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Article
Employing Calcination as a Facile Strategy to Reduce
the Cytotoxicity in CoFe2O4 and NiFe2O4 Nanoparticles
Débora Lima, Ning Jiang, Xin Liu, Jiale Wang, Valcinir Vulcani, Alessandro Martins,
Douglas Machado, Richard Landers, Pedro HC Camargo, and Alexandre Pancotti
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13103 • Publication Date (Web): 23 Oct 2017
Downloaded from http://pubs.acs.org on October 25, 2017
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ACS Applied Materials & Interfaces is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
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ACS Applied Materials & Interfaces
Employing Calcination as a Facile Strategy to Reduce the
Cytotoxicity in CoFe2O4 and NiFe2O4 Nanoparticles
Débora R. Lima1a, Ning Jiang2a, Xin Liu3a, Jiale Wang4*, Valcinir A. S. Vulcani1,
Alessandro Martins1, Douglas, S. Machado1, Richard Landers5, Pedro H. C.
Camargo6, and Alexandre Pancotti1*
1
Universidade Federal de Goiás, Regional Jataí, Unidade Acadêmica Especial de
Ciências Exatas and Unidade Acadêmica Especial de Ciências da Saúde, Rod. Br
364, km 168, Jataí-GO, Brazil
2
Department of Oral and Craniomaxillofacial Science, Ninth People’s Hospital,
College of Stomatology, Shanghai Jiao Tong University School of Medicine,
Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
3
Shanghai Biomaterials Research & Testing Center, Shanghai Key Laboratory of
Stomatology, Ninth People’s Hospital, Shanghai Jiaotong University School of
Medicine, No. 427, Ju Men Road, Shanghai 200023, China
4
College of Science, Donghua University, Shanghai 201620, China
5
Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin, Campinas-
SP, Brazil
6
Departamento de Química Fundamental, Instituto de Química, Universidade de São
Paulo, Av. Lineu Prestes, 748, 05508-000 São Paulo, SP, Brazil
*Corresponding authors: Email: jiale.wang@dhu.edu.cn (J.W.) and
apancotti@gmail.com (A.P.)
a
These three authors contributed equally to this article.
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ABSTRACT
CoFe2O4 and NiFe2O4 nanoparticles (NPs) represent promising candidates for
biomedical applications. However, in these systems, the knowledge over how
various physical and chemical parameters influence their cytotoxicity remains limited.
In this paper, we investigated the effect of different calcination temperatures over
cytotoxicity of CoFe2O4 and NiFe2O4 NPs, which were synthesized by a sol-gel
proteic approach, towards L929 mouse fibroblastic cells. More specifically, we
evaluated and compared CoFe2O4 and NiFe2O4 NPs presenting low crystallinity (that
were calcined at 400 and 250
o
C, respectively) with their highly crystalline
counterparts (that were calcined at 800 oC). We found that the increase in the
calcination temperature led to the reduction in the concentration of surface defect
sites and /or more Co or Ni atoms located at preferential crystalline sites in both
cases. A reduction in the cytotoxicity towards mouse fibroblast L929 cells was
observed after calcination at 800 oC. Combining with ICP-MS data, our results
indicate that the calcination temperature can be employed as a facile strategy to
reduce the cytotoxicity of CoFe2O4 and NiFe2O4, in which higher temperatures
contributed to the decrease in the dissolution of Co2+ or Ni2+ from the NPs. We
believe these results may shed new insights into the various parameters that
influence cytotoxicity in ferrite NPs, which may pave the way for their wide spread
applications in biomedicine.
Keywords: ferrites, nanoparticles, cytotoxicity, NiFe2O4, CoFe2O4
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ACS Applied Materials & Interfaces
INTRODUCTION
Magnetic nanoparticles (NPs) have been extensively investigated towards
biomedical applications.1 They present dimensions that are comparable to bioentities, including cells (10-100 µm), viruses (20-450 nm), and proteins (5-50 nm).2
As a result of their strong magnetization, they can be manipulated by external
magnetic fields, allowing for the remote control over their location at relatively long
distances.3 Therefore, combined with the large penetration capability of magnetic
fields in human tissues, magnetic NPs offer many opportunities related to the
transport, immobilization, and labeling of biological entities.4 This can be employed,
for example, as an alternative way to carry anticancer drugs to specific regions of the
human body presenting tumor cells.5 Magnetic NPs have also shown great promise
towards cancer hyperthermia, as their interaction with alternating magnetic fields
allow the generation of controlled/localized heating which is enough to kill
neighboring cancer cells.6 However, in order to achieve the full potential of magnetic
NPs in the biomedical field, they must present size less than 50 nm, chemical
stability, biocompatibility, high magnetic saturation, and no magnetic agglomeration.7
In this context, systematic investigations towards their synthesis, characterization,
manipulation, cytotoxicity, and assembly are required in order to unravel how various
physical and chemical features influence these properties.
