Multifunctional iron-based Metal?Organic framework as biodegradable nanozyme for microwave enhancing dynamic therapy

Article abstract of DOI:10.1016/j.biomaterials.2019.119223

Nanozymes with excellent enzyme-mimicking catalytic property are playing an increasingly significant role in tumor diagnosis and therapy. Fe-metal organic framework nanoparticles (MIL-101(Fe) NPs) are prepared as nanozymes to generate reactive oxygen spec

Full text of DOI:10.1016/j.biomaterials.2019.119223

Accepted Manuscript
Multifunctional iron-based Metal−Organic framework as biodegradable nanozyme for
microwave enhancing dynamic therapy
Xiaoyan Ma, Xiangling Ren, Xudong Guo, Changhui Fu, Qiong Wu, Longfei Tan,
Hongbo Li, Wei Zhang, Xudong Chen, Hongshan Zhong, Xianwei Meng
PII:
S0142-9612(19)30314-X
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119223
Article Number: 119223
Reference:
JBMT 119223
To appear in: Biomaterials
Received Date: 28 January 2019
Revised Date: 1 May 2019
Accepted Date: 21 May 2019
Please cite this article as: Ma X, Ren X, Guo X, Fu C, Wu Q, Tan L, Li H, Zhang W, Chen X, Zhong
H, Meng X, Multifunctional iron-based Metal−Organic framework as biodegradable nanozyme
for microwave enhancing dynamic therapy, Biomaterials (2019), doi: https://doi.org/10.1016/
j.biomaterials.2019.119223.
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ACCEPTED MANUSCRIPT
MultifunctionalIron-Based Metal−Organic
Framework as Biodegradable Nanozyme for
Microwave Enhancing Dynamic Therapy
Xiaoyan Ma,ab Xiangling, Ren,a Xudong Guo,b Changhui Fu,a Qiong Wu,a Longfei
Tan,a* Hongbo Li,c Wei Zhang,d Xudong Chen,d Hongshan Zhong,e Xianwei Menga*
aLaboratory of Controllable Preparation and Application of Nanomaterials, Laboratory
of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of
Sciences, 29 Zhongguancun East Road, Beijing, 100190, China
E-mail: mengxw@mail.ipc.ac.cn, longfeitan@mail.ipc.ac.cn
bState Key Laboratory of Reproductive Regulation & Breeding of Grassland
Livestock, Inner Mongolia University, 24 Zhaojun Road, Hohhot, 010070, China
cBeijing Key Laboratory of Construction-Tailorable Advanced Functional Materials
and Green Applications, School of Materials Science & Engineering, Beijing Institute
of Technology, Beijing 100081, China
d First Affiliated Hospital of Southern University of Science and Technology, Shenzhen
People's Hospital & Shenzhen Interventional Medical Engineering Research and
Development Center, Shenzhen 518020, China
eDepartment of Radiology, First Hospital of China Medical University, Shenyang
110001, People's Republic of China
KEYWORDS: nanozyme, metal-organic framework, enzyme mimics, microwave,
cancer therapy

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ABSTRACT
Nanozymes with excellent enzyme-mimicking catalytic property are playing an
increasingly significant role in tumor diagnosis and therapy. Fe-metal organic
framework nanoparticles (MIL-101(Fe) NPs) are prepared as nanozymes to generate
reactive oxygen species (ROS) and induce cancer cell death by catalyzing endogenous
substances in tumor microenvironment. Abundant stimuli-responsive hydroxyl
radicals (·OH) are accelerated to generate in the presence of microwave irradiation,
realizing microwave enhancing dynamic therapy (MEDT). Moreover, MIL-101(Fe)
NPs possess biodegradability and bioresponsibility, which exhibit favourable
properties of metabolism and non-toxic accumulation comparing with inorganic
nanozymes. Fluorescent gold nanoclusters (BSA-Au NCs) are rapidly coupled with
the surface of MIL-101(Fe) NPs to obtain MIL-101(Fe)@BSA-AuNCs NPs.
MIL-101(Fe)@BSA-AuNCs NPs with magnetic resonance imaging (MRI) and
fluorescent imaging (FI) not only image accurately for the site of tumor, but also
monitor dynamic distribution process of MIL-101(Fe) in vivo. The signal intensity of
FI and MRI reaches maximum at 1 h in the liver and 5 h in the tumor. Ionic liquid (IL)
is also loaded into MIL-101(Fe)@BSA-AuNCs NPs as microwave sensitive reagents
for microwave thermal therapy (MTT). This work synthesizes the nanozymes that
possess degradability, microwave sensitivity and dual-mode imaging to achieve the
combination of MTT and MEDT against tumor. Experiment result in vivo confirms
that the kill rate of tumor is up to 96.65 %, showing an outstanding anti-tumor
efficacy.

