Journal Pre-proofs

Trienzyme-like Iron Phosphate Nanozyme for Enhanced Anti-tumor Efficien‐

cy with Minimal Side Effects

Zhongqiang Wang, Guoliang Li, Yang Gao, Ying Yu, Bing Li, Xingkai
Wang, Jinjian Liu, Kezheng Chen, Jianfeng Liu, Wei Wang

PII: S1385-8947(20)31702-2
DOI: https://doi.org/10.1016/j.cej.2020.125574
Reference: CEJ 125574

To appear in: Chemical Engineering Journal

Received Date: 6 February 2020

Revised Date: 27 April 2020
Accepted Date: 18 May 2020

Please cite this article as: Z. Wang, G. Li, Y. Gao, Y. Yu, B. Li, X. Wang, J. Liu, K. Chen, J. Liu, W. Wang,
Trienzyme-like Iron Phosphate Nanozyme for Enhanced Anti-tumor Efficiency with Minimal Side Effects,
Chemical Engineering Journal (2020), doi: https://doi.org/10.1016/j.cej.2020.125574

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 Trienzyme-like Iron Phosphate Nanozyme for Enhanced

Anti-tumor Efficiency with Minimal Side Effects

Zhongqiang Wanga,‡, Guoliang Lia,‡, Yang Gaob, Ying Yua, Bing Lia,c, Xingkai Wanga, Jinjian Liua, Kezheng

Chena,*, Jianfeng Liua,b,*, Wei Wanga,*

aLab of Functional and Biomedical Nanomaterials, College of Materials Science and Engineering, Qingdao

University of Science and Technology, Qingdao 266042, China

bTianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine,

Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China

cState Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of

Sciences, Lanzhou 730000, PR China

‡The authors contributed equally to this work

*Email: wangwei@qust.edu.cn; liujianfeng@irm-cams.ac.cn; kchen@qust.edu.cn

Abstract

Despite an ever-growing number of investigations and recent advances in nanozymes

and oxidative stress anti-tumor therapy, nanoparticles capable of maintaining the redox

balance as natural antioxidative system in normal tissues are scarce. Herein, iron phosphates

(FePOs)-based nanozyme, was synthesized via a simple hydrothermal method. We first

discovered that the FePOs nanozyme has trienzyme-like activities. It is peroxidase (POD)-like

in acidic tumor microenvironment and can mediate a highly efficient FePOs+H2O2 anti-tumor

protocol via catalyzing ·OH generation from H2O2. Quite different from any other reported

nanozymes, at physiological pH the FePOs is not only catalase (CAT)-like but also

superoxide dismutase (SOD)-like. It exerts combined SOD-CAT effects on normal tissues,

which is analogous to the synergistic effects of natural SOD-CAT in living organisms, and

hence protects normal tissues from injuries of the FePOs+H2O2 protocol. Apoptosis was

found to be the pathway by which cell death is induced. Notably, the FePOs+H2O2 protocol

led to no abnormal behavior, significant weight loss or obvious adverse effects on normal

tissues in mice as the results of the combined SOD-CAT effects. All these intriguing results
confirm that the FePOs nanozyme is an excellent candidate for nanomedicine and holds

promise for enhanced anti-tumor efficiency with minimal side effects.

Keywords: nanozymes; chemical dynamic therapy; oxidative stress; iron phosphates

1. Introduction

Oxidative stress caused by reactive oxygen species (ROS) is a common hallmark

in a multitude of human diseases. ROS, including superoxide anion radical (O2•-),
hydroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HOCl) and hydroxyl
radical (·OH), are metabolites generated by the antioxidant defense system through
partial reduction of oxygen in response to environmental stimulation [1]. This process
is essential for living organisms to protect themselves but may also lead to an
excessively high ROS level, termed “oxidative stress”, which plays important roles in
the occurrence of many diseases, such as tumors, Parkinson’s disease, and
Alzheimer’s disease [2, 3] Naturally, many enzymes, such as superoxide dismutase
(SOD) and catalase (CAT), involved in the antioxidant defense system regulate the
ROS level in organisms. SOD can catalyze O2•- dismutation into O2 and H2O2, and
CAT subsequently catalyzes H2O2 into water and O2.
Given that excessive ROS levels can cause severe damage in cells via oxidation,
oxidative stress therapy has been proposed for tumor ablation. Currently, clinical
application of ROS-induced apoptosis is a commonly used cancer treatment method
in the form of radiotherapy [4], photodynamic therapy (PDT) [5], sonodynamic
therapy (SDT) [6], and chemo-dynamic therapy (CDT) [7]. Nevertheless, from the
perspective of curative effects, only CDT is not limited by the hypoxia feature of
tumors [8-13]. During CDT, ·OH radicals are produced in large quantities from the
intrinsic excess H2O2 in solid tumors. Compared with H2O2, ·OH has a much stronger
oxidation capability (E(·OH/H2O)=2.80 V, E(H2O2/H2O)=1.78 V) and can cause
significantly higher oxidative stress in cancer cells, thus acting as the major
contributor to oxidative stress [1].
The use of catalytic nanoparticles, which were termed “nanozymes” [14], as a
new type of CDT agent has recently attracted considerable interest. Superior to
natural enzymes, nanozymes exhibit much better tolerance to environmental changes.
To date, a series of nanozymes have been applied as CDT agents to trigger ·OH
generation from H2O2 via Fenton or Fenton-like reactions. After uptake by cancer
cells, these nanoparticles react with H2O2 to produce ·OH burst in situ, which
damages important organelles, such as mitochondria, lysosomes, and DNA [15]. Due
to the different redox states between cancer and normal cells, cancer cells with higher
basal ROS levels are more susceptible to oxidative stress [16, 17]. Numerous studies
have shown that nanozyme-mediated CDT is an efficient protocol to kill cancer cells
through intracellular oxidative stress. Among all the metal-based nanozymes,
Fe-based nanomaterials are the most widely studied anti-tumor agents, not only due to
their high catalytic efficiency but also because of their high biocompatibility [18]. To
date, most nano-scaled CDT agents are Fe-based nanomaterials [19]. Nano-scaled
Fe3O4 was the first reported peroxidase (POD)-like nanomaterial. Based on the
POD-like activity of Fe3O4 nanoparticles reported by Yan et al. in 2007 [20], Gu and
coworkers succeeded in exploring Fe3O4 as a CDT anti-tumor agent [21].
In the past five years, the number of studies reporting cancer therapy using
metal-based nanozymes has dramatically increased. However, most of the reported
nanozymes cannot ablate tumors efficiently without side effects to normal tissues. As
is known, endogenous H2O2 levels are limited (10~50 μM) and vary in different solid
tumors [1]. In most cases, extensive death of cancer cells was only realized with the
assistance of exogenous H2O2. Since H2O2 molecules can easily diffuse across
biological membranes, side effects caused by migration of exogenous H2O2 from
tumors to healthy tissues are occur [22]. In addition, most nanozymes cannot
eliminate the toxicity of all ROS, and to date, few nanozymes have adequately
realized synergistic antioxidant effects and avoided side effects caused by their own
activity. Developing a multienzyme-like nanomaterial which can generate excessive
ROS in tumors while eliminating ROS in normal tissues may provide a powerful
means for simultaneously maximizing therapeutic effects and minimizing side effects
via maintenance of redox balance in normal cells. For example, SOD mimics can only
detoxify the oxidative damage induced by O2•-, but the H2O2 byproduct is still toxic
and may generate ·OH radicals, which are even more toxic. This additional toxicity
associated with SOD mimics has been noted by scientists. Several types of
nanoparticles that can carry or encapsulate natural SOD and CAT together have been
fabricated [23-28]. But to our knowledge, there has no POD/SOD/CAT
tri-enzyme-like nanoparticles were investigated to ablate tumors without side effects.
In this study, FePOs nanospheres with a small particle size suitable for cell
endocytosis were prepared by using different iron source, phosphate source and
capping agents. The prepared FePOs nanospheres were found to behave as a POD
mimic in an acidic environment and a SOD/CAT mimic at physiological pH. The
unique tri-enzyme mimetic properties inspired us to design a minimally invasive,
highly efficient anti-tumor strategy. We hypothesized that the prepared
POD/SOD/CAT-like FePOs nanozyme can kill cancer cells as a POD enzyme in the
acidic tumor environment, while protecting normal cells as a SOD/CAT enzyme,
thereby avoiding the side effects caused by H2O2 either injected exogenously or
generated as a byproduct of SOD-catalyzed O2•- dismutation in neutral or slightly
alkaline healthy tissues (Scheme 1). To verify the hypothesis, three different cancer
cell lines and one noncancerous cell line were used as cell models to evaluate both the
in vitro anti-tumor effects and side effects of this protocol. We also found that the
newly developed nanozyme exhibits robust therapeutic effects and good safety
profiles in mice and thus has potential for highly specific cancer therapy applications.
Scheme 1. Schematic illustration of FePOs-mediated anti-tumor therapy and protection to normal
tissues.

