Colloids and Surfaces B: Biointerfaces 97 (2012) 221–225

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Cytotoxicity evaluation of magnetite (Fe3 O4 ) nanoparticles in mouse embryonic

stem cells
Chigusa Shundo a , Hong Zhang b , Takuya Nakanishi a , Tetsuya Osaka a,∗
a
Department of Applied Chemistry, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
b
Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,
Koganei, Tokyo 184-8588, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Magnetite nanoparticles are expected to be applied in the medical field because of their biocompatibility
Received 30 January 2012 and high saturated magnetization. In this paper, magnetite nanoparticles with a diameter of approxi-
Received in revised form 28 March 2012 mately 40 nm were evaluated for their safety by using mouse embryonic stem (mES) cells. First, various
Accepted 4 April 2012
doses of magnetite nanoparticles were added to mES cells to find an optimal dose and to evaluate viabil-
Available online 13 April 2012
ity and keeping undifferentiated states of mES. The uptake of nanoparticles by mES cells was confirmed
by using cytospin and transmission electron microscopy. Next, mES cells containing magnetite nanopar-
Keywords:
ticles were collected by a magnet column 24 h after the addition of magnetite nanoparticles, and the
Magnetite nanoparticles
Embryonic stem cells change in the ratio of those mES cells to the total mES cells was assayed by FACS 0, 4, 8, 12, 16, 24, 48 and
Excretion 72 h after incubation. The result showed that the ratio decreased with time, indicating that the mES cells
Safety evaluation excreted the nanoparticles, for there was no change in the total number of cells. Based on these results,
it was concluded that magnetite nanoparticles were safe to mES cells.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction cells, and they have a self-renewability which leads to unlimited

proliferation, i.e., the role of stem cells is to make new cells [11].
Nanoparticles are defined as materials with sizes in the range of Especially, embryonic stem (ES) cells derived from the inner cell
1–100 nm [1]. The number of atoms comprising them is smaller mass of preimplantation embryos have an ability to be maintained
than that of bulk materials, and they have high specific surface as undifferentiated cells in culture and also have high capacity to
areas and small sizes which make them suitable for the fabrication differentiate into a broad range of cell types. Therefore, ES cells
of catalysts, magnetic recording media, and sensors [2]. Espe- provide a unique tool to study and evaluate the nanoparticles cyto-
cially, magnetite nanoparticles are being intensively investigated toxicity on various types of cells in vitro.
for applications in the medical field because they are superparam- In this study, we focus our attention on ES cells. We investigated
agnetic and biocompatible. They are also being developed in recent the safety of magnetite nanoparticles with mouse embryonic stem
years as contrasting agents in magnetic resonance imaging (MRI), (mES) cells. The final aim of this research is to establish a process
carriers in drug delivery systems and heating elements of hyper- of certification for safety and to feedback information to synthesize
thermia [3–7]. nanoparticles, which are ingested by targets selectively.
For practical applications of nanoparticles to the therapy in
vivo, “cellular uptake” and “intracellular behavior” are important.
2. Material and methods
Regarding the cellular uptake, the synthesis of magnetite nanopar-
ticles which easily go into cancer cells leads to an effective therapy
2.1. Preparation of magnetite nanoparticles
[8,9]. It should be noted that normal tissues and cells also inter-
nalize magnetite nanoparticles [10], and thus understanding their
Magnetite nanoparticles were synthesized from ferrous chlo-
intracellular behavior is also important for the purpose of safety. It
ride and 1,6-hexanediamine (Kanto Chemical Co., Japan) as
is apparent that stem cells are useful for the evaluation of safety.
reported in the previous paper [12].
Stem cells have an ability to differentiate between certain kinds of

2.2. Cell cultures

∗ Corresponding author. Tel.: +81 3 5286 3202; fax: +81 3 3205 2074. Mouse embryonic stem (mES) cell line employed in this study
E-mail address: [email protected] (T. Osaka). was CCE mouse ES cell line, purchased from Stemcell Technologies,

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.04.003
222 C. Shundo et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 221–225

