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Journal of Toxicology and Environmental Health, Part A
Current Issues
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/uteh20
Toxicological effects of the mixed iron oxide
nanoparticle (Fe3O4 NP) on murine fibroblasts LA-9
Karina Alves Feitosa, Ricardo de Oliveira Correia, Ana Carolina Maragno
Fattori, Yulli Roxenne Albuquerque, Patricia Brassolatti, Genoveva Flores
Luna, Joice Margareth de Almeida Rodolpho, Camila T. Nogueira, Juliana
Cancino Bernardi, Carlos Speglich & Fernanda de Freitas Anibal
To cite this article: Karina Alves Feitosa, Ricardo de Oliveira Correia, Ana Carolina Maragno
Fattori, Yulli Roxenne Albuquerque, Patricia Brassolatti, Genoveva Flores Luna, Joice Margareth
de Almeida Rodolpho, Camila T. Nogueira, Juliana Cancino Bernardi, Carlos Speglich & Fernanda
de Freitas Anibal (2022): Toxicological effects of the mixed iron oxide nanoparticle (Fe3O4
NP) on murine fibroblasts LA-9, Journal of Toxicology and Environmental Health, Part A, DOI:
10.1080/15287394.2022.2068711
To link to this article: https://doi.org/10.1080/15287394.2022.2068711
Published online: 25 Apr 2022.
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Toxicological eects of the mixed iron oxide nanoparticle (Fe
3
O
4
NP) on murine
broblasts LA-9
Karina Alves Feitosa
a
, Ricardo de Oliveira Correia
a
, Ana Carolina Maragno Fattori
a
, Yulli Roxenne Albuquerque
a
,
Patricia Brassolatti
a
, Genoveva Flores Luna
a
, Joice Margareth de Almeida Rodolpho
a
, Camila T. Nogueira
b
,
Juliana Cancino Bernardi
c
, Carlos Speglich
d
, and Fernanda de Freitas Anibal
a
a
Department of Morphology and Pathology, Inflammation and Infectious Diseases Laboratory, Federal University of São Carlos, São Carlos,
Brazil;
b
Department of Biochemistry, Federal University of São Paulo, Brazil;
c
Nanomedicine and Nanotoxicology Group, Physics Institute of São
Carlos, University of São Paulo, São Carlos, Brazil;
d
Leopoldo Américo Miguez de Mello Research Center CENPES/Petrobras, Rio de Janeiro, Brazil
ABSTRACT
The increase in large-scale production of magnetic nanoparticles (NP) associated with the
incomplete comprehensive knowledge regarding the potential risks of their use on environ-
mental and human health makes it necessary to study the biological eects of these particles
on organisms at the cellular level. The aim of this study to examine the cellular eects on
broblast lineage LA-9 after exposure to mixed iron oxide NP (Fe
3
O
4
NP). The following
analyses were performed: eld emission gun–scanning electron microscopy (SEM-FEG),
dynamic light scattering (DLS), zeta potential, ultraviolet/visible region spectroscopy (UV/
VIS), and attenuated total reactance-Fourier transform infrared (ATR-FTIR) spectroscopy ana-
lyses for characterization of the NP. The assays included cell viability, morphology, clonogenic
potential, oxidative stress as measurement of reactive oxygen species (ROS) and nitric oxide
(NO) levels, cytokines quantication interleukin 6 (IL-6) and tumor necrosis factor (TNF), NP
uptake, and cell death. The size of Fe
3
O
4
NP was 26.3 nm when evaluated in water through
DLS. Fe
3
O
4
NP did not reduce broblast cell viability until the highest concentration tested
(250 µg/ml), which showed a decrease in clonogenic potential as well as small morphological
changes after exposure for 48 and 72 hr. The NP concentration of 250 µg/ml induced
enhanced ROS and NO production after 24 hr treatment. The uptake assay exhibited time-
dependent Fe
3
O
4
NP internalization at all concentrations tested with no signicant cell death.
Hence, exposure of broblasts to Fe
3
O
4
NP-induced oxidative stress but not reduced cell
viability or death. However, the decrease in the clonogenic potential at the highest concen-
tration demonstrates cytotoxic eects attributed to Fe
3
O
4
NP which occurred on the 7
th
day
after exposure.
KEYWORDS
Nanotoxicology; SPION;
mixed iron oxide
nanoparticle; cytotoxicity;
murine fibroblasts LA-9
Introduction
Superparamagnetic iron oxide nanoparticles
(SPIONs) are iron oxide nanocrystals such as Fe
3
O
4
-magnetite or Fe
2
O
3
-maghemite, which gener-
ally show a diameter smaller than 30 nm (Ge et al.
2007; Hu et al. 2019; Samrot et al. 2021a; Teja and
Koh 2009). Superparamagnetic materials display
high magnetization in the presence of a magnetic
field, but these materials do not retain residual
magnetism after the magnetic field is removed.
This property possibility has numerous attractive
applications in industrial and biomedical areas
(Gupta and Gupta 2005; Hu et al. 2019; Samrot
et al. 2021b).
SPIONs have been frequently and reliably uti-
lized in biomedical applications as one of the first
approved nanoparticles (NP) employed for diagno-
sis of diseases as targeted magnetic resonance con-
trast agents (Fernández-Barahona et al. 2020; Israel
et al. 2020; Vangijzegem, Stanicki, and Laurent
2018; Wahajuddin 2012). Due to these superpara-
magnetic properties, these compounds might also
be used as powerful targeted drug delivery vehicles
in chemotherapy and radiotherapy to specific loca-
tions using a magnetic field. In cancer treatment,
magnetic hyperthermia using SPIONs, and an
alternating magnetic field generate local heat and
destroy the tumor by thermal ablation of cancer
CONTACT Karina Alves Feitosa karinaaf@estudante.ufscar.br Department of Morphology and Pathology, Inflammation and Infectious Diseases
Laboratory, Federal University of São Carlos, São Carlos, Brazil
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
https://doi.org/10.1080/15287394.2022.2068711
© 2022 Taylor & Francis
cells (Alphandéry 2020; Dadfar et al. 2019;
Musielak, Piotrowski, and Suchorska 2019;
Soetaert et al. 2020).
In industrial applications, iron oxide (II, II)
nanoparticles (Fe
3
O
4
), or magnetite, were trans-
formed into nanomagnetic fluids to enhance oil
recovery from rocks (Divandari et al. 2019; Zhou
et al. 2020). In addition, in a comparable manner to
magnetic resonance contrast agents used in biome-
dical science, these NPs may be used to analyze and
map oil reservoirs (Zhe and Yuxiu 2018). The NP of
the present study (Fe
3
O
4
NP) was synthesized with
the intention of use in the oil industry, and hence
terminal groups of sodium sulfonate (SO
3
) -Na
+
,
a surfactant, were added to its structure. Surfactants
are generally applied to stabilize nanofluids and to
aid in their solubility. The addition of this binder
reduced the surface tension of the fluids and
increased immersion of the particles, which are
responsible for specific functions such as controlled
surfactant delivery at the oil–water interface and
rock wettability (Caplan et al. 2019; I Rivera-
Solorio et al. 2013 Rosestolato et al. 2019; Silva-
Yumi, Romero, and Lescano 2021), thus helping
in oil recovery.
