|Year : 2019 | Volume
| Issue : 1 | Page : 42-51
Cytotoxicity and antimicrobial activity of naturally and chemically synthesized zinc oxide nanoparticles
Amr A El-Waseif PhD
Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt
|Date of Submission||15-Apr-2019|
|Date of Acceptance||12-May-2019|
|Date of Web Publication||27-Jun-2019|
Amr A El-Waseif
Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, Cairo, 11751
Source of Support: None, Conflict of Interest: None
Background/aim Zinc oxide (ZnO) is a polar inorganic compound with numerous applications, for example, as an antimicrobial agent. The present study aims to synthesize ZnO nanoparticles (NPs) by two different methods, and then determine the antimicrobial activity of ZnO NPs from both methods. It then focuses on comparison between the cytotoxicity of both ZnO NPs.
Materials and methods ZnO NPs were synthesized using natural and chemical methods. The synthesized and prepared ZnO NPs were detected by precipitation in both methods using de Man, Rogosa, and Sharpe broth and alkaline medium, respectively. The characterization of ZnO NPs was performed using ultraviolet spectroscopy, zeta potential, and transmission electron microscopy (TEM) to decide properties of NPs. Viability tests are essential for assessing the effect of toxicants on cells. To measure cell viability following NP exposure, MTT assay was used.
Results Results of ultraviolet and TEM experiments for both NPs indicated absorbance at 356–360 nm, which is typical for ZnO NPs. Results show that naturally prepared ZnO NPs had an average size within 7.8 nm; they were small spherical particles with a narrow size distribution relatively with smooth surfaces. The chemically prepared ZnO NPs’ TEM images showed an average size of 27.6 nm. Zeta value of naturally synthesized ZnO NPs was estimated to be −25.30 mV at pH=7. However, the value of zeta potential in chemical preparation strategy showed −18.6 mV. Results revealed that toxicity of naturally synthesized ZnO NPs was less than that of chemically prepared ZnO NPs. Furthermore, the decline in cytotoxicity attributed to ZnO NP exposure was dependent on the concentration of ZnO NPs. The antimicrobial activity results of ZnO NPs showed that the ZnO NPs produced from both methods recorded antimicrobial activities against the pathogenic strain models used.
Conclusion The microbial synthesized ZnO NPs within size 7.8 nm when used at concentration 625 μg/ml as antimicrobial agent recorded the lowest cytotoxicity when compared with chemically synthesized. So that natural synthesis of ZnO was recommended.
Keywords: antimicrobial activity, characterization, cytotoxicity, microbial zinc oxide nanoparticles
|How to cite this article:|
El-Waseif AA. Cytotoxicity and antimicrobial activity of naturally and chemically synthesized zinc oxide nanoparticles. J Arab Soc Med Res 2019;14:42-51
|How to cite this URL:|
El-Waseif AA. Cytotoxicity and antimicrobial activity of naturally and chemically synthesized zinc oxide nanoparticles. J Arab Soc Med Res [serial online] 2019 [cited 2019 Dec 6];14:42-51. Available from: http://www.new.asmr.eg.net/text.asp?2019/14/1/42/261617
| Introduction|| |
Antimicrobial agents have been used to control or inhibit microorganisms. However, microbial protection from these medications has created on a huge scale after some time, significantly decreasing their adequacy and is a consistently growing issue . Even though, many articles have demonstrated the inhibitory and cytotoxic activity of metallic nanoparticles (NPs), the explanations of how cellular damage is caused has yet to reach a consensus. The mechanisms related to NP toxicity are the generation of reactive oxygen species, release of toxic ions, absorptive capacity, etc. Several studies state that the medication-resistant diseases will kill an additional 10 million individuals every year around the world − more than as of now bite the dust from malignant growth by 2050 unless the move is made. In this way, a standout among the most encouraging systems for defeating microbial obstruction is the applicant of NPs.
