Biosynthesized copper oxide nanoparticles using aloe vera leaves extract and their evaluation of antibacterial, anticancer in human Hela cancer cells

Biosynthesized copper oxide nanoparticles using aloe vera leaves extract and their evaluation of antibacterial, anticancer in human Hela cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biosynthesized copper oxide nanoparticles using aloe vera leaves extract and their evaluation of antibacterial, anticancer in human Hela cancer cells Saima Rafique, Rizwan Akram, Tooba Javed, Shazia Bashir, Zobia Noreen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3530645/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This research work assessed the shape-dependent anti-cancer activity of copper oxide nanoparticles (CuO NPs) synthesized from aloe vera leaf extract on the human HeLa cell line (ATCCCCL-2TM) and their antimicrobial action against Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ). The CuO NPs were synthesized by varying the concentration of precursor and aging time to obtain different shapes of nanoparticles. The synthesized particles had rod-like, cube-shaped, triangular, and spherical morphologies. The CuO NPs were examined against gram-positive and gram-negative bacteria. The results showed that NPs inhibit the growth of these bacteria and antibacterial activity depends on the size and shape of CuO NPs. The minimum inhibition concentration obtained was 0.325 mgml − 1 for E. coli at 0.2 M and 24 hrs aging time. A dose-dependent reduction in cell viability was observed using CuO NPs. CuO NPs caused significant morphological alterations in Hela cell lines, including shrinkage, detachment, and distorted shape. These findings imply that different-shaped CuO NPs may inhibit bacterial growth, elevate oxidative stress, and induce apoptosis in addition shows cytotoxic effects on cancer cells. Nanoparticles CuO antibacterial activity anticancer activity Hela cell line Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Recently, nanoparticles have extensively been used in various fields such as medicine, diagnostics, gas sensing, etc. due to their distinct physical and chemical properties [ 1 – 2 ]. Nanoparticles come in a variety of sizes, shapes, and chemical compositions for human benefit [ 3 ]. Nanoparticles are synthesized via physical, chemical, and biological processes. In the chemical synthesis approach some toxins and chemicals absorbed at the surface have a variety of negative impacts on medical and environmental applications. An intriguing area of nanotechnology is biologically synthesized nanoparticles using plant extract, which is environmentally benign compared to chemical and physical methods [ 4 ]. Recent reports describe the use of various plants including cucumis prophetarum [ 5 ], origanum majorana [ 6 ], moringa oleifera [ 7 ], and cayratia pedata [ 8 ] to synthesize nanoparticles. Aloe vera is a significant medicinal plant that comes in two parts: the peel (leaf) and the gel. Numerous studies have shown that its leaf contains a wide variety of phenolic compounds, sterols, vitamins, saponins, proteins, lignin, flavonoids, enzymes, polysaccharides, and organic acids. All of these are crucial for the stabilization of synthesized nanoparticles and the reduction of ions to their constituent elements to form NPs [ 9 ]. There are over 75 potentially active components in aloe vera, and they have properties that can heal wounds or burns, suppress inflammation, and fight parasites [ 10 ]. In this connection, metal or metal oxide nanoparticles are particularly important in the field of nanomedicine, where nanoparticles are used as effective therapeutic agents, drug delivery vehicles, and nano biosensors [ 11 ]. These nanoparticles are strong stable, and highly effective against a variety of microbes. Besides this, copper oxide NPs have a shelf life that is too long compared to other organic antimicrobial agents. [ 12 ]. Nanoparticles show new or better properties based on certain characteristics such as morphology, size, and distribution. Therefore in nanoparticle synthesis, the main goal is the formation of nanoparticles with different shapes, minimum particle size, and maximum stability. Haocong et al., synthesized copper oxide nanoparticles (CuO NPs) using cinnamomum zelanicum extract [ 13 ]. They studied the antioxidant and anticancer activity for lung carcinoma. The synthesized nanoparticles have spherical morphology with size ranges of 19.55 to 69.70 nm. They came to the conclusion that there was no cytotoxicity on the normal cell line and that the copper nanoparticles exhibited very low cell viability and dose-dependent anti-lung cancer effects against different cell lines. Similarly, Vaitheeswari et al., used euphorbia hirta leaf extract for the synthesis of CuO NPs [ 14 ] and studied their antimicrobial and antioxidant activity. They synthesized spherical-shaped nanoparticles of uniform size. These nanoparticles showed prominent antibacterial activity and antioxidant activity. Resmi et al., synthesize CuO NPs using nilgirianthus ciliates plant extract [ 15 ]. They prepared spherical particles with uniform morphology and particle size in the range of 20 nm which shows a 13 mm zone of inhibition against E. coli . Similarly, Abbasi et al., studied the effect of the size of CuO NPs on antibacterial and anticancer properties [ 16 ]. They found that 30 nm CuO NPs showed a strong antibacterial effect against Acinetobacter baumannii and Staphylococcus epidermidis bacteria and more potential anticancer properties were obtained against 4T1 cell lines than 60 nm CuO NPs. Tabrez et al., used pumpkin seed extract to synthesize CuO NPs [I7] and evaluated their anticancer efficiency against the HCT-116 cell line. The data showed that spherical nanoparticles with an average size of 20 nm were formed. CuO NPs demonstrated 50% inhibitory concentration (IC50) against the HCT-116 cell line at 25 gml − 1 . Bhavana et al., uses vitex altissima leaf extract for CuO NPs synthesis and examined their anticancer activity against A549 cell lines. The synthesized CuO NPs were spherical and oval in shape [ 18 ]. The findings demonstrated that the CuO NP-treated cell lines A549 cells exhibited a considerable increase in the ratio of early to late apoptotic cells and a decrease in the percentage of viable cells. In the present study, we report the green synthesis of CuO NPs using an aloe vera leaf extract. The objective is to examine the morphology and size-dependent antibacterial and anticancer properties of CuO NPs by variation in aging time and concentration of precursor solution. The Agar Well-diffusion method was used to investigate the antibacterial impact of different shapes of CuO NPs on Escherichia coli, and Staphylococcus aureus. Finally, we have determined the concentration, shape, and time-dependent effect of CuO NPs exposure in human cervical cancer (HeLa) cells. 2. Materials and methods 2.1. Materials The copper nitrate (Cu(NO 3 ) 2 ) was purchased from Sigma Aldrich. The aloe vera was collected from Islamabad Pakistan. Double distilled (DI) water was used for the preparation of the extract and the solutions of metal salt. 2.2. Preparation of Aloe vera leaf extract Fully grown aloe vera leaves were washed with DI water, dried completely, and cut into small pieces. 100 ml of DI water and 40, 50, and 70 g of cleaned, dried, and chopped leaves were combined. The mixture was then placed in an oven at 110 o C for 30 minutes, or until the aqueous solution's color changed from transparent to light yellow. The mixture was then stirred for 30 minutes at 110 o C. The prepared solution was passed through a Whatman filter paper to remove any solid particles as shown in Fig. 1 (a). The solution was thereafter kept at 4 o C as a stock for the synthesis of copper oxide (CuO). 