2. Material and methods

2.1. Materials

Urea (H2NCONH2) was purchased from AIBI Chemistry Preparation CO. LTD.
(Shanghai, China). Sodium lauryl sulfate (CH3(CH2)11OSO3Na, SDS) was purchased
from Bodi Chemical Co., Ltd (Tianjin, China). Ferric sulfate (Fe2(SO4)3) was
purchased from Hongyan Chemical Reagent Factory (Tianjin, China).
3,3'5,5'-tetramethylbenzidine (TMB) was purchased from Sigma Reagent Company.
Phosphoric acid (20% H3PO4), 30% H2O2 solution, citric acid and disodium hydrogen
phosphate (Na2HPO4·12H2O) were obtained from Shanghai Chemical Corporation
(Shanghai, China). All other chemicals were reagent grade or better. Deionized (D.I.)
water was generated by a Milli-Q Plus system (Billerica, MA, USA) used in material
preparation. Ultrapure Milli-Q water (resistance >18 M cm-1) was used for cell
experiments and animal experiments. For cell culture experiments, the following
reagents were used: All cells were maintained in Tianjin Key Laboratory of Radiation
Medicine and Molecular Nuclear Medicine. Dulbeco’s Modified Eagle’s Medium
(DMEM) and penicillin/streptomycin were purchased from Gibco Corporation. Fetal
bovine serum (FBS) was purchased from Biological Industries Corporation.
Penicillin-Streptomycin, Trypsin-ethylene diamine tetraacetic acid (EDTA) solution,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and
4',6-diamidino-2-phenylindole (DAPI) were purchased from Solarbio (Beijing, China).
Calcein-AM/PI double stain kit was purchased from Yeasen Biotechnology (Shanghai,
China). (3,6-diamino-9-[2-(methoxycarbonyl) phenyl] xanthylium chloride)
(Rhodamine123, Rh123), 2’,7’-dichlorofluorescin diacetate (DCFH-DA) were
purchased from from Sigma-Aldrich Co. (St Louis, MO, USA). Anti-gamma H2AX
(phosphor A139) antibody and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)
antibody were purchased from Abcam Company.

2.2. Preparation of FePOs nanozyme

FePOs nanozyme was prepared via a simple hydrothermal method. In a typical

process, H2NCONH2 (6.0 g) and sodium lauryl sulfate (SDS, 0.5 g) were dissolved in
deionized water (84 mL) and then magnetically stirred for 10 minutes to obtain a
homogeneous solution. Afterwards, Fe2(SO4)3 (0.2 g) and phosphoric acid (20% wt
H3PO4, 0.490 g) were separately dissolved in deionized water (8 mL), and then added
dropwise to the above solution, which was then magnetically stirred for 20 minutes.
Next, the mixture was transferred into a 150-mL Teflon-lined autoclave, sealed and
maintained at 140 ºC for 2 h, and then cooled to room temperature naturally. The
obtained product was collected by centrifugation, washed with deionized water and
ethanol, and finally dried in a vacuum at 60 ºC.

2.3. Phase, composition and morphology characterization of FePOs nanozyme

XRD patterns of the products were obtained with a Rigaku D/max-2500 X-ray
powder diffractometer using Cu Kα radiation (λ=1.5406 Ǻ). The morphology of
FePOs nanozyme was observed with field-emission scanning electron microscopy
(FESEM; JEOL, JSM-6700F) and transmission electron microscopy (TEM; JEOL,
JEM-2000EX). XPS analyses were performed using an ESCALAB 250XI
spectrometer (Thermo Fisher, USA). The detailed chemical composition of FePOs
was determined with an Elementar Vario EL III element analyzer. Hydrodynamic size
was measured via dynamic light scattering (DLS; Zetasizer Nano, Malvern
Instruments).