Nanotechnology gave the answer for medicine in light of the fact that it can discover materials in the nanoscale distance across that have enhanced bioactivity . The principal explanation behind their significance is the expanded specific surface region of these NPs in contrast with their volume, which enables their connection with bioorganics present on the suitable cell surface . One of the celebrated NPs is zinc oxide (ZnO) NPs, which is one of metal oxide NPs. ZnO is a polar inorganic compound. It is a white powder, almost insoluble in water with numerous applications, for example, antimicrobial, wound recuperating, ultraviolet (UV) separating properties, high catalytic and photochemical action, owing to its extraordinary mix of fascinating properties, for example, nontoxicity, great electrical, optical and piezoelectric conduct, soundness in a hydrogen plasma atmosphere, and low cost .
The natural and chemical production of NPs is through different strategies: chemical, physical, and biological; the production of NPs through these strategies shows variation in sizes and states of NPs. The natural microbial method of production is more profitable than other synthetic strategies, as it is easy and does not use varying temperatures, toxic chemicals, high pressure, and energy ,. The synthesis of NPs by the microbial method is eco-accommodating, easy, and can be utilized as catalysts, which the traditional strategies cannot create, and NPs’ applications in drug and sensors are visualized. Moreover, bacterial NPs can be used to inhibit human pathogenic microorganisms .
The natural sources used for NPs synthesis are bacteria, yeast, fungi, and plant extract; the green science standards is good with using microorganisms: the microorganism is ecofriendly and lessens the operator utilized . Wide classes of gram-positive and gram-negative bacteria can be used to adsorb and catch up overwhelming metal particles. Bacterial framework focal points incorporate simple taking care of and innately manipulated effectively . The NPs incorporated organically have different applications like biolabelling, in the coating of medical products, and cancer management . The oxidized type of orchestrated NPs is increasingly valuable, in light of the fact that these NPs have great magnetic, electrical, and optical properties .
The present work aims to synthesize ZnO NPs by two different methods, and the resulted NPs were characterized using UV-vis spectroscopy, zeta potential, and transmission electron microscopy (TEM). Assaying of the cell viability was done to evaluate the cellular response to toxicants. The antimicrobial activity of ZnO NPs was determined from both methods against tested pathogenic strain models. The study also focused on the biological source for NPs synthesis, especially the ecofriendly bacteria Lactobacillus spp.
| Materials and methods|| |
Natural synthesis of zinc oxide nanoparticles
Bacterial strains used in this study were of human origin isolates and were identified in a previous research . They were grown in de Man, Rogosa, and Sharpe broth (10 g peptone, 8 g meat extract, 4 g yeast extract, 20 g D(+) glucose, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate trihydrate, 2 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g magnesium sulfate tetrahydrate, 1 l distilled water, final pH 6.2) at 30°C for 48 h .
A flask was filled with 40 ml of de Man, Rogosa, and Sharpe. Then 20 ml of zinc sulfate (0.1 g/ml) was added to the first and second flasks, respectively, and stirred for half hour on a magnetic stirrer. The final concentration ultimately would be equivalent. A fresh culture incubated for 24 h of Lactobacillus johnsonii was inoculated in the flask by 0.1 ml with optical density of 0.4 nm. Then, the flasks were transferred to the incubator at 37°C for 24 h. Then, they were centrifuged at 5000 rpm for 5 min and washed with distilled water several times to removed soluble impurities then dried in an oven at 50°C for 1 h and kept for further use .
Chemical preparation of zinc oxide nanoparticles
ZnO NPs were set up as indicated by Yadav et al.  by wet substance technique with some alteration, using zinc sulfate powder with sodium hydroxide as forerunners and starch as balancing out operator. Starch 0.1% was dissolved in 500 ml of refined water by utilizing microwave. At that point, 0.1 M of zinc sulfate was added to the aforementioned arrangement under consistent blending to totally dissolving the zinc sulfate. From that point onward, 0.2 M of sodium hydroxide arrangement was included drop by drop under consistent blending until bringing about a white arrangement. The reaction was permitted to continue for 2 h after total expansion of sodium hydroxide at that point permitted to settle overnight. From that point onward, the supernatant arrangement was disposed off cautiously and the white hasten was kept. The remaining white hasten was washed multiple times by utilizing refined water, centrifuged at 5000 rpm for 5 min Finally, the white accelerate was dried at 50°C and kept for further use.