Among several magnetic NPs, those based on transition metal-oxides such as
cobalt ferrite (CoFe2O4) and nickel ferrite (NiFe2O4) have shown excellent optical,
magnetic and electrical properties.8 CoFe2O4 presents excellent chemical stability9
and it is known as a hard magnetic material with high coercivity and moderate
magnetization.9 NiFe2O4, on the other hand, is a type of soft magnetic material with
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low coercivity10 and low saturation magnetization.1 Both hard and soft magnetic
nanomaterials have been proved to be promising candidates for applications such as
biosensors, drugs carriers, or contrast agents in magnetic resonance.11 However,
their widespread use has been hampered by their toxicity due to the remarkable
amount of cobalt (Co2+) or nickel (Ni2+) release in aqueous solutions, aggregation in
solution, and poor accessibility of the surface when surfactants are used during their
synthesis.12,13 Even though many studies have focused on the biomedical
applications of CoFe2O4 and NiFe2O4 NPs, the knowledge over how various physical
and chemical parameters influence their cytotoxicity remains limited.
In this paper, we investigated the effect of different calcination temperatures over
the physical and chemical features of CoFe2O4 and NiFe2O4 NPs, and how these
parameters influenced their cytotoxicity towards L929 mouse fibroblast cells. We
employed a sol-gel proteic approach to the synthesis of CoFe2O4 and NiFe2O4 NPs,
where the solution containing the precursor cations was mixed with a colorless
gelatin, forming a polymer network comprising the interconnected cations.14 The
CoFe2O4 and NiFe2O4 NPs were characterized by high-resolution electron
microscopy (HRTEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy
(XPS), vibrating sample magnetometry (VSM), and X-ray fluorescence (XRF).
Interestingly, we found that the calcination temperature can be employed as a facile
strategy to control the cytotoxicity of these systems, in which higher temperatures led
to reduction of cytotoxicity which could be assigned to the decrease of Co2+ or Ni2+
dissolution from the NPs.
Materials and methods
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Chemicals and Materials
The
commercial
reagents
Fe(NO3)3—9(H2O)
(Iron(III)
Co(NO3)2—6(H2O)
(Cobalt(II)
Gelatin
nitrate
nitrate
(40-50%
in
nonahydrate,
hexahydrate,
H2O,
Sigma-Aldrich),
>99.9%,
Sigma-Aldrich),
>98.0%,
Sigma-Aldrich),
Ni(NO3)2—6(H2O) (Nickel(II) nitrate hexahydrate, >98.0%, Sigma-Aldrich), H2O2
(Hydrogen peroxide solution, 35 wt. % in H2O, Sigma-Aldrich) were used as received
without further purification. Deionized
(18.2 MΩ) water was used throughout the
experiments.
Instrumentation
The XRD technique was used to evaluate the crystallinity and identify the
crystalline phases present in the samples. The measurements were performed at
XRD1 beamline at the National Synchrotron Light Laboratory (LNLS) in CampinasSP, Brazil. The detection system consists of a linear arc of 24 detectors, enabling a
measurement of 0.004º in step and 120° in range. The radiation passed by a
monochromator composed of double monocrystalline silicon to achieve a wavelength
of 1.033 Å.
The X-ray Photoelectron Spectroscopy experiments were performed using a
conventional Al Kα X-rays source with photon energy of 1486.7 eV. An Omicron
electron analyzer was used with 40 eV pass energy and 0.1 eV step, with an
acquisition time of 60 s / point. The total resolution was around 0.3 eV. The base
pressure in the analysis chamber was less than 5.0x10-9 mbar. The binding energy
(BE) scale was calibrated using the C 1s line at 284.6 eV as a reference. The data
were analyzed using the Winspec software. Shirley backgrounds were removed from
the data as part of the fitting process. For the XPS measurements, the powder
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samples containing the NPs were pressed to form tablets. All the samples were heat
to 100 ºC for 1 hour to remove water before being introduced into the XPS analysis
chamber.
The TEM analyses were carried out on an electron microscope operating at an
accelerating voltage of 100 kV (JEOL JEM-2100 EXII). CoFe2O4 or NiFe2O4 NPs
were dispersed in water, and their corresponding suspensions were drop cast onto a
Formvar-coated copper grid for TEM measurement. The TEM images were analyzed
using the ImageJ 1.46r software from which the size distribution histograms were
obtained.
The X-ray Fluorescence (XRF) measurements were performed on a Ray Ny
EDX-720 Shimadzo equipament. The base pressure inside the analysis chamber
was less than 2.0x10-2 mBar. The measurement was carried on both Kα and Kβ lines
for Co and Fe atoms, with 50 keV of photon energy from a Rh x-ray source, 25 mA
of emission current and a resolution of 0.2 keV.
