molecules

Article
In Vitro Toxicity Study of a Porous Iron(III)
Metal-Organic Framework
Gongsen Chen 1 , Xin Leng 1 , Juyuan Luo 1 , Longtai You 1 , Changhai Qu 1 , Xiaoxv Dong 1 ,
Hongliang Huang 3,4, *, Xingbin Yin 1, * and Jian Ni 1,2, *
1 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China;
gongsenchen@163.com (G.C.); 20160931927@bucm.edu.cn (X.L.); luojy0329@163.com (J.L.);
ylt_svip@163.com (L.Y.); quchanghai@bucm.edu.cn (C.Q.); dxiaoxv@163.com (X.D.)
2 Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine,
Beijing 100029, China
3 State Key Laboratory of Separation Membranes and Membrane Processes, Tianjin Polytechnic University,
Tianjin 300387, China
4 School of Chemistry and Chemical Engineering, Tianjin Polytechnic University, Tianjin 300387, China
* Correspondence: huanghl@mail.buct.edu.cn (H.H.); yxbtcm@163.com (X.Y.); njtcm@263.net (J.N.);
Tel.: +86-138-1147-5114 (H.H.); +86-10-8473-8616 (X.Y.); +86-10-6428-6407 (J.N.)




Received: 5 March 2019; Accepted: 27 March 2019; Published: 28 March 2019


Abstract: A MIL series metal-organic framework (MOF), MIL-100(Fe), was successfully synthesized
at the nanoscale and fully characterized by TEM, TGA, XRD, FTIR, DLS, and BET. A toxicological
assessment was performed using two different cell lines: human normal liver cells (HL-7702) and
hepatocellular carcinoma (HepG2). In vitro cytotoxicity of MIL-100(Fe) was evaluated by the MTT
assay, LDH releasing rate assay, DAPI staining, and annexin V/PI double staining assay. The safe
dose of MIL-100(Fe) was 80 µg/mL. It exhibited good biocompatibility, low cytotoxicity, and high
cell survival rate (HL-7702 cells’ viability >85.97%, HepG2 cells’ viability >91.20%). Therefore,
MIL-100(Fe) has a potential application as a drug carrier.

Keywords: MIL-100(Fe); in vitro toxicity; HL-7702 cells; HepG2 cells

1. Introduction
Metal-organic frameworks (MOFs) are the latest class of crystalline hybrid materials developed
over the last 20 years [1]. MOFs are built from virtually infinite combinations of metal subunits and
organic ligands (aromatic acids or bases) through coordination bonds, and are also named porous
coordination polymers (PCPs) [2–4]. MOFs are rich in composition, crystal structure, and crystal shape,
and have irreplaceable advantages compared with other types of materials [5]. The high versatility of
MOFs is shown by their various applications over the past 10 years, including catalysis [6], sensing [7],
gas storage [8], bio-imaging [9], and magnetism [10]. Because of their large specific surface area,
connectivity polyhedron, high pore volume, high stability, non-toxicity, biocompatibility, and small
size, MOFs have become the most promising nanocarriers in the biomedical field [11–14]. Therefore,
MOFs can be used in a variety of designs, such as imaging agents for early and minimally invasive
diagnostics [15], specific cell and tissue (more commonly tumor tissue) targeting [16], high drug
loading [17], increased local drug concentration [18], and developing drug delivery preparations [19].
As described above, nanomedical MOFs are considered promising candidates for developing more
effective therapies and reducing side effects.
However, the increasing potential uses of MOFs may pose health risks to the patient. Therefore,
this has led to controversies about whether MOFs are biocompatible in biomedical applications.

Molecules 2019, 24, 1211; doi:10.3390/molecules24071211 www.mdpi.com/journal/molecules

