Biomaterials 32 (2011) 195e205

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Cytotoxic effects of iron oxide nanoparticles and implications for safety in

cell labelling
Stefaan J.H. Soenen a,1, Uwe Himmelreich b, Nele Nuytten a, Marcel De Cuyper a, *
a
Lab of BioNanoColloids, Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, B8500 Kortrijk, Belgium
b
Biomedical NMR Unit/MoSAIC, Faculty of Medicine, Katholieke Universiteit Leuven, Campus Gasthuisberg, B3000 Leuven, Belgium

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

Article history: The in vitro labelling of cultured cells with iron oxide nanoparticles (NPs) is a frequent practice in
Received 11 August 2010 biomedical research. To date, the potential cytotoxicity of these particles remains an issue of debate. In
Accepted 27 August 2010 the present study, 4 different NP types (dextran-coated Endorem, carboxydextran-coated Resovist, lipid-
Available online 22 September 2010
coated magnetoliposomes (MLs) and citrate-coated very small iron oxide particles (VSOP)) are tested on
a variety of cell types, being C17.2 neural progenitor cells, PC12 rat pheochromocytoma cells and human
Keywords:
blood outgrowth endothelial cells. Using different NP concentrations, the effect of the NPs on cell
(Iron oxide) nanoparticle
morphology, cytoskeleton, proliferation, reactive oxygen species, functionality, viability and cellular
Magnetoliposome
Cytotoxicity
homeostasis is investigated. Through a systematic study, the safe concentrations for every particle type
Cell labelling are determined, showing that MLs can lead up to 67.37
5.98 pg Fe/cell whereas VSOP are the most toxic
Biomedical materials particles and only reach 18.65
2.07 pg Fe/cell. Using these concentrations, it is shown that for MRI up to
500 cells/ml labelled with VSOP are required to efficiently visualize in an agar phantom in contrast to only
50 cells/ml for MLs and 200 cells/ml for Endorem and Resovist. These results highlight the importance of
in-depth cytotoxic evaluation of cell labelling studies as at non-toxic concentrations, some particles
appear to be less suitable for the MR visualization of labelled cells.
Ó 2010 Published by Elsevier Ltd.

1. Introduction homeostasis. As FDA-approved, dextran-coated iron oxide cores

In the past two decades, labelling of cultured cells with iron
oxide nanoparticles (IONPs) has become a frequently employed
method in biomedical research. These magnetic IONPs can be used
to non-invasively visualize labelled cells by magnetic resonance
imaging (MRI) after transplantation, enhance the efficiency of drug
or gene delivery or be used as tools for magnetic hyperthermia
cancer treatment [1,2]. Recently, clinical trials have been set up
where cells are labelled in vitro with Endorem and subsequently
injected in human patients in order to track the migration and
distribution of these cells [3]. As the use of IONPs for cell labelling is
clearly increasing, the lack of information regarding the interaction
of these particles with cells also becomes more apparent [4].
Recently, it has been shown that despite the initial belief in the
non-cytotoxic properties of IONPs, the physico-chemical properties
of nanoparticles [5] and the high intracellular concentrations of
IONPs required for efficient MRI poses serious threats on cell
* Corresponding author. Tel.: þ32 56 246221; fax: þ32 56 246997.
E-mail address: Marcel.DeCuyper@kuleuven-kortrijk.be (M. De Cuyper).
1
Current address: Lab of General Biochemistry and Physical Pharmacy, Faculty of
Pharmaceutical Sciences, University of Gent, B9000 Gent, Belgium.
(Endorem) are intrinsically not well suited for cell labelling due to
their inefficient uptake, the low affinity-binding and possible
desorption of the dextran molecules, and their possible effects on
stem cell differentiation [6e10], many research groups have tried to
develop IONPs specifically suited for optimal cell labelling.
However, the lack of appropriate control particles, standard
procedures for cell labelling and the wide variety in types of
labelled cells have made it impossible to compare different IONPs in
terms of uptake efficiency and cytotoxic effects [6,11]. The disparate
data obtained from several studies have thus hardened making any
general conclusion regarding the safety of IONPs in cell labelling.
Prior to evaluating the clinical potential of these particles in cell
transplantation studies, the effects of IONPs on cultured cells
should be carefully evaluated.
Previously, it was shown by several groups that high intracel-
lular concentrations of IONPs diminished the proliferative capacity
of cultured cells [12,13]. These effects were recently ascribed by our
group to be the result of cytoskeletal changes induced by the high
IONP concentrations which impeded actin-mediated signalling
[14]. Furthermore, when present in the acidic endosomal
compartments of the cell, several types of particles have been
shown to be degraded, rendering them near useless in terms of MR
contrast generation [15,16]. The degradation of the particles also

0142-9612/$ e see front matter Ó 2010 Published by Elsevier Ltd.

