49 This work is licensed under a Creative Commons Attribution 4.0 International License IHJPAS. 37 (2) 2024 Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 Synergistic Inhibitory Effect of Biosynthesized ZnO-CuO Nanocomposite on Biofilm Formation of Proteus mirabilis Noor Hamza Faiq 1* and Mais E. Ahmed 2 1,2 Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq *Corresponding Author. Received 5 April 2023 Accepted 22 May 2023 Published 20 April 2024 doi.org/10.30526/37.2.3383 Abstract Proteus mirabilis is considered the third most common cause of catheter-associated urinary tract infections. With urease production, the potency of the catheter’s blockage due to the formation of biofilm is significantly enhanced. This study was aimed at exploring whether green synthesized ZnO-CuO nanoparticles (ZnO-CuO NPs) can function as an anti-biofilm agent produced by P.Mirabilis. The characterization of biosynthesized nanoparticles was carried out to determine the chemical and physical properties of the product using AFM, TEM, FESEM, XRD, and UV visible spectrometry. The hexagonal structure was confirmed by XRD, the size range was marked at 96.00nm by TEM, and FESEM was used to confirm the surface morphology. AFM analysis is used to reveal the roughness and distribution of nanoparticles. UV-visible spectra of the synthesized nanoparticles recorded a maximum peak at 287nm and 232nm. Zinc and Copper nanoparticles showed remarkable biofilm inhibitory effects on wild-type strains of multidrug-resistant Proteus mirabilis. Downregulation changes in LuxS expression using real- time PCR technology were detected after treatment with the ZnO-CuO nanocomposite of these strains. A cytotoxicity test of CuO-ZnO NPs-based nanocomposite showed that this nanocomposite is safe for use, where the IC50 was 161.6 µg/mL. Anti-cancer activity against urethral bladder cancer cell lines was recorded in vitro, where the IC50 was 107.4 µg/mL. Keywords: Proteus mirabilis, anti- biofilm, Zinc oxide nanoparticles, Anti-cancer, copper oxide nanoparticles. 1. Introduction Bacterial biofilms are well-organized communities with single or multi species enclosed by a defensive polysaccharide matrix containing extracellular DNA, lipids, and proteins [1]. The community of biofilm depends on complicated communications, enabling the pathogens to develop resistance against host defense mechanisms, different stress factors, and antibiotics [2]. Bacterial biofilms are ubiquitous in clinical settings since they can increase bacterial resistance up to 1000 fold in compression to planktonic cells. Subsequently, despite anticipation measures taken in hospitals and other clinical settings, nosocomial infections still result in major morbidity and mortality rates [3]. In this emerging situation, researchers are commended for improving new alternatives to traditional antibiotic treatment that can successfully combat multidrug resistance (MDR) and biofilm-producing bacteria. https://jih.uobaghdad.edu.iq/index.php/j/index#1609-4042 https://jih.uobaghdad.edu.iq/index.php/j/index#2521-3407 https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3983-4443 mailto:nour.hamza1102a@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-4961-4532 mailto:mais.emad@sc.uobaghdad.edu.iq IHJPAS. 37 (2) 2024 50 The green synthesis of metal oxide NPs has gained great interest since it is a clean, eco-friendly approach and has a wide range of applications in the biotechnology and medical fields [4]. ZnO and CuO nanoparticles display variable shapes and sizes with a minimum surface area that allows them to penetrate microbial cells; therefore, they are considered powerful antibacterial agents against a wide range of bacterial species [5]. ZnO NPs are one of the most important nanoparticles of metal oxides; they are a unique and inorganic material that can be used in several biological applications (anti-bacterial, anti- inflammatory). Several studies have described the quorum sensing system as a potential target for antimicrobial agents that limit biofilm formation [6]. Researchers in recent years have employed nanotechnology to improve antimicrobials that can target virulence factors without disturbing mammalian cells. Metal NP synthesis using microorganisms has represented a novel alternative