Molecules 2019,
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studies showed that, owing to differences in chemical formulations, the toxicity of Zn, Zr, Mg, and
To
Fe,date, the safety
as assessed by assessment
LD50, rose of MOFs
from has been
several μg/kg largely
to more limited
than by the metals
1 g/kg [20]. The andorganic
linkers.ligands,
Preliminarysuch
studies showed that, owing to differences in chemical formulations,
as the commonly used polycarboxylic acids, are less toxic and easily removed under physiological the toxicity of Zn, Zr, Mg, and Fe,
as assessed by
conditions LD50,of
because rose
theirfrom several
high µg/kg
polarity to more
[21,22]. Thethan 1 g/kgof[20].
diversity metal The organicand
clusters ligands,
organic such as the
ligands,
commonly used polycarboxylic acids, are less toxic and easily removed
as well as the functional modification inside and outside the surface, represents further advantages under physiological conditions
because
of MOFs,ofbut their
canhighalsopolarity
change [21,22]. The diversity
the chemical and physical of metal clustersofand
properties organic
materials ligands,
[23,24]. Theasrange
well as of
the functional modification inside and outside the surface, represents
cytotoxicity, hydrophobic/hydrophilic balance, host‒guest interaction, body distribution, further advantages of MOFs,
but can also changetissue
biodegradability, the chemical and physical
accumulation, propertiesdetermines
and excretion of materials their
[23,24]. The range
toxicity and of cytotoxicity,
whether they
hydrophobic/hydrophilic balance, host-guest
could be used for medical applications. Therefore,interaction,
before the body distribution,
practical biodegradability,
application of any new MOFs, tissue
accumulation,
a detailed study and of excretion
their toxicitydetermines their toxicity
and biosafety and whether
is urgently needed. In they could be itused
particular, for medical
is required that
applications. Therefore, before the practical application of any new
MOFs have minimal interference with the main effector cells [25], so the study of their possible MOFs, a detailed study of their
toxic
toxicity
effects isand
verybiosafety
important. is urgently needed. In particular, it is required that MOFs have minimal
interference with the
Up to this point, amongmain effector cells [25], so
the thousands ofthe
MOFsstudy of their possible
reported, the mosttoxic effects isused
commonly verybiomedical
important.
Up to this point, among the thousands of MOFs reported, the
materials are MIL (Material Institute Lavoisier) series [26]. MIL-100(Fe) is synthesized from most commonly used biomedical
materials
FeCl3.6Hare MIL H
2O and (Material
3BTC under Institute Lavoisier) series
hydrothermal [26]. MIL-100(Fe)
conditions. is synthesized
The molecular formulafrom is FeFeCl
3(H2O)3 .6H22O-BTC
O and
H
andBTC under hydrothermal conditions. The molecular formula is Fe (H2O)
3 the nanoporous structure is shown in Figure 1. There are two3 kinds 2of mesoporous cages in the O-BTC and the nanoporous
structure is shown in
skeletal structure Figure
with pore 1. diameters
There are two kinds
of 2.5 nmofand mesoporous cages in MIL-100(Fe)
2.9 nm [27–29]. the skeletal structure
has been with pore
widely
diameters of 2.5 nm and 2.9 nm [27–29]. MIL-100(Fe) has been widely studied
studied as an anticancer drug carrier owing to its high specific surface area (1700 BET), high porosity, as an anticancer drug
carrier owing to its high
large drug-loading specific
capacity, surface
stability inarea (1700 BET),
a biological high porosity,
medium, large drug-loading
and eco-friendly nature [30,31]. capacity,For
stability
instance,inMIL-100(Fe)
a biologicalhas medium,
a surfaceand functionalization
eco-friendly nature of [30,31].
maghemite For instance,
nanoparticles, MIL-100(Fe)
encapsulated has a
surface functionalization
doxorubicin with 14 wt % of loading,
maghemite andnanoparticles, encapsulated
progressive delivery of drug doxorubicin
(PBS, 37 °C) with 14 wt % loading,
exceeding 25 days
and ◦
[32].progressive
Doxycyclinedeliverymonohydrate of drug and (PBS, 37 C) exceeding
tetracycline hydrochloride, 25 days [32].separately
loaded Doxycycline monohydrate
on nano-MIL-100,
and
weretetracycline
able to givehydrochloride,
a sustainable releaseloadedatseparately
SGF and PBS on nano-MIL-100,
[33]. The effectwere of pH able
andtotemperature
give a sustainableon the
release
adsorption of cephalexin and release behavior of MIL-100(Fe) was studied [34]. cephalexin
at SGF and PBS [33]. The effect of pH and temperature on the adsorption of Docetaxel and was
release behavior
encapsulated of MIL-100(Fe)
in MIL-100(Fe) was
with studied
a drug [34]. Docetaxel
payload up to 57.2was wt encapsulated
%; the releasein MIL-100(Fe)
rate of the drug with
after a
drug payload up to 57.2 wt %; the release rate of the drug after
96 h was measured in neutral (pH 7.4, 5.08%) and acidic (pH 5.5, 15.53%) PBS that included 96 h was measured in neutral (pH 7.4,
5.08%) and acidic
polysorbate 80 (1% (pH 5.5,[35].
v/v) 15.53%)
To datePBSMOFs
that included polysorbate not
have, surprisingly, 80 (1%been v/v) [35]. To date MOFs
comprehensively have,
analyzed
surprisingly,
for their adversenot been
effectscomprehensively
on hepatic cells,analyzedbut havefor onlytheir
beenadverse effects on
preliminarily hepatic
studied forcells,
theirbut in have
vitro
only been preliminarily studied for their in vitro toxicity in cancer cells
toxicity in cancer cells (see Table 1). As is well known, the liver is a detoxifying organ, and (see Table 1). As is well known,
the liver is a detoxifying
nanomaterials organ, and nanomaterials
tend to accumulate in the liver tissue tend to accumulate
after entering in thethebody
liver[36].
tissueTherefore,
after entering it is
the body [36].
important Therefore,whether
to determine it is important
they causeto determine whether they cause hepatotoxicity.
hepatotoxicity.

Figure
Figure 1. Schematic 1. Schematic
illustration of theillustration
constructionof of
theMIL-100(Fe).
construction of MIL-100(Fe).
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Table
Table1.
1.The
TheEffect
Effectof
ofMIL-100
MIL-100(Fe)
(Fe)on
onVarious
VariousCells,
Cells, :biocompatible;
:biocompatible; :adverse
:adverseeffects.
effects.
Table 1. The Effect of MIL-100 (Fe) on Various Cells, : biocompatible; : adverse effects.