doi:10.1016/j.biomaterials.2010.08.075
196 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205
affected cell functionality and homeostasis, indicating that the
labelling procedure, if not tightly-controlled, could have grave
effects on the outcome of any cell transplantation study [17].
In the present study, four different types of IONPs (Endorem:
dextran-coat; Resovist: carboxydextran-coat; Magnetoliposomes
(MLs): lipid-coat; very small iron oxide particles (VSOP): citrate-
coat) which are frequently employed in cell labelling studies are
used to label C17.2 neural progenitor cells (NPCs), PC12 rat
pheochromocytoma cells and human blood outgrowth endothelial
cells (hBOECs). Using a systematic approach, the safe concentra-
tions of the IONPs were assessed by evaluating their effect on cell
morphology and cytoskeleton, cell proliferation, induction of
reactive oxygen species (ROS), cell homeostasis, viability and
functionality. The use of the different cell types, each with their
specific traits allowed to efficiently analyze the various parame-
ters. hBOECs are large, well-spread cells with a clear cytoskeleton,
C17.2 NPCs are very rapidly proliferating stem cells and were
previously used to evaluate ROS induction and TfR1 expression
and PC12 cells have been introduced as a model cell type for the
evaluation of cell functionality [18]. Upon establishing safe
concentrations of IONPs for cell labelling, the efficiency of the
different particles on MR visualization of cells labelled with the
IONPs at non-toxic concentrations was studied and compared for
the various IONPs.
2. Materials and methods
2.1. Nanoparticles
Resovist was purchased from Schering (Berlin, Germany), Endorem from Guer-
bet (Villepinte, France), VSOP C200 from Ferropharm (Teltow, Germany) and cationic
MLs (3.33% distearoyltrimethylammonium propane (DSTAP)-containing) were
produced and characterized by transmission electron microscopy, dynamic light
scattering, gas-liquid chromatography and electrophoretic mobility measurements
as described previously [13,19]. Physico-chemical data on all the particles used can
be found in Supplementary Table S1.
2.2. Cell culture conditions
C17.2 neural progenitor cells and PC12 rat pheochromocytoma cells were
cultured in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% fetal calf serum, 5% horse serum, 1 mM sodium pyruvate,
2 mM L-Glutamine and 1% penicillin/streptomycin (Gibco, Invitrogen, Belgium). C17.2
cells were passaged every 48 h and split 1/5. PC12 cells were passaged when
reaching near 70% confluency and split 1/5 in tissue culture dishes (Greiner Bio-One
BA/BV, Wemmel, Belgium) which were coated with synthetic laminin peptide
(Synthetic laminin peptide for Rat Neural Stem Cells, Millipore SA/NV, Brussels,
Belgium).
Human blood outgrowth endothelial cells were kindly donated by Dr. Simon De
Meyer (KULeuven Campus Kortrijk, Lab of Thrombosis Research). For culture, the
cells were maintained in endothelial basal/growth culture medium (EBM-2/EGM-2,
Clonetics, San Diego, CA) with medium changes every 48 h. Cells were passaged
when reaching near 80% confluency by lifting the cells with 0.05% trypsin (Gibco)
and were plated (1/5) onto tissue-culture flasks coated with collagen.
2.3. Cell exposure to IONPs
For cell exposure to IONPs, all cell types were usually seeded at 5  104 cells/well
unless indicated otherwise. Cells were then allowed to settle overnight in a humid
atmosphere at 37  C and 5% CO2. Prior to cell labelling, Resovist, Endorem, VSOPs
and MLs were diluted in 0.5 ml phosphate buffered saline (PBS; Gibco, Invitrogen,
Belgium) each, apart from the already cationic MLs, supplemented with 25 ml Lip-
ofectamine 2000 and incubated for 30 min at ambient temperature with light
shaking to allow complex formation. Next, 4.5 ml cell culture media was added and
the appropriate volume (0.2 ml per well in 96 well plates and 1 ml per well in 12 well
plates) could be given to the cells. As controls, cells were exposed to media not
containing any particles but having 10% PBS as was the case for the IONP suspen-
sions also. Cells were incubated for 24 h at 37  C and 5% CO2 prior to being further
analysed as described in the respective paragraphs below.
2.4. Staining of F-actin, a-tubulin and vinculin
To analyze the effects on cell morphology, hBOECs were labelled with the
different IONPs, kept in culture in IONP-free medium and then re-seeded at 2  104
cells/well in 12 well plates containing glass coverslips at 0, 3 and 6 days post IONP
incubation and allowed to settle for 24 h. Then, cells were fixed (2% para-
formaldehyde (PFA) for 15 min), permeabilized (1% Triton X-100 for 15 min) and
blocked for 30 min in PBS containing 10% goat serum (Gibco, Invitrogen, Belgium)
and 2% bovine serum albumin (BSA). Cells were then incubated with primary
antibody in blocking solution: anti-a-tubulin mouse monoclonal (no. 322500,
2.5 mg/ml; Molecular Probes, Leiden, Netherlands) or anti-vinculin mouse