The increasing application and larger-scale pro-
duction of magnetic NP might result in increased
usage and subsequent elevated waste generation,
leading to potential interactions with biological
systems and consequences for environmental and
human health (Laffon et al. 2018; Boccuni et al.
2020). At present sufficient knowledge for predict-
ing the risk of nanomaterials on health is lacking,
which consequently creates uncertainty in nano-
technology safety (Justo-Hanani and Dayan 2015;
Pandey and Jain 2020). Therefore, nanotoxicology
research is fundamental for understanding the
potential risks to human health and harm to the
environment (Hubbs et al. 2013; Kermanizadeh
et al. 2016; Kermanizadeh, Powell, and Stone
2020; Pandey and Jain 2020; Saifi, Khan, and
Godugu 2018).
Several studies indicated that these magnetic NP
might accumulate in aquatic organisms and crops,
and thus enter the food chain. In this manner,
humans have been increasingly exposed to these
NP, directly or indirectly (Kai et al. 2011). Due to
the small size of SPIONs, these might reach the
brain and produce neural function damage, in
addition to crossing the nuclear barrier, reaching
genetic material, and inducing mutations. Their
low solubility might also lead to clumping and
clogging of blood vessels. Several investigators
demonstrated adverse effects in organs such as
liver, spleen, kidneys, heart, lungs, and brain
(Kobos et al. 2020; Pandey and Prajapati 2018;
Patil et al. 2018).
Iron oxide nanoparticles produce important tox-
icology effects including decreased cell viability,
plasmatic membrane disruption, mitochondrial
alterations, cell death, oxidative damage, and cell
cycle impairments (Fernández-Bertólez et al. 2019).
Batista-Gallep et al. (2018) proposed that the
mechanism that triggers oxidative stress initiates
an inflammatory response that leads to the toxicity
attributed to these NP. In contrast, other investiga-
tors showed that there are few cytotoxic effects
induced by these NP (Mahdavi et al. 2013;
Valdiglesias et al. 2016; Patil et al. 2018; Crețu
et al. 2021; Guigou et al. 2021). It should be noted
that the cytotoxicity is associated with the type of
cell line, exposure duration, concentration-
dependent, and NP characteristics as size range
and chemical surface (Matahum et al. 2016).
Lazaro-Carrillo et al. 2020 found that Fe
3
O
4
NP
exhibited biocompatibility in macrophages of the
RAW 264.7 lineage. However, Hsiao et al. (2008)
found that macrophages of the same strain treated
with SPIONs accumulated NPs in their membrane,
which reduced their phagocytic capacity.
Different lineages of fibroblasts are found in the
literature derived from mesenchymal tissues, gas-
trointestinal tract, muscle, skin and adipose tissue
(Junqueira and Carneiro 2004; Kermanizadeh et al.
2016; Lynch and Watt 2018). Extracellular matrix
(ECM)-rich connective tissues performs a wide
range of essential functions for organs, providing
positional information to neighboring cells through
structural, biomechanical, and biochemical factors
and regulated secretion of soluble mediators such
as cytokines, growth factors, and metabolites
(Plikus et al. 2021). Given these properties, func-
tions, and characteristics of established culture,
fibroblasts became standards in nanotoxicological
studies. Furthermore, immune system cells such as
fibroblasts display a specific relationship with
inflammatory conditions, fibrosis, and genotoxicity
induced by NP (Koerich et al. 2020; Oberdörster
2K. ALVES FEITOSA ET AL.
2012; Oberdörster et al. 2005). Data from previous
studies by our research group also demonstrated
that the use of murine fibroblast LA-9 lineage as
a model for nanotoxicological analysis was reliable
when examining the exposure to carbon and tita-
nium dioxide (TiO
2
) NP (de Godoy et al. 2021, de
Almeida Rodolpho et al. 2021; Pedrino et al. 2022).
However, there are still no apparent studies asses-
sing the effects of exposure to Fe
3
O
4
NP with
ligands of terminal groups of sodium sulfonate
(SO
3
)-Na
+
, in the presence of fibroblast LA-9.
This led to the need to examine the underlying
mechanisms of the effects exerted by Fe
3
O
4
NP on
biological systems. By utilization of the in vitro
fibroblast cell model, the oxidative pathways were
examined by measurement of reactive oxygen spe-
cies (ROS) and reactive nitrogen species (RNOs)
with the association with synthesis and release of
pro-inflammatory molecules, such as tumor necro-
sis factor (TNF), resulting in apoptotic cell death
with the release of death cell markers such as mem-
brane enzymes and reduced cytotoxicity
(Santagostino et al. 2021). Thus, the objective of
this study was to examine those effects responsible
for cytotoxicity in fibroblast cells of the LA-9 line-
age following Fe
3
O
4
NP exposure to clarify the
relationship between production of oxidative stress
and cytotoxicity.
Materials and methods
Mixed iron oxide nanoparticle (Fe
3
O
4
NP)
The Fe
3
O
4
NP was received from Leopoldo
Américo Miguez de Mello Research Center
(CENPES/Petrobras) for biological analysis. It is
an NP of magnetic fluid nature, composed of
mixed iron oxide (magnetite) and ligands with
terminal groups of sodium sulfonate (SO
3
)
−
Na
+
,
which disperses in water between pH 4 and 10
and in saline concentration up to 0.15 mol/L
(approximately 1% NaCl).
Characterization
The DLS and zeta potential of the Fe
3
O
4
NP
suspended in Dulbecco’s modified Eagle’s med-
ium (DMEM) (Sigma-Aldrich®) supplemented
with 10% fetal bovine serum (FBS) and distilled
water were determined using a Malvern spec-
trometer Nano-ZS (Marvern Instruments). For
the DLS and zeta potential of the Fe
3
O
4
NP
suspended in DMEM (Sigma-Aldrich®) supple-
mented with 10% FBS the times of 0, 6, 24, 48
and 72 hr were evaluated. Polydispersity index
(PdI) value was also included. The experimental
protocol used to perform DLS and zeta poten-
tial was as follows: Water: viscosity = col0.8872
and Refractive Index (RI) = 1.330; DMEM 10%:
viscosity = 0.9400 and RI = 1.338. In both cases
the RI Fe
3
O
4
NP = 1.338 with abs = 0.029 were
maintained. The concentration and the size of
Fe
3
O
4
NP were evaluated with Nano Tracking
Analysis (NTA), Nanosight NS300, Malvern.
