IHJPAS. 36 (4) 2023 110 This work is licensed under a Creative Commons Attribution 4.0 International License *Corresponding Author: Intessar.Hameed1102a@ihcoedu.uobaghdad.edu.iq Abstract In the current study, gold nanoparticles were made using Acinetobacter baumannii broth cultures. UV-visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and zeta potential measurements were also used to study the properties of the Ab-AuNPs. The average size was 66 nm. The examination results proved that the Ab-AuNPs are semi-spherical and varied from 20 to 90 nm. MTT assays on the breast cancer cell line MCF-7 confirmed the anticancer activity in vitro. Cancer cells showed important cytotoxic activity for Ab-AuNPs. The mean lethal dose (IC50) was 11.45 µg/mL, and the maximal inhibitory concentration was 25.50 mg/mL (63.00% and 86.33%, respectively), but ineffective against the normal cell line. MCF-10. The results proved that Ab-AuNPs have DPPH scavenging activity, which increases with increasing concentration of Ab-AuNPs, where the concentrations (3.1, 6.25, 12.5, 25, and 50) mg/ml gave DPPH scavenging activity with the following values: 37.87%, 50.13%, 59.33%, 75.55%, and 85.13%, respectively. The present study concludes that gold nanoparticles synthesized using A. baumannii broth cultures are easy to prepare, inexpensive, and non-toxic to normal cells. Meanwhile, they possess antioxidant and anticancer activity. So, it can be used as an alternative treatment. Keywords: Gold nanoparticles, Acinetobacter baumannii, Antioxidant activity, Anticancer activities. 1. Introduction Article history: Received 13 December 2022, Accepted 20 Fabruary 2023, Published inOctober 2023. doi.org/10.30526/36.4.3141 Anticancer and Antioxidant activity of Gold Nanoparticles Biosynthesized Using Acinetobacter baumannii Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq Esam J. Al-kalifawi Department of Biology, College of Education for Pure Sciences Ibn-Al-Haitham University of Baghdad, Baghdad, Iraq. Intesar H. Al-Abdeli* Department of Biology, College of Education for Pure Sciences Ibn-Al-Haitham University of Baghdad, Baghdad, Iraq. https://creativecommons.org/licenses/by/4.0/ mailto:Intessar.Hameed1102a@ihcoedu.uobaghdad.edu.iq mailto:aesam365@yahoo.com mailto:Intessar.Hameed1102a@ihcoedu.uobaghdad.edu.iq IHJPAS. 36 (4) 2023 111 Humans develop cancer, a potentially fatal condition, when normal cells are repeatedly exposed to carcinogens. Malignant tumors are collections of cancerous cells that differ from healthy cells in that they develop and spread uncontrollably [1]. Due to its inherent anticancer action, the field of study on using nanoparticles (NPs) to prevent tumor formation, growth, and progression is expanding. Physicochemical properties of NPs that contribute to their anticancer activity include either intrinsic characteristics, like the antioxidant effect, or processes dependent on external stimuli, like hyperthermia, in response to the application of infrared rays or magnetic fields. [2]. The capacity to enter and accumulate in cancer cells, followed by heat therapy. Then, the physical and chemical properties of the built-up gold nanoparticles can help kill cancer cells without hurting normal cells in the body. This is the goal of the treatment compared to other methods like chemotherapy, which has a lot of bad side effects for the patient, like hair loss and damage to healthy blood cells, causing vomiting or infections. [3]. Breast cancer is the second most common cancer diagnosis in women worldwide, and it is thought to be one of the main reasons that breast cancer accounts for 10.4% of all cancers in women. Men, however, frequently have the illness. [4]. The chances of having breast cancer have been found to increase with age. [5]. Due to their shape, size, excellent optical and electronic properties, high biocompatibility, and stability, gold nanoparticles are the most commonly used metal nanoparticles [6]. The biosynthesis of gold nanoparticles (AuNPs) would be easier if microorganisms, like bacteria and fungi, used methods that were clean, non-toxic, and good for the environment. Bacteria have always been the organisms of choice because they have the unique ability to make enzymes that are involved in the decrease and stability of metallic nanoparticles [7]. After gold nanoparticles enter most cancer cells, a series of processes evolve from the loss of mobile stability and an unstable oxidative state, and waves of free radicals begin to destroy and expand the nuclear envelope of the nucleus, and mitochondria unfold and increase the oxidative stress in the cell [8]. The current study aims to synthesize gold nanoparticles using A. baumannii broth culture and studies their antioxidant and anticancer activity. 