG nanocomposites without radioisotope caused no
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toxic effect on 4T1 cells at our tested concentrations, indicating
the biocompatibility of rGO-MnO2 nanocomposites with
PEGylation. In addition, unlike to free 131I which was unable to
kill tumor cells effectively at our tested dose range, 131I-rGO-
MnO2-PEG with different radioactivity doses induced
significant death of cancer cell (Figure 4b). Therefore, 131I-
rGO-MnO2-PEG exhibited obvious toxicity to cancer cells,
likely owing to the ease penetration of 131I-rGO-MnO2-PEG
nanocomposites into the cells. Additionally, 4T1 cancer cells
were incubated with Cy5.5 labelled rGO-MnO2-PEG
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nanocomposites for 6 h, and then imaged by confocal
fluorescence microscopy. As expected, obvious red signal light
of Cy5.5 were observed in the cytoplasm (Figure 2d),
indicating that rGO-MnO2-PEG nanocomposites could easily
enter into cells. Moreover, flow cytometry analysis was also
conducted to measure the cellular uptake of nanocomposites
(Supporting information Figure S4a). The results exhibited
successful cellular uptake of nanocomposites, and satisfied the
confocal imaging study.
Considering the effective outcome of in vitro study, 131I
labelled rGO-MnO2-PEG nanocomposites were used for in vivo
study. Healthy Balb/c mice were intravenously (i.v.) injected
with 131I-rGO-MnO2-PEG nanocomposites (4 mg/kg of rGO-
MnO2-PEG corresponding to 20 µCi of 131I). Blood was taken
out from the tail veins of mice and the radioactivity was
measured by a gamma counter. The curve of blood circulation
displayed that the pharmacokinetics of the 131I-rGO-MnO2-PEG
were satisfied with a two-compartment model. Meanwhile, the
blood circulation of half-lives for first and second phase were
analysed to 1.49±0.27h, 20.05±1.22h, respectively (Figure 3a),
which possessed long-time blood circulation. Moreover, the
biodistribution was also conducted to estimate the accumulation
of 131I-rGO-MnO2-PEG nanocomposites in the major organs
including heart, liver, spleen, lungs, kidney, stomach, skin,
muscle, bone, intestine and tumor. After 1 day of injection,
major organs were collected, weighted and determined by
gamma counter. It was observed that tumor accumulation of
131
I-rGO-MnO2-PEG was sensibly high and seemed to greater
than other organs except spleen and liver, which are responsible
for the clearance of external particles upon complete
government by reticuloendothelial systems (RES) (Figure 3b).
131
I, which is generally used in clinical radiotherapy, could
be utilized as gamma emitting tracer for gamma imaging and
single photon emission computed tomography (SPECT).54, 55
Due to the limitation in our available facilities, in vivo gamma
imaging was conducted. The mice bearing 4T1 tumor model
were i.v. injected with free 131I (200 µCi of 131I) and 131I-rGO-
MnO2-PEG (10 mg/kg of rGO-MnO2-PEG corresponding to
200 µCi of 131I) respectively, and imaged by gamma camera at
different time intervals of post injection (p.i.). It turned out that
131
I-rGO-MnO2-PEG exhibited notable enrichment of 131I in the
tumor sites by the improved permeability and retention (EPR)
effect (Figure 3c), which matched well with the biodistribution
data. Free 131I, however, was removed quickly from the body
owing to the smaller in size (Figure 3c). In addition, after in
vitro demonstration on the capability of rGO-MnO2-PEG to
react with H2O2 for release of Mn2+ and the production of O2 at
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pH 5.8, we were encouraged to use these nanocomposites for
tumor MR imaging. Similar to gamma imaging, the mice of
bearing 4T1 tumor model were i.v. injected with rGO-MnO2-
PEG (10 mg/kg) and imaged by a 3.0-T clinical MR scanner
(Bruker Biospin Corporation, Billerica, MA, USA) at different
time points after injection. The rGO-MnO2-PEG
nanocomposites demonstrated strong T1-weighted in
comparison with the positive MR imaging of tumor tissues after
1h of post-intravenous administration. The signal intensity was
gradually increased with time and reached in a maximum
intensity in 6 h. This phenomenon could be explained as when
rGO-MnO2-PEG reached to the tumor tissue via the typical
EPR effect, MnO2 NPs disintegrated into Mn2+ under the
specific triggers of acidic environment and generated huge
Nanoscale Accepted Manuscript
amount of Mn2+ through catalytic pathway, thus it was possible
to image the tumor tissues in T1-weighted MRI (Figure 3d).
