Cerium Oxide nanoparticles: Biosynthesis, Cytotoxic and UV protection

In recent years, the nanoparticles applications have been well recognized in various fields. It is known that nanoparticles as an active ingredient in sunscreens are widely used. Zinc oxide and titanium oxide nanoparticles are common nanoparticles utilized in sunscreens. In this study, we aimed to suggest new nanoparticles for this purpose. Cerium oxide nanoparticles (CeO 2 -NPs) were synthesized by using Musa sapientum fruit peel extract. Synthesized nanoparticles were identified through Raman, Powder X-ray Diffraction (PXRD), Fourier Transform Infrared spectroscopy (FT-IR), Field Energy Scanning Electron Microscopy (FESEM) and Energy-Dispersive Spectroscopy (EDX). The results showed that size of synthesized nanoparticles are in range 4-13 nm. The cytotoxic activity of synthesized nanoparticles on lung (A549) cancer cell line was performed through MTT assay. The results showed that synthesized nanoparticles are non-toxicity against A549 cell line to below 500 μg/mL of nanoparticles concentration. The Sun protection factor (SPF) was estimated ~ 40 for synthesized CeO 2 -NPs. So, synthesized nanoparticles can be a good option for use in the cosmetics industry.


Introduction
Cerium is a lanthanide element with atomic number of 58 which has two oxide forms including cerium dioxide (CeO2) and di-cerium trioxide (Ce2O3) [1][2][3]. These forms can convert to each other's. The cerium oxide are suitable for many industrial applications due to the exchange of Ce 3+ and Ce 4+ ions and the presence of oxygen holes in its crystalline structure [4]. Hence, this material is widely used in the glass industry, the manufacture of alloys and catalysts [5][6][7]. It can also act as UV absorber due to its large bond-gap (~ 4 eV) [8].
Studies show the penetration of UV radiation into deep layers of the skin in a long time irritate and damage skin, and also it can lead to cancer. Irregularities of skin color, wrinkles and freckle are other complications of exposure of UV radiation. The sunscreen production industry has billions dollars per year turnover; so preparation more effective sunscreens with less skin stimulation, more transparent, and without changing appearance of consuming people is a demand of bazar [9][10][11]. To solve this problem, nanotechnology researchers have done studies. The results of these studies showed that titanium oxide (TiO2) and zinc oxide (ZnO) nanoparticles have the potential to improve the performance of sunscreens. Newer studies have shown that CeO2-NPs have a better ability than ZnO for utilizer in sunscreens as absorption of UV rays [12].
Cancer is a disease in which abnormal cells was proliferated uncontrollably and can involve nearby tissues. This can occur following several genetic events, including inactivation of tumor suppressor genes and activation of oncogenes [13]. Today, various methods of surgery, chemotherapy and radiation therapy are used to treat cancer, but one of the disadvantages and side effects of these methods is the destruction of healthy cells. This has led researchers to move toward the new treatments with reducing side effects [14,15]. Nanotechnology can provide a tool for direct targeting, selective cancer cells, and increasing effectiveness for doctors [16]. Nanoparticles are used in a variety of ways, such as delivering drugs to cancer cells, as well as for imaging cancer cells and observing them more closely, and are well used for the diagnosis and treatment of cancer [17]. One of these nanoparticles is cerium oxide nanoparticles. Recent studies have shown that this nanoparticle has cytotoxic effect on cancer cells; therefore, further studies are important to determine the side effects and its use in the treatment of cancers [18]. Synthetic methods of nanoparticles play an important role in determining their size, morphology and properties. Metal oxide nanoparticles are generally synthesized using physical and chemical methods such as spray pyrolysis, ultrasonication, chemical evaporation and sol-gel [19][20][21][22]. Physical methods have limitations such as non-uniformity in particle size and expensive equipment. Metal oxide nanoparticles can be chemically synthesized by oxidation-reduction reactions or precipitation of required metal ion precursor in an aqueous solution phase [23,24].
Today, biosynthesis of nanoparticles considers by researchers because of their costeffectiveness, environmentally friendly, faster, simple protocols, and mild reaction conditions. This method includes different biomaterials such as bacteria, fungi and plants as stabilizing and reducing agents. The nanoparticles biosynthesis is not unusual process in nature because some prokaryotes and eukaryotes are capable to produce of nanomaterials through intracellular and extracellular processes [25]. Biosynthesis methods can reduce the toxicity of produced nanomaterial, and produced nanoparticles have variety sizes and morphology [26][27][28][29][30][31].
Musa sapientum is belonging to Musaceae family. It is a plant with 6-8 meters height, and including helical and spiral shape of leaves. Its origin is Southeast Asia and well grows in humid tropics. M. sapientum are rich in potassium. Every part of it has pharmaceutical value, and includes a number of pharmaceutical compounds, such as sterol fatty acid, linoleic acid, fructose, xylose, galactose, glucose, mannose, and oleic acid. Also peel of M. sapientum having anthocyanins, carotenoids, phenolic compounds, amine compounds [32]. So, nontoxicity of nanoparticles is very important for application in industry and other fields. Hence, in this study, cerium oxide nanoparticles (CeO2-NPs) was synthesized using Musa sapientum fruit peel extract and survey its cytotoxicity and sunscreen properties.