2.Materials and Methods 2.1.Isolation and identification of A. baumannii A. baumannii isolates were obtained from the microbiology laboratories at the Yarmouk Teaching Hospital. It was confirmed by Gram staining, the growth on different media, and biochemical tests, including catalase, coagulase, urease, and oxidase. Finally, the use of the Vitek 2 compact system [9]. 2.2.Green synthesis of gold nanoparticles. IHJPAS. 36 (4) 2023 112 AuNPs were produced using the modified method described in [10]. Ten millilitres of stock solution of (HAuCl4.4H2O) were combined with (2,4,6,8 and10) mL of A. baumannii broth culture and heated at 50°C for 30 minutes on a magnetic hotplate stirrer. The resulting mixture [(HAuCl4.4H2O) +4mL of A. baumannii broth culture] was placed in a tube and subjected to a 30-minute ultrasonic bath. Following the development of red dots on the sides of the transparent tube inside the Ultrasonic Bath device, the colour of the solution changed from bright yellow to yellowish-orange. Each time the procedure is carried out, two ml of the bacterial broth culture is added to the gold salt solution while keeping the other parameters, such as the concentration of the gold salt solution, the temperature, the time, and a constant pH value, constant to produce an orange-coloured solution[(HAuCl4.4H2O) +6mL of A. baumannii broth culture]. It changed to a light purple[(HAuCl4.4H2O) +8mL of A. baumannii broth culture], then to a darker purple[(HAuCl4.4H2O) +10mL of A. baumannii broth culture], which showed that the gold nanoparticles in Figure 1 had been created. Figure 1. Illustration graphic of biosynthesis of gold nanoparticles using broth culture of A. baumannii. 2.3. Characterization of Gold Nanoparticles: Ab-AuNPs were characterized using many techniques, including UV-Vis spectroscopy (Shimadzu UV-160A), FTIR spectroscopy (IRAffinity1Shmadzo), FE-SEM, TEM (Carl Zeiss, Germany), XRD (Philips PW1730), AMF, and Zeta potential measurement. 2.4. Biological applications of prepared gold nanoparticles 2.4.1.Cancer Cell Lines The breast cancer cell line (MCF-7) was compared with the normal cell line (MCF-10) obtained from the cell bank of the Iraqi Center for Cancer and Medical Genetics Research. MCF-7 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 µg/mL, of streptomycin. The cells were then passaged using trypsin-EDTA, reseeded at 80% confluence twice a week, and incubated at 37°C [11, 12]. IHJPAS. 36 (4) 2023 113 2.4.2. Determination of cytotoxicity of Ab-AuNPs-using MTT assay Using the MTT assay, the cytotoxic effect of Ab-AuNPs was measured according to [13]. For the MTT test, 96-well plates were used. 1 × 104cells/well cells/well were used to start the cell lines. After 24 hours or when a confluent.monolayer was formed different concentrations of the tested compounds were added to the cells. After 72 hours of treatment, the cell's ability to live was tested by taking out the medium, adding 28 µL of 2 mg/mL of 2 mg/mL, MTT, and letting them stay at 37 °C for 2.5 hours. After taking out the MTT solution, the crystals still in the wells were broken up by adding 130 L of DMSO (Dimethyl Sulphoxide) and shaking the wells for 15 minutes at 37 °C. The absorbency was measured at 575 nm with a microplate reader. The test was done three times. The following equation was used to determine how much cell growth was stopped (the percentage of cytotoxicity). Inhibition rate = A- B/A*100" A represents the optical density "of the standard, and B represents the optical density of the samples. To examine the shape of cells through an inverted microscope, the cells were seeded into 424-well micro-titration plates at a density of 1×105 cells mL−1 and incubated for 24 h at 37 °C. Then, cells were exposed to Ab-AuNPs.at IC50 for 24 hours. After the exposure time, the plates were stained with crystal violet and incubated at 37 °C for 10–15 minutes. The stain was gently washed away with tap water until the dye was eliminated. At 40×magnification, the cells were observed with an inverted microscope, and images were captured with a digital camera attached to the microscope. 