Sparks fly between ascorbic acid and iron-based nanozymes: A study on Prussian blue nanoparticles

Article abstract of DOI:10.1016/j.colsurfb.2018.01.010

Herein we reported Prussian blue nanoparticles (PBNPs) possess ascorbic acid oxidase (AAO)- and ascorbic acid peroxidase (APOD)-like activities, which suppressed the formation of harmful H₂O₂ and finally inhibited the anti-cancer efficiency of ascorbic acid (AA). This newly revealed correlation between iron and AA could provide new insight for the studies of nanozymes and free radical biology.

Full text of DOI:10.1016/j.colsurfb.2018.01.010

Accepted Manuscript
Title: Sparks fly between ascorbic acid and iron-based
nanozymes: A study on Prussian blue nanoparticles
Authors: Wei Zhang, Yang Wu, Hai-Jiao Dong, Jun-Jie Yin,
Hui Zhang, Hao-An Wu, Li-Na Song, Yu Chong, Zhuo-Xuan
Li, Ning Gu, Yu Zhang
PII:
S0927-7765(18)30010-9
DOI:
Reference:
https://doi.org/10.1016/j.colsurfb.2018.01.010
COLSUB 9089
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Colloids and Surfaces B: Biointerfaces
Received date:
Revised date:
Accepted date:
29-7-2017
2-12-2017
9-1-2018
Please cite this article as: Wei Zhang, Yang Wu, Hai-Jiao Dong, Jun-
Jie Yin, Hui Zhang, Hao-An Wu, Li-Na Song, Yu Chong, Zhuo-Xuan
Li, Ning Gu, Yu Zhang, Sparks fly between ascorbic acid and iron-based
nanozymes: A study on Prussian blue nanoparticles, Colloids and Surfaces B:
Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.01.010
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Sparks fly between ascorbic acid and iron-based nanozymes:
A study on Prussian blue nanoparticles
Wei Zhanga,b,c, Yang Wu , Hai-Jiao Dong , Jun-Jie Yin , Hui Zhang , Hao-An Wu ,
d
a
e
e
a
Li-Na Song , Yu Chong , Zhuo-Xuan Li , Ning Gua,b,*, Yu Zhanga,b,*
f
e
a
a
State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and
Devices, School of Biological Science and Medical Engineering, Southeast University,
Nanjing 210096, P. R. China
b
Collaborative Innovation Center of Suzhou Nano Science and Technology, Southeast
University, Nanjing 210096, P. R. China
c
The Jiangsu Province Research Institute for Clinical Medicine, the First Affiliated
Hospital with Nanjing Medical University, Nanjing 210029, P.R. China
d
Research Centre of Clinical Oncology, Jiangsu Cancer Hospital, Nanjing 210009, P.
R. China
e
Division of Analytical Chemistry, Office of Regulatory Science, Center for Food
Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD
2
0740, USA
f
Department of Radiology, Affiliated Hospital of Nanjing University of Chinese
Medicine, Nanjing 210029, P. R. China
Corresponding authors
*
Phone/Fax：025-83272496, E-mail: zhangyu@seu.edu.cn; guning@seu.edu.cn
GRAPHICAL ABSTRACT

HIGHLIGHTS



PBNPs possess ascorbic acid oxidase (AAO)-like activity
PBNPs possess ascorbic acid peroxidase (APOD)-like activity
Unlike other iron-based NPs, PBNPs inhibited the anti-cancer efficiency of AA
Abstract: Herein we reported Prussian blue nanoparticles (PBNPs) possess ascorbic
acid oxidase (AAO)- and ascorbic acid peroxidase (APOD)-like activities, which
suppressed the formation of harmful H
2
2
O and finally inhibited the anti-cancer
efficiency of ascorbic acid (AA). This newly revealed correlation between iron and
AA could provide new insight for the studies of nanozymes and free radical biology.
Keywords: Prussian blue nanoparticles, ascorbic acid oxidase, peroxidase, electron
spin resonance, anti-cancer efficiency
1
Introduction
Ascorbic acid (AA) was found very early to be able to induce cancer cell death via
improving the intracellular reactive oxygen species (ROS) level. [1, 2] Mechanism
researches showed high-dose AA treatment induces ROS increment in cancer cells via
the auto-oxidation of AA (equations 1 and 2) and the enhanced cellular uptake
dehydroascorbic acid (DHA), the former generate by-product H
2
2
O , while the latter
can oxidize intracellular reduced enzymes during its reduction. [3-5] As shown in
equations 3-5, iron is considered to be able to reinforce the anti-cancer effect of AA
via Fenton reaction, which is the name given to the reaction of hydrogen peroxide and
a ferrous iron catalyst. [6]

