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2023.08.02.75
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Bio-Synthesis of zinc oxide nanoparticles and detect its antitumor activity against human skin cancer cell line (A375).
Abid W E1, Gdayea I A2 and Oraibi A G.3,*
1 Al-Iraqia University, College of Education, Biology Department, Baghdad, Iraq.
2 Al-Iraqia University, College of Education, Biology Department, Baghdad, Iraq.
3 Al-Nahrain University, College of Biotechnology, Baghdad, Iraq.
* Corresponding Email: [email protected]
Available from: http://dx.doi.org/10.21931/RB/2023.08.02.75

ABSTRACT

In this study, Lepidium sativum seeds were collected from the local markets in Baghdad. Zinc oxide nanoparticles were manufactured from the aqueous extract of  Lepidium sativum by adding zinc acetate in a green, safe and environmentally friendly manner. The formation of zinc oxide nanoparticles was inferred by changing the color of the extract from white to yellow, and for Detection of biosynthetic zinc oxide nanoparticles Examinations were conducted to detect these nanoparticles, including diagnosis using atomic force microscopy (AFM), which showed that the size of the nanoparticles (13) nm and the surface roughness (80.51) nm compared with the aqueous extract, which amounted to (23) nm and (57.22) nm respectively. The diagnosis using UV rays showed a peak absorption of nanoparticles at 350 nm compared with the aqueous extract, which reached 248 nm. As for the scanning electron microscope (SEM) examination, the nanoparticles' size ranged between 46.97 - 81.07 nanometers, compared with the aqueous extract, which reached 676 - 591.8 nanometers. To determine the toxic or inhibitory effect against A375 cancer cells and HdFn normal skin cells, MTT cytotoxicity assay was performed for aqueous extract and zinc oxide nanoparticles. The results showed that the aqueous extract was effective against the cancerous cell line A375, as the viability of the cells decreased with the increase in the concentration of the aqueous extract. The IC50 ratio was equal to 140.0 mg/ml for the A375 cancer cells, and the IC50 ratio was equal to 179.9 mg/ml for the normal HdFn cells. As for zinc oxide nanoparticles, the effectiveness was more substantial than that of the aqueous extract, and the vitality of cells was reduced in the cancerous line A375. The normal line HdFn, the higher the concentration of nanoparticles, and the IC50 ratio was equal to 59.46 mg/ml for the cancerous line. The IC50 ratio was equal to 196.9 mg/ml for the normal line.
Keywords: Zinc oxide nanoparticles, Lepidium sativum, antitumor activity or A375 cancer cells.
INTRODUCTION

Many plants have been used all or part of them in traditional medicines for thousands of years. Still, without sufficient scientific evidence, and since these materials contain effective natural substances such as phenols, flavonoids and other substances, this may lead to developing new natural drugs with low side effects or without effects 1. The seeds of the plant Lepidium sativum, which belongs to the Brassicaceae family, are substances used in folk remedies, as they are used in the treatment of many diseases such as scurvy, asthma, and coughing. They are also used in treating pimples and as a diuretic 2, 3. It was also found that the active components in the cress seeds stimulate apoptosis in cancer cells, thus killing cancer cells specifically without harming normal healthy cells. 4,5,6.    Over the past few decades, nanotechnology has been increasingly used in medicine, including applications for diagnosis, treatment, and targeting of tumors more safely and effectively. Nanoparticle-based drug delivery systems have shown many advantages in cancer treatment, such as precise targeting of tumor cells, reduction of side effects and drug resistance 7,8. It has been suggested that nanoparticles can play a role in treating cancer cells and the genes responsible for them 9. Studies on applying NP drugs in immunotherapy and ablation therapy for cancer 10,11 also supported this. This nanoparticle-based drug delivery system is believed to enhance immunotherapy 12. Many nanotherapeutic drugs have been commercialized and introduced into the clinical stage in recent years. A phase 1 clinical trial that used a targeted nanoparticle-based system to deliver small interfering RNA (siRNA) in patients with cancer was conducted in 2010 13. NPs have been shown to provide platforms for combination drug therapy and inhibit some drug resistance mechanisms, such as efflux vectors on cell membranes 14. This was also supported by studies on breast cancer (Alimoradi et al., 2018), ovarian cancer 15 and prostate cancer 16.
MATERIAL AND METHODS
Plant sample collection
The seeds of the Lepidium sativum plant were collected from the local markets in Baghdad during September 2021 and were confirmed by the Seed Examination and Certification Department / Ministry of Agriculture.

