Supporting Information

A pH-responsive platform combining chemodynamic therapy with limotherapy

for simultaneous bioimaging and synergistic cancer therapy

Jianmin Xiaoa,b,1, Guilong Zhangc,1, Rui Xue,1, Hui Chene, Huijuan Wangb, Geng Tianc,

Bin Wangc, Chi Yangd, Guo Baid, Zhiyuan Zhangd, Hongyi Yangf, Kai Zhongf,

Duohong Zoud,**, Zhengyan Wua,*

a Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei

Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, P.R.

China.

b Engineering and Materials Science Experiment Center, University of Science and

Technology of China, Hefei 230026, P.R. China

c Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai,

Shandong Province, 264003, P.R. China

d Department of Oral Surgery, Ninth People’s Hospital, Shanghai Jiao Tong

University School of Medicine, National Clinical Research Center for Oral Diseases,

Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of

Stomatology Shanghai 200001, P.R. China.

e Department of Dental Implant Center, Stomatologic Hospital & College, Anhui

Medical University, Key Laboratory of Oral Diseases Research of Anhui Province,

Hefei 230032, P.R. China

f High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese

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Academy of Sciences, Hefei 230031, P.R. China

* Corresponding author.

** Corresponding author.

E-mail addresses: zdhyy@ahmu.edu.cn (D. Zou), zywu@ipp.ac.cn (Z. Wu).

1Co-first authors.

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Methods

Materials. All chemical reagents were used as received without further purification.

Fe(acac)3, fluorescein isothiocyanate (FITC), N-hydroxysuccinimide (NHS), 3′,

3′, 5′, 5′-tetramethylbenzidine (TMB), polyethyleneimine (PEI), glutathione (GSH),

dulbecco’s modified eagle medium (DMEM), Mn(acac)2, and N-ethyl-N′-(3-

(dimethylamino)propyl) carbodiimide (EDC) were purchased from Aladdin

Chemical Co. Ltd. (Shanghai, China). Ethylene glycol (EG), hydrochloric acid,

sodium selenite, sodium hydrate, dimethylsulfoxide (DMSO), triethanolamine (TEA),

diethylene glycol (DEG), and anhydrous ethanol were provided by the Sinopharm

Chemical Reagent Co. Ltd. (Shanghai, China). In addition, polyvinylpyrrolidone

(PVP-K30) was provided by Fluka-Solarbio Co. Ltd. (Beijing, China). The cell

counting kit-8 (CCK-8) assay was obtained from Dojindo (Japan), and

reactive oxygen species (ROS) assay kit was obtained from Nanjing Jiancheng

Bioengineering Institute, and the dihydroethidium-ROS (DHE-ROS) assay kit was

purchased from BestBio Co.

Synthesis of ION. IONs were successfully synthesized based on our previous work

[1,2]. Firstly, Fe(acac)3 (0.5 g) was dissolved in the mixed solution of EG (10 mL) and

DEG (40 mL) under magnetic stirring at 80oC for 40 min. Next, PEI (1.5 g) was

added the resulting solution at 80oC for 30 min and formed homogeneous solution.

After that, TEA (5 mL) was added into the homogeneous solution, and the color of

solution gradually changed from turbid brown to transparent wine-red. Subsequently,

the obtained solution was transferred to an autoclave at 200oC for 12 h. Finally, the

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black solid products were collected using an external magnet, and washed at least

three times with distilled water and alcohol, respectively.

Synthesis of MCDIONs. The mixture of Fe(acac)3 and Mn(acac)2 were dissolved into

the solution containing EG (10 mL) and DEG (40 mL), under vigorous stirring at

80oC for 40 min. Then, PEI (1.5 g) was added into the resulting solution under

magnetic stirring for 30 min. Afterward, TEA (5 mL) was further added to the

resulting solution for 30 min. The obtained solution was transferred to an autoclave

and maintained at 200oC for 12 h. Finally, the black solid products were collected

using external magnet, and washed at least three times with distilled water and

alcohol, respectively.