2.4. Enzyme mimetic activity of FePOs nanozyme

Peroxidase-like activity assays of FePOs were carried out in a 1.25-mL

TMB-H2O2-FePOs reaction system. The oxidation product of TMB produces a blue
signal with characteristic absorption peaks at 370 nm and 652 nm, which can be
detected by a UV-Vis spectrophotometer (Cary 5000). In a typical test, the
pH-sensitive, peroxidase-like activity was characterized in 0.1 M citric acid-sodium
dihydrogen phosphate (Na2HPO4) buffer solution with pH values varying from 6.4 to
7.4. The TMB-H2O2-FePOs reaction system included H2O2 at a consistent
concentration of 7.5 mM, FePOs at a concentration of 50 µg/mL and TMB at a
concentration of 0.5 mM. All tubes were incubated for 0.5 h in the dark at room
temperature and then imaged and analyzed.
CAT-like activity assays of FePOs were carried out by measuring the rate of
oxygen production in an FePOs-H2O2 system using a SevenExcellenceTM Benchtop
Meter. Typically, the total volume of the reaction system was 4.5 mL, including H2O2
(75 mM), FePOs (100 µg/mL) and citric acid-Na2HPO4 buffer (0.1 M). Oxygen
production (unit: ppm) was detected at equal time intervals, and the pH sensitivity of
the CAT-like activity was evidenced by varying the buffer pH from 6.4 to 7.4.
SOD activity assays of the FePOs nanozyme were carried out at room
temperature by measuring the inhibition of autooxidation of pyrogallol. Pyrogallol is
prone to autooxidation under alkaline conditions, and the oxidation product has an
absorption peak at 320 nm, whereas the reaction can be greatly inhibited by the
enzyme SOD. In detail, chemicals were added into 1 mL of Tris-HCl (0.1 mM,
pH=8.2) buffer solution in the following order: 100 μL pyrogallol-HCl (final
pyrogallol concentration 0.15 mM, pH=2), and (or without) 100 μL FePOs (final
concentration 50 µg/mL) to form a pyrogallol-FePOs system. SOD-like activity was
assessed by inhibiting the autooxidation effect of pyrogallol, and the corresponding
oxidation products were measured using a spectrophotometer (Cary 5000).

2.5. In vitro toxicity of FePOs nanozyme

Three cancer cell lines (human cervical carcinoma cell line, HeLa; Michigan
Cancer Foundation-7 cell line, MCF-7; 4T1-GFP/mCherry-luc cell line, 4T1-Luc) and
one noncancerous cell line (mouse fibroblast cell line, L929) obtained from Tianjin
Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine were seeded
in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; BI) and 1%
penicillin/streptomycin (pen/strep; Gibco) at 37 ºC in 5% CO2. The in vitro
cytotoxicity of FePOs was evaluated using the classic MTT method. Cells were
seeded in 96-well plates at a density of 5×103 cells/well and grown in 100 µL
complete culture medium for 24 h. Then, the cells were treated with gradient
concentrations (0~200 µg/mL) of FePOs for another 24 h. After that, 10 µL MTT
solution (5 mg/mL) was added to each well, and the plates were further incubated for
4 h in an incubator. The absorbance of each well at 490 nm was read using a
full-wavelength scanning multifunction reader (Thermo Scientific Company, USA).

2.6. Cellular uptake of FePOs nanozyme

Cellular uptake of the FePOs was determined via cell ultrastructural analysis
using TEM. Briefly, HeLa cells were seeded in a 25-cm2 culture flasks (Costar) and
exposed to FePOs (125 µg/mL) for 6 h. The cells were then rinsed with cold PBS to
remove extra FePOs that were not taken up the cells, fixed with 2.5% glutaraldehyde
overnight at 4 ºC, and then scraped off and collected via centrifugation to obtain cell
clusters. Through a series of operations, including staining, dehydration, embedding,
frozen sectioning and other steps, cell section samples were observed with an 80 kV
transmission electron microscope (JEM-1200EX, JEOL).

2.7. In vitro anti-tumor effects of FePOs-mediated oxidative-stress therapy

 The anti-cancer effects of FePOs-mediated oxidative-stress therapy were
evaluated via the MTT method using HeLa and MCF-7 cells as cancer cell models.
Cells were seeded in 96-well plates and randomly assigned into control, FePOs or
FePOs+H2O2 groups. Cells in the FePOs and FePOs+H2O2 groups were both
incubated with FePOs (0~200 µg/mL in DMEM) for 12 h first. Then, the cells were
rinsed with PBS to remove excess FePOs. After that, FePOs group cells were exposed
to fresh DMEM for 4 h, but the FePOs+ H2O2 group cells were incubated with 1 mM
H2O2 in fresh DMEM (confirmed, safe concentration) for 4 h. Finally, the medium in
all wells was replaced with fresh DMEM folled by further incubation of the cells for
12 h. MTT analysis was performed to evaluate the in vitro anti-tumor effects.
Live/dead staining, which intuitively reflects the anti-cancer effects of the
FePOs, was also carried out. HeLa and MCF-7 cells randomly assigned into control,
FePOs and FePOs+H2O2 groups were seeded in six-well plates and allowed to adhere
for 24 h. After treatment with FePOs and H2O2 as described above, the cells were
stained with Calcein-AM (2 µM, 2 mL per well) at 37 °C for 15 min, followed by PI
staining (4.5 µM, 2 mL per well) in the dark for 5 min. Cellular fluorescence images
were captured with a fluorescence microscope (inverted Leica DMI 6000B) at
excitation wavelengths of 490 nm and 535 nm to detect the fluorescence signals of
Calcein-AM (green) and PI (red), representing live and dead cells, respectively.

2.8. Measurement of intracellular ROS

HeLa and MCF-7 cells were seeded in confocal cell culture dishes at a density of
3×105 cells/well and incubated with FePOs (0 µg/mL, 25 µg/mL and 50 µg/mL in
DMEM, 2 mL per well) for 12 h. After the excess FePOs were washed away with
cold PBS, the cells were exposed to 1 mM H2O2 (2 mL per well) for 4 h. Then, the
cells were incubated with fresh medium for 12 h and rinsed with cold PBS twice to
prepare for staining. The oxidant-sensitive fluorescent probe 2’,7’-dichlorofluorescin
diacetate (DCFH-DA) was used to detect intracellular ROS levels. Briefly, cells that
required staining were incubated with DCFH-DA (10 µM, 2 mL per well) in
serum-free DMEM for 30 min at 37 °C in the dark. The fluorescence intensity of
DCFH-DA was detected with a fluorescence microscope at excitation and emission
wavelengths of 480 nm and 525 nm, respectively.

