CC BY
6
AZERBAIJAN CHEMICAL JOURNAL № 3 2023 ISSN 2522-1841 (Online)
ISSN 0005-2531 (Print)
UDC 620.3. 573.6.546
Au@Fe3O4 NANOPARTICLES: PREPARATION, CHARACTERIZATION AND
CYTOTOXIC EVALUATION
Z.Kelgenbaeva1, S.Sulaimankulova2, E.Murzabekova3, T.Mashimo4
department of biochemistry, I.K.Akhunbaev Kyrgyz State Medical Academy, Bishkek, 720020,
Kyrgyzstan
2 3
, Institute of Chemistry and Phytotechnology, the National Academy of Sciences of the Kyrgyz
Republic, Bishkek, 720071, Kyrgyzstan 4Institute of pulsed power Sciences, Kumamoto University, Kumamoto, 860-8555, Japan
Received 05.12.2022 Accepted 05.04.2023
Gold-coated iron oxide nanoparticles are a class of potential theranostic nanoparticles that have been studied for their multitude of magnetic and optical properties with possible medical applications. In this work, spherical Au@Fe3O4 nanoparticles were successfully synthesized from Fe electrodes immersed in HAuCl4 solution using a very simple method - pulsed plasma in liquid, without any dopants or special conditions for stabilization. Vibrating sample magnetometer indicated ferromagnetic behavior of the particles at room temperature with coercivity and saturation magnetization of Hc=175 and Ms= 3.56 emu/g, respectively. Face-centered cubic Au and spinel inverse structure of FCC- Fe3O4 were determined by X-Ray diffraction analysis. High-resolution transmission electron microscopic analysis revealed that Fe3O4 particles with 14 nm in size are connected to 18 nm Au particles. The cytotoxicity of the nanoparticles was evaluated using a XTT assay on Hela cells, revealing very low (cell viability: 9889% with Fe3O4 and 99-91% for Au@Fe3O4 NPs). This indicates that Au improves biocompatibility of magnetic nanoparticles, enabling their application in biomedicine.
Keywords: nanoparticles, Au@Fe3O4, pulsed plasma in liquid, cytotoxicity, Hela cells.
doi.org/10.32737/0005-2531-2023-3-98-104 Introduction
Magnetic iron oxide exhibit unique magnetic properties that make them attractive for different biomedical applications, including drug delivery [1], magnetic resonance imaging [2-3], magnetic particle imaging [4] and magnetic hyperthermia [5]. Gold nanoparticles or gold - coated nanoparticles are also of great interest due to their extraordinary material properties that make them particularly interesting for biomedical applications. When gold is used to coat iron oxide nanoparticles, the outer gold shell acts as a barrier, preventing core oxidation and enzymatic degradation [6]. Hybridization of iron oxide nanoparticles with gold results in a multimodal platform which benefits from the unique properties of both materials [7].
Gold-coated iron oxide nanoparticles are a class of potential theranostic nanoparticles that have been studied for their multitude of magnetic and optical properties with possible medical applications [8-10]. Concerning the
synthesis of iron oxide nanoparticles coated with gold, Iancu et al. have prepared stable Fe3O4@Au nanoparticles and studied their cyto-toxicity and MRI tests on rats, showing a mild cytotoxic effect and optimal concentration of 6mg/100 g body weight to obtain high-quality images [11]. Nassireslami and Ajdarzade have produced surface modified Fe2O3 nanoparticles and MUC-1 aptamer, as a targeted moiety conjugated onto gold layer around nanoparticles [12]. Rizvi reports the cationic polyethylene-imine-functionalized superparamagnetic Fe3O4 nanoparticles deposition on the surface of anionic gold nanorods coated with bovine serum albumin [13].