−
2
퐴퐴 + 푂 → 2퐴퐴 · + 퐻 푂 (1)
ꢀ ꢀ ꢀ
−
2
퐴퐴 · + 푂 → 2퐷퐻퐴 + 퐻 푂
ꢀ ꢀ
(2)
ꢀ
퐹푒ꢀꢁ + 퐻 푂 → 퐹푒 +· 푂퐻 + 푂퐻 (ꢂ)
3ꢁ
−
ꢀ
ꢀ
퐹푒3ꢁ + 퐻 푂 → 퐹푒 +· 푂푂퐻 + 퐻 (4)
ꢀꢁ
ꢁ
ꢀ
ꢀ
퐴퐴 + 퐹푒3ꢁ → 퐴퐴 · + 퐹푒
−
ꢀꢁ
(5)
In good agreement with the data from other literatures, [7, 8] we found iron oxide
nanoparticles (IONPs) greatly strengthened the anti-cancer efficiency of AA in 4T1
and MCF7 cells. However, as an iron-based nanomaterial, Prussian blue nanoparticles
(PBNPs) inhibited the anti-cancer effect of AA. Further experiments suggested
PBNPs reduced the intracellular ROS level while IONPs increased the ROS level in
the cells incubated with AA. In our previous studies, [9, 10] we reported that PBNPs
are biologically active as antioxidants because of their peroxidase-, catalase- and
superoxide dismutase-like activities arising from their electron transfer ability.
Therefore, we inferred PBNPs may play as an AA-related nanozyme, which can
catalyze the oxidation of AA without producing H
2
O .
2
-
Ascorbyl radical (AA· ) is an intermediate having 10 min half-life formed during
the oxidation of AA and this radical can be directly detected by electron spin
resonance (ESR) method. ESR measurements showed PBNPs can catalyze the
oxidation of AA in the absence and presence of H
ascorbic acid oxidase (AAO)-like activity besides ascorbic acid peroxidase
APOD)-like activity. AAO and APOD are both ROS-scavenging enzymes in plants,
2
O
2
, indicating PBNPs possess
(
thus PBNPs may play as ROS-scavenging enzymes in the presence of AA in
mammalian cells. Our study show iron plays different roles in different chemical
environments such as in PBNPs and IONPs. Certainly, more specific explanation of
these phenomena should be given with more exploration.
2
Experimental Section
2
.1 Reagents and materials
All chemicals used in this experiment were of analytical reagent grade. Deionized
water was used throughout the study. Dimercaptosuccinic acid (DMSA) was
purchased from Shanghai Zhuorui Chemical Co., Ltd. (Shanghai, China). Polyvinyl
pyrrolidone (PVP), hydrogen peroxide (30%) and terephthalic acid (TA) was
purchased from from Aladdin Co., Ltd. (Shanghai, China). FeCl
3
·6H
2
O, FeSO
4
·7H
2
O,
K
4
[Fe(CN) ]·3H O, ammonia water, oleic acid (OA), ethanol, n-haxane and
6
2
dimethylsulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). Tert-butyl alcohol (TBA) was obtained from Shanghai
Lingfeng Chemical Reagent Co., Ltd. (China). Ascorbic acid (AA) was obtained from
Xilong Scientific Co., Ltd. (Shantou, Guangdong, China). Spin label 3-carbamoyl-2, 2,