MOFs and
andMOFs-
MOFsMOFs MOFs-
and Dose
Dose
Dose Cell
Cell
Cell Toxic
Toxic
Toxic
MOFs-Objects
Cells
Cells
Cells (µg/mL) Viability Grade
Reference
Reference
Reference
Objects
Objects (μg/mL)
(μg/mL) Viability
Viability Grade
Grade
MIL-100(Fe)
MIL-100(Fe)
MIL-100(Fe) PC3
PC3
PC3 100
100100
(24(24
(24 h)h)
h) 75%
75%
75% lowlow
low Saad et al.
Saad
Saad et[32]
et al.
al.[32]
[32]
MIL/USPIO-cit(10) PC3 100 (24 h) 75% low Saad et al. [32]
MIL/USPIO-cit(10)
MIL/USPIO-cit(10) PC3
PC3 100
100(24(24h)h) 75%
75% low
low Saad
Saadet etal.
al.[32]
[32]
HUVEC 200 (24 h) 100% none Stefan et al. [37]
HMEC 200 (72 Stefan
Stefan et
et al.
Stefan et al. [37]al.
HUVEC
HUVEC 200
200 (24
(24 h)h)
h) 85%
100%
100% lownone
none
MLE12 200 (24 h) 10% high Stefan et [37]
[37]
al. [37]
MIL-100(Fe)
MH-S 200 (24 h) 10% high Stefan et al. et
Stefan
Stefan [37]
etal.
al.
HMEC
HMEC
Gingival Fibroblasts 200
200 (72
200(72 h)
(24h)h) 85%
85%
100% low
nonelow Stefan et [37]
al. [37]
[37]
Human Schwann 200(24 h) 60% medium Stefan et al. [37]
HUVEC 200 (24
Stefan
Stefanet etal.
al.
MLE12
MLE12 200
200 (24
(24 h)h)
h) 100%
10%
10% none
high
high Stefan et al. [37]
HMEC 200 (72 h) 60% medium [37]
[37]
Stefan et al. [37]
MIL-100(Fe)
MIL-100(Fe)
MIL-100(Fe)@DOPC
MLE12 200 (24 h) 40% high Stefan
Stefan et al. et
Stefan etal.
[37]al.
MH-S
MH-S
MH-S 200
200 (24
200(24 h)
(24h)h) 10%
10%
20% high
highhigh Stefan et [37]
al. [37]
[37]
nanoMIL-100(Fe) MCF-7 100 (72 h) 94% none Mahsa et al. [35]
Gingival
Gingival Stefan
Stefanet etal.
al.
CCRF-CEM 200
200 (24
(24h)
5000(48 h)h) 100%
>85%
100% lownone
none Patricia et al. [38]
MIL-100(Fe) Fibroblasts
Fibroblasts
RPMI-8226 5000(48 h) >85% low [37]
[37]
Patricia et al. [38]
Human
Human
J744 5000(48 h) >85% low Stefan
Patricia et al.et
Stefan et al.
al.
[38]
200(24
200(24h)
h) 60%
60% medium
medium
Schwann
Schwann [37]
[37]
Stefan
Stefanetetal.
al.
HUVEC
The toxic effect of MIL-100(Fe) 200
200(24
HUVECon different (24 h) 100%
h) of cells
types 100% none
noneso one must ensure the
varies greatly, [37]
[37]
biocompatibility of MIL-100(Fe) to target cells and that its intrinsic toxicity does not exceed
Stefanthe
Stefan et benefits
etal.
al.
HMEC
HMEC 200
200 (72
(72 h)
h) 60%
60% medium
medium
of reducing the toxicity of drugs or treatments. The in vitro toxicity test is fast, effective, [37]and has a
[37]
MIL-100(Fe)@DOPC
MIL-100(Fe)@DOPC
low cost. It provides information about the possible interaction between cells and nanoparticles
Stefan
Stefan et
etal.
al.
MLE12
MLE12 200
200(24
(24h)
h) 40%
40% high
high
to evaluate the potential hazards of nanomaterials. Therefore, these assays are appropriate for [37]
[37]
HTS (high-throughput screening) of most new Stefan
Stefan et
etal.
al.
MH-S
MH-S 200medical
200 (24 h) nanomaterials
(24h) 20%
20% and are useful
high
high in establishing
cytotoxicity levels [39]. In accordance with the above statements, we synthesized MIL-100(Fe) [37]
[37] by the
Mahsa
Mahsaet etal.
al.
solventthermal method and focused
nanoMIL-100(Fe)
nanoMIL-100(Fe) MCF-7 on the 100
MCF-7 influence
100(72 h) of MIL-100(Fe)
(72h) 94%
94% on none
two
nonetypes of hepatocytes to
[35]
[35]
analyze its cytotoxicity in vitro. The synthesized MIL-100(Fe) was analyzed by TEM, TGA, XRD, FTIR,
Patricia
Patriciaetetal.
al.
DLS, and BET. The in vitro CCRF-CEM 5000(48
5000(48h)
CCRF-CEM of MIL-100(Fe)
cytotoxicity ＞85%
＞85% and HepG2
h) in HL-7702 low
low cells was evaluated
[38]
[38]
by the MTT assay, the LDH releasing rate assay, DAPI staining, and annexin V/PIPatricia double
Patricia etstaining
et al.
al.
MIL-100(Fe）
MIL-100(Fe）
assay. In conclusion, our studyRPMI-8226
RPMI-8226 5000(48
5000(48
evaluates the in vitro h)safety＞85%
h) ＞85%
of MIL-100(Fe)low
low
and provides [38] reference
a
[38]
for medical applications. Patricia
Patriciaetetal.
al.
J744
J744 5000(48
5000(48h) h) ＞85%
＞85% low
low
[38]
[38]
2. Results
The
The toxic
toxic effect
effect of of MIL-100(Fe)
MIL-100(Fe) on on different
different types
types of of cells
cells varies
varies greatly,
greatly, so so one
one must
must ensure
ensure the
the
2.1. Preparation and
biocompatibility
biocompatibility of Characterization
of MIL-100(Fe)
MIL-100(Fe) to toof MIL-100(Fe)
target
target cells
cells and
and that
that itsits intrinsic
intrinsic toxicity
toxicity does
does not not exceed
exceed thethe
benefits
benefits of
ofreducing
reducing
According the
thetoxicity
to previoustoxicity of
ofdrugs
studies,drugs or
ortreatments. wasThe
treatments.
MIL-100(Fe) The in
invitro
vitrotoxicity
synthesized by thetest
toxicity test isisfast,
fast,effective,
hydro-thermal effective, and
and
method.
has
has a
a low
low cost.
cost. It
Itprovides
provides information
information about
about the
the possible
possible interaction
interaction
The structure of MIL-100(Fe) was analyzed by TEM, TGA, XRD, FTIR, DLS, and BET. The TEM image between
between cells
cells and
and nanoparticles
nanoparticles
toofevaluate
to evaluate the
the potential
the synthesized potential hazards
hazards
materials, shownof
of nanomaterials.
nanomaterials.
in Figure 2a, shows Therefore,
Therefore, these
irregularthese assays
assays areare appropriate
polyhedron-shaped appropriate for
for HTS
HTS
nanoparticles
(high-throughput
(high-throughput screening)
screening) of
of most
most new
new medical
medical
for MIL-100(Fe) of 200–500 nm diameter, similar to the reported results [25]. nanomaterials
nanomaterials and
and are
are useful
useful in
in establishing
establishing
cytotoxicity
cytotoxicity
As shown
levels
levels [39].
[39].In
in Figure
accordance
In2b,accordance with
withthe
in the temperature the above
above statements,
range ofstatements,
40–600 ◦ C,we
we synthesized
thesynthesized
MIL-100(Fe)
first weight MIL-100(Fe)
by
bythe
loss occurs between the
solventthermal
solventthermal method
method and focused on the influence
influence of of MIL-100(Fe) on two types
types of of hepatocytes to
80 and 150 ◦ C (about 23andwt %)focused
due to onthe thevolatilization MIL-100(Fe)
of free wateron twomaterial.
in the hepatocytes
In the rangeto of
analyze
analyze its
its
◦ cytotoxicity
cytotoxicity in
in vitro.
vitro. The
The synthesized
synthesized MIL-100(Fe)
MIL-100(Fe) was
was analyzed
analyzed by
by TEM,
TEM, TGA,
TGA, XRD,
XRD,
150–350 C, there is a slow weight loss, which is due to the release of some anionic small molecules
FTIR,
FTIR, DLS,
DLS, and
andthe BET.
BET. The
The in in vitro
vitroandcytotoxicity
cytotoxicity of
of MIL-100(Fe)
MIL-100(Fe) in
in HL-7702
HL-7702 and
and HepG2
HepG2 cells was
used to balance skeleton charge bound water interrelated with the iron metal trimers.cells was
The third
evaluated
evaluated by the
the MTT
byoccurs MTT assay,
assay, the ◦ C,LDH
the LDH releasing rate assay,
assay, DAPI staining, and annexin V/PI V/PI double
weight loss at 350–500 with releasing
the largest rate
reduction DAPI staining,
in weight (aboutand 30annexin
wt %) mainly double
due to
staining
staining assay.
assay. In
In conclusion,
conclusion, our
our study
study evaluates
evaluates the
the in
in vitro
vitro safety
safety of
of MIL-100(Fe)
MIL-100(Fe) and
and provides
provides aa
the collapse of the sample frame structure and the decomposition of organic connectors [40]. The above
reference
reference for
formedical
medical applications.
applications. match MIL-100 (Fe).
thermogravimetric characteristics
The XRD patterns of the synthesized materials are presented in Figure 2c. Compared with
2.
2. Results
Results
the MIL-100(Fe) simulated pattern, the diffraction peaks of the experimental group match well,
which indicates that the synthesis of MIL-100(Fe) crystal is successful.
2.1.
2.1.Preparation
Preparation and
and Characterization
Characterizationof ofMIL-100(Fe)
MIL-100(Fe)
According to Figure 2d, peaks near 1624 and 1381 cm−1 belong to the stretching vibrations of C=O.
According
According
In addition, to
toprevious
bendingprevious studies,
studies,
vibration MIL-100(Fe)
MIL-100(Fe)
of aromatics was
wassynthesized
is observed at 760 and by
synthesized bythe
712 cmhydro-thermal
the −
hydro-thermal method.
method.The
1 , The wide absorption The
near
structure
structure of
ofMIL-100(Fe)
MIL-100(Fe)waswas analyzed
analyzed by by TEM,
TEM, TGA,
TGA, XRD,
XRD, FTIR,
FTIR, DLS,
DLS,andandBET.
BET. The
The TEM
TEM image
image of
of
the
thesynthesized
synthesizedmaterials,
materials,shown
shownin inFigure
Figure2a,
2a,shows
showsirregular
irregularpolyhedron-shaped
polyhedron-shapednanoparticles
nanoparticlesfor
for
MIL-100(Fe)
MIL-100(Fe)of of200–500
200–500 nm
nmdiameter,
diameter,similar
similartotothe
thereported
reportedresults
results[25].
[25].
%) mainly due to the collapse of the sample frame structure and the decomposition of organic
connectors [40]. The above thermogravimetric characteristics match MIL-100 (Fe).
The XRD patterns of the synthesized materials are presented in Figure 2c. Compared with the
MIL-100(Fe) simulated pattern, the diffraction peaks of the experimental group match well, which
indicates that24,the
Molecules 2019, synthesis of MIL-100(Fe) crystal is successful.
1211 4 of 14
According to Figure 2d, peaks near 1624 and 1381 cm−1 belong to the stretching vibrations of
C=O. In addition, bending vibration of aromatics is observed at 760 and 712 cm−1, The wide absorption
3500 cm −1 is due to the O–H stretching vibrations of H O molecules. All the above are characteristic
near 3500 cm −1 is due to the O–H stretching vibrations2 of H2O molecules. All the above are
peaks of MIL-100(Fe) [38].
characteristic peaks of MIL-100(Fe) [38].