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1. Introduction
Nanozymes, which possess desirable enzyme-mimicking catalytic property based
on nanomaterials, have sparked increasing interest from cross-discplinary scientific
community in recent years [1-5], for example, iatrology, environmental protection,
and materialogy, etc. In iatrology field, increasing nanozymes have been applied for
tumor diagnostic imaging [6-8], enhancing radiotherapy efficiency [9-10]. Moreover,
nanozymes have been reported for dynamic therapy, for example, enhancing
photodynamic therapy [11-12], and chemodynamic therapy (CDT) [13]. CDT has
been achieved by catalyzing endogenous chemical energy to generate reactive oxygen
species without external energy, which induce cell death. Although CDT is promising,
research teams still need find new strategies to improve the generation rate of
hydroxyl radicals (•OH) in tumor site and effectively control the process of tumor
treatment, realizing precise treatment ultimately. To achieve effective, controllable
and precise treatment, external stimulation is primary choice. Hence, it is of great
importance to find an idea external stimulus for future clinical application.
Microwave (MW), as a clinical tumor treatment method, possesses advantages of
low side effect and deep penetration, which is an ideal external stimulus [14-15]. Thus,
it is desired to apply external stimulus of MW to generate ·OH in a more efficient way.
Abundant ·OH in the tumor microenvironment are accelerated to generate in the
presence of MW irradiation, which is defined as microwave enhancing dynamic
therapy (MEDT). Hence, it is crucial to design and synthesize nanozymes with MW
response for the generation of ·OH and realizing MEDT.

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The metal-organic framework nanoparticles (MOF NPs) with tunable porous
structure and high specific surface area have great potential to act as MW sensitive
agents, which promote the MW energy conversion owing to ions collision in the
porous structure of MOF [14,16]. Meanwhile, it is well known that iron-based
compounds possess peroxidase-like catalytic activity [17]. Therefore, it is promising
to design and synthesize Fe based MOF NPs with both MW response and catalytic
property as nanozymes. What's more, MOF NPs possess good degradability, which
have unique application function for drug release as biosafe therapeutic nanoagents
[18,19]. Comparing with reported undegradable inorganic nanozymes (MnO2, Fe3O4),
degradable MOF nanozymes decrease significantly the toxicity in vivo. [20,21].
Furthermore, MOF nanozymes as a nanocarrier system possess bioresponsibility
based on the unique physicochemical property to accomplish enhanced therapeutic
specificity and simplicity [22-26]. The biocompatible and biodegradable nanozymes
promote great progress in biomedical applications of cancer theranostics [27]. MOF
NPs are composited initially multi-functional composite nanomaterials to apply for
tumor therapy [16,28-52]. Especially, MOF NPs as nanozymes can play high
multifunctional efficiency against tumor by combining with the properties of MOF
NPs [53-55].
To achieve the aforementioned aims for tumor therapy, we have designed
Fe-metal organic frameworks (MIL-101(Fe)) as nanozymes with degradability for
tumor therapy (Scheme 1). MIL-101(Fe) mimicing enzymes are capable of catalyzing
H2O2 in tumor microenvironment to generate ·OH. Under MW irradiation, amounts
of ·OH are generated into the tumor with an efficient and noninvasive manner, which

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realizes MEDT. Meanwhile, a rapid assembly method was adopted to achieve the
coupling of fluorescence gold nanoclusters (BSA-Au NCs) on surface of
IL@MIL-101(Fe) NPs without the presence of fluorescence quench to obtain
MIL-101@BSA-AuNCs NPs. Magnetic resonance imaging (MRI) and fluorescence
imaging (FI) of MIL-101(Fe)@BSA-AuNCs NPs are used for monitoring dynamics
distribution process of MIL-101(Fe) NPs in vivo and accurately diagnosing tumor site.
The signal intensity of FI and MRI becomes weak gradually because of significant
structural collapse of MIL-101(Fe) NPs. Due to the porous structure,
1-octy1-3-methylimidazolium hexafluorophosphate (IL) are loaded on MIL-101(Fe)
NPs to prepare IL@MIL-101(Fe)@BSA-AuNCs NPs as thermal sensitization agents,
achieving tumor synergetic therapy of microwave thermal therapy (MTT) and MEDT.
IL@MIL-101(Fe)@BSA-AuNCs NPs mainly set synergetic therapy and imaging
together, which have inestimable value for future clinical application.
2. Experimental Section
2.1 Synthesis of MIL-101(Fe) NPs
The MIL-101(Fe) NPs were prepared by solvothermal method. 0.0601 g of
H2BDC, 0.0611 g of H2BDC-NH2, and 0.0676 g of FeCl3·6H2O were dissolved in 100
mL of DMF, the mixed solution was packaged in Teflon-lined stainless-steel
autoclave and heated to 120 °C for 12 h in an oven. The precipitate obtained by
centrifugation was washed three times with ethanol after cooling to room temperature.
2.2 Synthesis of IL@MIL-101(Fe) NPs
10 mg of MIL-101(Fe) NPs were dispersed in 3 mL of diethylene dioxide solution,
then 100 ꢀL of IL were added. The mixture was stirred for 2 h in the dark.
IL@MIL-101(Fe) NPs were collected and washed with ethanol twice by

