Effect of Mg doping on morphology, photocatalytic activity and related biological properties of Zn1−xMgxO nanoparticles

The objective of this study is to synthesize ZnO and Mg doped ZnO (Zn1−xMgxO) nanoparticles via the sol-gel method, and characterize their structures and to investigate their biological properties such as antibacterial activity and hemolytic potential.Nanoparticles (NPs) were synthesized by the sol-gel method using zinc acetate dihydrate (Zn(CH3COO)2.2H2O) and magnesium acetate tetrahydrate (Mg(CH3COO)2.4H2O) as precursors. Methanol and monoethanolamine were used as solvent and sol stabilizer, respectively. Structural and morphological characterizations of Zn1−xMgxO nanoparticles were studied by using XRD and SEM-EDX, respectively. Photocatalytic activities of ZnO and selected Mg-doped ZnO (Zn1−xMgxO) nanoparticles were investigated by degradation of methylene blue (MeB). Results indicated that Mg doping (both 10% and 30%) to the ZnO nanoparticles enhanced the photocatalytic activity and a little amount of Zn0.90 Mg0.10 O photocatalyst (1.0 mg/mL) degraded MeB with 99% efficiency after 24 h of irradiation under ambient visible light. Antibacterial activity of nanoparticles versus Escherichia coli ( E. coli ) was determined by the standard plate count method. Hemolytic activities of the NPs were studied by hemolysis tests using human erythrocytes. XRD data proved that the average particle size of nanoparticles was around 30 nm. Moreover, the XRD results indicatedthat the patterns of Mg doped ZnO nanoparticles related to ZnO hexagonal wurtzite structure had no secondary phase for x ≤ 0.2 concentration. For 0 ≤ x ≤ 0.02, NPs showed a concentration dependent antibacterial activity against E. coli . While Zn0.90Mg0.10 O totally inhibited the growth of E. coli , upper and lower dopant concentrations did not show antibacterial activity.