The Ferromagnetic Resonance (FMR) measurements were performed with a
BRUKER ESP-300 spectrometer, which has a Klystron valve as source of
microwaves, operating at X-band (ν = 9.6 GHz) or Q-band (ν = 34 GHz) of
microwave frequency, whose maximum magnetic field is 16,500 Oe.
Determination of Fe, Co and Ni ions concentrations was performed by
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis (Thermo
FisheriCAPQ).
Synthesis of CoFe2O4 and NiFe2O4 NPs
The syntheses were performed by a sol-gel route. In a typical procedure, the
inorganic precursors comprising the respective metal salts, Fe(NO3)3—9(H2O),
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Co(NO3)2—6(H2O), and Ni(NO3)2—6(H2O) were mixed at a 2:1 ratio for obtaining
CoFe2O4 and NiFe2O4 NPs in a gelatin solution dissolved in distilled water, the
weight percentage of gelatin was 50 % of that of Co(NO3)2 or Ni(NO3)2.14 Then the
dissolution was kept at 40oC under vigorous magnetic stirring for 3 h.
Samples were then heated up to 100 °C in oven for approximately 24 hours.
After that, the samples were washed with water and collected by centrifugation. Then
they were washed 3 times with H2O2 to remove any residual organic matter. The
samples were then calcined at different temperatures, which corresponded to 400,
600, 800 and 1000ºC for CoFe2O4 NPs and 250, 500, 800 and 1000ºC for NiFe2O4
NPs. This was performed at a constant rate of 4 °C/min in order to preserve the
quality of the material. The calcination was carried out for 2 hours after the samples
achieved the desired temperatures, after which they were allowed to cool down
under ambient conditions.
Cell culture experiments, cytotoxicity assays and cells division assessments
The mouse fibroblast cell line (L929) was cultured in MEM medium (Gibco®;
Life Technologies, Carlsbad, CA, USA) supplemented with 10 % FBS and 100 U/mL
penicillin-streptomycin (GIBCO, CA, USA), at 37 °C and 5 % CO2 humidified
atmosphere. Cells without any exposure to nanoparticles served as controls.
Cytotoxicity was assessed by using the MTS assay (Cell Titer 96®Aqueous nonradioactive cell proliferation assay) (Promega, Madison, WI, USA). Briefly, to
evaluate mitochondrial function and cell viability of L929 cells treated with different
concentrations of CoFe2O4 and NiFe2O4 NPs (50, 100, 200, 400, 800, 1200 and 2000
µg/ml), cells were seeded at a density of 1×104 cells/well on 96-well plates, and then
treated with particles at different concentrations for 24 h. Followed by incubation with
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MTS reagent in serum-free culture medium for 3 h, absorbance at 490 nm of each
well was measured using a Microplate Reader (Multiskan GO, Thermo Fisher
Scientific; Waltham, MA, USA).
To further measure the impact of CoFe2O4 and NiFe2O4 NPs on cells division
and proliferation, the flow cytometric analysis of the fluorescence intensity of the vital
dye CFSE (carboxy fluoresce in diacetate, succinimidyI ester) was used by CFSE
Cell Division Assay Kit (Cayman Chemical, Michigan, USA) according to the
manufacturer’s instructions. CFSE consists of a fluoresce in molecule containing two
acetate moieties and a succinimidyl ester functional group. In this form, it is
membrane permeant and non-fluorescent. After diffusion into the intracellular
environment, endogenous esterases in live cells cleave the acetate groups, resulting
in the highly fluorescent molecule that is now membrane impermeant, which can be
detected by flow cytometry. In brief, CFSE labeled cells were cultured with different
concentrations of CoFe2O4 and NiFe2O4 NPs (50, 200, 800, and 2000 µg/ml) stimuli
for 24h, and then cells were harvest into FACS tubes, wash and read on a flow
cytometer (Becton Dickinson, San Jose, CA) with excitation at 488 nm and emission
at 525 nm. The mean fluorescence intensity (MFI) of 104 cells was quantified using
Cell Quest Software (Becton Dickinson).
Determination of Fe, Co and Ni ions concentrations in vitro
Briefly,
after
exposure
to
CoFe2O4
and
NiFe2O4
NPs
with
different
concentrations (50, 200, 800, and 2000 µg/ml) for 24 h, the cells-free supernatants in
the well were collected and centrifuged by 14000 r/min for 30 min to remove NPs,
and then the concentrations of Fe, Co and Ni ions were measured by ICP-MS.
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RESULTS AND DISCUSSION
We started our studies with the sol-gel proteic synthesis of CoFe2O4 and
NiFe2O4 NPs, followed by the calcination of these samples at different temperatures
in order to probe its effects over their crystallinities, particle size, surface
composition, magnetic properties, and cytotoxicity. Figures 1a and 1b show the
XRD diffractograms of the obtained CoFe2O4 and NiFe2O4 NPs, respectively, as a
function of the calcination temperature. For the CoFe2O4 NPs, the presence of broad
diffraction peaks assigned to the CoFe2O4 spinel structure was detected at 400 oC,
indicating that low crystallinity and/or small crystallite sizes. As the temperature was
increased, the intensity of the XRD peaks increased and they become narrower
because of the increase in the crystallite size as a result of sintering/agglomeration.