Preparation of MCDION-Se. Firstly, MCDION (10 mg) was dispersed into alcohol

(30 mL) solution containing PEI (0.1g) and then stirred for 6 h. Next, the resulting

solution was slightly shaken at 45oC bath for 30 min, and then the particles were

collected by external magnet. In addition, 20.4 mg of GSH and 8.467 mg of Na2SeO3

were dissolved into 20 mL of deionized water and stirred for 30 min. After that, the

obtained particles were uniformly dispersed into the resulting solution and NaOH (16

mg, 2 mL) was quickly added into the solution under stirring for 2 h. Finally, PVP

solution (w/w=5%, 5mL) was further added and continuously stirred for 6 h. The

product was collected and washed, and then dispersed into aqueous solution for

further use.

Release Behaviors of Mn2+ Ion from the MCDIONs. The MCDIONs (2 mg) with

different Mn contents were uniformly dispersed into different pHs of phosphate

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buffered saline (PBS, 10 mL) solution and shaken at 35oC. After that, the supernatant

was collected at different time intervals, and the Mn2+ ion concentration was

determined by ICP-MS (ThermoFisher 7200, USA).

Confocal Laser Scanning Observation. FITC was labeled to

MCDIONs-Se according to previously described methods [3,4]. HeLa cells were

seeded on CLSM-specific dish, incubated with different concentrations of FITC

labeled MCDION-Se (10, 20, and 40 µg/mL) for 4 h. Subsequently, the cells were

treated with PBS (pH=7.4) solution and stained by 4′, 6-diamidino-2-phenylindole

(DAPI) at 37oC in dark condition. Then, the fluorescence images of cells were

obtained via CLSM (Zeiss LSM710 NLO, Germany).

FITC Accumulation Assay. Firstly, HeLa cells were cultured with different

concentrations of FITC labeled MCDION-Se. Then, extracellular fluorescence was

quenched using the 0.4% trypan blue for 2 min. Finally, the cells were measured by

flow cytometry (CytoFLEX Beckman-Coulter, USA) and the corresponding

fluorescence intensity was calculated using FlowJo software (TreeStar).

Cell Culture and Cytotoxicity Assay. HeLa and HK-2 cells were seeded

into 96-well plates and further incubated in the DMEM supplemented with 10% fetal

bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2

at 37oC for 24 h. Meanwhile, the HeLa cells were incubated with different

concentrations of IONs, MCDION-1, Se, MCDION-1+Se, and MCDION-Se for 24 h

and 48 h. Subsequently, the culture media was removed from the 96-well plates and

the corresponding cells were washed using PBS. After that, the cells were treated with

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10% CCK-8 kit (120 μL) at 37oC for 2 h. Finally, the amounts of viable cells were

measured by microplate reader at a certain wavenumber (450 nm).

ROS Generation in Vitro. The MCDION-Se (100 µg) was dispersed into

PBS containing 200 µL of TMB and 20 µL of H2O2, subsequently the solution were

adjusted to pH 7.4, 6.5, 5.5, and 4.5. Meanwhile, different contents of MCDION-Se

were added into 4 mL of aqueous solution containing TMB (200 µL, DMSO solution,

1 mg/mL) and H2O2 (20 µL). Finally, the mixture solution was centrifuged and

hydroxyl radicals in the supernatant were measured via UV-vis spectroscopy.

In addition, the HeLa cells were seeded on glass coverslips placed in 12-well

plates (105 cell/well) and incubated with physiological saline (control group), ION

(Fe: 18.8 µg/mL), Se (12.4 µg/mL), MCDION-1 (Fe: 18.8 µg/mL, Mn: 12.4 µg/mL),

MCDION-1+Se (Mn+Se: 12.4 µg/mL), and MCDION-Se (Mn+Se: 12.4 µg/mL) for 4

h. Then, HeLa cells were treated with DCFH-DA prods (dilution 1:1500) for 30 min,

and then cells were washed with PBS for three times and fixed in paraformaldehyde

(4%) solution for 30 min. Subsequently, the cells were incubated with DAPI ((dilution

1:2000) fluorescent dye at 37oC for 10 minutes in the dark. Finally, the generation

of ROS in cell was directly observed by confocal laser scanning microscopy.

Meanwhile, the level of ROS was detected via measuring the fluorescence

intensity of DCFH-DA prods in HeLa cell.

The content of superoxide anion radicals in cells was further

measured using DHE-ROS assay kit [5,6]. Firstly, HeLa cells were seed on

glass coverslips placed in 12-well plates (105 cells/well) and incubated with

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physiological saline (control group), Se, MCDION-1+Se, and MCDION-Se at the

same concentration of Se for 4 h. Then, DEH-ROS prods (dilution 1:1000) were

added into media and further incubated for 30 min. After that, the levels of superoxide

anion radicals in cell media were detected using flow cytometry.