5
, 5-tetramethyl-3-pyrroline-1-yloxyl（CTPO, ≥98%）was obtained from Radical
Vision (Marseille, France). Fetal bovine serum (FBS) and the RPMI 1640 cell culture
medium were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
2
3
3
’,7’-Dichlorodihydrofluorescein
,3’,5,5’-Tetramethylbenzidine (TMB), nitroblue tetrazolium (NBT), and
-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
diacetate
(DCFH-DA)
,
purchased from Sigma-Aldrich (St. Louis, MO, USA). The murine breast cancer cell
line 4T1 and the human breast cancer cell line MCF7 were obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA).
2
.2 Synthesis of Prussian blue nanoparticles (PBNPs) and iron oxide nanoparticles
(IONPs)
PBNPs and IONPs were prepared according to our previous reported works.[9, 11]
Briefly, PBNPs were prepared by adding 20 mL of K
a 80 mL mixture of FeCl ·6H O (0.1 mmol) and PVP (K-30, 10 mmol in terms of
monomer) in water with vigorously stirring at 60ºC.[12] A micro-scale injection pump
WZS-50F6, Smiths Medical) was used to accurately control the adding speed at 40
4
[Fe(CN)
6
]·3H
2
O (0.1 mmol) to
3
2
(
mL/h. The reaction was kept for 30 min before cooled to room temperature under
ambient condition. The dialysis was carried out using a MILLIPORE TTF system
(
Tangential flow filtration system, MWCO: 130 kDa) in order to exclude remaining
impurity ions. The dialyzed solution was then filtrated with 220 nm filter to remove a
small quantity of aggregates. To prepare IONPs, 28 g of FeCl ·6H O and 20 g of
FeSO ·7H O were dissolved in 80 mL of deoxygenized water by bubbling nitrogen
3
2
4
2
and heated to in a 72ºC water bath. 40 mL of ammonia water was added under
vigorously stir, 9 mL of OA was added 5 min later and continued to stir for 3 h. Next,
remove the ammonia water at 85ºC and then cooled to room temperature, removed the
impurities by magnetic separation and ethanol lavation and dissolved the product in
n-hexane. 100 mL of Fe
3
4
O @OA solution (4 mg/mL, in n-hexane) was mixed with
1
00 mL of DMSA solution (2 mg/mL, in acetone). Then reflux condensation was
conducted at 60ºC. Finally, the product was washed with pure water and dissolved in
water, pH was adjusted to 10. Dialyze the solution at pH 7 and the dialyzed solution
was then filtrated with 220 nm filter and reserved at 4ºC. The concentration of PBNPs
was determined based on a standard curve, which was set up according to the
absorbance values at 710 nm of samples of known concentrations prepared by
freeze-dried PBNPs powder and calculated by the thermogravimetric analysis.
2
.3 Characterization of PBNPs
The particle morphology of the PBNPs and IONPs obtained above were
characterized by transmission electron microscopy (TEM, JEOL JEM-2100) with a
working voltage of 200 kV.
2
.4 Determination of cell viability

MTT method was used to evaluate the cytotoxicity and estimated IC50 (half
maximal inhibitory) of AA. 4T1 and MCF7 cells were added into each well in a
9
6-well plate and incubated for 24 h. The culture medium was replaced by fresh
medium containing different concentrations of AA, PBNPs and/or IONPs and
exposed for 24 h. After washing the cells with serum-free medium, 20 μL of 5 mg/mL
MTT solution in PBS-serum free medium was added to each well and incubated for 4
h. Finally, DMSO was added and the optical density (OD) was measured at 570 nm
with a microplate reader (Model 680, BIO-RAD, USA) after a 4 h-incubation.
Background values of PBNPs and IONPs were deducted, which were counted as the
OD at 570 nm of the cells incubated with 150 μL serum-free medium without MTT
solution.
2
.5 Detection of ROS
T1 and MCF7 cells were added into 35 mm dishes and incubated for 24 h. The
4
culture medium was replaced by fresh medium containing different concentrations of
AA in the presence and absence of PBNPs or IONPs and exposed for 24 h. The
concentration of AA and nanoparticles in MCF7 cells were 5 mM and 5 μg(iron)/mL,
respectively, while 4T1 cells were cultivated with 0.2 mM AA and 5 μg(iron)/mL
nanoparticles. The cells were collected and counted by a cell counting machine
(Cellometer Auto 1000, Nexcelom Bioscience, USA).
Intracellular ROS levels were evaluated using the oxidant-sensitivity dye
DCFH-DA as probe in a flow cytometry (BD FACS, USA) with fluorescence
intensity excited at 488 nm and recorded at 525 nm. Each sample containing 5 million
viable MCF7 or 4T1 cells was incubated in 1 mL 1640 serum-free medium with 10
μM DCFH-DA at 37°C for 20min in dark. Next, the cells were then washed with PBS
three times to thoroughly remove extracellular probe.
The hydroxyl radical (·OH) level was assessed using TA, a very specific detector
of hydroxyl radicals. [13-16] TA reacts with · OH to form fluorescent
hydroxyterephthaacid (HTA). 1 mL of 2 mM TA solution diluted with 50 mM
phosphate buffer (pH 7.5) was added into each sample containing 3 million cells and
incubated in dark for 30 min. The fluorescence of each solution was measured using a
multimode reader (Infinite 200 PRO, TECAN, Switzerland) with an excitation and
emission wavelengths of 315 and 425 nm, respectively.
-
The superoxide anion (O
2
· ) content was measured by NBT. [17-19] Firstly, 0.5
mg/mL NBT solution was prepared by dissolving 50 mg of NBT power in 100 mL of
PBS buffer (pH 7.4) and filtered with a 0.22 μm filter (Millipore Express). Secondly,
1
mL of NBT solution described earlier was added into each sample containing 1
million cells and incubated at 37°C for 30 min. Finally, the solutions were centrifuged
at 1000 rpm for 5 min and the precipitations were dissolved in DMSO and the OD at
5
60 nm was measured with a multimode reader (Infinite 200 PRO, TECAN,
Switzerland).
In pH 3.6 buffer, 0.4 mg/mL TMB, 1.2 M H
2
2
O , 0.1 μg[Fe]/mL PBNPs or IONPs
were reacted in the presence and absence of TBA. The absorbance (at 650 nm for