Characterization of zinc oxide nanoparticles
The UV-spectrum analysis was performed for both ZnO NPs products using: T80+UV/VIS Spectrometer (PG Instrument Ltd, range: 190–1000 nm).
Zeta potential measurements
Surface zeta potentials were measured using the laser zeta meter (Particle Sizing Systems Inc., Santa Barbara, California, USA). Liquid samples of the NPs (5 ml) were diluted with double distilled water (50 ml). The pH was then adjusted to the required value. The samples were shaken for 3 min. After shaking, the equilibrium pH was recorded and the zeta potential of the metallic particles was measured. Zeta potential was used to determine the surface potential of the ZnO NPs. In each case, an average of three separate measurements were reported. The criteria of stability of NPs are measured when the values of zeta potential ranged from +30 to −80 mV .
Transmission electron microscopy
This study was undertaken to know the size and shape of both ZnO NPs. The TEM image was performed using Electron probe micro-analyzer JEOL − JXA 840 A, Model (JEOL, Japan). Thin films of the sample were prepared on a coated copper grid by just placing a very small amount of the sample on the grid. Then the film on the TEM grid was allowed to dry and the images of NPs were taken.
Antimicrobial activity of zinc oxide nanoparticles
The media used for the antimicrobial activity of the strains under study have the following compositions (g/l): for growth of bacterial strains, nutrient agar medium was used, comprising D-glucose, 5.0; peptone, 5.0; meat extract, 5.0; NaCl, 5.0, and agar, 20.0; the pH was adjusted to 7 ), and for the growth of filamentous fungi, Czapek Dox agar medium was used, comprising sucrose, 20.0; NaNO3, 2.0; K2HPO4, 1.0; KCl, 0.5; MgSO4.7H2O, 0.001; and agar, 20.0; the pH was adjusted to 7 ).
The antimicrobial activity was assessed using various pathogenic microorganisms such as Staphylococcus aureus ATCC 29213 and Bacillus cereus, as models for gram-positive bacteria, Escherichia coli ATCC25922 and Pseudomonas aeruginosa as models for gram-negative bacteria, Candida albicans ATCC 10231 as a model for unicellular fungi and Aspergillus niger NRRL-363 as a model for filamentous fungi.
The antimicrobial activity of ZnO NPs was evaluated by the disc diffusion method by using the aforementioned test organisms. Samples were formed manually into disc shapes of 0.5 cm in diameter, dried, and subjected to UV sterilization for 2 h. Then, they were placed on the surface of agar plates freshly inoculated with the test microorganisms. The petri-dishes were kept in a refrigerator for one hour to permit homogenous diffusion of the antimicrobial agent before growth of the test microorganisms, and then plates were incubated at 37°C for 24 h for gram-positive and gram-negative bacteria and at 28°C for 72 h for filamentous fungi. The appearance of a clear inhibition zone around the sample in the inoculated Petri-dishes is an indication of the antimicrobial activity. Oxacillin 1 μg was used as reference drug with more than or equal to 1.3 cm inhibition zone for sensitive and less than or equal to 1.0 for resistance .