monoclonal (no. ab18058, 1:200; Abcam, Cambridge, UK) for 2 h at ambient
temperature followed by 1 h incubation at ambient temperature with secondary
Alexa Fluor 488-conjugated goat anti-mouse antibody (1:250; Molecular Probes,
Leiden, Netherlands) and Alexa Fluor 546-conjugated phalloidin (Molecular Probes,
Leiden, Netherlands). Subsequently, cells were washed three times with blocking
solution and mounted on microscope slides prior to being analysed by confocal
laser scanning microscopy (LSM510, Zeiss, Germany). To get quantitative data,
images were also collected at a 20 magnification by epifluorescence microscopy
using an Olympus BX51TF (Olympus, Tokyo, Japan) equipped with an Infinity 2
camera (BFi Optilas, Alphen aan den Rijn, Netherlands). Cell areas were calculated
using ImageJ (NIH, USA) for at least 150 cells per sample. For analysis of focal
adhesion areas, confocal images displaying vinculin were background-corrected,
thresholded, focal adhesions were identified and the total areas per cell were
calculated for 20 cells per condition.
2.5. Detection of reactive oxygen species
Induction of ROS was quantitated using nitroblue tetrazolium salt (NBT; Sig-
maeAldrich, Bornem, Belgium). C17.2 cells were seeded in 96 well plates and after
incubation with the IONPs in the absence or presence of 1 mM desferrioxamine
(SigmaeAldrich, Bornem, Belgium), media were removed, cells washed twice with
PBS (150 ml/well) and fresh medium containing NBT (1 mg/ml) was given, followed
by 6 h incubation at 37  C and 5% CO2. Media were then removed, cells washed once
with PBS after which 95 ml lysis solution (0.04 N HCl in isopropanol) was added to
each well. Plates were shaken at 1200 rpm for 30 min at ambient temperature. Then,
105 ml 10 N KOH was added, and plates were again shaken at 1200 rpm for 30 min
prior to measuring absorbance at 620 nm (FluoStar Optima plate reader, BMG
Labtech, Isogen Life Sciences, Sint-Pieters-Leeuw, Belgium).
2.6. Cell viability, proliferation and cellular iron content
Cell viability was assessed by means of a lactate dehydrogenase assay (CytoTox
96 non-radioactive cytotoxicity assay, Pierce, Rockford, US) and intracellular iron
content was determined spectrophotometrically using Tiron (SigmaeAldrich, Bor-
nem, Belgium) as described previously [14]. Cell proliferation was assessed by
manual counting using a Bürker Chamber. To this end, C17.2 cells were seeded at
1  106 cells/culture flask and labelled with the IONPs for 24 h. After labelling, cells
were kept in culture and were re-seeded at 1  106 cells/culture flask every 48 h. The
average cell division time for three independent tests is given.
2.7. PC12 neurite formation
PC12 cells were seeded at 2  104 cells per well in 12 well plates containing
laminin-coated glass coverslips. After IONP incubation, cells were kept in culture in
IONP-free medium overnight. Then, cells were given nerve growth factor (NGF)
induction medium, consisting of high glucose DMEM, supplemented with 1% fetal
calf serum, 5% horse serum, 1 mM sodium pyruvate, 2 mM L-Glutamine, 1% penicillin/
streptomycin and 100 ng/ml NGF (Sigma Aldrich, Bornem, Belgium). Cells were kept
in this medium for 48 h where after 24 h, half the medium was replaced by fresh
induction medium. Samples were then fixed, permeabilized and stained for F-actin
and a-tubulin and images were collected as described in Section 2.4. Image pro-
cessing occurred using ImageJ software and using the epifluorescence images, the
number of neurites e defined as having twice the length of the cell body e could be
calculated using an ImageJ plug-in, specifically developed for the analysis of neurites
[20]. Neurite length was also calculated and data are expressed as the average
number of neurites of a certain length per cell. At least 150 cells were analysed per
sample to allow statistical analysis.
2.8. Transferrin receptor-1 expression
To analyze expression of transferrin receptor-1 (TfR1), C17.2 cells were seeded at
2  105 cells/dish in 6 cm diameter tissue culture dishes. After IONP labelling (4
dishes per type of IONP), cells were kept in culture for 24 h in IONP-free medium.
Cells were then re-seeded at 2  105 cells/dish and kept in culture for another 72 h.
This step was repeated once, meaning that in total, the cells had been further
cultivated for 1 week. Cells were then lifted by scraping and counted using a Bürker
chamber followed by repeated centrifugation and washing with PBS (2 repeats).
Cells were resuspended in 1 ml PBS and equal portions (0.5 ml) of every tube was
divided over 2 tubes followed by centrifugation. One tube was incubated for 30 min
with FITC-tagged rat anti-mouse TfR1 antibody (BD Biosciences Pharmingen,
Erembodegem, Belgium; 1 mg/ml in FACS buffer: PBS containing BSA (1%) and fetal
calf serum (0.1%)); the other tube was treated with FITC-tagged rat anti-mouse
 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205 197

