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Synthesis and characterization of highly efficacious Fe-doped ceria nanoparticles for cytotoxic and antifungal activity
Ceramics International 45 (2019) 7950–7955
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Ceramics International journal homepage: www.elsevier.com/locate/ceramint
Synthesis and characterization of highly efficacious Fe-doped ceria nanoparticles for cytotoxic and antifungal activity
T
Abbas Rahdara, , Mousa Aliahmadb, , Masoume Samanib, Mostafa HeidariMajdc, ⁎ Md. Abu Bin Hasan Susand, ⁎
⁎
a
Department of Physics, University of Zabol, Zabol 98613-35856, Iran Department of Physics, University of Sistan and Baluchestan, Zahedan, Iran c Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran d Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh b
ARTICLE INFO
ABSTRACT
Keywords: Green synthesis Ceria nanoparticles Xanthan gum Cytotoxicity
Ceria nanoparticles (NPs) are promising materials for their superior anticancer and antifungal activities. In this work, we synthesized Fe-doped ceria NPs by co-precipitation method using xanthan gum (XG) as a green capping agent. The NPs were calcined at different temperatures ranging from 200 to 800 °C. The crystallinity, crystallite size, structure, particle size, morphology, and magnetic property of the NPs were investigated by using x-ray diffraction, field emission scanning electron microscopy, vibrating sample magnetization, Fourier transform infrared spectroscopy, and dynamic light scattering. The influence of calcination temperature on the crystallite size was critically examined. The crystallize size of the Fe-doped ceria NPs was dependent on the calcination process and increased with increasing temperature. Nanocrystalline ceria particles with spherical morphology was chosen for cytotoxic studies. Anticancer activities were investigated against Michigan Cancer Foundation-7 breast cancer cell lines through 3–4,5-dimethylthiazol-2-yl)− 2,5diphenyltetrazolium bromide (MTT) assay while antifungal activities against clinical isolates of C. albicans. Fe-doped ceria NPs reduced the cell viability to about 21% of the control after 72 h of exposure. The NPs have the necessary promises for use as an effective agent toward therapy of cancer cells. The antifungal study indicated that Fe-doped ceria NPs are effective anti-fungal agent and exhibit superior activity compared to an antibiotic, fluconazole.
1. Introduction Ceria, one of the most intriguing metal oxide of the lanthanide series, has attracted significant and wide-spread attention in current research. The nanoparticles (NPs) of ceria, due inter alia to their outstanding physical and chemical properties, have been promising for biomedical applications for their antibacterial and antifungal activities [1–3]. They have found technological applications either directly or as an inevitable ingredient of ultraviolet absorbers, gas sensors, polishing agents, catalysts, cosmetic products, alloys, phosphors, and magnetic devices [4,5]. Research to-date experiences numerous attempts to synthesize nanometer-sized ceria particles both as undoped or doped materials. The notable approaches include the uses of plant extracts, gums, nutrients or use of chemical methods such as, co-precipitation, hydrothermal, microwave, reverse microemulsion, and sono-chemical techniques [1–3]. With a view to accomplishing biomedical objectives, there have been concerns of safety, biocompatibility and environmental benignity for synthesis of such NPs. It is therefore not surprising that recent bias in this
⁎
regard has been shifted to greener approaches through overcoming the disadvantages associated with the use of traditional chemical methods [1–3]. There are numerous reports in the literature on ceria NPs focusing mainly on the synthesis and characterization ceria NPs with emphasis on the effect of the NPs on different living organism models such as rats, mice, human cancer cell lines and non-rodent models [1–3]. It is worth exploring the possibility of the use of these NPs against infectious diseases and exploiting the possible antifungal activity. Candidiasis is a well-known fungal infection caused by Candida albicans in humans [6,7]. However, there have been reports of failure of treatments in recent years for patients receiving long-term antifungal therapy against candidiasis and the use of the agent in chemotherapy has been facing setback [6,7]. It has therefore been necessary to develop and design new antifungal drugs for effective treatment of this infection and eyes have naturally been pointed on ceria NPs. However, our knowledge on the effect of ceria NPs on lines cells and so on is still in a very rudimentary stage. In addition, there are lots of controversies about positive and negative impacts of ceria NPs on human health. Biocompatibility related to such NPs still thereby remains a very demanding but challenging task.
Corresponding authors. E-mail addresses: [email protected] (A. Rahdar), [email protected] (M. Aliahmad), [email protected] (Md. A.B.H. Susan).
https://doi.org/10.1016/j.ceramint.2019.01.108 Received 8 January 2019; Received in revised form 15 January 2019; Accepted 15 January 2019 Available online 16 January 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
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In the pursuit of biocompatible nanomaterials of high efficacy, we have devoted our attention on the synthesis of a range of nanomaterials for potential biomedical applications [8–12]. In continuation of such efforts, we report here the synthesis of Fe-doped ceria NPs by co-precipitation method in the presence of a polysaccharide of xanthan gum (XG) as a green capping agent. Notably, XG, a biopolymer capable of stabilizing NPs, has outstanding and unique properties such as high water-solubility and effective biocompatibility [13] to provide added advantage to the use of ceria NPs. The focus has however been on the in vitro cytotoxicity analysis of the NPs against Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines using a colorimetric assay for assessing cell metabolic activity. The 3–4,5-dimethylthiazol-2-yl)− 2,5diphenyltetrazolium bromide assay knows as MTT assay has been used in this regard. The antifungal activity study of the NPs was studied against clinical isolates of a dimorphic fungus, Candida albicans, which is a major opportunistic pathogen in immune-compromised patients.
Fig. 1. XRD patterns of ceria NPs.