2.3. Copper oxide nanoparticle (CuO NPs) synthesis CuO NPs with diverse morphologies were synthesized using copper nitrate as a precursor. Figure 1 (b) shows the complete biosynthesizing process. The copper precursor aqueous solutions were combined with aloe vera extract, mixed for 30 minutes at 110°C, and then placed in an oven for 45 minutes at 100°C. In order to age the prepared solutions, they were kept for 4 and 24 hours (aging time). The obtained solutions were then turned brown by gradually adding an aqueous solution of NaOH. CuO NPs of different shapes and sizes were prepared by varying the precursor solution's concentration (0.2 M, 0.4 M, and 0.6 M) and the ageing period (4 hrs, 24 hrs). The as-precipitated CuO NPs were then removed after 10 minutes of centrifuging and subjected to analysis. The chemical processes used to synthesize CuO NPs are shown in Fig. 1 (c). For the preliminary evaluations, the CuO NPs were synthesized at room temperature and assessed using UV-Visible spectroscopy. 2.4. Antibacterial activity of copper oxide nanoparticles (CuO NPs) 2.4.1. Test microorganisms A wide variety of multi-drug resistant pathogenic bacteria, such as gram-negative enteropathogenic Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus), were examined for the antibacterial activity of all the synthesized nanoparticles. The bacteria was obtained from Microbiology and Public Health Laboratory CIIT, Islamabad, Pakistan. 2.4.2. Screening of nanoparticles for antibacterial activity The Agar well diffusion method was used to screen the antibacterial activity of the nanoparticles [ 19 ]. Briefly, stock solutions of each synthesized nanoparticle were prepared at a concentration of 100 mgml − 1 in DMSO. Overnight culture of bacteria was spread on Muller Hinton Agar plates and 10 mm well were made by using a sterile corker borer. 100 µL of stock solutions of nanoparticles was added to each well and the petri plates were incubated at 37ºC for S. aureus and E. coli for 24 hrs. The zone of inhibition was measured in mm after the specified incubation period Ampicillin was used as a control. 2.4.3 Determination of minimum inhibitory concentration The micro-titer well plate test was used to determine the minimum inhibitory concentration (MIC) of each of the synthesized nanoparticles. 2, 3, 5-Triphenyl tetrazolium chloride (TTC) was utilized as a metabolic indicator to determine if the bacterial cells were alive or dead after being exposed to the nanoparticles [ 20 ]. Each synthesized nanoparticle's stock solution was made at a concentration of 100 mgml − 1 . After that, a series of dilutions were produced by serially diluting these stock solutions, with concentrations ranging from 100 to 1.5 mgml − 1 . Briefly, 180µ L of Muller Hinton broth was used to inoculate each micro-titer plate well with overnight bacterial suspension (roughly 107 CFUmL − 1 ). Next, 20 µl of nanoparticles from each of the stock solutions were added, resulting in final concentrations of 10 mgml − 1 , 5 mgml − 1 , 2.5 mgml − 1 , 1.25 mgml − 1 , 0.625 mgml − 1 , 0.325 mgml − 1 , and 0.15 mgml − 1 of synthesized nanoparticles. The 96 well plate was incubated at 37°C for 24 h S. aureus and E. coli . Untreated bacterial cells served as positive control and Ampicillin-treated cells as a negative control, respectively. While no color change was regarded as dead bacteria, any change from pink to pale red was noted as active bacteria. 