However, it was observed that the T1 signal strength was
weakened in tumor tissues after 6 h, likely owing to the
metabolic effects of manganese ions in mice.
Encouraged by the nuclide imaging and MR imaging, we
next studied the in vivo cancer RIT using 131I-rGO-MnO2-PEG
as therapeutic agents. Balb/c mice bearing 4T1 tumors were
randomly divided into four groups (n=5 per group) including:
(1) PBS, (2) rGO-MnO2-PEG (10 mg/kg), (3)131I-rGO-PEG
(200 µCi of 131I corresponding to 10 mg/kg of GO-PEG), and
(4) 131I-rGO-MnO2-PEG (200 µCi of 131I corresponding to 10
mg/kg of rGO-MnO2-PEG). The nanocomposites were
administrated via the tail vein. The therapeutic processes were
repeated at the day of 0, 4 and 8. The tumor volumes were
measured by a digital calliper each two days intervals. We have
found that the tumors of mice treated with rGO-MnO2-PEG,
showed no notable change in their growth as similar to the mice
group treated with PBS, indicating that the rGO-MnO2-PEG
nanocomposites without radiolabelling have no substantial
antitumor therapeutic effect. It was worth to note that the tumor
growth of the mice treated with 131I-rGO-PEG was partly
inhibited whereas, for 131I-rGO-MnO2-PEG nanocomposites
treated mice, tumor growth was notably inhibited. (Figure 6a).
In order to enhance the therapeutic efficiency of 131I-rGO-
MnO2-PEG, DMXAA (5,6-dimethylxanthenone-4-acetic acid),
a representative hydrophobic vascular disrupting agent 56, was
used. Owing to the strong hydrophobic property, DMXAA was
very hard to load onto the surface of rGO-MnO2-PEG
nanocomposites (Supporting information Figure S7). Therefore,
DMXAA was dissolved in dimethyl sulfoxide (DMSO) and
then used directly in our experiments. Similar to the
abovementioned RIT, the mice of bearing 4T1 tumors were i.v.
injected with the mixture of 131 I-rGO-MnO2-PEG and DMXAA
(Figure 6b). The tumor volume was recorded every other day.
As expected, owing to the ability of vascular disrupting of
DMXAA, 131I-rGO-MnO2-PEG showed relatively high tumor
uptake and exhibited significant inhibition of tumor growth
compared to other control group (Figure 6b). Therefore,
DMXAA could significantly enhance the therapeutic efficiency
of RIT via inducing the vascular disruption. Meanwhile, tumors
of mice received different treatments were collected for
Hematoxylin and Eosin (H&E) staining. It was revealed that as-
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made 131I-rGO-MnO2-PEG plus DMXAA showed obvious cell
apoptosis and necrosis compared to other control groups
(Supporting information Figure S5c). Furthermore, the major
organs of mice treated with 131 I-rGO-MnO2-PEG or PBS were
obtained for H&E staining after 15 days of injection. No
obvious toxicity was found of the 131I-rGO-MnO2-PEG treated
group contrasted with the control group. (Supporting
information Figure S6). Moreover, the body weight of mice
received different groups showed no apparent change
(Supporting information Figure S5a&b).
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Such excellent therapeutic efficiency of 131I-rGO-MnO2-
PEG with or without DMXAA was attributed to overcome the
tumor hypoxic microenvironments via the decomposition of
MnO2 in the presence of H2O2. In order to further verify the
efficacy of rGO-MnO2-PEG nanocomposites to overcome
tumor hypoxic microenvironment, immunofluorescence
staining assay was tested. Mice bearing 4T1 tumors were i.v.
injected with PBS or rGO-MnO2-PEG. The mice were i.v.
injected with pimonidazole hydrochloride at 24h postejection.
Tumors were removed after 90 min, and then sliced for
immunofluorescence staining. The hypoxia positive signals
(green) in tumors of mice treated with rGO-MnO2-PEG were
much weaker than that in tumors of mice treated with PBS,
indicating that the treatment with rGO-MnO2-PEG was useful
for the tumor oxygenation and to overcome tumor hypoxia
(Figure 6c). Therefore, our as-made rGO-MnO2-PEG
nanocomposites could be severed as suitable therapeutic agents
for enhanced RIT of cancer via overcoming tumor hypoxia.