There is a number of methods and techniques for the synthesis of gold-coated iron oxide nanoparticles. Pulsed plasma in liquid (PPL) can be considered as one of the simplest, non -toxic, and cheapest tool for nanomaterial fabrication among other well-known techniques [14]. Pulsed plasma is an excellent tool for the
nanostructuring of solid matter produced in liquids between two electrodes of a suitable conductive element. The energy of a single pulse is efficient for the nanostructuring of any refractory conductive material. Materials produced by PPL are very active, owing to the large surface area of particles and the high surface energy provided by high dispersion [15]. A key advantage of PPL are its simplicity and cost effectiveness. Up-to-date various types of nanostruc-tures were successfully synthesized by PPL, and their properties and applications have been studied [16, 17]. Unlike previous works, this study applied aqueous solution of chloroauric acid (HAuCl4) as a liquid. The objectives of this study is to synthesize Au@Fe3O4 nanoparticles using the PPL and to investigate their physico-chemical properties. In addition, cytotoxicity effects of obtained Au@Fe3O4 and previously synthesized Fe3O4 (magnetite) nanoparticles on Hella cells were evaluated.
Experimental part
The experimental setup of PPL was applied as described previously [14-17], by changing the medium and some experimental conditions. Iron rods with a purity of 99.9%, a diameter of 5 mm and length of 150 mm were purchased from Rare Metallic and were immersed in a 200 mL Pyrex beaker filled with a liquid: aqueous solution (1.0 mM) of hydrogen tetra-chloroaurate (HAuCl4) (Kanto Chemical Co., Ltd). Pulsed plasma was applied for 1 h at room temperature followed by voltage of 125 V, current of 4 A, and frequency of 60 kHz. After experiment, the sample was separated from the liquid (solution of HAuCl4) using centrifuge, and prepared product was subjected to structural and morphological characterizations. The resulting powder-like Au@Fe3O4 sample was dark and sparkled, indicating the presence of gold content.
Atomic emission spectra of the plasma discharge during the synthesis were collected by an optical spectrometer, SEG2000 UV-vis, installed close to the plasma discharge zone outside the quartz beaker. Emission spectrum peaks were identified using the NIST database [18].
Powder X-ray diffraction (XRD) was recorded on a Rigaku RINT-2500VHF dif-fractometer using Cu Ka radiation (X=0.15406 nm) and operated at 40 kV and 200 mA. For the measurement, powder-like samples were placed on a glass sample holder. High-resolution transmission electronic microscopy (HR-TEM) analysis was performed with a Philips Tecnai F20 S-Twin instrument at 200 keV to study the size and morphology of the sample. Samples for HR-TEM measurements were suspended in eth-anol and ultrasonically dispersed. Drops of the suspension were placed on a copper grid coated with carbon. Size distribution of nanoparticles was determined by measuring the diameters of about 200 particles randomly selected from the HR-TEM images. Magnetization studies were performed at room temperature using a vibrating sample magnetometer (VSM) (Riken Denshi Co., Ltd., Japan).
Cytotoxicity of the nanoparticles synthesized using pulsed plasma in a liquid was evaluated using a mammalian endothelial cell line (HeLa cell), commonly used for testing the tox-icity and trafficking of nanoparticles. Magnetite nanoparticles were exposed for cytotoxicity measurements in order to compare the toxicity with Au@Fe3O4 nanoparticles. HeLa cell line was seeded onto 96-well plate three days before the measurement and incubated at 370C in a 5% CO2 humidified incubator. These cells were maintained as monolayer cultures in Dulbec-cos's modified eagle medium (D-MEM) solution (Wako pure Chemical Industries., Ltd) supplemented with L-Glutamine, low Glucose, Phenol Red, Sodium Pyruvate and Standard Serum Supplementation. Cell viability was measured by sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assays kit (molecular formula: C22H16N7NaO13S2). After incubation, (3*103 cells/100 ml of growth medium/well) supernatants from the wells were aspirated out, and fresh aliquots of growth medium containing Fe3O4 and Au@Fe3O4 nanoparticles were added in various concentrations: 25, 50, 100, and 200 ^l ml-1. After 24 h, supernatants were aspirated out and the cell monolayers in
the wells were washed, then XTT reagent was added to each well and incubated again. Cell viability was measured two times (4 h and 24 h after incubation) using a microplate reader (Bio Rad, model no 350).
Results and discussion
Figure 1 shows XRD pattern of Au@Fe3O4 nanoparticles synthesized by PPL. The diffraction peaks at (20) 38.2, 44.4, 64.5, and 77.60 are attributed to gold, which can be indexed to (111), (200), (220), and (311) lattice planes of gold in cubic phase (JCPDS card no 65-2870) with cell parameter of a= 0. 4079 nm. Peaks at 35.42, 43.05, 53.49, 57.03, 62.74, and 74.240 corresponded to cubic spinel structure of magnetite (JCPDS card no 19-0629: a=0.8396 nm), indicating the presence of both Fe3O4 (magnetite) and Au (gold) in the sample.