2. Materials and methods
help of DLS. In fact DLS has been an effective tool to determine the size distribution of NPs. We have exploited the technique using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a He−Ne laser beam (633 nm) with vertically polarized light. For nano-colloidal solution, the time-dependent scattered light intensity is a fluctuating quantity and is dependent on number factors which include: size, Brownian motion and diffusive behaviour of NPs in solution and viscosity of the continuous phase. The normalized autocorrelation function, g1(τ) of the scattered electrical field for a given delay time, τ characterizes such fluctuations very well and provides useful information about the structure and dynamics of the scattered particles [14,15].
2.1. Materials Cerium nitrate (Ce(NO3)3·6H2O), xanthan gum (C35H49O29) and sodium hydroxide were analytical grade reagents from Sigma-Aldrich and were used as received without further purification. The breast cancer cell lines, MCF-7 were purchased from the Pasteur Institute (Iran). Fluconazole (Amin Pharmaceutical Company, Iran). CHROMagar Candida medium (Paris, France), and Corn meal agar (Difco, USA) were also used as received without further purification. Deionized water (DI, specific conductivity = 0.055 μS cm−1) was used as solvent.
g1 (q , ) =
2.2. Synthesis of nanoparticles
E (q , t ) E *(q, t + ) I (q , t )
(1)
Where, E* is the complex conjugate of E. In a DLS experiment, the intensity autocorrelation function, g2(q, τ), is experimentally determined as [14,15]
Fe-doped ceria (CeO2: Fe) NPs were synthesized by co-precipitation method under ambient condition. Cerium nitrate (10.965 g) was dissolved in DI water and stirred for 10 min. The solution was added to a clear xanthan gum solution prepared by dissolving 0.0471 g of XG in 48 mL of DI water. The mixture was stirred for 30 min followed by dropwise addition of 8 mL of 0.02 M of Fe(NO3)3 solution to it. The pH value of 9.0 was adjusted by adding a 3 M ammonia solution slowly under stirring condition. The particles formed were collected by centrifugation at 5000 rpm for 10 min followed by washing with water and further centrifugation at 5000 rpm for 3 min. The ceria particles were dried in an oven at 70 °C for 24 h and were ground to powder using mortar and pestle. The sample was divided into 4 parts and heated at different temperatures. The samples were labelled as A, B, C, D for the temperature of heat treatment applied of 200, 400, 600 and 800 °C, respectively.
g 2 (q , ) =
E (q , t ) E *(q, t ) E (q, t + ) E *(q , t + I 2 (q , t )
(2) 2
The normalized autocorrelation function, g (q,τ), is related to the autocorrelation function of the scattered electrical field, g1(q, τ) and can be easily converted by the Siegret relationship [14,15].
g2 (q, ) = 1 + A exp(
)
2
(3)
Here, A is an instrumental constant. A single exponential decay curve usually represents the function of g1(q, τ) for a colloidal system containing monodisperse micelles as given by [14,15]
g1(q, ) = A exp(
2.3. Characterization of nanoparticles
)
(4)
The decay rate, Γ, is related to the diffusion coefficient by D=Γ/q2. Finally, the hydrodynamic diameter is obtained from the diffusion coefficient according to the Stokes-Einstein relation.
X-ray powder diffraction (XRD) patterns of the samples were recorded on a D8 Advance X'pert X-ray diffractometer (Bruker). Fouriertransform infrared (FT-IR) spectroscopic measurements of NPs were performed on a JASCO 640 plus instrument in the range 4000–400 cm−1 at room temperature using KBr pellets. A Kavir Precise Magnetic instrument (MDKFT, Iran) was used for vibrating sample magnetometer (VSM) studies of NPs; while a Mira 3-XMU instrument with a 700,000x magnification allowed recording field emission scanning electron microscopic (FE-SEM) images. A UV–Visible (UV–vis) spectrophotometer (PerkinElmer) was used for recording UV–vis spectra. A pinhole small-angle x-ray scattering (SAXS) (KEFA Nano Laboratory, Iran) was used for SAXS measurements of NPs.
rh =
KB T 6 D
(5)
where KB is Boltzmann's constant, T is the temperature in Kelvin, and η is the viscosity of solvent. 2.5. Cell culture and in vitro anticancer analysis of Fe-doped ceria nanoparticles Cytotoxic study of Fe-doped ceria NPs was conducted using MCF-7 breast cancer cells through MTT cell viability assay [16,17]. Cells were cultured in a control medium of RPMI 1640, 10% phosphate buffer solution (PBS) and 100 µL of penicillin G/streptomycin mixture for two weeks in a pellet culture system. Trypsinization was used for cell passage while PBS served the purpose of cell washing during this period. It
2.4. Dynamic light scattering (DLS) measurements The dynamic parameters of NPs including the diffusion coefficient and particle size in a colloidal system are well characterized with the 7951
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40 (ahv)2 (eV.cm-1)
Absorbance (a.u.)
35 0.4
0.3
30 25 20 15 10 5
300 400 500 600 700 800 Wavelength (nm)
0
2
3
4
5
6
hv (ev) Fig. 2. UV–vis spectrum (left) and band gap energy (right) of Fe-doped ceria NPs (sample D).
0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -8000 -6000 -4000 -2000
0
2000 4000 6000 8000
Fig. 3. VSM of Fe-doped ceria NPs. Fig. 5. SEM image of synthesized Fe-doped ceria NPs (sample D).
2
g (t)-1
1
0.001
Fig. 4. FT-IR spectrum of Fe-doped ceria NPs (sample D).
0.01
0.1
Time (ms)
was followed by seeding the in 96-well plates at a cell density of 1.3 × 104 cells per well. The cells were then incubated for 24 h at 37 °C and 5% CO2. The cells were treated with sample A at six different concentrations (ranging from 0.39 to 12.5 mg/mL). The period was either 48 or 72 h for each case. Upon treatment for the designated period, medium of each well was replaced with 150 µL of fresh medium plus 50 µL of MTT solutions (prepared as 2 mg/mL in PBS) and incubated for 4 h. MTT solutions were removed and 200 µL of DMSO was added to the each well to solubilize formazan crystals in the system. The absorbance at 570 nm was monitored using a spectrophotometer (BioTek Instruments, Inc., Bad Friedrichshall, Germany).