Cytotoxic assay of zinc oxide nanoparticles
For determination of sample cytotoxicity on cells, MTT protocol according to Senthilraja and Kathiresan  was performed. The 96-well tissue culture plate was inoculated with 1×105 cells/ml (100 μl/well) and incubated at 37°C for 24 h to develop a complete monolayer sheet. Growth medium was decanted from 96-well microtiter plates after confluent sheet of cells were formed; cell monolayer was washed twice with wash media. Two-fold dilutions of the tested sample were made in RPMI medium with 2% serum (maintenance medium). Overall, 0.1 ml of each dilution was tested in different wells, leaving three wells as control, which received only maintenance medium. The plate was incubated at 37°C and examined. Cells were checked for any physical signs of toxicity, for example, partial or complete loss of the monolayer, rounding, shrinkage, or cell granulation. MTT solution was prepared (5 mg/ml in PBS) (Bio Basic Canada Inc.). Overall, 20 μl of MTT solution was added to each well. The plate was place on a shaking table, 150 rpm for 5 min, to thoroughly mix the MTT into the media, and then incubated (37°C, 5% CO2) for 1–5 h to allow the MTT to be metabolized. Dumping of the media (dry plate on paper towels to remove residue if necessary) was done. Resuspension of formazan (MTT metabolic product) in 200 μl DMSO was done. The plate was placed on a shaking table, 150 rpm for 5 min, to thoroughly mix the formazan into the solvent. Optical density was read at 560 nm and subtract background at 620 nm. Optical density should directly correlate with cell quantity.
| Results|| |
Characterization of zinc oxide nanoparticles
UV-spectrum was used to confirm the presence of ZnO NPs. Absorption peaks obtained at 356 and 360 nm, as appeared in [Figure 1], confirmed the presence of ZnO NPs in culture filtrate in case of natural synthesis and chemical preparation, respectively. In detail, in the natural synthesis, there was an absorption peak observed at 360 nm, indicating the formation of NPs in nanosize, relatively. The absorption peak was shifted to 356; this could be attributed to the formation of smaller NPs.
|Figure 1 UV-spectrum of ZnO NPs in natural and chemical synthesis forms. NP, nanoparticle; UV, ultraviolet; ZnO, zinc oxide.|
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Zeta potential estimation
[Figure 2] and [Figure 3] summarize the zeta potential estimations of the samples in a solution structure. In the natural strategy for NPs synthesis, zeta values were estimated and observed to be −25.30 mV at pH=7. The value of the zeta potential of strategy one using naturally synthesized ZnO NPs gives full explanation about their little tendency toward aggregation.
|Figure 2 Zeta potential of naturally synthesized ZnO NPs. NP, nanoparticle; ZnO, zinc oxide.|
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|Figure 3 Zeta potential of chemically prepared ZnO NPs. NP, nanoparticle; ZnO, zinc oxide.|
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This conduct unambiguously recommends the presence of strong electric charges on the ZnO NPs surfaces to hinder agglomeration. These values were found to fall in the negative side which demonstrated the productivity of the topping materials in stabilizing the NPs by providing intensive negative charges that keep all the particles away from each other.
Transmission electron microscopy
The TEM images at 100 and 200 nm scales of natural ZnO NPs are shown in [Figure 4]. Results revealed that ZnO NPs sizes within average 7.8 nm were small spherical particles with a narrow size distribution from 5 to 9 nm relatively with smooth surfaces. The chemically prepared ZnO NPs’ TEM images showed an average size of 27.6 nm at 100 and 200 nm scales . It is obvious that NPs have a larger grain size, uniform shape, and polycrystalline in nature ([Figure 5]).
|Figure 4 TEM images at 100 and 200 nm scales of naturally synthesized ZnO NPs. NP, nanoparticle; TEM, transmission electron microscopy; ZnO, zinc oxide.|
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|Figure 5 TEM images at 100 and 200 nm scales of chemically prepared ZnO NPs. NP, nanoparticle; TEM, transmission electron microscopy; ZnO, zinc oxide.|
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Results of both ZnO NPs at the same magnification revealed complete difference between transparent and smaller naturally produced ZnO NPs and dark and enormous chemically produced ZnO NPs in morphological characters. Therefore, natural synthesis of ZnO was recommended.