Introduction
Synthesis, characterization, and application of nanoscale materials lead to the design of new functional materials and devices with unique properties. Among nanosized materials, metal-oxide nanoparticles have an incredibly big potential in many research areas due to their small particle size, large surface area and advanced chemical, optical, morphological and antibacterial properties. Metal oxide NPs can be synthesized by using biological systems as bacteria, fungi and yeast [1][2][3][4] or synthetic pathways like hydrothermal method [5,6], sol-gel method [7][8][9][10][11] and chemical vapor deposition [12]. Metal-oxide NPs find applications in textile industry [13,14], electronics [15,16,17], gas sensing applications [18][19][20], in medical applications like selective destruction of tumor cells [21], intracellular drug delivery [22] and some diagnostic applications.
Among metal-oxide NPs, zinc oxide (ZnO) has a special biological and medical importance [23][24][25][26]. ZnO NPs are regarded as biosafe due to their low toxicity in some researches [27,28] and they are also assigned as GRAS (generally recognized as safe) by FDA (US Food and Drug Administration) [29,30]. In a related study, the effects of ZnO nanowires (average diameter of 1 µ m and the average length of 200 µ m) on Hela cell line (a type of epithelial cell) and L-929 cell line (connective tissue cells) were investigated by measuring the activity of succinate dehydrogenase (SDH). ZnO nanowire concentrations of 0.1, 1, 10, and 100 µ g/mL were studied, and it was stated that ZnO nanowires were completely biocompatible and biosafe at cellular level at concentrations lower than 100 µ g/mL [23]. Moreover, zinc is one of the essential elements both in plants and animals [31][32][33].
It has an important role in enzyme activity and acts as a cofactor of some enzymes such as carbonic anhydrase, carboxypeptidases, dipeptidases, DNA and RNA polymerases, pyruvate carboxylase and alkaline phosphatase [34,35].
Synthetic materials in contact with blood may cause damage to the walls of erythrocyte cells and may trigger various cell reactions, such as thrombus formation [36][37][38]. The hemolytic potential of a material is a measure of hemolysis that is caused by any material in contact with blood. ZnO NPs also show disruptive effect on biofilm formation and inhibit hemolysis so that they are assigned as hemocompatible [39]. Zhang et al. have shown that ZnO NP suspensions with 0.75 mg/ml (mg ZnO/ml physiological saline) concentration cause no apparent hemolysis [40]. Other studies on the healing of skin wounds showed that ZnO is highly effective in promoting wound healing by increasing cell reepithelization [41].ZnO NPs are currently used in pain easing and itch relieving commercial ointments [42][43][44].
The antibacterial activities of metal-oxide nanoparticles are noteworthy as a new technique that can replace the traditional methods in which organic antibiotics are used. The most remarkable advantages of inorganic antimicrobial agents are being of safe and stable, as compared totheir organic equivalents.
Metal-oxide nanoparticles which contain essential mineral elements like Zn, Mg and Ca (ZnO, MgO and CaO) also show antimicrobial action which their equivalent micro and macro sized materials do not possess [45][46][47]. In some studies, it was shown that, ZnO NPs show selective toxicity against bacteria and act as an inert nanomaterial on human cells [48][49][50][51]. Several mechanisms were introduced to the literature about the antibacterial action of ZnO NPs. In some researches, release of metal ions like Zn 2+ from NP structure was held responsible from the antibacterial activity [52]. However, the solubility of metal oxides like ZnO is very low and concentration dependent. Besides, some researches showed that as an essential mineral, low concentrations of Zn 2+ ion (0.01-1.00 mM) may act as a nutrient and can enhance the growth of bacteria [53]. Another accepted mechanism of antibacterial action is the electrostatic interaction between cell walls and nanoparticles and the accumulation of nanoparticles on cell membrane [54]. According to Stoimenov, the total electrical charge of bacteria cells is negative at biological pH because of the presence of negatively charged carboxylate groups on cell wall. On the contrary, ZnO NPs are positively charged [54]. These opposite charges cause an attraction between bacteria cell and ZnO NPs which results in cell death. Pati et al. [55] have shown the disruptive effect of ZnO NPs on the integrity of bacteria cell wall and oxidative stress resistant genes in cells. ZnO NPs are known as stable at neutral or biological pH. On the contrary, in acidic media (pH 4.5), they dissolve to form Zn 2+ ions [56]. ZnO particles accumulate on cell wall and introduce into the cell, dissolve in lysosomes having a pH range of 4.5-5.0 [57] and cause necrotic cell death [58,59]. Thus, although Zn is essential for the continuity of metabolic activities in the cell, the uncontrolled increase in Zn concentration as a result of the dissolution of ZnO nanoparticles in the cell is stated to be areason of cell death.
Besides the other mechanisms, the main responsible effect related to the antibacterial activity of nano sized ZnO and its derivatives causing intracellular death of bacteria is the light catalyzed formation of reactive oxygen species (ROS) such as hydroxyl radical ( * OH), superoxide anion radical (O − * 2 ) and hydrogen peroxide ( [60][61][62]. ZnO nanoparticles may cause light catalyzed formation of ROS like O − * 2 , * OH and H 2 O 2 . When the concentration of ROS exceeds the defense capacity of the cell, adverse biological consequences result [63]. In this study, the effects of Mg doping ratios on morphology, photocatalytic activity and biological effects of ZnO nanoparticles were investigated. ZnO and Mg-doped ZnO(Zn 1−x Mg x O) nanoparticles were synthesized via the sol-gel method and examined for their antibacterial activity and hemolytic potential.The effects of high doping levels (30%) of Mg on Zn 1−x Mg x O NPs were investigated and results were compared with pure ZnO nanoparticles.  To form the gel phase, aging solutions were evaporatedat room temperature under magnetic stirring. Then, heat treatment was applied at 400°C for 10 min to combust the organic residues in the gel phase and at 600°C for 30 min to obtain an accurate crystal orientation.