2.9. Measurement of mitochondrial membrane potential (Δψm)

Changes in mitochondrial membrane potential (Δψm) were assessed with an

inverted fluorescence microscope using the fluorescent probe Rhodamine123
(Rh123). For staining, the cells were incubated with Rh123 solution (20 μΜ, 50 μL
per well) for 30 min in confocal cell culture dishes and then rinsed with PBS several
times before fluorescence imaging.

2.10. DNA double-strand break detection

Pretreated cells receiving FePOs-mediated therapy were fixed with 4%

paraformaldehyde for 30 min at 4 °C. Cold methanol was added to permeate the fixed cells

for 15 min. After incubation in 1% bovine serum albumin for 2 h to prevent nonspecific

protein interactions, the fixed cells were immunostained with anti-gamma H2AX (phosphor

A139) antibody (2 µg/mL, 500 µL per well) overnight at 4 °C and then incubated with goat

anti-rabbit IgG H&L (Alexa Fluor® 488) antibody (1 µg/mL, 500 µL per well) for 1 h at

room temperature. Finally, the cells were stained with DAPI (10 µg/mL, 500 µL per well) for

15 min and visualized using a fluorescence microscope.

2.11. In vivo hemocompatibility evaluations of FePOs nanozyme

In vivo hemocompatibility evaluations were conducted by performing hemolysis tests

and hematology analysis. In the hemolysis test, red blood cells (RBCs) were obtained by

centrifugation of CD rat whole blood cells in heparinized tubes at a speed of 5000 r/min for 5

min, followed by three washes with cold PBS (pH 7.4). Immediately, the pellets were

resuspended and diluted to 2% (by volume) with the same buffer. FePOs nanozyme were

dispersed in PBS at gradient concentrations of 25~1000 µg/mL. The same volume of blood

cells and FePOs-PBS dispersion were added to a centrifuge tube for a total volume of 1 mL.

Negative (PBS only) and positive controls (including 2% Triton X-100) were used as
references. RBCs were incubated with FePOs upside down for 3 h in a 37 °C shaking water

bath. Afterwards, the optical absorption of the supernatant was detected at 540 nm using a

Varioskan Flash multimode reader (Thermo Scientific Company, USA) after centrifugation

(5000 r/min, 5 min). Hemolysis occurred when the hemolysis rate exceeded 5%. The

hemolysis rate was calculated using the following formula:

(absorbance of sample) - (absorbance of negative control)
hemolysis (%) =  100% (eqn.1)
(absorbance of positive control) - (absorbance of negative control)

For hematology analysis, female BALB/c mice were randomly assigned into three

groups (n=5 per group): a control group (PBS, 200 µL), low dose group (20 mg/kg), and high

dose group (40 mg/kg). The indicated dose of FePOs was intravenously injected twice on two

consecutive days. Blood samples were collected from all mice by extracting the blood from

the eye on the first and seventh day after injection. Afterwards, the blood samples (100 µL)

were mixed with anticoagulant (EDTA-3K, 20 µL, 30 mg/mL), and hematological analysis

was performed using an automatic blood analyzer (Celltace, Japan).

2.12. In vitro protection of normal cells from H2O2 damage by FePOs nanozyme

To investigate the interactions of FePOs and H2O2 in normal cells, a high concentration

of H2O2 was directly delivered to L929 cells to induce massive cell death. L929 cells were

seeded into a 96-well plate (5×103 cells/well) and randomly assigned into control, H2O2 and

FePOs+H2O2 groups. After 24 h, the H2O2 group cells were co-cultured with H2O2 (5 mM in

DMEM) and FePOs+H2O2 group cells were co-cultured with gradient concentrations of

FePOs (12.5~200 µg/mL) in addition to H2O2 (5 mM in DMEM) for another 2/4/6/8 h. Then,

cell survival rates were measured using the MTT method as described above. The protection

mechanism was investigated via cell morphology observation, cellular ROS level

characterization and Δφm measurement.

2.13. Tumor model

Female BALB/c Nude mice (5 weeks old, 19~21 g) were purchased from Beijing Vital

River Laboratory Animal Technology Co., Ltd. To obtain a tumor-bearing mouse model, the

prepared 4T1-Luc tumors were cut into pieces the size of rice grains and then carefully placed
subcutaneously into the right rear flank of the mice. The in vivo tumor inhibition experiment

started when the tumor volume grew to 150~200 mm3.

2.14. Tumor inhibition experiments

For in vivo evaluation of the catalytic activity of FePOs, tumor-bearing mice were

randomly divided into 4 groups: PBS, H2O2, FePOs, and FePOs+H2O2. In the first 7 days, the

mice were intratumorally injected with PBS (100 µL), FePOs (2 mg/mL, 100 µL), or H2O2

(20 mM, 100 µL) three times at an interval of 3 days. On the fifth and twentieth days, two

4T1-Luc tumor-bearing mice in each group were intraperitoneally injected with D-Luciferin

(150 μg/mL, 100 μL), which is a fluorescent substrate used for in vivo imaging.

Bioluminescence occurs in 4T1-Luc tumors when D-Luciferin converts into oxyluciferin

under the action of oxygen and ATP. Six minutes after injection, the mice were anesthetized

with isoflurane for in vivo imaging using a KODAK IS in vivo FX system. The tumor volume

and body weight were measured every day. The tumor volume was calculated as V=(tumor

length)×(tumor width)2/2, and the relative tumor volume was defined as VR=Vi/V0 (V0: tumor

volume on the first day; Vi: daily measured tumor volume). After 21 days of evaluation, all

the mice were sacrificed and dissected, and the tumor sizes in different groups were compared

to evaluate the in vivo anti-tumor effects mediated by the FePOs nanozyme.

H&E staining: On the tenth day after treatment, one mouse per group was sacrificed.