TMB[20]) of the color reaction was recorded at a certain reaction time via a
Microplate Reader (BIO-RAD model 680) to express the generation of ·OH
-
2
.6 Detection of Ascorbyl radical (AA· ) by electron spin resonance (ESR) method
To detect the APOD- and AAO-like activities of PBNPs, ESR was used to record
-
the AA· signals in pH 7.4, 5.0 and 3.9 buffer solutions. Sample solutions contained
different concentrations of AA and PBNPs in the presence or absence of H
spectra of AA· were recorded under the following instrumentation conditions:
microwave power 20 mW; field modulation 1 G; sweep width 30 G.
2
O . ESR
2
-
2
.7 Detection of oxygen content
ESR oximetry was chosen to monitor the oxygen content of the reaction system.[9]
Oxygen concentrations were thus obtained by the ESR spectra of spin label CTPO.
PBNPs, AA and H were mixed in a buffer solution of pH 7.4, and then CTPO was
used to trap the proton super hyperfine interaction. Sample solutions contained 0.1
mM CTPO, 5 mM AA, 10 mM PBS (pH 7.4), 5 mM H and 500 μM PBNPs. ESR
2
O
2
2
O
2
spectra were recorded under the following instrumentation conditions: microwave
power 1 mW; field modulation 0.04 G; sweep width 5 G. The AAO-like activity
detection of PBNPs was also carried out at room temperature by direct measurement
of the oxygen content using a specific oxygen electrode on a Multi-Parameter
Analyzer (DZS-708, Cany Precision Instruments Co., Ltd). 5 mM AA, 10 mM PBS
(pH 7.4), 5 mM H
2
O
2
and 500 μM PBNPs were mixed and the O solubility (unit:
2
mg/L) was measured at different reaction time (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20
min).
3
. Results and Discussion
PBNPs and IONPs were prepared according to earlier described methods and
characterized using TEM (Figure 1). The average sizes of IONPs and PBNPs were
calculated to be approximately 8.5 nm and 31.4 nm, respectively. Before formal
experiments, possible cytotoxicity of IONPs and PBNPs had been checked and half
maximal inhibitory (IC50) of AA to MCF7 and 4T1 cells was measured. The cell
viability results (Figure S1) showed IONPs and PBNPs have no significant
cytotoxicity to 4T1 and MCF7 cells at concentrations below 12.5 μg(iron)/mL. IC50
values for 24 h of AA treatment of MCF7 and 4T1 cells were 5.13 mM and 0.22 mM,
respectively (Figure S2). As shown in Figures 2a and b, 5 mM and 0.2 mM of AA can
remarkably harm the MCF7 and 4T1 cells, leading to ca. 55% of cell viability if AA is
used alone. When 5 mM AA was added to the culture medium in the presence of 1.25
μg(iron)/mL IONPs, the MCF7 cell viability was dramatically decreased (p<0.05).
Moreover, a successive increase in the concentration of IONPs induced escalating
cytotoxicity. However, the addition of PBNPs made the opposite impact. From the
free radical biology perspective, we hypothesized the different effects of IONPs and
PBNPs could be attributed to their different roles in the AA-mediated production of
ROS. Intracellular ROS (Figures 2c and d) suggested IONPs do play as a