Synthesis of CeO2-NPs
The dried and crushed Musa sapientum fruit peel was extracted using water as a solvent (ratio to 1:10) through the soaking method. In the next step results was filtered and filtrate was used for the synthesis of nanoparticles. 10

Cytotoxicity evaluation of CeO2-NPs
The lung cancer cell line (A549) was prepared from Pasteur Institute of Iran. Cells were incubated in RPMI culture medium supplemented with 10% FBS, 100 μg/ml of Streptomycin, and 100 U/ml of Penicillin, at 37 ºC and CO2 atmosphere with 5% moisture.
The cytotoxicity of synthesized CeO2-NPs was survey through 3-(4,5-Dimethylthiazol-2-yl)- was added to each well and optical absorbance of each well was measured at 570 nm by using a micro-plate reader. Cell viability was presented as a percent compared to untreated control cells.

Sun Protection Factor (SPF) assay
SPF was matured using spectrophotometry method [33]. The solution of 100, 1000 and 10000 ppm of synthesized nanoparticles at four sintered temperature was prepared using ethanol as a solvent, and their absorbance were measured at 290 to 320 nm.

Characterization
Structure, morphology and size of synthesized CeO2-NPs were identified through Raman

Discussion and conclusion
The phenolic compound of Musa sapientum fruit peel can act as a reducing and stabilizer agent for cerium ions. Then oxidation transfer between Ce +3 to Ce +4 simultaneously accomplished due to oxygen of air, which leads to CeO2-NPs product. Figure 2 shows PXRD pattern of the synthesized CeO2-NPs using aqueous extract of M. sapientum fruit peel at 300, 400, 500 and 600 ˚C. Accordance with JCPDS standard, all appearing peaks in diffraction pattern confirm the structure of fluorite cubic structure for synthesized nanoparticles [34].
Size of synthesized nanoparticles at 300, 400, 500 and 600 ˚C were estimated by using the Scherrer equation (D=0.9*λ/βcosθ; where λ is the X-ray wavelength, β is the full width at half the maximum (FWHM) and θ is the Braggs' angle) [  The Raman spectrum of the synthesized CeO2-NPs is shown in Figure 3 at 400 °C. This data illustrate structural irregularities and sample topologies [34]. The active Raman mode for synthesized CeO2-NPs shows a very strong band at 453 cm -1 , which it conforms to F2g mode.
The crystalline fluorite cubic structure confirms for synthesized nanoparticles [35].   Figure 4A. The appearance of synthesized particles is snowflakes, and the grading particles are not well defined, it is foam-like. This issue may be due to CO2 and H2O fast exit from raw martial during calcination presses [36,37]. Figure 4B shows  synthesized CeO2-NPs by using P. fracta [33], gum [38], agarose [39] against HT-29, Nero2A and L929 cell lines, respectively. So, synthesized nanoparticles can utilize for applications such as drug delivery, cosmetic and ceramics. Today, there are over 300 sunscreens containing zinc oxide and titanium oxide nanoparticles in the market [40]. Previous studies have shown that CeO2-NPs have a better ability to absorb UV than to zinc oxide nanoparticles [12].  Table 1.  Table 2). As shown in Figure 6, UV protection values of synthesized nanoparticles reduce by reducing the particle size. The particle size is an important factor for adsorption and transmission of UV ray. So reducing the particles size reduces the distance between them, so the light is more refracted towards its path. The synthesized CeO2-NPs have different sizes at four temperatures, and analytic of SPF results showed that SPF levels increased as the particle size gets smaller. The synthesized CeO2-NPs have a good ability to absorb UV rays.

Conclusion
Due to the importance of sunscreens in cosmetic products, efforts have been done to improve their quality by researchers. The results of the studies show that some nanoparticles have a good ability to absorb UV rays. CeO2-NPs can be suggested as an option for this purpose due to its high band-gap. These nanoparticles are synthesized through a quick and low-cost method using aqueous extract of M. sapientum peel. The size of synthesized nanoparticles was in the range of 4-13 nm. The cytotoxic study of synthesized nanoparticles show that they are non-cytotoxic on A549 cell line at below 500 µg/ml. So, synthesized nanoparticles are suitable for medicinal applications. The results of the SPF measurement of synthesized nanoparticles show that by decreasing the particle size, the SPF level increases.

Compliance with ethical standards
Conflict of interest the authors declare that they have no conflict of interest.

Ethical approval
This study was done on synthesis of CeO2-NPs by using extract of M. sapientum peel. In following, cytotoxic activity of synthesized nanoparticles was performed using MTT assay against lung (A549) cell line. The lung cell line was prepared from Pasteur Institute of Iran.
Therefore, this research does not require the approval of Animal Experimentation Committee.