Figure 2.
Figure Characteristics of
2. Characteristics of MIL-100(Fe):
MIL-100(Fe): (a)
(a) TEM,
TEM, (b)
(b) TG,
TG, (c)
(c) XRD,
XRD, (d)
(d) FTIR.
FTIR.

The particle
The particle size
size distribution
distribution ofof MIL-100(Fe)
MIL-100(Fe) waswas measured
measured byby aa Malvern
Malvern Zetasizer
Zetasizer Nano
Nano ZS ZS 90
90
(Malvern, UK). The particle size analysis (Figure 3a) shows an average particle size of 345.3 nm
(Malvern, UK). The particle size analysis (Figure 3a) shows an average particle size of 345.3 nm (Pdi: (Pdi: 0.181;
Intercept:
0.181; 0.956). 0.956).
Intercept: The porosity of MIL-100(Fe)
The porosity was characterized
of MIL-100(Fe) by N2 adsorption–desorption
was characterized isotherms
by N2 adsorption–desorption
at 77 K, as shown in Figure 3b. The Brunauer–Emmett–Teller (BET) specific surface
isotherms at 77 K, as shown in Figure 3b. The Brunauer–Emmett–Teller (BET) specific surface areaarea is 1750 m2 /g
is
and the pore volume is 0.83 cc/g, large enough for efficient mass transfer of drug loading
1750 m /g and the pore volume is 0.83 cc/g, large enough for efficient mass transfer of drug loading
2 and releasing.
Molecules 2019, 24, x FOR PEER REVIEW 5 of 15
and releasing.