A similar behavior was detected for NiFe2O4 NPs (Figure 1b), in which the
appearance of crystalline phases as manifested by the presence of broad peaks in
the XRD diffractograms took place at 250 oC. Higher crystallinitie and/or larger
crystallite sizes were detected after the sample was calcined at higher temperature.
In both CoFe2O4 and NiFe2O4 NPs, the XRD data showed that they were obtained as
the only crystalline phase,15-19 and Table S1 depicts the calculated lattice
parameters from the XRD data. It is important to note that it has been reported by
Fontanive et al.15 that the CoFe2O4 NPs presented a secondary phase during the
synthesis. In this study, only one crystalline phase was observed in the samples,
which is also consistent with reported studies that only one crystalline phase was
obtained for CoFe2O4 even after calcination at 1000 °C.16
As we were particularly interested in investigating the effect of particle size and
surface composition over the cytotoxicity of the NPs, we decided to focus on
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CoFe2O4 NPs that were calcined at 400 and 800 oC; and NiFe2O4 NPs that were
calcined at 250 and 800 oC for our further studies. These samples were chosen due
to their significant differences in particle size and crystallinity, and therefore may
serve as excellent model systems to investigate the relationship between their
morphological, compositional, structural features with their cytotoxic effects.
Figures 2a and b show HRTEM images of the obtained CoFe2O4 and NiFe2O4
NPs, respectively, after calcination at 800 °C for 2 h. Their corresponding size
histograms are depicted in Figures S1a and b, respectively. These HRTEM images
indicate that the CoFe2O4 and NiFe2O4 NPs were relatively uniform, were 35 and 30
nm in diameter, respectively, and displayed a polyhedral morphology in which the
presence of surface facets could be detected (zoomed in images are shown in
Figure S2a and b). The TEM images of CoFe2O4 and NiFe2O4 NPs that were
calcined at 400 and 250 oC are shown in Figure 2c and d, respectively. Here, the
NPs sizes were smaller as compared to calcination at 800 oC, corresponding to
around 10 and 5 nm for CoFe2O4 and NiFe2O4 NPs, respectively. Phase-contrast
HRTEM images (Figure S2c and d) for these samples confirm their smaller
crystallite sizes, which is in agreement with the XRD results.
Figure S3 shows the typical XRF spectra for CoFe2O4 and NiFe2O4 NPs that
were calcined at 400 and 800 oC (CoFe2O4) and 250 and 800 oC (NiFe2O4). The
chemical compositions of the samples show that the relative ratio for the metals
corresponded to 1:2 for Co:Fe and Ni:Fe in all cases, which is in agreement with the
formation of the ferrites as pure samples.
In order to get further insights into the surface compositions of the samples as a
function of the annealing temperature, we performed XPS analyses on the CoFe2O4
and NiFe2O4 NPs that were obtained after calcination at 400 and 800 oC (CoFe2O4)
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and 250 and 800 oC (NiFe2O4). Figure 3a shows the Co 2p3/2 core-level spectra for
CoFe2O4 NPs calcined at 400 °C (top trace) and 800 °C (bottom trace). The Co 2p3/2
core-level of CoFe2O4 NPs calcined at 400 °C has 4 components with binding
energies (BE) corresponding to 778.1, 779.8, 782.5 and 786.3 eV. The BE of 779.8
and 782.5 eV were related to Co2+ ions at octahedral and tetrahedral sites,
respectively.9,20 The peak with BE of 786.3 eV corresponded to the shake-up satellite
peak of Co 2p3/2 main line.9,21 However, a weak, low-binding-energy (LBE)
component at 778.1 eV assigned to reduced Co sites was observed.9,22 The latter
could occur as a result of the presence of surface Co defect sites, or corresponding
to the low crystallization of the sample, in which the Co atoms didn’t migrate to the
most preferred crystalline sites in the lattice.9,22 This is in agreement with XRD and
HRTEM images, which shown that this sample was comprised of small NPs sizes
and broad diffraction peaks (Figures 1 and 2).