Fig. 6. Autocorrelation function versus time for ceria NPs (sample D) at room temperature. Table 1 Diffusion coefficient and hydrodynamic radius of the ceria NPs calcined at 800 °C (sample D) according to analysis the autocorrelation function.
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Sample
Diffusion coefficient (m2/s)
Hydrodynamic radius (nm)
Ceria NPs calcined at 800 °C (sample D)
9.58 × 10–12
29.90
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sharpness of XRD peaks of Fe-doped NPs increased and the FWHM decreased as the calcination temperature increased from 200 to 800 . For instance, for a calcination temperature of 800 , the peaks in the XRD patterns were sharper to infer the crystallinity and relatively larger size. The crystallinity index of Fe-doped ceria NPs is thus dependent on the calcination process [19–23]. Fe-doped ceria NPs could be successfully prepared by a simple coprecipitation method using XG as a biopolymer capable of stabilizing the NPs. Fe-doped ceria NPs were stabilized due to the steric repulsion force originated from XG, that they form a thin film around the ceria NPs in solutions [19,20]. Fig. 2 shows the UV–vis spectrum and the evaluation of the band gap energy of the ceria NPs. It is worth mentioning that the optical direct band gap of the NPs could be calculated using Tauc's relation [1,24]:
Fig. 7. Cytotoxicity results of Fe-doped ceria NPs (sample D) in MCF-7 breast cancer cell lines for 48 and 72 h. Data represents mean (n = 4) ± SD.
h =
Ceria NPs were subjected to the minimal inhibitory concentration (MIC) assays against 20 clinical strains of Candida albicans. The assays were performed following the methods developed by the document, M27-A3 of the Clinical and Laboratory Standards Institute (CLSI). The final concentration of the fungal inocula was in the range of 1.5 × 103 cells/mL. The reproducibility of the data was checked by replicate measurements; at least three measurements were conducted in each case. 3. Results and discussion 3.1. Characterization of physical properties and magnetic behaviour The XRD technique was used to assess the crystalline nature of the NPs. The XRD patterns for Fe-doped ceria NPs at different calcination temperatures are shown in Fig. 1. The XRD patterns clearly show that the synthesized Fe-ceria NPs are identical in crystallographic structure in all cases and could be indexed to the standard CeO2 with face centred cubic structure in agreement with JCPDS#0957–076-01 [18]. Scherer equation was successfully used for determination of the average crystallite size of Fe-doped ceria NPs [13].
K Bhklcos
(6)
hkl
(7)
Eg )1/2
where hν, αo and Eg are photon energy, a constant and optical band gap of the nanoparticles, respectively. Absorption coefficient (α) of the powders at different wavelengths were calculated from the absorption spectra. Finally, the values of the Eg was determined by extrapolations of the linear regions of the plot ( h )2 of versus h . Fig. 2 shows an absorption peak at 307 nm in the UV–vis spectrum for Fe-doped ceria NPs. This is attributed to the intrinsic optical band-gap absorption of ceria NPs and is originated from the electron transitions from the valence band to the conduction band [19,20]. The Eg of ceria NPs is much higher than the value for bulk ceria (3.19 eV). This is attributed to quantum confinement effect in nanometer-sized structures [4,18]. NPs were further characterized using VSM technique. Fig. 3 shows the curves of magnetization of the NPs at room temperature. Fe-doped ceria NPs calcined at 800 show magnetic behaviour originated from the magnetic metal ions present in their structure [4]. FT-IR spectra were also recorded for Fe-doped ceria NPs. Fig. 4 shows the spectra for Fe-doped ceria NPs calcined at 800 °C (sample D). The FTIR spectrum and assignment of bands are consistent with literature [21,22]. The broad band in the frequency range 3750–3000 cm−1 is an absorption band assigned to O–H stretching from residual alcohols, water and Ce–OH. Ceria NPs also gives an absorption band at 3400 cm−1 [23,24]. Moreover, absorption at 2900 cm−1 corresponds to the C–H bond. Other typical bands at 1600 cm−1 are assigned to C = O bond [21,22]. Band observed around 550 cm−1 is assigned to Ce-Fe or Ce-O stretching. Band around 1100 cm−1 corresponds to Ce-Fe-O and Fe-O-Ce stretching. The antisymmetric Ce-O-Ce stretching mode of the surfacebridging oxide gives rise to the intense band at 500 cm−1 [21,22]. The morphology of the calcined Fe-doped ceria particles at 800 °C was examined by FESEM and the image is presented in Fig. 5. The image shows the spherical morphology of the sample. The average particle size estimated from the image is about 26 nm. The particle size compares well with the crystallize size. The larger size of particles is not surprising since crystallites in general form larger particles. The autocorrelation function versus time for NPs (sample D) from DLS measurements carried out at 25 °C is shown in Fig. 6. Table 1 summarizes the data related to diffusion and hydrodynamic radius of the ceria NPs (sample D). The fact that hydrodynamic radius is greater than the particle size estimated from FESEM and crystallize size determined from XRD pattern can be ascribed to scattering of light by the hydrated NPs in aqueous medium, which are larger in volume due to solvation.
