Calero et al.

Journal of Nanobiotechnology (2015) 13:16

DOI 10.1186/s12951-015-0073-9

RESEARCH Open Access

Characterization of interaction of magnetic

nanoparticles with breast cancer cells
Macarena Calero1,5†, Michele Chiappi2†, Ana Lazaro-Carrillo1,5, María José Rodríguez2, Francisco Javier Chichón2,
Kieran Crosbie-Staunton3, Adriele Prina-Mello3,4, Yuri Volkov3,4, Angeles Villanueva1,5* and José L Carrascosa2,5*

Abstract
Background: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in
cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for
relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of
dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and
negative surface charge in MCF-7 breast cancer cells.
Results: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for
different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively
slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic
acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly
removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis
and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and
clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these
nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution,
reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells.
Conclusions: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles
have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cells.
Keywords: MCF-7 cells, Superparamagnetic iron oxide nanoparticles, Intracellular trafficking, Transmission electron
microscopy, Cellular uptake, Endocytosis, Cytotoxicity

Background and targeted therapy of cancer by hyperthermia and/or

Although huge efforts have led to worldwide advances in releasing anti-cancer molecules, which can be combined
cancer treatment, this multifactorial and heterogeneous in theranostic approaches [3-5].
disease is still one of the major causes of death in devel- Factors such as size, shape and surface charge of nano-
oped countries [1,2]. In the recent years, several reports particles (NPs) can determine their cellular internalization
have focused on the potential use of superparamagnetic and distribution and, thus, their effective performance
iron oxide nanoparticles (SPION) in cancer research. [6,7]. Furthermore, colloidal stability can be achieved,
These reports have raised great expectations because which is essential to ensure reproducibility, as well as
SPION are a promising tool for biomedical applications, to influence the amount of cellular loading and toxicity.
including diagnosis by magnetic resonance imaging (MRI) The possibility to modify the surface of these particles
with biologically active compounds enables transport of
therapeutic agents into specific target cells, increasing
* Correspondence: [email protected]; [email protected]
†
Equal contributors
specificity and avoiding the access of cytotoxic agents
1
Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, to non-target tissues during the delivery process [8].
28049 Madrid, Spain Different SPION have been tested for potential use in
2
Department of Macromolecular Structure, Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
cancer treatment by hyperthermia, as they enter into
Full list of author information is available at the end of the article cells with high effectiveness and without any cytotoxicity,
© 2015 Calero et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 2 of 15

and they are retained for relatively long periods of time charged magnetic nanoparticles (MNP) showed a higher
inside the cells [3]. The evaluation of the potential use of degree of internalization than neutral and negatively
these nanoparticles requires a precise knowledge of sur- charged MNP due to their effective attachment to nega-
face modified SPION internalization mechanisms at the tively charged cell-membrane surface [3,14,16]. Although
ultrastructural level and resulting intracellular pathways, there are somewhat contradictory findings about cyto-
as well as on the fate of SPION inside the cells. Factors toxicity levels between positively or negatively charged
such as uptake rate and internalization dynamics are the nanoparticles [3,17-19], the latter ones are favored due
key to understand how an insufficient cellular accumula- to their overall lower toxicity levels.
tion of nanoparticles can lead to usage limitations, for Incorporation of DMSA-SPION into MCF-7 cells can
example as imaging probes [9]. be followed by bright field microscopy after 24 h incuba-
In the past few years, there has been a great interest tion (Figure 1A), where SPION are observed inside living
in applying nanotechnology for biomedical studies, in cells, distributed as brown cytoplasmic spots of different
particular for diagnostic and therapeutic purposes. How- sizes, always outside of the nucleus. Similar results have
ever, the possible toxicity of nanoparticles to humans and been previously described for iron oxide nanoparticles
environment has become a question of absolute priority with different coatings and different sizes in HeLa (human
in Nanomedicine [4-6,10]. cervical adenocarcinoma) cell line [3,17].
In this regard, cell cultures are important first line In depth qualitative and quantitative studies on the
tools to screen therapeutic efficiency and safety of drugs internalization of DMSA-SPION in MCF-7 cancer cells
(nanoparticles included) and provide essential information were performed by both Prussian blue staining and
to understanding cell-nanoparticle interactions, before ferrozine-based assay. Figure 1B shows cells incubated with
moving to in vivo analysis [11]. Hence, any new magnetic DMSA-SPION for different times (0.5-72 h) by Prussian
nanoparticle formulation with potential biomedical appli- blue staining. An increase of intracellular DMSA-SPION
cations should be accompanied by a detailed study that accumulation was visualized as blue cytoplasmic granular
ensures both its effectiveness and safety. In this sense, sev- stain within cells directly correlating with incubation times.
eral specific parameters and experimental protocols for However, the uptake of nanoparticles seems to reach a
assessing nanomaterial toxicity have been developed [10]. saturation point at 24 h. It is important to note that 100%
We have studied the interaction of dimercaptosuccinic cell labeling efficiency (Prussian blue positive staining)
acid-coated superparamagnetic iron oxide nanoparticles was achieved after 12 h nanoparticles incubation.
(DMSA-SPION) with breast cancer cells (MCF-7) in These results were confirmed by colorimetric ferrozine-
culture. Monodisperse nanoparticles (around 15 nm in based assay, a widely recognized test to quantify iron in
diameter) with a high saturation magnetization value, cultured cells [20]. Figure 1C shows intracellular iron con-
were surface modified by meso-2,3-dimercaptosuccinic centrations after 24 and 48 h incubation at 0.4 mg ml−1
acid (DMSA) to ensure their dispersion and stability in DMSA-SPION (20.67 pg cell−1 and 28 pg cell−1, respect-
aqueous buffers and media [12]. Interaction, uptake of ively). There is abundant literature with regard to
the particles (0.05-0.4 mg ml−1), as well as their accumu- SPION-labeling efficiency, although results are difficult
lation and persistence inside cells after prolonged incu- to compare because the experimental protocols are differ-
bation (up to 72 h), were assessed by combining optical ent (size and surface coating of the SPION, incubation
light and electron microscopy methods. This approach time, concentration, cell line type, etc.). Generally, pro-
allowed us to correlate the overall cell visualization with longed incubation times, as well as elevated iron doses
the precise localization of SPION inside the cell, their enable to reach higher intracellular loading of SPION and
relationship to cell organelles and the analysis of particle increase labeling efficiency [21,22]. However, overexposure
shapes and sizes. Furthermore, several cytotoxicity assays, to high concentrations of SPION for extended times may
including cell morphology, analysis of cytoskeleton and cause cytotoxicity [23]. Therefore, sufficient intracellular
adhesion proteins, cell cycle distribution, measurement of uptake of nanoparticles for efficient diagnosis and/or
intracellular reactive oxygen species (ROS) levels and two treatment must be balanced with their biocompatibility
viability tests, have been carried out to evaluate biocom- [17]. In this sense, our results with ferrozine assay
patibility of these nanoparticles. indicate that DMSA-SPION accumulate effectively
(20.67 pg cell−1) within MCF-7 cells. Previously, we had
Results and discussion detected 37.1 pg cell−1 (into HeLa cells), after 24 h of
DMSA-SPION uptake and internalization in cultured cells incubation at 0.5 mg ml−1 DMSA magnetic nanoparticles
Size, shape and charge of iron oxide nanoparticles, as with lower core diameter (9 nm). The small difference in
well as cell type, are important parameters which affect the amount of accumulated iron could be either due to
effective internalization of nanoparticles into cells in cul- different of SPION diameters (15 vs 9 nm) or to the type
ture [13-16]. It has been well documented that positively of cell line (HeLa vs MCF-7) [17]. Much lower amounts
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 3 of 15