(Au). The Cu and C peaks appeared due to the sample containing TEM micro-grid made of cupper mesh coated with carbon.
50 60 2Theta (deg) Fig. 1. X-ray diffraction pattern of Au(®Fe304 nanoparticles synthesized by pulsed plasma in liquid.
The morphology and size of the nanopar-ticles were imaged using the HR-TEM system equipped with an EDX detector for elemental analysis. Evidence for the presence of gold and magnetite in the sample (Au@Fe3O4) is from energy dispersive spectroscopy (EDS), where the elements gold (Au), iron (Fe), and oxygen (O2) have been found as presented in Figure 2. Composition of the magnetite phase (Fe3O4) is 43 at% of iron and 57 at% of oxygen. Thus, EDS results confirms existence of nanoparticles composed of iron (Fe), oxygen (O), and gold
Fig. 2. Energy dispersive spectra of Au@Fe3O4 nanopar-ticles synthesized by pulsed plasma in liquid.
Figure 3 a presents typical high-resolution TEM images of Au@Fe3O4 nanoparticles with Fe3O4 at around 14 nm and Au at 18 nm, respectively. Au particles appear black; Fe3O4 are light colored in the image because Au has a higher electron density and allows fewer electrons to transmit. The lattice fringes in each of the particles correspond to atomic planes within the particle, indicating that both particles are single crystals as given in inset (Figure 3b). Figure 3c-d show the line profile of the image contrast along the lines marked in the image. The inter-planar d-spacing of the magnetite (Fe3O4) was found to be 0.29 nm, in a good agreement with (220) planes in the inverse spinel structured Fe3O4. That of the gold (Au) is measured as 0.234 nm, very close to the (111) planes of face centered cubic Au.
Atomic emission spectroscopy (AES) is an established laboratory diagnostic technique for plasma processes. Detection of light from electronic transitions of atoms enables identification and monitoring chemical species in plasma. In process development efforts, AES provides major improvement in process understanding as it is an in-situ method to study the process. Figure 4 shows atomic emission spectra collected from the plasma discharge zone
between two Fe electrodes submerged in 1.0 mM HAuCl4 solution.
0.5 1 1.5 2
Distance (nm)
Fig. 3. High-resolution TEM: a) high-resolution TEM image of one Au particle connected with two Fe3O4 particles; b) high-resolution TEM image of one 18-14 nm Au@Fe3O4 nanoparticles. The insets show a fast Fourier transform of the image showing of reflections corresponding to [220] and [111] crystallographic zone of Fe3O4 and Au; c-d) the gray contrast profiles for the selected regions of Fe3O4 and Au, respectively.
From the atomic emission spectrum, we identified peaks of Fe I, O II, Au II, and H. Based on these identified atoms, we illustrate a possible pathway of Au@Fe3O4 nanoparticles formation mechanism:
H2O
high-energy electrons
e" №, OH, H2O2, H2, H3O
The major ion components in water (aqueous solution of HAuCl4) during the plasma process are superoxides (O2-), which further formed H2O2 and hydroxyl (OH-) created by the reaction between plasma and water molecules [19]. These strong oxides are considered to be oxygen sources for the formation of iron oxide (a-Fe2O3, y-Fe2O3, and Fe3O4) particles. Also, generated hydrated electrons e-aq can serve as the reducing agent for the formation of gold nanopar-ticles [20].
Magnetic properties are strongly influenced by many parameters, including crystallini-ty, size, shape, and crystal defect density of the material. A magnetic property of the Au@Fe3O4
nanoparticles is shown by magnetization hysteresis loop in Figure 5.
Fig. 4. Atomic emission spectra from plasma between two Fe electrodes submerged in 1.0 mM HAuCl4 solution.