2.6. Antifungal study of Fe-doped ceria NPs and determination of minimal inhibitory concentration
Dhkl =
0 (h
where Dhkl is the crystallite size perpendicular to the normal line of (hkl) plane, k is a constant (0.9 for spherical sample), Bhkl is the full width at half maximum (FWHM) of the (hkl) diffraction peak, hkl is the Bragg angle of (hkl) peak and is the wavelength of x-ray radiation. The average crystallite size of the NPs was 2.32, 2.57, 4.18 and 20.86 nm for sample A, B, C and D, respectively. The variation of the average crystallite size of the NPs is consistent with the variation in the calcinations temperature. The average crystallite size of Fe-doped NPs is below 10 nm for samples A, B, and C, which causes the broadening of the peaks as reported in the literature [19,20]. The properties of the NPs were dependent on the calcination temperature. The average crystallite size of the NPs varied from 2 to 21 nm due to the variation in the calcination temperature (vide supra). The Table 2 In vitro susceptibility testing of clinical isolates of C. albicans. Sample
MIC50 (µg/mL)
MIC90 (µg/mL)
MIC range (µg/mL)
Ceria NPs calcined at 800 °C (sample D) Fluconazole
0.12 3.12
0.48 6.25
0.12–0.48 1.75–2512
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3.2. Toxicity and cell growth inhibition analysis of nanoparticles
Conflicts of interests
The MCF-7 breast cancer cell lines were used to investigate the cytotoxic effects of the Fe-doped ceria NPs using MTT assay [16,17]. The cytotoxicity results of Fe-doped ceria NPs (sample D) in MCF-7 breast cancer cell lines are summarized in Fig. 7. Fig. 7 clearly demonstrates that the viability of breast cancer cell lines is dependent on concentrations of the Fe-doped ceria NPs and time of incubation. The high concentrations of Fe-doped ceria NPs allow reduction of both cell proliferation and viability to about 21% of the control after 72 h of treatment. The half maximal inhibitory concentration (IC50) which is widely known as a measure of the potency in inhibiting a specific biological or biochemical function was compared for different time periods. The IC50 was achieved as 6.1 and 3.02 mg/ mL for the Fe-doped ceria NPs following 48 and 72 h exposure, respectively.
The authors declare that there are no conflicts of interest. References [1] S. Rajeshkumar, P. Naik, Synthesis and biomedical applications of Cerium oxide nanoparticles–A Review, Biotechnol. Rep. 17 (2018) 1–5, https://doi.org/10.1016/ j.btre.2017.11.008. [2] L. Rubio, R. Marcos, A. Hernández, Nanoceria acts as antioxidant in tumoral and transformed cells, Chem. Biol. Inter. 291 (2018) 7–15, https://doi.org/10.1016/j. cbi.2018.06.002. [3] U. Carlander, T.P. Moto, A.A. Desalegn, R.A. Yokel, G. Johanson, Physiologically based pharmacokinetic modeling of nanoceria systemic distribution in rats suggests dose-and route-dependent biokinetics, Int. J. Nanomed. 13 (2018) 2631–2646, https://doi.org/10.2147/IJN.S157210. [4] E.K. Goharshadi, S. Samiee, P. Nancarrow, Fabrication of cerium oxide nanoparticles: characterization and optical properties, J. Colloid Interf. Sci. 356 (2011) 473–480, https://doi.org/10.1016/j.jcis.2011.01.063. [5] T.N. Ravishankar, T. Ramakrishnappa, G. Nagaraju, H. 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3.3. Determination of antifungal minimal inhibitory concentration Clinical strains of C. albicans were used for investigation of the antifungal activity of the ceria NPs. Table 2 compares the experiment results of MIC of fluconazole drug and ceria NPs (sample D) against clinical isolates of C. albicans. In our previous study, MIC range of fluconazole for clinical isolates of C. albicans was demonstrated as 1.75–25 µg/mL [12]. In the present work, MIC for ceria NPs (sample D) against clinical strains of C. Albicans was determined using broth microdilution method. The value was evaluated in the range of 0.12–0.48 µg/mL (Table 2). Table 2 concludes that the Fe-doped ceria NPs have the potent and effective antifungal activity against isolates of C. albicans compared to fluconazole. It is worth mentioning that reported studies by different groups confirm major growth inhibition and antibacterial activity of ceria NPs against the gram-negative bacterium-Staphylococcus aeruginosa and gram-positive bacterium, Staphylococcus aureus [25,26]. 4. Conclusions In conclusion, we have successfully synthesized and characterized XG-stabilized Fe-doped ceria NPs calcined at different temperatures by a simple co-precipitation method and demonstrated the cytotoxicity and antifungal activities of the NPs. XG serves as a green capping agent and can stabilize ceria NPs. This may be exploited for synthesis of stable NPs of this kind both as doped and undoped materials for their desired applications. The Fe-doped ceria NPs have fcc structure. The crystallite sizes of Fe-doped ceria NPs were in the range of ~2–20 nm. Fe-doped ceria NPs exhibited cytotoxic effect against MCF-7 breast cancer lines and showed the potential of an anticancer agent. The inhibitory effect of Fe-doped ceria NPs on MCF-7 breast cancer cells has been highly dependent on the time of treatment. Fe-doped NPs showed antifungal activity with MIC90 value of 0.48 μg/mL against clinical isolates of C. albicans. A greener approach followed in the synthesis would ensure safety, biocompatibility and environmental benignity for synthesis of such NPs to accomplish biomedical objectives. The results on anticancer and antifungal activities are very promising. These are definite improvement over previous reports on effect of such NPs on different living organisms as well as non-rodent models and against infectious diseases. A fundamental knowledge-base established would help to underpin further success to develop highly efficacious stable ceria NPs with proper doping for use as anticancer or antifungal agent. Acknowledgments The authors would like to thank the University of Zabol and Sistan and Baluchestan for financial support for this work. 7954
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[25] C.M. Magdalane, K. Kaviyarasu, J.J. Vijaya, B. Siddhardha, B. Jeyaraj B, Photocatalytic activity of binary metal oxide nanocomposites of CeO2/CdO nanospheres: Investigation of optical and antimicrobial activity, J. Photochem. Photobio. : B, 163, 2016,pp. 77–86. https://doi.org/10.1016/j.jphotobiol.2016.08.013. [26] M. Balouiri, M. Sadiki, S.K. Ibnsouda, Methods for in vitro evaluating antimicrobial activity: a review, J. Pharm. Anal. 6 (2016) 71–79, https://doi.org/10.1016/j.jpha. 2015.11.005.