Conclusions
In summary, we have developed rGO-MnO2 nanocomposites
with PEG modification as multifunctional therapeutic agents
for multimodal imaging and enhanced RIT of cancer via
overcoming tumor hypoxic microenvironments. The significant
amount of the oxygen formation was possible due to the
decomposition of MnO2 from rGO-MnO2-PEG. 131I
radiolabelled rGO-MnO2-PEG possessed long time blood
circulation and high tumor accumulation via intravenous
injection into mice. After the decomposition of MnO2
nanoparticles, the released Mn2+ ions from rGO-MnO2-PEG
nanocomposites acted as strong T1-weighted MR contrast
agent. Meanwhile, tumor hypoxia had been reduced remarkably
through rGO-MnO2-PEG triggered oxygen formation in the
presence of H2O2 in the solid tumor. With the help of reversing
tumor hypoxic microenvironments, 131I-rGO-MnO2-PEG
exhibited significant inhibition of tumor growth. Importantly,
DMXAA we used significantly caused the tumor vascular
disruption, and increase tumor uptake of 131I-rGO-MnO2-PEG
nanocomposites, further enhancing the therapeutic efficiency of
RIT. Therefore, our study provided an appropriate strategy to
overcome tumor hypoxia and enhance RIT efficiency, further
highlighting the promising application prospect of graphene-
based functional nanocomposites in tumor diagnosis and
treatment
Experimental Section
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The Preparation and modification of rGO-MnO2
nanocomposites
Firstly, graphene-oxide (GO) was manufactured using the
modified Hummer’s method as followed in our previous work.
53
The obtained GO was dissolved in deionized (DI) water for
next experiments. For preparation of rGO-MnO2
nanocomposites, 10 mg of potassium permanganate (KMnO 4)
were added into the 10 ml of water-solution containing 10 mg
of GO under magnetic stirring at room temperature. After 5 min,
2 ml of borane morpholine complex (5 mg/ml) solution was
added dropwise into the above mixture to trigger the formation
of MnO2 nanoparticles on the surface of reduced GO. The
above compound was reacted at room temperature overnight.
The yielded rGO-MnO2 nanocomposites were collected and
Nanoscale Accepted Manuscript
washed with DI water for several times.
Owing to the limited water soluble of rGO-MnO2
nanocomposites, surface coating was essential for promoting
the biomedical application of rGO-MnO2 nanocomposites. In
this work, PEG linked poly (maleic anhydride-alt-1-octadecene)
(C18PMH-PEG5000), which was prepared according to our
earlier reported protocol45, was selected to modify rGO-MnO2
nanocomposites. Briefly, the mixture containing 2 mg of rGO-
MnO2 and 20 mg of C18-PMH-PEG dissolved in 5 ml of water
solution was ultrasonicated for 90 min. Afterwards, the above-
mentioned solution was centrifuged at 21000 ×g for 3 h to
remove any unsteady aggregates. This uncoated C18PMH-PEG
was removed by centrifugation through 100 nm filter
membrane and the obtained PEGylated rGO-MnO2
nanocomposites was kept at 4℃ for further experiments.
131
I labelling
In this work, radionuclide 131I, which has been commonly used
in clinic, was used to radiolabel rGO-MnO2-PEG to study its in
vivo behaviour. The detailed process of radiolabelling could be
found in our previously published protocol.50, 57 The
radiolabelling yield of 131I-rGO-MnO2-PEG was measured to
be 60% after purification. The radiolabelling stability was
tested in serum at 37°C.
Cell experiments
4T1 murine breast cancer cells were cultured following the
standard method.58 For cytotoxicity analysis, 4T1 cells pre-
seeded in 96-well plate were treated with different
concentrations of rGO-MnO2-PEG, 131I and 131I-rGO-MnO2-
PEG for 24 h. The cells relative viabilities were tested by
CCK8 (cell counting kit-8). To investigate the cell uptake of
nanocomposites, 4T1 cells incubated with Cy5.5 labelled rGO-
MnO2-PEG-NH2 at different time points (0.5, 2, 4, 6 h) were
washed with phosphate buffered saline (PBS) for two or three
times, and then visualized by confocal fluorescence microscope
(OLYMPUS).