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centrifugation.
2.3 Synthesis of BSA-Au NCs
300 mg of bovine serum albumin (BSA) were dissolved in 6 mL of H2O, and 8 mL
of 10 mM chloroauric acid was added to the mixture. After stirring for 2 min, 0.65
mL of 1 M NaOH was added. The mixture was placed in oven at 100 °C and allowed
to stand for 1 h after stirring.
2.4 Synthesis of IL@MIL-101(Fe)@BSA-AuNCs NPs
2 mL of fluorescent gold nanoclusters were dispersed in 15 mL of H2O and
activated by stirring with 1 mL of EDC and NHS for 15 min. When the temperature
reached 60 °C, 5 mg of IL@MIL-101(Fe) NPs were added with stirring for 60 s.
IL@MIL-101(Fe)@BSA-AuNCs NPs were separated by centrifugation and washing
with H2O.
2.5 IL@MIL-101(Fe) NPs MW Heating Experiment in Vitro
Microwave heating experiment confirmed that the IL@MIL-101(Fe) NPs
possessed MW thermal sensitizing property. The IL@MIL-101(Fe) NPs were
formulated with saline into different concentrations of 1, 3, 5, 7, and 10 mg mL-1, and
then normal saline was used as a control group, and 1.0 mL each was added to the
reaction hole and irradiated at 0.9 W for 5 min. The forward-looking infrared (FLIR)
imaging instrument was used to monitor the temperature change while accompanied
by infrared imaging. Meanwhile the thermal sensitivity of IL@MIL-101(Fe) NPs with
gold clusters coupled with the outer layer (IL@MIL-101(Fe)@BSA-AuNCs NPs)
were not changed.

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2.6 Microwave Enhancing Dynamics Experiment of MIL-101(Fe) NPs in Vitro
In this experiment, the MIL-101(Fe) NPs were dispersed in H2O to avoid the
heating effect, exhibiting no increase in temperature under MW irradiation. For the
detection of ·OH, TMB detection solution was prepared by using 1 mL of DMSO
liquid to disperse 10 mg of TMB. 20 ꢀL of TMB, 25 ꢀg mL-1 of MIL-101(Fe) NPs
and 5 ꢀL of H2O2 were dispersed in water and then supernatants were analyzed by
UV-vis spectroscopy every 5 min. Under the same condition, the mixed solution in
reaction vessel was irradiated with 0.9 MW MW for 5 min. The mixed solution was
then analyzed every 5 min by UV-vis spectroscopy. In the same manner, the
ultraviolet absorption of 2, 4, 6, and 8 ꢀL of different volumes of H2O2 before and
after MW was measured. Finally, in the same condition, the ultraviolet absorption of
0.02, 0.04, 0.06, and 0.08 ꢀg mL-1 of MIL-101(Fe) NPs before and after MW
irradiation was measured.
10 mg of terephthalic acid (TA) were dispersed in DMSO to prepare TA solution.
50 ꢀg mL-1 of MIL-101(Fe) NPs were dispersed in water with 20 ꢀL TA solution and
10 ꢀL of H2O2, and then the intensity of fluorescence was tested. In the same situation,
the intensity of fluorescence was tested under MW irradiation at 0.9 W for 5 min. The
intensity of fluorescence under MW irradiation and no MW irradiation was compared,
and the intensity of fluorescence was recorded under MW irradiation for 3, 5, 10, 15
min by fluorescence spectrophotometry.
ESR was applied to test the generation of •OH by 5, 5′-dimethylpyrroline-N-oxide.
5 ꢀL of H2O2 was dispersed in H2O with MW irradiation, and 5 ꢀL of H2O2 and 50 ꢀg

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of MIL-101(Fe) NPs were dispersed in H2O with MW irradiation. ESR spectrums
from two groups were collected.
2.7 Experimental Study on Degradation of MIL-101(Fe) NPs in Vitro
It has been confirmed that the tumor site is acidic by consulting other research
literature. Therefore, the degradation experiment of MIL-101(Fe) NPs was taken carry
out in acidic PBS solution. The appropriate amount of MIL-101(Fe) NPs were
dispersed in acidic PBS solution and placed in a constant temperature water bath at
37 °C. The sample was taken different time points for TEM characterization and size
measurement. Degradation experiment proved that the MIL-101(Fe) NPs were
degradable.
2.8 Cytotoxicity Experiment in Vitro
In order to investigate the toxicity of the as-made IL@MIL-101(Fe)@BSA-AuNCs
NPs, the MTT assay was used to explore on HepG2, L929, H22 cells. The cytoxicity
of IL@MIL-101(Fe)@BSA-AuNCs NPs was judged by the cell viability after
incubated with different concentrations of IL@MIL-101(Fe)@BSA-AuNCs NPs.
Firstly, the cells were uniformly dispersed in 96-well plate. After the cells were
adherent, then cells were incubated with different concentrations (1.5625, 3.125, 6.25,
12.5, 50, 100, 200 ꢀg mL-1) for 24 h. Cell viability was evaluated as the absorbance of
formazan at 492 nm. The control group cells (without nanomaterials) were considered
to be 100 % of survival rate.
2.9 Cell Experiments in Vitro
To access the efficacy by combination of MEDT and MTT, cell experiment was