2.5. Cell proliferation test to assess anticancer efficacy The MTT assay and direct microscope strategy have opted to study the in vitro anticancer activity of synthesized CuO NPs. 2.5.1. Cell line and cell culture The HeLa cervical cancer cells (ATCCCCL-2TM) were grown in a DMEM medium supplemented with 5% fetal bovine serum (FBS) at 37°C in a 5% CO 2 atmosphere. Hela cells were cultured at a density of 2 ×10 4 cells into 96 well microplates. Next, the cell was treated with synthesized CuO NPs at a fixed concentration of 0.5 µgml − 1 for 12 hrs. 2.5.2 MTT assay methodology The vitality of cells was evaluated using MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-DiphenyltetrazoliumBromide). After incubating the cells with MTT at 37°C, the formazan crystals produced were dissolved with HCl. An ELISA plate reader was used to measure the absorbance at 540 nm in order to assess the vitality of the cells. The percentage of cell viability was evaluated by using the following equation: $$\text{%} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}= \frac{Average OD of sample}{Average OD of control} \times 100$$ 2.6. Characterization of CuO NPs Different characterization techniques were used to investigate the structural, morphological, and optical properties of synthesized CuO NPs. The structural evaluation of CuO NPs was conducted using the Panalytical X'pert Pro. The FEI-Nova Nano SEM 430 scanning electron microscope was used to examine the morphology of composite materials. The FT-IR, JASCO 6600 was used to capture the FTIR spectra in the range of 500–4000 cm − 1 . 3. Results and discussion 3.1. Parameter optimization for plant extract preparation Figure 2 shows the UV spectra of aloe vera leaf extract for different optimization conditions. Figure 2 (a-b) represents the UV results for weight optimization and time optimization (1 and 3 hr) and Fig. 2 (c) for temperature optimization (70, 80, and 110℃), respectively. The extract had two significant resonances in every sample, one between 265 and 285 nm and another weak but wide resonance between 330 and 380 nm. It might be as a result of the polyphenolic components found in the leaf extract, which suggest the formation of aloe vera leaf extract [ 21 ]. Whereas the peak at 280 nm is due to the presence of flavonoids compound of plant extract. The highest intensity of UV spectra is obtained for 70 g which was kept on stirring and heating for 3 hrs at 80 ℃. This may be due to the fact that at low temperatures, time is insufficient to produce a homogeneous mixture. The primary metabolites will change into secondary metabolites when the temperature rises, and at that specific temperature, the phenolic molecule has acquired. With a further increase in temperature to 110 ℃, the water from the solution evaporates quickly which does not form a homogenous mixture. Therefore, 70 g of aloe vera heated at 80 ℃ for 3 hrs will be used for further experimentation. 3.2. Characterization of copper oxide nanoparticles (CuO NPs) 3.2.1. Ultra-violet visible spectroscopy (UV-vis) The presence of phytochemicals in aloe vera plant extracts is responsible for the creation of complexes with copper salt, which reduces the ions to synthesize the nanoparticles. Since the color change in the produced solutions could be seen, UV-vis spectroscopy was carried out in the 200–700 nm range. Figure 2 (d) shows UV spectra of green synthesized CuO NPs with a different molar ratio of copper nitrate (0.2 M, 0.4 M, and 0.6M) with an aging time of 4 and 24 hours. In all samples, a prominent peak at 288 nm is observed that is the characteristic of CuO nanoparticles arising due to the interband transition of the core electrons of the CuO NPs [ 22 ]. One sign that the narrow-band gap CuO NPs were effectively synthesized is the green synthesised CuO NPs' strong absorbance throughout the visible wavelength range. 3.2.2. X-Ray diffraction (XRD) analysis X-ray powder diffraction was used to analyze the phase composition and crystallite structure of the synthesized CuO NPs. CuO NPs XRD pattern is displayed in Fig. 3 (a). It shows diffraction peaks at 2θ = 29.66°, 32.72°, 35.78°, 38.97°, 46.60°, 49.10°, 53.81°, 58.78°, 61.88°, 66.70°, 68.52°, 72.80°, and 75.47° having miller indices (200), (110), (-111), (111), (-112), (-202), (020), (202), (-113), (-311), (222), (311) and (004) respectively. These crystal planes and the 2θ values were in very close agreement with the JCPDS card no 01-074-1021. This shows that it has a monoclinic structure [ 23 ]. The peak