Mg-doped NiO Nanoparticles Decorated MultiWalled Carbon Nanotube (MWCNT) Nanocomposite and their Biological Activities

Here we report a novel nanocomposite composed from Mg-doped NiO and Mg-doped\MWCNT using a facile sol-gel method. The synthesized Mg-doped NiO and Mg-doped\MWCNT nanocomposite was characterization by XRD diffraction (XRD), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and UV–Vis spectrophotometer. The X-Ray analysis revealed that the formation of nanocomposites, which has a cubic phase and a high crystalline nature. The FE-SEM images conrmed the success of decoration the 6%Mg- doped NiO on the surface of the treated MWCNT through the emergence of spherical shapes over the cylindrical tubes. Conversely, optical measurements reveal that the energy gap value for the Mg-NiO and the Mg/NiO-MWCNT nanocomposite are 3.28 and 2.82 eV, respectively. This indicates decreasing the zone between conduction band and valance band. Moreover, it found that Mg doped NiO\MWCNT nanocomposite showed high removal eciency towards the lead element compared with the Mg-doped NiO. Also, MTT test was employed to study antitumor activity against MCF-7 and WRL68 cells. Our results showed that the Mg doped NiO-MWCNT had cell viability of 66.7 and 71.9% against MCF-7 and WRL68, respectively. Whereas, Mg-doped NiO sample showed cell viability of 70.2 and 71.9% against MCF-7 and WRL68, respectively.


Introduction
Nanotechnology started to be used fty years ago because it easy to deal with things at the nanometer scale. Recently, nanotechnology has entered many areas of life, such as biomedicine, food, energy, electronics, textile, environment, solar cells, and hydrogen fuel cells [1]. Carbon is one of the most common elements found on the surface of the earth, and it appears in a variety of forms, namely, carbon nanotubes (CNTs), uorine, and graphene [2]. In state of Multi-Walled Carbon Nanotubes (MWCNTs), that concerned grate consideration due to the remarkable properties such as extremely light weight, high chemical and thermal stability, and high tensile strength, resistance to basic and acidic media. The MWCNTs also play an important role in preparing nanocomposites and treating water pollution [3][4][5][6]. The MWCNTs are produced from various methods including arc chemical vapor deposition (CVD) [6], hydrothermal processes [7], discharge [8], laser ablation [9], etc. To improve the performance of MWCNTs, the decorating them with metal oxides has been widely used due to good merits such as thermal stability and, high mechanical strength. Moreover, the dopants of MWCNTs reduces the band gap, to produce an effective metal oxide nanoparticle in the ultraviolet and visible region [10]. Among the many metal oxides, nickel oxide (NiO) nanoparticle was used as dopant with MWCNTs to produce a more effective compound. The multi walled carbon nanotubes MWCNTs act as a dispersing agent that prevents NiO nanoparticles from accumulation, which result in increasing the surface area compared to pure NiO. NiO is a transition metal oxide has a cubic structure. It is also a p-type semiconductor material with stable band gap in range of 3.6-4.0 Ev. Although most NiO is used as an antiferromagnetic insulator [11]. The nanoparticle oxides have a high surface area, so they use good carriers, absorbents and catalysts [12].
Recently, the cancer cell is one of the most di cult diseases that the world has faced in recent years due to the increase in the number of cases up to 25 million annually in a year 2015. There are many common methods of treating cancer, such as radiotherapy, surgery, and chemotherapy [13]. MWCNTs work to make cancer drugs accumulate at the tumor site by enhanced penetration effect. Since CNT has high elasticity, good stability, and biocompatibility, it can easily penetrate biological barriers and destroy the contents of the cancer cells [14,15].

Treatment of MWCNTs
Treated MWCNTs was achieved by adding o.5 g of Raw-MWCNTs in a mixture of sulfuric acid (95% H 2 SO 4 ) and nitric acid (65% HNO 3 ) 3:1 in ask of 500 ml. The ask was placed in ultrasonic bath for 30min at temperature 30 o C to improve dispersion bandy functionalized MWCNTs (F-MWCNTs). Afterwards, the mixture was then diluted with 400 ml of distilled water and washed several times by using vacuum ltered through micro membrane (0.22μm) made from cellulose nitrate to remove any impurities from F-MWCNTs. Finally, the ltered products were dried for overnight at 100 o C to form treated MWCNTs powder.

Synthesis of Mg-doped NiO nanoparticles
In this step, the 6% Mg-doped NiO nanoparticle was synthesized by sol-gel process. In this proses, Added 2 ml of NaOH to the mixture and stirred for another 2h. After that, the resulted mixture was washed and ltered with absolute ethanol and deionized water tow times. The product was dried at 120 o C for 3h and calcinated for 2 h at 450 o C in an oven.