In vitro oxygen generation
We investigated the O2 generation ability of rGO-MnO2-PEG
nanocomposite (100 µM of [Mn2+]) in a H2O2 solution (100
µM). Amount of dissolved oxygen generation was measured by
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an oxygen probe (JPBJ-608 portable Dissolved Oxygen Meters, group, (6) 131I-rGO-MnO2-PEG plus DMAXX (20 mg/kg)
Shanghai REX Instrument Factory). treated group (200 µCi of 131I corresponding to 10 mg/kg rGO-
MnO2-PEG). Those nanomaterials were i.v. injected at day 0, 4,
In vitro MRI measurement 8. The tumor volumes were measured using a caliper every
rGO-MnO2-PEG solutions with different manganese other day, and calculated the volume to tumor according our
concentrations treated with pH 7.4 or pH 5.8 were scanned previously published method.52
under a 3.0-T clinical MR scanner (Bruker Biospin Corporation,
Billerica, MA, USA) at room temperature. After acquiring the
T1-weighted MR images, the signal intensity values were Acknowledgements
measured for each sample in the region of interest (ROI). T This work was partially supported by National Natural
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Relaxation rates r1 (1/T1) was calculated from T1 values at Science Foundation of China (81471716, 81302383, 81672430,
different Mn concentrations. 31400861, 11575124), the National Natural Science Foundation
of Jiangsu Province (BK20140320), Fund for Young medical
Tumor model Scholars of Juangsu province (QNRC2016232), a Project

Nanoscale Accepted Manuscript

Female Balb/c mice were purchased from Nanjing Peng Sheng Funded by Science and Technology Bureau of
Biological Technology Co, Ltd. All animal procedures were Suzhou(SYS201653), the Anhui polytechnic university youth
performed in accordance with the Guidelines for Care and Use talent support program (2016BJRC006), a Project Funded by
of Laboratory Animals of Soochow University and approved by the Priority Academic Program Development of Jiangsu Higher
the Animal Ethics Committee of Soochow University Education Institutions (PAPD), and Funds of Science
Laboratory Animal Center. The in vivo tumor models were Technology Department of Zhejiang Province (No.
generated by subcutaneous injection of 4T1 cells (2 × 106) into LGF18H160026).
the right shoulder of mice. When the tumor volume reached
about 100 mm3, the mice were randomly divided into six References
groups to start in vivo experiments. 1. A. L. Harris, Nature Reviews Cancer, 2002, 2, 38-47.
2. I. Lohse, C. Lourenco, E. Ibrahimov, M. Pintilie, M.-S.
Multimodal imaging Tsao and D. W. Hedley, Cancers, 2014, 6, 459-471.
4T1 tumor-bearing mice were intravenous (i.v.) injected with 3. W. Zeng, P. Liu, W. Pan, S. R. Singh and Y. Wei, Cancer
rGO-MnO2-PEG (10 mg/kg) and imaged by a 3.0-T clinical letters, 2015, 356, 263-267.
MR scanner (Bruker Biospin Corporation, Billerica, MA, USA) 4. J. M. Brown and W. R. Wilson, Nature Reviews Cancer,
at different time points post injection. Similar to MR imaging, 2004, 4, 437-447.
tumor-bearing mice were i.v. injected with 131I labelled rGO- 5. S. J. Turley, V. Cremasco and J. L. Astarita, Nature reviews
MnO2-PEG (10 mg/kg of rGO-MnO2-PEG corresponding to immunology, 2015, 15, 669-682.
200 µCi of 131I) and imaged by small animal imaging system 6. J. Lin, S. Wang, P. Huang, Z. Wang, S. Chen, G. Niu, W. Li,
(Kodak). J. He, D. Cui and G. Lu, ACS nano, 2013, 7, 5320-5329.
7. S. Stapleton, D. Jaffray and M. Milosevic, Advanced drug
Biodistribution Measurement and Blood Circulation delivery reviews, 2017, 109, 119-130.
Blood samples collected from mice i.v. injected with 131I-rGO- 8. B. G. Wouters and J. M. Brown, Radiation research, 1997,
MnO2-PEG (4 mg/kg of rGO-MnO2-PEG corresponding to 20 147, 541-550.