O)
E
a>-
c o
"-4—»
03 N
"S
c O) CD
3 0 1 2 I S1/ 5/
-800 -600 -400 /OC ( -3 f 200 400 600 BOO Magnetic field (Oe)
Au@Fe304
He=210 Oe
Mr= 0.86 emu/g
Ms = 3.56 emu/g
M= Ms/Mr= 4.14
-6000 -4000 -2000 0 2000 4000 Magnetic field / H (Oe)
6000
Fig. 5. Hysteresis loops for magnetic behavior of Au@Fe3O4 nanoparticles synthesized by pulsed plasma in liquid.
The Au@Fe3O4 nanoparticles showed ferromagnetic behavior at room temperature though the part of samples, Au in our case, is non-magnetic material. This reveals that gold (Au) not only acts as protecting material for Fe3O4 from oxidation, agglomeration and aggregation, but also maintains strong magnetic properties of magnetite. High coercivity (Hc=210 Oe) shows suitability of Au@Fe3O4 nanoparticles synthesized by PPL for use as magnetic recording materials suitable for such
procedures as cancer treatment and magnetic resonance imaging (MRI).
Magnetic nanoparticles have been of significant use in disparate technological areas, particularly in biomedical applications. However, use in biological systems requires biocom-patibility with cells and tissues, among other properties. Cytotoxicity of Fe3O4 and Au@Fe3O4 nanoparticles synthesized by PPL was measured. Representative results obtained from these measurements are given in Figure 6.
0 25 50 100 200 Concentration (ng/ml)
Fig. 6. Cytotoxic effect of Fe3O4 and Au@Fe3O4 nano-particles synthesized by PPL on HeLa cells.
HeLa cells were exposed to nanosized Fe3O4 and Au@Fe3O4 particles for 24 h. Nano-particles suspensions with concentrations of 25, 50, 100 and 200 ^g/ml were prepared by serial dilution. Cytotoxic effects were determined by using XTT assay. The organics used in the XTT assay are more stable and give more accurate results. Cell viability (%) was calculated according to the following Eq.:
Cell viability (%) =OD (sample)/ OD (control) x100.
Here, OD (sample) represents optical density of wells treated with various concentrations of the Fe3O4 and Au@Fe3O4 nanoparticles, and OD (control) represents that of wells treated with D-MEM. Cell viability (%) was calculated after 4h and 24 h incubation; all results are listed in
Table. Thus, the cytotoxicity assay results indicate that Fe3O4 and Au@Fe3O4 nanoparticles have low effect on the cells when they are used in low concentration. However, with increasing nanoparticle concentration, cell viability decreased in a concentration-dependent manner. In fact, the toxicity of Au@Fe3O4 nanoparticles was below that of Fe3O4 nanoparticles, meaning that Au improves biocompatibility of magnetic nanoparticles. This enables them to be applicable further in biomedicine.
Cytotoxicity of Fe3O4 and Au@Fe3O4 nanoparticles in HeLa cells by XTT assay_
Concentration (Mg ml-1) Cell viability (%)
Fe3O4 Au@Fe3O4
4 h 24 h 4 h 24 h
25 87 86 95 92
50 79 78 94 89
100 73 69 92 88
200 71 66 89 85
Conclusions
Ferromagnetic Au@Fe3O4 nanoparticles with coercivity of 210 Oe synthesized from Fe electrodes submerged in 1.0 mM solution of HAuCl4 by a simple route - pulsed plasma. X-ray diffraction and energy-dispersive spectro-scopy analysis showed that the sample was composed of face-centered cubic Au and spinel inverse structure of face-centered cubic Fe3O4. High-resolution TEM revealed that Fe3O4 particles with about 14 nm in size were connected to 18 nm Au particles; both of which were spherical in shape. VSM indicated the ferromagnetic behavior of samples at room temperature in spite of their small size (<20 nm). Cytotoxicity measurements showed very low toxicity of Au@Fe3O4 nanoparticles (cell viability 85-95%) indicating that samples are suitable candidate for biomedical applications in cancer treatment and magnetic resonance imaging (MRI).
Acknowledgements
Authors of this study thank Mashimo Laboratory staff (Kumamoto University, Japan) for their contribution in performing analysis and their interpretations. This work partially was
supported by MEXT (Monbukagakusho Scholarship, Japan).
References
1. Karimi M., Ghasemi A., Sahandi Zangabad. Smart micro/nanoparticles in stimulus-responsive drug/ gene delivery systems. Chem Soc Rev. 2016. 45 (5), 1457-1501.