The absorbances reading of the UV/VIS of Fe
3
O
4
NP (Laurent et al. 2008) was performed
using a spectrophotometer (U-2900) in the
wavelength range 190 nm to 800 nm. The ATR-
FTIR spectroscopy was conducted on a Bruker
Alpha-P instrument (Germany) equipped with
diamond crystal windows as the reflective ele-
ment of the 4 mm
2
. The spectrum was prepared
from 400 cm
−1
to 4000 cm
−1
, at room tempera-
ture (25°C), the resolution of 4 cm
−1
was
selected and a measurement considered 128
scans was performed in the solid phase with dry
Fe
3
O
4
NP. The microphotography of the Fe
3
O
4
NP was obtained by Scanning Microscopy
Analysis equipped with field emission gun elec-
tron (SEM-FEG) (Philips XL-30 FEG-Field
Emission Gun) at 50000x magnification, oper-
ated at an accelerating voltage of 25 KV and
a secondary detector was used.
Fibroblasts LA-9 cell culture
Fibroblasts (Lineage LA-9/Rio de Janeiro Cell
Bank (BCRJ) code 0142) are cells originating
from the connective adipose tissue of mice
(Mus musculus/Vulgar Name: Mouse; C3H/
An). The cells were cultivated in DMEM (Sigma-
Aldrich®) supplemented with 10% FBS and 1%
antibiotic (streptomycin/penicillin) (LGC
Biotecnologia) and incubated at 37°C in a 5%
CO
2
atmosphere.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 3
Cell viability and morphology
The MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphe-
nyl tetrazoilium bromide) assay method was used
for measurement of cell viability (Mosmann 1983).
To select the working concentrations, fibroblasts
LA-9 were seeded in a 96-well plate at a density of
6 × 10
3
cells/well where these cells were exposed to
different concentrations of Fe
3
O
4
NP (0.1, 0.5, 1,
10, 50, 100, 250, 500 or 600 µg/ml) (data not
shown), for 24, 48 or 72 hr. To avoid the color
interference in the reading of optical density
(OD), the concentrations 50, 100 and 250 µg/ml
NP were selected and incubated for 24, 48 or 72 hr
and performed blank for each concentration (Fe
3
O
4
NP concentration without cells). After exposure,
the supernatant was removed and cells were
washed three times with phosphate-buffered saline
(PBS) – (80 g NaCl, 2 g KCl, 11,5 g Na
2
HPO
4
, 2 g
KH
2
PO
4
for 1 L) and 100 µl MTT (Sigma-Aldrich®)
added. The formazan crystals formed were diluted
with 100 µl dimethyl sulfoxide (DMSO) (Synth)
and measured spectrophotometrically at 570 nm
(Multiskan GO). Cell viability (%) values were
determined as follows:
Cell viability %ð Þ ¼ OD of sample Mean OD blank concentration
Mean OD value of the negative control Mean OD blank MTT x100
Previous to addition of MTT, microphotographs
referring to LA-9 morphology were obtained after
incubation with different concentrations of Fe
3
O
4
NP using a microscope (Zeiss) at 100x magnifica-
tion. Three independent experiments were carried
out in quadruplicate for cell viability and in tripli-
cate for obtaining the images.
Clonogenic potential
The clonogenic assay consists of the ability of
a single cell to form colonies after exposure to
a material (Franken et al. 2006). At a density of
100 cells/well, fibroblast LA-9 were cultured in
6-well plates and were exposed to 50, 100 or
250 µg/ml Fe
3
O
4
NP for 24 hr. After this period,
the cell medium containing different concentra-
tions of Fe
3
O
4
NP was replaced with fresh
DMEM, and cells incubated for 7 days. After this
period cells were fixed with cold methanol (CH
3
OH) and stained with violet crystal (C
24
H
28
N
3
Cl –
0.1%). The wells were photographed, and colonies
counted using the ImageJ 1.53a software (Wayne
Rasband, National Institutes of Health, USA).
Three independent experiments were performed
in duplicate. The plating efficiency (PE) and survi-
val fraction (SF) values were determined as follows:
PE ¼Number of colonies
Number of cellsinitially seeded
SF ¼Mean PE of cells exposed to nanoparticle
Mean PEof negative control
Reactive Oxygen Species (ROS)
To detect formation of intracellular ROS, the probe
DCFH-DA (2′,7′-dichlorodihydrofluorescein dia-
cetate) was used (Wan, Myung and Lau 1993).
Fibroblasts LA-9 were cultured in 96-well plates at
a cell density of 1 × 10
4
cells/well and were exposed
to 50, 100 or 250 µg/ml Fe
3
O
4
NP for 24 hr. For the
positive control, 100 µl hydrogen peroxide (H
2
O
2
)
was employed. After exposure, DCFH-DA (Sigma-
Aldrich®) was added for 30 min protected in the
dark and cells washed three times with PBS.
Intracellular ROS production determination was
performed using 100 µl with PBS to avoid extra-
cellular interferences. The analysis was performed
in a fluorescence reader for microplates at 330–
485 nm (Spectra MAX i3 (Molecular Devices).
Three independent experiments were performed
in triplicate.Intracellular ROS production (%) was
calculated using the formula:
ROS %ð Þ ¼ Mean fluorescence emission of samples
Mean fluorescence emission of the negative control x100
Nitric oxide (NO)
The measurement of NO production was performed
utilizing the Griess reaction (Green et al. 1982;
Saltzman 1954). Fibroblasts LA-9 were seeded in
a plate of 96 wells at density of 1 × 10
4
cells/well
and exposed to 50, 100 or 250 µg/mL of Fe
3
O
4
NP for
24 hr. Blank was used for each concentration (Fe
3
O
4
NP concentration without cells) to avoid color inter-
ference in the reading of absorbances. After exposure
to Fe
3
O
4
NP concentrations, the supernatant of cells
was collected and 50 µl Griess reagent (naphthy-
lethylenediamine dihydrochloride (NEDD) added
4K. ALVES FEITOSA ET AL.
(0.1% diluted in distilled water + 1% sulfanilamide –
C
6
H
8
N
2
O
2
S diluted in 5% phosphoric acid (H
3
PO
4
).
The absorbance at 550 nm was measured using
a plate spectrophotometer (Multiskan Go) after
10 min reaction. The concentration of nitrite in the
supernatant was quantified from a standard curve
with known concentrations of sodium nitrite. Three
independent experiments were performed in
triplicate.
Cytokine quantication
The levels of IL-6 and TNF from the supernatant of
fibroblasts LA-9 were determined using ELISA
(enzyme-linked immunosorbent assay) detection
kit according to the manufacturer’s instructions
(BD Biosciences). The fibroblasts were seeded in
a plate of 96 wells at density of 1 × 10
4
cells/well
and were exposed to 50, 100 or 250 µg/ml Fe
3
O
4
NP
for 24 hr. After this period cell supernatants were
collected. Absorbance measurement was conducted
at 450 nm using a plate spectrophotometer
(Multiskan Go) and concentrations of the cytokines
calculated from a standard curve for each cytokine.
Three independent experiments were conducted in
triplicate.