Figure3.3.(a)
Figure (a)Size
Sizedistribution
distributionof
ofMIL-100(Fe);
MIL-100(Fe);(b)
(b)BET
BETisotherm
isothermofofMIL-100(Fe)
MIL-100(Fe)atat77
77K.
K.

2.2. Effect of MIL-100(Fe) on HL-7702 Cells

The MTT assays were carried out on HL-7702 cells. The cells were treated with different
concentrations of MIL-100(Fe) and the toxicity was determined (Figure 4a). The results showed that
MIL-100(Fe) particles with a concentration of less than 80 μg/mL were nontoxic (cell viability >85%).
Moreover, the amount of LDH was measured in the medium, which was regarded as an important
indicator of cell membrane integrity, as illustrated in Figure 4b. MIL-100(Fe) of high concentrations
(160 μg/mL) was toxic to HL-7702 cells and would destroy the cell membrane, leading to significant
LDH release. Fortunately, cells treated with 160 μg/mL MIL-100(Fe) for 48 h retain a cell survival rate
of 79.81 ± 3.01%, and IC50 is up to 1.37 mg/mL. DAPI staining for HL-7702 cells did reveal a high
concentration of MIL-100(Fe)-induced morphological changes associated with apoptosis, as
Molecules 2019, 24, 1211 5 of 14

2.2. Effect of MIL-100(Fe) on HL-7702 Cells

The MTT assays were carried out on HL-7702 cells. The cells were treated with different
concentrations of MIL-100(Fe) and the toxicity was determined (Figure 4a). The results showed
that MIL-100(Fe) particles with a concentration of less than 80 µg/mL were nontoxic (cell viability
>85%). Moreover, the amount of LDH was measured in the medium, which was regarded as an
important indicator of cell membrane integrity, as illustrated in Figure 4b. MIL-100(Fe) of high
concentrations (160 µg/mL) was toxic to HL-7702 cells and would destroy the cell membrane, leading
to significant LDH release. Fortunately, cells treated with 160 µg/mL MIL-100(Fe) for 48 h retain a cell
survival rate of 79.81 ± 3.01%, and IC50 is up to 1.37 mg/mL. DAPI staining for HL-7702 cells did
reveal a high concentration of MIL-100(Fe)-induced morphological changes associated with apoptosis,
as demonstrated in Figure 4c.
Molecules 2019, 24, x FOR PEER REVIEW 6 of 15

Figure 4. Effects of MIL-100(Fe) on HL-7702 cells’ viability and morphology: (a) MTT assay data,
Figure 4. Effects of MIL-100(Fe) on HL-7702 cells’ viability and morphology: (a) MTT assay data,
presented as mean ± SD of viability% of three independent experiments; (b) the effect of MIL-100(Fe)
presented as mean ± SD of viability% of three independent experiments; (b) the effect of MIL-100(Fe)
on LDH release of HL-7702 cells; (c) an evaluation of HL-7702 cells nuclear morphology by DAPI
on LDH release of HL-7702 cells; (c) an evaluation of HL-7702 cells nuclear morphology by DAPI
staining. (* p < 0.05 vs. Control.)
staining. (* p < 0.05 vs. Control.)
Molecules 2019, 24, 1211 6 of 14

After the treatment of HL-7702 cells with MIL-100(Fe) for 48 h, the higher dose of MIL-100(Fe)
(160 µg/mL) had a significant effect on the apoptosis of HL-7702 cells (Figure 5). The percentage
of necrotic cells only reached 6.07%. The results indicated that MIL-100(Fe) particles had good
biocompatibility with HL-7702 cells.
Molecules 2019, 24, x FOR PEER REVIEW 7 of 15

Figure 5.
Figure 5. Effects of MIL-100(Fe) on apoptosis
apoptosis inin HL-7702
HL-7702 cells.
cells. (a) Flow cytometry
cytometry detection
detection ofof
apoptosis with
apoptosis with FITC-annexin
FITC-annexin V/PI V/PI double staining. (b) (b) The
The percentages
percentages of viable, necrotic,
necrotic, and
and
apoptotic HL-7702
apoptotic HL-7702cells
cellsafter
afterincubation
incubation with
with different
different concentrations
concentrations of MIL-100(Fe)
of MIL-100(Fe) forh.48
for 48 h.data
The The
are
dataexpressed as means
are expressed ± S.D.± from
as means threethree
S.D. from independent experiments.
independent (* p <(*0.05
experiments. vs. Control.)
p < 0.05 vs. Control.)

2.3.
2.3. Effect
Effect of
of MIL-100(Fe)
MIL-100(Fe) on
on HepG2
HepG2 Cells
Cells
The
The MTT
MTT toxicity
toxicity test
test was
was performed
performed by by treating
treating HepG2
HepG2 cells
cells with
with different
different concentrations
concentrations of of
MIL-100(Fe).
MIL-100(Fe). AsAs depicted
depicted inin Figure
Figure 6a,
6a, MIL-100(Fe)
MIL-100(Fe) shows
shows nono cytotoxic
cytotoxic effect
effect on
on the
the cells.
cells. It
It reveals
reveals
89.98 ±±4.96%
89.98 4.96%cell
cellviability
viability
at at
thethe high
high concentration
concentration (160(160 µg/mL).
μg/mL). Remarkably,
Remarkably, the LDH
the LDH release release
assay
assay showed that HepG2 cells had good tolerance to MIL-100(Fe), even at higher
showed that HepG2 cells had good tolerance to MIL-100(Fe), even at higher doses (Figure 6b). At doses (Figure 6b).
a
At a MIL-100(Fe) concentration of 160 µg/mL, LDH release was significantly increased,
MIL-100(Fe) concentration of 160 μg/mL, LDH release was significantly increased, but there was no but there
was no significant
significant decreasedecrease in cellular
in cellular metabolicmetabolic
activity,activity, indicating
indicating thatwere
that they theywell
weretolerated.
well tolerated.
DAPI
DAPI staining of HepG2 cells indicated that high concentrations of MIL-100(Fe)
staining of HepG2 cells indicated that high concentrations of MIL-100(Fe) did induce nucleus did induce nucleus
morphological
morphological changes,
changes, as as shown
shown in in Figure
Figure 6c.
6c.
After a 48-h treatment of HepG2 cells with MIL-100(Fe), there was no significant change in the
percentage change of viable cells and early apoptotic cells. The late apoptotic cells were significantly
affected at concentrations of 40–80 μg/mL, but the overall difference was small (Figure 7). MIL-
100(Fe) had little effect on cells’ morphological changes, indicating the good biocompatibility of MIL-
100(Fe) with HepG2 cells.
Molecules 2019, 24, 1211 7 of 14