at 29.66° belongs to the cubic phase of Cu 2 O with miller indices (200) matched with card no (JCPDS # 00-002-1067). All the diffraction peaks correspond to typical monoclinic structure and no other phase was observed. It can be observed that CuO NPs synthesized at 0.4 M with 4 and 24 hrs aging time give the highest peak intensity and for CuO NPs prepared at 0.2, 0.4 M with 4 hrs aging time, the value was the lowest. Additionally, it was noted that as the concentration from 0.2 to 0.4 M and aging time, The strength of the major diffraction peaks was raised, as was the degree of crystallinity of CuO NPs. It was observed that by increasing the concentration from 0.2 M to 0.4 M the full width at half the maximum intensity increased. With a further increase in concentration (0.6 M), it decreases as shown in Fig. 3 (b). It can be observed that in Fig. 3 (c) increasing the aging time for 0.2 M concentration makes the peaks tend to shift to smaller diffraction angles. Whereas for 0.4 M, 4, and 24 hrs aging time no shift was observed. With further increase of concentration, first blue shift is observed for 4 hrs aging time and redshift for 24 hrs aging time. These peaks shift via XRD diffraction; the left side shift demonstrates lattice relaxation, whereas the right side shift reveals lattice strain [ 24 ]. These variations in lattice characteristics, as indicated in Table 1 , also affect the crystalline size by causing it to contract or shrink (right side) and expand (left side). Table 1 Lattice parameters (a, b, c), average crystallite size and full width at half the maximum intensity, β for CuO NPs synthesized with 0.2, 0.4 and 0.6 M concentration and aging time 2 and 24 hours. Sample a (Å) b (Å) c (Å) β (°) Average crystallite size (nm) 0.2 M, 4 hour 4.66 3.40 5.07 0.305 22.79 0.2 M, 24 hour 4.65 3.41 5.08 0.274 27.35 0.4M, 4 hour 4.68 3.42 5.12 0.427 19.53 0.4 M, 24 hour 4.68 3.40 5.11 0.366 20.36 0.6 M, 4 hour 4.67 3.40 5.08 0.244 28.38 0.6 M, 24 hour 4.66 3.38 5.06 0.305 22.18 3.2.3. Scanning electron microscopy analysis The precursor concentration and aging time play an effective role in the size and morphology of CuO NPs. The morphology of the synthesized samples was studied using FESEM shown in Fig. 4 (a-f). For 0.2 M concentration and 4 hrs aging time, the triangular + cube shaped (Fig. 4 (a)) morphology of particles was observed. With the increase in aging time to 24 hrs, cube and rod-like shapes (Fig. 4 (b)) are observed. When the concentration is increased to 0.4 M and for 4 hrs aging time, the particle morphology was spherical (Fig. 4 (c)). As the aging time increases to 24 hrs, morphology changes to leaf/spherical (Fig. 4 (d)) like structure. As the concentration is increased to 0.6 M, the particle size increases and gets agglomerated (Fig. 4 (e)) showing spherical plate-particles with a smooth surface. Whereas the morphology changes to cube/spherical (Fig. 4 (f)) structure when aging is increased to 24 hrs. According to the findings, increasing the aging time from 4 to 24 hours resulted in a change in the morphology of CuO NPs. It might be because surface energy was reduced in order to achieve thermodynamic stability. A longer aging period might give adequate time for the system's surface energy to be reduced by switching the particle morphology from cube to rod and leaf/spherical type [ 25 ]. Besides this, for fixed aging time, while the concentration has increased an aggregation/ increment in the size of nanoparticles was clearly observed when the concentration increased from 0.2 M to 0.6 M. The high NP aggregation/agglomeration may be induced by the polarity and electrostatic attraction of CuO NPs as a result of the higher concentration of the salt precursor [ 26 ]. Therefore, it could be concluded that the aging time and precursor concentration both affect the size and shape of CuO NPs. Energy dispersive X-ray (EDX) spectroscopy was used to analyse the elemental makeup of the synthesized nanoparticles. Figure 4 (g) displays the copper oxide nanoparticles' EDX spectrum. The outcomes show that the reaction product is made up of very pure CuO NPs, which is consistent with the XRD result (Fig. 2 ). The weight composition of the normalized spectrum obtained from EDS analysis was Cu (75.62%) and oxygen (24.38%). Additionally, nonstoichiometric CuO NPs containing oxygen vacancies were discovered by EDS, which may improve their antibacterial activity. 