After calcination at 800 °C, the Co 2p3/2 core-level of CoFe2O4 NPs spectra also
displayed 4 components with BE of 778.1, 779.8, 782.5 and 786.3 eV. The
components present at 779.8 and 782.5 eV were associated with Co2+ ion at
octahedral and tetrahedral sites, respectively.9,20 The binding energy of 786.3 eV
corresponds to the shake-up satellite peak.9,21 A weak LBE component at 778.1 eV
corresponding to reduced Co, was also observed.9,22 However, the intensity of this
LBE component decreased as compared with CoFe2O4 NPs calcined at 400 °C,
which demonstrates the decrease in the number of Co surface defect sites, or the
migration of Co atoms to the preferred crystalline site during the annealing at high
temperature.21
The top trace of Figure 3b shows the Fe 2p core-level of CoFe2O4 NPs calcined
at 400 °C. Here, 3 components were detected. The BE of 709.4 eV (Fe 2p3/2) and
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722.9 eV (Fe 2p1/2) refers to the Fe3+ ions at octahedral site. The BE of 712.2 eV and
725.5 eV, were related to Fe3+ ions at tetrahedral sites. The high binding energy
(HBE) components with BE of 717.3 eV (Fe 2p3/2) and 731.2 eV (Fe 2p1/2) might be
the satellite shake-up structure of tetrahedral and octahedral ions.20 In this case,
satellite shake-up structures in Fe 2p core-level XPS spectrum for the tetrahedral
and octahedral ions around 717.7 eV for Fe 2p3/2 and 731.6 eV for Fe 2p1/2 have
been reported22. Interestingly, after calcination at 800 °C, no significant changes
were detected in the Fe 2p core-level of CoFe2O4 NPs (Figure 3b, bottom).
Figure3c shows the O 1s core-level of CoFe2O4 NPs calcined at 400 °C (top
trace), which has 2 components with BE of 528.0 and 530.1 eV. The component
present at 528.0 eV was attributed to bulk oxygen, and the component at 530.1 eV
corresponds to carbonate or hydroxyl groups chemically bound to the surface of the
NPs.21 After calcination at 800 oC (Figure3c, bottom trace), an inversion in the
relative peak intensities was detected, indicating more carbonate or hydroxyl groups
chemically bound to surface cations of NPs, which might be due to the stock
condition.
Figure 4 depicts the Ni 2p (Figure 4a), Fe 2p (Figure 4b), and O 1s (Figure 4c)
core-level XPS spectra for NiFe2O4 calcined at 250 and 800 oC (top and bottom
traces, respectively). The Ni 2p3/2 core-level spectrum for NiFe2O4 NPs calcined at
250 °C has 2 components (Figure 4a, top trace). The peaks with BE of 855.3 eV
refer to the Ni2+ ions in the lattice.21 Meanwhile, there is a LBE component at 852.7
eV corresponding to reduced Ni2+, which is known to occur as a result of the poor
crystallization of the sample at this temperature, where there exist the surface Ni
defect sites, or corresponding to the low crystallization of the sample, in which the Ni
atoms didn’t migrate to the most preferred crystalline sites.21 This LBE component
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disappears in the NiFe2O4 NPs calcined at 800 °C (Figure 4a, bottom trace), which
confirms the hypothesis. The peaks at 862.5 and 859.1 eV correspond to the satellite
peaks of bulk and reduced Ni2+ ions, respectively.20,21 It is noteworthy that after the
sample was calcined at 800 °C (Figure 4a, bottom trace), only one component was
detected due to the Ni2+ preferred occupation of octahedral sites. The peak present
in the BE equal to 855.2 eV and 861.3 eV refers to the Ni 2p3/2 main and satellite
peaks, respectively.20,21 Similarly to what was discussed for CoFe2O4 NPs, the
presence of 2 components for the sample calcined at low temperature was a result
of poor crystallization, which led to the formation of reduced Ni2+ sites.
The XPS spectrum of Fe 2p core-level calcined at 250 °C is depicted in Figure
4b, top trace. 3 components were identified: at BE of 709.8 eV (723.0 eV for Fe
2p1/2) and 712.8 eV (725.4 eV for Fe 2p1/2) correspond to Fe3+ ions present in the
tetrahedral and the octahedral site of spinel structure, respectively. The high binding
energy (HBE) component with BE of 717.7 eV (731.6 eV for Fe 2p1/2) might be the
satellite shake-up structure of tetrahedral and octahedral ions.23 After calcination at
800 °C (Figure 4b, bottom trace), no significant changes were observed in the Fe 2p
core-level of NiFe2O4 NPs.
The O 1s core-level XPS spectra for NiFe2O4 (Figure 4c) displayed similar
features as a function of the calcination temperature as compared to the CoFe2O4
NPs. In this case, the presence of lattice oxygen sites and carbonate or hydroxyl
groups chemically bound to surface cations of NPs were detected.
The hysteresis loops for the CoFe2O4 and NiFe2O4 NPs that were calcined at
400 and 800 oC (CoFe2O4) and 250 and 800 oC (NiFe2O4) measured using VSM at
room temperature are presented in Figure 5. In this case, a strong dependence on
the magnetic properties as a function of the calcination temperature was detected.