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explored. Tumor cells were incubated with 100 ꢀg mL-1 of
IL@MIL-101(Fe)@BSA-AuNCs NPs for 24 h. The cells were irradiated with 0.9 W
for 3, 5 and 8 min, then the cell relative activity was accessed by MTT assay. MW
group and IL@MIL-101(Fe)@BSA-AuNCs + MW group verified the anti-tumor
effect of IL@MIL-101(Fe)@BSA-AuNCs by combining MEDT with MTT.
2.10 Animal Toxicity Experiment
To further evaluate to the maximum safe dose of IL@MIL-101(Fe)@BSA-AuNCs
NPs in mice, animal toxicity experiment was studied. After the preliminary
experiment, 20 experimental mice were selected and divided into 4 groups.
IL@MIL-101(Fe)@BSA-AuNCs NPs were injected into the mice at doses of 100,
150 and 200 mg kg-1, mice injected PBS were used as control. After 15 days, mouse
blood was collected for further routine blood and blood chemistry examinations. At
the same time, the heart, spleen, liver, lungs and kidneys were soaked in formalin
solution for further histological examination. Tissue sections of mouse organs with
different doses of material were contrasted to observe the difference.
2.11 Tumor Therapy Experiment
The anti-cancer effect of IL@MIL-101(Fe)@BSA-AuNCs NPs by combining
with MDT and MTT at mice was further evaluated. When the tumor diameter of
female mice bearing H22-tumor was about 8 mm, experimental mice were randomly
divided into the following four groups: (1) control group, (2) MW, (3)
IL@MIL-101(Fe)@BSA-AuNCs, (4) IL@MIL-101(Fe)@BSA-AuNCs + MW (The
control group was injected with PBS). The injection dose was 50 mg kg-1 and the

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above materials were injected into the mice through the tail vein. After 5 h of
injection, microwave irradiation was performed for microwave experimental groups
with MW irradiation (0.9 W) for 5 min, and the process of temperature change was
recorded by FLIR imaging instrument. The mice were recorded daily for body weight
and tumor volume. At the end of the experiment, heart, spleen, liver, lungs, and
kidneys of mice were soaked in formalin solution for further histological examination
to observe the histological section of the mouse organs.
2.12 Animal Experiments
All animal studies were approved by Guide for the Care and Use of Laboratory
Animals, Eighth Edition (International Publication No: 978-0-309-15400-0) and was
approved by the Institutional Animal Care and Use Committee (IACUC) of the
Chinese PLA General Hospital Animal Care and Use Committee.
3. Results and Discussion
3.1 Synthesis and Characterization of IL@MIL-101(Fe)@BSA-AuNCs
IL@MIL-101(Fe)@BSA-AuNCs NPs were synthesized through three steps: first,
MIL-101(Fe) NPs were prepared by the solvothermal method [54-56]. MIL-101(Fe)
NPs possessed good dispersibility, uniform size. The size of MIL-101(Fe) NPs
measured by scanning electron microscope (SEM) (Fig. 1a) and transmission electron
microscopy (TEM) was around 150 nm (Fig. 1b). The dynamic light scattering was
used to measure the average hydrodynamic diameter of MIL-101(Fe) NPs, showing
170.6 nm (Fig. S1a). In the second step, IL with excellent MW heating effect was
loaded into MIL-101(Fe) NPs to prepare IL@MIL-101(Fe) NPs [59]. As-made

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IL@MIL-101(Fe) NPs also possessed the property of MW sensitization. The element
mapping of F and P in IL@MIL-101(Fe) NPs manifested IL was successfully loaded
into MIL-101(Fe) NPs (Fig. S2). Au NPs had more and more application in catalytic
reaction, sensitive detection, and diagnostics [59-62]. Thence, BSA-Au NCs were
chose basing on the hydrophilicity and security of BSA-Au NCs. In the third step,
BSA-Au NCs were prepared according to the initial literature, which showed
excitation and emission peaks at 544 nm and 640 nm [63]. BSA-Au NCs were
coupled with the surface of the as-made IL@MIL-101(Fe) NPs by coupling method to
synthesize IL@MIL-101(Fe)@BSA-AuNCs NPs, which possessed the fluorescence
property. The shape of IL@MIL-101(Fe)@BSA-AuNCs NPs was approximate sphere,
which could be observed from SEM (Fig. 1c) and TEM (Fig. 1d). The hydrodynamic
diameter of IL@MIL-101(Fe)@BSA-AuNCs was 261.9 nm by dynamic light
scattering (Fig. S1b). To further investigate whether the BSA-Au NCs were coupled
with the surface of IL@MIL-101(Fe), the element mapping were carried out. The
element mapping of Au, Fe, O, and C in IL@MIL-101(Fe)@BSA-AuNCs was
showed in Fig. 1e, it demonstrated the BSA-Au NCs were successfully coupled with
the surface of IL@MIL-101(Fe). The fluorescent IL@MIL-101(Fe)@BSA-AuNCs
NPs were similar to BSA-Au NCs with emission peak at 640 nm. The fluorescence
spectrums of IL@MIL-101(Fe)@BSA-AuNCs NPs were gradually increased with the
concentration from 25 to 400 ꢀg mL-1. As the concentration increased, the
fluorescence intensity also increased (Fig. 1f, S3a). IL@MIL-101(Fe)@BSA-AuNCs
NPs possessed good stability in PBS ( pH=7.4) and DMEM (with 5 % serum) at 0 to