Intracellular Fe
3
O
4
NP accumulation
For this assay Prussian Blue method was
applied. Intracellular ferric ions (Fe
3+)
are
detected when combined with the ferrocyanide
[Fe (CN)
6
]
4−
and results in the formation of
a blue pigment (Zhu et al. 2012; Feng et al.
2018). The fibroblasts LA-9 were seeded in
a plate of 24 wells in the density of 5 × 10
3
cells/well, and exposed to 50, 100 or 250 µg/ml
Fe
3
O
4
NP for 24, 48, or 72 hr. After treatment
cells were washed three times with PBS, fixed
with cold methanol, and stained with a solution
(1:1) of potassium ferrocyanide (K
4
[Fe (CN)
6
] –
4%) and hydrochloric acid (HCl – 4%) for
15 min. The micrographs were obtained at
400x in the microscope (Zeiss) using the soft-
ware Future WinJoe. For the Prussian blue
assay, three independent experiments were con-
ducted in triplicate.
Apoptosis detection
The assay was performed using the PE Annexin
V Apoptosis Detection kit (BD Biosciences)
according to the manufacturer’s instructions. The
fibroblast LA-9 were seeded in a plate of 24 wells in
the density of 1 × 10
5
cells/well, and exposed to 50,
100 or 250 µg/ml Fe
3
O
4
NP for 24 hr.
Camptothecin (500 µM) (Sigma-Aldrich®) was
used as a positive control. The analysis was per-
formed utilizing a flow cytometer Accuri C6 (BD
Biosciences), with 10,000 events per gate analyzed
using the software FlowJo version X (BD
Biosciences). Two independent experiments were
carried out in quadruplicate.
Statistical analysis
All data were analyzed in the GraphPad Prism
program, version 7 (San Diego, California, USA).
Results were expressed as means ± SD (Standard
Deviation). Parametric data were analyzed using
the One-way ANOVA test (One-way Analysis of
Variance) and posttest by Tukey’s multiple-
comparison test. For the non-parametric data, the
Kruskal–Wallis test and posttest by Dunn’s multi-
ple-comparison test were used. Statistical signifi-
cance was set at * p < 0,05 compared to negative
control.
Results
Characterization of Fe
3
O
4
NP
Figure 1 illustrates the SEM-FEG of the dry Fe
3
O
4
NP sample with its UV–visible absorption spec-
trum and ATR-FTIR spectrum. The SEM-FEG
microphotography (Figure 1a) shows a compacted
heterogeneous distribution with presence of clus-
ters of particles and a spherical shape, but due to
their magnetic property, these tend to form larger
spherical aggregates and clusters, characteristic of
high magnetization, while UV–vis spectra indicate
a scattering of the absorption light around 350 nm
typically for Fe
3
O
4
NP (Figure 1b). Figure 1c pre-
sents the ATR-FTIR spectrum collected of a dry
sampler of Fe
3
O
4
NP. The spectrum shows peaks
at 532; 960; 1025; 1088; 1161; 1262; 1449; 1542;
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 5
1742; 2371, 3236, 3653, and 3753 cm
−1
. All these
peaks may be associated with the stretching group’s
presence in Fe
3
O
4
NP. These groups are described
in the discussion section.
As noted in Table 1, in time zero (t = 0), Fe
3
O
4
NP exhibited a hydrodynamic diameter of
26.3 ± 3.1 nm and zeta potential of −19 ± 1.6 mV
in water dispersion. The average PdI of Fe
3
O
4
NP
dispersion displayed a value of 0.604 ± 0.082, indi-
cating a population of polydisperse nanostructures
typical of magnetic nanostructure. In contrast, pro-
longed storage of Fe
3
O
4
NP in culture DMEM
medium might initiate significant changes in their
physicochemical properties, as detected in Table 1.
The size of the Fe
3
O
4
NP in culture medium
increased over time (0, 6, 24, 48 and 72 hr). At
time zero (t = 0), immersion of the Fe
3
O
4
NP in
a protein-rich medium resulted in changes in their
potential zeta from −19 ± 1.6 mV to −8.7 ± 0.5 mV
and a rise in size from 26.3 ± 3.1 nm to
35.2 ± 3.2 nm. The observed changes were probably
due to formation of a protein corona. However, the
PdI of these particles decreased to 0.29 ± 0.01 at
time 72 hr (t = 72 hr) in this medium. The latter
may influence the biodistribution and circulation
time of Fe
3
O
4
NP in biological media. NTA
revealed that Fe
3
O
4
NP has a standard concentra-
tion of 4.72 × 10
10
± 1.46 x 10
8
particles/ml.
Viability, clonogenic potential, and morphology of
broblasts LA-9
Data in Figure 2a–c demonstrated that none of the
Fe
3
O
4
NP concentrations reduced fibroblast cell
viability. All concentrations reached viability
above 70%, therefore Fe
3
O
4
NP was considered
not cytotoxic to fibroblasts under conditions tested.
Subsequently, cytotoxicity in fibroblasts was deter-
mined after 24 hr exposure to Fe
3
O
4
NP and then
measurement on day 7 post-exposure for clono-
genic assay. Figure 2d represents cell colonies
formed by fibroblasts LA-9 showing that concen-
tration of 250 µg/ml Fe
3
O
4
NP decreased the clo-
nogenicity potential. There was a smaller number
of colonies at this concentration compared to nega-
tive control. Figure 2e demonstrates fibroblasts LA-
9 survival fraction, where only the highest concen-
tration, 250 µg/ml, was significant in diminishing
reproductive viability (clonogenic capacity) com-
pared to negative control.
When observing the fibroblast morphology
(qualitative data), it was noted that the negative
control presented normal morphology and
growth (Figure 3a,e,i). For 250 µg/ml at 24 hr,
there was no marked morphological change
(Figure 3b), but after 48 hr (Figure 3f), less
elongated cells were detected compared to nega-
tive control and at 72 hr (Figure 3j), a possible
Figure 1. SEM- FEG, UV-VIS absorption and ATR-FTIR spectroscopy of the Fe
3
O
4
NP. (a) SEM-FEG micrograph; (b) UV-VIS absorption
spectra; (c) ATR-FTIR spectrum. Magnification: 50000x/500 nm scale.
Table 1. Z-average values obtained by Dynamic Light Scattering (DLS), Polydispersity index (PdI) and Zeta potential obtained by Fe
3
O
4
NP
in water and DMEM medium.
Water
DMEM 10% FBS
t = 0 t = 6 hr t = 24 hr t = 48 hr t = 72 hr
Z-average (nm) 26.3 ± 3.06 35.2 ± 3.25 43.7 ± 1.50 47.3 ± 3.60 48.4 ± 0.73 48.9 ± 1.70
PdI 0.604 ± 0.082 0.491 ± 0.087 0.372 ± 0.021 0.322 ± 0.043 0.271 ± 0.001 0.291 ± 0.010
Zeta potential
(mV)
−19 ± 1.6 −8.7 ± 0.5 −9.3 ± 0.3 −7.0 ± 0.4 −8.9 ± 1.9 −7.2 ± 1.2
6K. ALVES FEITOSA ET AL.
retraction of the cell cytoplasm was found. There
were no marked morphological changes
observed for the concentrations of 50 and
100 µg/ml Fe
3
O
4
NP at any incubation time
tested. Despite the morphological changes
described for the concentration of 250 µg/ml,
for all concentrations tested, cell density was
similar to negative control, indicating growth
and viability not significantly affected.