After a 48-h treatment of HepG2 cells with MIL-100(Fe), there was no significant change in the
percentage change of viable cells and early apoptotic cells. The late apoptotic cells were significantly
affected at concentrations of 40–80 µg/mL, but the overall difference was small (Figure 7). MIL-100(Fe)
had little effect on cells’ morphological changes, indicating the good biocompatibility of MIL-100(Fe)
with HepG2 cells.
Molecules 2019, 24, x FOR PEER REVIEW 8 of 15

Figure6.6.Effects
Figure EffectsofofMIL-100(Fe)
MIL-100(Fe)onon HepG2
HepG2 cells’
cells’ viability,
viability, membrane,
membrane, andand morphology:
morphology: (a)vitro
(a) In In vitro
cell
cell viabilities of HepG2 cells after being incubated for 48 h with MIL-100(Fe); (b) LDH
viabilities of HepG2 cells after being incubated for 48 h with MIL-100(Fe); (b) LDH release of HepG2 release of
HepG2
cells cells incubated
incubated for 48 hfor 48 MIL-100(Fe);
with h with MIL-100(Fe); (c) nuclear
(c) nuclear morphology
morphology imagesimages of HepG2
of HepG2 cells 48
cells after after
h
48 h exposure
exposure to MIL-100(Fe).
to MIL-100(Fe). (* p <(*0.05
p <vs.
0.05Control.)
vs. Control.)
Molecules 2019, 24, 1211 8 of 14
Molecules 2019, 24, x FOR PEER REVIEW 9 of 15

Figure 7.7.Effects
Figure Effectsof of
MIL-100(Fe)
MIL-100(Fe) on apoptosis in HepG2
on apoptosis cells. (a)
in HepG2 Flow(a)
cells. cytometry detection detection
Flow cytometry of apoptosis
of
with FITC-annexin
apoptosis V/PI doubleV/PI
with FITC-annexin staining. (b) The
double percentages
staining. (b) Theof viable, necrosis
percentages of and apoptotic
viable, HepG2
necrosis and
cells after incubation
apoptotic HepG2 cellswithafterdifferent concentrations
incubation of concentrations
with different MIL-100(Fe) forof48MIL-100(Fe)
h. The datafor
are48
expressed as
h. The data
means ± S.D. from three independent experiments. (* p < 0.05 vs. Control.)
are expressed as means ± S.D. from three independent experiments. (* p < 0.05 vs. Control.)

3. Discussion
3. Discussion
This study was designed to assess the potential toxicity of MIL-100(Fe) and explore its safe dose.
This study was designed to assess the potential toxicity of MIL-100(Fe) and explore its safe dose.
To date, few studies have used normal cells to study the toxic effects of MIL-100(Fe), so there is
To date, few studies have used normal cells to study the toxic effects of MIL-100(Fe), so there is
currently no comparison of the effects of MIL-100(Fe) between normal hepatocytes and liver cancer
currently no comparison of the effects of MIL-100(Fe) between normal hepatocytes and liver cancer
cells. Recently, Wang et al. [41] demonstrated a low toxicity of zinc-based MOFs by MTT experiments,
cells. Recently, Wang et al. [41] demonstrated a low toxicity of zinc-based MOFs by MTT experiments,
and also tested drug loading and evaluated the efficacy. Another report by Sun et al. [42] used a novel
and also tested drug loading and evaluated the efficacy. Another report by Sun et al. [42] used a novel
Gd(III) MOF-loaded anticancer drug, and, using MTT, evaluated its toxicity to both cell lines and
Gd(III) MOF-loaded anticancer drug, and, using MTT, evaluated its toxicity to both cell lines and its
its anticancer activity after drug delivery. The cytotoxicity of these materials depends not only on
anticancer activity after drug delivery. The cytotoxicity of these materials depends not only on the
the composition of the MOFs, but also on the choice of cell model. In other words, different MOFs
composition of the MOFs, but also on the choice of cell model. In other words, different MOFs have
have different toxicities to different cell lines. The current study aimed to determine the toxicity of
different toxicities to different cell lines. The current study aimed to determine the toxicity of MIL-
MIL-100(Fe) on normal human liver cells and liver cancer cells and compare them.
100(Fe) on normal human liver cells and liver cancer cells and compare them.
We recently reported the in vitro hepatotoxicity of MIL-53(Fe), loaded the antitumor drug
We recently reported the in vitro hepatotoxicity of MIL-53(Fe), loaded the antitumor drug
oridonin, and conducted in vitro sustained release and pharmacodynamic studies [43]. Although
oridonin, and conducted in vitro sustained release and pharmacodynamic studies [43]. Although in
in vitro cell models are not a substitute for animal experiments, they can serve as a basis for further
vitro cell models are not a substitute for animal experiments, they can serve as a basis for further
evaluation of the potential toxicity.
evaluation of the potential toxicity.
Our results showed that both cell lines, which were treated with the same concentration gradient
of MIL-100(Fe) for 48 h, showed a dose-dependent increase in LDH leakage and a decrease in cell
viability. Compared with HepG2 cells, HL-7702 cells were significantly affected by the dose change
Molecules 2019, 24, 1211 9 of 14

