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Monodisperse polyvinylpyrrolidone-coated CoFe2O4 nanoparticles: Synthesis, characterization and cytotoxicity study
Accepted Manuscript Title: Monodisperse polyvinylpyrrolidone-coated CoFe2 O4 nanoparticles: Synthesis, characterization and cytotoxicity study Author: Guangshuo Wang Yingying Ma Jingbo Mu Zhixiao Zhang Xiaoliang Zhang Lina Zhang Hongwei Che Yongmei Bai Junxian Hou Hailong Xie PII: DOI: Reference:
S0169-4332(16)00045-3 http://dx.doi.org/doi:10.1016/j.apsusc.2016.01.031 APSUSC 32263
To appear in:
APSUSC
Received date: Revised date: Accepted date:
11-10-2015 23-12-2015 5-1-2016
Please cite this article as: G. Wang, Y. Ma, J. Mu, Z. Zhang, X. Zhang, L. Zhang, H. Che, Y. Bai, J. Hou, H. Xie, Monodisperse polyvinylpyrrolidone-coated CoFe2 O4 nanoparticles: Synthesis, characterization and cytotoxicity study, Applied Surface Science (2016), http://dx.doi.org/10.1016/j.apsusc.2016.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Monodisperse polyvinylpyrrolidone-coated CoFe2O4 nanoparticles: Synthesis, characterization and cytotoxicity study Guangshuo Wang*, Yingying Ma*, Jingbo Mu, Zhixiao Zhang, Xiaoliang Zhang, Lina Zhang,
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Hongwei Che, Yongmei Bai, Junxian Hou, Hailong Xie
College of Equipment Manufacturing, Hebei University of Engineering, Handan, 056038, China
Correspondence to: [email protected] (Wang GS); [email protected] (Ma YY)
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*
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Abstract
In this study, monodisperse cobalt ferrite (CoFe2O4) nanoparticles were prepared successfully with
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various additions of polyvinylpyrrolidone (PVP) by sonochemical method, in which PVP served as a stabilizer and dispersant. The effects and roles of PVP on the morphology, microstructure and
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magnetic properties of the obtained CoFe2O4 were investigated in detail by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray
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photoelectron spectroscopy (XPS) and superconducting quantum interference device (SQUID). It
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was found that PVP-coated CoFe2O4 showed relatively well dispersion with narrow size distribution.
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The field-dependent magnetization curves indicated superparamagnetic behavior of PVP-coated CoFe2O4 with moderate saturation magnetization and hydrophilic character at room temperature. More importantly, the in vitro cytotoxicity testing exhibited negligible cytotoxicity of as-prepared PVP-CoFe2O4 even at the concentration as high as 150 µg/mL after 24 h treatment. Considering the superparamagnetic properties, hydrophilic character and negligible cytotoxicity, the monodisperse CoFe2O4 nanoparticles hold great potential in a variety of biomedical applications.
Keywords: CoFe2O4 nanoparticles; Polyvinylpyrrolidone-coated; Monodisperse; Sonochemical method; Superparamagnetism; Low cytotoxicity
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1. Introduction In recent years, cobalt ferrite (CoFe2O4) nanoparticles with unique properties have shown great potential in a wide range of fields, including biomedicine, hydrogenation catalysis, supercapacitor
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electrodes, environmental remediation and ferrofluids [1-5]. One of the fast developing applications of CoFe2O4 nanoparticles is in biomedical areas, such as magnetic resonance imaging contrast agents,
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targeted drug delivery, cancer hyperthermia and biological separation [6-8]. For practical purposes,
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CoFe2O4 nanoparticle surfaces must be tailored to improve biocompatibility and reduce aggregation. Without any surface modification, CoFe2O4 nanoparticles with large surface area to volume ratios
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tend to agglomerate and form larger clusters, resulting in increased particle sizes [9]. These agglomerations have strong dipole-dipole interactions and ferromagnetic behavior [10]. Clusters will
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be further magnetized when in a external magnetic field, causing a stronger attraction between the nanoparticles and consequently, creating increased aggregation and cytotoxicity. Moreover, CoFe2O4
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nanoparticles with proper surface coatings not only can avoid such agglomerations, but also can stay
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longer in circulation and are less recognized by the body’s biological particulate filters, such as the reticulo-endothelial system (RES) which is a part of the immune system that consists of the
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phagocytic cells located in the reticular connective tissue [9, 11]. Although several methods have been attempted and developed to reduce aggregation and improve biocompatability, such as the formation of a biocompatible polymeric coating on the surface of CoFe2O4 nanoparticles, using poly(ethyleneglycol) (PEG) and poly(vinyl alcohol) (PVA) [12-14], it is still a big challenge to improve size uniformity and biological safety. Except for PEG and PVA, polyvinylpyrrolidone (PVP) as coating materials has been studied by Sivakumar’s group [15]. In their study, CoFe2O4 particles were prepared by thermal treatment method, and the effects of PVP on the phase composition, morphology and magnetic properties of CoFe2O4 were investigated. However, the obtained CoFe2O4 showed extensively aggregated and lacked superparamagnetic properties and cytotoxicity test, which affect significantly the potential applications of CoFe2O4 nanoparticles in
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biomedical fields. Herein, superparamagnetic CoFe2O4 nanoparticles were prepared successfully with different concentrations of polyvinylpyrrolidone (PVP) by sonochemical method. This polymer is considered
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as a promising candidate for modification by adsorption or covalent bonding, which is expected to help solving the problems of dispersion and biocompatability for CoFe2O4 nanoparticles. Then, the
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effects and roles of PVP on the morphology, microstructure and magnetic properties of CoFe2O4 were investigated by XRD, TEM, FTIR, XPS and SQUID. In addition, the in vitro cytotoxic activity
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was investigated using MTT assays on HeLa cell lines under different sample concentrations. As
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expected, the as-prepared PVP-coated CoFe2O4 nanoparticles exhibited relatively well dispersion, superparamagnetic behavior and low cytotoxicity, indicating great potential of such functionalized
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CoFe2O4 nanoparticles in biomedical applications.