Production of ROS, NO and pro-inammatory
cytokines of broblasts LA-9
Figure 4a illustrates intracellular ROS generation in
fibroblast LA-9 after exposure to concentrations of 50,
100 or 250 µg/ml Fe
3
O
4
NP for 24 hr. A significant
elevation was noted for 250 µg/ml Fe
3
O
4
NP com-
pared to negative control, and for positive control
compared to negative control, affirming the effective-
ness of H
2
O
2
in inducing ROS production.
Figure 2. Viability cells and clonogenic potential of fibroblasts LA-9 after exposure to Fe
3
O
4
NP (50, 100 or 250 µg/mL). (a) MTT 24 hr;
(b) MTT 48 hr; (c) MTT 72 hr; (d) Colonies formed by fibroblasts LA-9 with 7 days of recovery after exposure to different concentrations
of Fe
3
O
4
NP for 24 hr; (e) fibroblasts LA-9 survival fraction expressed as %. Negative control (C-): cells + culture medium; Positive control
(C+): cells + 5% dextran. Data represents the mean ± SD of 3 independent experiments, where *p < .05.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 7
Figure 4b demonstrates NO generation evalu-
ated in the cell supernatant of fibroblasts, exposed
to different concentrations of Fe
3
O
4
NP for 24 hr.
There was a significant increase for all concentra-
tions tested in relation to negative control. Also,
there was a significant rise in NO production at
a concentration of 250 µg/ml of Fe
3
O
4
NP.
Cytokines, IL-6 and TNF levels in the fibroblast
LA-9 supernatant after treatment with 50, 100 or
250 µg/ml Fe
3
O
4
NP for 24 hr are presented in
Figure 4c,d. There was no significant difference
compared to negative control.
Uptake of Fe
3
O
4
NP in broblast LA-9
Cellular internalization of Fe
3
O
4
NP at 24, 48 or
72 hr is depicted in Figure 5, which was found to be
time dependent. For all concentrations of Fe
3
O
4
NP, a process of NP cluster formation around the
cell membrane was observed. This process became
more extensive overtime for all concentrations
tested and may have contributed to morphological
damage found in cells exposed to 250 µg/ml after
48 hr.
Fibroblast LA-9 cell death
Qualitative analysis of the expression proportion of
PE Annexin V probe and 7AAD marker, for all
concentrations of Fe
3
O
4
NP (50, 100 and 250 µg/
ml), negative control (cells and medium) and posi-
tive control (camptothecin) are presented in
Figure 6a. As for the determination of cell death
(Figure 6b), the % of both markers showed an
apoptotic process, and the correlation between
apoptosis and necrosis (Figure 6c) demonstrated
a significant increase in positive control in the
apoptotic process. All Fe
3
O
4
NP concentrations
produced similar levels compared to negative con-
trol. Figure 6d,e (early and late apoptosis) indicates
no significant findings for all Fe
3
O
4
NP concentra-
tions. The existing correlation between early and
Figure 3. Morphology of fibroblasts LA-9 post exposure within 24, 48 and 72 hr of Fe
3
O
4
NP. (a, e, i) C- 24, 48 and 72 hr; (b, f, j) 250 µg/
ml 24, 48 and 72 hr; (c, g, k) 100 µg/ml 24, 48 and 72 hr; (d, h, l) 50 µg/ml 24, 48 and 72 hr. Negative Control (C-): cells + culture
medium. Qualitative analysis performed in three independent experiments. Magnification: 100x.
8K. ALVES FEITOSA ET AL.
late apoptosis (Figure 6e) once again illustrated
a significant rise in positive control suggesting late
apoptosis.
Discussion
The in vitro model for nanotoxicological studies
has been widely used, since cell cultures might be
better controlled and data obtained more quickly.
The most used cells for cytotoxicity studies are
macrophages, fibroblasts, and epithelial cells (Clift
et al. 2014; Kermanizadeh et al. 2016). Our study is
the first to determine the cytotoxicity of Fe
3
O
4
NP
(Fe
3
O
4
/SO
3
Na
+
ligand), of interest in the oil indus-
try, in murine fibroblasts of the LA-9 lineage. The
surfactants are generally applied to aid in their
solubility (Mieloch et al. 2020). For this purpose,
the characterization of this NP was performed, as
well as the observation of internalization in cells
and the effects derived from cell/nanoparticle
interaction, such as cell morphology, production
of ROS, NO and pro-inflammatory cytokines, cell
death and viability. Furthermore, the effect post
exposure on LA-9 cells was assessed by examining
colony formation by clonogenic assay.
At the physiological level, the concentrations
of Fe
3
O
4
NP used in our study (50, 100, and
250 µg/ml) are considered high, since the use of
this NP for magnetic resonance range from 0.2 to
0.8 mg Fe/kg body weight, which is similar to
concentrations of 2.5–10 μg/ml (Fernández-
Bertólez et al. 2019; Reimer and Balzer 2003).
However, the toxicity of this NP is related to
the high amount of Fe, which might induce oxi-
dative stress. At low concentrations, toxicity is
not observed, as SPIONs are removed from the
body (Natarajan et al. 2019; Patil et al. 2018).
Hence, concentrations selected were reported
based upon studies of Fe
3
O
4
NP used as drug
delivery and/or cancer therapy, which employed
Figure 4. Production of ROS, NO and pro-inflammatory cytokines of fibroblasts LA-9 post exposure to Fe
3
O
4
NP within 24 hr. (a)
Production of Reactive Oxygen Species (ROS) through the DCFH-DA probe. Negative Control (C-): cells + culture medium; Positive
Control (C+): (Cells + 1 µm H
2
O
2
). (b) Concentration of Nitric Oxide (NO) in the fibroblasts LA-9 supernatant; IL-6 (c) and TNF (d)
concentrations in pg/ml in the fibroblasts LA-9 supernatant. Data represents the mean ± SD of 3 independent experiments, where *
p < .05.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 9
an in vitro model and aimed at potential applica-
tion in humans (Yusefi et al. 2020; Hussein et al.
2021; Ebadi et al. 2021).
The characterization of NP allows a better
understanding of how these agents behave in dif-
ferent solutions. Nanoparticle surface characteris-
tics such as charge, surface area and surface
reactivity may be considered as potential modula-
tors of toxicity, attributed to interaction with cell
membrane (Sharma, Madhunapantula, and
Robertson 2012). The Fe
3
O
4
NP in the form of
ferrofluid, in addition to exhibiting high magneti-
zation, display interaction dependent upon their
physicochemical characteristics, composition, mor-
phology and their surface binders (Ma et al. 2004;
Gubin et al. 2005; Ramya and Mahadevan 2012).