Fig. 1. High intracellular nanoparticle concentrations affect cell spreading. Confocal micrographs of hBOECs incubated for 24 h either untreated (A) or incubated with (B) VSOP, (C)
Endorem, (E) MLs or (G) Resovist at 600 mg Fe/ml for 24 h, (D) Endorem at 400 mg Fe/ml, (F) MLs or (H) Resovist at 300 mg Fe/ml for 24 h. Cells were kept in culture for an additional 3
days. F-actin is coloured red, the nucleus is coloured blue by DAPI staining and a-tubulin is indicated in green. Green and red channels are shown separately for every image. Scale
bars: 50 mm.

antibody (BD Biosciences Pharmingen, Erembodegem, Belgium; 1 mg/ml) and served
as an isotype control. Cells were centrifuged and washed with FACS buffer repeat-
edly (3 repeats) and then resuspended in 0.4 ml FACS buffer and analysed using
fluorescence activated cell sorting (EPICSÒ XL-MCL, Coulter, Analis, Gent, Belgium).
2.9. Magnetic resonance imaging and sample preparation
To prepare samples for MRI, C17.2 cells at 70% confluency in culture flasks were
labelled with the IONPs at the non-toxic concentrations indicated in the main text.
After labelling, cells were kept in culture with IONP-free medium for an additional
8 h. Cells were then lifted by trypsin, fixed by 2% PFA and stock suspensions were
made of all the cells in PBS at a density of 2  105 cells/ml. Cell samples are trans-
ferred to eppendorf tubes, reaching densities of 2000, 500, 200, 50, 20 cells/ml. For all
the cells, a total volume of 150 ml was taken, which was then mixed and solidified in
a 1.5% agarose gel (Invitrogen, Merelbeke, Belgium). After solidifying, the eppendorfs
were completely filled with 1.5% agarose gel. For control samples, unlabelled cells at
a density of 2000 cells/ml were taken. For every type of IONP, the samples were put
together in a plastic cylinder which was filled with 1.5% agarose gel. Upon solidi-
fying, these agar cups were analysed in a Bruker Biospec 9.4 T small animal MR
scanner (Bruker Biospin, Ettlingen, Germany; horizontal bore, 20 cm) equipped with
198 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205

Fig. 2. High intracellular nanoparticle concentrations affect focal adhesion formation and maturation. Confocal micrographs of hBOECs incubated for 24 h either untreated (A) or
incubated with (B) VSOP, (C) Endorem, (E) MLs or (G) Resovist at 600 mg Fe/ml for 24 h, (D) Endorem at 400 mg Fe/ml, (F) MLs or (H) Resovist at 300 mg Fe/ml for 24 h. Cells were kept
in culture for an additional 3 days. F-actin is coloured red, the nucleus is coloured blue by DAPI staining and vinculin is indicated in green. Green and red channels are shown
separately for every image. Scale bars: 50 mm.

actively shielded gradients (600 mT m1). A quadrature RF resonator (transmit/
receive; inner diameter 7 cm, Bruker Biospin) was used. 2D multi-slice-multi-echo
(MSME) experiments were acquired for the calculation of T2-maps (TR ¼ 3000 ms
and 16 TE increments of 10 ms, 2562 matrix, 275  275 mm in plane resolution,
0.35 mm slice thickness). T2* maps were acquired similarly to MSME experiments
using a gradient echo pulse sequence and 16 TE increments (first TE ¼ 4.5 ms with
increments of 6.7 ms). Three-dimensional, high-resolution T2*-weighted MR
images were acquired using a gradient echo sequence (FLASH, TR ¼ 200 ms,
TE ¼ 15 ms, flip angle 30 ). The resolution was usually 75  75  75 mm.
Images were processed using Paravision 5.0 (Bruker Biospin). Relaxation times
(T2/T2*) were determined as mean values of homogeneous sections of the cell
loaded areas in the agar phantoms. Data were expressed as mean
standard
deviation.
2.10. Statistical analysis
All data are expressed as mean
s.e.m. unless indicated otherwise and analysed
using one-way analysis of variance (ANOVA). For cellular iron content, multiple
comparisons were analysed using the Tukey-HSD post-hoc method. When
comparing the different NPs to the same control group (reference), the Dunnett
post-hoc analysis method was used. In all cases, the degree of significance is indi-
cated when appropriate (*: p < 0.05; **: p < 0.01; ***: p < 0.001).
 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205 199

Day 1 Day 3
A 35 B 35
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Cell doubling time (h)

Cell doubling time (h)

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Fig. 3. High intracellular nanoparticle concentrations impede cell cycle progression. Cell doubling times for C17.2 NPCs, either as control cells or after incubation with the particles
at the indicated concentrations for 24 h. Cell numbers were counted after (A) 1, (B) 3 or (C) 6 days after incubation with the nanoparticles. Values are represented as
mean
standard error to the means (n ¼ 3). Where appropriate, statistical significance is indicated: *: p < 0.05; **: p < 0.01.