2. Experimental
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2.1. Materials
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Ferric chloride hexanydrate (FeCl3·6H2O, 98%), cobalt chloride hexahydrate (CoCl2·6H2O, 98 %), sodium hydroxide (NaOH, AR) and polyvinylpyrrolidone (PVP K30, Mn = 30000) were
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purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals were used directly without further purification.
2.2. Synthesis of PVP-coated CoFe2O4 nanoparticles The PVP-coated CoFe2O4 nanoparticles were synthesized by sonochemical method according to the following procedure. In a typical experiment, 1.6 g FeCl3·6H2O and 0.81 g CoCl2·6H2O were dissolved in 150 mL deionized water, and followed by the addition of different amounts of PVP. The mixture solution was added dropwise into 250 mL three-necked flask containing 3.5 M NaOH solution in an ultrasonic bath and allowed to sonicate for 1 h. Power and frequency of the ultrasonic bath was 100 W and 20 kHz, respectively. During the reaction, pH was maintained at 11 and the temperature rose to 80 °C. After that time, the reaction mixture was cooled to room temperature and
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the precipitate was magnetically separated, washed three times with deionized water and three times with ethanol, respectively. The prepared PVP-coated CoFe2O4 nanoparticles with PVP/FeCl3·6H2O weight ratio of 1/10, 1/1 and 2/1 were designated as PVP-CoFe2O4-1/10, PVP-CoFe2O4-1/1 and
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PVP-CoFe2O4-2/1, respectively. For comparison, bare CoFe2O4 nanoparticles were prepared by the same procedure in the absence of PVP.
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2.3. Characterization
The crystalline phase was identified by means of Rigaku Dmax-Ultima+ X-ray diffraction with
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Ni-filtered Cu/K-α radiation (λ = 0.15418 nm). The operating target voltage and the tube current
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were 40 kV and 100 mA, respectively. The transmission electron microscope (TEM) images, energy dispersive X-ray (EDX) analysis, high-resolution TEM (HRTEM) image and selected area electron
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diffraction (SAED) pattern were taken on a Tecnai G2 F20 transmission electron microscope. The TEM samples were made by placing a drop of powder solution on a carbon-coated copper grid. The
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Fourier transform infrared spectroscopy (FTIR) spectra were acquired using a Nicolet Magna 750
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spectrometer. The specimens were mixed with KBr pellet and then pressed into flakes for FTIR measurements. The X-ray photoelectron spectroscopy (XPS) spectra were performed on an Thermo
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Scientific Escalab 250Xi multifunctional imaging electron spectrometer. Magnetic properties were investigated in a Quantum Design MPMS-XL-7 superconducting quantum interference device (SQUID) from -20000 Oe to 20000 Oe at room temperature. 2.4. In vitro cytotoxicity
HeLa cell lines were obtained from the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). All cell culture related reagents were purchased from HyClone. Cells were grown in normal RPMI-1640 culture medium with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin and the cells were maintained at 37 °C in a humidified and 5 % CO2 incubator. They were routinely harvested by treatment with a trypsin-ethylene diamine tetraacetic acid (EDTA) solution (0.25 %). The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT,
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Sigma-Aldrich) assay. HeLa cells were seeded into 96-well cell-culture plates at a density of 2 × 104 cells per well and then incubated in 5 % CO2 at 37 °C for 24 h. After the medium was replaced with a fresh medium containing PVP-CoFe2O4 with different concentrations (0~150 µg/mL) and further
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incubated for 12 h and 24 h, respectively. The standard MTT assay was carried out to determine the cell viabilities relative to the control untreated cells.
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3. Results and discussions
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It is well known that ultrasonic irradiation in liquid and liquid-solid systems can produce a series of unique chemical and physical effects. When ultrasonic waves propagate through a liquid
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medium, its power will not only be a driving force for mass transfer, but it will also initiate an important phenomenon known as cavitation. That is, the nucleation, growth and collapse of bubbles
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occur as a result of transition of acoustic waves in the liquid [16]. Hence, the sonochemical method is considered as one of the most promising techniques for the synthesis of various nanoparticles. The
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advantages of this approach in the synthesis of metal oxide nanoparticles, including faster reaction
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time, more uniform size distribution and higher specific surface area, have been recognized by many research groups [17, 18]. In this study, the illustration process of PVP modification of CoFe2O4
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nanoparticles is shown in Fig. 1. As NaOH solution was added, rapid color change from light yellow to dark black occurred and CoFe2O4 nanocrystals were generated following a sonochemical mechanism in aqueous medium. During this reaction process, cobalt (II) and iron (III) ions are bound by the strong ionic bonds between the metallic ions and the amide group in a polymeric chain. PVP acts as a stabilizer for dissolved metallic salts through steric and electrostatic stabilization of the amide groups of the pyrolidine rings and the methylene groups. For comparison, the bare CoFe2O4 nanoparticles were prepared by the same procedure in the absence of PVP. The crystalline structure of the CoFe2O4 functionalized with PVP was characterized by XRD, as shown in Fig. 2. The XRD patterns of PVP-CoFe2O4 with different additions of PVP display obvious diffraction peaks of pure CoFe2O4, which indicates that the functionalization does not affect the core
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structure of cobalt ferrite. It is found that the d-spacing values of significant diffraction peaks match well with data from the JCPDS card (22-1086) for CoFe2O4. The diffraction peaks at 2θ = 18.6°, 30.4°, 35.7°, 43.4°, 53.8°, 57.2° and 62.9° can be assigned to the (111), (220), (311), (400), (422),
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(511) and (440) planes, respectively, which indicates the cubic spinel crystal structure of CoFe2O4 [1, 8]. It should be noted that an additional weak peak is observed at 2θ value of 33.1° in the XRD
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patterns of PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1, which can be attributed to the impurity phase of α-Fe2O3 [15]. Moreover, the average crystallite sizes of PVP-coated CoFe2O4 nanoparticles can be
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estimated by XRD patterns using the Scherrer Equation. The sharp diffraction peak located at 35.7o
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is chosen to calculate the crystallite size, and the estimated average crystallite sizes of bare CoFe2O4, PVP-CoFe2O4-1/10, PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1 were obtained to be 12.8 ± 1.1 nm,
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11.4 ± 1.2 nm, 10.9 ± 0.9 nm and 8.6 ± 0.7 nm, respectively. The decrease in the particle size can be attributed to the fact that the interactions between PVP coating and metal ions have inhibitory effects
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of the particle size [12, 15].