Our results showed that both cell lines, which were treated with the same concentration gradient
of MIL-100(Fe) for 48 h, showed a dose-dependent increase in LDH leakage and a decrease in cell
viability. Compared with HepG2 cells, HL-7702 cells were significantly affected by the dose change
of MIL-100(Fe), which showed strong cytotoxicity (cell viability <85%) at a high dose (160 µg/mL).
With the same treatment, HepG2 cells had less damage. Both cell types showed obvious membrane
damage (LDH leakage) at the highest concentration. LDH enzymes are highly sensitive indicators of
cellular metabolic status, aerobic or anaerobic directions of glycolysis, activation status, and malignant
transformation [44]. It is unclear whether the rapid sedimentation of MIL-100(Fe) particles in the
medium has an effect on the state of the cells. Normal hepatocytes and liver cancer cells have different
tolerances to MIL-100(Fe) particles, and it is unclear whether this is related to cell metabolism or cell
membrane changes after carcinogenesis [45]. Overall, based on MTT and LDH data, MIL-100(Fe)
showed low toxicity to HepG2 and HL-7702 cells at a dose of 80 µg/mL.
Additional studies were performed using DAPI staining to observe changes in the nuclear
morphology of the two cell lines after MIL-100(Fe) treatment. In a study on the role of polyadenylation
in apoptosis and drug resistance of K562 cells, Lallas used DAPI staining to detect apoptotic cell
nuclear division [46]. DAPI (40 ,6-diamidino-2-phenylindole) is a fluorescent dye that binds strongly
to DNA and can be utilized for staining living and fixed cells [47]. Our results show that the
nuclear morphological changes of the two cell lines gradually became obvious with an increased
dose, indicating that a high concentration of MIL-100(Fe) induced morphological changes related to
apoptosis. annexin V combined with PI can distinguish early apoptotic cells from late apoptotic and
dead cells, and is the ideal quantitative detection method for apoptosis at present [48]. Han et al. [49]
used the MTT assay to detect the biocompatibility of the Zr-based MOF, UiO-66, against SMMC-7721
and HeLa cells, and evaluated the apoptosis-inducing effect of AgNPs@UiO-66 by annexin V/PI
staining. To more clearly elucidate the effects of apoptosis, we performed annexin V/PI staining. Our
results indicate that the percentage of apoptotic normal hepatocytes increases in a dose-dependent
manner as the concentration of MIL-100(Fe) increases, with significant differences at the highest dose.
It has been demonstrated that MIL-100(Fe) particles at a dose of 80 µg/mL had little effect on the
degree of apoptosis in the two cell models. There are several problems with determining how high
concentrations of MIL-100(Fe) particles induce toxicity. For example, we need to address the questions
of whether the particles enter the cell or adhere to the cell membrane, and whether the toxicity of
materials with different shapes and sizes is the same. MIL-100(Fe) is one of the most studied MOFs
today, and its toxicity and safe dose are currently unclear. It is therefore necessary to study their
toxicology, and this report provides some preliminary information in this regard.
Due to the complexity of toxicity, the next step is to study whether the toxicity of MIL-100(Fe)
is related to intracellular pathways such as autophagy, mitochondrial membrane potential change,
lysosomal membrane permeability, and endoplasmic reticulum stress, so as to more comprehensively
evaluate the toxicity mechanism of MIL-100(Fe).

4. Conclusions
In conclusion, high concentrations of MIL-100(Fe) particles resulted in decreased viability of
HepG2 and HL-7702 cells, LDH leakage, and induction of apoptosis. The safe dose of MIL-100(Fe)
was determined to be 80 µg/mL by the MTT assay, the LDH release assay, the DAPI staining assay,
and the annexin V/PI double staining assay. MIL-100(Fe) particles had excellent biocompatibility with
HL-7702 and HepG2 cells, low cytotoxicity, and allowed a high cell survival rate. Therefore, the metal
organic framework material MIL-100(Fe) has a potential application as a drug carrier.
Molecules 2019, 24, 1211 10 of 14

5. Experimental Section

5.1. Materials
All general reagents and solvents (AR grade) were commercially available. Ethanol, nitric acid
(HNO3, 65%~68%), and hydrofluoric acid (HF ≥ 40%) were purchased from Beijing Chemical Works
(Beijing, China). 1,3,5-benzenetricarboxylic acid (H3BTC, 99%) was supplied by TCI (Shanghai, China).
Iron powder (Fe ≥ 99.5%) was obtained from Aladdin Chemistry Co. Ltd. (Aladdin, Shanghai,
China). DMEM, FBS, penicillin-streptomycin solution and 0.25% trypsin were purchased from Corning
(Corning, NY, USA). PBS was obtained from Solarbio (Beijing, China). MTT was acquired from
Biotopped (Beijing, China). The LDH Assay Kit, DAPI Assay Kit, and annexin V-FITC Apoptosis
Assay Kit were purchased from Beyotime (Nanjing, China).

5.2. Synthesis and Characterization of MIL-100(Fe)

MIL-100(Fe) was synthesized and activated according to previous studies [50,51]. Typically,
Fe powder (0.82 g), H3BTC (2.06 g), HF (40%, 0.6 mL), HNO3 (65%, 1.14 mL), and deionized water
(80 mL) were mixed in a Teflon liner (Yushen, Shanghai, China), which was placed in an oven,
and progressively heated to 150 ◦ C within 8 h. The temperature was maintained at 150 ◦ C for 96 h
and then the oven was cooled down to room temperature for 24 h. The resulting orange powder was
collected after filtration and then washed with water. To remove the residual ligand and metal ions
from the pore of the MOF, the sample was dispersed in hot water (80 ◦ C) for 8 h (about 1 g sample in
300 mL water) and then in hot ethanol for 8 h. Finally, the solid was dried overnight in a vacuum at
120 ◦ C.