Preparation of the plant aqueous extract of the seeds of the Lepidium sativum plant
The seed Lepidium sativum plant extract was prepared using the traditional method 18. By washing the seeds with water well to remove the contaminants from the surface and soaking them for 30 minutes, then removing the moisture from them and drying them well with dry air, 10 g of dried flaxseeds were ground and placed in a 250 ml glass beaker containing 100 ml of deionized water, then The mixture was heated in a water bath at 45 °C for 30 min, after which the extract was filtered using Whatman No. 1 filter paper. The filtrate was stored at 4 °C for later use.

Manufacture of zinc oxide nanoparticles
Zinc oxide particles were prepared for the extract of Lepidium sativum by melting 50 ml of aqueous plant extract at a temperature of 40-45 °C, then 5 grams of zinc acetate were added to the solution with continuous heating until it turned into a cohesive yellow paste. The paste was collected in a glass Petri dish and dried at 45°C for 12 hours; then, the dried material was ground to obtain a powder that was stored for detection, characterization and other studies.

Detection and characterization of biosynthetic zinc oxide nanoparticles.
The prepared samples were diagnosed and compared to the aqueous extract using the following methods:

Atomic Force Microscopy (AFM)
Atomic force microscopy (AFM) assay was used to determine the surface morphology of the as-prepared ZnO nanoparticles and their size and diameter. A small drop of the sample solution, prepared in advance, was placed on an 11x cm glass slide, left to dry at room temperature, and ready for testing 19.

Ultraviolet-Visibie spectrometer (UV)
1 ml of the aqueous extract of thyme seeds and zinc oxide nanoparticles was taken and diluted with deionized water and then centrifuged for 5 minutes in a centrifuge at a rate of 3000 rpm, then 1 ml was taken and examined within the wavelength 200-1100 nm by a UV spectrometer according to the method 20.

Energy-dispersive X-ray spectroscopy
Energy-dispersive X-ray spectroscopy (EDX) confirms that the synthesis process produces pure nanoparticles 21.

Scanning Electron Microscopy (SEM)
The Japanese Meiji SEM scanner was used to determine the shape and size of the particles in the prepared samples 22. Place approximately 5 μl of ready-to-examine solutions onto a clamped gold-carbon electron microscope holder, let them dry at room temperature, and test them with different magnifying powers.

Cell lines
In this study, the natural skin cell line HdFn and the cancer cell line A375 (melanoma) were used, which were obtained from the University of Malaya/ College of Medicine/ Department of Pharmacy/ Center for Natural Product Research and Drug Discovery/ Department of Malaysia Pharmacology/ Faculty of Medicine University of Malaya Kuala Lumpur/Malaysia The cancer line was maintained and developed. Tests were conducted on it at the Biotechnology Research Center at Al-Nahrain University.

Sterilization methods:
Wet heat sterilization
The solid and liquid culture media and the used solutions were sterilized by osmosis at a temperature of 121°C and at a pressure of 15 pounds/ing2 for 15 minutes 23.

Dry heat sterilization
The needles used to grow and activate the bacteria until they reached redness were sterilized by direct Bunsen flame, and the glassware was sterilized for two hours at a temperature of 150 ° C in an electric oven 24.

Sterilization with chemicals
The culture room (Laminar air flow cabinet) was sterilized with 70% ethyl alcohol and 30% chlorine.