2. Kudr J., Haddad Y., Richtera L. Magnetic nanoparticles: from design and synthesis to real world applications, Nanomaterials, 2017. 7. 243.
3. Woodard L.E., Dennis, C.L., Borchers J.A. Nano-particle architecture preserves magnetic properties during coating to enable robust multi-modal functionality. Sci. rep. 2018. 8. 1-13.
4. Bauer L.M., Situ S.F., Griswold M.A., Samia A.C., Magnetic particle imaging tracers state-of-the-art and future directions. J. Phys. Chem. Lett., 2015, 6, 2509-17.
5. Pardo A., Pelaz B., Gallo J. Synthesis, characterization, and evaluation of superparamagnetic doped ferrites as potential therapeutic nanotools. Chem Mater. 2020. 32. 2220-32.
6. Wang L, Luo J, Fan Q, Suzuki M, Suzuki I.S, Engelhard M.H, Lin Y, Kim N, Wang J.Q and Zhong C.J. Monodispersed core-shell Fe3O4@Au nanoparticles. J. Phys. Chem. B. 2005. V. 109. P. 21593-601.
7. Huang C, Jiang J, Muangphat C, Sun X and Hao Y. Trapping iron oxide into Hollow gold nanoparticles. Nanoscale Res. Lett. 2011. V. 6. P. 1-5.
8. Nassireslami E, Ajdarzade M. Gold coated super-paramagnetic iron oxide nanoparticles as effective nanoparticles to eradicate breast cancer cells via photothermal therapy. Adv Pharm Bull. 2018;8(2):201-209. doi:10.15171/apb.2018.024.
9. Neshastehriz A, Khosravi Z, Ghaznavi H, Shakeri-Zadeh A. Gold-coated iron oxide nanoparticles trigger apoptosis in the process of thermoradiotherapy of U87-MG human glioma cells. Radiat Environ Biophys. 2018;57(4):405-418. doi:10.1007/s00411-018-0754-5.
10. Carneiro MFH, Machado ART, Antunes LMG, et al. Gold-coated superparamagnetic iron oxide na-
noparticles attenuate collagen-induced arthritis after magnetic targeting. Biol Trace Elem Res. 2019. doi:10.1007/s12011-019-01799-z
11. Iancu S.D., Albu C., Chiriac L., Moldovan R et al, Assessment of gold-coated iron oxide nano-particles as negative T2 contrast agent in small animal MRI studies, International Journal of nano-medicine. 2020:15. 4811-4824.
12. Nassireslami E., Ajdarzade M., Gold coated superparamagnetic iron oxide nanoparticles as effective nanoparticles to eradicate breast cancer cells via photothermal therapy. Adv Pharm Bull. 2018. 8(2). 201-209.
13. Rizvi M.H., Wang R., Schubert J., Crumpler W.D., et al, Magnetic alignment for plasmonic control of gold nanorods coated with iron oxide nanoparticles. Adv Mater. 2022. 34. 2203366.
14. Tamura S., Mashimo T., Yamamoto K., Kelgen-baeva Z., et al. Synthesis of Pd-Fe system alloy nanoparticles by pulsed plasma in liquid. Nano-materials (Basel) 2018. 18:1068.
15. Mashimo T., Tamura Z., Yamamoto K., Kelgen-baeva Z. Synthesis of Pd-Ru solid-solution nano-particles by pulsed plasma in liquid method, RSC Adv. 2020. 10. 13232-236.
16. Tamura S., Kelgenbaeva Z., Yamamoto K., Chen L., Mashimo T., Preparation of FePt nanoparticles by pulsed plasma in liquid method, Key Eng Mater. 2017.730. P. 248-252.
17. Kelgenbaeva Z., Khandaker J. I., Ihara H., Omur-zak E., Sulaimankulova S., Mashimo T. Thermal and optical properties of In and In2O3 nanoparti-cles synthesized using pulsed plasma in water. Phys Stat Solidi (a). 2018. V. 11. P. 1700910 -16.
18. Ralchenko Y, Kramida AE, J. Reader, and NIST ASD Team: National Institute of Standards and Technology. Gaithersburg, MD, Version 4.1.0 (2011).