to chemical and physical approaches as antibacterial, antioxidant, and antifungal agents, along with anticancer properties, in many different applications [7]. Several studies were interested in investigating the antibacterial activity of CuO. Noor in 2020 revealed in her study a moderate antibacterial activity of fungal-mediated CuO NPs against E.coli, K. pneumoniae, and other gram-negative bacteria [8]. The anti-cancer activity of zinc nanoparticles was previously approved against HeLa cell lines in cervical cancer. Zinc NPs can induce apoptosis through ROS production inside cancer cells. Induced cell cycle arrest, decreased antioxidants, and increased lipid peroxidation were also observed [9]. 2.Materials and methods 2.1.Sample collection A total of 100 urine samples were collected from patients with urinary tract infections and resident hospital patients with urine catheters presented to the AL Yarmouk Teaching Hospital. Isolation and identification: P.mirabilis was identified primarily by culturing on MacConkey and Blood agar at 37 °C for 24 h and confirmed by the VITIC 2 system. 2.2.Antibiotic susceptibility test According to CLSI 2021, the susceptibility test takes place using the disk method on Mueller- Hinton agar plates. The antibiotics used were (Levofloxacin 5 μg, Ciprofloxacin 15 μg, Ampicillin 25 μg, Gentamicin 10 μg, Cefoxitin 30 μg, Ticarcilline + Clavulanic Acid μg, Piperacillin-Tazobactam 100/10 μg, Cefepime μg, Nitrofurnation 300 μg). 2.3.Nanocomposite preparation As precursors, copper (II) acetate Cu (CH3COO) 2⋅H2O) and Zinc acetate (Zn (O2CCH3)2) were used in this research. 2.4.Preparation of bacterial suspension P. mirabilis was grown in 250 ml of Brain Heart broth at 37 °C for 24 hours in a non-shaking incubator. The bacterial broth was then centrifuged, the supernatant was collected, and the sediment was discarded. 2.5. Synthesis of CuO-ZnO NPs The nanocomposites were synthesized by the precipitation method: (200ml) of P. mirabilis broth filtrate, 10 g of copper (II) acetate, and 10 g of Zinc acetate were added at room temperature (37 °C), sealed, and covered with a dark plastic bag, and then incubated in a shaker incubator for 24 h. The mixture was then centrifuged, and the precipitate was collected and washed thrice with deionized water. The nanoparticles were synthesized by the extracellular route, where metal ions get reduced on the cell surface by the action of bacterial reducing IHJPAS. 37 (2) 2024 51 enzymes. Next, NPs synthesis can be detected by the color change of the solution; the precipitant is then air dried and kept in a dry place for further characterization [10]. 2.6.Characterization of CuO-ZnO nanocomposite Characterization is essential for understanding nanoparticle properties. The following methods were used to determine the characteristics of NPs: for determining shapes and sizes, SEM and TEM were used. Atomic force microscopy (AFM) is used to define the height and volume of NPs in 3D vision, and UV-visible (UV-DRS) is used to study the optical properties of the samples. (XRD) is a technique used in materials science to determine the crystallographic structure of a material [11]. 2.7.MIC determination We used the broth microdilution method in Eppendorf tubes to figure out the minimum inhibitory concentration (MIC) and the lowest concentration of synthesized nanoparticles that stop bacterial growth. We prepared the bacterial inoculum in MHB and modified the concentration to 100 UFC/mL. Double serial dilutions of nanocomposite were prepared in (16, 32, 64,128, 256,and 512 μg/ml) using MHB in six tubes, which were then incubated for 24 hours at 37°C. 2.8.Quantitative determination of biofilm formation was determined by a colorimetric microtiter plate assay [12]. 1- Brain heart infusion Broth supplemented with an additional 1% glucose was used for this assay. 2-Biofilm inoculums for cultivation were made from bacteria cultivated in broth, diluted 1:100, and poured into the well with 200 μl. Only broth is used in the negative control wells (200 μl of BHI supplemented with 1% glucose per well). Each strain was tested in three different ways. 3- Under static conditions, the inoculated plate was covered with a lid and incubated aerobically for 24–30 hours at 35–37°C. 4- The contents of the wells were decanted, and each well was washed three times with (300 μl) of PBS (2.2.5.1). The plates were then drained, inverted, and fixed with 150 of ml methanol. 