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Synthesis and characterization of highly efficacious Fe-doped ceria nanoparticles for cytotoxic and antifungal activity
T
Abbas Rahdara, , Mousa Aliahmadb, , Masoume Samanib, Mostafa HeidariMajdc, ⁎ Md. Abu Bin Hasan Susand, ⁎
⁎
a
Department of Physics, University of Zabol, Zabol 98613-35856, Iran Department of Physics, University of Sistan and Baluchestan, Zahedan, Iran c Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran d Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh b
ARTICLE INFO
ABSTRACT
Keywords: Green synthesis Ceria nanoparticles Xanthan gum Cytotoxicity
Ceria nanoparticles (NPs) are promising materials for their superior anticancer and antifungal activities. In this work, we synthesized Fe-doped ceria NPs by co-precipitation method using xanthan gum (XG) as a green capping agent. The NPs were calcined at different temperatures ranging from 200 to 800 °C. The crystallinity, crystallite size, structure, particle size, morphology, and magnetic property of the NPs were investigated by using x-ray diffraction, field emission scanning electron microscopy, vibrating sample magnetization, Fourier transform infrared spectroscopy, and dynamic light scattering. The influence of calcination temperature on the crystallite size was critically examined. The crystallize size of the Fe-doped ceria NPs was dependent on the calcination process and increased with increasing temperature. Nanocrystalline ceria particles with spherical morphology was chosen for cytotoxic studies. Anticancer activities were investigated against Michigan Cancer Foundation-7 breast cancer cell lines through 3–4,5-dimethylthiazol-2-yl)− 2,5diphenyltetrazolium bromide (MTT) assay while antifungal activities against clinical isolates of C. albicans. Fe-doped ceria NPs reduced the cell viability to about 21% of the control after 72 h of exposure. The NPs have the necessary promises for use as an effective agent toward therapy of cancer cells. The antifungal study indicated that Fe-doped ceria NPs are effective anti-fungal agent and exhibit superior activity compared to an antibiotic, fluconazole.
1. Introduction Ceria, one of the most intriguing metal oxide of the lanthanide series, has attracted significant and wide-spread attention in current research. The nanoparticles (NPs) of ceria, due inter alia to their outstanding physical and chemical properties, have been promising for biomedical applications for their antibacterial and antifungal activities [1–3]. They have found technological applications either directly or as an inevitable ingredient of ultraviolet absorbers, gas sensors, polishing agents, catalysts, cosmetic products, alloys, phosphors, and magnetic devices [4,5]. Research to-date experiences numerous attempts to synthesize nanometer-sized ceria particles both as undoped or doped materials. The notable approaches include the uses of plant extracts, gums, nutrients or use of chemical methods such as, co-precipitation, hydrothermal, microwave, reverse microemulsion, and sono-chemical techniques [1–3]. With a view to accomplishing biomedical objectives, there have been concerns of safety, biocompatibility and environmental benignity for synthesis of such NPs. It is therefore not surprising that recent bias in this
⁎
regard has been shifted to greener approaches through overcoming the disadvantages associated with the use of traditional chemical methods [1–3]. There are numerous reports in the literature on ceria NPs focusing mainly on the synthesis and characterization ceria NPs with emphasis on the effect of the NPs on different living organism models such as rats, mice, human cancer cell lines and non-rodent models [1–3]. It is worth exploring the possibility of the use of these NPs against infectious diseases and exploiting the possible antifungal activity. Candidiasis is a well-known fungal infection caused by Candida albicans in humans [6,7]. However, there have been reports of failure of treatments in recent years for patients receiving long-term antifungal therapy against candidiasis and the use of the agent in chemotherapy has been facing setback [6,7]. It has therefore been necessary to develop and design new antifungal drugs for effective treatment of this infection and eyes have naturally been pointed on ceria NPs. However, our knowledge on the effect of ceria NPs on lines cells and so on is still in a very rudimentary stage. In addition, there are lots of controversies about positive and negative impacts of ceria NPs on human health. Biocompatibility related to such NPs still thereby remains a very demanding but challenging task.
Corresponding authors. E-mail addresses: [email protected] (A. Rahdar), [email protected] (M. Aliahmad), [email protected] (Md. A.B.H. Susan).
https://doi.org/10.1016/j.ceramint.2019.01.108 Received 8 January 2019; Received in revised form 15 January 2019; Accepted 15 January 2019 Available online 16 January 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
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In the pursuit of biocompatible nanomaterials of high efficacy, we have devoted our attention on the synthesis of a range of nanomaterials for potential biomedical applications [8–12]. In continuation of such efforts, we report here the synthesis of Fe-doped ceria NPs by co-precipitation method in the presence of a polysaccharide of xanthan gum (XG) as a green capping agent. Notably, XG, a biopolymer capable of stabilizing NPs, has outstanding and unique properties such as high water-solubility and effective biocompatibility [13] to provide added advantage to the use of ceria NPs. The focus has however been on the in vitro cytotoxicity analysis of the NPs against Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines using a colorimetric assay for assessing cell metabolic activity. The 3–4,5-dimethylthiazol-2-yl)− 2,5diphenyltetrazolium bromide assay knows as MTT assay has been used in this regard. The antifungal activity study of the NPs was studied against clinical isolates of a dimorphic fungus, Candida albicans, which is a major opportunistic pathogen in immune-compromised patients.