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10 h and exhibited subsidence at 20 h (Fig. S4). Fourier Transform Infrared
Spectroscopy was used to analyze the functional groups of IL, BSA-Au NCs,
MIL-101(Fe) and IL@MIL-101(Fe)@BSA-AuNCs. The mainly characteristic peak of
IL was 1567 cm-1, showing the vibration of the imidazole skeleton. The normal
characteristic peak of IL at 674 cm-1 was showed. Especially, the characteristic peak
of IL appeared at 845 cm-1, which showed the stretching vibration of P-F bond. Above
all, it indicated IL was loaded successfully into MIL-101(Fe) NPs. Mainly
characteristic peak of BSA-Au NCs was 2877 cm-1, which exhibited in
IL@MIL-101(Fe)@BSA-AuNCs. The characteristic peaks of MIL-101(Fe) NPs were
3402 cm-1 and 1396 cm-1, which represented amino group and vibration absorption of
benzene ring. The main characteristic peaks of IL, BSA-AuNCs and MIL-101(Fe)
were exhibited in IL@MIL-101(Fe)@BSA-AuNCs (Fig. 1g). As Figure 1h shown
that the specific surface area of MIL-101(Fe) NPs was 1861.5 m2/g and the specific
surface area of IL@MIL-101(Fe) NPs was 827.2 m2/g (Fig. S3b). The total weight
losses of MIL-101(Fe) and IL@MIL-101(Fe) were 36.94 % and 31.27 %, thus the
loading amount of IL was 5.67 % by calculating the TGA curves of MIL-101(Fe) and
IL@MIL-101(Fe) (Fig. S3c). BET and TGA results indicated that IL were loaded
successfully into MIL-101(Fe) NPs. The pattern of MIL-101(Fe), IL@MIL-101(Fe)
and IL@MIL-101(Fe)@BSA-AuNCs detected by X-ray diffractometry showed
powder pattern of MIL-101(Fe) at low angles of 5.3°, 9.2° and IL@MIL-101(Fe) at
low angles of 10.5°, IL@MIL-101(Fe)@BSA-AuNCs with weak diffraction broad
peaks.
It
indicated
MIL-101(Fe)
NPs
were
synthesized
and

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IL@MIL-101(Fe)@BSA-AuNCs NPs have their low crystallinity (Fig. S5)
[56,58,65-66]. Above all, IL@MIL-101(Fe)@BSA-AuNCs NPs were successfully
synthesized.
3.2 IL@MIL-101(Fe) and IL@MIL-101(Fe)@BSA-AuNCs NPs Microwave Thermal
Therapy Experiment in Vitro
The hyperthermy in tumor site can effectively destroy tumor cells, IL with
excellent MW heating effect is loaded into MIL-101(Fe) NPs. Thus MW sensibility of
IL@MIL-101(Fe) NPs need be confirmed under MW irradiation. It has been
confirmed that MW sensitizer dispersed in saline could cause ions high collision
frequency under MW irradiation, resulting in the temperature increasing obviously
[14,15]. During the experiment, different concentrations of IL@MIL-101(Fe) NPs (1,
3, 5, 7, and 10 mg mL-1) were prepared. 1 mL mixed solution was under 0.9 W MW
irradiation for 5 min, 1 mL of saline was used as the control group. From Fig. 2a, the
temperature of control group was increased to 19.1 ℃ after MW irradiation. In the
same condition, the temperature of IL@MIL-101(Fe) NPs was increased to 22.2, 23.9,
27.5, 32.6 and 35.6 ℃, when the concentration was 1, 3, 5, 7, 10 mg mL-1. Compared
with the control group, the temperature was increased by 3.1, 4.8, 8.4, 13.5 and
16.5 ℃ , the data proved that IL@MIL-101(Fe) NPs possessed MW heating
sensibility (Fig. 2b). The FLIR imaging instrument was used to record the change
process of corresponding temperature (Fig. 2c). The MW heating sensibility of
IL@MIL-101(Fe)@BSA-AuNCs NPs did not change with BSA-Au NCs coupling
with the surface of IL@MIL-101(Fe) NPs (Fig. S6). Therefore, the

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IL@MIL-101(Fe)@BSA-AuNCs NPs possessed MW sensitivity to increase
temperature under MW irradiation and provide energy for generation of ROS, which
could be used for MTT in vivo [67-68].
3.3 Evaluation of Microwave Enhancing Dynamic Therapy Effect in Vitro
Interestingly, it was found that MIL-101(Fe) NPs with peroxidase-like catalytic
activity could catalyze H2O2 to generate ·OH. Meanwhile, abundant ·OH could be
accelerated to generate under MW irradiation. The generation of ·OH was tested by 3,
3', 5, 5'-tetramethylbenzidine (TMB) with emerging blue and absorption peak at 652
nm, because TMB could be oxidized by ·OH [14,69]. To avoid accelerative
generation of · OH due to thermal effect under MW irradiation, MIL-101(Fe) NPs
were dispersed in water, there was no obvious increase in temperature even under
MW irradiation for 5 min (Fig. S7).
When 10 ꢀL H2O2 were added in 50 ꢀg mL-1 of MIL-101(Fe) NPs and TMB
mixing solution and the UV spectrumer was allowed to measure immediately. As the
time went on, the intensity of ultraviolet absorption at 652 nm was gradually
increased (Fig. 3a). Under the same condition, the intensity of ultraviolet absorption
at 652 nm became stronger (Fig. 3b) under MW irradiation at power of 0.9 W for 5
min than that of no MW irradiation. It could be seen obviously that the absorbance
after MW irradiation was higher than that without MW irradiation at the same time
point (Fig. 3c). Abundant ·OH were accelerated to generate under the MW irradiation,
the production rate gradually was slow as time went on. MEDT effect was also
demonstrated from different volume of H2O2 and amount of MIL-101(Fe) NPs (Fig.