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on the growth of CoFe2O4 crystal nucleus during nucleation stage and consequently lead to reduction
The morphology and structure of bare CoFe2O4 and PVP-coated CoFe2O4 were characterized by
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TEM. The typical TEM image (Fig. 3a) indicates that the obvious agglomeration can be observed with the unmodified CoFe2O4 nanoparticles. In contrast, it can be seen clearly from Fig. 3b that the ultrafine PVP-coated CoFe2O4 nanoparticles show relatively well dispersion and homogeneous shape, with a narrow size distribution. It should be noted that the steric and electrostatic interaction between amide groups of PVP coated on the surface prevents the aggregation of CoFe2O4 nanoparticles. Moreover, the EDX spectrum (inset of Fig. 3b) shows the presence of Fe, Co, C and O elements, which further confirms the formation of CoFe2O4 in the sample. The crystal structure of the PVP-coated CoFe2O4 nanoparticles was further revealed by high-resolution TEM. Fig. 3c shows that the magnetic nanoparticles are structurally uniform with a lattice fringe spacing about 0.29 nm, which corresponds to the index of (220) reflections of CoFe2O4. In addition, SAED pattern reveals
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that the sample is satisfactory polycrystalline of cubic spinel crystal structure (Fig. 3d). The six most-distinct concentric diffraction rings from the centre can be assigned to the (111), (220), (311), (400), (511) and (440) planes of cubic spinel CoFe2O4, which agrees well with the results obtained
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from the XRD analysis (Fig. 2). The FTIR spectra of PVP and CoFe2O4 nanoparticles coated with different contents of PVP are
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shown in Fig. 4. From the IR spectra of bare CoFe2O4, the peaks at 3431 cm-1 and 1636 cm-1 are attributed to the stretching vibrations and deformed vibrations of -OH band, while the characteristic
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peak corresponding to the stretching vibration of Fe-O band appears at 592 cm-1. Compared with
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bare CoFe2O4, the absorption peaks of PVP located at 847 cm-1, 1294 cm-1 and 1461 cm-1 can be found in all the FTIR spectra of PVP-CoFe2O4. Moreover, for the samples of PVP-CoFe2O4-1/10,
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PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1, the C=O absorption peaks introduced from the PVP are observed at 1641 cm-1, 1652cm-1 and 1654 cm-1, respectively. Compared with C=O absorption peak
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located at 1657 cm-1 for PVP, the shift of characteristic peaks for PVP-coated CoFe2O4 is found with
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the increasing concentrations of PVP, which indicates that CoFe2O4 is modified via coordination interaction through carbonyl groups of PVP [19]. Overall, FTIR results reveal that the surface of the
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CoFe2O4 nanoparticles have been successfully modified with PVP polymer. Although FTIR results indicate that PVP molecule coordinates with CoFe2O4 nanoparticles via its active carbonyl groups, more detailed information regarding the surface binding situation remains important to be revealed. To fulfill this task, XPS measurements were carried out to determine the chemical composition and electronic state of PVP-CoFe2O4, as shown in Fig. 5. The wide spectrum of XPS survey is shown in Fig. 5a, from which C 1s, N 1s, O 1s, Co 2p, Fe 2p core photoionization signals and O KLL, Fe LMM Auger signals are clearly displayed. To evaluate the oxidation states of iron and cobalt in PVP-CoFe2O4 nanoparticles, the Fe 2p and Co 2p narrow scan spectra were also investigated. In the Fe 2p spectrum (Fig. 5b), there are two peaks at 711.2 eV and 724.8 eV without no obvious shakeup satellite at higher binding energy side of both main peaks, which can be assigned
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to Fe 2p3/2 and Fe 2p1/2 in CoFe2O4, respectively [20]. In the Co 2p spectrum (Fig. 5c), the binding energy peaks at 780.8 eV and 786.4 eV are corresponding to Co 2p3/2 and its shake-up satellite, while another two peaks at 796.2 eV and 802.7 eV can be attributed to Co 2p1/2 and its shake-up satellite,
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respectively. The presence of Co 2p3/2 shakeup satellite indicates that there exists a number of Co2+ oxidation states in the PVP-CoFe2O4 sample. It can be attributed to the fact that the low spin Co3+
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cations can only lead to much weaker satellite features than high spin Co2+ cations with unpaired valence 3d electron orbitals [21].