Characterization
The crystal structure and crystallite size of the prepared Mg-doped NiO and Mg-doped NiO\MWCNT nanocomposite were identi ed by X-ray spectrum (XRD-6000, Shimadzu) with Cu Kα radiation source (wavelength of 1.54056 ˚A) at diffraction angle (2θ) from 20° to 80°. The chemical composition and functional groups was determined through the FT-IR technique (8400S, Shimadzu) in the range 4000-400 cm −1 . Also, the morphological of the prepared samples was identi ed by eld emission scanning electron microscopy (FESEM) (Hitachi Type S-4160) and quickening voltage (20-30 kV). The UV-Vis absorption spectra of Mg-doped NiO and Mg-doped NiO\MWCNTs hybrid were determined on UV.Vis spectroscopy (1800, Shimadzu, Kyoto, Japan).

Removal of lead (Pb +2 ) by using doped NiO and nanocomposite
In this test, stock solution of 1000ppm Pb +2 was prepared by dissolving a suitable amount of Pb(NO 3 ) 2 in deionized water. The obtained concentrations were resulted from dilution stock solution of Pb +2 . In batch adsorption test, 800 mg of Mg-doped NiO and Mg-doped NiO\MWCNT nanocomposites were added to 50 ml of 20 ppm solution of Pb +2 , separately, with stirring at room temperature. After adsorption, the nanocomposite was taken from the solution and the residual Pb +2 concentrations were measured by ame atomic adsorption spectroscopy. Samples withdraw at 0, 3, 6, 9 and 24 h to measure the amount of adsorption and removing percentage (R) of Pb +2 by the equation in following [16]: here, C o and C e are the initial and equilibrium concentrations of Pb +2 (mg L -1 ).

MTT Assay of Determination of antitumor activity
The MTT assay has been used to assess the cytotoxic effect of Mg-doped NiO and nanocomposite materials on different concentrations of L.camara crude extracts. The preparation MTT solution was achieved using following procedure: A) Kit content contain 1 ml in 10 vials and solubilization solution 50 ml in 2 bottles. Then B) Protocol of Tumor cells (1x10 4 -1x10 6 cells\ml) were grown in 96 at well microtiter plates, in a nal volume of 200 ml culture medium per each well. The using microplate is covered with sterilizing para lm and shacked gently. The plates were incubated at 37 o C for 24h and 5%CO 2 .
Afterwards, the medium was removed and two-fold serial dilutions of suitable nanostructures (12.5, 25, and 50,100,200,400) μg\ml were addition to the wells. Triplicates were used per each concentration and controls (the cells treatment with serum medium). Then, the plates were incubation at 37 o C, 5% CO2 at exposure time 24h. 10μl of prepared MTT solution was addition to each well. Moreover, the plates were incubation at 37 o C, 5%CO 2 for 4h. Then, removing the media gradually and 100 μl of solubility solution were added per each well doe 5 min. the absorbance of samples was measured by using the ELISA reader at wavelength 575nm. The resulted data of optical density was submitted to statistical analysis in order to calculate the concentration of required nanostructure to result 50% limitation in cell viability for each cell line. It can be seen that the sample is high crystallized and cubic phase is the only component of the hybrid material. Also, XRD pattern shows diffraction peak at 2θ = 58.54 ∘ for (311) with less intensity, corresponding to magnesium ions. This small peak reveals the impregnation of Mg ions on the cubic surface of NiO structure. Besides, the XRD pattern of nanocomposite reveals a diffraction peak at 2θ = 26.6 ∘ of (002) crystal plane related to the graphitic structure of MWCNTs [4,18].  Figure (3a,b) represents the FE-SEM images of pure NiO NPs and NiO doped with Mg element, respectively. It is observed that pure NiO NPs displays a smooth surface covered with particles of spherical shapes and asymmetric sizes distributed over the surface [25]. FESEM images of the nanocomposite Mg-doped NiO/MWCNTs are shown in Figure (3c,d) with different magni cations. It can be seen that the Mg-doped NiO nanoparticles specially made and attached to the surfaces of treated MWCNTs rather than to other regions without MWCNTs. The FESEM analysis demonstrates the light spots which relate to doped NiO nanoparticles decorated the tubes. As display in these gures, the side walls of MWCNTs are evenly decorated with doped NiO nanoparticles and there are NPs aggregates on the wall of MWCNTs which are so dense and not uniform, that it is hardly to see the hollow cavity of the tubes, which may result from the application of heat for a long time [28]. The particle size mean of Mg-NiO and Mg-doped NiO\MWCNT nanocomposite are about 78.26 nm and 35.02 nm. This con rms the Mg doped NiO nanoparticles and its heterogeneous dispersed on the surface of the F-MWCNTs. The results obtained from the FESEM analysis are similar to a study prepared by Saravanakkumar et al. [26], Karnaukhov et al. [27], and Mustafa et al. [28].