3. Results Resovist and MLs at 600 mg Fe/ml clearly displayed a diminished

3.1. Physico-chemical properties of the IONPs
In the present work, four highly differing IONPs were used,
providing a broad scala in different physico-chemical properties. An
overview of the most important features (core size, hydrodynamic
diameter, surface charge) can be found in the supplementary
information (see Supporting Table S1). The MLs used throughout this
work consist of 14 nm diameter iron oxide cores each individually
surrounded by a lipid bilayer (96.67% dimyristoylphosphatidylcho-
line and 3.33% distearoyltrimethylammonium propane). As MRI
requires high doses of contrast agent due to the intrinsic low
sensitivity of the method, uptake of all particles here apart from the
already cationic MLs, was enhanced by combining the IONPs with
Lipofectamine 2000 (5 ml/ml total incubation medium), a frequently
used transfection agent in cell labelling procedures [21].
3.2. Effects of high intracellular IONP concentrations on cell
cytoskeleton
In line with previous findings [13,14], cellular cytoskeleton and
morphology were affected at high intracellular concentrations of all
types of IONPs. As can be seen in Fig. 1, hBOECs incubated with
cell spreading, whereas Endorem-treated cells showed slight
effects but VSOP-treated cells were not affected. The effects seen
were only transient (data not shown) and after 6 days, no signifi-
cant changes in cell surface area between control cells and IONP-
treated cells could be observed. Reducing the concentration of
Endorem to 400 mg Fe/ml and Resovist and MLs to 300 mg Fe/ml
abolished any effects on cell cytoskeleton as can be seen in Fig. 1D,
F, H. Histograms representing the global distributions of the
respective cell surface areas for the different conditions can be
found in Supporting Figs. S1 and S2.
3.3. Effects of high intracellular IONP concentrations on focal
adhesions and proliferation
Apart from the cell surface area, the total area occupied by focal
adhesions is also affected when hBOECs are incubated with
Resovist and MLs at 600 mg Fe/ml, but not at 300 mg Fe/ml as can be
seen in Fig. 2. In line with the minor (Endorem) or lack (VSOP) of
any effect on the actin cytoskeleton and microtubule network,
Endorem led to slight effects whereas VSOP did not have any effects
on focal adhesion complexes (see Supporting Fig. S3 for quantita-
tive data). As a diminished signalling through focal adhesion
complexes along actin fibers was previously found to result in
200 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205

A 900%
particles displayed a pronounced increased level of TfR1, indicative
of an altered iron metabolism. For MLs, the increase in ROS levels
*** Normal and the augmentation in TfR1 expression levels were only minimal,
750% +Desf suggesting the least intracellular degradation.
Relative ROS levels

3.5. Effects of intracellular IONPs on cell functionality

600%
To verify whether the effects on cell homeostasis have any
450% consequences for cell functionality, PC12 cells were first incubated
*** with the different IONPs and subsequently exposed to nerve
300% growth factor (NGF) after which the responsiveness of the cells to
their biological cue was assessed by means of analysing neurite
outgrowth (Fig. 6). The results indicate a clear effect of all particles
150% on the functionality of PC12 cells at the concentrations used. Using
a concentration-series for all IONPs, PC12 cell functionality was
0% found to be unaffected when incubating the cells with MLs at
VSOP (600) Endo (400) ML (300) Reso (300) 250 mg Fe/ml; Resovist: 150 mg Fe/ml; Endorem: 200 mg Fe/ml and
VSOP: 200 mg Fe/ml. The respective length and number of neurites
found in PC12 cells incubated with the different particles at the
B 900% concentrations used can be found in Fig. 6KeO.
Normal
750% +Desf 3.6. Efficacy of labelling cells with IONPs at non-toxic
concentrations
Relative ROS levels