5- Staining was done with (150 μl) of crystal violet at room temperature for 15 min, then washed and dried at room temperature. 6- For dye re-solubilizing, 150 μl of 95 percent ethanol was added, and the microtiter plate was covered with the lid and left at room temperature for at least 30 min. without shaking. 7- Using a microtiter-plate reader (GloMax/ Promega-USA), the optical density (OD) of each well was measured at 630 nm, and the results were as follows: OD ≤ ODc = no biofilm producer; ODc < OD ≤ 2 ODc= weak biofilm producer; 2 ODc < OD≤ 4 ODc = moderate biofilm producer; 4 ODc < OD= strong biofilm producer The cut-off value for the negative control is ODc, which is defined as three standard deviations (SD), above the mean OD of the negative control: ODc= average OD of negative control+ (3× SD of negative control). 2.9.Effect of CuO-ZnO nanocomposites on Biofilm formation This assay was carried out on a 96-well microtiter plate, as performed by [13]. Tested isolates were cultured in brain-heart infusion broth at 37 o C for 24 h. 100 ml of bacterial inoculum was adjusted to 0.5 McFarland and added to each well. 100 ml of nanocomposite suspension was added to each well at the sub-inhibitory concentration, and the tubes were incubated at 37 o C for IHJPAS. 37 (2) 2024 52 48 h. A positive control was presented as bacterial culture without Nano formulations, while clear broth was considered a negative control. The contents of each well were discarded after incubation, and the microplate was rinsed thrice with sterile saline and dried for 45 minutes at 60 °C200 ml of 0.1% crystal violet were used in staining, followed by incubation at room temperature for 15 min. The microplate was rinsed three times with sterile normal saline. Then acetic acid at 30% was added to each well by 200 ml, and the optical density (OD) was read at 630 nm for all wells using a microtiter plate reader. 2.10. RT-qPCR protocol The presence of LuxS and rpo in P.mirabilis was determined by employing the thermal cycler to amplify the isolated genomic DNA (Thermo Fisher Scientific, USA). This main step is separated into two stages. The first stage is the synthesis of cDNA from RNA using specific primers for LuxS and rpo transcripts, as mentioned in Table 1. and the proto-script cDNA synthesis kit. This procedure has been performed as detailed in the manufacturing procedure. The second stage of this protocol is done by taking cDNA samples from tested isolates and controls at the same time. For each sample, there are three PCR tubes, and one tube for the LuxS gene, rpo, which is considered a housekeeping gene in this study. The fluorescent power of Syber Green is used for quantity estimation. The reaction mix of components with their quantity is mentioned in Table 1. below: Table 1. Synthesis of cDNA from RNA using primers for LuxS, rpo transcripts Component 20 ul Luna Universal Master Mix 10 ul Forward primer 1 ul Reverse primer 1 ul Template DNA 5 ul Nuclease-free Water 3 ul The difference in cycle thresholds (Ct) and fold changes between the treated groups and the calibrators for each gene were evaluated [14]. The rpo values were used to normalize the data. PCR tubes were spanned and the liquid was collected for 1 minute at 2000g, then the program for real-time PCR was setup with the indicated thermos cycling protocol. The result was collected and analyzed by the Livak formula. Relative quantitation was used to determine expression levels. 2.11. Anti-cancer activity of CuO-ZnO nanocomposite 2.11.1. Determination of Cell Viability The cytotoxic effect of CuO/ZnO NPs synthesis from P. mirabilis was performed by using MTT ready-to-use kit contents [15], UBC-40, and HdFn cell lines in 96-well microtiter plates at a concentration of 4000 cells per well. After 12 hours of incubation, the growth medium was replaced with a fresh medium containing various concentrations of nanocomposite (25, 50, 100, 200, and 400 μg/ml) to treat both cell lines. According to the provider’s instructions, the cells were cultured with the recommended medium at 37 °C. A microplate reader from Bio-Tek, Georgia, USA, was used to detect absorbance at 450 nm. Curves were generated with GraphPad Prism 7 software from GraphPad Software, Inc., USA, in a dose-dependent manner. IHJPAS. 