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The magnetic properties of bare CoFe2O4 and PVP-coated CoFe2O4 were measured out from
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-20000 Oe to 20000 Oe at room temperature using SQUID magnetometer, as shown in Fig. 6. It can be seen that all magnetization curves appear S-shaped over the applied magnetic field. The values of
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saturation magnetization (Ms) of bare CoFe2O4 and PVP-CoFe2O4 are 43.4 emu/g, 38.7 emu/g, 35.0 emu/g and 28.1 emu/g, respectively, which is lower than that of corresponding pure bulk CoFe2O4
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(94 emu/g) [3, 22]. The values of Ms for PVP-coated CoFe2O4 nanoparticles decrease with increasing
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the concentrations of PVP, which can be attributed to the smaller particle size and the presence of non-magnetic PVP coating. In addition, the field-dependent magnetization curves show negligible
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remanence and coercivity, indicating superparamagnetic behavior of all the samples at room temperature. Compared with sonochemical method, CoFe2O4 nanoparticles prepared by conventional chemical co-precipitation method show ferrimagnetic state with high coercivity even up to 4000 Oe [23, 24]. The feature of superparamagnetism is an important parameter for biomedical applications, which can make magnetic nanoparticles easily disperse in the solution with negligible magnetic interactions between each other and avoid magnetic clustering [6, 25]. Similarly, the hydrophilic character of the materials is another vital factor for this type of application because they can avoid being coated with plasma components and being removed from circulation rapidly [26]. As shown in the inset of Fig. 6, PVP-CoFe2O4 can be well dispersed in the deionized water and a homogeneous suspension is obtained prior to magnetic separation. When an external magnetic field was applied for
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a while, the as-prepared nanoparticles were attracted to the cuvette wall in a short time, and a nearly colorless solution was obtained. The phenomenon of manipulating superparamagnetic nanomaterials is extremely important for biomedical applications with the ability of carrying drugs, gene and
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contrast agents to targeted positions under an external magnetic field. Generally, safety and toxicity are major concerns for magnetic nanoparticles in biomedical
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applications. So far, many studies have been performed to evaluate the in vitro cytotoxic effects of different magnetic nanoparticles, and the results show that the cytotoxicity might depend on many
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factors, such as their physical form, degrees of functionalization and agglomeration state [27-29].
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The correlative toxicity investigations have revealed that the CoFe2O4 nanoparticles with appropriate surface modification show relatively low cytotoxicity [30, 31]. Herein, MTT assays were utilized to
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evaluate the cytotoxicity effect the obtained PVP-CoFe2O4 on HeLa cell lines. The cells were incubated at 37 °C for 12 h and 24 h under various sample concentrations. As shown in Fig. 7, the
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cell livability maintained greater than 80% after 24 h of treatment with PVP-CoFe2O4 even at the
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concentration as high as 150 µg/mL. The above data indicate that as-prepared PVP-coated CoFe2O4 has a low level of cytotoxicity on HeLa cells. Therefore, considering the superparamagnetic behavior,
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moderate saturation magnetization and good dispersibility, the obtained monodisperse PVP-coated CoFe2O4 nanoparticles are promising candidate for various biomedical applications.
4. Conclusions
In summary, the present work reported the preparation of PVP-coated CoFe2O4 nanoparticles using a facile and controllable sonochemical method and the structure, morphology, magnetization properties and in vitro cytotoxicity were examined in detail using different characterization methods. It was found that the PVP-coated CoFe2O4 showed well dispersion and homogeneous shape with narrow size distribution. The field-dependent magnetization curves indicated superparamagnetic behavior of the PVP-coated CoFe2O4 with moderate saturation magnetization and hydrophilic character at room temperature. More importantly, the in vitro cytotoxicity experiments exhibited 9
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negligible cytotoxicity of PVP-coated CoFe2O4 at high sample concentration after 24 h treatment. The results indicated that the monodisperse PVP-coated CoFe2O4 nanoparticles could be a promising candidate for widespread biomedical applications.
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Acknowledgements The authors gratefully acknowledge the National Natural Science Foundation of China (No.
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51405131) and Natural Science Foundation of Hebei Province (No. E2015402088 and E2015402111)
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for providing the financial support.
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Figure captions
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Fig. 1. Schematic illustration for the synthesis process of PVP-coated CoFe2O4. Fig. 2. XRD patterns of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c) and
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PVP-CoFe2O4-2/1 (d)
Fig. 3. TEM image of bare CoFe2O4 (a), TEM image (b), HRTEM image (c) and SAED pattern (d)
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of PVP-CoFe2O4-1/1. The inset of Fig. 3b shows the EDX spectrum of PVP-CoFe2O4-1/1. Fig. 4. FTIR spectra of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c),
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PVP-CoFe2O4-2/1 (d) and PVP (e)
Fig. 5. XPS spectra of survey scan (a) Fe 2p narrow scan(b) and Co 2p narrow scan (c) of
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PVP-CoFe2O4-1/1.
Fig. 6. Magnetization curves of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c),
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PVP-CoFe2O4-2/1 (d). The inset shows the dispersion and separation process of the aqueous
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(right).
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solution of PVP-CoFe2O4-1/1 in the absence (left) and presence of an external magnetic field
Fig. 7. Effect of PVP-CoFe2O4-1/1 on the viability of HeLa cells after incubation for 24 h at different concentrations.
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Highlights:
polyvinylpyrrolidone (PVP) by a facile sonochemical method.
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► Monodisperse cobalt ferrite (CoFe2O4) nanoparticles were prepared with various additions of
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► PVP-coated CoFe2O4 showed relatively well dispersion and homogeneous shape with narrow size distribution.
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► PVP-coated CoFe2O4 exhibited superparamagnetism with moderate saturation magnetization and
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hydrophilic character at room temperature.
► Negligible cytotoxicity of PVP-coated CoFe2O4 was observed even at high sample concentration
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after 24 h treatment.