Figure 1 Uptake and accumulation of DMSA-SPION into cells. (A) MCF-7 living cells visualized by bright field microscopy. (a) Control cells.
(b) Cells incubated with 0.4 mg ml−1 SPION for 24 h. Scale bar represents 10 μm. (B) Cells incubated with 0.4 mg ml−1 SPION for different time,
stained with Prussian blue reaction and visualized by bright field microscopy. (a) Control cells. (b-i) Cells incubated for 0.5, 1, 3, 6, 12, 24, 48 and
72 h, respectively. Scale bar represents 10 μm. (C) Intracellular iron content quantification by ferrozine assay (expressed as weight of iron per cell),
after 24 and 48 h of incubation. (D, E) Untreated and incubated MCF-7 (area, red filter), cell with DMSA-SPION. Representative images (D) and
quantitative box-plot of 100 cells per treatment (E). Details of x-axis: 1) Untreated cell only (background red filter), 2) Untreated, cell only (blue
filter), 3) Cell + SPION (total SPION), 4) Cell + SPION (total cell area, blue filter).

(5.3 ± 1.1 pg cell−1) have been detected over 48 h of incu- was carried out by automated epifluorescence imaging
bation with SPION (Feridex®) at 0.075 mg ml−1 in labeled with multichannel acquisitions (bright field, blue and red
NPC (neural progenitor cells) [24]. channels). From the overlapping and thresholding against
Quantitative and statistical population analysis of total the iron content it was possible to identify and quantify
iron oxide area per total cell area of 100 MCF-7 cells the ratio of inorganic iron content versus the total cell
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 4 of 15

area. Figure 1D shows representative microscopy images bright field microscopy showed that uptake and accumu-
of untreated and exposed MCF-7 cells to 0.4 mg ml−1. lation of nanoparticles in MCF-10A cells was equivalent
Samples from the same experiments were processed to MCF-7 cancer cells (see Additional file 1). This was
for observation by electron transmission microscopy confirmed by Prussian blue staining. Analysis by electron
(Figure 2). Even after very short incubation times (0.5 h), microscopy clearly revealed that aggregates of particles
it was possible to detect SPION clusters within cell cyto- were accumulated inside MCF-10A cells near nucleus with
plasm (Figure 2a). DMSA-SPION were found surrounded similar kinetics to that found in carcinoma cells (Additional
by a membrane and no free cytoplasmatic nanoparticles file 1). The overall response of these non-cancerous cells
were detected. Incubations of 1 and 3 h revealed a small was similar to carcinoma cells (see Additional file 1).
increment in the presence of vesicles containing DMSA- Results obtained for nanoparticles internalization in
SPION (Figure 2b, c). During longer incubation times malignant (MCF-7) and non-malignant (MCF-10A) cell
(6, 12 and 24 h), the number of vesicles with larger lines are not entirely surprising. It is important to recall
DMSA-SPION aggregates increased and they were accu- that all established cell lines, including non-malignant
mulated close to the nuclei (Figure 2d-f and inset in f). cells, have alterations in their genome, which make them
Together with an increment in the number of vesicles, different from healthy cells of an organism. Therefore,
prolonged incubation time also resulted in important MCF-10A cannot be considered as a fully “normal” hu-
morphological changes of DMSA-SPION containing vesi- man cell line [25,26]. In this sense, quantum dot (QD)
cles. While analysis of sectioned cells revealed a small in- nanoparticles with different surface coatings can be
crement in their size, the most important change however internalized within human mammary non-tumorigenic
was related to their morphology, where a clear evolution epithelial cell line MCF-10A as well as in human mam-
from translucent vesicles with nanoparticles towards a mary adenocarcinoma epithelial cell line MCF-7 [27].
much denser and multivesicular aspect has been detected Zhang et al. [28] have described that both (MCF-7 and
(Figure 2 a-f). MCF-10A) cells can internalize iron oxide nanoparticles
As MCF-7 cells are derived from a human breast by vesicular transport after incubation for different times
adenocarcinoma, we decided to study also DMSA-SPION (30 min, 4 and 24 h). This research was carried out
uptake and accumulation in a non-malignant breast cell using commercial iron oxide nanoparticles (maghemite
line MCF-10A. Cells were incubated with DMSA-SPION γ-Fe2O3 with diameter around 30 nm) from Alfa Aesar®
under the same conditions as MCF-7 cells. Analysis by (Karlsruhe, Germany) without any coating.