Structural analysis
Phase compositionsand crystal structures were characterized by the X-ray diffraction (XRD-Bruker D8 Advance) measurements. Surface morphologies were identified using scanning electron microscopy (SEM-FEI Quanta

Evaluation of photocatalytic properties
The photocatalytic performances of ZnO and Zn 1−x Mg x O nanoparticles were investigated by measuring the photocatalytic degradation rate of MeB similar to the procedure given by Ali et al. [64]. Initial dye and nanoparticle concentrations were 1.0 ×10 −5 M and 1.0 mg/mL respectively. In a typical photocatalytic degradation experiment, selected samples from Mg doped ZnO nanoparticles and pure ZnO were dispersed in 100 mL of aqueous MeB solution and magnetic stirring was applied for 30 min in the dark to ensure adsorption/desorption equilibrium. Aqueous nanoparticle-MeB dispersions were then irradiated under ambient visible light while being continuously stirred. Also, characteristics of ambient light were examined using CCS200compact spectrometer and a mixture of light in different wavelengths (436 nm, 546 nm, and 611 nm) were detected.
At appropriate time intervals, constant volume aliquots were taken and then centrifuged at 3000 rpm for 5 min to remove suspended ZnO or Zn 1−x Mg x O nanoparticles. The instant concentrations of MeB were determined usinga UV/vis spectrophotometer (Shimadzu UV mini 1240, Shimadzu Corp., Kyoto, Japan) at a wavelength of 664 nm using DDW as reference.
The photocatalytic degradation of MeB followed the first order kinetics and corresponding rate constants were determined by the following equation [65]; where A o and A were the absorbances of MeB solution after adsorption equilibrium and after the time (t) respectively. k was the first order rate constant for the photocatalytic degradation. Moreover, the decolorization efficiency of MeB was estimated by the following equation [66]; where C o and C represent the initial concentration of MeB before irradiation, and the concentration of MeB after a certain irradiation time (t), respectively.

Antibacterial activity tests
Antibacterial activity of Zn 1−x Mg x O NPs was determined by the standard agar plate count method using E.
coli as model microorganism. Three different concentrations (0.5, 1.0, and 5.0 mg/mL) of ZnO and Zn 1−x Mg x O nanoparticle suspensions were prepared in sterile test tubes by dispersing the NPs in DDW. Escherichia coli K12 strain (Invitrogen, France) was used as a model organism in this study. Bacteria were cultivated in tyriptic soy broth containing 5 g/L of yeast extract, 10 g/L bactotryptone, and 10 g/L NaCl. After an overnight cultivation at 37 o C, optical density (OD) of bacterial suspension was determined at 600 nm. Then suspension was diluted using fresh tyriptic soy broth to final concentration of 1.0×10 5 cell forming unit(cfu)/mL. 150 µL of this bacterial suspension was inoculated onto each nanoparticle suspension and samples were incubated at 37 o C for 20 h. Test tubes were stirred at 200 rpm under magnetic stirring to keep the contact of NPs and bacteria during incubation period. At the end of incubation time, 50 µL from each test sample was spreaded out onto a TSA plate uniformly, then, plates were incubated at 37 o C for 20 h and the colonies were counted as a final step. Four replicates of each nanoparticle concentration were tested.

Blood compatibility tests
Human whole blood samples were used in the experiments and hemolytic activity of NPs were investigated according to earlier reports [67]. 4 mL of human blood anticoagulated with trisodium citrate solution (0.108 mM) was added to 10 mL of Ca and Mg free PBS solution and centrifuged at 2000 rpm for 5 min to separate the erythrocytes from plasma. After the supernatant was decanted, RBCs were diluted to 100 mL with PBS. 1.25 mL of this RBC stock solution was added to 5.0 mL of NPs suspensions in test tubes with different concentrations. RBCs suspended in DDW were used as positive control test (100% hemolysis), while RBCs solution dispersed in PBS (pH 7.4) was selected as negative control test (0% hemolysis). All dispersions were incubated under magnetic stirring at 200 rpm at 37 o C for 3 h and then centrifuged at 10,000 g for 3 min using a microcentrifuge. Three replicates of each sample were tested. Hemolysis ratios were calculated by measuring the absorbance value (ABS) of the supernatant solution at 545 nm using a UV-vis spectrophotometer.