600% ***
***
***
450%
300% ** **
** **
**
150%
0%
VSOP (600) Endo (400) ML (300) Reso (300)
Fig. 4. Nanoparticle internalization induces ROS. Relative levels of ROS in C17.2 NPCs
incubated with the various particles at the concentrations indicated for (A) 4 h or (B)
24 h, in the absence (light grey) or presence of 1 mM desferrioxamine. Values are
represented as mean
standard deviation (n ¼ 20). Where appropriate, statistical
significance is indicated: *: p < 0.05; **: p < 0.01; ***: p < 0.001.
a slowing down of cell cycle progression, cell proliferation was
assessed for C17.2 NPCs and found to transiently decrease when
incubated with Resovist and MLs at 600 mg Fe/ml but not at 300 mg
Fe/ml (Fig. 3).
3.4. Effects of intracellular IONPs on cell homeostasis
Based on the above-mentioned findings, VSOP was further used
at 600 mg Fe/ml, Endorem at 400 mg Fe/ml, whereas MLs and
Resovist were used at 300 mg Fe/ml. To further evaluate the effect of
the IONPs at these concentrations, the level of ROS produced by the
particles was measured by means of an NBT assay after 4 and 24 h
(Fig. 4). Citrate-coated VSOP particles led to the highest induction of
ROS and also reached the fastest maximum (4 h incubation), which
could lead to toxic effects. The generation of free radicals hints at
the possible degradation of the particles and the release of ferric
ions from the IONPs into the acidic endolysosomal compartments.
This could be validated by the use of desferrioxamine, an iron
chelator, which significantly reduced the induction of ROS levels in
all setups apart from MLs (Fig. 4). To further verify this, the
expression of transferrin receptor-1 (TfR1), involved in iron
metabolism, was investigated as shown in Fig. 5. In line with the
induced generation of ROS, C17.2 NPCs incubated with the different
To verify the lack of any toxic effects of the IONPs at the
concentrations described in the paragraph above, a lactate dehy-
drogenase assay was performed on C17.2 cells as shown in Fig. 7A.
The results indicate that IONP labelling at the non-toxic concen-
trations indeed did not cause any acute toxicity. However, as for
most biomedical applications concerning the use of IONPs, not the
administrated dose of IONPs, but rather the intracellular concen-
tration of these particles is an essential parameter in determining
the efficacy of the envisioned application. To this end, the intra-
cellular IONP concentration in C17.2 cells was determined as shown
in Fig. 7B. The results show that although the IONP concentrations
in the incubation media do not vary much, the intracellular IONP
levels are significantly different. The VSOPs are only poorly inter-
nalized, reaching 18.65
2.07 pg Fe/cell, whereas Resovist and
Endorem reach levels of 31.99
2.99 and 46.59
4.70 mg Fe/cell.
Cationic MLs display the greatest cellular internalization at non-
toxic levels and finally lead to 67.37
5.98 pg Fe/cell.
3.7. Effect of safe IONP concentrations on MR contrast
As the amount of intracellular iron was quite variable for the
different IONPs, their effect on MR contrast generation of labelled
cells was investigated, as shown in Fig. 8. The data clearly indicate
that at least 500 cells/ml loaded with VSOPs can are needed for clear
MR detection, whereas Endorem and Resovist require 200 cells/ml
and MLs only 50 cells/ml.
4. Discussion
From a historical perspective, IONPs were considered safe, due
to the high levels of Fe-ions which could be tolerated (up to 4 mM)
and the high LD50 of the original dextran-coated IONPs in first
animal trials [22,23]. Based on the assumption of non-toxic IONPs,
these particles were widely used in cell labelling strategies as well,
without paying much attention to the possible effects of IONPs on
cell homeostasis [24]. Later on, it became more apparent that
IONPs and NPs in general do possess several specific traits which
makes them different from bulk material or free ions, in terms of
surface reactivity and toxic effects [5,25]. Due to these insights, the
number of studies focussing on IONP toxicity has steadily increased
over the last 5 years [24], but due to the wide variability in types of
 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205
A B
256
256
Control
Events
Events
0
0
100 101 102 103 104 100 101
FL1 LOG
D E
256
ML
(300)
Events
0
100 101 102 103 104
FL1 LOG
Fig. 5. Iron oxide nanoparticle internalization and degradation affects cellular iron metabolism. Flow cytometric analysis of TfR1 expression in (A) untreated control cells or cells
incubated with (B) VSOPs, (C) Endorem, (D) MLs or (E) Resovist at the indicated concentrations for 24 h. After 1 week of continuous culture post NP-exposure, where cell densities
between the different groups were kept identical, samples were collected and analysed by flow cytometry. Expression levels were normalized using isotype control antibody. Data
are given for 10,000 events per condition.
IONPs or cells used and differences in incubation conditions (IONP
concentrations varying from a few mg/ml over 1 mg/ml), many
confusing data have been obtained and the need for more infor-
mation is primordial to advance the use of NPs in biomedical
applications [4]. By combining different assays to assess multiple
parameters important for determining IONP safety, the present
study aims to determine the safe concentrations of some of the
most widely used IONPs for cell labelling purposes.
Recently, our group showed that high intracellular concentra-
tions of IONPs affected the actin cytoskeleton which resulted in
a diminished cell proliferation [14]. The important role of actin
fibers in maintaining cell morphology and their effect on signalling
pathways monitoring several parameters such as cell death,
migration and differentiation is well known [26]. Recently, the
effect of NPs on focal adhesion-mediated cell signalling pathways
has also been confirmed by the work of Miller et al. [27] who found
that depending on the nature of the NP coating, the NPs could
interact more or less with the extracellular domain of integrins and
thereby trigger their activation and intracellular signalling cascade.
The results obtained in the present study are in line with the
intracellular concentration-dependent effects as MLs and Resovist
elicited the gravest effects, but were previously found to lead to the
highest uptake efficiencies. The results presented here show that
Resovist and MLs significantly affect cellular morphology and cell
cytoskeleton when incubated at 600 mg Fe/ml (Fig. 1). Lowering the
concentration of the IONPs diminished its effect, leading to the
absence of any effect when incubated at 300 mg Fe/ml. The finding
that only MLs and Resovist significantly affected cell morphology
was likely due to the fact that these particles are internalized most
avidly and leads to the highest amount of cell-associated iron [17].
In line with our previous study [14], the effect on actin cytoskeleton
and tubulin network also resulted in a diminution of focal adhesion
formation and maturation as can be seen in Fig. 2. This further
resulted in a transiently decreased proliferative capacity (Fig. 3).
201
C
VSOP Endo
256
(600) (400)
Events
0
102 103 104 100 101 102 103 104
FL1 LOG FL1 LOG
256
Reso
(300)
Events
0
100 101 102 103 104
FL1 LOG
The low proliferation rate of VSOP-labelled cells at day one seems,
at first sight, somewhat contradictory with the above-mentioned
reasoning that the effects are all caused by high intracellular
concentrations of IONPs. However, unlike the effects seen for
Resovist, MLs and Endorem, where proliferation was minimal after
three days, the effect of VSOPs was limited to the first time point
only. This could be ascribed to the acute toxicity of VSOP particles at
the high concentration used [14] and is therefore not the result of
an affected cell signalling.
Here, the high ROS levels and the increases in TfR1 expression for
all IONP types were significant, but in terms of toxicity of IONPs it is
yet unclear what the precise effect of these factors is on cell viability
as all cells have defence mechanisms which allows them to deal
with certain levels of oxidative stress [28,29]. Regarding the use of
IONPs, the term “biocompatibility” is often employed, as being
a necessary asset of any IONP to be used in a biological context.
Quite often, IONPs are named biocompatible when no acute cyto-
toxic effect is apparent. In its full definition “biocompatible” should
however signify a particle which, when taken up by the cells, has no
effect whatsoever on cell homeostasis meaning that no difference
should be observable in any cellular parameter before or after
labelling. In this context, the induction of high ROS levels, although
perhaps not toxic to the cells as such, could have long-term effects
and should be considered as a hazard. The induction of ROS, which
could be diminished by the use of an iron chelator, and the alter-
ations in TfR1 expression indicate the intracellular degradation of
the IONPs, where free ferric ions are generated in endolysosomal
structures and shuttled towards the cytoplasmic iron pool. This
intracellular degradation of IONPs and the generation of free ferric
ions have recently been described by Huang et al. [30] who noticed
an altered expression of protein regulators of the cell cycle which
was ascribed to Resovist degradation.
The intracellular degradation of IONPs and the associated release
of free iron can have profound effects on cell homeostasis, as has
202 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205