ATR-FTIR is a useful technique to observe the
surface groups at the NP level due to their stretch-
ing vibration (Lee, Liong, and Jemainb 2017;
Naseer, Ali, and Qazi 2021). Our results showed
peaks at 532; 960; 1025; 1088; 1161; 1262; 1449;
1542.; 1742; 2371, 3236, 3653 and 3753 cm
−1
. The
band at 532 showed a peak at cm
−1
due to Fe-O
stretching vibration, according to Ni et al. (2009)
and Medeiros et al. (2015). Other stretching vibra-
tion peaks were related to the polymer used to
stabilize the particles that are rich in sulfonate
groups. Aghazadeh et al (2017a) observed peaks
at 960 cm
−1
associated with C-C stretching vibra-
tions and peaks at 1025, 1088 and 1161 cm
−1
attributed to the presence of sulfonate group
(Toma et al. 2022). According to Medeiros et al.
(2015), band around 1035 and 1195 cm
−1
repre-
sents the asymmetric and symmetric stretching
vibration of SO
3
, thus confirming the ligands of
Fe
3
O
4
NP. Furthermore, in the spectrum peaks
approximately 1262 and 1449 cm
−1
were found
which are ascribed to -CH
2
twisting and scissoring
vibrations (Aghazadeh et al. 2017b; Masoudi et al.
2012). The peaks around 1542 cm
−1
are character-
ized by ring vibration in aromatic skeleton or
C-C double bands (Liu, Zhang, and Sasai 2010;
Toma et al. 2022). While the peaks observed
around 1742 cm
−1
are indexed to C-O double
band, the peak at 2371 is indicative of
C-O bonds (Islam and Peng 2020; Kadik et al.
2006; Nadimi et al. 2019). The bands at 3200 to
3700 cm
−1
have been described in the literature as
O-H vibrations (Karimzadeh et al. 2017; Medeiros
et al. 2015).
Sulfonate groups, as mentioned above, might aid
in the solubility of Fe
3
O
4
NP and solubility is also
related to the toxicity of this NP. Brunner et al.
(2006)found that toxic effects may be related either
Figure 5. Uptake of nanoparticles in fibroblasts LA-9 post exposure to Fe
3
O
4
NP within 24, 48 and 72 hr. (a, e, I) C- 24, 48 and 72 hr; (b, f,
j) 250 µg/ml 24, 48 and 72 hr; (c, g, k) 100 µg/ml 24, 48 and 72 hr; (d, h, l) 50 µg/ml 24, 48 and 72 hr. Negative Control (C-): cells +
culture medium. Prussian blue coloration. Qualitative analysis performed in three independent experiments. Yellow arrows indicate the
formation of Fe
3
O
4
NP clusters. Magnification: 400x.
10 K. ALVES FEITOSA ET AL.
Figure 6. Assay for quantifying the type of cell death in fibroblasts LA-9 using Annexin V and 7AAD markers. (a) Dot Plot Density
diagrams, with the percentages of apoptosis and necrosis induced by camptothecin (C+) and different concentrations of Fe
3
O
4
NP (50,
100 or 250 µg/ml). Negative control (C-): cells and culture medium. Positive control (C+): cells and 500 µM camptothecin. (b) Bar
diagram representing % fibroblasts LA-9 in a state of apoptosis or necrosis; (c) Correlation between the levels found for apoptosis and
necrosis; (d) Bar diagram representing % fibroblasts LA-9 in early apoptosis or (e) late apoptosis; (f) Correlation between the levels
found for early and late apoptosis. Data represents the mean ± SD of 2 independent experiments, where *p < .05.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 11
based upon chemical composition or due to oxida-
tive stress initiated during the exposure. When
these NPs do not dissolve or partially dissolve in
the culture medium, it becomes more difficult to
differentiate the types of toxicity. The Fe
3
O
4
NP
used by Brunner et al. (2006) was described as
partially soluble, but toxicity was attributed to
some specific mechanism of these NPs, and not
only to the chemical effects of undissolved Fe
ions. When SPIONs were analyzed without any
ligand, these compounds showed low solubility,
which might lead to clumping that clog blood ves-
sels (Patil et al. 2018; Thorat et al. 2016). A possible
solution to obtain water solubility is to add surfac-
tant chemical binders to these NPs, as was done in
our study (Mieloch et al. 2020).
Through DLS analysis, the size and zeta potential
of Fe
3
O
4
NP were estimated in the culture medium
used and in distilled water. The zeta potential is
important for understanding the stability of the
suspended NP. As for analysis in cell culture med-
ium, the zeta potential was −8.7 mV, indicating
a tendency for particles to aggregate. Nanoparticle
suspensions are considered stable when the zeta
potential measurement is greater than ±30 mV
(Nurdin, Ridwan, and Satriananda 2016). There
was no UV–VIS absorption peak for the Fe
3
O
4
NP sample. Nanoparticles that form clusters or
samples with a high PdI may not form the absorp-
tion peak (Sarma et al. 2009). Furthermore, clusters
might scatter light, masking absorption.
The NP used in our study underwent changes in
contact with the culture medium. The size of the
Fe
3
O
4
NP in culture medium increased over time
(0, 6, 24, 48 and 72 hr). Several studies observed
a tendency of inorganic nanoparticles, as well as Fe
3
O
4
NP, to absorb proteins from the cell culture
medium, creating a corona effect of proteins
around these NPs (Calatayud et al. 2014; Casals
et al. 2011). In in vivo studies, the formation of
the corona effect might alter the surface of the NP
thus modifying the interaction with cells and result-
ing in an undirected distribution of NP, which may
lead to immune responses (Park 2020). Maiorano
et al. (2010) compared DMEM and Roswell Park
Memorial Institute (RPMI) culture media and
found that DMEM induced a greater formation of
corona protein in a time-dependent manner com-
pared to RPMI in HeLa and U937 cell lines. At the
intracellular level, the toxic effects of NP can also be
initiated through formation of the corona effect.
Cellular proteins are directly affected, resulting in
interruptions in important mechanisms such as
transcription, proliferation, signal transduction,
cell cycle regulation, metabolism, and apoptosis
(Park 2020).
Possible mechanisms of intracellular uptake of
Fe
3
O
4
NP involve passive diffusion, receptor-
mediated endocytosis or clathrin and caveolin, or
phagocytosis (Hillaireau and Couvreur 2009;
Sakhtianchi et al. 2013; Singh et al. 2010).
Phagocytosis is carried out by only a few cells,
such as macrophages. When facing the exposure
to NP, these specialized cells try to phagocytose
these particles; however, macrophages may
agglomerate resulting in cellular infiltrates and
a consequent inflammatory process that, if pro-
longed, might lead to more severe cases, producing
lesions, fibrosis and even tissue tumors (Arick et al.
2015; Radaic et al. 2016).