Sterilization by filtration
The plant extracts and the heat-sensitive filtration-prepared nanosolutions were sterilized using 0.22 µm osmotic microfilters 24.
Preparation of solutions and reagents used in tissue culture
Prepared solutions according to 25.
Phosphate buffer solution Phosphate – brine
This solution was prepared by dissolving 8 g of NaCl salt, 0.2 g of KCL, 1.15 g of Na2H2PO4 and 0.2 g of Na2HPO4 in 900ml of distilled water, and the pH was adjusted to 7.2. the use.
Trypsin Solution
The Preparation was prepared by dissolving 1g of trypsin powder in 100 ml of PBS phosphate buffer solution and then sterilizing it with a filter with holes with a diameter of 0.22 µm. Then it was divided into 10ml tubes and stored at -20°C.
Trypan Blue dye
The Preparation was made by dissolving 1 g of dye powder in 100 ml of phosphate buffer solution to prepare a 1% solution; then, it was filtered using a filter with holes with a diameter of 22.0 μm and kept at a temperature of 4 °C until use.
EDTA solution
The Preparation was prepared by dissolving 1g of Ethylene Diamine - Tetra Acetic Acid (EDTA) in 100 ml of PBS phosphate buffer solution and sterilizing the solution by oxidation for 10 minutes. The resulting solution was divided into 10ml doses in tubes and kept at 4°C until use.
Trypsin - EDTA. Solution
The Preparation was prepared by mixing 20ml of trypsin solution, 10ml of EDTA and 370ml of phosphate buffer PBS. The mixture was kept at 4°C.
Serum Free Medium
It is a ready-made food medium (RPMI-1640) extracted from fetal bovine serum.
Development and maintenance of the A375 and HdFn cell lines.
The method (Freshney, 2015) followed the following steps.
The cells were treated individually using a water bath at a temperature of 37 °C.
The cells were placed in an animal cell culture vessel with a diameter of 25 cm 2, which contained RPMI=1640 culture medium on 10% bovine calf serum.
Incubating the container containing the cell suspension as well as the culture medium in a CO2 5% incubator at a temperature of 37 °C for 24 hours and after a day of incubation and after making sure that the cell farm was growing and free from pollution, secondary farms were conducted
The cells were examined using an inverted microscope to ensure their viability, contamination-free, and growth to the required number (700-600) cells/ml.
The cells were transferred to the growth cabin, and the used culture medium was disposed of.
The cells were washed with PBS solution and discarded. The process was repeated twice for 15 minutes each time.
A sufficient amount of trypsin enzyme was added to the cells, incubated for 60-30 seconds at 37 °C, and monitored until they changed from a monolayer of cells to single cells. Then, the enzyme was stopped by adding a new medium (BSA) Bovine Serum albumin-containing serum.
The cells were collected in centrifugal tubes and placed in a centrifuge machine at a speed of 2000 rpm for 10 minutes at room temperature to precipitate the cells and eliminate the trypsin and the used culture medium.
The filtrate was discarded, and the cells were suspended in fresh culture medium containing 10% serum.
Examination of the number of cells by taking a specific volume of the cell suspension and adding the same volume of Trypan Blue dye to determine the number of cells and their vitality percentage using a Hemacytometer chip and according to the following equation
C= N×104×F/ml
whereas:
C:   The number of cells in one ml of solution
F:    mitigating factor
N:    The number of cells in the slide
104:   Slide dimensions.
The percentage of cell vitality in the sample was calculated using a Hemacytometer chip according to the following equation:

The cell suspension was distributed in new containers and incubated in a CO2 5% incubator at 37°C for 24 hours.
2.6. MTT assay to check cell viability after treatment with zinc oxide particles.
The values ​​of samples IC50 were calculated using the logarithmic dose inhibition curve or assay to determine the cytotoxic effect of the nano-extract of the seeds of thyme plant on the cancer cell line A375 and HdFn to use it in other assays High content screening (HCS).
The contents of the MTT examination kit.
MTT solution 1 ml × 10 glass vials
Solubilization solution 50 ml × 2 bottles
This method was carried out according to Rashid et al. (2017).
The cancer cells were prepared (1 x 104-1 x 1106 x cell/ml-1) according to the section on cell development (3-2-7-1), then flat base and covered. The dishes were gently covered with sterile parafilm, stirred, and incubated in a 5% CO2 incubator at 37 °C for 24 hours. After incubation, the medium was removed.
After incubation, 100 µl of cell suspension was added to each pit of the same plate.
The prepared concentrations of the aqueous extract of turmeric seeds and zinc oxide nanoparticles (400, 200, 100, 50, 25, 12.5) μl were added to the pits in the plate.
The plate was incubated for 24 hours at a temperature of 37 °C.
10 μl of MTT solution was added to each hole at a 0.45 mg/ml concentration.
The plate was incubated for 4 hours at 37°C.
100 μl of solubilization solution was added to each hole to dissolve Formazan crystals and then incubated for 5 minutes.
I was reading the absorbance of the sample at a wavelength of 570 nm using an ELISA device (Bio-rad Germany) and at a wavelength of 575 nm.
Statistical analysis of optical density readings to calculate IC50 according to the following equation:

In the same way, the toxicity of zinc oxide nanoparticles was investigated on the normal HdFn cell line.
RESULTS
.
Phenotypic characteristics of aqueous extracts of flaxseed and biosynthesized zinc oxide nanoparticles.
In Figure 1, the color of the aqueous extract of the plant appeared in white. It was noted that after adding zinc acetate solution to the aqueous extract of the plant, the color of the extract turned yellow. This color change is the first sign of the formation of nanoparticles made of zinc oxide particles from plant leaf extract, where they manufactured zinc nanoparticles in safe and environmentally friendly ways.
     

Figure 1. It shows the color change of the aqueous extract during the formation of nanoparticles
Detection and characterization of zinc oxide nanoparticles for the extract of the plant in comparison with the aqueous extract:
Zinc oxide nanoparticles and aqueous extract were diagnosed using atomic force microscopy (AFM).
The results of the AFM technique showed that the zinc nanoparticles manufactured from the sage plant extract had different surface topography measurements. Figure 2 shows that the size of the biosynthesized nanoparticles was 13 nanometers, the size of the particles of the aqueous extract of the plant extract was 23 nanometers, and the roughness rate of the minute Nanoparticles was 80,51 nm, and the aqueous extract (57,22) nm. The result of our research was consistent with the research line of Daphedar and Taranath (2018). The size of ZnO NPs nanoparticles ranged between 10 and 85 nanometers.
     
Figure 2. Diagnosing biosynthetic nanoparticles using an atomic force test device, where A: aqueous extract B: biomanufactured nanoparticles.
Diagnosis of nanoparticles and aqueous extract using UV spectroscopy.
By using a spectrophotometer to detect zinc oxide nanoparticles, it was shown in Figure 4-3 that the highest absorption peak of nanoparticles at a wavelength ranged between 210-350 nm and it is within the absorbance limits of zinc oxide nanoparticles, which confirms the success of the biosynthesis process of zinc oxide particles.
Figure 3. Characterization of zinc oxide nanoparticles and aqueous extract using a UV absorber
Diagnosis of zinc oxide nanoparticles and aqueous extract using X-ray Distributed Energy Spectrometry (EDX).
The results showed in Figure (4a) that the components of the aqueous extract from the elements included C, Cl, O, Na, K, S, Mg, Al, according to the weight ratios shown (21.7, 20.7, 18.6, 14.1, 11.2, 10.2, 1.8) sequentially, while the results in Figure 4b of the nano-extract showed the appearance of the following elements (Zn, C, O, Ca) and in the following weight ratios (44.9, 25.5, 25.1, 4.5) respectively.
     