Figure 2 Electron microscopy analysis of uptake kinetics. Images from thin sections of MCF7 cells incubated with DMSA-SPION. (a) Cells incubated
for 0.5 h, (b) 1 h, (c) 3 h, (d) 6 h, (e) 12 h and (f) 24 h. The inset in (f) shows the overall cell shape and morphology. Scale bars represent 1 μm for each
image, 200 nm for insets in a to e, and 2 μm for the inset in f, respectively.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 5 of 15

In summary, it is rather difficult to compare our results we performed transmission electron microscopy (TEM)
with those reported in the literature previously, because studies. The high contrast of the magnetic particles
nanoparticles used in other studies have very different allowed for their clear identification (Figure 4). Small
characteristics. It is well known that parameters such as groups of particles were seen near cell membranes.
nanoparticle size and particle surface coating are crucial Actually, SPION incubated in culture media present a
on nanoparticle-cell interactions [3,8,17,29]. relatively wide size distribution (ranging between 50 to
more than 400 nm, see Additional file 2). Although we
Internalization mechanism and accumulation of did not make an attempt to sort the SPION by size, we
DMSA-SPION inside cells found significant differences in the way the SPION were
To analyze internalization mechanism, cells were incu- incorporated in the cells according to the aggregate size.
bated with particles at different temperatures. At 4°C, in- Smaller aggregates were seen adjacent to distinct clathrin-
ternalization of DMSA-SPION was inhibited and coated patches (Figure 4A). Closed clathrin vesicles con-
nanoparticles were attached at the cell surface, while up- taining small DMSA-SPION aggregates (smaller than 200
take was developed successfully after 3 h at 37°C (Fig- nm) were seen in the cytoplasm, near membrane. Larger
ure 3A). This result indicated that an active energy- DMSA-SPION aggregates were seen near cell periphery,
dependent transport was implicated in the SPION intern- in most cases engulfed by cell membrane extensions, indi-
alization process [13,14,17,21]. cating the existence of a macropinocytic DMSA-SPION
To get insight into these nanoparticles subcellular uptake process (Figure 4B a, b). Other studies have also
localization, MCF-7 cells were incubated with DMSA- proposed a macropinocytic process for cationic iron oxide
SPION for 24 h and then incubated with LysoTracker nanoparticles internalization [30], as well as for other
Red to stain the lysosomal compartment and finally nanoparticles [31].
visualized by bright field and fluorescence microscopy. Following short incubation times, particles were found
Figure 3B show SPION into MCF-7 living cells using near the cell membrane, showing SPION-containing
fluorescence microscopy. As can be seen in the same vesicles closely resembling early endosomes (Figure 4C a).
figure lysosomes were labeled with LysoTracker Red. At later incubation stages, there were denser SPION-
Merged images displayed a substantial fraction of red containing vesicles resembling multi-vesicular bodies con-
fluorescence from LysoTracker which colocalizes with in- taining intraluminal vesicles (Figure 4C b). Subsequently,
ternalized nanoparticles, strongly suggesting that DMSA- the vesicles adopted a multi-lamellar lysosome aspect
SPION were accumulated in endosome/lysosome fraction. containing large numbers of DMSA-SPION clusters
To identify the precise mechanism of endocytosis (Figure 4C c,d).
(phagocytosis, pinocytosis, macropinocytosis, clathrin- The same type of analysis has been carried out with
mediated endocytosis, or caveolae-mediated endocytosis), the non-malignant MCF10-A cells. The results clearly

Figure 3 Internalization mechanism and accumulation of DMSA-SPION inside cells. (A) Temperature dependence of DMSA-SPION uptake.
(a, a’) Control cells. (b) Cells incubated at 4 °C for 3 h with DMSA-SPION. (b’) Cells incubated for 3 h with same nanoparticles at 37 °C. Scale bars
10 μm. (B) Subcellular localization. (a, b) Visualization of control cells and cells incubated with nanoparticles for 24 h by bright field microscopy,
respectively. (a’, b’) Lysosomes labeled with LysoTracker Red probe in the same cells, respectively. (a”, b”) Overlay images of control and treated
cells, respectively. Scale bar 20 μm.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 6 of 15

Figure 4 Electron microscopy study of SPION interaction and uptake. (A) Electron microscopy images of thin sections of cells interacting
with DMSA-SPION by clathrin mediated uptake (<200 nm in diameter aggregates). Scale bar represents 200 nm. (B) Two images by electron
microscopy of thin sections of cells showing typical images of macropinocytosis for DMSA-SPION uptake (>200 nm in diameter aggregates).
Scale bars represent 200 nm. (C) Electron microscopy images of different types of endosomes containing SPION aggregates: (a) Early endosome.
(b) Multivesicular body containing intraluminal vesicles. (c) Late endosome characterized by a multilamellar morphology. (d) Late endosomes and
lysosomes with multivesicular structure and large electron-dense areas. Scale bar represents 200 nm.

showed that incorporation of DMSA-SPION and their carried out using electron microscopy. Cells containing
intracellular trafficking feature the same overall character- DMSA-SPION evolved and divided in a similar way as
istics in the case of the non-cancerous breast epithelial control cells without DMSA-SPION. Multi-vesicular
cells (see Additional file 1). bodies and lysosomes containing nanoparticles did not
change much, even after extended incubation intervals
Intracellular persistence of SPION (Figure 5B a-d). SPION clusters were retained inside
Other important questions related to the incorporation the vesicles and these vesicles further evolved towards
of nanoparticles into cells are to establish how long they late endosomal or lysosomal morphology, but neither
remain inside cells and to disclose their eventual release their number nor their localization in cell cytoplasm
mechanism. To get an insight into these questions, after underwent significant changes, thereby indicating that
24 h incubation, nanoparticles were removed and cultures DMSA-SPION were not massively released from cells.
were further incubated up to 72 h at 37°C. Samples, taken These results suggest that, although cells keep dividing,
at 24, 48 and 72 h, were stained with Prussian blue and iron oxide nanoparticles persist inside them for a long
observed by bright field microscopy. Figure 5A shows that time. These qualitative results were confirmed by quantifi-
SPION remain within MCF-7 cells in vesicles up to 72 h. cation of intracellular iron content in ferrozine-based
To get more detailed information on the evolution of assay (Figure 5C), which confirmed that the amount of
the intracellular vesicles after prolonged incubation iron remains substantially unaltered inside the cells after
times, a parallel analysis to that described above was 48 h post-incubation interval.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 7 of 15