Fig. 1. XRD patterns of ceria NPs.
2. Materials and methods
help of DLS. In fact DLS has been an effective tool to determine the size distribution of NPs. We have exploited the technique using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a He−Ne laser beam (633 nm) with vertically polarized light. For nano-colloidal solution, the time-dependent scattered light intensity is a fluctuating quantity and is dependent on number factors which include: size, Brownian motion and diffusive behaviour of NPs in solution and viscosity of the continuous phase. The normalized autocorrelation function, g1(τ) of the scattered electrical field for a given delay time, τ characterizes such fluctuations very well and provides useful information about the structure and dynamics of the scattered particles [14,15].
2.1. Materials Cerium nitrate (Ce(NO3)3·6H2O), xanthan gum (C35H49O29) and sodium hydroxide were analytical grade reagents from Sigma-Aldrich and were used as received without further purification. The breast cancer cell lines, MCF-7 were purchased from the Pasteur Institute (Iran). Fluconazole (Amin Pharmaceutical Company, Iran). CHROMagar Candida medium (Paris, France), and Corn meal agar (Difco, USA) were also used as received without further purification. Deionized water (DI, specific conductivity = 0.055 μS cm−1) was used as solvent.
g1 (q , ) =
2.2. Synthesis of nanoparticles
E (q , t ) E *(q, t + ) I (q , t )
(1)
Where, E* is the complex conjugate of E. In a DLS experiment, the intensity autocorrelation function, g2(q, τ), is experimentally determined as [14,15]
Fe-doped ceria (CeO2: Fe) NPs were synthesized by co-precipitation method under ambient condition. Cerium nitrate (10.965 g) was dissolved in DI water and stirred for 10 min. The solution was added to a clear xanthan gum solution prepared by dissolving 0.0471 g of XG in 48 mL of DI water. The mixture was stirred for 30 min followed by dropwise addition of 8 mL of 0.02 M of Fe(NO3)3 solution to it. The pH value of 9.0 was adjusted by adding a 3 M ammonia solution slowly under stirring condition. The particles formed were collected by centrifugation at 5000 rpm for 10 min followed by washing with water and further centrifugation at 5000 rpm for 3 min. The ceria particles were dried in an oven at 70 °C for 24 h and were ground to powder using mortar and pestle. The sample was divided into 4 parts and heated at different temperatures. The samples were labelled as A, B, C, D for the temperature of heat treatment applied of 200, 400, 600 and 800 °C, respectively.
g 2 (q , ) =
E (q , t ) E *(q, t ) E (q, t + ) E *(q , t + I 2 (q , t )
(2) 2
The normalized autocorrelation function, g (q,τ), is related to the autocorrelation function of the scattered electrical field, g1(q, τ) and can be easily converted by the Siegret relationship [14,15].
g2 (q, ) = 1 + A exp(
)
2
(3)
Here, A is an instrumental constant. A single exponential decay curve usually represents the function of g1(q, τ) for a colloidal system containing monodisperse micelles as given by [14,15]
g1(q, ) = A exp(
2.3. Characterization of nanoparticles
)
(4)
The decay rate, Γ, is related to the diffusion coefficient by D=Γ/q2. Finally, the hydrodynamic diameter is obtained from the diffusion coefficient according to the Stokes-Einstein relation.
X-ray powder diffraction (XRD) patterns of the samples were recorded on a D8 Advance X'pert X-ray diffractometer (Bruker). Fouriertransform infrared (FT-IR) spectroscopic measurements of NPs were performed on a JASCO 640 plus instrument in the range 4000–400 cm−1 at room temperature using KBr pellets. A Kavir Precise Magnetic instrument (MDKFT, Iran) was used for vibrating sample magnetometer (VSM) studies of NPs; while a Mira 3-XMU instrument with a 700,000x magnification allowed recording field emission scanning electron microscopic (FE-SEM) images. A UV–Visible (UV–vis) spectrophotometer (PerkinElmer) was used for recording UV–vis spectra. A pinhole small-angle x-ray scattering (SAXS) (KEFA Nano Laboratory, Iran) was used for SAXS measurements of NPs.
rh =
KB T 6 D
(5)
where KB is Boltzmann's constant, T is the temperature in Kelvin, and η is the viscosity of solvent. 2.5. Cell culture and in vitro anticancer analysis of Fe-doped ceria nanoparticles Cytotoxic study of Fe-doped ceria NPs was conducted using MCF-7 breast cancer cells through MTT cell viability assay [16,17]. Cells were cultured in a control medium of RPMI 1640, 10% phosphate buffer solution (PBS) and 100 µL of penicillin G/streptomycin mixture for two weeks in a pellet culture system. Trypsinization was used for cell passage while PBS served the purpose of cell washing during this period. It
2.4. Dynamic light scattering (DLS) measurements The dynamic parameters of NPs including the diffusion coefficient and particle size in a colloidal system are well characterized with the 7951
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40 (ahv)2 (eV.cm-1)
Absorbance (a.u.)
35 0.4
0.3
30 25 20 15 10 5
300 400 500 600 700 800 Wavelength (nm)
0
2
3
4
5
6
hv (ev) Fig. 2. UV–vis spectrum (left) and band gap energy (right) of Fe-doped ceria NPs (sample D).
0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20 -8000 -6000 -4000 -2000
0
2000 4000 6000 8000
Fig. 3. VSM of Fe-doped ceria NPs. Fig. 5. SEM image of synthesized Fe-doped ceria NPs (sample D).
2
g (t)-1
1
0.001
Fig. 4. FT-IR spectrum of Fe-doped ceria NPs (sample D).