2.4.3.Determination of DPPH Scavenging Assay of Ab-AuNPs Free Radical Scavenging (DPPH) Activity Assay was used to test the antioxidant activity of the biosynthesized Ab-AuNPs. [14]. 2.5. Statistical analysis GraphPad.Prism 6 was used to analyze the data which was presented as the mean standard deviation±(SD) for three replicates per experiment. [15]. 3.Results Figure 2 shows the change in colour from yellow to dark violet, evidence of the biosynthesis of Ab-AuNPs using A. baumannii broth culture. IHJPAS. 36 (4) 2023 114 Figure 2. Exhibit solutions after changing their colour from yellow to dark violet. 3.1. UV-Visible analysis Figure 3 shows the UV-Visible spectrum of Ab-AuNPs made with an A. baumannii broth culture. Ab-AuNPs had surface plasmon resonance (SPR) bands around 574 nm in their UV- Visible spectra While the absorbance peak of A. baumannii broth culture was at 301.5 nm by UV- Visible spectrophotometer. Figure 3. UV-Vis spectrum of Ab-AuNPs colloidal and A. baumannii broth culture. FT-IR spectra of Ab-AuNPs in Figure 4 indicate the primary peaks at (3321.42, 2823.79, 2144.84, and 1631.78) cm-1. The large1peak at 3321.421cm-1 is relativity O–H2stretching vibration.of hydrogen-bonded alcohols, phenol, and N-H stretching of amines or amides, Alkanes, aldehydes, and corrosive carboxylic compounds all exhibit the C = H stretch independently at IHJPAS. 36 (4) 2023 115 2823.79 cm-1, where C=O stretching vibrations are present at 1631.78 cm-1. The vibration of the FT-IR spectrum of synthesized Ab-AuNPs illustrated a decrease in the peak intensities of the functional groups compared with the functional groups in A. baumannii broth culture. It is possible to observe the appearance and the disappearance of some bonds and that some of the absorbances have decreased due to their consumption as a capping agent during the synthesis process. The absence of these functional groups in the synthesized Ab-AuNPs indicates the formation of Ab- AuNPs. Identified sharp peaks in the range of (578.64–748.38) cm-1 refer to the vibration of Au- NPs. This result means that Ab-AuNPs were formed successfully [16]. Figure 4. FT-IR spectrum of (read) A. baumannii broth culture and (blue) the Ab-AuNPs colloidal. 3.2.X-ray diffraction (XRD) analysis Figure 5 displays the primary peaks at"111, 200, 220, and 311, which respectively, correspond to reflections with 2 values of the Bragg angles"38.18°, 44.51°, 64.80°, and 77.72°. These findings demonstrate that the tested substance is high-purity AuNPs. Using the.Debye-Scherrer, equation, the average crystallite size of the Ab-AuNPs in the arrangement was calculated to be 26.82 nm. [17]. Table 1. Structural parameter of Ab-AuNPs Average Crystallite D (nm) Crystallite size (nm) FWHM (hkl) Peak position(20°) 26.82211084 23.72694466 0.35431 (111) 38.18363 23.26486114 0.36896 (200) 44.51299 24.70709167 0.38084 (220) 64.80844 35.58954589 0.28667 (311) 77.72309 IHJPAS. 36 (4) 2023 116 Figure 5. XRD of Ab-AuNPs biosynthesized using A. baumannii broth culture. D = 𝐾𝜆𝛽𝐶𝑂𝑆𝜃 It elaborates on the connection between XRD peak broadening and crystallite size. Where D is the average size of nanoparticles, K is the Scherrer constant, which has a value of 0.9, Bragg's angle, and the wavelength of the X-ray radiation source, 0.15406 nm". 3.3. Transmission Electron Microscope (TEM) Examination Ab-AuNPs had sizes that varied from 20 to 90 nm, with an average of 66 nm. Figures 6, 7 demonstrate this. The reaction begins with the generation of semi-spherical Ab-AuNPs. Due to the high concentration of reducing factors in the A. baumannii broth culture, they subsequently cluster together along with rectangular, triangular, pentagonal, cylindrical, irregular, and polymorphic shapes. Figure 6. TEM image of diverse shapes of biosynthesized AuNPs using A. baumannii broth culture. IHJPAS. 36 (4) 2023 117 Figure 7. TEM image of biosynthesized Ab-AuNPs using A. baumannii broth culture, (a) Average diameters of gold nanoparticles, (b) diverse shapes. 3.4. Field Emission Scanning Electron Microscope analysis (FESEM) As shown in Figure 8, the results show that Ab-AuNPs have a spherical form, a high degree of aggregation, and a size that ranges from (66-363) nm. Figure 8. FESEM images of biosynthesized Ab-AuNPs using A. baumannii broth culture, a, b, c shows shapes of Ab-AuNPs. 