Structural analysis
Structural, electrical, magnetic and antibacterial properties of ZnO and ZnO doped with transtion metals, strongly depend on the single phase of resulting materials with growth condition. The X-ray diffraction was   Table by using XRD analyzes. The detailed evaluations of D, u, V, and L parameters were given below.  Table. The average crystallite size was calculated from the XRD peak width of (101) based on the Debye-Scherrer equation [7,9],  where β hkl is the integral half width, K is a constant equal to 0.90, λ is the wavelength of the incident Xray ( λ = 0.1540 nm), D is the crystallite size, and θ is the Bragg angle. The particle size calculated for synthesized ZnMgO nanoparticles was in the range of 21.6-26.88 nm.

Samples A(Å) C(Å) C/A D (NM)
The lattice constants a and cwere calculated with the following formula [7,9]: The volume of the unit cell of hexagonal system was calculated by the following equation: ZnO bond length was calculated by the following equation [7,9]: where a and c are lattice constants of ZnO and u is the wurtzite structure which can be found as In a structure, both doping ratio and annealing temperature, are predominantly affecting strain. Therefore, microstrain ( ε ) was calculated by the following equation: These parameters were presented in Table. As can be seen from Table,  As shown in Table, [70,71]. It has also been reported that surface area of NPs decreases with increasing particle size and this causes a decrease in the concentration of ROS generated from nanoparticles [72].
The morphology and element concentrations of Mg doped ZnO NPs were characterized by SEM and EDX, respectively.  Figure 11, the highest agglomeration and a pellet like dense morphology were observed.         The kinetics of MeB photo degradation shown in Figure 12d could be described by a first order kinetics in agreement with the Langmuir-Hinshelwood model:

Photocatalytic measurements
where k 1 is the adsorption coefficient, k 2 is the specific rate constant for degradation and C o is the initial concentration of MeB. The integrated form of the above equation can be written as follows: If the concentration (C) is very low, then the second term of the equation becomes very small compared to the first one and the equation can be simplified to: To form hydrogen peroxide, hydroperoxyl radicals may collide with each other, or they can also interact with CB electrons and H + ions (Eqs. 5-7). H 2 O 2 will then react with O * − 2 to form OH * which is a powerful oxidizing agent (Eq. 8).ROS would be responsible from both antibacterial properties and degradation of organic molecules like MeB into less toxic degradation products such as CO 2 and H 2 O (Eq. 9) [53].

Antibacterial properties
Antibacterial activity of ZnO NPs synthesized by the hydrothermal and the vapor deposition methods is well known in literature [82][83][84][85]. Nevertheless, concentration dependent antibacterial activity of ZnO NPs synthesized by the sol-gel method still have unsolved issues.
In this study, activity of Zn 1−x Mg x O NPs against bacteria was examined with different routes. To determine the antibacterial activity of NPs, the Kirby-Bauer disc diffusion method and the agar well diffusion method was first applied. In disc diffusion method, powder form of nanoparticles was pelleted using a hydraulic press, then they were transferred carefully on to the agar plates inoculated with E. coli. Similar to the procedure used in the Kirby-Bauer disc diffusion method, the agar plate surfaces were inoculated with the 1×10 6 cfu/mL E. coli suspension. Then, a hole for each test sample was punched aseptically with a sterile cork borer, and NPs were placed into wells with three different concentrations in two ways; as solid powder form and as colloidal nanoparticle suspensions in water (30 µL each). Then, agar plates were incubated at 37 o C for overnight. In both tests, the zone of inhibition could not be formed. As a third route, standard agar plate count method was used.
Antibacterial activities of ZnO and Zn 1−x Mg x O nanoparticles were studied against gram-negative Escherichia coli K12 strains. In our study, nanoparticle concentrations were determined as 0.5 mg/mL, 1.0  this lowest concentration may not trigger the defense mechanism of bacteria. As nanoparticle concentration increases antibacterial activity that was observed at low NP concentration disappeared at 1.0 mg/mL. It may be due to some upregulations in bacterial genome as it was stated in literature [87]. Nevertheless, at high ROS concentration due to increased NP concentration (5.0 mg/mL) bacteria could not survived. In a previous study conducted by Khan et al. [88], the effect of type and concentration of ROS on leading to cell death was  [87]. Researchers indicated that bacteria can modify its proteomic structure when they are exposed to harsh environmental conditions. Mg is an essential element for both prokaryotic and eukaryotic organisms.