peroxidase-like enzyme, [21] which enhanced the generation of ·OH when exposed
to the oxidative stress environment induced by AA, leading to an enhanced
cytotoxicity to 4T1 and MCF7 cells in a dose-dependent manner. Moreover, the
-
intracellular O
2
·and ·OH levels were determined by NBT and TA, respectively. The
results (Figure S3a) show ·OH level was significantly elevated by IONPs (p<0.05).
However, PBNPs do function as an antioxidant in the same oxidative stress
environment (Figures 2c, d and S3). The function of IONPs was thought to be induced
by Fenton reaction and the mechanism of PBNPs does need further exploration.
[21-23] To further verify the role of·OH , ·OH trapping agent was introduced to the
pH 3.6 buffer systems containing TMB, H in the presence of PBNPs or IONPs. It
2
O
2
can be seen from the results (Figure S4) that unlike IONPs, the peroxidase-like
activity of PBNPs did not exert via the generation·OH generation, consistent with our
earlier data. [21]
Figure 1. Characterizations of IONPs and PBNPs. (a) TEM image of IONPs, (b) size
distribution of IONPs, (c) TEM image of PBNPs and (d) size distribution of PBNPs.

Figure 2. Effects of the concentration of IONPs and PBNPs on MCF7 (a) and 4T1 (b)
cell viability with treatment of 5 mM and 0.2 mM AA, respectively. The data were
normalized to control values (no particle and AA exposure), which were set as 100%
cell viability; # indicates p<0.05 as compared with control group and * indicates p <
0
.05 as compared to AA group (no particle exposure), mean SEM, n = 5. Treated
MCF-7 (c) and 4T1 (d) cells were assessed for intracellular ROS generation by
measuring DCF-fluorescence and analyzed by a flow cytometer. The concentration of
AA and nanoparticles in MCF7 cells were 5 mM and 5 μg(iron)/mL, respectively,
while 4T1 cells were cultivated with 0.2 mM AA and 5 μg(iron)/mL nanoparticles.
To further study the reaction of PBNPs, H
the intermediate products. As shown in Scheme 1, AA· is a free radical generated
2
O and AA, ESR was used to monitor
2
-
during the oxidation of AA, which can be directly recorded by ESR. As shown in
-
Figure 3, the AA· signals in reaction solutions without PBNPs (-PBNPs groups)
-
indicate the auto-oxidation of AA is quite slow. Comparing the signals of AA· in pH
7
.4, 5.0 and 3.9 buffer solutions, we can conclude that PBNPs catalyzed the oxidation
-
of AA by H
2
O . Moreover, the AA· signal intensity was positively associated with
2
the concentration of PBNPs and the pH levels. Meanwhile, the data in Figure S5 show
-
there is also a positive correlation between the AA· signal intensity and AA
concentration. These results further verify that PBNPs are peroxidase mimics taking
AA as substrate (equations 6 and 7).
푃퐵푁푃푠/ꢃ푃ꢄꢅ
−
2
퐴퐴 + 퐻 푂 ꢆꢇꢇꢇꢇꢇꢇꢇꢇꢇꢈ 2퐴퐴 · + 2퐻 푂 (6)
ꢀ ꢀ ꢀ