Antimicrobial activity of zinc oxide nanoparticles
The disc diffusion experiment was performed against S. aureus ATCC 29213, B. cereus, E. coli ATCC25922, P. aeruginosa, C. albicans ATCC 10231 and A. niger NRRL-363. The result proved that both natural and chemical produced ZnO NPs at 1 mg/ml of have antimicrobial activity against the pathogenic tested strains under study. No critical difference was observed between the activity of naturally and chemically produced ZnO NPs. The zone of inhibition is obtained in [Table 1]. To determine the minimum inhibition concentrations of both ZnO NPs, four dilution from 1 mg/ ml were performed. Results showed that ZnO NPs at concentration 250 μg/ml was the lowest concentration that can inhibit the pathogens growth in both ZnO NPs synthesized, but in this study, the concentration of ZnO NPs was determined depending on the cytotoxicity results.
|Table 1 The antimicrobial activity of zinc oxide nanoparticles against tested pathogens|
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Cytotoxicity of ZnO nanoparticles
Results showed in [Figure 6] explain how cell viability decreases when NP concentration increases. Nevertheless, all eight concentrations of chemically prepared ZnO NPs recorded cytotoxicity effect, as shown by the percentage of viability obtained. After 5 h of exposure at 10 000, 5000, 2500, 1250, 625, 312.5, 156.25, and 78.125 μg/ml the percentages were 2.6, 2.9, 4.2, 27.6, 43.8, 71.5, 86.3, and 99.6% respectively. The results in [Figure 7] of natural synthesis ZnO NPs obtained were completely different in cytotoxicity. The viability percentages of cells at the previous concentrations were 6.3, 24.0, 43.2, 84.6, 98.2, 100, 100, and 100% respectively. These results suggest that naturally synthesized ZnO NPs cause less cytotoxicity than that of chemical prepared ZnO NPs. Furthermore, the decline in cytotoxicity resulting from ZnO NP exposure was dependent on the concentration of ZnO NPs.
|Figure 6 Cytotoxicity percentages of chemically prepared ZnO NPs. NP, nanoparticle; ZnO, zinc oxide.|
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|Figure 7 Cytotoxicity percentages of naturally synthesized ZnO NPs. NP, nanoparticle; ZnO, zinc oxide.|
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ZnO NPs treatment results in a noticeable change in morphology of cells. Therefore, examination was performed using microscopy. As appeared in [Figure 8], cell shape in the control remained normal; the cells adhered well, with most of them attaching. Most cells were polygonal, with transparent cytoplasm, had better scattering, and had a few new cells during the process of adhering. After treatment with the lowest concentration of chemically prepared ZnO NPs, morphology of cell significantly changed. Although cells adhered, they could not spread, and some lost the polygonal and became rounded shape. When the concentration of ZnO NPs was increased, the treated cells atrophied and could not adhere, suggesting that cell viability was lower than that of the control cells. These results indicate that the chemically produced ZnO NPs is adequate mostly like less than 625 μg/ml. In contrast, cell morphology after treatment with naturally synthesized ZnO NPs in [Figure 9] was not significantly different from that of the control group, and most of the cells could adhere and spread. A comparative pattern was seen in cells treated with ZnO NPs in both treatment groups increased to approximately above 1250 μg/ml, respectively, with fewer dead cells observed after treatment with natural NPs, indicating that natural ZnO NPs are less cytotoxic than chemical ZnO NPs.
|Figure 8 Morphology changes in Vero cells after 5 h exposure to chemically prepared ZnO NPs at concentrations of 10 000, 5000, 2500, 1250, 625, 312.5, 156.25, and 78.125 μg/ml in a, b, c, d, e, f, g, h, and i as a control with normal cell shape respectively. NP, nanoparticle; ZnO, zinc oxide.|
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|Figure 9 Morphology changes in Vero cells after 5 h exposure to naturally synthesized ZnO NPs at concentrations of 10 000, 5000, 2500, 1250, 625, 312.5, 156.25, and 78.125 μg/ml in a, b, c, d, e, f, g, h, and i as a control with normal cell shape, respectively. NP, nanoparticle; ZnO, zinc oxide.|
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| Discussion|| |
Characterization of ZnO NPs using UV-spectrum performed and absorption peaks at 356 and 360 nm confirmed the presence of ZnO NPs in culture filtrate in case of natural synthesis and chemical preparation, respectively.