19. Fridman A, (2008) Plasma Chemistry: (Cambrdige University Press, Cambridge, U. K.)
20. Liang X, Wang Z and Liu C. Size-controlled synthesis of colloidal gold nanoparticles at room temperature under the influence of glow discharge. Nanoscale Res Lett 2010. V. 5. P. 124-2.
Au@Fe3O4 NANOHiSSOCiKLORi: ALINMASI, XARAKTERiSTiKASI VO SÎTOTOKSÎKLÎK DOROCOSi
J.Kelgenbayeva, S.Sulaymankulova, E.Mirzabekova, T.Maçimo
Qara örtüklü damir oksid nanohissaciklari, tibbda mümkün tatbiqlarla maqnit va optik xüsusiyyatlarin paylanmasi ûçûn tadqiq edilmiç sarbast teranistik nanohissaciklar sinfidir. Bu içda sferik Au@Fe3O4 nanohissaciklari HAuCl4 mahluluna batirilmiç Fe elektrodlarindan çox sada üsulla, mayelarda plazma impulsundan istifada etmakla, heç bir xüsusi çarait va ya xüsusi konservasiya çartlari olmadan ugurla sintez edilmiçdir. Titrama maqnitometri) macburi qüvva va doyma maqnitlaçmasi ila otaq temperaturunda hissaciklarin ferromaqnit davraniçini müvafiq olaraq Hc=175 va Ms=3.56 emu/g göstardi. Au-nun til markazli kub quruluçu va FCC-Fe3O4-ün çpinel tars strukturu rentgen §üalarimn difraksiya analizi ila müayyan edilmiçdir. Yüksak ayirdetma qabiliyyatina malik transmissiya elektron mikroskopu (TEM) 14 nm Fe3O4 hissaciklarinin 18 nm Au hissaciklarina baglandigini göstardi. Nanohissaciklarin sitotoksiklik daracasi Hela
hüceyralarina XTT nümumsi gotürmakla qiymatlandirilmiíjdir ki, bu da gox a§agi olmu§dur (hüceyra canliligi: Fe3O4 ügün 98-89% va Au@Fe3O4 NP-lar ügün 99-91%). Bu, qizilin maqnit nanohissaciklarin biouygunlugunu yax§ila§dirdigini gostarir ki, bu da onlan biotibbda istifada etmaya imkan verir.
Agar sozlar: nanohissaciklar, Au@Fe3O4, mayeda impulslu plazma, sitotoksiklik, Hela hüceyralari.
НАНОЧАСТИЦЫ Au@Fe3O4: ПОЛУЧЕНИЕ, ХАРАКТЕРИСТИКА И ОЦЕНКА ЦИТОТОКСИЧНОСТИ
Ж.Келгенбаева, С.Сулайманкулова, Э.Мирзабекова, Т.Машимо
Наночастицы оксида железа с золотым покрытием представляют собой класс потенциальных тераностических наночастиц, которые были изучены на предмет их множества магнитных и оптических свойств с возможным применением в медицине. В данной работе сферические наночастицы Au@Fe3O4 были успешно синтезированы из Fe-электродов, погруженных в раствор HAuCl4, с использованием очень простого метода - импульсной плазмы в жидкости, без каких-либо добавок или специальных условий для стабилизации. Вибрационный магнитометр) показал ферромагнитное поведение частиц при комнатной температуре с коэрцитивной силой и намагниченностью насыщения Hc=175 и Ms=3.56 emu/g соответственно. Гранецентрированная кубическая структура Au и шпинельная инверсная структура FU,K-Fe3O4 были определены методом рентгеноструктурного анализа. Просвечивающий электронный микроскоп (ПЭМ) с высокой разрешающей способностью показал, что частицы Fe3O4 размером 14 нм связаны с частицами Au размером 18 нм. Степень цитотоксичности наночастиц оценивали путем взятия пробы ХТТ на клетках Hela, которая оказалась очень низкой (жизнеспособность клеток: 98-89% для Fe3O4 и 99-91% для НЧ Au@Fe3O4). Это свидетельствует о том, что золото улучшает биосовместимость магнитных наночастиц, что позволяет использовать их в биомедицине
Ключевые слова: наночастицы, Au@Fe3O4, импульсная плазма в жидкости, цитотоксичность, клетки Hela.
