Employing Calcination as a Facile Strategy to Reduce the Cytotoxicity

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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

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: [email protected] (J.W.) and [email protected] (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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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)39(H2O)

(Iron(III)

Co(NO3)26(H2O)

(Cobalt(II)

Gelatin

nitrate nitrate

(40-50%

in

nonahydrate, hexahydrate,

H2O,

Sigma-Aldrich),

>99.9%,

Sigma-Aldrich),

>98.0%,

Sigma-Aldrich),

Ni(NO3)26(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)39(H2O),


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Co(NO3)26(H2O), and Ni(NO3)26(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

Employing Calcination as a Facile Strategy to Reduce the Cytotoxicity

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