3.3. Antibacterial activity The potential antibacterial applications of CuO NPs produced under different conditions were also studied. Antibacterial activity of the synthesized CuO NPs of different shapes was determined using Gram-positive bacteria Staphylococcus aureus and Gram-negative pathogenic bacteria- Escherichia coli following the disc diffusion method. The CuO NPs were subjected to these bacteria and then the diameter of the zone of inhibition was measured. The inhibition zone (ZOI) obtained is shown in Fig. 5 . It can be seen that all the samples exhibit effective bacterial inhibitory activity. However, the size and shape of the CuO NPs had a substantial impact on the growth of the two studied bacteria. The CuO NPs synthesized at 0.2 M/24 hr revealed the highest antibacterial activity against E. coli and S. aureus , sample0.6M/24 hr showed minimum inhibition. In the present work, cube-shaped, rod-like, and spherical particles showed the highest inhibition effect against E. coli . It can be seen that the ZOI for E. coli increased from 22 to 27 mm as 0.2 M and 4 hr is not enough to synthesize enough amount of particles but as the aging time increased the particles are grown to rod-like and their antibacterial activity increased to 27 mm. As the concentration is increased to 0.4 M and 4 hr aging time the concentration of particles (average particle size 21 ± 2 nm) is increased which gives a high value of ZOI of 26 mm. However, as the concentration of precursor is increased to 0.6 M the size of particles increased which affects the antibacterial activity as well. These results are in good agreement with the literature [ 27 – 28 ]. So, the antibacterial activity depends on the size and shape of CuO NPs. The minimum inhibition concentration (MIC) obtained was 0.325 mgml − 1 for E.coli at 0.2 M and 24 hr aging time (Table 2 ). From here three sets of CuO NPs synthesized at 0.2 M/4 hr, 0.2 M/24 hr, and 0.4 M/2 hrs were selected to examine the anticancer activity. In addition to their size and morphology, CuO-NPs antibacterial activity was also influenced by the type of microbe. Gram-negative was more vulnerable to CuO NPs, whereas Gram-positive was more resistant to them. Similar results were reported by Sania et al., [ 29 ] demonstrating that the action of CuO-NPs is more pronounced against Gram-negative than Gram-positive bacterial strains. The different cell membrane composition and structure could be the cause of the differential in the activity against these two different types of bacteria. Compared to Gram-negative bacteria, Gram-positive bacteria have thicker peptidoglycan wall, so CuO NPs are unable to break this wall and showed weak antibacterial activity. Table 2 Minimum inhibition concentration (MIC) against E. coli and S. aureus MIC (mgml − 1 ) Samples 0.2 M/2hrs 0.2 M/24hrs 0.4 M/2hrs 0.4 M/24hrs 0.6M/2hrs 0.6M/24hrs E. coli 0.625 0.325 1.25 2.5 5 5 S. aureus 5 5 5 20 20 20 3.4. Anti-cancer activity of CuO NPs In the anticancer assay, several parameters such as size, shape, texture, and type of surface functions affect the anticancer activity. According to earlier studies, small-size particles have more anticancer effectiveness due to their improved capacity to penetrate cell lines [ 30 ]. Similarly, it is also reported that different shapes of the nanoparticles showed different anticancer activity [ 31 ]. The cytotoxicity of copper nanoparticles (synthesized at 0.2 M/4 hrs, 0.2 M/24 hrs, and 0.4 M/2 hrs) was investigated in this study by using the MTT assay for 12 hr on Hela cancer cell lines. The interaction is expressed as cell viability (%) was observed at different CuO NPs concentrations (0-180 ngml − 1 ) with the cell line and shown in Fig. 6 (a-d). When CuO NPs were used to treat the Hela cell line, cytotoxicity increased in a concentration-dependent manner. MTT assay has shown that CuO NPs (0.2 M/4 hrs) significantly decreased the viability in the concentration range of 60–180 ngml − 1 . CuO spheres exerted the smallest cytotoxic effect compared to other analyzed nanoparticles. The cell viability decrease with an increase in time. Comparing the irregularly shaped CuO NPs to the rod-like and spherical CuO NPs, the current study demonstrates that the irregularly shaped CuO NPs have the greatest anti-cancer effect on the cell line. The irregularly shaped nanoparticles can move through unevenly distributed neoplastic cells with more ease; it is plausible to conclude that these unique unpredictable cube/triangle forms have a stronger anti-proliferative effect [ 32 ]. Furthermore, after incubation for 12, 24, and 48 hours, the morphological alterations caused by treatment with the CuO NPs (180 ngml − 1 ) were observed. As can be seen, the untreated (control) cells are widely dispersed and have adherent epithelial cells as their morphology under the inverted microscope. At high temperatures, the epithelial monolayer sheets are destroyed partially or entirely. Moreover, several morphological modifications were seen in Fig. 6 (A-D). Figure 6 (A-D) represents the morphological changes observed for control (Fig. 6 A), 0.4 M/2 hrs (Fig. 6 B), 0.2 M/24 hrs (Fig. 6 C), and 0.2 M/4 hrs (Fig. 6 D) for 48 hours of incubation. Furthermore, compared with the untreated cells several morphological changes like rounding, migrating, buoyancy, shrinkage, and granulations are observed. In the case of 0.2 M/4 hrs synthesized CuO NPs, apoptotic and dead cells were found in large numbers throughout the cultivated plates, causing significant changes in cell structure and quantity. CuO NPs ability to inhibit the growth of cancer cells may be directly related to their ability to produce ROS [ 33 ]. When a cell is exposed to CuO NPs, it dissociates to generate Cu 2+, which binds to important macromolecules such as proteins, nucleic acids, and ribosomes. This causes the molecules to become distorted or broken down, which ultimately causes cell death. Moreover, CuO-NPs in the cell interact with the lysosome's acidic environment or the mitochondria, producing ROS that harms the cells. According to studies that have been published, cancer cells produce more ROS than healthy cells do as a result of their high metabolic activity [ 34 ], and this characteristic is advantageous for the use of NPs in the treatment of cancer. 4. Conclusion The present work reports a green synthesis approach by co-precipitation method using aloe vera leaf extract in the production of copper oxide nanoparticles. The CuO NPs were characterized by UV Visible, XRD, and SEM. The SEM analysis showed that the morphology of CuO NPs changes from spherical to agglomerated flake-like form as the concentration of precursor and aging time changes. The crystallite size for 4 hr aging time was 22.35, 19.53, and 28.35 for 0.2 M, 0.4 M, and 0.6 M respectively. However, for 24 hr aging time, the crystallite size changed from 27.35, 20.79, and 22.18 for 0.2 M, 0.4 M, and 0.6 M respectively. The elemental composition analysis showed that the prepared CuO NPs of high purity as no other compound was found in EDX. The prepared CuO NPs were used to examine the antibacterial activity against E. coli and S. aureus . The maximum zone of inhibition (28 mm) attained by CuO NPs 0.2 M with an aging time of 24 hr against multidrug-resistant E.coli . The dose-dependent cytotoxicity of CuO NPs against human Hela cells points to the possibility of using these particles as an anticancer