5.3. Cell Culture

HL-7702 cells and HepG2 cells were purchased from Guangzhou Jeniobio Biotechnology (Beijing,
China). The cells were cultured in a high-glucose DMEM medium supplemented with 10% (v/v) FBS
and 1% (v/v) penicillin (100 µg/mL)-streptomycin (100 µg/mL) at 37 ◦ C in a humidified atmosphere
containing 5% CO2 . Cells were passaged at 1:3 after 3–4 days. MIL-100(Fe) was dissolved in DMEM to
a stock concentration of 2 mg/mL, and after being dispersed by ultrasonic and ultraviolet disinfection
was stored at 4 ◦ C. The cells were treated with different doses of MIL-100(Fe) after dilution in the
basal medium.

5.4. Cell Viability Assay

The effect of MIL-100(Fe) on HL-7702 and HepG2 cells was assessed by the MTT assay. Cells
were seeded into 96-well plates at a density of 3.0 × 103 cells/well overnight. The cells were treated
with 0, 10, 20, 40, 80, and 160 µg/mL MIL-100(Fe) for 48 h. Fresh basal medium was used as the
untreated control. Then, 20 µL MTT working solution (5 mg/mL) was added. After incubation for
4 h at 37 ◦ C, culture supernatant was removed from all the wells, and the formazan crystals were
dissolved in 150 µL DMSO. The supernatant (100 µL) was transferred into new 96-well plates after
centrifugation (1000 g, 5 min). A microplate reader (Multiskan GO, Thermo, Waltham, MA, USA) was
used to measure the absorbance at 570 nm. All the experimental results were carried out three times.

5.5. Cell Membrane Damage Determination

LDH is an enzyme that predominantly exists in the cytoplasm, and is released into the extracellular
medium when the cell membrane is damaged. Therefore, the cell membrane damage was determined
by LDH leakage (LDH%). For the measurement of LDH leakage, the LH-7702 and HepG2 cells were
seeded into 96-well plates at a density of 5.0 × 103 cells/well overnight and then treated with various
concentrations of MIL-100(Fe) for 48 h. The supernatant was collected and LDH activity was measured
Molecules 2019, 24, 1211 11 of 14

with a commercial kit following the manufacturer’s instructions. The experiments were conducted
in triplicate.

5.6. DAPI Fluorescence Staining

Morphological changes of chromatin condensation and nuclear fragmentation could be observed
by DAPI staining. HL-7702 and HepG2 cells at a density of 3.5 × 105 cells/well were plated in six-well
plates and treated with 0, 10, 20, 40, 80, and 160 µg/mL MIL-100(Fe) for 48 h. The culture supernatant
was discarded and the cells were fixed with 4% paraformaldehyde (500 mL) for 10 min at room
temperature. Then the fixed cells were washed two times with PBS and stained with DAPI solution
(2.5 µg/mL, 0.8 mL) for 10 min. After being washed twice with PBS, the morphological changes were
photographed using an inverted Olympus IX71 fluorescence microscope (Tokyo, Japan) at 200×.

5.7. Apoptosis Analysis

Apoptosis usually refers to a type of programmed cell death in the process of development or
under the action of various factors, through the regulation of genes and their products in cells, and
can be detected by an annexin V-FITC detection kit [52]. HL-7702 and HepG2 cells were plated in a
six-well plate (3.5 × 105 cells/well) and incubated with gradient concentrations (0, 10, 20, 40, 80, and
160 µg/mL) of MIL-100(Fe) for 48 h at 37 ◦ C. The cells were gathered and washed with PBS. Then,
the cells were resuspended in 295 µL binding buffer and incubated with 5 µL annexin V-FITC and 10
µL PI at room temperature, hatching 20 min and blocking the light ray. All the samples were analyzed
by flow cytometry (BD FACS Canto II, Franklin Lakes, NJ, USA) immediately.

5.8. Statistical Analysis

Each experiment was repeated three times, and the results were expressed as mean ± SD. By using
SAS 9.4 software (Beijing, China), the statistical analysis was carried out with the further statistical
methods of single factor analysis of variance, multivariate comparison, and a non-parametric test.
A value of p < 0.05 was regarded as significant.

Author Contributions: X.Y. and J.N. designed the research. G.C., X.L., J.L., and L.Y. performed the experiments.
C.Q. and X.D. conducted the data analyses. G.C., X.L., and H.H. wrote the paper. All authors have reviewed and
approved the final version of the manuscript.
Funding: This work was financially supported by the National Natural Science Foundation of China (No.
81703715 and 21606007), the Training Program Foundation for the Beijing Municipal Excellent Talents (No.
2017000020124G295), and the fundamental Research Funds for the Central Universities (Beijing University of
Chinese Medicine Scientific Research Project for Distinguished Young Scholar (No. 2018-JYB-XJQ005)).
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
MOFs Metal-organic frameworks
PCP Porous coordination polymer
LD50 Lethal dose 50%
MIL Material Institut Lavoisier
H3 BTC 1,3,5-Benzenetricarboxylic acid
PBS Phosphate-buffered saline
SGF Simulated gastric fluid
PC3 Human prostate cancer cell line
HUVEC Human umbilical vein endothelial cells
HMEC Human microvascular endothelial cells
MLE12 Murine alveolar epithelial cells
MH-S Mouse alveolar macrophage cell line
MCF-7 Human breast adenocarcinoma cell line
Molecules 2019, 24, 1211 12 of 14

CCRF-CEM Human leukemia

RPMI-8226 Human multiple myeloma
J774 Human macrophages
SEM Scanning electron microscopy
XRD X-ray diffraction
TGA Thermogravimetric analysis
FITC Annexin V-fluoresce isothiocyanate
LDH Lactate dehydrogenase
HL-770 Normal liver cells
HepG2 Liver hepatocellular carcinoma
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide
DAPI 4’,6-diamidino-2-phenylindole
PI Propidium iodide
IC50 Half-maximal inhibitory concentrations
DRG Rat neonatal organotypic dorsal root ganglion
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethyl sulfoxide
FBS Fetal bovine serum

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Sample Availability: Samples of the compounds are available from the authors.

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