Fig. 6. Iron oxide nanoparticle internalization and degradation affects cell functionality. (AeJ) Representative micrographs of PC12 cells at 2 days of NGF-exposure, showing
a-tubulin (green) and G-actin (red) staining. Scale bars ¼ 10 mm. Controls cells (A) and non-NGF-exposed control cells (B) are shown as well as cells incubated with VSOP (C,D),
Endorem (E,F), ML (G,H) or Resovist (I,J) at the concentrations indicated for 24 h. Figures KeO show the number of neurites of a certain length per cell for control cells (K), VSOP (L),
Endorem (M), ML (N) or Resovist (O)-treated cells. Data are expressed as mean
standard error to the means. When appropriate, the degree of significance when compared with
untreated controls cells is indicated (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

recently been illustrated in the study by Chen and colleagues [31].
There, it was shown that labelling of human mesenchymal stem cells
by Resovist resulted in a dose-dependent inhibition of osteogenic
differentiation and altered cell migration. All these effects were
suppressed when desferrioxamine, an iron chelator was used, which
emphasized the importance of free iron on the observed effects. In
a different study, it was shown that various types of IONPs were
prone to degradation at pH-levels which can be found in
endolysosomal structures of the cell [14]. This degradation had clear
effects on cell functionality as could be shown by the PC12 cell model
system [17]. The use of the PC12 cells as a model system to assess cell
functionality offers many advantages as this is a very sensitive cell
system which allows to easily compare the effects of different IONPs
and the data can easily be quantified [18]. In terms of IONP uptake,
PC12 cells internalize IONPs less avidly than C17.2 NPCs or hBOECs
[17]. This effect could possibly be due to the small size of the cells as
 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205 203

A 120
4h
it is well known that NP uptake between different cell types can lead
to large variations, but when NP uptake is expressed in terms of cell
24 h surface area available for NP binding, internalization levels become
100
more comparable. This rule does, however, not apply to all cells, as
Relative viability (%)

the C17.2 NPCs internalized similar amounts of IONPs than hBOECs,

80 although the latter are much larger.
The cationic MLs used in this work appear to be the least toxic,
60 despite the inherent toxicity of cationic lipids [32] and resulted in
the highest amount of cell-associated iron. This is in line with
previous data showing a highly reproducible production of these
40
MLs and an optimized liposomal coating suited for cell labelling
[20]. This lipid coat also provides a good protection of the iron oxide
20 core from endolysosomal degradation in contrast with (carboxy)
dextran or citrate coatings [17]. The VSOPs were found to have the
0 gravest effects on cell functionality and viability and could not be
VSOP Endo ML Reso Triton used to reach high intracellular levels of iron oxide. Next to the
possible degradation of the citrate-coat, the small size of the VSOPs
B 90 4h
could also play a very important role in their higher toxicity. As the
radius of the iron oxide core of VSOPs is approximately three times
24 h smaller than the radius of the ML core, it follows that the volume
75 and thus the mass of iron oxide for a single VSOP will be nearly 27
times lower than for a single ML. Although the amount of iron
internalized is 3.6 times less than is the case for MLs, this means
Iron per cell (pg)