Figure 5 Persistence of internalized DMSA-SPION. (A) MCF-7 cells incubated with nanoparticles for 24 h, stained with Prussian blue reaction
after different post-incubation times and visualized by bright field microscopy. (a) Untreated control cells. (b-d) Cells incubated for 24 h and
stained 24, 48 and 72 h after incubation, respectively. Scale bar represents 10 μm. (B) Study of persistence by electron microscopy: (a) Cells were
incubated with DMSA-SPION for 24 h. The cells were further incubated in medium without particles for additional (b) 24 h, (c) 48 h and (d) 72 h.
Insets show larger magnification details of the endosomes. Scale bars represent 5 μm in overall areas and 500 nm in larger magnification insets,
respectively. (C) Intracellular iron content quantification by ferrozine assay (expressed as weight of iron per cell) in control (non-treated) cells (c),
and immediately (0) or 48 h after incubation with DMSA-SPION.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 8 of 15

Cytotoxicity of DMSA-SPION of DMSA-SPION, followed by localization of them in-

Exposure to SPION has been associated with significant side the cells by bright field microscopy, did not affect
toxic effects due to the generation of ROS, which result the organization of stress fibers or focal adhesions.
in deleterious cellular consequences eventually leading
to cell death [32-35]. There are contradictory results (ii) Cell morphology
related to biocompatibility of DMSA-coated magnetic MCF-7 cells were exposed to 24 h incubation with
nanoparticles. Several reports have described some cyto- DMSA-SPION and then they were stained with neutral
toxicity for DMSA magnetic iron oxide nanoparticles in red or Hoechst-33258 to observe cytoplasmic and nuclear
different cell lines [19,36]. On the contrary, little effects morphology, respectively. Cells stained with neutral red
on cell viability, oxidative stress, cell cycle or apoptosis have a similar morphology to control cells after nanoparti-
have been reported for these magnetic nanoparticles by cle incorporation in cytoplasm and, nuclei also presented
other authors [17,37]. the same characteristics as control cells (Figure 7A).
Taking into account such a contradictory background,
we decided to analyze the biocompatibility of these (iii) Cell cycle
nanoparticles using several complementary approaches, As shown in Figure 7B, cell cycle distribution was not
such as (i) studies of cytoskeletal components, (ii) cell affected after incubation of cells with 0.4 mg ml−1
morphology observations by bright field microscopy SPION for 24 h when compared to untreated control
(neutral red and Hoechst-33258 staining), (iii) analysis of populations. A broad overview of the effects of magnetic
the cell cycle, (iv) detection of ROS generation and, (v) and nonmagnetic nanoparticles on the cell life cycle has
two alternative viability tests. been recently compiled by Mahmoudi et al. [39].

(i) Analysis of cytoskeletal components (iv) Detection of ROS generation

Two components of cytoskeleton were analyzed: micro- To analyze whether DMSA-SPION produce ROS, cells
tubules (MTs) and actin filaments (F-actin). MTs are were preincubated with SPION for 24, 48 and 72 h and
highly dynamic fibers of the cytoskeleton, with critical then treated with the fluorochrome probe DCFH-DA
functions in eukaryotic cells including intracellular [40]. As shown in Figure 7C, no significant fluorescent
transport, organization of cell structural dynamics and signal was detected after 72 h incubation with SPION at
cell division. We have evaluated the effects of nanoparti- 0.4 mg ml−1. Several in vitro studies have suggested that
cle internalization on MTs during interphase and mitosis a range of iron oxide nanoparticles with different
by means of indirect immunofluorescence analysis to physico-chemical characteristics induce ROS formation
α-tubulin (DNA counterstained with Hoechst-33258). which can lead to cellular injury and death [32-35].
Figure 6A shows fluorescence images of MTs (green)
and DNA (blue) for interphase and metaphase MCF-7 (v) Cell viability studies
control cells. After 24, 48 or 72 h of incubation with MTT assay showed that cell viability was not signifi-
nanoparticles, interphase microtubules maintain their cantly affected by the presence of DMSA-SPION at 24 h
normal morphology and distribution. In the same sam- of treatment (>96% viability in relation to the control
ples, DMSA-SPION were visualized inside the cells by sample), even at the highest concentration (0.4 mg ml−1)
bright field microscopy. Distributions of mitotic spindles (Figure 7D a). The results obtained using Trypan blue
and chromosomes were also similar to metaphase control assay (Figure 7D b), confirmed the biocompatibility of
cells up to 72 h after incubation. DMSA-SPION, and cell survival was > 90% after 24 h
We also investigated the effects of SPION on F-actin incubation. It is important to note that Trypan blue
and vinculin, a protein implicated in cell adhesion as a exclusion test has been proposed as the gold standard
focal adhesion complex component. F-actin builds the method to validate the cell viability after magnetic nano-
thinnest filaments of cytoskeleton in the cytoplasm of particle incubation [41]. These results were further con-
eukaryotic cells. They are involved in cell morphology, firmed using a multiparametric High Content Screening
transport of vesicles and organelles, positioning of cellu- Cytotoxicity Assay in agreement with a previously pub-
lar components, cytokinesis, cell motion, cell-cell and lished report [42], (data not shown).
cell-substrate interactions, and signal transduction. Focal In summary, the results presented here justify a deeper
adhesions are specialized sites containing a complex net- research on the synthesis and biological characterization of
work of proteins, included vinculin, favoring interactions iron oxide nanoparticles. The complementary approaches
between cell and extracellular matrix through the actin recommended for risk assessment of nanoparticles [43,44]
cytoskeleton [38]. Figure 6B shows fluorescence images indicate that DMSA-SPION are safe and efficient nanopar-
of actin microfilaments (red), vinculin protein (green) ticles for possible biomedical applications. This is a crucial
and DNA (blue) for MCF-7 control cells. Incorporation fact, before further functionalization of these SPION for
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 9 of 15