When these Fe
3
O
4
NP are degraded by lyso-
somes, Fe ions are generated (Gupta and Curtis
2004b), which are identified intracellularly. Zhu
et al. (2012)observed that the internalization effi-
ciency of SPIONs is dependent upon the character-
istics that each cell lineage exhibits, as well as the
nature of the NP, including its coating. Our results
of Prussian Blue confirm internalization of Fe
3
O
4
NP in fibroblasts LA-9 at all concentrations and
experimental times performed. A time-dependent
correlation was noted. Gu et al. (2011) found inter-
nalization of SPIONs in macrophages RAW 264.7
at different periods up to 48 hr and showed that this
also occurs in a time-dependent manner, thus a rise
in SPIONs uptake was detected at longer incuba-
tion times.
The morphological damages found in our study
may be related to the tendency for formation of NP
clusters confirmed by characterization and
Prussian Blue. Zhu et al. (2012), who performed
the Prussian Blue in different cells including fibro-
blasts L929 lineage, did not find cellular morpho-
logical changes in fibroblasts after exposure to NP
within 24 hr. However, other investigators demon-
strated that internalization of Fe
3
O
4
NP in dermal
fibroblasts resulted in formation of vacuoles, and in
the L929 lineage granules and organelle damage
were observed in exposure periods up to 72 hr
12 K. ALVES FEITOSA ET AL.
(Berry et al. 2004; Mahmoudi et al. 2012, 2009),
suggesting that long periods of exposure promote
cell damage.
The internalization of Fe
3
O
4
NP might be a key
factor to describe the toxicity, and in this sense for
some intrinsic physiological mechanisms. The che-
mical reaction via Fenton is well described when
Fe
3
O
4
NP are evaluated in the biological environ-
ment and occurs from an accumulation of free Fe
+
ions in the cell cytoplasm, which induces an
increase in the production of H
2
O
2
produced pre-
dominantly by SOD, which results in the genera-
tion of hydroxyl radicals that are toxic by impairing
cellular homeostasis. Another factor involved is the
imbalance in cellular protection system through
activities of oxidant/antioxidant enzymes, and
finally, mitochondrial dysfunction and oxidative
stress via ROS production, which in excess induces
toxicity, accompanied by NO that furthermore
exacerbates this response, inhibits adhesion mole-
cules or even induce cell death (de Jesus and Kapila
2014; Wu et al. 2014; Radaic et al. 2016; Maurizi
et al. 2018; Gholinejad, Ansari, and Rasmi 2019; Yu
et al. 2019; Nakamura, Naguro, and Ichijo 2019;
Bonadio et al. 2020).
Our findings indicate that internalization of
these NP induced significant production of ROS
and NO in fibroblasts LA-9 at a concentration of
250 µg/ml, possibly derived from accumulation of
intracellular Fe ions detected by the Prussian Blue
methodology. Our results are in agreement with
Radu et al. (2015), who also identified accumula-
tion of Fe ions inside lung fibroblasts MRC-5 after
exposure to magnetic NP generating elevated levels
of ROS, NO and PGE2 prostaglandins E2 (PGE2),
and resulting in significant cellular dysfunctions.
Pongrac et al. (2016) investigated the action of
SPIONs in neural cells, and noted oxidative stress
triggered by imbalance between the excess produc-
tion of ROS and the cellular protection system
mediated by antioxidant enzymes.
The oxidative state of iron (Fe
2+
or Fe
3+
) enables
to predict cytotoxicity, since Fe
2
O
3
is more toxic
than Fe
3
O
4
. Further, concentration may also be
considered a crucial factor in determining effects
on the cellular environment. Interestingly, Wu,
Ding, and Sun (2013) investigated the action of
Fe
3
O
4
NP on PC12 cells and observed
concentration-dependent cytotoxicity with the pre-
sence of oxidative stress as evidenced by increased
ROS levels. In addition, Wu, Ding, and Sun (2013)
found that in just 24 hr of exposure, NP impaired
the cell cycle, inducing the cell to remain in the G2/
M phase with the intention of triggering a possible
repair mechanism for damaged DNA. These find-
ings corroborate the results of our study regarding
oxidative stress, also suggesting that not only the
oxidative state and NP concentration, but also the
cell type examined might interfere with the toxic
response arising from the biological environment.
Data demonstrate that the damage initiated by
enhanced oxidative stress might lead the cell to
a process of senescence, as a reduction in colony
formation in the period of 7 days was observed,
which suggests a reduction in the cellular metabolic
activity in a longer period of exposure. Berry et al.
(2004) and Soenen et al. (2009) reported that mag-
netoliposomes with FeO nuclei affected the forma-
tion of focal adhesion complexes and impaired
long-term cell proliferation in fibroblasts 3T3, and
that SPION-mediated oxidative stress may be
related to disruption of a protein from the cytoske-
leton (tubulin) in human fibroblasts hTERT-BJ1,
thus affecting cell reproducibility. Furthermore,
Prijic et al. (2010) corroborated our findings, as
SPIONs were noted to reduce reproducibility of
fibroblasts L929 after 8 days of treatment.
Unlike the senescence process, Basuroy et al.
(2011) showed that increased oxidative stress and
imbalance in redox regulation might trigger cellular
mechanisms related to apoptotic cell death.
Apoptosis is characterized by a series of cellular
events such as alterations in the cytoskeleton that
initiate cell contraction, DNA fragmentation, chro-
matin condensation and vesicle formation without
the occurrence of loss of plasma membrane integ-
rity. Another important feature is usually non-
activation of inflammatory responses, once cells
undergo apoptotic process to not release their cel-
lular constituents to the extracellular environment
or, if this occurs, substances are rapidly phagocy-
tosed, avoiding necrosis and immunologically
silent (Kurosaka et al. 2003; Elmore 2007; Szondy
et al. 2017). Significant alterations in pro-
inflammatory cytokines, TNF and IL-6 were not
detected in cells exposed to Fe
3
O
4
NP.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 13
Some studies linked cell death induced by Fe
3
O
4
NP to oxidative stress, which led to mitochondrial
and genetic material damage, and consequent death
by apoptosis (Sadeghi, Tanwir, and Babadi 2015;
Valdiglesias et al. 2015; Gaharwar, Meena, and
Rajamani 2017; Paunovic et al. 2020). Mahmoudi
et al. (2009) reported that in fibroblasts L929
exposed to uncoated SPIONs, significant levels of
apoptosis occurred, possibly due to damage to cel-
lular DNA. Another approach used in studies
aimed at cancer treatment, applied hyperthermia
and Fe
3
O
4
NP to promote apoptosis as conducted
by Ferraz et al. (2020), who designed SPIONs to
induce apoptosis in human lung fibroblasts MRC-
5. Other cell types were also susceptible to apopto-
sis when exposed to NP with Fe in their composi-
tion. Wani et al. (2014) employed MCF7 and
MDAMB-231 lineage cells, two tumor lines
exposed to cinnamaldehyde functionalized with
Fe
3
O
4
NP and noted a loss of mitochondrial mem-
brane potential and caspase-3 activation resulting
in apoptosis in both strains. In addition, neuronal
cells (PC12) when treated with Fe
3
O
4
NP resulted
in reduced cell viability and induced apoptosis (Liu
et al. 2018). However, despite the relation between
Fe
3
O
4
NP and cell death initiated by apoptosis and
data in our study, demonstrating an increase in
production of ROS and NO at the highest concen-
tration analyzed, this toxicity that failed to lower
cell survival frequency may be related to absence of
sufficient apoptotic changes.