Morphological Measurements
Besides, the EDS spectrum of Mg-doped NiO displays the appearance of oxygen, nickel and magnesium elements, and this indicates that Mg is successfully doped in NiO sites during the chemical reaction formation Mg-NiO. We observe a clear increase in the weight ratio of Mg from 2.78% to 3.48% at high concentration of Mg (except at 4% Mg). In contrast, EDS analysis for Mg-doped NiO\MWCNT nanocomposite is demonstrate in Figure (4 a,b). The EDS analysis con rms the appearance of nickel and magnesium components in the hybrid sample beside to carbon and oxygen. These results indicate the success of forming the Mg-doped NiO\MWCNT nanocomposite. Moreover, the appearance of weak peaks in the EDS spectrum were due to the presence of small content of contaminants such as aluminum (Al, 3.01%), calcium (Ca, 1.21%), and zinc (Zn, 5.26 %) [26].

Optical Measurements
The optical properties of the prepared colloidal solutions can be identi ed via UV-Vis spectroscopy in the wavelength range of 200-800 nm, as shwon in Figure (5). The absorption spectrum of Mg-doped NiO shows a broad absorption peak at wavelength of 265 nm. In contrast, the UV-Vis spectra of the Mg-doped NiO\MWCNTs nanocomposite display a shift towards the higher wavelength (red shift) at absorption peak at 361 nm with an apparent higher band intensity [29,30]. In state of measurement the optical band gap energy (E g ) for all samples can be calculated by Tauc's equation [31]: Where hν is the photon energy, E g is the optical band gap energy, and A is a constant. Figure (6 eV. An obvious decrease in the E g was observed, which related to the capability of the treated MWCNTs to become as photogenertaed of electron assepters and SPR [32].

Biological Treatments
Removal of lead nitrate (200 ppm) had been determined by atomic absorption spectroscopy, after incubation at room temperature with 16 g/L of Mg-doped NiO and Mg doped NiO\MWCNTs nanocomposites within 24 h. The percentage of lead nitrate was removed by using the synthesized nanoparticles as shown in Figure ( Industrial wastewater commonly includes lead heavy toxic metal that not easily degrade and threat the environment even when presence at low concentration. There are many chemical and physical treatment methods to removal heavy metals from wastes. Adsorption by natural and nanomaterials is one of the attractive approaches to remediate heavy metals. Adsorption e ciency of lead by nanoparticles depends on the concentration of lead and adsorbent, incubation time, adsorption kinetic and a nity between pollutant and nanomaterial. A signi cant removal percentage of 65 was observed at pH of 6.0 when the effect dose of nanocomposite was 4 g/L and lead concentration was 100 ppm [33]. On the other studies, equilibrium between lead and NiO was performed (45 % removal) within two hours when lead concentration used at 50 mg /L and NiO, prepared by either organic solvent or precipitate methods, at 25 g/L [15], while 99 % of Pb removal can be performed by 15 g/L of Cr doped NiO NPs, and lead adsorbed higher than other examined ions [34]. On the other hand, many nanomaterials had been developed to remove heavy metals, such as; SWCNTs and MWCNTs, clay, chitosan, and natural zeolite from industrial wastewater [35]. Based on the results, the tumor can be treated either physically by radiation and hyperthermia, or chemically by intracellular entry of nanoparticles to induce reactive oxygen spices (ROS), and most importantly, the apoptotic, programmed cell death, and necrotic, direct cell damage, in tumor cells populations [13]. The results also reveal capability of ROS generation of the Mg-doped NiO and nanocomposite.
As shown in Figure 9, the increasing ROS levels induce signi cant damage to the DNA of the cells, resulting in the arrest of cell-cycle and subsequently cell death. The high concentration of Mg-doped NiO might have increased the production of oxygen free radicals within the cells which causes cell death.
Besides, the cytotoxic effects of doped NiO NPs are generally resulted by the high level of (ROS) and or less Mg ions. The formation of ROS results in more oxidative stress and oxidant damage in cells. The results suggested that cytotoxicity is related to release Mg-doped NiO from the extracellular degradation of nanocomposite. Cells could also phagocytize high content of 6% Mg-doped, and the MWCNTs released from the intracellular degradation of nanocomposite in the acidic environment of lysosome could also induce cytotoxicity, as shown in Figure 10.

Conclusions
The Mg-doped NiO and Mg-doped NiO/MWCNTs were successfully prepared by sol-gel method. The UV-Vis spectroscopy showed that all the prepared samples had a high absorbance and an energy band gap of up to 2.82 eV. It was observed that the Mg-doped NiO/MWCNTs nanocomposite had good physical stability and a high zeta potential value up to -31mV. The XRD and EDX analysis displayed that the formation of the Mg-doped NiO/MWCNTs nanocomposite, which has a cubic phase and a high crystalline nature. The FE-SEM images con rm the success of deposition of the Mg-NiO on the surface of the treated MWCNTs through the appearance of spherical shapes over the cylindrical tubes. The test for removing lead from water contaminated with Mg-doped NiO and Mg-doped NiO/MWCNTs nanocomposite reveal that the removal percentage of pollutant reaches 43 and 60%, respectively. The Mg-doped NiO and Mg-doped NiO/MWCNTs nanocomposites were showed low cytotoxicity against MCF-7 and WRL68 cell lines.

Declarations
Compliance with ethical standards Con ict of interest: The authors declare that they have no con ict of interest.