Figure 6 Analysis of cytoskeleton. (A) Representative images of cells immunostained for α-tubulin (green) and DNA counterstained with
Hoechst-33258 (blue). (a) Interphase control cells. (a’) Metaphase control cell. (b-b’) Interphase cells incubated for 24 h with DMSA-SPION and observed
by fluorescence and bright-field microscopy, respectively; (c-c’) cells incubated for 48 h; (d-d’) cells incubated for 72 h. (e-e’) Mitotic spindle of cells
incubated for 24 h, (f-f’) 48 h and (g-g’) 72 h. (B) Merged images of F-actin labeled with rhodamine-phalloidin (red), vinculin immunostaining (green)
and DNA counterstained with Hoechst-33258 (blue). (a) Control untreated cells. (b-b’) Cells treated with DMSA-SPION for 24 h and observed by
fluorescence and bright-field microscopy, respectively. (c-c’) Cells incubated for 48 h. (d-d’) Cells incubated for 72 h. Scale bar 10 μm.

medical applications (drug delivery and/or hyperthermia), to confirm the absence of toxicity induced by nanoparti-
that require high levels of intracellular accumulation for cles (non-functionalized), to ensure their biocompatibility,
effective treatment. even if they were accumulated by non-tumor cells. This is
It is important to point out that the purpose of our especially important, taking into account the pressing
study was twofold: i) to analyze the effectiveness of need to identify any potential cellular damage associated
DMSA-SPION accumulation within tumor cells and ii) with SPION [32]. In the broader context, following such
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 10 of 15

Figure 7 Cytotoxic studies. (A) Cell morphology by neutral red and Hoechst-33258 staining. (a-a’) Control cells. (b-b’) Cells incubated with
DMSA-SPION for 24 h. Scale bar represents 10 μm. (B) Cell cycle analysis of control (untreated) and cells incubated with SPION for 24 h. (C) Analysis of
ROS generation by DCFH-DA assay. Cells incubated with DMSA-SPION for different times and loaded with DCFH-DA were visualized under bright field
or fluorescence microscopy, respectively. (a, a’) Cells incubated with nanoparticles for 24 h. (b, b’) Cells incubated for 48 h. (c-c’) Cells incubated for
72 h. Scale bar represents 50 μm. (D) Cytotoxicity analysis in MCF-7 cells incubated DMSA-SPION for 24 h. (a) MTT cell viability assay after 24 h of
treatment with 0.05, 0.1 and 0.4 mg ml−1. (b) Trypan blue exclusion test immediately after incubation at 0.4 mg ml−1.

work carried out these “marginally toxic nanoparticles” dependent path, while larger aggregates were incorporated
will be further functionalized with biologically active by macropinocytosis. In all cases, SPION aggregates were
molecular moieties such as peptides and antibodies for found surrounded by endocytotic membrane, which local-
breast cancer targeting. From this prospective, our study ized in perinuclear areas after long incubation times,
is relevant to the safe development of nanoparticles for but never inside the cell nucleus. Following cellular
biomedical applications, as well as to understanding their uptake, SPION showed a slow release rate and continu-
biological behavior in the “bare” or non-functionalized ous persistence over extended intervals inside the cells.
state, since once delivered inside the cells, nanoparticles These characteristics are relevant for the rational design
can be processed by intracellular pathways (e.g. distinct and subsequent utilization of SPION for biomedical appli-
endocytic pathways) and “stripped” or separated from the cations, both for diagnosis by magnetic resonance imaging
molecules they have been originally conjugated with. (MRI) and for targeted therapy of cancer by hyperthermia
and releasing anti-cancer molecules with significantly
Conclusions reduced side effects.
Dimercaptosuccinic acid surface coating of SPION en-
hanced their cellular uptake efficiency without inducing Methods
either cytotoxicity, alteration of the major cytoskeletal Magnetic nanoparticles
components, vinculin protein dynamics, cell cycle or ROS Superparamagnetic iron oxide nanoparticles of uniform
formation in MCF-7 breast cancer cell line. Incorporation size (15 nm) were obtained by thermal decomposition of
of DMSA-SPION inside the cells followed two endocytic an iron oleate complex in 1-octadecene [12]. These
pathways depending on the size of the particle aggregates: particles, with a coating of DMSA that make them stable
smaller aggregates were incorporated using a clathrin- in aqueous buffers, were kindly provided by Dr. Puerto
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 11 of 15