Blood compatibilities
Nano sized materials may change or deform the morphology of red blood cells (RBCs or erythrocytes) and cause hemolysis, i.e. lysis of cell membrane when they interact with erythrocytes while, ZnO nano particles are regarded as non-toxic, biosafe and possibly biocompatible [28,78]. NPs including ZnO are usually administered intravenously in drug delivery and medical imaging applications [89,90]. However, catalytic activity of the nanoparticle surfaces may cause ROS generation and resulting an increase in oxidative stress leading to cellular damage [53,72,61].
Determination of the hemolytic potential of nanoparticles on human erythrocytes is an alternative method for in vivo testing of biological properties. In a literature study [78], human RBCs were found to be very stable after treatment with 0.100 mg/mL of ZnO NPs (~25 nm in diameter) synthesized by homogeneous gas phase condensation method. In another study, ZnO nanoparticles (~30 nm in diameter) synthesized by solution precipitation method showed no apparent hemolysis up to 0.600 mg/mL [41].
In this study, human erythrocytes drawn from healthy volunteers were used to investigate the hemolytic potentials of ZnO and Zn 1−x Mg x O nanoparticles. Figure 15 shows the hemolysis percentages of blood samples in contact with ZnO and Zn 1−x Mg x O nanoparticles. Results indicated that hemolysis ratio and nanoparticle concentration were directly proportional in case of ZnO and lower than the acceptable level of 5% [91] up to 5.0 mg/mL. In this study, nanoparticle concentrations were 5 times higher compared to the relevant literature [41,85,86].
In the study of Iqbal et al. [92], biocompatibility of cobalt doped ZnO NPs synthesized by coprecipitation method were investigated. They found concentration dependent increments in hemolysis percentages and relatively low hemolysis ratios up to 0.25 mg/mL nanoparticle concentration.In the present study, low The results of blood compatibility tests showed acceptable (<5%) levels [91] of hemolysis ratios for most of our nanoparticle applications. In case of ZnO, hemolysis ratios were below the acceptable level up to 5.0 mg/ml and nanoparticle concentration was also directly proportional to the hemolysis ratios. Additionally, in this study, antibacterial activity of NPs wasobtained in both 0.5 mg/mL and 5.0 mg/mL concentrations for ZnO, Zn 0.99 Mg 0.01 O and Zn 0.90 Mg 0.10 O against E. coli. Among these, Zn 0.90 Mg 0.10 O showed both higher antibacterial properties and lower hemolytic ratios. As known, in addition to low and/or acceptable hemolytic activity, broad spectrum, high stability, minimum drug resistance and side effects are desired features for antimicrobial drugs. In this respect, Mg doped ZnO nanoparticles and especially Zn 0.90 Mg 0.10 O could have commercial applications in UV sterilization systems [93].The studies and analyses in this study should give valuable ideas to design and develop new antibacterial nanosized materials for pharmaceutical industry that looking for alternative antibacterial agents in today's world while the effects of classical antibiotics are continuously decreased in recent years.