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For the CoFe2O4 NPs calcined at 400 °C, the saturation magnetization (Ms) and
coercivity (Hc) correspond to 1.0 emu/gand 568Oe, respectively (Figure 5a), for
CoFe2O4 NPs calcined at 400 °C. However, these values increase to 66.7 emu/g and
1010 Oe for the CoFe2O4 NPs after calcination at 800 °C (Figure 5b). For NiFe2O4
NPs, the similar variation can be observed, where the saturation magnetization (Ms)
and coercivity (Hc) change from 4.4 emu/g and 98 Oe, respectively, for the NiFe2O4
NPs calcined at 250 °C, to 32.1 emu/g and 175 Oe for the NiFe2O4 NPs calcined at
800 °C (Figure 5c and d, respectively). The variation saturation magnetization and
coercivity with respect to the calcination temperature occurs due to two reasons.
First, the increase in NPs size with higher calcination temperature leads to better
magnetic properties. This size dependent effect has been well-established in
magnetite NPs, and has been assigned in terms of the presence of magnetic dead
layer on the surface of a nanoparticle.24 Second, the annealing temperature affected
cation migration to the preferred crystalline sites and decrease the number of surface
defect sites.25 Furthermore, annealing at high temperatures can result in the
structural distortion and increase the anisotropy of the Co or Ni ions, in accordance
with large coercivities that were noticeable in the NPs samples with higher calcined
temperature.23
After the synthesis and investigation on the effect of calcination temperature
over the NPs sizes, surface composition, and magnetic properties for CoFe2O4 and
NiFe2O4 NPs with low and high crystallinities, we turned our attention to probing how
these parameters affect their in vitro cytotoxicity. In this context, cells were exposed
to different concentrations (0, 50, 100, 200, 400, 800,1200 and 2000 µg/mL) of
particles for 24 hours and cell viability was measured using the colourimetric MTS
assay, a well-established photometric method to determine the cell mitochondrial
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function as described in Figure 6. Figure 6a shows the decrease in reduction
percentage of cell viability after L929 cells had been exposed to different
concentrations of CoFe2O4 that had been calcined at 400 and 800 oC. Interestingly,
CoFe2O4 NPs that were calcined at 400 °C showed higher cytotoxic effect than that
were calcined at 800 °C across all concentrations in a dose-dependent manner. In
detail, at the concentration of 400 µg/mL CoFe2O4 that were calcined at 400 °C, a
slight decrease in cell viability was observed which reduced the percentage of viable
cells from 100% to 81%, followed by a moderate decrease to 64.4 % at the
concentration of 800 µg/mL, and a severe decrease to 26.7% at concentration of
2000 µg/mL (Figure 6a). However, no less than 80% of the viable cells was
observed even at the highest concentrations of CoFe2O4 NPs that were calcined at
800 °C (Figure 6a). Likewise, NiFe2O4 NPs that were calcined at 800 °C had no
dramatic cytotoxic effects on cell viability from 50 to 2000 µg/mL, while cell viability in
the NiFe2O4 that were calcined at 250 °C was also lower than that in the NiFe2O4
NPs calcined at 800 °C from 800 to 2000µg/mL (P<0.05) concentrations in a dosedependent manner (Figure 6b).
These results indicate that the calcination temperature, and thus crystallinity and
NPs size had a significant influence over the cytotoxicity. In this case, CoFe2O4 and
NiFe2O4 NPs calcined at lower temperatures presented higher cytotoxicity. Regaring
morphological and structural features, L929 cells treated with CoFe2O4 calcined at
400 °C or NiFe2O4 calcined at 250 °C at high concentrations displayed a large
percentage of cells showing dead cell features under the optical microscope. They
appeared bright, circular in shape, and started to detach from the bottom of the well.
When cells were treated with CoFe2O4 calcined at 800 °C or NiFe2O4 calcined at
800 °C, the majority of cells displayed features of live cells: they appeared dark,
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fibrous in shape, and adherent to the well. In these materials, the most significant
source of toxicity which could influence the cell viability was the dissolution of Co2+,
Ni2+, and Fe3+ ions from the surface of the NPs which then could interact with the
cells.12,13 The described XRD and XPS results demonstrate that the CoFe2O4 NPs
that were calcined at 400 °C and the NiFe2O4 NPs that calcined at 250 °C had more
defect sites at their surface as a result of their lower crystallinity, which was also
supported by our TEM analysis. Moreover, TEM results revealed smaller NPs sizes
for the samples calcined at lower temperatures. This leads to increased surface
areas and thus easier dissolution of ions from the NPs surface. This statement is
further supported by our ICP-MS to quantify the concentrations of Fe, Co and Ni ions.
After exposure to CoFe2O4 and NiFe2O4 NPs at different concentrations (50, 200,
800, and 2000 µg/ml) for 24 h, the cells-free supernatants were collected and
centrifuged by 14000 r/min for 30 min to remove NPs. Then the concentrations of Fe,
Co and Ni ions in the supernatant was determined by ICP-MS.