Major organs (heart, liver, spleen, lung, and kidney) and tumors were dissected, rinsed with

PBS, fixed in 4% formaldehyde, embedded in paraffin, and sectioned into 5-µm slices, and

pathological examination was performed after hematoxylin and eosin staining.

3. Results and discussion

3.1. Phase, composition and morphology of FePOs nanozyme

The FePOs nanozyme was prepared through a facile hydrothermal method with
Fe2(SO4)3 as the iron source in the reaction. TEM showed that the as-prepared FePOs
nanozyme exhibited a spherical morphology (Figure 1a and Figure S1). The average
hydrodynamic diameter (Dh) of FePOs measured by DLS was approximately 420~430
nm (Figure S2). The XRD pattern (wide peaks, Figure S3) and SAED pattern (diffuse
scattering halo, inset in Figure 1a) showed that the prepared FePOs particles were
amorphous. The chemical state of the FePOs surface was investigated by XPS
analysis, and the results are shown in Figure 1b-1f. The XPS survey spectrum (Figure
1b) demonstrated the existence of Fe, P and O in the FePOs nanozyme. In the
high-resolution spectrum of Fe 2p (Figure 1c), the peak centered at 711.1 eV can be
attributed to Fe 2p3/2, which has been investigated by many researchers, with reported
values between 710.6 and 711.2 eV [29-31]. The other peak centered at 724.6 eV
corresponds to Fe 2p1/2, which is narrower than the Fe 2p3/2 peak due to fact that Fe
2p3/2 has 4 degeneracy states while Fe 2p1/2 has only 2 [29]. The weak peak centered
at 56.3 eV can be attributed to Fe 3p (Figure 1d), but detailed information regarding
Fe 3p3/2 or Fe 3p1/2 could not be obtained, possibly due to the low resolution of the
XPS instrument. After careful comparison with the standard binding energy values of
Fe3+ and Fe2+, we designated the valence of the Fe element in the prepared FePOs as
Fe3+. As shown in Figure 1e and 1f, the P 2p and O 1s peaks at binding energies of
132.3 eV and 530.2 eV, respectively, which are in good agreement with reported
values [29], further verified the presence of phosphate groups. The FTIR spectrum in
Figure S4 provides more evidence of the presence of –OH and Fe-P-O groups in the
FePOs nanozyme. For more chemical composition information, the C/H/N element
contents in the FePOs products were characterized using an element analyzer. The
results presented in Table S1 show that the prepared FePOs consisted of 3.723% C,
3.093% H and 2.374% N elements (by weight).
Fig. 1. (a) TEM image and SAED pattern (inset) of FePOs, (b) XPS survey spectrum of FePOs,

and (c-f) high-resolution XPS spectra of Fe 2p, Fe 3p, P 2p and O 1s in FePOs.

Table 1. Chemical state information for FePOs nanozyme.

Ratio of peak
Peak energy FWHV Peak area [eV Sensitivity area to
Element
[eV] [eV] counts] factor sensitivity factor
[eV]

Fe 2p 711.1/724.6 3.37/4.65 335095.39 2.11 158812.98

Fe 3p 56.3 3.03 41319.62 2.11 19582.76

P 2p 132.3 1.24 42367.64 0.35 121050.4

O 1s 530.2 1.27 480316.99 0.71 676502.81

N 1s 400.2 3.37 18288.78 0.47 38912.30

Na 1s 1070.0 1.78 26680.74 1.66 16072.73

3.2. Enzyme mimetic properties of FePOs

Accumulating reports have shown that nano-sized materials have enzyme-like

activities, which regulate ROS levels and produce corresponding biological effects
[32, 33]. It is well known that Fe ions can catalyze degradation of H2O2 through a
Fenton reaction [19]. In this study, we used TMB as a substrate to detect the POD-like
activity of FePOs, and the resulting color changes can be read from the UV-vis
spectra at wavelengths of 370 nm and 652 nm. As shown in the inset in Figure 2a,
colorless TMB was oxidized to produce a blue product in the presence of FePOs at
lower pH conditions. The absorption peak at 652 nm, corresponding to the blue
product, increased with decreasing pH, indicating pH-sensitive, POD-like activity of
FePOs. In contrast, the TMB-H2O2 system without FePOs exhibited no blue color
with other conditions unchanged. Figure 2b-2c show the results of the SOD-like
activity investigation of the FePOs nanozyme using the autooxidation of pyrogallol
method described by S. Marklund and G. Marklund [34]. Pyrogallol is a compound
that can be autooxidized under alkaline conditions, and the autooxidation product has
an absorption peak at 320 nm. This process can be greatly inhibited by SOD, which
eliminates the O2•- radicals released as byproducts. Our results show that the
characteristic absorption of oxide pyrogallol at 320 nm increased significantly in the
absence of FePOs but was greatly inhibited if FePOs was present. This inhibition
effect by FePOs is very similar to that of natural SOD, thus indicating SOD-like
activity of the FePOs nanozyme. Moreover, we found that FePOs possessed CAT-like
activity by monitoring dissolved oxygen in FePOs-H2O2 reaction systems. The results
in Figure 2d demonstrate that the CAT-like activity was also pH responsive. In the
presence of an equal amount of H2O2, FePOs generated more O2 under neutral
conditions (pH=7.4) than under acidic conditions (pH=6.4~6.8), suggesting a more
robust CAT-like activity in tumor tissue than normal tissues.
Together, the prepared FePOs possesses pH-sensitive, multienzyme activities,
i.e., it is POD-like under acidic conditions, CAT-like under neutral conditions and
SOD-like under alkaline conditions.
Fig. 2. (a) UV-Vis absorption spectra and photos (inset) of the TMB-H2O2-FePOs reaction system.

(b, c) UV-Vis spectra of the pyrogallol autoxidation system in timescan mode: (c) without FePOs,

0.002 A/min and (d) with 50 μg/mL FePOs, 0.042 A/min. Insets (b) and (c) show pictures of the

corresponding pyrogallol autoxidation system in 0.1 M Tris-HCl. (d) Oxygen generation test

results for the H2O2-FePOs reaction system in 0.1 M citric acid-NaH2PO4 buffer with pH values

ranging from 6.4 to 7.4.