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Graphical Abstract
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Title: Monodisperse polyvinylpyrrolidone-coated CoFe2O4 nanoparticles: Synthesis, characterization and cytotoxicity study
Authors: Guangshuo Wang, Yingying Ma, Jingbo Mu, Zhixiao Zhang, Xiaoliang Zhang, Lina Zhang, Hongwei Che, Yongmei Bai, Junxian Hou, Hailong Xie
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S0169-4332(16)00045-3 http://dx.doi.org/doi:10.1016/j.apsusc.2016.01.031 APSUSC 32263
To appear in:
APSUSC
Received date: Revised date: Accepted date:
11-10-2015 23-12-2015 5-1-2016
Please cite this article as: G. Wang, Y. Ma, J. Mu, Z. Zhang, X. Zhang, L. Zhang, H. Che, Y. Bai, J. Hou, H. Xie, Monodisperse polyvinylpyrrolidone-coated CoFe2 O4 nanoparticles: Synthesis, characterization and cytotoxicity study, Applied Surface Science (2016), http://dx.doi.org/10.1016/j.apsusc.2016.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Monodisperse polyvinylpyrrolidone-coated CoFe2O4 nanoparticles: Synthesis, characterization and cytotoxicity study Guangshuo Wang*, Yingying Ma*, Jingbo Mu, Zhixiao Zhang, Xiaoliang Zhang, Lina Zhang,
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Hongwei Che, Yongmei Bai, Junxian Hou, Hailong Xie
College of Equipment Manufacturing, Hebei University of Engineering, Handan, 056038, China
Correspondence to: [email protected] (Wang GS); [email protected] (Ma YY)
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*
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Abstract
In this study, monodisperse cobalt ferrite (CoFe2O4) nanoparticles were prepared successfully with
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various additions of polyvinylpyrrolidone (PVP) by sonochemical method, in which PVP served as a stabilizer and dispersant. The effects and roles of PVP on the morphology, microstructure and
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magnetic properties of the obtained CoFe2O4 were investigated in detail by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray
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photoelectron spectroscopy (XPS) and superconducting quantum interference device (SQUID). It
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was found that PVP-coated CoFe2O4 showed relatively well dispersion with narrow size distribution.
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The field-dependent magnetization curves indicated superparamagnetic behavior of PVP-coated CoFe2O4 with moderate saturation magnetization and hydrophilic character at room temperature. More importantly, the in vitro cytotoxicity testing exhibited negligible cytotoxicity of as-prepared PVP-CoFe2O4 even at the concentration as high as 150 µg/mL after 24 h treatment. Considering the superparamagnetic properties, hydrophilic character and negligible cytotoxicity, the monodisperse CoFe2O4 nanoparticles hold great potential in a variety of biomedical applications.
Keywords: CoFe2O4 nanoparticles; Polyvinylpyrrolidone-coated; Monodisperse; Sonochemical method; Superparamagnetism; Low cytotoxicity
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1. Introduction In recent years, cobalt ferrite (CoFe2O4) nanoparticles with unique properties have shown great potential in a wide range of fields, including biomedicine, hydrogenation catalysis, supercapacitor
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electrodes, environmental remediation and ferrofluids [1-5]. One of the fast developing applications of CoFe2O4 nanoparticles is in biomedical areas, such as magnetic resonance imaging contrast agents,
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targeted drug delivery, cancer hyperthermia and biological separation [6-8]. For practical purposes,
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CoFe2O4 nanoparticle surfaces must be tailored to improve biocompatibility and reduce aggregation. Without any surface modification, CoFe2O4 nanoparticles with large surface area to volume ratios
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tend to agglomerate and form larger clusters, resulting in increased particle sizes [9]. These agglomerations have strong dipole-dipole interactions and ferromagnetic behavior [10]. Clusters will
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be further magnetized when in a external magnetic field, causing a stronger attraction between the nanoparticles and consequently, creating increased aggregation and cytotoxicity. Moreover, CoFe2O4
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nanoparticles with proper surface coatings not only can avoid such agglomerations, but also can stay
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longer in circulation and are less recognized by the body’s biological particulate filters, such as the reticulo-endothelial system (RES) which is a part of the immune system that consists of the
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phagocytic cells located in the reticular connective tissue [9, 11]. Although several methods have been attempted and developed to reduce aggregation and improve biocompatability, such as the formation of a biocompatible polymeric coating on the surface of CoFe2O4 nanoparticles, using poly(ethyleneglycol) (PEG) and poly(vinyl alcohol) (PVA) [12-14], it is still a big challenge to improve size uniformity and biological safety. Except for PEG and PVA, polyvinylpyrrolidone (PVP) as coating materials has been studied by Sivakumar’s group [15]. In their study, CoFe2O4 particles were prepared by thermal treatment method, and the effects of PVP on the phase composition, morphology and magnetic properties of CoFe2O4 were investigated. However, the obtained CoFe2O4 showed extensively aggregated and lacked superparamagnetic properties and cytotoxicity test, which affect significantly the potential applications of CoFe2O4 nanoparticles in
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biomedical fields. Herein, superparamagnetic CoFe2O4 nanoparticles were prepared successfully with different concentrations of polyvinylpyrrolidone (PVP) by sonochemical method. This polymer is considered
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as a promising candidate for modification by adsorption or covalent bonding, which is expected to help solving the problems of dispersion and biocompatability for CoFe2O4 nanoparticles. Then, the
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effects and roles of PVP on the morphology, microstructure and magnetic properties of CoFe2O4 were investigated by XRD, TEM, FTIR, XPS and SQUID. In addition, the in vitro cytotoxic activity
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was investigated using MTT assays on HeLa cell lines under different sample concentrations. As
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expected, the as-prepared PVP-coated CoFe2O4 nanoparticles exhibited relatively well dispersion, superparamagnetic behavior and low cytotoxicity, indicating great potential of such functionalized
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CoFe2O4 nanoparticles in biomedical applications.