60
that in total, still more VSOPs (11.85 times, see Supporting
Information for a calculation of the number of MLs and VSOPs
45 per cell) are internalized. When the size of an NP decreases, the
surface area over volume ratio greatly increases accordingly [33].
30 This suggests that the total surface area of VSOPs available for
interaction with the cellular structures could be larger than for MLs,
but the relative amount of iron oxide present at the surface and
15
thus prone to degradation and cellular interactions over total iron
oxide present in the particles is vastly larger for VSOPs. This could
0 greatly influence toxic effects which have been described to be
VSOP Endo ML Reso dominated by NP surface properties [25]. For Endorem and
Resovist, this comparison cannot be made as these NPs are beads,
Fig. 7. Effects of iron oxide nanoparticles on cell viability and internalization efficiency.
The results of (A) an LDH assay or (B) cellular iron determination for C17.2 NPCs
meaning that several small iron oxide cores are embedded within
incubated with VSOP (200 mg Fe/ml), Endorem (200 mg Fe/ml), ML (250 mg Fe/ml) or a larger matrix. The number of cores and the size of the matrices are
Resovist (150 mg Fe/ml) after 4 and 24 h incubation. For (A), data are normalized quite heterogeneous [34], which hardens the analysis and renders
relative to the values obtained for untreated control cells (¼ 100% viability). For the it impossible to view any effects in terms of NP numbers.
LDH assay, cells treated with 1% Triton X-100 for 15 min prior to the LDH assay were
Of further interest for MRI is the actual size of the iron oxide
used as negative controls. Data are expressed as mean
standard error to the means
(n ¼ 10). core. For a similar amount of iron, larger cores will have a bigger
effect on negative contrast enhancement than smaller ones. For

Fig. 8. The effect of safe IONP concentrations on MR contrast generation. (A) Colour-coded T2*-maps of C17.2 cells labelled with VSOP (200 mg Fe/ml), Endorem (200 mg Fe/ml), ML
(250 mg Fe/ml) or Resovist (150 mg Fe/ml) for 24 h. MR images were acquired for cells placed in an agar tube at a density of 0, 20, 50, 200, 500 or 2000 cells/ml, respectively. (B)
Calculated T2* relaxation times for C17.2 cells incubated with VSOP (200 mg Fe/ml), Endorem (200 mg Fe/ml), ML (250 mg Fe/ml) or Resovist (150 mg Fe/ml) for 24 h. Data are
expressed as mean
standard deviation (n ¼ 3). Where appropriate, the degree of significance with respect to control samples (no cells or particles) is indicated: *: p < 0.05; **:
p < 0.01; ***: p < 0.001.
204 S.J.H. Soenen et al. / Biomaterials 32 (2011) 195e205
maghemite particles with core sizes of 3.5 and 12 nm, the satura-
tion magnetization values were determined to be 41 and 83 emu/g
Fe, respectively [35]. For MLs, with a core size of approximately
14 nm compared with 4 nm for Endorem, Resovist and VSOP
particles, this, next to a higher intracellular iron oxide levels, will
further enhance the MR contrast. The beneficial MR properties of
MLs for cell labelling compared with common dextran-coated iron
oxide NPs have recently been shown to be enhanced by intracel-
lular clustering on the MLs [17]. Upon exposure to the degradative
lysosomal environment, the outer leaflet of the lipid bilayer is easily
degraded, leaving a monolayer-coated ML which has a hydrophobic
surface and tends to cluster together, further enhancing transversal
MR contrast [17].
5. Conclusion
Assessing the effects of IONPs on cell homeostasis requires
a multidisciplinary approach where many parameters should be
analysed as IONPs can lead to a wide variety of toxicological effects.
Furthermore, apart from toxicological aspects, the final usefulness
of the IONPs in terms of MR contrast generation of labelled cells is
also evaluated. The results obtained highlight the importance of
toxicity assessments in evaluating nanoparticle efficiency as at non-
toxic concentrations of IONPs, several of them might not be well
suited for their original purpose. The data shown here could
provide a good reference point for other researchers who would
want to safely label mammalian cells with IONPs or, alternatively,
could be used as a model system for the further toxicological
analysis of IONPs.
Acknowledgements
SJHS is a post-doctoral fellow from the FWO-Vlaanderen. M.D.C.
and U.H. are recipients of an IWT grant sponsoring project
SBO80017. U.H. received financial support by the European
Commission for EC-FP6 network DiMI (LSHB-CT-2005-512146),
EC-FP7 network ENCITE (2008-201842), and the K.U. Leuven
Centers of Excellence ‘MoSAIC’.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.biomaterials.2010.08.075.
Appendix
Figure with essential color discrimination. Most of the figures in
this article are difficult to interpret in black and white. The full color
images can be found in the on-line version, at doi:10.1016/j.
biomaterials.2010.08.075.
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