µCi of 131I) at indicated time points were analysed by a gamma 9. A. I. Minchinton and I. F. Tannock, Nature Reviews Cancer,
counter (Berthold Inc.). Meanwhile, tissues including spleen, 2006, 6, 583-592.
liver, kidney, lung, heart, stomach and intestine were collected 10. Y. Liu, Y. Liu, W. Bu, Q. Xiao, Y. Sun, K. Zhao, W. Fan, J.
at 24 h post injection for biodistribution measurement. Liu and J. Shi, Biomaterials, 2015, 49, 1-8.
11. Y.-L. Hu, M. DeLay, A. Jahangiri, A. M. Molinaro, S. D.
Hypoxia Staining Rose, W. S. Carbonell and M. K. Aghi, Cancer research, 2012,
Mice injected with or without rGO-MnO2-PEG (10 mg/kg) 72, 1773-1783.
for 24h were then intraperitoneal injected with the hypoxia 12.W.-C. Huang, W.-H. Chiang, Y.-H. Cheng, W.-C. Lin, C.-F.
probe of pimonidazole hydrochloride (60 mg/kg). After Yu, C.-Y. Yen, C.-K. Yeh, C.-S. Chern, C.-S. Chiang and H.-C.
injection of 90 min, tumors were collected for frozen sections, Chiu, Biomaterials, 2015, 71, 71-83.
and imaged by fluorescence microscope (OLYMPUS). 13.W. R. Wilson and M. P. Hay, Nature Reviews Cancer, 2011,
11, 393-410.
In vivo enhanced cancer radiotherapy 14. P. Huang, J. Lin, X. Wang, Z. Wang, C. Zhang, M. He, K.
4T1 tumors of mice were randomly divided into different Wang, F. Chen, Z. Li and G. Shen, Advanced Materials, 2012,
groups, including (1) Control group (PBS), (2) rGO-MnO2- 24, 5104-5110.
PEG treated group, (3) 131I-rGO-PEG treated group (200 µCi of 15. Z. Liu, P. He, H. Zhao, B. Jia and F. Wang, Journal of
131
I corresponding to 5 mg/kg of rGO-PEG), (4) 131I- rGO- Nuclear Medicine, 2012, 53, 174-174.
MnO2-PEG treated group (200 µCi of 131I corresponding to 10 16. C. A. Hoefnagel, Annals of nuclear medicine, 1998, 12, 61-
mg/kg rGO-MnO2-PEG), (5) free DMAXX (20 mg/kg) treated 70.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2017, 00, 1-3 | 5

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 Please doNanoscale
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ARTICLE
17. R. G. Bristow and R. P. Hill, Nature Reviews Cancer, 2008,
8, 180-192.
18. L. Bodei, M. Cremonesi, M. Ferrari, M. Pacifici, C. M.
Grana, M. Bartolomei, S. M. Baio, M. Sansovini and G.
Paganelli, European journal of nuclear medicine and molecular
imaging, 2008, 35, 1847-1856.
19. H. E. Barker, J. T. Paget, A. A. Khan and K. J. Harrington,
Nature reviews Cancer, 2015, 15, 409-425.
20. R. L. Jensen, B. T. Ragel, K. Whang and D. Gillespie,
Journal of neuro-oncology, 2006, 78, 233-247.
Published on 01 February 2018. Downloaded by University of Reading on 02/02/2018 00:22:18.
21. T. M. Ashton, E. Fokas, L. A. Kunz-Schughart, L. K.
Folkes, S. Anbalagan, M. Huether, C. J. Kelly, G. Pirovano, F.
M. Buffa and E. M. Hammond, Nature communications, 2016,
7.
22. G. Song, C. Liang, X. Yi, Q. Zhao, L. Cheng, K. Yang and
Z. Liu, Advanced Materials, 2016, 28, 2716-2723.
23. M. Guan, T. Qin, J. Ge, M. Zhen, W. Xu, D. Chen, S. Li, C.
Wang, H. Su and C. Shu, Journal of Materials Chemistry B,
2015, 3, 776-783.
24. E. Di Gregorio, G. Ferrauto, E. Gianolio, S. Lanzardo, C.
Carrera, F. Fedeli and S. Aime, ACS nano, 2015, 9, 8239-8248.
25. P. Prasad, C. R. Gordijo, A. Z. Abbasi, A. Maeda, A. Ip, A.
M. Rauth, R. S. DaCosta and X. Y. Wu, Acs Nano, 2014, 8,
3202-3212.