Inc., USA. We maintained the cells at the low passage, between

passage 25 and passage 30. They were cultured in Dulbecco’s Mod-
ified Eagle Medium (DMEM, Sigma) containing 15% fetal bovine
serum (Hyclone), MEM non-essential amino acid (0.1 mM, Gibco),
sodium pyruvate (1 mM, Sigma), l-glutamine (2 mM, Invitrogen),
antibiotic–antimycotic (Gibco), 2-mercaptoethanol (100 M) and
leukemia inhibitory factor (10 ng/mL, Millipore) in a gelatin-coated
dish. The human umbilical vein endothelial cells were purchased
from Cambrex, Inc., USA (Cell lot number: 1F1804). They were
grown in Endothelial Cell Growth Medium-2 Bullet Kit (Cambrex,
Inc., USA). All cells were incubated at 37 ◦ C in an atmosphere with
5% CO2 .

2.3. Measurements of cellular uptake of magnetite nanoparticles

Cells were prepared at the density of 1.5 × 104 cells/mL in 6-

well gelatin-coated plates for co-culture with 125, 250, 500, 750,
and 1000 ␮g of magnetite nanoparticles. After incubation for 24 h,
excess magnetite nanoparticles were removed by rinsing with
phosphate buffered saline (PBS). Next, cells were harvested and
treated by 0.25% trypsin–EDTA for measuring the amount of incor-
porated magnetite nanoparticles. Cells were dissolved in 500 ␮L of Fig. 1. TEM images of magnetite nanoparticles.
HCl (12 M). Then, 2.5 mL of trichloroacetic acid solution was added
and the mixture stored at 4 ◦ C for 30 min. After centrifugation, a
prepared by slicing with a diamond cutter, and they were observed
400 ␮L portion of the supernatant liquid was collected, to which
with TEM (Hitachi, Japan).
10 mL of H2 O2 and 4 mL of potassium thiocyanate solution (1 mol/L)
were added. One milliliter of this mixture was diluted with 2 mL of
2.7. Cytospin observation
ultrapure water to measure the absorbance at 480 nm by a spec-
trophotometer.
Cells were collected with centrifugation after co-culture
with magnetite nanoparticles for 24 h and dissolved in PBS
2.4. Viability assay of mES cells co-cultured with magnetite (>107 cells/mL PBS). Next, samples were dispensed to a cuvette
nanoparticles with slide glass and they were centrifuged (860 rpm, 5 min) with
Cytospin 4 (Thermo, Japan). The cells on the slide glass were dried,
For measurement of the viability of mES cells, those mES cells co- and the magnetite nanoparticles were stained with potassium fer-
cultured with magnetite nanoparticles were collected and stained rocyanide (Sigma). Magnetite nanoparticles turned blue 10 min
with 10 ␮L of propidium iodide (PI, BD). The cells were mea- later, at which time the residues were rinsed away with PBS. Then,
sured with flow cytometry (FACSCanto II, BD, CA). Dead cells were the cells were stained red with pararosaniline chloride solution
recognized by PI positive response, and the uptake of magnetite (Sigma), and after 5 min the residues were rinsed away with PBS.
nanoparticles by mES cells was derived in the height SSC (side The sample on the slide glass was observed with an optical micro-
scatter) section. scope.