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S8).
To evaluate the MEDT effect of MIL-101(Fe) NPs overall, TA was also used
as ·OH trapping agent to synthesize 2-hydroxyterephthalic acid with excitation
wavelength at 315 nm and emission peaks at 450 nm by fluorescence
spectrophotometry [70]. As shown in Figure 3d that the fluorescence intensity was
improved under MW irradiation at power of 0.9 W for 5 min comparing with no MW
irradiation. The intensity of fluorescence was gradually increasing with the
microwave time extending, but fluorescence intensity increased slowly (Fig. 3e, f).
The results confirmed that MIL-101(Fe) NPs could catalyze H2O2 to generate
abundant ·OH with the rapid under MW irradiation, showing the excellent MEDT
effect.
ESR was also used to detect more accurately the generation of ·OH. The ESR
signal was not detected after H2O2 dispersing in H2O with MW irradiation. While the
ESR signal was observed after H2O2 and MIL-101(Fe) NPs dispersing in H2O after
MW irradiation (Fig. S9).
3.4 Biodegradability Experiment of MIL-101(Fe) NPs in Vitro
The biodegradation and biosafety of MIL-101(Fe) NPs determine clinical
application in the future [2]. The degradability of MIL-101(Fe) NPs was verified in
vitro by incubating MIL-101(Fe) NPs in neutral phosphate buffer (pH=7.4 PBS) and
acid phosphate buffer (pH=5.5 PBS) [71]. As shown in Fig. 4a, the structure of
MIL-101(Fe) was not almost degraded in neutral phosphate buffer, indicting
MIL-101(Fe) were stable. While MIL-101(Fe) NPs had the degradable tendency in

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acid phosphate buffer, and the structure evolution of MIL-101(Fe) NPs was monitored
by TEM observations at different time points (1, 5 and 10 h) (Fig. 4b). After
incubation for 1 h, the morphology of MIL-101(Fe) NPs remained its original shape.
When incubation time reached 5 h, the structure of MIL-101(Fe) NPs started to
exhibit degradable tendency. While incubating for 10 h, the structure of MIL-101(Fe)
NPs exhibited significant structural collapse, the intact structure of MIL-101(Fe) NPs
could hardly be seen. The results manifested that MIL-101(Fe) NPs were degradable,
which is promising for the future clinic application.
3.5 Toxicity Experiment of IL@MIL-101(Fe)@BSA-AuNCs NPs
The biosafety of nanomaterials was crucial in biomedical application, so the
biosafety and compatibility were evaluated at cells and mice respectively. In order to
detect the toxic effect of IL@MIL-101(Fe)@BSA-AuNCs NPs, cellular activity of
different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 ꢀg mL-1) of the
IL@MIL-101(Fe)@BSA-AuNCs NPs were evaluated in the HepG2 cells, H22 cells
and L929 cells. The toxicity of IL@MIL-101(Fe)@BSA-AuNCs NPs at cells was
accessed by methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 5a, the
relative activity of the cells hardly decreased obviously as the concentration of
IL@MIL-101(Fe)@BSA-AuNCs NPs increasing. The relative activity of cells was
approximately
80
%
at
the
NPs.
maximum
The result
concentration
confirmed
of
IL@MIL-101(Fe)@BSA-AuNCs
that
IL@MIL-101(Fe)@BSA-AuNCs NPs possessed good biocompatibility [72], which
could be further used in vivo.

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To evaluate the biological toxicity of IL@MIL-101(Fe)@BSA-AuNCs NPs in vivo,
the dose of 100, 150, 200 mg kg-1 of IL@MIL-101(Fe)@BSA-AuNCs NPs was
injected in mice by tail vein (PBS was used as the control). The result of body weight
was shown in Fig. 6b. During the experimental period, the body weight of mice
exhibited a slowly increasing trend. Compared with the control group, experimental
groups showed no significantly different in body weight indexs. In order to further
evaluate the toxicity of IL@MIL-101(Fe)@BSA-AuNCs NPs and their effect on the
health of mice, blood routine test and blood biochemical test were performed as
shown in Fig. S10. From the test results, the experimental groups were normal
comparing with control group. The viscera index of liver and kidney in the group
injected with 200 mg kg-1 of IL@MIL-101(Fe)@BSA-AuNCs NPs was significantly
higher than that of the control group, indicating that higher concentration of
IL@MIL-101(Fe)@BSA-AuNCs NPs affected the health of mice. While there was no
abnormality when the injected dose was 150 mg kg-1, manifesting that the maximum
tolerated dose of IL@MIL-101(Fe)@BSA-AuNCs NPs was approximately 150 mg
kg1. As shown in Fig. S11, the H&E staining results of tissue sections (heart, liver,
lung, spleen, and kidney) showed no obvious abnormalities in the experimental
groups treated with different concentrations of the IL@MIL-101(Fe)@BSA-AuNCs
NPs
comparing
with
the
control
group.
Above
all,
the
IL@MIL-101(Fe)@BSA-AuNCs NPs possessed low toxicity and good
biocompatibility characteristics.
3.6 Tumor Diagnosis and Degradation Monitoring by Dual-modality Imaging in Vivo