Morales (ICMM-CSIC) as part of MULTFUN FP7 NMP Quantification of iron in cultured cells
project (see details in Additional file 2). Colorimetric ferrozine-based method
DMSA-SPION were sterilized by 0.22 μm pore size fil- This sensitive assay permits the quantification of iron in
tration (Millipore Corp., Bedford, USA). SPION stock cultured cells [46]. In time-dependent studies, MCF-7
at 4 mg ml−1 was dispersed by sonication for 5 min in a cells seeded in 24-well plates were incubated with
40 kHz sonicator bath (Branson 3510 ultrasonic cleaner, DMSA-SPION at a fixed concentration of 0.5 mg ml−1
Thomas Scientific, Swedesboro, USA). SPION were then for 24 or 48 h. For intracellular persistence studies,
resuspended in complete cell culture media at a final con- cells were incubated 24 h and intracellular iron content
centration of 0.4 mg ml−1. The mixture was then soni- was evaluated 48 h after removing DMSA-SPION from
cated for 1 min and incubated with cells at different times. culture media by three washes with PBS. After that, in
both cases, cells in three wells were trypsinized and cell
Cell cultures concentrations per well were determined by hemocy-
Human breast cancer MCF-7 cells were grown as mono- tometer with 0.4% Trypan blue solution. Cells grown in
layer cultures in Dulbecco’s modified Eagle’s medium other 24-well dishes were frozen at −20°C for 1 h and
(DMEM), supplemented with 10% (v/v) fetal bovine then, 500 μl of 50 nM NaOH (Panreac Química) were
serum (FBS), 50 units ml−1 penicillin and 50 μg ml−1 added to each well for 2 h in movement. Aliquots of
streptomycin. All products were purchased from Gibco cell lysates were then transferred to 1.5 ml eppendorf
(Paisley, Scotland, UK) and sterilized by means of 0.22 and mixed with 500 μl of 10 mM HCl, and 500 μl of
μm filters. Cell cultures were grown in an incubator with iron-releasing reagent (a freshly mixed solution of equal
5% CO2 plus 95% air at 37°C. Depending on the purpose volumes of 1.4 M HCl and 4.5% (w/v) KMnO4 (Merck,
of experiment, cells were seeded on 24-well plates (with Germany) in distilled H2O. These mixtures were incu-
or without 10 mm square coverslips) or 25 cm2 flasks. bated for 2 h at 60°C within a fume hood, since
Sub-confluent cell cultures were used. All sterile plastics chlorine gas is produced during the reaction. After the
were sourced from Corning (Corning Inc., New York, mixtures had cooled to room temperature, 150 μl of
USA). iron-detection reagent (6.5 mM ferrozine (Sigma-Aldrich,
Non-tumorigenic human breast epithelial cell line St Louis, USA), 6.5 mM neocuproine (Sigma-Aldrich),
MCF-10A was used for comparison in some experi- 2.5 M ammonium acetate (Panreac Química), and 1 M
ments (see Supporting Information). Cell lines used in ascorbic acid (Sigma-Aldrich) dissolved in water) were
this study were obtained from American Type Culture added to each tube. After 30 min, 500 μl of the solution
Collection (ATCC)®. obtained in each tube was transferred into a well of a
24-well plate, and absorbance was measured at 570 nm
DMSA-SPION internalization in a SpectraFluor spectrophotometer (Tecan Group
Live cell imaging Ltd., Männedorf, Switzerland). Iron content of the sam-
In order to analyze internalization of nanoparticles, ple was calculated by comparing its absorbance to that
MCF-7 cells were grown on coverslips and incubated for of a range of standard concentrations of equal volume,
24 h with DMSA-SPION. After incubation, culture that had been prepared in a way similar to that of the
medium was removed and samples were washed three sample (mixture of 100 μl of FeCl3 standards (0–300
times with phosphate-buffered saline (PBS, pH 7.4). μM) in 10 mM HCl, 100 μl 50 mM NaOH, 500 μl
Then, cells were observed immediately under bright releasing reagent, and 1500 μl detection reagent). The
light microscopy without being processed, to avoid po- determined intracellular iron concentration for each
tential fixation artifacts. well of a cell culture was normalized against number of
cells per well.
Prussian blue staining
Cells preincubated with nanoparticles for different periods
of time (0.5, 1, 3, 6, 12, 24, 48 or 72 h), were visualized by High content screening
Prussian blue staining for iron detection [17,45]. Briefly, Quantification of iron oxide content was based on auto-
cells were fixed in methanol (at −20°C) for 5 min, stained mated epifluorescence images taken from stained cell
with an equal volume of 4% hydrochloric acid and 4% monolayer cultured on slides. On average 100 cells were
potassium ferrocyanide trihydrate for 15 min, and coun- selected from the two cell line provided. Images were
terstained with 0.5% neutral red for 2 min. Preparations analyzed by single channel, filtered and threshold of each
were then washed with distilled water, air dried, and channel was identified. Composite rebuilt in order to
mounted in DePeX (Serva, Heidelberg, Germany). All identify localization of SPION against cellular staining.
other reagents were purchased from Panreac Química Filtering was applied on the red and blue filter in order
(Montcada i Reixac, Spain). to account for the SPION or the cell only.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 12 of 15