2.5. Selection of cells containing magnetite nanoparticles 2.8. FACS analysis of undifferentiated state of mES

To study the internalization of magnetite nanoparticles into For staining, mES cells and mES cells co-cultured with mag-
mES cells, the cells were inoculated in a 10-cm dish at the den- netite nanoparticles were collected by 0.25% trypsin–EDTA solution
sity of 9.0 × 104 cells/mL. The cells were cultured until they grew and suspended in PBS with 0.1% bovine serum albumin (BSA) at a
to a sufficient density in the dish. Next, magnetite nanoparticles concentration of 1 × 105 cells per sample. In all experiments, cell
were mixed with the medium at the concentration of 1 mg/mL samples were preincubated in 0.1 mL PBS supplemented with 10 ␮L
added to the dish with mES cells at the ratio of 500 ␮g per normal rabbit serum (Funakoshi, Tokyo, Japan) for 30 min to block
1.5 × 105 cells, which were then incubated for 24 h. After washing nonspecific binding. After a wash with PBS, cells were stained for
with PBS to remove excess nanoparticles, cells containing mag- 40 min on ice with monoclonal antibodies rat antimous SSEA-1 con-
netite nanoparticles were sorted by a magnet column (Miltenyi jugated by phycoerythrin (PE). Stained cells were washed by PBS
Biotec, Germany) and a magnet stand (Miltenyi Biotec, Germany). and analyzed by using FACSCanto II (BD, CA). PE-conjugated rat
Then the nanoparticle-containing cells were cultured, and the ratio antimous SSEA-1 were purchased from BD.
of magnetite nanoparticles to positive cells was evaluated using a
flow cytometer and a spectrophotometer at 0, 4, 8, 16, 24, 48, and 3. Results and discussion
72 h.
3.1. Shape and size of magnetite nanoparticles
2.6. Transmission electron microscopic observation
After the synthesis of magnetite nanoparticles, their shape and
Cells were evaluated with TEM to confirm the uptake of mag- size were observed by TEM. The TEM images in Fig. 1 reveal
netite nanoparticles. The mES cells with added nanoparticles in that magnetite nanoparticles were spherical or truncated cubic in
a 15-mL tube were centrifuged. Next, mES cells were fixed with shape, measuring 42.7 ± S.D. 16.8 nm from randomly selected 100
2% of glutaraldehyde, dehydrated through 50%, 60%, 70%, 80%, 90% nanoparticles. Those results were essentially same as reported in
and 100% ethanol, and firmed to slice by resin. Then, samples were the previous paper [12].
 C. Shundo et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 221–225 223

Fig. 2. Optical microscopic images of mES cells internalizing magnetite nanoparticles (a) and cytospin images of mES cells only (control) (b) and mES cells containing
magnetite nanoparticles (c). In the cytospin images, mES cells were stained red and magnetite nanoparticles were stained blue by iron stain. Black arrows point at magnetite
nanoparticles, which were added to mES (500 ␮g/mL). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the
article.)

3.2. Toxicity of magnetite nanoparticles to mES with dose suggested that magnetite nanoparticles were internalized into mES
dependence cells (Fig. 2(b) and (c)). In addition, the TEM image of Fig. 3 clearly
shows that magnetite nanoparticles are internalized in mES cell.
Fig. 2a shows an optical microscopic image of mES cells co- This image indicates that the nucleus does not internalize mag-
cultured with magnetite nanoparticles. As pointed at by white netite nanoparticles, and that mES cells use an endocytic system
arrows, magnetite nanoparticles were observed in the mES colony. for uptake.
Generally, mES cells grown on the dish to consist of many layers Fig. 3 shows that mES cells internalized magnetite nanopar-
called colonies [10], which make it difficult to observe mES. To find ticles, and no nanoparticles were present in the nucleus of mES.
out whether magnetite nanoparticles were internalized in a cell
or between cells, cytospin observation was used. Monolayer sam-
ples were made on slide glasses by centrifugation. Cytospin images

Fig. 4. Dose dependence of magnetite nanoparticles uptake for mES. Magnetite

Fig. 3. TEM image of mES cell internalizing magnetite nanoparticles. White arrows nanoparticles were added to mES (0, 125, 250, 500, 750, and 1000 ␮g/mL), mES
point at magnetite nanoparticles. Magnetite nanoparticles were added to mES cells were incubated 24 h and the uptake was evaluated based on the absorbance at
(500 ␮g/mL). 480 nm.
224 C. Shundo et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 221–225

Scheme 1. Routes of reduction in proportion of mES cells containing magnetite nanoparticles.