26. L. Liu, S. Chang, J. Sun, S. Zhu, M. Yin, Y. Zhu, Z. Wang
and R. X. Xu, Cancer letters, 2015, 361, 147-154.
27. H. Chen, J. Tian, W. He and Z. Guo, Journal of the
American Chemical Society, 2015, 137, 1539-1547.
28. W. Zhu, Z. Dong, T. Fu, J. Liu, Q. Chen, Y. Li, R. Zhu, L.
Xu and Z. Liu, Advanced Functional Materials, 2016, 26, 5490-
5498.
29. I. A.-M. Nanoparticles as pH, Journal.
30.X. Yi, L. Chen, X. Zhong, R. Gao, Y. Qian, F. Wu, G. Song,
Z. Chai, Z. Liu and K. Yang, Nano Research, 2016, 9, 3267-
3278.
31. S. Gao, G. Wang, Z. Qin, X. Wang, G. Zhao, Q. Ma and L.
Zhu, Biomaterials, 2017, 112, 324-335.
32. G. Song, C. Liang, H. Gong, M. Li, X. Zheng, L. Cheng, K.
Yang, X. Jiang and Z. Liu, Advanced Materials, 2015, 27,
6110-6117.
33. J. Chen, M. Li, X. Yi, Q. Zhao, L. Chen, C. Yang, J. Wu
and K. Yang, Particle & Particle Systems Characterization,
2017, 34.
34. H. Liu, Y. Liu, C. Chu, H. Tong, W. Xue, J. Wang, G. Liu
and W. Zhang, Journal of Biomedical Nanotechnology, 2017,
13, 1321-1332.
35. Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts and
R. S. Ruoff, Advanced materials, 2010, 22, 3906-3924.
36. Y. Zhang, T. R. Nayak, H. Hong and W. Cai, Nanoscale,
2012, 4, 3833-3842.
37. Y. Chen, P. Xu, Z. Shu, M. Wu, L. Wang, S. Zhang, Y.
Zheng, H. Chen, J. Wang and Y. Li, Advanced Functional
Materials, 2014, 24, 4386-4396.
38. A. M. Pinto, I. C. Goncalves and F. D. Magalhães, Colloids
and Surfaces B: Biointerfaces, 2013, 111, 188-202.
39. Y. Yang, A. M. Asiri, Z. Tang, D. Du and Y. Lin, Materials
Today, 2013, 16, 365-373.
6 | J. Name., 2017, 00, 1-3
Please do not adjust margins
Page 6 of 12
View Article Online
DOI: 10.1039/C7NR08747K
Journal Name
40. G. Wang, F. Zhang, R. Tian, L. Zhang, G. Fu, L. Yang and
L. Zhu, ACS applied materials & interfaces, 2016, 8, 5608-
5617.
41. D. Chen, C. A. Dougherty, K. Zhu and H. Hong, Journal of
controlled release, 2015, 210, 230-245.
42. X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang and Y. Chen,
The Journal of Physical Chemistry C, 2008, 112, 17554-17558.
43. L. Zhang, J. Xia, Q. Zhao, L. Liu and Z. Zhang, Small,
2010, 6, 537-544.
44. T. Yin, J. Liu, Z. Zhao, Y. Zhao, L. Dong, M. Yang, J.
Zhou and M. Huo, Advanced Functional Materials, 2017, 27.
45. K. Yang, J. Wan, S. Zhang, B. Tian, Y. Zhang and Z. Liu,
Biomaterials, 2012, 33, 2206-2214.
46. K. Yang, S. Zhang, G. Zhang, X. Sun, S.-T. Lee and Z. Liu,
Nanoscale Accepted Manuscript
Nano letters, 2010, 10, 3318-3323.
47. H. Hong, K. Yang, Y. Zhang, J. W. Engle, L. Feng, Y.
Yang, T. R. Nayak, S. Goel, J. Bean and C. P. Theuer, ACS
nano, 2012, 6, 2361-2370.
48. K. Yang, L. Hu, X. Ma, S. Ye, L. Cheng, X. Shi, C. Li, Y.
Li and Z. Liu, Advanced materials, 2012, 24, 1868-1872.
49. S. Shi, K. Yang, H. Hong, H. F. Valdovinos, T. R. Nayak,
Y. Zhang, C. P. Theuer, T. E. Barnhart, Z. Liu and W. Cai,
Biomaterials, 2013, 34, 3002-3009.