2. Experimental
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2.1. Materials
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Ferric chloride hexanydrate (FeCl3·6H2O, 98%), cobalt chloride hexahydrate (CoCl2·6H2O, 98 %), sodium hydroxide (NaOH, AR) and polyvinylpyrrolidone (PVP K30, Mn = 30000) were
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purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals were used directly without further purification.
2.2. Synthesis of PVP-coated CoFe2O4 nanoparticles The PVP-coated CoFe2O4 nanoparticles were synthesized by sonochemical method according to the following procedure. In a typical experiment, 1.6 g FeCl3·6H2O and 0.81 g CoCl2·6H2O were dissolved in 150 mL deionized water, and followed by the addition of different amounts of PVP. The mixture solution was added dropwise into 250 mL three-necked flask containing 3.5 M NaOH solution in an ultrasonic bath and allowed to sonicate for 1 h. Power and frequency of the ultrasonic bath was 100 W and 20 kHz, respectively. During the reaction, pH was maintained at 11 and the temperature rose to 80 °C. After that time, the reaction mixture was cooled to room temperature and
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the precipitate was magnetically separated, washed three times with deionized water and three times with ethanol, respectively. The prepared PVP-coated CoFe2O4 nanoparticles with PVP/FeCl3·6H2O weight ratio of 1/10, 1/1 and 2/1 were designated as PVP-CoFe2O4-1/10, PVP-CoFe2O4-1/1 and
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PVP-CoFe2O4-2/1, respectively. For comparison, bare CoFe2O4 nanoparticles were prepared by the same procedure in the absence of PVP.
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2.3. Characterization
The crystalline phase was identified by means of Rigaku Dmax-Ultima+ X-ray diffraction with
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Ni-filtered Cu/K-α radiation (λ = 0.15418 nm). The operating target voltage and the tube current
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were 40 kV and 100 mA, respectively. The transmission electron microscope (TEM) images, energy dispersive X-ray (EDX) analysis, high-resolution TEM (HRTEM) image and selected area electron
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diffraction (SAED) pattern were taken on a Tecnai G2 F20 transmission electron microscope. The TEM samples were made by placing a drop of powder solution on a carbon-coated copper grid. The
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Fourier transform infrared spectroscopy (FTIR) spectra were acquired using a Nicolet Magna 750
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spectrometer. The specimens were mixed with KBr pellet and then pressed into flakes for FTIR measurements. The X-ray photoelectron spectroscopy (XPS) spectra were performed on an Thermo
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Scientific Escalab 250Xi multifunctional imaging electron spectrometer. Magnetic properties were investigated in a Quantum Design MPMS-XL-7 superconducting quantum interference device (SQUID) from -20000 Oe to 20000 Oe at room temperature. 2.4. In vitro cytotoxicity
HeLa cell lines were obtained from the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). All cell culture related reagents were purchased from HyClone. Cells were grown in normal RPMI-1640 culture medium with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin and the cells were maintained at 37 °C in a humidified and 5 % CO2 incubator. They were routinely harvested by treatment with a trypsin-ethylene diamine tetraacetic acid (EDTA) solution (0.25 %). The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT,
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Sigma-Aldrich) assay. HeLa cells were seeded into 96-well cell-culture plates at a density of 2 × 104 cells per well and then incubated in 5 % CO2 at 37 °C for 24 h. After the medium was replaced with a fresh medium containing PVP-CoFe2O4 with different concentrations (0~150 µg/mL) and further
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incubated for 12 h and 24 h, respectively. The standard MTT assay was carried out to determine the cell viabilities relative to the control untreated cells.
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3. Results and discussions
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It is well known that ultrasonic irradiation in liquid and liquid-solid systems can produce a series of unique chemical and physical effects. When ultrasonic waves propagate through a liquid
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medium, its power will not only be a driving force for mass transfer, but it will also initiate an important phenomenon known as cavitation. That is, the nucleation, growth and collapse of bubbles
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occur as a result of transition of acoustic waves in the liquid [16]. Hence, the sonochemical method is considered as one of the most promising techniques for the synthesis of various nanoparticles. The
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advantages of this approach in the synthesis of metal oxide nanoparticles, including faster reaction
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time, more uniform size distribution and higher specific surface area, have been recognized by many research groups [17, 18]. In this study, the illustration process of PVP modification of CoFe2O4
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nanoparticles is shown in Fig. 1. As NaOH solution was added, rapid color change from light yellow to dark black occurred and CoFe2O4 nanocrystals were generated following a sonochemical mechanism in aqueous medium. During this reaction process, cobalt (II) and iron (III) ions are bound by the strong ionic bonds between the metallic ions and the amide group in a polymeric chain. PVP acts as a stabilizer for dissolved metallic salts through steric and electrostatic stabilization of the amide groups of the pyrolidine rings and the methylene groups. For comparison, the bare CoFe2O4 nanoparticles were prepared by the same procedure in the absence of PVP. The crystalline structure of the CoFe2O4 functionalized with PVP was characterized by XRD, as shown in Fig. 2. The XRD patterns of PVP-CoFe2O4 with different additions of PVP display obvious diffraction peaks of pure CoFe2O4, which indicates that the functionalization does not affect the core
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structure of cobalt ferrite. It is found that the d-spacing values of significant diffraction peaks match well with data from the JCPDS card (22-1086) for CoFe2O4. The diffraction peaks at 2θ = 18.6°, 30.4°, 35.7°, 43.4°, 53.8°, 57.2° and 62.9° can be assigned to the (111), (220), (311), (400), (422),
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(511) and (440) planes, respectively, which indicates the cubic spinel crystal structure of CoFe2O4 [1, 8]. It should be noted that an additional weak peak is observed at 2θ value of 33.1° in the XRD
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patterns of PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1, which can be attributed to the impurity phase of α-Fe2O3 [15]. Moreover, the average crystallite sizes of PVP-coated CoFe2O4 nanoparticles can be
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estimated by XRD patterns using the Scherrer Equation. The sharp diffraction peak located at 35.7o
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is chosen to calculate the crystallite size, and the estimated average crystallite sizes of bare CoFe2O4, PVP-CoFe2O4-1/10, PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1 were obtained to be 12.8 ± 1.1 nm,
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11.4 ± 1.2 nm, 10.9 ± 0.9 nm and 8.6 ± 0.7 nm, respectively. The decrease in the particle size can be attributed to the fact that the interactions between PVP coating and metal ions have inhibitory effects
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of the particle size [12, 15].
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on the growth of CoFe2O4 crystal nucleus during nucleation stage and consequently lead to reduction
The morphology and structure of bare CoFe2O4 and PVP-coated CoFe2O4 were characterized by
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TEM. The typical TEM image (Fig. 3a) indicates that the obvious agglomeration can be observed with the unmodified CoFe2O4 nanoparticles. In contrast, it can be seen clearly from Fig. 3b that the ultrafine PVP-coated CoFe2O4 nanoparticles show relatively well dispersion and homogeneous shape, with a narrow size distribution. It should be noted that the steric and electrostatic interaction between amide groups of PVP coated on the surface prevents the aggregation of CoFe2O4 nanoparticles. Moreover, the EDX spectrum (inset of Fig. 3b) shows the presence of Fe, Co, C and O elements, which further confirms the formation of CoFe2O4 in the sample. The crystal structure of the PVP-coated CoFe2O4 nanoparticles was further revealed by high-resolution TEM. Fig. 3c shows that the magnetic nanoparticles are structurally uniform with a lattice fringe spacing about 0.29 nm, which corresponds to the index of (220) reflections of CoFe2O4. In addition, SAED pattern reveals
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that the sample is satisfactory polycrystalline of cubic spinel crystal structure (Fig. 3d). The six most-distinct concentric diffraction rings from the centre can be assigned to the (111), (220), (311), (400), (511) and (440) planes of cubic spinel CoFe2O4, which agrees well with the results obtained
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from the XRD analysis (Fig. 2). The FTIR spectra of PVP and CoFe2O4 nanoparticles coated with different contents of PVP are
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shown in Fig. 4. From the IR spectra of bare CoFe2O4, the peaks at 3431 cm-1 and 1636 cm-1 are attributed to the stretching vibrations and deformed vibrations of -OH band, while the characteristic
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peak corresponding to the stretching vibration of Fe-O band appears at 592 cm-1. Compared with
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bare CoFe2O4, the absorption peaks of PVP located at 847 cm-1, 1294 cm-1 and 1461 cm-1 can be found in all the FTIR spectra of PVP-CoFe2O4. Moreover, for the samples of PVP-CoFe2O4-1/10,
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PVP-CoFe2O4-1/1 and PVP-CoFe2O4-2/1, the C=O absorption peaks introduced from the PVP are observed at 1641 cm-1, 1652cm-1 and 1654 cm-1, respectively. Compared with C=O absorption peak
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located at 1657 cm-1 for PVP, the shift of characteristic peaks for PVP-coated CoFe2O4 is found with
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the increasing concentrations of PVP, which indicates that CoFe2O4 is modified via coordination interaction through carbonyl groups of PVP [19]. Overall, FTIR results reveal that the surface of the
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CoFe2O4 nanoparticles have been successfully modified with PVP polymer. Although FTIR results indicate that PVP molecule coordinates with CoFe2O4 nanoparticles via its active carbonyl groups, more detailed information regarding the surface binding situation remains important to be revealed. To fulfill this task, XPS measurements were carried out to determine the chemical composition and electronic state of PVP-CoFe2O4, as shown in Fig. 5. The wide spectrum of XPS survey is shown in Fig. 5a, from which C 1s, N 1s, O 1s, Co 2p, Fe 2p core photoionization signals and O KLL, Fe LMM Auger signals are clearly displayed. To evaluate the oxidation states of iron and cobalt in PVP-CoFe2O4 nanoparticles, the Fe 2p and Co 2p narrow scan spectra were also investigated. In the Fe 2p spectrum (Fig. 5b), there are two peaks at 711.2 eV and 724.8 eV without no obvious shakeup satellite at higher binding energy side of both main peaks, which can be assigned
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to Fe 2p3/2 and Fe 2p1/2 in CoFe2O4, respectively [20]. In the Co 2p spectrum (Fig. 5c), the binding energy peaks at 780.8 eV and 786.4 eV are corresponding to Co 2p3/2 and its shake-up satellite, while another two peaks at 796.2 eV and 802.7 eV can be attributed to Co 2p1/2 and its shake-up satellite,
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respectively. The presence of Co 2p3/2 shakeup satellite indicates that there exists a number of Co2+ oxidation states in the PVP-CoFe2O4 sample. It can be attributed to the fact that the low spin Co3+
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cations can only lead to much weaker satellite features than high spin Co2+ cations with unpaired valence 3d electron orbitals [21].
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The magnetic properties of bare CoFe2O4 and PVP-coated CoFe2O4 were measured out from
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-20000 Oe to 20000 Oe at room temperature using SQUID magnetometer, as shown in Fig. 6. It can be seen that all magnetization curves appear S-shaped over the applied magnetic field. The values of
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saturation magnetization (Ms) of bare CoFe2O4 and PVP-CoFe2O4 are 43.4 emu/g, 38.7 emu/g, 35.0 emu/g and 28.1 emu/g, respectively, which is lower than that of corresponding pure bulk CoFe2O4
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(94 emu/g) [3, 22]. The values of Ms for PVP-coated CoFe2O4 nanoparticles decrease with increasing
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the concentrations of PVP, which can be attributed to the smaller particle size and the presence of non-magnetic PVP coating. In addition, the field-dependent magnetization curves show negligible
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remanence and coercivity, indicating superparamagnetic behavior of all the samples at room temperature. Compared with sonochemical method, CoFe2O4 nanoparticles prepared by conventional chemical co-precipitation method show ferrimagnetic state with high coercivity even up to 4000 Oe [23, 24]. The feature of superparamagnetism is an important parameter for biomedical applications, which can make magnetic nanoparticles easily disperse in the solution with negligible magnetic interactions between each other and avoid magnetic clustering [6, 25]. Similarly, the hydrophilic character of the materials is another vital factor for this type of application because they can avoid being coated with plasma components and being removed from circulation rapidly [26]. As shown in the inset of Fig. 6, PVP-CoFe2O4 can be well dispersed in the deionized water and a homogeneous suspension is obtained prior to magnetic separation. When an external magnetic field was applied for
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a while, the as-prepared nanoparticles were attracted to the cuvette wall in a short time, and a nearly colorless solution was obtained. The phenomenon of manipulating superparamagnetic nanomaterials is extremely important for biomedical applications with the ability of carrying drugs, gene and
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contrast agents to targeted positions under an external magnetic field. Generally, safety and toxicity are major concerns for magnetic nanoparticles in biomedical
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applications. So far, many studies have been performed to evaluate the in vitro cytotoxic effects of different magnetic nanoparticles, and the results show that the cytotoxicity might depend on many
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factors, such as their physical form, degrees of functionalization and agglomeration state [27-29].
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The correlative toxicity investigations have revealed that the CoFe2O4 nanoparticles with appropriate surface modification show relatively low cytotoxicity [30, 31]. Herein, MTT assays were utilized to
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evaluate the cytotoxicity effect the obtained PVP-CoFe2O4 on HeLa cell lines. The cells were incubated at 37 °C for 12 h and 24 h under various sample concentrations. As shown in Fig. 7, the
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cell livability maintained greater than 80% after 24 h of treatment with PVP-CoFe2O4 even at the
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concentration as high as 150 µg/mL. The above data indicate that as-prepared PVP-coated CoFe2O4 has a low level of cytotoxicity on HeLa cells. Therefore, considering the superparamagnetic behavior,
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moderate saturation magnetization and good dispersibility, the obtained monodisperse PVP-coated CoFe2O4 nanoparticles are promising candidate for various biomedical applications.
4. Conclusions
In summary, the present work reported the preparation of PVP-coated CoFe2O4 nanoparticles using a facile and controllable sonochemical method and the structure, morphology, magnetization properties and in vitro cytotoxicity were examined in detail using different characterization methods. It was found that the PVP-coated CoFe2O4 showed well dispersion and homogeneous shape with narrow size distribution. The field-dependent magnetization curves indicated superparamagnetic behavior of the PVP-coated CoFe2O4 with moderate saturation magnetization and hydrophilic character at room temperature. More importantly, the in vitro cytotoxicity experiments exhibited 9
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negligible cytotoxicity of PVP-coated CoFe2O4 at high sample concentration after 24 h treatment. The results indicated that the monodisperse PVP-coated CoFe2O4 nanoparticles could be a promising candidate for widespread biomedical applications.
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Acknowledgements The authors gratefully acknowledge the National Natural Science Foundation of China (No.
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51405131) and Natural Science Foundation of Hebei Province (No. E2015402088 and E2015402111)
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for providing the financial support.
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287-292.
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Figure captions
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Fig. 1. Schematic illustration for the synthesis process of PVP-coated CoFe2O4. Fig. 2. XRD patterns of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c) and
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PVP-CoFe2O4-2/1 (d)
Fig. 3. TEM image of bare CoFe2O4 (a), TEM image (b), HRTEM image (c) and SAED pattern (d)
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of PVP-CoFe2O4-1/1. The inset of Fig. 3b shows the EDX spectrum of PVP-CoFe2O4-1/1. Fig. 4. FTIR spectra of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c),
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PVP-CoFe2O4-2/1 (d) and PVP (e)
Fig. 5. XPS spectra of survey scan (a) Fe 2p narrow scan(b) and Co 2p narrow scan (c) of
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PVP-CoFe2O4-1/1.
Fig. 6. Magnetization curves of CoFe2O4 (a), PVP-CoFe2O4-1/10 (b), PVP-CoFe2O4-1/1 (c),
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PVP-CoFe2O4-2/1 (d). The inset shows the dispersion and separation process of the aqueous
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(right).
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solution of PVP-CoFe2O4-1/1 in the absence (left) and presence of an external magnetic field
Fig. 7. Effect of PVP-CoFe2O4-1/1 on the viability of HeLa cells after incubation for 24 h at different concentrations.
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Fig. 1.
Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 7.
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Highlights:
polyvinylpyrrolidone (PVP) by a facile sonochemical method.
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► Monodisperse cobalt ferrite (CoFe2O4) nanoparticles were prepared with various additions of
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► PVP-coated CoFe2O4 showed relatively well dispersion and homogeneous shape with narrow size distribution.
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► PVP-coated CoFe2O4 exhibited superparamagnetism with moderate saturation magnetization and
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hydrophilic character at room temperature.
► Negligible cytotoxicity of PVP-coated CoFe2O4 was observed even at high sample concentration
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after 24 h treatment.
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Graphical Abstract
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Title: Monodisperse polyvinylpyrrolidone-coated CoFe2O4 nanoparticles: Synthesis, characterization and cytotoxicity study
Authors: Guangshuo Wang, Yingying Ma, Jingbo Mu, Zhixiao Zhang, Xiaoliang Zhang, Lina Zhang, Hongwei Che, Yongmei Bai, Junxian Hou, Hailong Xie
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