Figure 4a,b. Diagnostics of printed nanoparticles using EDX; A- Aqueous extract of currant seeds; B- Biosynthesized ZnoNPs
Analysis of nanoparticle images and aqueous extract using scanning electron microscopy (SEM).
It was shown in Figure 5a that the size of zinc oxide nanoparticles ranged between 46.97 and 81.07 nanometers, and this agrees with the accepted sizes of biosynthetic nanoparticles. The results in Figure 5b showed that the size of the particles of the water extract of the aqueous extract ranged between 591.8 and 676 nanometers, and this shows the clear difference between the size of the nanoparticles and the size of the particles of the water extract. It was scanning electron microscopy of ZnO NPs with a size ranging between 62 and 94 nm 31.
     
Figure 5a. Image showing the nano sizes of zinc oxide nanoparticles by SEM
     


Figure 5b. Image showing the nano sizes of the aqueous extract of Lepidium sativum by SEM

MTT cytotoxicity test
The results of the test showed that the nanoparticles showed effectiveness against A375 skin cancer cells, as the viability of the cells was 52.83%, 63.70%, 77.62%, 90.59%, 95.95% at concentrations of 400, 200, 100, 50 and 25, respectively.
At the same time, the nanoparticles showed cytotoxicity on HdFn normal cells, as the cell viability was 66.47%, 74.46%, 90.97%, 94.64%, 95.29% at concentrations 400, 200, 100, 50, 25, respectively, as shown in Table (1).

Table 1. shows the effect of zinc oxide nanoparticles on cancer cell line A375 and normal line HDFn using MTT assay.
   
 

In addition, the results showed significant differences P ≤ 0.0001 when calculating IC 50 for both normal cells and cancer cells, where the IC value of 50 for A375 cancer cells reached 129.3 µg/ml after being treated with nanoparticles, and the IC value of 50 for normal cells HdFn 160.1 µg/ml, as shown in Figure 6.

Figure 6. shows the IC50 values ​​for the cancer cell line A375 and the normal line HdFn when treated with zinc oxide nanoparticles.
Also, the MTT toxicity assay was used to determine the toxic effect of the plant extract on the skin cancer cell line A375 and the normal line HdFn. The results showed that the aqueous plant extract showed effectiveness against the cell line A375, as the cell viability was 54.78%, 61.92%, 75.81, 82.99, 85.03 at concentrations 400, 200, 100, 50, and 25, respectively. At the same time, the plant extract showed an effect on the HdFn cell line, as the cell viability ranged by 70.80, 79.78, 89.35, 95.33, and 95.22 at concentrations 400, 200, 100, 50, 25 respectively, as shown in Table 2.

Table 2. shows the effect of the aqueous extract on the cancer cell line A375 and the normal line HdFn using the MTT test.
   
In addition, the results showed significant differences P ≤ 0.0001 when calculating the IC50 when the plant extract was treated with A375 cancer cells, the IC50 ratio was (140.0 µg/ml) and the normal HdFn cells, the IC50 was equal to (179.7 µg/ml), as shown in Figure (7).