It can be observed from Figure S4 that the dissolution of Fe3+ ions is similar for
CoFe2O4 and NiFe2O4 NPs before and after calcination. The solubilities of Fe3+ ions
for CoFe2O4 NPs calcined at 250 °C were 83.92, 84.91, 83.14 and 80.95 ppb,
respectively, corresponding to supernatants treated with different concentrations of
50, 200, 800, and 2000 µg/ml, respectively. For CoFe2O4 NPs calcined at 800 °C,
the solubilities of Fe3+ ions were 73.25, 74.53, 76.20 and 72.24, respectively (Figure
S4a). Similarly, for NiFe2O4 NPs, the solubilities of Fe3+ ions were 80.75, 77.52,
80.08 and 71.16, respectively, for samples calcined at 250 °C; as well as 62.29,
65.74, 70.63 and 61.01, respectively, for samples calcined at 800 °C (Figure S4b).
The solubilities of Fe3+ ions were similar for the samples calcined at different
temperatures, and the solubilities decreased a little when calcined at higher
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temperature. Thus it notes that the source of toxicity influencing the cell viability
could not be due to the dissolution of Fe ions. The decrease of solubility with higher
calcination temperature suggests the easier dissolution of Fe ions at tetrahedral site.
However, this interesting aspect is beyond the scope of the present study.
Figure 7a show the solubilities of Co2+ ions for CoFe2O4 NPs calcined at
different temperatures. For the supernatants treated with different concentrations (50,
200, 800, and 2000 µg/ml, respectively), the solubilities of Co2+ ions for CoFe2O4
NPs calcined at 800 °C were 0.28, 0.81, 3.50 and 8.89 ppb, respectively. However,
the solubilities increased to 10.24, 33.27, 134.49 and 241.29 ppb, respectively, when
the CoFe2O4 NPs were calcined only at 400 °C. Similarly, the solubilities of Ni2+ ions
for NiFe2O4 NPs calcined at 800 °C were 0.75, 0.81, 2.25 and 9.75 ppb, respectively,
and the solubilities elevated to 2.96, 10.28, 46.65 and 122.40, respectively, for
NiFe2O4 NPs calcined at 250 °C (Figure 7b). Based on a reasonable estimate of the
photoelectron inelastic mean free path using the National Institute of Standards and
Technology (NIST) database,26 the detection depth of the XPS is limited to the first
few atomic layers of the surface, providing information on the local atomic structure
around 1.2 and 1.08 nm for Co and Ni, respectively. As demonstrated by our XRD,
TEM, and XPS results, the CoFe2O4 NPs that were calcined at 400 °C and the
NiFe2O4 NPs that calcined at 250 °C had a lower crystallinity, smaller NPs sizes, and
more defect sites at their surface. All these factors could contribute to a higher Co2+
or Ni2+ susceptibility towards dissolution from the NPs surface as compared to the
samples calcined at 800 oC,27 which resulted in a higher toxicity.
In order to further investigate the biological effects of the ferrite NPs, cell division
in L929 cells was assessed by flow cytometry. Cell division was tracked and
detected by labeling the fluoresce in related dye CFSE, which is partitioned with
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remarkable fidelity between daughter cells. As the cells divide, the Mean CFSE
intensity per cell decreases and the dye is diluted amongst the daughter cells.
Therefore, by tracking mean CFSE intensity per cell over time, cell proliferation could
be quantitated. On the 2nd day after ferrite NPs stimulation, the Mean CFSE
intensityof CFSE-labelled populations in CoFe2O4 NPs group that were calcined at
400 °C was significantly higher than that in the negative control group and the
CoFe2O4 NPs group were calcined at 800 °C, in a dose-dependent manner (Figure
8a), but no significantly differences was observed in NiFe2O4 NPs group that were
calcined at 250 °C and 800°C (Figure 8b), indicating that CoFe2O4 NPs group that
were calcined at 400 °C could effectively inhibit cells division from the dose of 200 to
2000 µg/mL. The results suggest that the solubilities of Co2+ ions could obviously
inhibit the cell division when its concentration is more than 33.3 ppb. However, cell
division is tolerant to the solubilities of Ni2+ ions even when its concentration is as
high as 122.4 ppb.