37 (2) 2024 53 3.Results and discussion 3.1. Isolation and identification: Out of 100, 46 isolates were confirmed as P. mirabilis, the conformation was carried out by the VITEK 2 system, which was considered a reliable identification by Biochemical tests; in addition, swarming on blood agar as shown in Figure 1. and colony morphology on MacConkey agar. Figure 1. Proteus mirabilis on blood agar at 37C for 24h of incubation. 3.2. Antibiotic susceptibility test 3.2.1. MDR strains screen: Forty out of sixty-four isolates (62.5%) were confirmed as MDR strains. After incubation at 37 °C for 24 hours, all tested isolates were resistant to Piperacillin-Tazobactam, six isolates (15%) were only resistant to levofloxacin; while six isolates (15%) were found to be resistant to ciprofloxacin; ampicillin did not inhibit the growth of 15 isolates (37.5%); gentamicin resistance was found in two strains only (5%); six isolates (15%) were recorded as cefoxitin resistant; and 17 isolates (42.5%) were resistant to nitrofurantoin. A ten-year study in 2019 shows a growing concern about antibiotic resistance in proteae (the proteae tribe includes the genera Proteus, Morganella and Providencia) Based on the results of the study, Resistance rates to third- generation cephalosporins developed significantly (the indicator during the study was ceftriaxone). Resistance to Ceftriaxone has increased by three times in hospitalized patient samples [15]. Another study carried out in China stated that 25 isolates of 54 P. mirabilis were classified as MDR. The samples were collected from animals and humans. This study highlighted that P. mirabilis produces AmpC β-lactamases and extended spectrum β-lactamases, which makes this bacterium a serious public health risk. P. mirabilis uses an efflux pump to resist the main classes of antibiotics [16]. 3.3. Biosynthesis of ZnO-CuO Nanoparticles Zinc oxide nanoparticles were biosynthesized from P. mirabilis. The formation of Nanoparticles was indicated by a color change from yellow to blue, in addition to forming a light blue precipitate. After centrifugation, the precipitate appeared in a greenish-blue color. After drying with the microwave, we obtained a shiny blue powder, as shown in Figure 2. In recent years, an obvious coordination to use bacteria to synthesize nanomaterials (mainly silver, zinc, gold, and nanoparticles) with remarkable properties has been observed for the development of antimicrobials with in vitro activities against pathogenic bacteria [17]. Bacteria are easy- IHJPAS. 37 (2) 2024 54 cultivating microorganisms with short generation times; these characteristics make bacteria a great candidate for nanoparticle synthesis as they have extra cellular reduction enzymes [18]. The anti-biofilm activity of zinc oxide NPs was previously reported on S. aureus and P. aeruginosa [19]. Figure2. Biosynthesis of CuO-ZnO Nanocomposite Powder 3.4. Characterization of Nanocomposite 3.4.1. UV–Vis Spectral Analysis For this typical analysis, the sample was dissolved in deionized water. The results confirmed the formation of freshly prepared ZnO and CuO. Biosynthesized CuO-ZnO NPs exhibited absorption peaks at 287nm and 232 nm, as shown in Figure 3. Two peaks were detected, representing the presence of zinc oxide and copper oxide, as reported by [20]. Figure 3. UV visible of nanocomposite. 3.4.2. Atomic Force Microscopy (AFM) analysis AFM analysis of CuO-ZnO NPs was performed with CSPM to identify and characterize the distributions of nanoparticles. The estimated grain size and mean square roughness were determined. As shown in Figure 4. Portraits of a three-dimensional profile of nanocomposite and a 3D image of the nanoparticles show that the average grain size is 96.00nm. Different magnification ranges were conducted to give an insight into the roughness and topography of nanoparticles at 1.93 μm, 3.3 μm, and 3.46 μm. IHJPAS. 37 (2) 2024 55 Figure 4. Atomic Force Microscopy analysis of CuO-ZnO nanocomposite. 