The lack of toxicity that results in diminished
cell viability might also be associated with mito-
chondrial activity. None of the concentrations
tested showed significant cytotoxicity when eval-
uated by the MTT assay, regardless of time and
concentrations tested. Studies in different cell lines
also did not find cytotoxicity of Fe
3
O
4
NP against
dermal fibroblasts, mouse fibroblasts L929 and
human fibroblasts HDF (Alili et al. 2015; Auffan
et al. 2006; Prijic et al. 2010). Keshtkar et al. (2018)
reported fibroblasts of the lineage HFFF-PI6 were
exposed for 12 and 24 hr at concentrations of 10,
50, 100, 200, 300 or 400 μg/ml Fe
3
O
4
NP. Data
showed that only concentrations of 300 and
400 μg/ml were cytotoxic to fibroblasts. This
study corroborates our data, where concentrations
close to those used also demonstrated diminished
cell survival rates. It is known that Fe participates
in cell metabolism as a nutrient, which like phos-
pholipids, stimulates cell growth and proliferation.
Further, Fe deficiency may even affect some pro-
teins such as cyclin-dependent kinases (CDKS)
and cyclins, and thus impair the cell cycle
(Ghasempour et al. 2015; Gupta and Curtis
2004a; Mahmoudi et al. 2009; Mao et al. 2013). It
should be noted that the toxicity of NP is related to
dose and time and any concentration might be
toxic depending upon cell type and NP (Keshtkar
et al. 2018).
The clonogenicity potential of fibroblasts ana-
lyzed on the 7
th
day after exposure revealed
a significant reduction in reproductive viability at
a concentration of 250 µg/ml and apoptosis results
are related to observations in the MTT assay, where
cells proved to be viable. Thus, as the clonogenic
assay evaluates a longer time interval compared to
the viability assay, accumulation of NP internalized
by fibroblasts and consequent increase in oxidative
stress might result in significant cytotoxic effects
that interfere with reproductive potential and con-
sequent colony formation.
Conclusions
Fe
3
O
4
NP enhanced oxidative stress pathways
(ROS and NO) in fibroblasts LA-9 when exposed
within 24 hr but did not generate a significant
increase in number of apoptotic cells. However, it
was possible to observe that despite morphological
changes, cellular internalization of NP and oxida-
tive stress but no reduction in cell viability.
Cytotoxic effects were found measuring clonogenic
potential of fibroblasts LA-9 on the 7
th
day after
exposure to Fe
3
O
4
NP. Our study suggests that the
increase in oxidative stress led the cell to a process
of senescence, associated with low cellular metabo-
lism and consequent reduction in formation of
colonies in the 7-day period. Further, future studies
with antioxidants, such as N-acetylcysteine (NAC),
might serve as a valuable tool in understanding the
relationship between ROS production and mechan-
isms of cellular senescence in the face of Fe
3
O
4
NP
exposure. It is evident that NP exert varying effects
on different cell types. More studies following expo-
sure to Fe
3
O
4
NP are needed to better understand
molecular and genetic mechanisms that trigger
cytotoxicity.
14 K. ALVES FEITOSA ET AL.
LIMITATIONS
Fe
3
O
4
NP toxicology using colorimetric assays
There are investigations reporting interferences
that Fe
3
O
4
NP produce in some tests, especially
those using the colorimetric principle. The main
limiting factors are related to the optical properties
or the agglomeration tendency of these NPs
(Gonzales et al. 2010; Kong et al. 2011; de Simone
et al. 2020).
One alternative would be the use of more
than one method with the same objective for
comparison and confirmation of the results;
however, data show that the interference
initiated by NP might occur regardless of the
method. Holder et al. (2012) performed
a comparative study between two common fea-
sibility assay methods, MTT and lactate dehy-
drogenase (LDH) and demonstrated that both
methods are subjected to NP interference.
Thus, Holder et al. (2012) recommended that
toxicity assessments need to be performed prior
to characterization of the NP, also carrying out
controls with the concentrations that need to be
used in the tests, in order to eliminate possible
interferences. Kroll et al. (2012) also compared
NP through the methods dichlorofluorescein
(DCF), MTT, LDH and ELISA. Interferences
were found between the methods according to
the NP type, but when changes were made in the
protocols with reduction of concentrations of
carbon black NP interferences could be avoided.
Kroll et al. (2012) also suggested developments
in the classical methods of cytotoxicity and that
adaptations in the protocols need to be made
using controls for the NP.
Other studies used Fe
3
O
4
NP with adaptations in
the assay protocols, especially regarding control of
the concentrations employed to eliminate possible
interferences and obtain reliability of the results
(Häfeli et al. 2009; Ying and Hwang 2010;
Kenzaoui et al. 2012; Costa et al. 2016; Fazio et al.
2016; Svitkova et al. 2021). In view of the methods
used in our study, there was a need for changes in
the protocols of the MTT assay (greater number of
washes before adding the MTT, controls for the
concentrations and blanks) and changes in the
method of evaluating the production of NO (con-
centration of blanks). The complete protocols with
the modifications are described in the material and
methods section. Our results showed no interfer-
ence of Fe
3
O
4
NP in the analysis.
Highlights
• New Fe
3
O
4
NP with sodium sulfonate ligands of interest in
the oil industry.
• Cytotoxicity against fibroblasts LA-9 never evaluated
before against Fe
3
O
4
NP.
• Significant increase in ROS, NO and possible cell senes-
cence process.
• Importance of furthermore in vitro studies to assess the
cytotoxicity of Fe
3
O
4
NP in longer periods after exposure.
Acknowledgments
We thank Doctor Márcia Regina Cominetti (Department of
Gerontology, UFSCar) for the cytometer available for our analy-
sis, Doctor Iran Malavazi (Department of Genetics and Evolution,
UFSCar) for all his help in obtaining the images by SEM-FEG,
Doctor Eduardo Henrique Martins Nunes and M.Sc. Himad
Ahmed Alcamand (Department of Metallurgical and Materials
Engineering, UFMG) for the help in our analysis ATR-FTIR.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by Leopoldo Américo Miguez de
Mello Research Center CENPES/ Petrobras /Project: Proc. No
2017/00010-7.
Data Availability Statement
The data that support the findings of this study are available from
the corresponding author, [K.A.F], upon reasonable request.
Data available at https://drive.google.com/drive/folders/
1YgRjJlGHR-D0Ixe82EKfA4YMyzj-0HsQ.
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