Zeta potential is a physical property which is given the net surface charge of the NPs when these particles inside the arrangement repulsing each other’s since produced Coulomb explosion between the charges of the NPs offering ascend to no tendency for the particles to agglomerate. The criteria of stability of NPs are measured when the estimations of zeta potential ranged from higher than +30 mV to lower than −80 mV .
This outcome recommends that the ZnO NPs and thus their solution are stable which is also in accordance with the result reported before at [Figure 2] and [Figure 3] NPs scattering conduct. The value of zeta potential in strategy two using chemical preparation ZnO NPs was −18.6 mV at pH=7 likewise negative charge with a diameter about 27.6 nm, which indicate that no basic contrast between two strategies in zeta potential measurements. Estimations of the zeta potentials of the natural synthesis ZnO NPs in addition to their narrow size distributions give acceptable evidence about their little tendency toward aggregation. Disruption of the surface charges assumes a foremost work in the assembly of the NPs. Therefore, using these NPs as medication may provide a new horizon in avoiding microbial resistance. In the examined arrangement the electric charges were sufficiently able to ruin agglomeration and to give adjustment of the NPs .
Several researchers discussed the effect of zinc NPs on the microorganisms and proved that, the activity of ZnO NPs against the pathogen was due to a response of the surface of ZnO NPs with water which led to formation of hoisted levels of receptive oxygen species, to be specific hydroxyl radicals and thus actuate as oxidative anxiety. Moreover, a presentation of microorganisms with ZnO NPs results in an expanded cell disguise of the NPs and microbial cell harm. Among metal oxide powders, ZnO demonstrates very significant growth inhibition of a broad spectrum of bacteria. The suggested mechanism for the antibacterial activity of ZnO is based mainly on the catalysis of formation of the reactive oxygen species from water and oxygen that disrupt the integrity of the bacterial membrane, although additional mechanisms have also been suggested. As the catalysis of radical formation occurs on the particle surface, particles with larger surface area demonstrate stronger antibacterial activity.
Consequently, as a result, the antibacterial activity of the ZnO particles increased when the size of particles decreases . The synthesis of NPs by the biological method is ecofriendly, simple and can be used as catalysts composition, which the classical methods cannot be produced it, NPs applications in medicine and sensors are envisaged. Also, bacterial NPs can be used to control human pathogens .
Viability assays are essential for evaluating the cellular response to toxicants. To measure total cell viability following NP exposure, we used MTT assay . These results show highly cytotoxicity values as reported by Brunner et al.  in ZnO NPs prepared chemically which used the same cell line, lower concentrations, and similar particle size. However, other authors reported significant lower cytotoxicity (with larger or smaller particles size than this study) using the same concentrations and cell line. ZnO NPs cytotoxicity is still controversial and the mechanism has not been well identified ,. In comparison indicating that natural ZnO NPs are less cytotoxic than chemical ZnO NPs .
| Conclusion|| |
ZnO NPs synthesized using natural and chemical methods were characterized by UV spectroscopy, zeta potential, and TEM to decide properties of NPs. ZnO NPs showed antimicrobial activity against pathogenic strains models used. However, the natural ZnO NPs was less cytotoxic than that of chemical prepared ZnO NPs. So that natural synthesis of ZnO was recommended.
The author acknowledges Prof. Dr Abd Alsalam El-Mohamady professor of nanotechnology and president of Arab Center for Nanotechnology who supported this study and for kindly advices and for providing some chemical materials; the unit of tissue culture at Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt, for supporting the cytotoxicity experiment with experience; and the National Research Centre, Egypt, without the support of which this study could not have been possible.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]