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Tumor diagnosis is a prerequisite for cancer treatment. However, single-mode
imaging of the tumor site cannot provide sufficient evidence for the exact tumor site
and adequate features for different tumors. As traditional single-mode imaging
technology, FI holds outstanding sensitivity at molecular level but has inherent
drawbacks such as low penetration depth and poor spatial resolution. MRI overcomes
the optical diffusion limitation, owns high spatial resolution but has shortcomings of
low sensitivity. To solve these problems, many researchers have focused on
developing multi-mode imaging probes and integrating various imaging enhancement
behaviors into a single system to realize complementary imaging functions [37,
73-76]. It is well-known that Fe3+ can quench the fluorescence of dyes and quantum
dots [77]. In this work, a rapid assembly and coupling method was adopted to avoid
the quench of BSA-Au NCs. The BSA-Au NCs were coupled on surface of
IL@MIL-101(Fe) NPs to endow IL@MIL-101(Fe)@BSA-AuNCs NPs with
dual-modality
imaging
function.
Fig.
6a,
e
showed
that
IL@MIL-101(Fe)@BSA-AuNCs NPs possessed MRI and FI property in vitro. We
measured plot of T2 relaxation rate (1/T2) under different concentrations of
IL@MIL-101(Fe)@BSA-AuNCs with r2=7.93086 (Fig. 6a). The signal intensity of
fluorescence and MR imaging of IL@MIL-101(Fe)@BSA-AuNCs NPs reached
maximum at 5 h in the tumor after IL@MIL-101(Fe)@BSA-AuNCs NPs were
injected by tail vein. The tumor site could be imaged, which was expected that
IL@MIL-101(Fe)@BSA-AuNCs NPs could promote precise diagnosis of tumor after
injecting for 5 h (Fig. 6e).

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To explore the degradation and distribution process of MIL-101(Fe) NPs in vivo,
the dual-mode imaging was also employed to study the degradation and distribution
of MIL-101(Fe) NPs in vivo. The MRI signal intensity could directly determine
degradation and distribution process of IL@MIL-101(Fe)@BSA-AuNCs NPs. When
IL@MIL-101(Fe)@BSA-AuNCs NPs degraded slowly in vivo, Fe3+ was produced
gradually, causing the fluorescence of IL@MIL-101(Fe)@BSA-AuNCs NPs to
slowly disappear. With fluorescence of IL@MIL-101(Fe)@BSA-AuNCs NPs
disappearing, the degradation of MIL-101(Fe) NPs was indirectly determined. As
shown in Fig. 6b, MR images showed the dynamic distribution of
IL@MIL-101(Fe)@BSA-AuNCs NPs in liver and tumor of tumor-bearing mice. Fig
6c showed the T2-MR signal of liver and tumor, the T2-MR signal intensity reached
maximum at 1 h in the liver and 5 h in the tumor during test for 10 h. Fig. 6f showed
the biodistribution of IL@MIL-101(Fe)@BSA-AuNCs in liver, lung, spleen, kidney,
heart, tumor, indicating that the fluorescence signal intensity is consistent with the
intensity of MRI in vivo. Furthermore, the results confirmed that
IL@MIL-101(Fe)@BSA-AuNCs NPs were degradable in vivo, which could be
degraded in vivo by observing FI and MRI of IL@MIL-101(Fe)@BSA-AuNCs NPs.
Moreover, the main organ for metabolism and distribution was liver, which was also
confirmed by alanine aminotransferase (ALT), aspartate aminotransferase (AST) in
blood biochemistry test. And tumor site could be imaged accurately. These results
indicated that as-obtained IL@MIL-101(Fe)@BSA-AuNCs NPs were biodegradable,
and expected to be in vivo excreted out of the body after therapeutic functioning.

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3.7 Evaluation of Microwave Enhancing Dynamic Effect and Combination Treatment
of MTT and MEDT in Tumor Cells
In order to further verify the effect of MEDT in cells, cells were incubated with
extracorporeal H2O2. Due to cells at tumor site lose regulation and control of H2O2,
superfluous H2O2 would be generated. In this study, in order to simulate excess H2O2
generated at tumor site, we employed a method from the literature to explore a certain
concentration that has no effect on cell growth by MTT assay [78-81]. As Fig. 7a
shown that 60 ꢀM was the criticality concentration, which was consistent with
literature result that neither promoted cell growth nor inhibited cell growth. In the
MEDT cell experiment, there were 9 groups being divided, HepG2 cells were
incubated with H2O2 at 60 ꢀM for 24 h in the groups containing H2O2, then HepG2
cells were incubated with 100 ꢀg mL-1 of MIL-101(Fe) NPs for 24 h in the groups
containing MIL-101(Fe). Then 1.0 mL of cells were treated under 0.9 W MW
irradiation for 3 min at 5 % glucose solution to avoid the heating effect. The cell
viability of MIL-101(Fe) +H2O2+MW group as experiment group was 47.7 %, while
H2O2 group, H2O2+MW group, H2O2+MIL-101(Fe) group were 102.8 %, 90 %,
85.6 %, the data confirmed that MIL-101(Fe) NPs were cable of catalyzing H2O2 to
generate ·OH in a more efficient under MW irradiation, amount of ·OH produced
could destroy tumor cells effectively (Fig. 7b). As shown in Fig. 7c, it was further
evaluated anti-cancer effect by combination of MEDT and MTT in cells. The 100 ꢀg
mL-1 of IL@MIL-101(Fe)@BSA-AuNCs NPs were incubated into cells overnight,
the MW group and IL@MIL-101(Fe)@BSA-AuNCs + MW group were treated with