CONCLUSIONS
We described here in an investigation on the influence of the particle size,
crystallinity, and surface composition as a function of the calcination temperature in
CoFe2O4 and NiFe2O4 NPs over their cytotoxicity, which is an important parameter to
enable their biomedical applications. We started by synthetizing CoFe2O4 and
NiFe2O4 NPs by the sol-gel proteic method, followed by the calcination of the
samples at different temperatures. We then systematically evaluated and compared
CoFe2O4 and NiFe2O4 NPs presenting low crystallinities (calcined at 400 and 250 oC,
respectively) with their highly crystalline counterparts (calcined at 800 oC). In these
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systems, the increase in the calcination temperature yielded larger NPs sizes, lower
concentration of surface defect sites, and more Co or Ni atoms located at the
preferred crystalline sites in the lattice. A reduction in the cytotoxicity towards mouse
fibroblast L929 cells was detected after calcination at 800 oC relative to their
counterparts that had been calcined at 400 and 250 oC, respectively. Confirmed by
ICP-MS results, the main sources of cytotoxicity in these samples that influenced the
cell viability coming from the dissolution of Co2+ or Ni2+ ions from the surface of the
NPs. Our results suggest that calcination at higher temperatures can be employed
as an efficient strategy to reduce the cytotoxic effect in these NPs. In this case,
increased crystallinities, sizes, and reduced concentration of surface defect sites
contributed to a lower Co2+ or Ni2+ dissolution from the NPs. We believe our results
shed new insights into the various parameters that influence cytotoxicity in ferrite
nanoparticles, which may pave the way in the future for their widespread biomedical
applications.
Supporting Information:
:
Histrograms of size distribution, HRTEM images, XRF Spectra, Fe ion solubility and lattice
parameters calculated from the XRD data.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China
(21703031), Shanghai Talent Development Funding, Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) (grant number 441963/2015-3),
and Fundação de Amparo à Pesquisa do Estado de São Paulo (grant numbers
2015/21366-9 and 2015/26308-7). D.R.L. thanks the CAPES (42629) for fellowship.
J.W. thanks the funds from Donghua University for Distinguished Research Fellow.
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23. Giri, A. K.; Kirkpatrick, E. M.; Moongkhamklang, P.; Majetich, S. A.
Photomagnetism and structure in cobalt ferrite nanoparticles. Appl. Phys. Lett. 2002,
80, 2341.
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24. Kumar, E. R.; Jayaprakash, R.; Kumar, S. Effect of annealing temperature on
structural and magnetic properties of manganese substituted NiFe2O4 nanoparticles.
Materials Science in Semiconductor Processing 2014, 17, 173-177.
25. Roongtao, R.; Vittayakorn, N.; Klysubun, W.; Vittayakorn, W. C. Effect of
annealing time on the cation distribution in Mn doped CoFe2O4. Ferroelectrics 2016,
492, 43-53.
26. Powell, C. J.; Jablonski, A. Evaluation of calculated and measured electron
inelastic mean free paths near solid surfaces. J. Phys. Chem. Ref. Data 1999, 28,
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27. Wanger, C. D.; Riggs, W. M.; Davis, L. E.; Moulder, J. F.; Muilenberg, G. E.
Handbook of X-ray Photoelectron Spectroscopy 1979, Perkin-Elmer Corporation.
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Figure 1. XRD diffractograms for CoFe2O4 (a) and NiFe2O4 (b) NPs as a function of
calcination temperature.
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Figure 2. HRTEM images for CoFe2O4 NPs after calcination at 800 oC (a), NiFe2O4
NPs after calcination at 800 oC (b), CoFe2O4 NPs after calcination at 400 oC (c), and
NiFe2O4 NPs after calcination at 250 oC (d).
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Figure 3. XPS spectra of the Co 2p (a), Fe 2p (b) and O 1s (c) core level for
CoFe2O4 NPs that were calcined at 400 and 800 °C (top and bottom traces,
respectively).
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Figure 4. XPS spectra of the Ni 2p (a), Fe 2p (b) and O 1s (c) core level for NiFe2O4
NPs that were calcined at 400 and 800 °C (top and bottom traces, respectively).
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Figure 5. Hysteresis curves for CoFe2O4 NPs after calcination at 400 oC (a),
CoFe2O4 NPs after calcination at 800 oC (b), NiFe2O4 NPs after calcination at 250 oC
(c), and NiFe2O4 NPs after calcination at 800 oC (d). All the samples were analyzed
at room temperature.
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Figure 6. Cytotoxicity measured by the MTSassay for (a) CoFe2O4 NPs after
calcination at 400 and 800 °C, and (b) NiFe2O4 NPs after calcination at 250 and 800
°C. Normal cells without nanoparticles treatment served as control. Data represents
mean±SD, n =5.*p < 0.01 for CoFe2O4:800 °C or NiFe2O4:800 °C vs. control. #p <
0.01 for CoFe2O4:400 °C or NiFe2O4:250 °C vs. control.
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Figure 7. (a) Co ion solubility of CoFe2O4 calcined at 400 and 800 °C, (b) Ni ion
solubility of NiFe2O4 calcined at 250 and 800 °C.
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Figure 8. Analysing cell division using flow cytometric measurement of CFSE dye
dilution for CoFe2O4 NPs after calcination at 400 and 800 °C (a) and NiFe2O4 NPs
after calcination at 250 and 800 °C (b). As the cells divide, the Mean CFSE intensity
per cell decreases and the dye is diluted amongst the daughter cells. Normal cells
without nanoparticles treatment served as control. Data represents mean ± SD, n
=5.*p < 0.05, **p < 0.01 vs control.
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