3.3. Cytotoxicity assessment and internalization of FePOs by cells

The cytotoxicity of FePOs was further evaluated with MTT method using HeLa,
MCF-7, 4T1-Luc and L929 cell lines as cell models. As shown in Figure S5, no
significant cytotoxicity was observed after the cells were incubated with FePOs
(12.5~200 μg/mL) for 24 h. On the one hand, this indicates that the material is safe for
in vitro dispersed cells; on the other hand, the material did not catalyze a highly
efficient Fenton reaction, which might be due to the low level of endogenous H2O2 in
the in vitro dispersed cells. Therefore, we next tried to raise the H2O2 level in cells by
introducing exogenous H2O2 to verify whether FePOs could accelerate cancer cell
death via the Fenton reaction.
The uptake of FePOs was investigated via TEM using the HeLa cell line as a cell
model. After the cells were incubated with FePOs at a concentration of 125 μg/mL for
10 h, remarkable cell uptake was observed. Figure 3a-3b show that FePOs particles
were encapsulated into cell vesicles, which were dispersed throughout the cytoplasm.
The main organelle and membrane structure of the cells did not change significantly
even when many FePOs particles entered the cells, indicating low cytotoxicity of the
FePOs. Additionally, Fe, O, and P signals in EDS mapping data obtained from the
black particles located in cytoplasm (Figure 3c) further confirmed the uptake of
FePOs by cells.

Fig. 3. (a, b) Sectional TEM images of HeLa cells incubated with FePOs (125 μg/mL) for 10 h,

demonstrating the uptake behavior of the cells towards FePOs nanozymes. (c) EDS data of FePOs

enriched in HeLa cytoplasm; the inset clearly reflects the distribution of iron. (d-g) Sectional TEM

images of HeLa cells in the H2O2 group (1 mM H2O2, 4 h) and FePOs+H2O2 group (1 mM H2O2

for 4 h and then 50 μg/mL FePOs for 12 h); untreated HeLa cells were used as controls.

3.4. In vitro anti-tumor effects by FePOs

To investigate the in vitro anti-tumor effects of FePOs, we first optimized the

doses of FePOs and H2O2, as well as the treatment programs. Well-growing HeLa
cells were exposed to FePOs at concentrations of 12.5~200 μg/mL for 12 h and then
washed with PBS to expel excess FePOs. Next, 1 mM H2O2 was added and incubated
with cells for 4 h and then replaced with fresh culture medium, followed by
cultivation for another 12 h. Finally, the viability of the cells was measured using
MTT reagent. As shown in Figure S6, neither FePOs nor H2O2 showed cytotoxicity at
the set dosages. However, due to the acidic pH in HeLa cells [35-36], incubation of
FePOs together with H2O2 significantly enhanced the cytotoxicity of H2O2 and
reduced cell viability to less than 50%, even at the minimum concentration of 12.5
μg/mL, demonstrating that FePOs played an important role in inducing cell death.
Cell structure characterization by TEM is the most reliable method to determine
cell death pathways. The main features of apoptotic cells, such as nuclear chromatin
concentration, edge accumulation and mitochondria deformation, can be clearly
observed via TEM [37]. In this study, we employed TEM to further investigate cell
death pathways. HeLa cells in the control, H2O2 and FePOs+H2O2 groups were sliced
into ultrathin sections and then dyed with uranyl acetate to obtain more resolvable cell
microstructural details, and the results are shown in Figure 3d-3g. After careful
comparison, no significant difference was observed between the control and H2O2
groups, indicating minimal damage to cell structures was caused by H2O2 treatment at
the set dosage. However, a many vacuoles were found in the cytoplasm of HeLa cells
treated with FePOs+H2O2 (post-time 0 h), which is a typical feature of cell autophagy.
Autophagy is a catabolic process by which excessive or damaged endogenous
components as well as foreign matter are degraded by cells. It is regarded as an initial
cytoprotective mechanism in response to cell injury to rescue cells [38-39]. However,
the injury caused by ROS was so serious that the cells experienced excessive damage
and could not survive despite initiation of autophagy. As a result, as time went by,
apoptosis proceeded, and cells sampled at a post-treatment time of 12 h presented a
morphology typical of apoptosis in which nuclear chromatin developed into highly
condensed black blocks.
To visually understand the catalytic properties of FePOs, we carried out a
LIVE/DEAD cell viability assay in which the cells were stained with Calcein-AM/PI
and then observed with a fluorescence microscope. This assay can distinguish live
cells from dead cells, live cells are green, while dead cells are red. The obtained
fluorescence images shown in Figure 4a-4c indicated the addition of exogenous H2O2
(1 mM, 4 h) had no influence on cell viability, whereas FePOs promoted the death of
a large number of cells in the presence of exogenous H2O2. These results, which were
consistent with the MTT results, revealed that FePOs accelerated the cell death with
the assistance of exogenous H2O2.
It is well known that cytotoxicity is primarily the result of elevated levels of ROS
in cells [3]. In this study, we stained cells with DCFH-DA, an ROS-sensitive
fluorescent probe, and detected the intracellular ROS level by comparing fluorescence
intensity in different cell groups. As shown in Figure 4d-4f, the FePOs+H2O2 group
HeLa cells stained with DCFH-DA emitted much brighter fluorescence than both
control and H2O2 group cells, suggesting a higher ROS level in FePOs+H2O2-treated
HeLa cells. The extremely faint fluorescence signal observed in the H2O2 group was
only slightly higher than that in the control group, indicating that the effect of a safe
H2O2 dose was weak and transient and did not cause significant elevation in
intracellular ROS levels. Therefore, it is reasonable to attribute the in vitro anti-tumor
mechanism of FePOs to its peroxidase-like activities, which accelerated the
intracellular Fenton reaction and elevated the ROS level by promoting H2O2
decomposition into ·OH radicals.
Mitochondrial dysfunction has been confirmed to participate in the induction of
apoptosis and has even been considered to be the core of the apoptotic pathway [40].
In this paper, cytofluorimetric analysis of mitochondrial membrane potential (Δψm)
was performed using a Rhodamine123 probe, which can selectively accumulate in the
mitochondria of living cells and emit bright green fluorescence to denote a normal
Δψm. As shown in Figure 4g-4i, full, bright green fluorescence was observed in both
control group and H2O2 group cells but decreased sharply under the joint action of
H2O2 and FePOs, indicating a sharp decrease in Δψm and the occurrence of apoptosis
induced by FePOs+H2O2 treatment.
γ-H2AX staining assay was used to visualize DNA double-strand breaks (DSBs)
in tumor cells during the FePOs-mediated anti-tumor protocol [41-42]. The results are
shown in Figure 4j-4l, and cell nuclei were labeled with the blue fluorescent probe
4',6-diamidino-2-phenylindole (DAPI). Obviously, a minimal amount of DSBs (green
fluorescent spots) were observed in the H2O2 group, while severe DNA damage
occurred in the nuclei of FePOs+H2O2 group cells (whether the low or high dose
group). These results further confirmed that cell death was caused by FePOs+H2O2
treatment via enhancement of the Fenton reaction.