50. L. Chen, X. Zhong, X. Yi, M. Huang, P. Ning, T. Liu, C.
Ge, Z. Chai, Z. Liu and K. Yang, Biomaterials, 2015, 66, 21-28.
51. H. Hong, Y. Zhang, J. W. Engle, T. R. Nayak, C. P. Theuer,
R. J. Nickles, T. E. Barnhart and W. Cai, Biomaterials, 2012,
33, 4147-4156.
52. D. Schmidt, A. Szikszai, R. Linke, W. Bautz and T. Kuwert,
Journal of Nuclear Medicine, 2009, 50, 18-23.
53. Z. Liu, J. T. Robinson, X. Sun and H. Dai, Journal of the
American Chemical Society, 2008, 130, 10876-10877.
54. L. Tian, Q. Chen, X. Yi, J. Chen, C. Liang, Y. Chao, K.
Yang and Z. Liu, Small, 2017.
55. X. Yi, K. Yang, C. Liang, X. Zhong, P. Ning, G. Song, D.
Wang, C. Ge, C. Chen and Z. Chai, Advanced Functional
Materials, 2015, 25, 4689-4699.
56. W. Song, Z. Tang, D. Zhang, M. Li, J. Gu and X. Chen,
Chemical Science, 2016, 7, 728-736.
57. Z. Liu, C. Jin, Z. Yu, J. Zhang, Y. Liu, H. Zhao, B. Jia and
F. Wang, Bioconjugate chemistry, 2010, 21, 314-318.
58.K. Tao, M. Fang, J. Alroy and G. G. Sahagian, BMC cancer,
2008, 8, 228.
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131
Figure 1. Schematic illustration of the formation of I-rGO-MnO2-PEG nanocomposite for reducing tumor hypoxia and
enhancing radioisotope therapy efficiency.

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Figure 2. Characterization of rGO-MnO2 before and after PEG coating (a) TEM images of the as-made rGO-MnO2 and rGO-
MnO2-PEG. (b&c) Energy disperse X-ray (EDX) spectra (b) and TEM elemental mapping images (c) of the rGO-MnO2-PEG. (d)
AFM images of rGO-MnO2 and rGO-MnO2-PEG. (e) Dynamic light scattering of rGO-MnO2 and rGO-MnO2-PEG

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Figure 3. Oxygen formation of rGO-MnO2-PEG. (a) Oxygen formation in H2O2 solutions (100 µM) in presence and absence of
rGO-MnO2-PEG under room temperature. (b&c) T1 relaxivity (b) and T1-weighted solution magnetic resonance imaging of rGO-
MnO2-PEG with different concentrations under two pH values (red line: 5.8 and black line: 7.4) at room temperature (c). (d)
Radiolabelling stability of 131I-rGO-MnO2-PEG in mouse plasma at 37 °C for 7 d.

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Figure 4. In vitro cell experiments. (a)&(b) The relative cell viabilities of 4T1 cells treated with different concentrations of rGO-
MnO2-PEG, 131I and 131I-rGO-MnO2-PEG for 24 h. (c) Confocal fluorescence images of 4T1 cells incubated with Cy5.5-labeled
rGO-MnO2-PEG for 6 h.

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Figure 5. In vivo experiments. (a&b) The blood circulation (a) and biodistribution of 131I-rGO-MnO2-PEG (b). (c) Gamma
imaging of mice i.v. injected with free 131I or 131I-rGO-MnO2-PEG. (d) T1-weighted MRI of mice tumor before and after treated
with rGO-MnO2-PEG. Red circle indicates the position of the tumor.

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Figure 6. In vivo cancer radioisotope therapy. (a&b)Tumor growth curves of grouped mice given at day 0th, 4th and 8th. Doses
for each injection: 131I (200 µCi), rGO-MnO2-PEG (10 mg/kg), rGO-PEG (5 mg/kg), DMXAA (20 mg/kg). The tumor volumes
were normalized to their initial sizes. (c) Representative immunofluorescence images of tumor slices collected from mice at 24 h
post i.v. injection of PBS or rGO-MnO2-PEG. The cell nuclei, blood vessels, and hypoxia areas were stained with DAPI (blue),
anti-CD31 antibody (red), and antipimonidazole antibody (green), respectively. P values in panel (a&b) were analysed by two-
tailed Student’s t test using GraphPad Prism 5.0 (**p < 0.01, *p < 0.05).

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