3.4.3. Transmission Electron Microscopy analysis (TEM) TEM imaging was used to explore the morphology and size of the nanocomposite. The core structure of the nanoparticles is confirmed by TEM analysis [21]. TEM images in Figure 5. demonstrate the formation of spherical and semi-spherical-shaped particles with agglomeration. The TEM images show that CuO-ZnO NPs, formed by the reduction of copper and zinc ions using P. mirabilis Filtrate, are spherical . Metallic nanoparticles are well protected against chemical oxidation by the organic shell of bacterial growth medium, which makes them constant and suitable for coatings or biotechnology applications [22]. These results are consistent with those of FESEM and XRD studies. Figure 5. The Transmission Electron Microscopic TEM images of CuO-ZnO NPs 3.4.4. Scanning electron microscopy SEM Scanning electron microscopy (SEM) provided further insight into the surface morphology of the Cu-Zn NPs. Images reveal that CuO-ZnO NPs are crowded together to look like combined cauliflower Figure 6. Higher magnification for further observations reveals that these crowded NPs are groups of smaller nanoparticles of copper and zinc that exhibit spherical-shaped structures with good homogeneity. IHJPAS. 37 (2) 2024 56 Figure 6. FESEM images with 60000x and 15000x magnification powers. 3.4.5. X-ray Diffraction (XRD) The crystalline structure and purity of synthesized nanoparticles (NPs) have been investigated with the help of dispersive X-ray diffraction. The XRD spectra of Cu-Zn NPs powder are shown in Figure 7. which confirms the presence of Cu-Zn NPs by revealing 19 prominent peaks that correspond to the diffraction peaks at 2θ values: 11.794, 13.056, 16.766, 18.12, 19.525, 20.539, 22.494, 24.312, 35.725, 39.148, 42.864, 54.37, 57.254, 58.473, 59.689, 61.492, 72.206, 73.681, and 77.314. According to JCPDS file No. 79-0207, all peaks have been indexed via the wurtzite hexagonal phase. No peaks were present in the spectrum related to any impurities or bulk remnant materials detected except the characteristic peaks related to the wurtzite hexagonal phase of CuO-ZnO, indicating the high purity of synthesized uncapped NPs. Figure 7. X-ray Diffraction XRD of nanocomposite shows 19 peaks at 2θ values. 3.5. Determination of biofilm formation before treatment with Nanocomposite A total of 20 MDR isolates were tested for their biofilm production ability using a plastic micro titer plate; nine isolates were recorded as strong biofilm producers, as detailed in Figure 8 and Table 2. To estimate the effect of Cu-Zn NPs on the biofilm production of these isolates, five strong producers’ strains were selected. The OD of each well was read at 630 nm using a micro titer plate reader. IHJPAS. 37 (2) 2024 57 Figure 8. Micro titer plate assay for biofilm production 3.6. Determination of biofilm formation after treatment with Nanocomposite Biofilm production was significantly reduced after 48 hours of incubation at 37 °C with a sub- MIC of Cu-Zn NPs. The OD measurement of the same nine strains of P. mirabilis lowered from strong to weak production, as detailed in Table 2. and Figure 9. Figure 9. Microtiter plate assay for biofilm production after treatment with CuO-ZnO NPs Sub MIC Table 2. O.D values before and after treatment with biogenic CuO- ZnO. NO. of isolate O.D Before treatment O.D after treatment with CuO-ZnO NPs 1. 5.75 1.0 2. 9.19 1.45 3. 6.30 1.11 4. 7.25 1.14 5. 7.15 1.17 Antimicrobial action can be explained by the small particle size of ZnO NPs because smaller materials can enter bacterial cell membranes and cause cell damage. The anti-biofilm activity of zinc oxide NPs was previously reported in several studies; one study showed a significant decrease in the biofilm formation of pseudomonas aeruginosa after treatment with zinc oxide NPs in a concentration-dependent manner [23]. Biofilm formation is considered a target for different antimicrobial agents, such as nanoparticles from different sources and silver NPs synthesized from Date palm extract against E. coli and Klebsiella pneumonia [24]. This study also agreed with other studies that approved the Anti-biofilm activity against methicillin resistant Staphylococcus Aureus [24]. This action can be explained by the ROS generated by nanoparticles that impose oxidative stress, causing bacterial cell destruction [25]. Studies also found that treatment with nanomaterials induced a major reduction in membrane potential, signifying it may target the membranes of bacterial cells [26]. IHJPAS. 