Western Blot. HeLa cells were incubated in 6-well plates and the number of cells

was adjusted to 5×105 cells/well. Then the cells were treated with physiological saline

(control group), Se, MCDION-1, and MCDION-Se at the same concentration for 24

h. The cells were collected and further lysed, and then the total proteins were

extracted. Meanwhile, the content of the total proteins was measured using BCA

(bicinchoninic acid) protein quantitative kit. Subsequently, the total proteins were

separated by SDS-PAG (dodecyl sulfate, sodium salt (SDS)-polyacrylamide gel

electrophoresis) and transferred to poly(vinylidene difluoride) membranes.

Afterwards, the membranes were then treated with SOD antibodies (dilution 1:500, 5

mL, 12 h) and β-actin (dilution 1:2000, 5 mL, 12 h). Finally, the membranes were

visualized via the chemiluminescence system.

Intracellular ATP Contents Measuring. HeLa cells were incubated on 6 cm glass

coverslips and the number of cells were adjusted to 106 cells/well., Subsequently, the

cells were treated with physiological saline (control group), ION (Fe: 18.8 µg/mL), Se

(Se: 12.4 µg/mL), MCDION-1 (Fe: 18.8 µg/mL, Mn: 12.4 µg/mL), and MCDION-Se

(Mn+Se: 12.4 µg/mL) for 24 h. Afterwards, the cells were collected and divided into

the two groups. One group was used to measure the content of total protein using

BCA protein quantitative kit, and the other group was treated with ATP assay kit for 2

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h. After that, corresponding absorbance of tube was measured via a

spectrophotometer. Finally, ATP content in cells could be calculated via the following

equation:

ATP content (umol/gprot) = [(Tm - Tc)/(Ts-Tb)]×Cs×A/Cp

where Tm and Tc were respectively the concentration of ATP absorbance in the

measuring tube and control tube, and Ts and Tb were respectively standard tube and

blank tube. Cs was standard concentration, A was dilution multiple of samples, and

Cp was total protein concentration of samples.

MR Experiment in Vitro and in Vivo. In vitro and in vivo MR

experiments were conducted on a 9.4 T/400 mm wide bore scanner

(Agilent Technologies, Inc., Santa Clara, CA, USA). For in vitro MR

experiments, longitudinal relaxation time (T1) of samples was measured via a series of

inversion-prepared fast spin-echo images. This series of parameters were identical in

all aspects (TR 6000 ms, effective TE 5.6 ms, BW 25 kHz, slice thickness 1 mm,

matrix 96×96, 1 average) except for the 20 different inversion times (TIs) that were

varied linearly from 10 to 2500 ms. The r1 values were preciously calculated via a

linear fit of the relaxation time as a function of metal ions concentration.

For the in vivo MR experiments, the tumor-bearing nude mice were placed in a

prone position on a specially designed cradle and inserted into the magnet. For the

duration of the experiment, the animal’s body temperature was maintained at 36.5°C

with a homemade heating pad. T1-weighted MR images of tumor were acquired,

typically along the coronal orientation, using a spin-echo sequence. The acquisition

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parameters were as follows: repetition time (TR) = 370 ms, echo time (TE) = 11.6 ms,

field of view (FOV) = 40 mm × 40 mm, matrix size = 192 × 192, slice thickness = 1

mm (12 slices, gap = 0), 1 average, and bandwidth (BW) = 50 kHz. A series of T1-

weighted MR images were obtained before CA injections. Mice were scanned at post-

injection CAs 15 min, 30 min, 60 min, 90 min and 120 min. T1-weighted images were

analyzed using ImageJ software. For each mouse, ROIs were manually drawn around

the tumor on the coronal MR images, and the signal intensities were measured and

normalized to the noise values in the corresponding MR slices for comparison

between pre-injection and post-injection.