Fig. 4. Fluorescence microscopy images of HeLa cells stained with different fluorescent

probes. The scale bars are 100 μm for Calcein-AM/PI group, and 30 μm for the other

groups. All the fluorescence images were taken at a post-treatment time of 12 h.

The data shown in Figure S6-S8 demonstrate similar in vitro anti-tumor effects
of the FePOs+H2O2 protocol in 4T1-Luc and MCF-7 cells, indicating that this therapy
is also highly effective on other types of cancer cells.
 The TEM images presented in Figure 5a and 5b demonstrate the L929 cell
microstructures after exposure to 5 mM H2O2 for 6 h. Both apoptotic cells and
necrotic cells, characterized by highly condensed nuclear chromatin and broken cell
membranes, respectively, were observed, indicating severe damage caused by the
high dose of H2O2. It is very exciting to find that this damage could be completely
eliminated by FePOs. The TEM images in Figure 5b and 5d show that L929 cells
exposed to FePOs+H2O2 treatment presented a normal morphology, without apoptosis
or necrosis features, suggesting minimal side effects of the protocol, as we expected.
The MTT assay, Δψm measurement and ROS level detection results further confirmed
our expectations. In Figure S9, the viability of L929 cells in the FePOs+H2O2 group
was higher than that of cells in the H2O2 group (72.43% vs. 15.76%). In fluorescence
microscopy images (Figure 5e and 5f), Rh123-stained L929 cells emitted extremely
faint fluorescence after H2O2 treatment but turned brilliant green when FePOs was
added, which indicated a much higher Δψm in FePOs+H2O2 group cells than in H2O2
group cells. The ROS levels detected by DCFH-DA are shown in Figure 4g and 4h.
The significantly brighter fluorescence observed in H2O2 group cells provides clear
evidence that H2O2 treatment induced a high level of oxidative stress in L929 cells,
resulting in mitochondrial injury and a decrease in Δψm. These results further
confirmed that the adverse effects of the FePOs+H2O2 anti-tumor protocol in vitro
were minimal because the FePOs nanozyme protected noncancerous cells from the
injury induced by H2O2 due to its antioxidative activities at physiological pH. This
protection could be ascribed not only to the CAT-like activity but also to the
SOD-like activity of FePOs because it is well established that mitochondria undergo
spontaneous bursts of O2•- generation, termed ‘‘superoxide flashes’’, under oxidative
stress [43]. With superoxide anion as the primary form, ROS are produced as
byproducts of mitochondrial respiration when electrons leak from the electron transfer
chain (ETC) in quiescent cells. Metabolic stress causes massive increases in localized
O2•- production, which ultimately contributes to apoptotic or necrotic cell death. In the
present study, L929 cell death via apoptosis and necrosis caused by exogenous H2O2
was also observed. However, most cells survived when FePOs was added to the
incubation medium. We found that mitochondria generated massive O2•- in L929 cells
that were exposed to exogenous H2O2, which then caused apoptotic or necrotic cell
death. In the physiological pH of L929 cells, FePOs first served as a SOD-mimic to
dismutate O2•- into H2O2, and then decomposed H2O2 into O2 and H2O as a
CAT-mimic, thus protecting L929 cells from exogenous H2O2 injury.

Fig. 5. (a-d) TEM images of ultrathin sections of L929 cells treated with H2O2 (5 mM) for 6 h

without or with FePOs (50 μg/mL). (e-h) Corresponding fluorescence microscopy images of L929

cells stained with the ROS detector DFCH-DA (e, f) and MMP detector Rh123 (g, h).

Low toxicity is a necessary characteristic for biological application of

nanomaterials. To evaluate the in vivo safety of FePOs, we performed hemolysis tests
and blood cell analysis. All animal experiments were performed according to the
guidelines of the Administration of Experimental Animals (Tianjin, revised June
2004) and adhered to the national standard of the People’s Republic of China (GB/T
16886.6-1997). The hemolysis test, which primarily detects the membrane-damaging
properties of materials, was conducted according to protocols described by Parnham
and Wetzig [44] and Fan Huang [45]. Typically, an RBC suspension was exposed to a
series of pre-designed concentrations of FePOs and then centrifuged, and the optical
absorption of the supernatants at 540 nm was measured to evaluate the level of
hemolysis, an intense absorption at 540 nm suggests severe hemolysis. As shown in
Figure S10, minimal absorption was measured for all groups, indicating that there was
indistinct hemolysis even at the highest FePOs concentration of 500 μg/mL, further
demonstrating the desired hemolytic resistance of RBCs to FePOs.
 For a non-endogenous inorganic material, the potential risk of inducing
inflammation can be reflected by hematological indicators in a hematology analysis.
To detect acute toxicity and chronic toxicity of FePOs nanozyme, we performed two
consecutive FePOs injections into model mice and collected blood samples from the
eye for hematology analysis on the first and seventh days after injection. As shown in
Figure S11, all typical blood indicators, including red blood cells (RBCs), hemoglobin
(HGB), white blood cells (WBCs), hematocrit (HCT), mean corpuscular volume
(MCV), platelets (PLTs), mean corpuscular hemoglobin concentration (MCHC), and
mean corpuscular hemoglobin (MCH) were detected in each group. Compared with
the PBS group, all the blood indexes in both the high and low dose groups were found
to be normal, demonstrating that no obvious infection and inflammatory reaction
occurred throughout the evaluation period. Taken together, the results indicated
satisfactory biocompatibility of the FePOs nanozyme, which favors its further
bio-application.