3.5. AFM Analysis The size distribution of Ab-AuNPs is seen in the 2D and 3D AFM pictures. No aggregation or agglomeration was observed in the slide sample of the AFM sample, which shows the great stability of the Ab-AuNPs produced after two months of AuNPs synthesis. The Ab- Figure 9 shows that the size of the Ab-AuNPs particles ranged from 15 to 125 nm, with an average length of 63.82 nm. IHJPAS. 36 (4) 2023 118 Figure 9. AFM images of biosynthesized Ab-AuNPs using A. baumannii broth culture, (a) Two-Dimensional, (b) Three-Dimensional, and (c) Average particle size. 3.6. Zeta Potential Measurements Figure 10 shows that the biosynthesized Ab-AuNPs produced by A. baumannii broth culture had a Zeta Potential of -22 mV and less than 30 mV, indicating that the particles are stable and do not aggregate. Figure 10. Zeta potential analysis of biosynthesized Ab-AuNPs using A. baumannii broth culture. 3.7. The Cytotoxic Effect of synthetic Ab-AuNPs Figures 11, 12, 13 show that the cytotoxicity of the Ab-AuNPs was unaffected at 3.1 μg/ml, while the cytotoxicity increased significantly with increasing concentrations of Ab-AuNPs. Moreover, this assay showed that the minimum inhibitory concentration of MCF-7 cells was obtained by interaction with Ab-AuNPs at 6.25 mg/ml and 12.5 mg/ml, giving a reduction ratio (23.33% and 52.33%), respectively. However, the maximum concentration of inhibition was at 25 IHJPAS. 36 (4) 2023 119 mg/ml and 50 mg/ml giving a reduction ratio (63.00% and 86.33%), respectively. This study showed a very important cytotoxic activity of Ab-AuNPs against the breast cancer cell line MCF- 7 but ineffective against the normal cell line MCF-10 as shown in Figs. (15, 16). The results indicate the ability of Ab-AuNPs to suppress the growth of cell lines; this effect depends on the concentration. The median (IC50) was 11.45 mg/ml. Figure 11. Cytotoxic effect of Ab-AuNPs in MCF-7 cells. IC50=11.45 µg/ml Figure 12. Control untreated MCF-7 cells IHJPAS. 36 (4) 2023 120 Figure 13. Morphological changes in MCF-7 cells after being treated with AuNPs 3.8. Cytotoxic Effect of synthetic Ab-AuNPs using Acinetobacter bumannii solution on MCF- 10 normal cell lines: Significant inhibition was demonstrated for the proliferation of the MCF-7 line opposite to the MCF-10 cells, the MCF-7 line, and the MCF-10 cells treated with Ab-AuNPs under the same time and conditions, "as shown in Figure 14. Figure 14. Cytotoxic effect of Ab-AuNPs in MCF-10 cells, IC50=11.45 µg/ml IHJPAS. 36 (4) 2023 121 Figure 15. Control untreated MCF-10 cells. Figure 16. Morphological changes in MCF-10 cells after being treated with Ab-AuNPs 3.9. Antioxidant 3.9.1. DPPH Scavenging Assay The results proved that Ab-AuNPs have DPPH scavenging activity, which increases with increasing concentrations of Ab-AuNPs. The concentrations (3.1, 6.25, 12.5, 25, and 50) mg/mL gave DPPH scavenging activity of 37.87%, 50.13%, 59.33%, 75.55%, and 85.13%, respectively, Figure 17. IHJPAS. 36 (4) 2023 122 Figure 17. Antioxidant activity of Ab-AuNPs, Ascorbic acid (10µg/ml) as a positive control. The results are represented as the mean ± SD. 4.Discussion This study biosynthesized Ab-AuNPs for the first time, using the gold solution and A. baumannii broth culture. Bacterial broth culture contains polysaccharides, amino acids, peptides, proteins, and enzymes. Ab-AuNPs were synthesized when the broth culture changed color from pale yellow to deep purple. These compounds can act as capping and reducing agents. These findings support previous research that showed metal biotransformation might involve capping proteins orpeptides and reductases, quinines, cytochromes, or electron shuttles to decrease metals and metal oxides [18, 19]. This study added a fixed volume of gold solution to many broth cultures. As the broth culture volume increased, the color shift rose, indicating that AuNPs had more reducing and capping agents. IHJPAS. 