푃퐵푁푃푠/ꢃ푃ꢄꢅ
−
2
퐴퐴 · + 퐻 푂 ꢆꢇꢇꢇꢇꢇꢇꢇꢇꢇꢈ 2퐷퐻퐴 + 2퐻 푂 (7)
ꢀ
ꢀ
ꢀ
Scheme 1. Oxidation of AA in the presence of O
2
and catalyzed by AAO.
Figure 3. APOD-like activity of PBNPs detected by ESR spectrum of the ascorbyl
radical. Sample solutions contained 5 mM AA, buffers (pH 7.4, 5.0, 3.9), 5 mM H
2
O
2
and different concentrations of PBNPs. ESR spectra were collected at 1 min after
sample mixing.
As shown in our previous study, PBNPs possess catalase-like activity (equation 8)
at pH 7.4. [9] In this study, we monitor the oxygen content in pH 7.4 reaction system
to investigate the catalase-like activity of PBNPs. CTPO is quite sensitive to oxygen
concentration, thus ESR oximetry was usually used to monitor the small variations of
oxygen concentration. [9, 24] Less evident resolution of the super hyperfine structure
of the low-field line of ESR spectra of CTPO represents higher oxygen concentration.
As shown in Figure 4a, at pH 7.4, the resolution of super hyperfine structure of CTPO
in the solution containing AA and PBNPs gradually became evident in 20 min,
indicating the consumption of O
S6, the oxygen content as also monitored by a specific oxygen electrode and the
results verify the consumption of O
2
in a closed capillary. Similarly, as shown in Figure
2
.
푃퐵푁푃푠/푐푎푡푎푙푎푠ꢉ
퐻 푂 ꢆꢇꢇꢇꢇꢇꢇꢇꢇꢇꢇꢇꢈ 2퐻 푂 + 푂
2
(8)
(9)
(10)
ꢀ
ꢀ
ꢀ
ꢀ
푃퐵푁푃푠/ꢃꢃꢄ
−
2
퐴퐴 + 푂 ꢆꢇꢇꢇꢇꢇꢇꢇꢈ 2퐴퐴 · + 퐻 푂
ꢀ ꢀ ꢀ
푃퐵푁푃푠/ꢃ푃ꢄꢅ
−
2
퐴퐴 · + H 푂 ꢆꢇꢇꢇꢇꢇꢇꢇꢇꢈ 2퐷퐻퐴 + 2퐻 푂
ꢀ ꢀ ꢀ

The disappearance of oxygen in solution during oxidation of AA catalysed by
PBNPs hints PBNPs may catalyse the oxidation of AA by oxygen. ESR signal
-
intensity of AA· (Figure 4b) in the reaction solution contained only AA and PBNPs
(
without H
2
O ) is dependent on the concentration of PBNPs, with the strongest signal
2
-
at pH 7.4. The amount of AA· in the pH 7.4 solution reaches a plateau 300 s later
while the auto-oxidation of AA is quite slight during 300 s (Figure 4c). The reaction
process of AAO-like activity can be expressed by equations 9 and 10. Altogether,
PBNPs can catalyze reactions consuming H
2
2
O due to their APOD and AAO-like
activities, which subsequently lead to the protective effect on MCF7 and 4T1 cells
from the killing of AA.
Figure 4. AAO-like activity of PBNPs. (a) ESR spectrum of the spin label CTPO
during the oxidation of AA catalyzed by PBNPs. Sample solutions contained 0.1 mM
CTPO, 5 mM AA, 10 mM PBS (pH 7.4) and 500 μM PBNPs. ESR spectra were
obtained at 0, 5, 10, 15 and 20 min after addition of PBNPs. (b) AAO-like activity of
PBNPs detected by ESR spectrum of the ascorbyl radical. Sample solutions contained
0
.1 mM CTPO, 5 mM AA, 10 mM PBS (pH 7.4) and 50 μM PBNPs. (c) The signal
intensity was measured as the peak-to-peak value of the first line of the spectrum.
ESR spectra were collected at 1 min after sample mixing.
4
Conclusions
In conclusion, we demonstrated that PBNPs possess APOD- and AAO-like activities.
The results of cellular experiment taking AA as a model molecule show iron plays
different roles when placed in the different chemical environments of PBNPs and
IONPs. These data provide new insights in the study of iron-based enzymes and
iron-based nanomaterials.

Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This research was supported by the National Key Research and Development
Program of China (No. 2017YFA0205502), the National Basic Research Program of
China (973 program No. 2013CB733800), National Natural Science Foundation of
China (No. 81571806, 81671820, 81301870), the Jiangsu Provincial Special Program
of Medical Science (No. BL2013029), the Fundamental Research Funds for the
Central Universities. This work was also partially supported by a regulatory science
grant under the FDA Nanotechnology CORES Program. This paper is not an official
US FDA guidance or policy statement. No official support or endorsement by the US
FDA is intended or should be inferred.
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