0.01
0.1
Time (ms)
was followed by seeding the in 96-well plates at a cell density of 1.3 × 104 cells per well. The cells were then incubated for 24 h at 37 °C and 5% CO2. The cells were treated with sample A at six different concentrations (ranging from 0.39 to 12.5 mg/mL). The period was either 48 or 72 h for each case. Upon treatment for the designated period, medium of each well was replaced with 150 µL of fresh medium plus 50 µL of MTT solutions (prepared as 2 mg/mL in PBS) and incubated for 4 h. MTT solutions were removed and 200 µL of DMSO was added to the each well to solubilize formazan crystals in the system. The absorbance at 570 nm was monitored using a spectrophotometer (BioTek Instruments, Inc., Bad Friedrichshall, Germany).
Fig. 6. Autocorrelation function versus time for ceria NPs (sample D) at room temperature. Table 1 Diffusion coefficient and hydrodynamic radius of the ceria NPs calcined at 800 °C (sample D) according to analysis the autocorrelation function.
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Sample
Diffusion coefficient (m2/s)
Hydrodynamic radius (nm)
Ceria NPs calcined at 800 °C (sample D)
9.58 × 10–12
29.90
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sharpness of XRD peaks of Fe-doped NPs increased and the FWHM decreased as the calcination temperature increased from 200 to 800 . For instance, for a calcination temperature of 800 , the peaks in the XRD patterns were sharper to infer the crystallinity and relatively larger size. The crystallinity index of Fe-doped ceria NPs is thus dependent on the calcination process [19–23]. Fe-doped ceria NPs could be successfully prepared by a simple coprecipitation method using XG as a biopolymer capable of stabilizing the NPs. Fe-doped ceria NPs were stabilized due to the steric repulsion force originated from XG, that they form a thin film around the ceria NPs in solutions [19,20]. Fig. 2 shows the UV–vis spectrum and the evaluation of the band gap energy of the ceria NPs. It is worth mentioning that the optical direct band gap of the NPs could be calculated using Tauc's relation [1,24]:
Fig. 7. Cytotoxicity results of Fe-doped ceria NPs (sample D) in MCF-7 breast cancer cell lines for 48 and 72 h. Data represents mean (n = 4) ± SD.
h =
Ceria NPs were subjected to the minimal inhibitory concentration (MIC) assays against 20 clinical strains of Candida albicans. The assays were performed following the methods developed by the document, M27-A3 of the Clinical and Laboratory Standards Institute (CLSI). The final concentration of the fungal inocula was in the range of 1.5 × 103 cells/mL. The reproducibility of the data was checked by replicate measurements; at least three measurements were conducted in each case. 3. Results and discussion 3.1. Characterization of physical properties and magnetic behaviour The XRD technique was used to assess the crystalline nature of the NPs. The XRD patterns for Fe-doped ceria NPs at different calcination temperatures are shown in Fig. 1. The XRD patterns clearly show that the synthesized Fe-ceria NPs are identical in crystallographic structure in all cases and could be indexed to the standard CeO2 with face centred cubic structure in agreement with JCPDS#0957–076-01 [18]. Scherer equation was successfully used for determination of the average crystallite size of Fe-doped ceria NPs [13].
K Bhklcos
(6)
hkl
(7)
Eg )1/2
where hν, αo and Eg are photon energy, a constant and optical band gap of the nanoparticles, respectively. Absorption coefficient (α) of the powders at different wavelengths were calculated from the absorption spectra. Finally, the values of the Eg was determined by extrapolations of the linear regions of the plot ( h )2 of versus h . Fig. 2 shows an absorption peak at 307 nm in the UV–vis spectrum for Fe-doped ceria NPs. This is attributed to the intrinsic optical band-gap absorption of ceria NPs and is originated from the electron transitions from the valence band to the conduction band [19,20]. The Eg of ceria NPs is much higher than the value for bulk ceria (3.19 eV). This is attributed to quantum confinement effect in nanometer-sized structures [4,18]. NPs were further characterized using VSM technique. Fig. 3 shows the curves of magnetization of the NPs at room temperature. Fe-doped ceria NPs calcined at 800 show magnetic behaviour originated from the magnetic metal ions present in their structure [4]. FT-IR spectra were also recorded for Fe-doped ceria NPs. Fig. 4 shows the spectra for Fe-doped ceria NPs calcined at 800 °C (sample D). The FTIR spectrum and assignment of bands are consistent with literature [21,22]. The broad band in the frequency range 3750–3000 cm−1 is an absorption band assigned to O–H stretching from residual alcohols, water and Ce–OH. Ceria NPs also gives an absorption band at 3400 cm−1 [23,24]. Moreover, absorption at 2900 cm−1 corresponds to the C–H bond. Other typical bands at 1600 cm−1 are assigned to C = O bond [21,22]. Band observed around 550 cm−1 is assigned to Ce-Fe or Ce-O stretching. Band around 1100 cm−1 corresponds to Ce-Fe-O and Fe-O-Ce stretching. The antisymmetric Ce-O-Ce stretching mode of the surfacebridging oxide gives rise to the intense band at 500 cm−1 [21,22]. The morphology of the calcined Fe-doped ceria particles at 800 °C was examined by FESEM and the image is presented in Fig. 5. The image shows the spherical morphology of the sample. The average particle size estimated from the image is about 26 nm. The particle size compares well with the crystallize size. The larger size of particles is not surprising since crystallites in general form larger particles. The autocorrelation function versus time for NPs (sample D) from DLS measurements carried out at 25 °C is shown in Fig. 6. Table 1 summarizes the data related to diffusion and hydrodynamic radius of the ceria NPs (sample D). The fact that hydrodynamic radius is greater than the particle size estimated from FESEM and crystallize size determined from XRD pattern can be ascribed to scattering of light by the hydrated NPs in aqueous medium, which are larger in volume due to solvation.