37 (2) 2024 58 3.7. Gene expression of LuxS LuxS is a transcript of the structural operon luxCDABF that is responsible for the production of autoinducer 2 AI-2, which predominantly participates in cell-to-cell signaling in bacteria. The transcription of luxS is enhanced when luxR is bound to an autoinducer. After luxS was produced, AI-2 cast signals that were used to sense interspecies interaction and its cell density in multi-microbial populations that have essential roles in the regulation of virulence factors [27]. RT-PCR reveals a major downregulation in LuxS expression after exposure to CuO-ZnO NPs suspension compared to normal gene expression in bacterial broth with no NPs involved, as detailed in Table 3. A fold change in gene expression reveals that LuxS was down-regulated in response to copper oxide and zinc oxide NPs in three out of five isolates of proteus mirabilis; the other two isolates were almost not affected, as entailed in Table 3. This result corresponds with the phenotypic changes in biofilm formation in the microtiter plate experiment carried out in this study, as shown in Figure 10. above. This finding was accepted with other reports by Rajalakshmi and Sangeetha [28]. The anti-biofilm activity of metal oxide nanoparticles has gained great interest in the last few decades since biofilm is an important virulence factor that enables bacteria to resist antibiotics [29]. Several studies have approved that these particles have bactericidal and bacteriostatic activity. Khan et al. confirm the downregulation effect of ZnO NPs on las and pqs systems that control quorum sensing and biofilm production in P. aeruginosa [30]. Other reports estimate the ability of Nano oxides to inhibit QS systems of S. agalactiae [31]. Table 3. Ct before and after treatment with CuO-ZnO. Ct before treatment with CuO-ZnO NPs Ct after treatment with CuO-ZnO NPs Isolate No. Ct rpo Ct LuxS ΔCt Ct rpo Ct LuxS ΔCt ΔΔCt Fold Change P.1A 13.73 12.32 -1.41 12.59 12.31 -0.28 1.13 0.456 P.2B 13.77 11.75 -2.02 16.75 17.05 -0.3 1.72 0.303 P.3C 10.91 12.4 -1.49 11.01 11.68 -0.67 0.82 0.566 P.4D 11.03 11.67 0.64 11.96 10.89 -1.12 -1.76 0.29 P.5E 10.31 10.47 0.16 12.66 12.06 -0.6 -0.76 1.693 The effect of metal-based nanoparticles depends on particle size, morphology, and composition. Dispersion, aggregation, or agglomeration also play a key role in the nanoparticle effect [32]. Most studies attributed the effect of zinc oxide NPs on the quorum sensing system to signal perception and response rather than the synthesis of autoinducers [33]. Other reports revealed the effect of polymeric nanoformulations on the attachment of biofilm cells to Pseudomonas aeruginosa (PAO1) [34]. Our results demonstrate that Cu-Zn NPs exhibit selective toxicity towards prokaryotic cells, prompting further investigation into NP toxicity in eukaryotic cells. 3.8. Cytotoxic effect of CuO-ZnO nanocomposite Human Dermal Fibroblasts, neonatal (HDFn) cell line used to measure the cytotoxic effect viability, were reduced to half after treatment with the nanocomposite (161.6 mg/L IC50). Figure 10. shows cell viability getting lower with the nanocomposite concentration increment. The cytotoxic effect of copper nanoparticles generally depends on their physicochemical properties, including particle shape, size, aggregation, crystallinity, and surface coating. Reports have been recorded to be ten times lower than the toxic dose of ionic copper and copper (I) oxide IHJPAS. 37 (2) 2024 59 CuO to some plants, rodents, and cell cultures, thus classifying it as more cytotoxic. This fact can be exploited by proposing the use of copper nanoparticles as a drug carrier in chemotherapy [35]. Several studies take place to estimate the toxic effect of nanoparticles on human cells. There is a significant decrease in the human T cell count after treatment with ZnO NP; 77% of the cells remain viable at 5 mM, and 43% are kept at 10 mm. [36]. On the other hand, copper toxicity has been under investigation in the last few years. Neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, have been linked to copper toxicity. Wilson and Menken diseases are also labeled as copper intolerance examples [37]. The nanoparticle interaction can also cause inflammation in tissues, oxidative stress, and histopathological lesions [38]. 