Generally, cells internalize extracellular materials by endocytosis nanoparticles was close to 100%. Interestingly, from Fig. 4, the
[9,13]. In the method, extracellular materials are not taken up by amount of uptake at the dose of 1000 ␮g was lower than that at
nucleus, and they are blended with lysosome and degraded. It is 750 ␮g. Two reasons can be considered for this finding: (i) mES
emphasized then that magnetite nanoparticles do not cause dam- cells excreted or did not take up excess nanoparticles, and (ii)
ages to mES, and this result agrees with that of the previous study mES cells died due to the addition of a high dose. From Fig. 5,
on the safety of magnetite nanoparticles. however, the viability at 1000 ␮g was not lower than that for
The dose dependence of the uptake of magnetite nanoparticles the control experiment, which suggests that the low uptake was
by mES cells was evaluated by measuring the absorbance at 480 nm. caused by the reason (i). On the other hand, regarding the ratio
Fig. 4 was obtained by performing the experiments with the addi- of undifferentiated cells after co-culture with magnetite nanopar-
tion of 125, 250, 500, 750, and 1000 ␮g of magnetite nanoparticles tilces for 24 h shown in Table 1, 91.9% of mES and 92.1% of mES
per 1 mL of medium. The result shows that there is a limit of cel- with added magnetite nanoparticles were stained with SSEA-1.
lular uptake by mES cells. The cellular uptake increased with an These ratios were almost identical to each other, indicating that
increase of dose, and the value of approximately 120 pg of nanopar- there is no influence of the dose of magnetite nanoparticles on
ticles per cell was observed at the dose of 500 ␮g, after which mES.
the uptake decreased. The value of 120 pg/cell seems slightly high
considering the mass density of magnetite, which would depend 3.3. Excretion of magnetite nanoparticles by mES cells
on the evaluation method. To consider the dose-dependent influ-
ence of magnetite nanoparticles on mES cells further, the viability To search magnetite nanoparticles which were internalized in
of cells was examined next. Fig. 5 shows the dose dependence mES cells, the ratio of the number of cells containing magnetite
of viability measured by FACS. It is seen that nanoparticles have nanoparticles to the total number of cells was evaluated by FACS.
no toxicity because the survival rate of mES cells containing the To discuss the behavior specific to mES cells, the experiments
were carried out under the same conditions as those used for
human umbilical vein endothelial cells (HUVEC). The results are
shown in Fig. 6(a) and (b). In Fig. 6(a) the number of mES cells
did not changed (4.0 × 105 ) for 12 h, but the ratio of mES con-
taining magnetite nanoparticles decreased from 53.7% to 34.7%
during the same period of time. To explain the reason why the
ratio of mES cells containing magnetite nanoparticles decreased,
the following three routes are considered: (i) the number of
mES cells increased, (ii) magnetite nanoparticles were lost with
cell division, and (iii) magnetite nanoparticles were excreted by
mES cells (see Scheme 1). Because the number of mES cells was
kept constant from 0 to 12 h, the first and second routes can

Table 1
The ratio of undifferentiated cells. mES and mES with added magnetite nanoparticles
were stained.

Condition mES (%) mES with added magnetite

Fig. 5. Dose dependence of viability of mES cells. Magnetite nanoparticles were nanoparticles (%)
added to mES (0, 125, 250, 500, 750, and 1000 ␮g/mL). mES cells were incubated
SSEA-1(+) 91.9 92.1
for 24 h and stained by ropidium iodide, and then viability was determined by flow
SSEA-1(−) 8.1 7.9
cytometry.
 C. Shundo et al. / Colloids and Surfaces B: Biointerfaces 97 (2012) 221–225 225

4. Conclusion

Magnetite nanoparticles synthesized by the method used in

our previous study were added to mES to confirm the safety of
magnetite nanoparticles. It was found that the nanoparticles and
the mES internalized magnetite nanoparticles were not present
in the nucleus of mES, that there is a limit of cellular uptake by
mES, and that the dose of magnetite nanoparticles does not influ-
ence their ability of retaining the undifferentiated state. It was
also found that the ratio of mES containing magnetite nanoparti-
cles decreased, indicating that mES excreted nanoparticles. For this
reason, excess nanoparticles were excreted by mES, which makes
magnetite nanoparticles safe to mES. We expect a widespread
application of magnetite nanoparticles in the medical field.

Acknowledgment

This work was financially supported by Grant-in-Aid for Spe-

cially Promoted Research “Establishment of Electrochemical Device
Engineering” and Global COE program “Practical Chemical Wis-
dom”, from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan. This work was partly carried out at the
Consolidated Research Institute for Advanced Science and Medical
Care, Waseda University (ASMeW).
Fig. 6. Change in the ratio of cells containing magnetite nanoparticles to the total
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