Figure 7. shows the IC50 values ​​for each cancer cell line A375 and the normal line HdFN when treated with aqueous extract.
Several studies have confirmed the toxicity of zinc oxide nanoparticles and their ability to inhibit many types of cancer, with different levels of inhibition depending on the concentration of zinc oxide nanoparticles and according to the type of cancer cell line used in the study. Significant acid damage was also observed. Nuclear in cells treated with the highest concentration of ZnO-NPs These results indicate that ZnO-NPs have toxic and inhibitory potential in A375 cells and may induce oxidative stress.
To analyze the anticancer efficacy of ZnO NPs, human hepatocellular carcinoma (HepG2) and normal murine hepatocytes (hepatocytes) cells were used, and the cytotoxicity was determined using the MTT assay. Concentrations up to 5 μg/ml have not been shown to lead to a significant loss of cell viability. However, concentrations of 10-15 μg/ml are beneficial. HepG2 vitality was reduced by 33%, while normal hepatocytes remained unaffected 45.
DISCUSSION
This result was consistent with 27, who synthesized zinc oxide particles from aloe vera and starch extract, and 28, who synthesized zinc oxide particles from Lippia adoensis and 29. Another research path was the size of ZnO NPs. In another research path, the size of ZnO NPs was 50-82 nm 39. In another study, zinc oxide nanoparticles were synthesized from garlic skin extract. The particle size was found to be about 25 nm, and the maximum surface particle size of ZnO NPs was 23.34 nm for the study carried out by 32. Nanoparticles because the values ​​between 300-380 nm are among the diagnostic properties of zinc oxide nanoparticles due to the plasmon surface absorption, while the ultraviolet measurement showed that the highest absorption peaks of the aqueous extract molecules were 248 nm. 33. and 375 nm 34. While the absorption peak was close to our study by researcher 35, it was 351 nm. This slight difference in the absorbance values ​​may be due to several factors, including the conditions and method of manufacturing and the plant or its parts from which the nanoparticles were manufactured. These results showed the disappearance of some elements during the bio-manufacturing process of nanoparticles with the presence of zinc at the highest weight percentage, and this is evidence of the formation of nanoparticles and the entry of some influential groups and elements in the aqueous extract into the manufacturing process. This result was within the research line of 36. Whereas EDX analysis confirmed the presence of O and Zn elements on the sample's surface, similar peaks were reported 37. In a study by 38 the EDX of NPs revealed a precise formation of zinc oxide nanoparticles where the atomic weight of oxygen was 48.83% while its previous weight was 21%. On the other hand, the atomic weight of Zn was 37.16%, while its last weight was 65.35%, while the other minor components present in ZnO-NPs are due to the presence of ginger root extract. In another study, the diameter of ZnO NPs was determined using SEM technique in the range of 20-80 nm 36. In another line of research, the shape and size of zinc oxide nanoparticles were determined by electronic scanning, and the SEM image showed zinc oxide nanoparticles manufactured from Cassia alata leaf extract that most of the nanoparticles are rod-formed within the diameter range of 30-50 nm. 40 This is consistent with our current study. The toxic effect of the aqueous extract on cancer cells may be due to secondary metabolites; for example, phenolic compounds inhibit several enzymes responsible for DNA replication in cancer cells, such as the enzyme Topoisomerase I, II 41. Several studies have indicated that eating food containing such compounds leads to a healthy lifespan and prevents heart attacks and cancer 42. In a study compatible with ours, 43. They revealed the toxic effects of ZnO-NPs on human melanoma A375 cells. The results showed induction of apoptosis as confirmed by tests for chromosomal condensation and caspase-3 activation. In a study, ZnO NPs were exposed to a liver cancer cell line, and the results indicate that ZnO NPs stimulate autophagy, downregulate the expression of caspase 3 and p53 markers, and trigger apoptosis in HCC cells, thus limiting HCC cell growth and proliferation 44. In another study, ZnO nanorods were prepared with album Santalum and dose-dependent cytotoxicity against MCF-7 cells was found. The results revealed that ZnO nanorods induced apoptosis via an intrinsic mitochondrial pathway dependent on caspase activation 46. Similarly, another study using ZnO nanorods manufactured from Leea asiatica extract against MCF-7 cancer cells reported similar results 47. ZnO NPs prepared from aerial parts of Deverra Tortusa were evaluated against human colon cancer (Caco-2) and A549 cell line. Through ROS stimulation, ZnO NPs showed cytotoxicity against these two cell lines, while the human lung fibroblast line (WI38) did not show significant cytotoxicity 48.
CONCLUSION

It can be concluded from this study that the vitality of cells in the cancerous line and the normal line decreases with increasing concentration of nanoparticles. As well as that the viability of cells in the cancerous line and the normal line decreases with increasing concentration of the aqueous extract.
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Received: May 15, 2023/   Accepted: June 10, 2023 / Published: June 15, 2023


Citation: Abid W E, Gdayea I A, Oraibi A G. Bio-Synthesis of zinc oxide nanoparticles and detect its antitumor activity against human skin cancer cell line (A375). Revis Bionatura 2023;8 (2) 75. http://dx.doi.org/10.21931/RB/2023.08.02.75
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