3.9. Anti-cancer activity of CuO-ZnO nanocomposite Urethra bladder cancer cells used in this research show reduced viability after treatment with CuO-ZnO nanoparticles in dose-dependent matter. As Figure 10. clarifies, the IC50 value of nanocomposite that inhibits cell line viability is 107.4 mg/l. According to the data collected by this test, cancer cell viability was much lower than HdFn cells in the same concentrations. This leads to the conclusion that CuO-ZnO nanocomposite had a greater effect on cancer cells. The anti-cancer activity of nanoparticles was extensively studied to estimate the capability of nanoparticles to act as anti-cancer agents in humans and animals. Apoptosis is the most frequently mentioned model of cell death after exposure to metal-based nanoparticles; the second model is necrosis, with approximately half as much. The third path of nanoparticle cytotoxicity in cancer cells is also reported, and it involves the stimulation of autophagy, in which cell organelles and proteins are degraded. In one study recorded using MTT, apoptosis, and gene expression methods, nanoparticles with a 30nm size were found to have more anti-cancer potential than 60 nm CuO NPs against the 4T1 cell line as malignant breast cancer cells. [39]. Another study revealed the significant role of CuO NPs in inhibiting TIC-enriched PANC1 human pancreatic cancer cell cultures [40]. On the other hand, Zinc Oxide NPs were also under investigation as anti-cancer material. The human melanoma cell line (A375) was used to estimate this role in one study. The mRNA expression of apoptotic genes like caspase 3, 8, and 9 was elevated, followed by exposure to ZnO. Nanoparticles also stimulate apoptotic cell necrosis at the transcriptional stage [41]. Other results showed the effect of zinc oxide on cancer cell lines and toxicity assays [42]. The genotoxic effect of nanoparticles on cancer cells was also investigated. The suggested mechanisms of action that cause cellular damage by nanoparticles are Oxidative stress, ROS generation, and reducing mitochondrial membrane potential [41]. Figure 10. The IC50 value of nanocomposite that inhibits cell line viability IHJPAS. 37 (2) 2024 60 4. Conclusions CuO-ZnO NPs were successfully synthesized following a direct, eco-friendly, low-cost, high- yield, and green method. CuO-ZnO NPs showed exceptional antimicrobial activity against several bacterial strains. Thus, NPs can be used for external purposes as antibacterial agents by coating surfaces on various substrates to prevent biofilm-producing microorganisms from attaching and colonizing in indwelling medical devices. The application of nanoparticles is increasing nowadays due to their effectiveness in all fields of science. Green synthesized nanomaterials that are mostly used in drug delivery and medical approaches have some downsides to using these metal oxides because of their higher toxicity when used in higher concentrations. All the previous conclusions highlight that there is an actual need for more investigation in order to use CuO-ZnO NPs as encouraging alternate antibacterial materials and anti-cancer therapeutic settings. Acknowledgment Many thanks to editor-in-chief and members of the editorial committee in Ibn Al-Haitham Journal for Pure and Applied Sciences. Conflict of Interest There are no conflicts of interest. Funding There is no funding for the article. Ethical Clearance This research was subjected to ethical considerations, and the research was approved by the Committee of Ethical Standards in the college Science, University of Baghdad, in line with the form issued for this purpose by the Iraqi Ministry of Health. References 1. Spirescu, V.A.; Șuhan, R.; Niculescu, A.-G.; Grumezescu, V.; Negut, I.; Holban, A.M.; Oprea, O.-C.; Bîrcă, A.C.; Vasile, B. Ștefan; Grumezescu, A.M. Biofilm-Resistant Nanocoatings Based on ZnO Nanoparticles and Linalool. Nanomaterials (Basel) 2021, 11, 2564. https://doi.org/10.3390/nano11102564. 2. Balaure, P.C.; Grumezescu, A.M. Recent Advances in Surface Nanoengineering for Biofilm Prevention and Control. 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