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MW irradiation at 0.9 W for 0, 3, 5, 8 min, then the relative viability of cells was
calculated by MTT assay respectively. It could be clearly seen from the Fig. 7c that as
the
microwave
time
increased,
the
cell
viability
of
the
IL@MIL-101(Fe)@BSA-AuNCs + MW group became less and less, and the relative
viability of the cells decreased from 86.5 % to 33.6 %. When the microwave time is
the same, the cell viability of the IL@MIL-101(Fe)@BSA-AuNCs + MW group was
significantly lower than the MW group and and IL@MIL-101(Fe)@BSA-AuNCs
group, which demonstrated that IL@MIL-101(Fe)@BSA-AuNCs NPs could destroy
effectively the cancer cells by combination of MEDT and MTT.
3.8 Tumor Therapy Experiment
The IL@MIL-101(Fe)@BSA-AuNCs NPs could promote the increase in
temperature and the generation of ·OH under MW irradiation in vitro, thus the
anti-tumor effect of IL@MIL-101(Fe)@BSA-AuNCs NPs by combining MTT with
MEDT need be further verified in vivo. The tumor therapy effect of
IL@MIL-101(Fe)@BSA-AuNCs NPs was further validated in mice. 20 experimental
mice were divided into control, IL@MIL-101(Fe)@BSA-AuNCs, MW,
IL@MIL-101(Fe)@BSA-AuNCs + MW group. In addition, the optimal tumor
treatment time was 5 h after intravenous injection according to the strongest signal
intensity of MRI, FI and the maximum content of Au at tumor site by ICP analysis
(Fig. S12). In Fig. 8a, the FLIR imaging instrument imaging instrument was used to
record the change process of corresponding temperature of the MW group and the
IL@MIL-101(Fe)@BSA-AuNCs + MW group. The IL@MIL-101(Fe)@BSA-AuNCs

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+ MW group was significantly higher than that of the MW group in tumor region. The
maximum temperature difference between the MW group and the
IL@MIL-101(Fe)@BSA-AuNCs +MW group was 5 ℃ (Fig. 8b). It indicated that
IL@MIL-101(Fe)@BSA-AuNCs NPs with excellent heating sensitivity also had
better effect in vivo. In Fig. 8c, it showed the change in body weight of tumor-bearing
H22 mice after MTT and MEDT, each group of mice weight slowly rose. The tumor
volume in the IL@MIL-101(Fe)@BSA-AuNCs + MW group was the smallest by
comparing with mice in different experimental groups. It has shown that the control
group had the largest tumor volume. The IL@MIL-101(Fe)@BSA-AuNCs group only
exhibited chemical therapy effect comparing with the control group, the MW group
only exhibited hyperthermia effect, IL@MIL-101(Fe)@BSA-AuNCs + MW group
exhibited the treatment effect of by combining MTT and MEDT (Fig. 8d). As shown
in Fig. 8e, the survival rate of MW group was 20 %, the survival rate of control group
and IL@MIL-101(Fe)@BSA-AuNCs group was 0 %. While the survival rate of
IL@MIL-101(Fe)@BSA-AuNCs + MW group reached 100 %, indicating that had the
excellent tumor-inhibitory effect (We determined that the mice have been dead when
the tumor volume exceeded 2000 mm3 due to animal welfare). The synergy of MTT
and MEDT proved that IL@MIL-101(Fe)@BSA-AuNCs + MW had excellently
therapeutic effect on tumor-bearing mice. Images of tumors in different groups after
treatment for 14 days were consistent with the line chart of tumor volume (Fig. 8f).
The tissues of the mice were finally collected, including spleen, liver, kidney, lung
and tumor. The obtained tissues were stained with hematoxylin and eosin (H&E).

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Histologic section was no significant difference by comparing with the other groups
as shown in Fig. S13. Compared with the other groups, the tumor necrosis area of
IL@MIL-101(Fe)@BSA-AuNCs + MW group was the largest, which exhibited
distinct anti-cancer effect (Fig. 8g).
4. Conclusion
In conclusion, we fabricated biodegradable IL@MIL-101(Fe)@BSA-AuNCs NPs
possessing peroxidase-like catalytic activity and dual-modality imaging property.
MIL-101(Fe) NPs as nanozymes, enabled to catalyze H2O2 to generate ·OH and
accelerate to generate abundant ·OH under MW irradiation in tumor
microenvironment due to good MW conversion capability and intrinsic
enzyme-mimicking property. As-made IL@MIL-101(Fe)@BSA-AuNCs NPs with
MRI and FI function not only monitored dynamic distribution process in the body but
also diagnosed accurate tumor site. Accurate diagnosis and synergistic treatment of
MEDT and MTT are concentrated on IL@MIL-101(Fe)@BSA-AuNCs NPs, which
shows great potential for improving tumor treatment effect.
Acknowledgements
The authors acknowledge financial support from the National Key R&D
Program (2018YFC0115503), National Natural Science Foundation of China (Nos.
81630053, 61671435, 61571426), CAS-DOE program (No. GJHZ1705), and Beijing
Natural Science Foundation (No. 4161003).

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Scheme 1.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.