3.6. In vivo anti-tumor effects by FePOs

Inspired by the excellent biocompatibility and desirable in vitro cancer cell

killing efficiency, we further performed tumor inhibition studies in vivo using 4T1
tumor-bearing mouse model. As schematically illustrated in Figure 6a, the tumor
model was initially obtained via growth of embedded tumor grains over a ten-day
period. In the pretreatment period, corresponding doses of H2O2 and FePOs were
administered three times via intratumoral injection. Ten days later, one mouse per
group was sacrificed and dissected, and H&E staining was performed. During the
tumor inhibition experiment, tumor volume and body weight were recorded, and in
vivo fluorescence imaging was also performed.
In tumor inhibition experiments, we divided the tumor-bearing mice into four
groups: PBS, H2O2, FePOs, and FePOs+H2O2 groups. To obtain better therapeutic
effects, exogenous H2O2 was introduced into the tumor via intratumoral injection,
which was consistent with the in vitro experiments. As shown in Figure 6b, the tumor
growth curve of the H2O2 group was very similar to that of the PBS group, indicating
that the H2O2 injection at a dosage of 20 mM, 100 μL, and 3 administrations at an
interval of 3 days did not significantly affect the growth of the tumors. Notably both
the FePOs group and FePOs+H2O2 group showed remarkably delayed tumor growth,
and tumor growth in the FePOs+H2O2 group was significantly more prominent than
that in the FePOs group. According to statistical analysis, compared with the PBS
group, the tumor volume percentages measured on the 21st day in the H2O2, FePOs,
and FePOs+ H2O2 groups were 109.0%, 46.1%, and 15.6%, respectively (Figure 6c),
which suggests a high tumor ablating efficiency of FePOs+H2O2 therapy.
In vivo fluorescence imaging of the mouse tumor was also carried out in order to
visualize the tumor cell activity. In this study, the 4T1-luc cell line, which has been
developed for optical monitoring of tumors, was used to establish the tumor model.
As shown in Figure 6d, tumors in the FePOs+H2O2 group exhibited the lowest signal
intensity. Tumor inhibition effects could be observed from the fifth day of the
protocol for both the FePOs group and FePOs+H2O2 group. Comparison of the
resected tumor volume (Figure 6e) further identifies the admirable anti-tumor
potentials of FePOs+H2O2, in which the tumors were almost completely ablated after
21 days of the treatment. Taken together, these results clearly show that the
anti-tumor effects can be attributed to FePOs catalysis of the Fenton reaction.
Exogenous H2O2 is rapidly converted to ·OH in the presence of FePOs, which then
irreparably damaged cancer cells. Moreover, the FePOs group showed a certain
anti-cancer activity due to the well-known microenvironmental conditions of tumor
tissues, i.e., higher endogenous H2O2 levels and lower pH values than found in
healthy tissues. It is very exciting to find that over the duration of the treatment
period, the body weight, an indicator of systemic toxicity, of mice in all groups did
not show significant differences (Figure S12), suggesting low systemic toxicity of the
FePOs nanozyme and exogenous H2O2. In fact, the average body weights of mice in
the FePOs+H2O2 and FePOs groups were even slightly higher than those of mice in
the H2O2 and PBS groups at the end of the treatment, which further indicated the
therapeutic effect of FePOs+H2O2 treatment or even FePOs-only treatment. No black
blots could be found on the skin of FePOs+H2O2 group mice (Figure S13). This result
clearly demonstrates that the side effects accompanying FePOs+H2O2 therapy are
extremely minimal, which might be due to the SOD-CAT synergistic effects of FePOs
in normal tissues. The potential danger of exogenous H2O2 was eliminated by FePOs
through CAT-like activity.
H&E staining of tumor tissue slices was performed on the tenth day after
treatment to observe the fate of cancer cells and damage to major organs. The
microscopy images in Figure 6f show that the cancer cells in the PBS and H2O2
groups retained an intact cell structure and clear nuclear boundaries, without obvious
apoptosis or necrosis. However, in both the FePOs group and FePOs+H2O2 group,
serious apoptosis, evidenced by cell membrane lysis and tissue shriveling, was
observed for the tumor cells. This sharp contrast provides more evidence supporting
the notion that the in vivo anti-tumor mechanism of the FePOs nanozyme involves
promotion of the Fenton reaction and the cancer apoptotic cell death pathway.
Furthermore, H&E staining of major organs and the skin from all experimental groups
showed no significant morphological differences compared with the PBS group
(Figure S14), which further confirmed the minimal side effects of FePOs-mediated
therapy.
Fig.6. In vivo anti-tumor effect of FePOs NPs against 4T1 tumor xenografts. (a) Schematic

of tumor model establishment, administration and H&E staining. (b) Relative tumor

volume after intratumoral injection of PBS, H2O2, and FePOs according to the

experimental design (n=7). (c) Standardized tumor volume percentages in different groups.

(d) In vivo bioluminescence imaging of 4T1-Luc tumor-bearing mice on the fifth and
twentieth days, D-Luciferin substrate was intraperitoneally injected at a dose of 15 μg/mL,

100 μL per mouse. (e) Photographs of the resected tumors from each group after 21 days of

treatment. (f) H&E staining of tumor tissues on the tenth day. Scale bars: 100 µm.

4. Conclusions
In summary, we have presented a novel iron phosphate nanozyme that shows
POD/SOD/CAT tri-enzyme-like activities and can be used as a highly efficient
anti-tumor agent with minimal side effects. A tumor inhibition efficiency as high as
84.4% can be achieved by intratumoral injection of FePOs+H2O2, with no significant
physical toxicity observed. The mechanism behind the minimal adverse effects might
be ascribed to the ability of the FePOs nanozyme to maintain the redox balance in
normal cells due to its combined SOD-CAT activity, which is analogous to the
synergistic effects of natural SOD-CAT in living organisms. This work provides a
new type of Fe-based nanozyme that can trigger the Fenton reaction via tumor
microenvironment regulation, which holds promise for enhanced anti-tumor
efficiency with the advantages of selective killing of tumors and minimal side effects

Acknowledgements

This research was financially supported by the Natural Science Foundation of

Shandong Province (CN) (grant no. ZR2018MEM016), the National Natural Science
Foundation of China (81722026), the CAMS Innovation Fund for Medical Sciences
(CIFMS, 2016-I2M-3-022), the Non-profit Central Research Institute Fund of
Chinese Academy of Medical Sciences (2018PT35031), the Drug Innovation Major
Project (2018ZX09711-001) and the National Science Fund for Distinguished Young
Scholars of Tianjin (18JCJQJC47300).

Appendix A. Supplementary data

The following are the supplementary data related to this article.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: