Synthesis and cytotoxicity study of magnesium ferrite-gold core-shell nanoparticles

Materials Science and Engineering C 61 (2016) 123–132

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Synthesis and cytotoxicity study of magnesium ferrite-gold core-shell nanoparticles Jeeranan Nonkumwong a, Phakkhananan Pakawanit b, Angkana Wipatanawin c, Pongsakorn Jantaratana d, Supon Ananta b, Laongnuan Srisombat a,⁎ a

Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Physics, Faculty of Science, Kasetsart University, Bangkok 11900, Thailand b c

a r t i c l e

i n f o

Article history: Received 27 August 2015 Received in revised form 11 November 2015 Accepted 10 December 2015 Available online 11 December 2015 Keywords: Magnesium ferrite Gold Core-shell Nanoparticles Magnetic properties Cytotoxicity

a b s t r a c t In this work, the core-magnesium ferrite (MgFe2O4) nanoparticles were prepared by hydrothermal technique. Completed gold (Au) shell coating on the surfaces of MgFe2O4 nanoparticles was obtained by varying core/ shell ratios via a reduction method. Phase identification, morphological evolution, optical properties, magnetic properties and cytotoxicity to mammalian cells of these MgFe2O4 core coated with Au nanoparticles were examined by using a combination of X-ray diffraction, scanning electron microscopy, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, UV–visible spectroscopy (UV–vis), vibrating sample magnetometry and resazurin microplate assay techniques. In general, TEM images revealed different sizes of the core-shell nanoparticles generated from various core/shell ratios and confirmed the completed Au shell coating on MgFe2O4 core nanoparticles via suitable core/shell ratio with particle size less than 100 nm. The core-shell nanoparticle size and the quality of coating influence the optical properties of the products. The UV–vis spectra of complete coated MgFe2O4-Au core-shell nanoparticles exhibit the absorption bands in the near-Infrared (NIR) region indicating high potential for therapeutic applications. Based on the magnetic property measurement, it was found that the obtained MgFe2O4-Au core-shell nanoparticles still exhibit superparamagnetism with lower saturation magnetization value, compared with MgFe2O4 core. Both of MgFe2O4 and MgFe2O4-Au core-shell also showed in vitro non-cytotoxicity to mouse areola fibroblast (L-929) cell line. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Recently, there have been enormous publications related to magnetic nanoparticle-based hyperthermia materials [1] due to heat generating ability of these filled nanoparticles in tumor cells when applying alternating current magnetic field from external coils [2]. Specific magnetic properties like superparamagnetism and defined size of magnetic nanoparticles (MNPs) are important properties for use in this biomedical application to create heating spot for destroying the tumor cells and/or to release the drugs either attached to the MNPs or embedded inside the matrix of thermosensitive polymer matrices at the target area [3]. In addition, superparamagnetism prevents the aggregation of MNPs [4]. The MNPs size also plays an important role in the penetration to target tissue (cancerous tissue) through small vascular pores between tumor endothelial cells of blood vessels [4] and in the uptake by phagocytes system when the MNPs are too large [5]. Due to these important criterions, the total size of MNPs should be smaller than ⁎ Corresponding author. E-mail address: [email protected] (L. Srisombat).

http://dx.doi.org/10.1016/j.msec.2015.12.021 0928-4931/© 2015 Elsevier B.V. All rights reserved.

100 nm [5–7]. In addition, biocompatibility improvement of synthesized MNPs must be considered in order to reduce toxicity for healthy cells when the MNPs are injected into the patient body. Consequently, surface modification or coating of the synthesized MNPs by biocompatible materials should be considered. From our previous work [8], we successfully synthesized MgFe2O4 nanoparticles (MgFe2O4 NPs), one kind of the soft magnetic materials [9], by hydrothermal technique with superparamagnetism and the smallest size of about 65 nm. Finding out the optimal method to synthesize the biocompatible MgFe2O4 NPs followed by characterization for further utilization is therefore interesting. Moreover, core-shell systems or core-shell structures are challenging due to their multifunctionality originated from combination of different properties generated by core and shell (i.e. different material compositions can be accumulated into single particles) [3]. So far, there are many kinds of shell materials normally be used to coat MNPs, such as silica [10] and gold nanoparticles (AuNPs) [11]. Even though, both of them are biocompatible but silica could affect the saturation magnetization value of MNPs because of the diamagnetic contribution of the thick silica shell [12]. Thus, the AuNPs seem to be a better choice for coating on MNPs because AuNPs are inert and non-

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toxic [13], ease of synthesis [14], ready functionalization via amine/thiol linkages [15,16] and have photophysical properties that could stimulate drug release at remote area [17]. In general, tissue and blood are transparent in the NIR electromagnetic (EM) spectrum. Thus, some gold shells which can display absorbance properties at this EM region will be the possible candidates for the applications of therapeutics [18, 19]. In connection with this, many researchers have focused on synthesis of Fe3O4-Au core-shell NPs with appropriate properties for drug delivery and hyperthermia treatment applications [20–22]. However, to date, the potential of MgFe2O4-Au core-shell NPs for these applications is not available in literatures. Similar to other gold-coated magnetic nanoparticles [23,24], gold shells are considered as oxidation and corrosion protecting layers for MgFe2O4 core, exhibiting good biocompatibility and providing further functionalization on their surfaces. Where superparamagnetic MgFe2O4 provides magnetic inducibility facilitated for delivery and hyperthermia properties converted from incident alternating magnetic field, AuNPs also could absorb the incident energy from alternating magnetic field, NIR light, etc. and then convert the energy to heat. Thus, a combination of MgFe2O4 and Au in the form of core-shell structure is promising as high performance hyperthermia materials and/or drug release materials induced from thermosensitive encapsulating polymer degradation [3,20]. In addition, some related works had been only focused on using MgFe2O4 NPs as supporting materials, due to their capability of magnetic separation, for AuNPs decorating in case of catalytic application [25,26]. In continuation of our prior studies where we had successfully synthesized MgFe2O4 NPs as mentioned earlier, the aim of the present work therefore is to prepare MgFe2O4-Au NPs with core-shell structure and then investigate their physical, chemical and biological properties. A combination of X-ray diffraction, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, UV–visible spectroscopy, vibrating sample magnetometry and resazurin microplate assay techniques were used to reveal phase identification, morphological evolution, chemical compositions, optical properties, magnetic properties and in vitro cytotoxicity to mammalian cells (L-929) of the synthesized core-shell nanoparticles as a function of core/shell ratio.

2.2. Synthesis of MgFe2O4-Au core-shell NPs A schematic illustration of the synthesis is provided in Fig. 1. The details of synthesis are described as following sections.

2.2.1. Surface modification of MgFe2O4 NPs 0.05 g of synthesized MgFe2O4 NPs obtained from 2.1 was dispersed in 50 ml of absolute ethyl alcohol (J.T. Baker, 99.9% purity) in threenecked round bottom flask. After N2 gas flow for 15 min, 0.5 ml of 3Aminopropyltriethoxysilane, APTES (Sigma-Aldrich, 99% purity) was slowly added to the suspension. The mixture was refluxed at 70 °C for 1.5 h then cooled to room temperature. The precipitates were collected by centrifugation and washed at least 2 times with absolute ethyl alcohol. The final product was kept in 10 ml of absolute ethyl alcohol. The Fourier transform infrared spectroscopy (FTIR; Bruker TENSOR 27) was used to characterize the functionalization of APTES on the surface of MgFe2O4 NPs.

2.2.2. Attachment of AuNPs on surface modified MgFe2O4 NPs Small colloidal AuNPs with average size ~2 nm were synthesized by using Duff and Baiker's method [30]. Briefly, 0.5 ml of 1 M NaOH (Ajax Finechem, 99% purity) and 1.0 ml of 0.067 M Tetrakis (hydroxymethyl) phosphonium chloride, THPC solution (Sigma-Aldrich, 80% in water) were added into 45 ml of deionized water. The solution was stirred for 5 min and then 2.00 ml of 1% HAuCl 4 (Sigma-Aldrich, 99.999% purity) was added and continuously stirred for 30 min providing aqueous AuNPs. To attach small colloidal AuNPs on MgFe2 O 4 NPs, 0.5 ml of surface modified MgFe 2O 4 NPs obtained from 2.2.1 was added into 30 ml of the as-synthesized aqueous AuNPs. The mixture was left overnight and then centrifuged for 10 min. The precipitates were washed once by centrifugation and redispersed into 25 ml with deionized water, defined as MgFe2 O 4-Au NPs. UV–visible spectroscopy (UV–vis; PerkinElmer LAMBDA 25) was employed to monitor the optical properties of the MgFe2 O 4-Au NPs. A combination of the SEM, TEM and EDX techniques was performed to reveal their morphologies and chemical compositions.

2. Experimental 2.1. Synthesis of MgFe2O4 NPs In this work, pure phase of the MgFe2O4 NPs with superparamagnetism and smallest average size (65 ± 8 nm), as reported earlier [8] was synthesized by using a hydrothermal method [27]. In brief, 1 mmol of Mg(NO3)2 ⋅6H2O (Loba Chemie, 99% purity) and 2 mmol of Fe(NO3)3 ⋅9H2O (Carlo Erba, 98% purity) were dissolved in 30 mL ethylene glycol (Carlo Erba, 98% purity). The 15 mmol of CH3COONa (Loba Chemie, 99.5% purity) was added into the mixture solution and then transferred to a Teflon-lined stainless steel autoclave (HP series 5500 compact reactor). After heating inside the autoclave at 180 °C for 14 h [8], the precipitates were collected by magnetic separator, washed three times with deionized water, another three times with ethyl alcohol and dried at 70 °C for 12 h [28]. A combination of X-ray diffraction (XRD; Bruker D2 phaser diffractometer) technique, scanning electron microscopy (SEM; JEOL JSM6335F), transmission electron microscopy (TEM; JEOL JEM-2010) and energy-dispersive X-ray (EDX) analyzer was used to reveal their phases, morphologies, selected area electron diffraction (SAED) patterns and chemical compositions of the obtained products. Finally, magnetization measurements were carried out at room temperature using an in-house developed vibrational sample magnetometer (VSM). Crystallinity degree by XRD amorphous subtraction method is calculated as the ratio between the area of the crystalline contribution and the total area (crystalline + amorphous) [29].

2.2.3. Growth of Au shell The MgFe2O4-Au NPs cores were coated with the Au shell by following Pham et al. work [31]. The K-gold solution was used as gold source. The K-gold solution was prepared by dissolving 0.18 mmol of K2CO3 (Fisher Scientific, 99.87% purity) in 100 ml of deionized water. After stirring the solution for 10 min, 2.00 ml of 1% HAuCl4 was added into the stirred solution. The solution mixture changed from yellow to colorless within 30 min. Various amounts of MgFe2O4-Au NPs core were then mixed with the 4 ml of K-gold solution by using homemade mechanical stirrer, following by adding 20 μl of formaldehyde (Ajax Finechem, 38% w/w). In order to optimize the preparation condition of shell coating, various amounts of MgFe2O4-AuNPs core i.e. 0.05, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 ml were designed. The mixture color changes from yellow brown to purple within 15 min. The mixture was stirred for 30 min with final color in red to red brown. The precipitates were separated by magnetic separation and washed twice by deionized water. The final product was redispersed in deionized water to give black colloidal MgFe2O4-Au core-shell NPs. XRD technique was performed for phase identification. A combination of the SEM, TEM and EDX techniques was employed to identify their morphologies, SAED patterns and estimated chemical compositions of the products. Room temperature magnetization measurements were carried out using an in-house developed VSM. UV–vis technique was carried out to reveal the optical properties of the samples.

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Fig. 1. A Schematic illustration for synthesis of MgFe2O4-Au core-shell NPs.

2.3. Cytotoxicity of MgFe2O4 and MgFe2O4-Au core-shell NPs in mouse areola fibroblast cell line (NCTC clone 929; L-929) by resazurin microplate assay (REMA) L-929 cells were cultivated in Dulbecco's Modified Eagle's Medium, DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 3.7 g/L sodium bicarbonate (Sigma-Aldrich, 99.5% purity) and 100 unit/ml penicillin-100μg/ml streptomycin (Gibco) and incubated at 37 °C humidified incubator with 5% CO2. Cells at a logarithmic growth were harvested and diluted to 1 × 104 cells/ml in medium prior to assessing cytotoxicity by REMA [32]. First, 96-well plates were seeded with 200 μl of cell suspension or blank medium into well, and incubated at 37 °C humidified incubator with 5% CO2 for 48 h. Subsequently, culture medium was replaced with 200 μl of fresh medium containing MgFe2O4 NPs and MgFe2O4-Au core-shell NPs (test-compounds) at different concentrations (15.6– 500.0 μg/ml), and plates were further incubated for 24 h. After incubation period, the plates was added with 50 μl of 125 μg/ml resazurin solution (Sigma-Aldrich, 75% purity) and incubated at 37 °C humidified incubator with 5% CO2 for 4 h. Fluorescence is measured at 530 nm excitation and 590 nm emission wavelengths by using the bottom-reading mode of fluorometer (PerkinElmer VICTOR3 V Multilabel Plate Counter model 1420). The signal is subtracted with the blank before calculation. Three independent experiments for each assay were carried out in four replicates in order to obtain the precise results. The cell relative growth rate

(RGR) and the percentage of survival (%survival) of cells are calculated by the following equations: RGR ¼ FUT =FUC

ð1Þ

%Survival ¼ ðFUT =FUC Þ  100

ð2Þ

Whereas FUT and FUC are the mean fluorescent unit from cells treated with test-compound and that treated with DMEM (negative control), respectively. The RGR value can be interpreted to cytotoxic activity by these cutoff criteria: 1) if the RGR value fits in grade 0 (RGR value ≥1) or grade 1 (RGR value = 0.75–0.99), the activity is reported as “non-cytotoxic” and 2) if the RGR value fits in grade 2 (RGR value = 0.50–0.74), the activity may be reported as “cytotoxic” considering with the morphological imaging of cell death [21]. 3. Results and discussion The powder XRD pattern of MgFe2O4 NPs is shown in Fig. 2. The result indicated that all of the main peaks are indexed as the spinel MgFe2O4, which could be matched with the JCPDS file no. 88-1935 [33] with 100% crystallinity. To be able to making shell on the MgFe2O4 NPs, the surfaces of these NPs have to be modified to be suitable for the attachment of Au. The FTIR technique is known as a technique to confirm the attachment of ligand on the surface of magnetic

Fig. 2. XRD patterns of MgFe2O4 NPs compared with representative MgFe2O4-Au core-shell NPs.

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Fig. 3. FTIR spectra of unmodified and modified surface of MgFe2O4 NPs by APTES.

nanoparticles [34]. From the FTIR spectra of surface unmodified and modified MgFe2O4 NPs by APTES shown in Fig. 3, it is seen that two peaks at 441 and 596 cm− 1 in the spectrum of unmodified surface MgFe2O4 NPs indicate the formation of Fe–O stretching mode from MgFe2O4 which corresponds to spinel ferrite structure reported by Gherca et al. [35]. Surface modified and unmodified MgFe2O4 NPs exhibit FTIR peaks at 1046 cm−1 which is best described by the Si–O–

Si and/or C–O stretching (from ethylene glycol). Also, the vibrational mode at 3440 cm−1 could be attributed to N–H stretching for surface modified MgFe2O4 NPs and/or O–H stretching from moisture presented in both surface modified and unmodified MgFe2O4 NPs. It is clearly seen that only the functionalized MgFe2O4 NPs show the vibrational modes of N–H bending at 1516 cm−1. This observation indicates the existence of APTES on the MgFe2O4 NPs surfaces, consistent with the

Fig. 4. SEM images of (a) MgFe2O4 NPs, (b) AuNPs attached MgFe2O4 NPs, MgFe2O4-Au core-shell NPs with (c) 0.05, (d) 0.2, (e) 0.3, (f) 0.4, (g) 0.5, (h) 1.0 and (i) 2.0 ml of MgFe2O4-Au NPs core.

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previous study on APTES functionalized BaTiO3 [36] and Fe3O4 [34,37] nanoparticles. The morphological evolution of MgFe2O4 core (~ 65 nm), AuNPs attached MgFe2O4 NPs and MgFe2O4-Au core-shell NPs as a function of MgFe2O4 core concentration are shown in Figs. 4 and 5. It can be observed that contrast of TEM images increased when AuNPs had been attached on MgFe2O4 core (Fig. 5(b)) compared with bare MgFe2O4 core (Fig. 5(a)). This observation could be determined by the high electron density of Au [38]. Moreover, it is obvious that a lot of small particles dispersed on MgFe2O4 surface with comparable size of MgFe2O4 NPs as shown in Fig. 5(b) and also the inset. Growth of Au shell on MgFe2O4 core provides changing to bigger size of particles, indicated in Figs. 4(c–i) and 5(c–i). MgFe2O4-Au core-shell NPs with 0.05, 0.5, 1.0 and 2.0 ml of core contents demonstrate the significant degree of particle's aggregation (Fig. 4(c) and (g–i)), resulting in the cluster size larger than 100 nm. For MgFe2O4-Au core-shell NPs with 0.2, 0.3 and 0.4 ml of colloidal core NPs, the average particle size is around 79, 72 and 65, respectively. These three conditions were therefore optimal for shell coating due to providing the total size of particles smaller than 100 nm as aimed. In addition, it is seen that the MgFe2O4Au core-shell NPs with 1.0 and 2.0 ml cores exhibit some incomplete coated particles or defined as incomplete coated MgFe2O4-Au coreshell NPs (Fig. 5(h,i)). On the other hand, the complete surface coating is found for these MgFe2O4-Au core-shell NPs with 0.05, 0.2, 0.3, 0.4 and 0.5 ml of colloidal core NPs. Based on the obtained SEM and TEM results, the MgFe2O4-Au core-shell NPs with 0.2, 0.3 or 0.4 ml of colloidal cores were selected for further characterizations. As shown in Fig. 2, only Au (JCPDS file no. 04-0784 [39]) with 100% crystallinity was indexable for MgFe2O4-Au core-shell NPs. This could be due to the sufficient thickness of Au shell coating [40,41]. In order to confirm the co-existence of MgFe2O4 and Au in MgFe2O4-Au core-

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shell NPs with complete surface coating, the comparison SEAD patterns of MgFe2O4 NPs, AuNPs attached MgFe2O4 NPs, complete and incomplete coated MgFe2O4-Au core-shell NPs are performed, as shown in Fig. 6. The results show that when AuNPs attached on MgFe2O4 NPs, the SAED pattern (Fig. 6(b)) still exhibit MgFe2O4 similar to SAED pattern of MgFe2O4 NPs (Fig. 6(a)) without the existence of Au phase. This observation is attributed to the small amount of AuNPs in comparison with MgFe2O4. When the growth of Au shell is increased, the SAED patterns of both complete (Fig. 6(c)) and incomplete (Fig. 6(d)) coated MgFe2O4-Au core-shell NPs show only Au phase without MgFe2O4 phase. This could be explained that the interaction volume of incident electron beam to generate X-ray decreases with an increasing in atomic number due to backscattering process [42]. Therefore, unlike the case of AuNPs attached MgFe2O4 NPs, the results of MgFe2O4-Au core-shell NPs, with larger amount of Au contents tend to exhibit Au predominantly. EDX analysis also had been used to characterize the elemental compositions of the as-synthesized particles. Fig. 7(a) shows the existence of Mg, Fe and O in the MgFe2O4 NPs. Moreover, EDX spectra of the Au attached MgFe2O4, incomplete and complete coated MgFe2O4-Au core-shell NPs (Fig. 7(b-d)) reveal the presence of Mg, Fe, O and Au in those samples. In order to examine the optical properties of the synthesized nanoparticles, the UV–vis spectra of AuNPs, AuNPs attached MgFe2O4 NPs and MgFe2O4-Au core-shell NPs containing various amounts of MgFe2O4-Au NPs core are displayed in Fig. 8. The small AuNPs exhibit the maximum intensity peak at ~510 nm because of their characteristic plasmon resonance for gold colloidal spheres [43]. While, the absorption band of small AuNPs attached MgFe2O4 NPs has no strong peaks in this UV–vis region which is similar to the causes of AuNPs attached SiO2 [44] and small amount of AuNPs attached Fe3O4 [45]. After growing the Au shell on the surface of MgFe2O4-Au NPs particles, the

Fig. 5. TEM images of (a) MgFe2O4 NPs, (b) AuNPs attached MgFe2O4 NPs, MgFe2O4-Au core-shell NPs with (c) 0.05, (d) 0.2, (e) 0.3, (f) 0.4, (g) 0.5, (h) 1.0 and (i) 2.0 ml of MgFe2O4-Au NPs core.

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Fig. 6. SAED patterns of (a) MgFe2O4 NPs, (b) AuNPs attached MgFe2O4 NPs, (c) complete coated MgFe2O4-Au core-shell NPs and (d) incomplete coated MgFe2O4-Au core-shell NPs.

absorption peak appears in this UV–vis region. This observation indicates the coating of gold shell on MgFe2O4 NPs from a red-shifting in plasmon resonance benchmarked with such resonance of a solid gold sphere. It is believed that this shift is contributed from an electromagnetic coupling phenomenon between the plasmon oscillations on the inner and the outer surfaces of gold shell [46]. For MgFe2O4-Au coreshell NPs obtained by using 0.05, 0.2, 0.3, 0.4, 0.5 and 1.0 ml MgFe2O4AuNPs cores, there are only two peaks, one around 520 and the other at ~670 to 900 nm. In connection with this, two possible assumptions are considered. First, these features might indicate the absorption spectra of smooth nanoshell that has several multipole plasmon resonances i.e. the coating of gold shell on MgFe2O4 NPs from a redshifting in plasmon resonance benchmarked with such resonance of a solid gold sphere, where the shorter wavelength might correspond to a quadrupole and the longer wavelength corresponds to a dipole [12]. Alternatively, these two peaks may arise from the surface plasmon resonance of solid gold and the gold-coated nanostructure, respectively [46]. The latter postulation is consistent with the literatures reported on Au2S-Au core-shell [43,47], γ-Fe2O3-Au core-shell [46] and Fe3O4Au core-shell [48]. With further reduction of Au shell, increasing of MgFe2O4-AuNPs core, the second plasmon resonance band shifts toward the red, consistent with the thickness reduction of the Au shell. Until the employed MgFe2O4-AuNPs core is reached to 2.0 ml, the plasmon band then shifts backward to blue shift indicating the occurrence of an incomplete shell coating circumstance [46], as demonstrated in TEM image (Fig. 5(i)). The second broaden peak which is in the NIR region suggests that these as-synthesized particles are proper for therapeutic applications because, in general, tissue and

Fig. 7. EDX spectra of (a) MgFe2O4 NPs, (b) AuNPs attached MgFe2O4 NPs, (c) complete coated MgFe2O4-Au core-shell NPs and (d) incomplete coated MgFe2O4-Au core-shell NPs.

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Fig. 8. UV–vis absorption spectra of AuNPs, AuNPs attached MgFe2O4 NPs and MgFe2O4-Au core-shell NPs with various amount of MgFe2O4 core.

blood are transparent for this region as earlier stated in the Introduction [18,19]. The VSM was performed (Fig. 9) here to investigate the magnetic properties of synthesized nanoparticles. As demonstrated in Fig. 9(a), the magnetization curves at 300 K of both MgFe2O4 NPs and MgFe2O4Au core-shell NPs exhibit superparamagnetism due to near-zero values coercivity (0.07 and 2.41 Oe) and remnant magnetization (0.03 and 0.04 emu/g) observed from the curves [49–51], respectively. After growing the Au shell on the surface of MgFe2O4-AuNPs particles, the observed values for saturation magnetization of MgFe2O4-Au coreshell NPs are dropped from 53.92 emu/g of MgFe2O4 core to 2.52. This observation is similar with those reported by Zhai et al. [52] and Maleki et al. [53] where the diamagnetism of thick Au shell is believed to be the cause. However, the MgFe2O4-Au core-shell NPs still display superparamagnetism, as shown in the enlarged view (Fig. 9(b)). By placing the magnet beside the tube of MgFe2O4-Au core-shell NPs in order to confirm the magnetic properties of these MgFe2O4-Au coreshell NPs (Fig. 10), it is clearly seen that these NPs move toward to the magnet and leaving clear solution as a result. Moreover, this observation can also be considered as the supporting evidence for the co-existence of MgFe2O4 and Au in the MgFe2O4-Au core-shell structure.

Further confirmation of the cytotoxicity to mammalian cells by the synthesized MgFe2O4 NPs and MgFe2O4-Au core-shell NPs was also important to prove that whether the synthesized particles are toxic or non-toxic to healthy cells when introducing them into patient body [20,21]. The mammalian cells from mouse areola fibroblast (L-929) cell line were therefore chosen to be tested by our products. Fig. 11 shows the in vitro cytotoxicity of MgFe2O4 NPs and MgFe2O4-Au coreshell NPs tested on L-929 cell line by REMA test and the capped marks indicate the standard deviation (SD) from three independent experiments. The reduction of cell viability, compared with control set, was found most obviously at 500 μg/ml of MgFe2O4 NPs and MgFe2O4-Au core-shell NPs to 12.36% and 0.73%, respectively. Although the cells were treated with maximum concentration of samples (500 μg/ml), the results showed that both samples are non-toxic to the cells due to the criteria of RGR values [21], as tabulated in Table 1. Our noncytotoxic results from MgFe2O4 NPs treatment are consistent with Yang et al. [54] research which human osteosarcoma cell line (Saos-2) was tested with 10 nm MgFe2O4 particles in the range of 12.5 to 75 μg/ml. However, cytotoxicity was found when the MCF-7 human breast cancer cell line was treated with 20 nm MgFe2O4 particles in 200–800 μg/ml concentration as reported by Kanagesan et al. [55]. The

Fig. 9. Magnetization curve at 300 K of (a) MgFe2O4 NPs and representative MgFe2O4-Au core-shell NPs and (b) enlarged view of representative MgFe2O4-Au core-shell NPs.

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Fig. 10. Appearance of (a) black colloidal MgFe2O4-Au core-shell NPs (final product) and (b) magnetization properties of MgFe2O4-Au core-shell NPs dispersed in water.

latter case was described as high concentration of magnetic nanoparticles induced apoptosis (cell death due to cell committed suicide) and necrosis (cell death due to bio-incompatible chemicals or outside factors) which are possibly attributed to free radical generation occurred at high intracellular concentrations of iron through Fenton and/ or Haber–Weiss reactions [55,56]. The reasons for non-similarity of cytotoxicity results between our case and Kanagesan et al. [55] could be due to different radically toxicity profile for each cell type as described by Huang et al. [57]. In addition, it seems like MgFe2O4 NPs showed more toxic to the cells than that obtained from MgFe2O4-Au core-shell NPs at the maximum concentration from our present work. This might be attributed to more biocompatible of the coated gold on MgFe2O4 that prevented leaching of iron which can induced cell death due to intracellular acidification, in agreement with many works that explored the cytotoxicity of gold-coated Fe3O4 [20,21,58]. In comparison with the same cell type (L-929), our products (MgFe2O4Au) showed less toxicity than that of Li et al. (Fe3O4-Au) [21] for each maximum concentration of samples (500 and 100 μg/ml, respectively). Thus, the synthesized MgFe2O4-Au core-shell NPs obtained here

demonstrate high potential for further utilization as magneticplasmonic nanoparticle-based materials. 4. Conclusions Magnetic-plasmonic nanoparticle-based materials with biocompatibility and total size less than 100 nm, in the form of MgFe2O4-Au nanoparticles with core-shell structure are developed. Superparamagnetic MgFe2O4-core nanoparticles with diameter of 65 nm were successfully prepared by hydrothermal technique. Gold-shell coating is carried out on the surface of these nanoparticles. The surfaces of MgFe2O4 nanoparticles were successfully modified by using APTES providing good attachment of small Au particles (~ 2 nm) on MgFe2O4 surfaces. As a result, complete coating of shell with various core/shell ratios was prosperously achieved by using a reduction method. The complete coated MgFe2O4-Au core-shell nanoparticles are obtained when 0.2, 0.3 or 0.4 ml of MgFe2O4 core: 4 ml of shell is used as core/shell ratio. These particles provide different size due to varying of shell thickness with total size of all conditions smaller than 100 nm with superparamagnetic

Fig. 11. In vitro cytotoxicity of MgFe2O4 NPs and MgFe2O4-Au core-shell NPs tested on L-929 cell line by REMA test.

J. Nonkumwong et al. / Materials Science and Engineering C 61 (2016) 123–132 Table 1 Activity of MgFe2O4 NPs and MgFe2O4-Au core-shell nanoparticles on L-929 cell line resulted from RGR value consideration at 500 μg/ml of samples. Sample code Cells + DMEM MgFe2O4 MgFe2O4-Au

RGR (Average ± SD)

Toxicity grade

Activity

1 0.88 ± 0.11 0.99 ± 0.08

– Grade 1 Grade 1

– Non-cytotoxic Non-cytotoxic

[20]

Remarks [21] Negative control [22]

[23]

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