36 (4) 2023 123 The UV-Visible spectra of AuNPs within the visible absorbance band agree with [20]. Ab- AuNPs had SPR bands at 574 nm in their UV-Visible spectra (500 nm-600 nm). FTIR analysis of A. baumannii broth culture-produced Ab-AuNPs revealed the solution-stabilizing biomolecules. A. baumannii broth culture AuNPs produced strong bands at (3321.42, 2823.79, 2144.84, and 1631.78) cm-1. These bands are amide III, polypeptides, and proteins [21]. A. baumannii broth culture polypeptides capped AuNPs. Strong peaks at (578.64-748.38) cm-1indicate AuNP vibration. AuNPs were created [22]. The XRD spectrum has four major peaks, according to Bragg's reflection of AuNPs described in a prior work using external and intracellular culture supernatants of bacteria and yeast [23]. The primary peaks at 111, 200, 220, and 311 match reflections with Bragg angles 38.18°, 44.51°, 64.80°, and 77.72°. These results indicate high-purity AuNPs. The Debye-Scherrer equation estimates Ab-AuNPs' average crystallite size at 26.82 nm. Our results match yeast-produced AuNPs, bacteria [30], and plant extracts [24]. According to Transmission Electron Microscope (TEM) analysis, AuNPs biosynthesized using the A. baumannii broth culture ranged from (20-90) nm to 66 nm. Grouped and semi-spherical. These findings match previous research. [27]. The shape and size of the AuNPs biosynthesized using the A. baumannii broth culture are similar to the transmission electron microscopy findings, according to the field emission scanning electron microscopy (FESEM). Bacteria broth culture constituents are principally magnesium, chloride, and potassium, sodium salts of remnants of bacterial cells, and nutrient broth. Bacteria broth culture in water dissociates the salts. It reveals the negative charge, which allows the interaction with the positive direction of gold nanoparticles. This caused accumulation, which was also observed in the examination of FESEM. Even though, the bacteria broth culture acted as a reducing agent and capping agent, the collection can still be hindered by keeping the solution at a low temperature. These findings concur with numerous researches [28]. An atomic force microscope (AFM) measures the size and shape of biosynthesized Ab-AuNPs from A. baumannii broth culture (AFM). 2D and 3D AFM images and AuNP size distributions are shown. Ab-AuNPs ranged in size from 15–125 nm, averaging 63.82 nm. The results match the research of Vitosha et al. [29]. and are close to the result you have reached in Jafarizad et al. [30]. In contrast, another study synthesized AuNPs of different sizes [40]. This size disparity could result from other synthesis techniques, bacteria, plant extracts, or different synthesis circumstances. And the Zeta Potential analysis showed -22 mV and values below 30 mV, showing that the particles are stable and do not aggregate. These findings concur with many studies. [31, 32]. The cytotoxicity results of Ab-AuNPs showed that proliferation was significantly increased depending on concentration; the different concentrations of Ab-AuNPs used in this study are as follows: (3.1, 6.25, 12.5,25, and 50 mg/ml). The results indicate that Ab-AuNPs are a precious source of effective anti-proliferative and cytotoxic substances. Rajan et al. [33] showed that MCF- 7 cells treated with Ab-AuNPs were significantly reduced compared with the control cells; AuNPs killed >70 % of the cells. It is also compatible with our study. In comparison, AuNPs did not show any significant effect on MCF-10 cells, which is also in agreement with the findings of a previous study that AuNPs inhibited the proliferation of human breast cancer cells (MCF7 cell line) [34, 35]. Through changes in the shape of MCF7 cell lines, the biologically prepared gold nanoparticles were more toxic than those chemically prepared in studies with PC-3, HCT116, and HepG2 tumor cells [36]. The normal cells were the controls. MCF-10 cells showed that the treated cells kept their shape before being treated. On the other hand, MCF7 cell lines that were treated with Ab- AuNPs showed changes in their shape and permeability. [37]. IHJPAS. 36 (4) 2023 124 DPPH radical scavenging activity matches the researcher's previous study [38]. Lifestyle changes, radiation, and pollutants disrupt the balance between antioxidant action and free radical production. Still, "Green" synthesis AuNPs have promising anticancer and antioxidant properties [39, 40]. 5.Conclusion We conclude from the present study that gold nanoparticles synthesized using A. baumannii broth cultures are easy to prepare, inexpensive, and non-toxic to normal cells. 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