Endocytic mechanisms Cell morphology analysis

In order to analyze the degree of involvement of the endo- Neutral red staining
cytic mechanisms in internalization of nanoparticles, cells MCF-7 cells grown on coverslips in 24-well plates were
were preincubated with nanoparticles for 3 h at either 4°C incubated with DMSA-SPION for 24 h, fixed in methanol
or 37°C, washed three times with PBS, and stained with at −20°C for 5 min and then stained with 0.5% neutral red
Prussian blue technique (as described above). for 2 min. After that, samples were washed with distilled
water, air dried, mounted in DePeX and visualized by light
Subcellular localization of nanoparticles microscopy.
To determine DMSA-SPION subcellular location inside
cells, endocytic compartments of MCF-7 cells were labelled Hoechst-33258 staining
with 50 nM LysoTracker® Red DND-99 (Molecular Probes, Cells seeded on coverslips and treated with nanoparticles
Eugene, Oregon, USA) fluoroprobe in culture medium, at for 24 h were fixed in methanol at −20°C for 5 min and
37°C for 30 min. Cells incubated with nanoparticles for 24 stained with Hoechst-33258 for 5 min. Samples were
h were labelled with LysoTraker Red and then coverslips washed with distilled water, air dried, and mounted in
were washed with PBS and cells were observed immediately DePeX for observation using fluorescence microscopy.
under bright field and fluorescence microscopy.
Cell cycle analysis
Analysis of the cytoskeleton and adhesion proteins MCF-7 cells were plated in 25-cm2 flasks and incubated
Immunofluorescence staining of α-tubulin with DMSA-SPION for 24 h. Analysis of cell cycle was
Cells grown on glass coverslips were incubated with nano- performed by flow cytometry using propidium iodide
particles for 24, 48 and 72 h and then, immunostained for (PI) labeling of DNA cell content. Cells were trypsinized
α-tubulin. Briefly, cells were fixed with cold methanol for (also harvesting possible detached cells) and centrifuged
5 min, washed three times for 5 min with PBS, and then at 1200 rpm for 5 min. After centrifugation, pellet was
permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in resuspended in 100 μl of culture medium without phe-
PBS for 5 min. After Triton removal, cells were incubated nol red. Then, it was added 50 μl of Coulter DNA Prep
with primary monoclonal mouse anti-α-tubulin antibody Reagents Kit (Beckman-Coulter Inc, California, USA), 1
(Sigma-Aldrich) diluted 1:100 at 37°C in a wet chamber ml of PI solution with RNase and incubated for 30 min
for 1 h. Three washings with PBS were then carried out at 37°C. Both reagents were purchased from Sigma-
before addiction of Triton X-100 for 5 min. Incubation of Aldrich. Distribution of cells in different phases of cell
the secondary antibody (Fab specific goat anti-mouse cycle was determined using a Coulter Epics XL-MCL
FITC-IgG; Sigma-Aldrich) was identical to that of the first flow cytometer (Beckman-Coulter Inc.) with an argon
one. Then, DNA was counterstained by Hoechst-33258 laser line (488 nm), complemented with appropriate
(0.05 mg ml−1 in distilled water) for 5 min. Finally, cells filters, and a minimum of 104 labeled cells per sample
were washed with PBS and mounted with ProLong Gold were analyzed in each experimental condition. Percent
(Molecular Probes) antifade reagent. of cells in each phase of the cell cycle was compared
with that of control cells (without nanoparticles incuba-
Vinculin immunofluorescence and F-actin staining tion). At least 10000 fluorescent events were counted
For vinculin immunostaining, cells grown on coverslips per sample.
were fixed with formaldehyde in PBS (1:10 v/v), for 20
min at 4°C, washed three times for 5 min with PBS and Measurement of intracellular ROS
permeabilized with 0.5% Triton X-100. After incubation Intracellular ROS levels were determined using 2′,7′-
with a blocking solution (5% bovine serum albumin, 5% dichlorodihydrofluorescein diacetate (DCFH-DA) assay.
FBS, 0.02% Triton X-100 in PBS) at room temperature Cells were seeded on coverslips and, after exposure to
for 30 min, cells were incubated with 1:50 solution nanoparticles for 24, 48 and 72 h, were washed with PBS
mouse monoclonal anti-vinculin (Sigma-Aldrich) at 37°C and incubated with 10 μM DCFH-DA (Sigma-Aldrich)
in a wet chamber for 1 h. Primary antibody binding was for 30 min. Then, cells were washed with PBS again and
detected using Fab specific goat anti-mouse FITC-IgG visualized immediately by fluorescence microscopy.
diluted 1:50. F-actin was visualized in the same sam- Bright field microscopy was also used to corroborate
ples by incubation with rhodamine-labeled phalloidin accumulation of nanoparticles. For control induction of
(Sigma-Aldrich) diluted 1:200 at 37°C in a wet chamber oxidative stress, cells were treated with 800 μM H2O2
for 25 min. Then, samples were washed three times with (Panreac Química) for 1 h 30 min in complete medium.
PBS, counterstained with Hoechst-33258 for 5 min, ROS production was observed in cells, 1 h 30 min
washed with PBS and mounted with Prolong Gold anti- after that H2O2 was removed, with 10 μM DCFH-DA
fade reagent. for 30 min.
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 13 of 15