Anticancer Activity Assay. The cervical tumor-bearing mice were employed to

investigate the therapeutic efficacy of nanoparticles for tumor, and the mice were

treated in accordance with the ethics committee guidelines in University of Science

and Technology of China. Mice with approximately 100 mm3 tumor were randomly

divided into 5 groups (n= 5/groups) and then were respectively injected with saline,

MCDION-1, Se, MCDION-1+Se, MCDION-Se at a dosage of 2 mg/kg via tail vein

with 2 days intervals. The volume of tumor was calculated according to the following

formula: V=a×b2/2, where “a” and “b” were the longest and shortest diameter of the

tumor, respectively. Meanwhile, the weight of mice was recorded at two days

intervals. Approximately post-injection 24 days, the mice were sacrificed and the vital

tissues (heart, liver, spleen, lung, kidney, and tumor) were collected and nitrated.

After that, Mn and Se content in the nitrated solution were determined via inductively

coupled plasma mass spectrometry (ICP-MS).

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Pathological Analysis. The vital tissues and tumor of mice treated with samples were

fixed in 10% buffered formalin, and embedded in paraffin. Then, the tissue sections

were cut and stained with H&E for histopathological study. The slices of vital tissues

and tumor were observed using light microscopy at 400X magnification. Besides, the

vital tissue and tumor sections were analyzed via the immunohistochemical detection

of caspase 3. The tissue sections were hydrated in PBS for 5 min, and then treated

with 10 mM sodium citrate buffer (pH=6.0) at 80oC for 10 min. After that, the

sections were washed using PBS when cooling to ambient temperature, and then

treated with PBS solution containing 1% hydrogen peroxide. Afterwards, the sections

were washed using PBS and further treated with PBS containing 1.5% normal serum.

Subsequently, the sections were treated with primary antibodies against caspase 3 at

4oC in a humidified chamber, and the sections were then treated with the horseradish

peroxidase-conjugated secondary antibodies at 37oC for 30 min (dilution: 1:100). The

color development with DAB was employed to visualize the immune reaction, and

sections were observed by the inverted fluorescence microscope system.

Characterization. The morphology and element mapping of particles were observed

by transmission electron microscope (JEM-ARM200F, JEOL Co., Japan). The size

distribution of particles was measured via dynamic light scattering (DLS) detector

(Nanotrac WaveII, Microtrac Co. USA). The crystal structure measurement was

performed on X-ray diffraction (XRD) (TTR-III, Rigaku Co., Japan). The

measurements of composition and structure of samples were conducted on an FT-IR

spectrometer (iS10, Nicolet Co., USA).

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Fig. S1 The interplanar spacing in MCDION-1.

Fig. S2 (A) The hydrodynamic size distribution of IONs and MCDION and (B) The

change of particles size with the Mn content increasing.

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Fig. S3 (A) FT-IR spectra of nanoparticles. The release behavior of Mn2+ ion from

nanoparticles in (B) pH 7.4 PBS or (C) pH 6.5 PBS solution.

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Fig. S4 MR T1-weighted maps of MCDIONs under different pH PBS solution.

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Fig. S5 Schematic illustration of MCDION-1 as T1 contrast agent with magnetic

switch function in acidic environment.

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Fig. S6 (A) The hydrodynamic size distribution and (B) EDS of MCDION-Se. (C)

Zeta potential of nanoparticles in neutral solution. (D) XRD pattern of MCDION-Se.

(E) Average sizes of MCDION-Se with the increase of standing time. (F) Digital

pictures of MCDION-Se aqueous solution in external magnet environment.

Fig. S7 (A) The release behavior of Mn2+ ion from MCDION-Se under different pH

PBS solution, (B) The analysis of relaxation rate r1 of MCDION-Se under different

pH PBS solution.

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Fig. S8 (A) The CLSM images of HeLa cells treaded with FITC-labeled MCDION-Se

at different condition, and the green color was assigned to the FITC-labeled

MCDION-Se, the corresponding nucleus was stained with DAPI (blue), all images

shared the same scale bar; (B) the corresponding flow cytometric analysis.

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0

20

40

60

80

100

MCDION-1 Se MCDION-1+Se MCDION-Se

0 1.5 3.0 6.1 12.3 24.6

Cell v

iabilit

y (%

)

Mn+Se concentration (g/mL)49.3

48h

Fig. S9 Viabilities of HeLa cells treated with nanoparticles for 48 h.

Fig. S10 The photographs of tumor-bearing mice treated with nanoparticles for 24

days.

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Fig. S11 The biodistribution of Mn and Se ion in vital tissue of mice treated with

various nanoparticles for 24 days.

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