2.6. Antifungal study of Fe-doped ceria NPs and determination of minimal inhibitory concentration
Dhkl =
0 (h
where Dhkl is the crystallite size perpendicular to the normal line of (hkl) plane, k is a constant (0.9 for spherical sample), Bhkl is the full width at half maximum (FWHM) of the (hkl) diffraction peak, hkl is the Bragg angle of (hkl) peak and is the wavelength of x-ray radiation. The average crystallite size of the NPs was 2.32, 2.57, 4.18 and 20.86 nm for sample A, B, C and D, respectively. The variation of the average crystallite size of the NPs is consistent with the variation in the calcinations temperature. The average crystallite size of Fe-doped NPs is below 10 nm for samples A, B, and C, which causes the broadening of the peaks as reported in the literature [19,20]. The properties of the NPs were dependent on the calcination temperature. The average crystallite size of the NPs varied from 2 to 21 nm due to the variation in the calcination temperature (vide supra). The Table 2 In vitro susceptibility testing of clinical isolates of C. albicans. Sample
MIC50 (µg/mL)
MIC90 (µg/mL)
MIC range (µg/mL)
Ceria NPs calcined at 800 °C (sample D) Fluconazole
0.12 3.12
0.48 6.25
0.12–0.48 1.75–2512
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3.2. Toxicity and cell growth inhibition analysis of nanoparticles
Conflicts of interests
The MCF-7 breast cancer cell lines were used to investigate the cytotoxic effects of the Fe-doped ceria NPs using MTT assay [16,17]. The cytotoxicity results of Fe-doped ceria NPs (sample D) in MCF-7 breast cancer cell lines are summarized in Fig. 7. Fig. 7 clearly demonstrates that the viability of breast cancer cell lines is dependent on concentrations of the Fe-doped ceria NPs and time of incubation. The high concentrations of Fe-doped ceria NPs allow reduction of both cell proliferation and viability to about 21% of the control after 72 h of treatment. The half maximal inhibitory concentration (IC50) which is widely known as a measure of the potency in inhibiting a specific biological or biochemical function was compared for different time periods. The IC50 was achieved as 6.1 and 3.02 mg/ mL for the Fe-doped ceria NPs following 48 and 72 h exposure, respectively.
The authors declare that there are no conflicts of interest. References [1] S. Rajeshkumar, P. Naik, Synthesis and biomedical applications of Cerium oxide nanoparticles–A Review, Biotechnol. Rep. 17 (2018) 1–5, https://doi.org/10.1016/ j.btre.2017.11.008. [2] L. Rubio, R. Marcos, A. Hernández, Nanoceria acts as antioxidant in tumoral and transformed cells, Chem. Biol. Inter. 291 (2018) 7–15, https://doi.org/10.1016/j. cbi.2018.06.002. [3] U. Carlander, T.P. Moto, A.A. Desalegn, R.A. Yokel, G. Johanson, Physiologically based pharmacokinetic modeling of nanoceria systemic distribution in rats suggests dose-and route-dependent biokinetics, Int. J. Nanomed. 13 (2018) 2631–2646, https://doi.org/10.2147/IJN.S157210. [4] E.K. Goharshadi, S. Samiee, P. Nancarrow, Fabrication of cerium oxide nanoparticles: characterization and optical properties, J. Colloid Interf. Sci. 356 (2011) 473–480, https://doi.org/10.1016/j.jcis.2011.01.063. [5] T.N. Ravishankar, T. Ramakrishnappa, G. Nagaraju, H. 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3.3. Determination of antifungal minimal inhibitory concentration Clinical strains of C. albicans were used for investigation of the antifungal activity of the ceria NPs. Table 2 compares the experiment results of MIC of fluconazole drug and ceria NPs (sample D) against clinical isolates of C. albicans. In our previous study, MIC range of fluconazole for clinical isolates of C. albicans was demonstrated as 1.75–25 µg/mL [12]. In the present work, MIC for ceria NPs (sample D) against clinical strains of C. Albicans was determined using broth microdilution method. The value was evaluated in the range of 0.12–0.48 µg/mL (Table 2). Table 2 concludes that the Fe-doped ceria NPs have the potent and effective antifungal activity against isolates of C. albicans compared to fluconazole. It is worth mentioning that reported studies by different groups confirm major growth inhibition and antibacterial activity of ceria NPs against the gram-negative bacterium-Staphylococcus aeruginosa and gram-positive bacterium, Staphylococcus aureus [25,26]. 4. Conclusions In conclusion, we have successfully synthesized and characterized XG-stabilized Fe-doped ceria NPs calcined at different temperatures by a simple co-precipitation method and demonstrated the cytotoxicity and antifungal activities of the NPs. XG serves as a green capping agent and can stabilize ceria NPs. This may be exploited for synthesis of stable NPs of this kind both as doped and undoped materials for their desired applications. The Fe-doped ceria NPs have fcc structure. The crystallite sizes of Fe-doped ceria NPs were in the range of ~2–20 nm. Fe-doped ceria NPs exhibited cytotoxic effect against MCF-7 breast cancer lines and showed the potential of an anticancer agent. The inhibitory effect of Fe-doped ceria NPs on MCF-7 breast cancer cells has been highly dependent on the time of treatment. Fe-doped NPs showed antifungal activity with MIC90 value of 0.48 μg/mL against clinical isolates of C. albicans. A greener approach followed in the synthesis would ensure safety, biocompatibility and environmental benignity for synthesis of such NPs to accomplish biomedical objectives. The results on anticancer and antifungal activities are very promising. These are definite improvement over previous reports on effect of such NPs on different living organisms as well as non-rodent models and against infectious diseases. A fundamental knowledge-base established would help to underpin further success to develop highly efficacious stable ceria NPs with proper doping for use as anticancer or antifungal agent. Acknowledgments The authors would like to thank the University of Zabol and Sistan and Baluchestan for financial support for this work. 7954
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