Cytotoxicity assays and distilled water, post-fixed for 45 minutes with 1%

MTT test osmium tetroxide (TAAB Laboratories Equipment Ltd.)
Cytotoxicity was assessed by MTT colorimetric assay 24 in PBS, washed with distilled water, treated during 45
after incubation with DMSA-SPION. Immediately prior minutes with 1% aqueous uranyl acetate (Electron Micros-
to use, a stock solution of dimethylthiazolyl-diphenyl- copy Sciences, Hatfield, USA), washed again and dehy-
tetrazolium bromide (MTT; Sigma-Aldrich, 1 mg ml−1) drated with growing quantities (50%, 75%, 95% and 100%)
in PBS was prepared. Five hundred microliters of this of ethanol seccosolv (Merck KGaA, Darmstadt, Germany).
MTT solution (50 μg ml−1 MTT in culture medium) The samples were maintained in coverslips throughout
was added to each culture dish without coverslip. Cells the process and finally embedded in epoxy resin 812
were incubated for 3 h, then reduced formazan was (TAAB Laboratories Equipment Ltd.) contained in gel-
extracted with 500 μl dimethylsulfoxide and absorbance atine capsules (Electron Microscopy Sciences). The epoxy
measured at 570 nm in a SpectraFluor spectrophotom- resin was polymerized for 2 days at 60°C. Resin was
eter (Tecan Group Ltd, Männedorf, Switzerland). Cell detached from the coverslips by successive immersions in
survival was expressed as the percentage of absorption liquid nitrogen and hot water. Ultrathin, 70-nm-thick
of treated cells in comparison with that of control cells. sections were obtained with an Ultracut UCT ultramicro-
tome (Leica Microsystems), transferred to 200 mesh
Trypan blue exclusion test Nickel EM grids (Gilder, Lincolnshire, UK) and stained
Cell viability was quantified by Trypan blue dye exclusion with 3% aqueous uranyl acetate (10 minutes) and lead cit-
method. Briefly, after 24 h of incubation with DMSA- rate (2 minutes) (Electron Microscopy Science). Sections
SPION, trypsin was added to control and treated cells. were visualized on a JEOL JEM 1200 EXII electron micro-
After cells were detached from the plate, they were scope operating at 100 kV (JEOL Ltd., Tokyo, Japan).
resuspended in culture media. Equal volumes of each cell
suspension and trypan blue solution (0.2% in PBS) were Additional files
mixed and used for cell counting by hemocytometer.
Blue-stained cells were counted as nonviable cells and Additional file 1: Uptake, accumulation and cytotoxicity of
unstained cells as viable cells. DMSA-SPION into non oncogenic MCF-10A cells.
Additional file 2: Nanoparticle concentration and stability
characterization by Nanoparticle Tracking and Analysis (NTA).
Bright field and fluorescence microscopy
Observation of samples processed for bright field and
Abbreviations
fluorescence microscopy were made with an Olympus SPION: Superparamagnetic iron oxide nanoparticles; DMSA: meso-2,3-
BX61 epifluorescence microscope, equipped with an dimercaptosuccinic acid; DMSA-SPION: Dimercaptosuccinic acid coated
Olympus DP50 digital camera (Olympus, Tokyo, Japan), superparamagnetic iron oxide nanoparticles; MNP: Magnetic nanoparticles;
NP: Nanoparticles; TEM: Transmission electron microscopy; PBS: Phosphate
and processed using the Adobe Photoshop 7 software buffered saline buffer; FBS: Fetal bovine serum; DMEM: Dulbecco’s modified
(Adobe Systems, San Jose, CA, USA). The following filters Eagle’s medium; HCl: Hydrochloric acid; MTs: Microtubules; F-Actin: Actin
were used to visualize the fluorescence signal of probes: filaments; FITC-IgG: Fluorescein isothiocyanate conjugated immunoglobulin-G;
ROS: Reactive oxygen species; DCFH-DA: 2′,7′-dichlorodihydrofluorescein
UV excitation light (365–390 nm) for Hoechst-33258, diacetate; PI: Propidium iodide; MRI: Magnetic resonance imaging;
blue (460–490 nm) for FITC, and green (510–550 nm) for MTT: Dimethylthiazolyl-diphenyl-tetrazolium bromide.
TRITC.
Competing interests
The authors declare that they have no competing interests.
Statistical analysis
Statistical analysis was performed by GraphPad Prism Authors’ contributions
Software (GraphPad Inc., CA, USA) using one-way AV and JLC conceived and designed the study and wrote the manuscript.
MC (M Calero) and MCh (M Chiappi) have carried out the majority of
ANOVA and Tukey’s test. The threshold for significance experimental techniques and contributed equally to this paper under
was P = 0.05 and P values < 0.05 (*), < 0.01 (**) and < supervision of AV and JLC respectively. ALC have contributed to cell cycle
0.005 (***) were considered as significant. experiments and ferrozine assay and performed statistical analysis. MJR
processed the electron microscopy samples. FJC participated in microscopy
and interpretation. KCS, APM and YV completed quantification by High
Sample preparation for transmission electron microscopy Content Screening, measured DMSA-SPION size in water and culture
MCF-7 cells were incubated with SPION at different medium by Nanoparticle Tracking and Analysis (NTA) and partly wrote the
manuscript. All authors read and approved the final manuscript.
times, as described above, washed with PBS and treated
with a mixture of 2% formaldehyde (Ultra Pure EM Grade, Acknowledgements
Polysciences Inc., Philadelphia, USA) and 2.5% glutaralde- The research leading to these results have received partial funding from the
hyde (EM Grade, TAAB Laboratories Equipment Ltd., European Seventh Framework Programme (FP7/2007-2013) under the project
MULTIFUN grant agreement no. 262943, and the project Nanofrontmag-CM
Berks, UK) in PBS for 1 h at room temperature. The cell (S2013/MIT-2850) from the Comunidad de Madrid. Additional grants were
monolayer on the coverslips was then washed with PBS obtained from BFU 2011–29038 and CTQ2013-48767-C3-3-R from the Ministerio
Calero et al. Journal of Nanobiotechnology (2015) 13:16 Page 14 of 15

de Economia y Competitividad and S2009/Mat 1507 from the Comunidad de 17. Calero M, Gutierrrez L, Salas G, Luengo Y, Lazaro A, Acedo P, et al. Efficient and
Madrid (to JLC), from EU FP7 project NAMDIATREAM (ref 246479) and from “la safe internalization of magnetic iron oxide nanoparticles: two fundamental
Caixa” / CNB International PhD Programme Fellowships. We acknowledge requirements for biomedical applications. Nanomedicine. 2014;10:733–43.
Dr. Puerto Morales and Dr. Gorka Salas for providing the SPION samples. 18. Liu Y, Wang J. Effects of DMSA-coated Fe3O4 nanoparticles on the
The encouragement and continuous support of Rodolfo Miranda is deeply transcription of genes related to iron and osmosis homeostasis. Toxicol Sci.
recognized. Authors recognize the valuable contribution of Carmen 2013;131:521–36.
Moreno-Ortiz (Flow Cytometry, Centro Nacional de Biotecnología, Madrid). 19. Pisanic 2nd TR, Blackwell JD, Shubayev VI, Finones RR, Jin S. Nanotoxicity of
iron oxide nanoparticle internalization in growing neurons. Biomaterials.
Author details 2007;28:2572–81.
1
Departamento de Biología, Universidad Autónoma de Madrid, Cantoblanco, 20. Wang Z, Cuschieri A. Tumour cell labelling by magnetic nanoparticles with
28049 Madrid, Spain. 2Department of Macromolecular Structure, Centro determination of intracellular iron content and spatial distribution of the
Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, intracellular iron. Int J Mol Sci. 2013;14:9111–25.
28049 Madrid, Spain. 3Department of Clinical Medicine, Trinity Centre for 21. Gu J, Xu H, Han Y, Dai W, Hao W, Wang C, et al. The internalization
Health Science, James’s Street, Dublin 8, Ireland. 4Centre for Research on pathway, metabolic fate and biological effect of superparamagnetic iron
Adaptive Nanostructures and Nanodevices (CRANN), and AMBER Centre, oxide nanoparticles in the macrophage-like RAW264.7 cell. Sci China Life
Trinity College Dublin, College Green, Dublin 2, Ireland. 5Instituto Madrileño Sci. 2011;54:793–805.
de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 22. Mahajan S, Koul V, Choudhary V, Shishodia G, Bharti AC. Preparation and
28049 Madrid, Spain. in vitro evaluation of folate-receptor-targeted SPION-polymer micelle
hybrids for MRI contrast enhancement in cancer imaging. Nanotechnology.
Received: 3 October 2014 Accepted: 28 January 2015 2013;24:015603.
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Concentration-dependent toxicity of iron oxide nanoparticles mediated by
increased oxidative stress. Int J Nanomedicine. 2010;5:983–9.
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