treatment. Results of this study point to the possibility of using biosynthesized CuO NPs as antibacterial and anticancer products for commercial and medicinal uses. Declarations Conflict of interest The authors declare that they have no financial or other conflicts of interest. Funding This research received no specific grant from any funding agency. Acknowledgment The Functional Material Laboratory (FML), Air University, Islamabad, provided the synthesis facilities, for which the authors are grateful. The Pakistan Institute of Engineering and Applied Sciences (PIEAS) Islamabad and the Microbiology and Public Health Laboratory at CIIT in Islamabad, Pakistan, are also acknowledged by the authors for providing characterization facilities. Contribution All authors contributed to the study conception and design. The main concept was designed by Dr. Saima Rafique. Material preparation and data collection were performed by Ms. Tooba Javed. The analysis was performed by Dr. Shazia Bashir and Dr. Rizwan Akram. The biological test were performed by Dr. Zobia Noreen and Ms. Zeenat Haq. The first draft of the manuscript was written by Dr. Saima Rafique and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Y. Khan, H. Sadia, S.Z. Ali Shah, M.N. Khan, A.A. Shah, N. Ullah, M.F. Ullah, H. Bibi, O.T. Bafakeeh, N. Ben Khedher, S.M. Eldin, B.M. Fadhl, M.I. Khan, Classification, Synthetic, and Characterization Approaches to Nanoparticles, and Their Applications in Various Fields of Nanotechnology: A Review, Catalysts 2022, Vol. 12, Page 1386. 12 (2022) 1386. https://doi.org/10.3390/CATAL12111386 . N. Baig, I. Kammakakam, W. Falath, I. Kammakakam, Nanomaterials: a review of synthesis methods, properties, recent progress, and challenges, Mater Adv. 2 (2021) 1821–1871. https://doi.org/10.1039/D0MA00807A . H. Hadji, K. Bouchemal, Effect of micro- and nanoparticle shape on biological processes, J Control Release. 342 (2022) 93–110. https://doi.org/10.1016/J.JCONREL.2021.12.032 . C. Pandit, A. Roy, S. Ghotekar, A. Khusro, M.N. Islam, T. Bin Emran, S.E. Lam, M.U. Khandaker, D.A. Bradley, Biological agents for synthesis of nanoparticles and their applications, J King Saud Univ Sci. 34 (2022) 101869. https://doi.org/10.1016/J.JKSUS.2022.101869 . Hemlata, P.R. Meena, A.P. Singh, K.K. Tejavath, Biosynthesis of Silver Nanoparticles Using Cucumis prophetarum Aqueous Leaf Extract and Their Antibacterial and Antiproliferative Activity Against Cancer Cell Lines, ACS Omega. 5 (2020) 5520–5528. https://doi.org/10.1021/ACSOMEGA.0C00155 . M.T. Yassin, F.O. Al-Otibi, A.A.F. Mostafa, A.A. Al-Askar, Facile Green Synthesis of Silver Nanoparticles Using Aqueous Leaf Extract of Origanum majorana with Potential Bioactivity against Multidrug Resistant Bacterial Strains, Crystals (Basel). 12 (2022). https://doi.org/10.3390/CRYST12050603/S1 . J.S. Moodley, S.B.N. Krishna, K. Pillay, Sershen, P. Govender, Green synthesis of silver nanoparticles from Moringa oleifera leaf extracts and its antimicrobial potential, Advances in Natural Sciences: Nanoscience and Nanotechnology. 9 (2018) 015011. https://doi.org/10.1088/2043-6254/AAABB2 . A. Jayachandran, A. T.R., A.S. Nair, Green synthesis and characterization of zinc oxide nanoparticles using Cayratia pedata leaf extract, Biochem Biophys Rep. 26 (2021) 100995. https://doi.org/10.1016/J.BBREP.2021.100995 . B. Fardsadegh, H. Jafarizadeh-Malmiri, Aloe vera leaf extract mediated green synthesis of selenium nanoparticles and assessment of their in vitro antimicrobial activity against spoilage fungi and pathogenic bacteria strains, Green Processing and Synthesis. 8 (2019) 399–407. https://doi.org/10.1515/GPS-2019-0007/MACHINEREADABLECITATION/RIS . A. Surjushe, R. Vasani, D. Saple, ALOE VERA: A SHORT REVIEW, Indian J Dermatol. 53 (2008) 163–166. https://doi.org/10.4103/0019-5154.44785 . Z. Vaseghi, A. Nematollahzadeh, O. Tavakoli, Green methods for the synthesis of metal nanoparticles using biogenic reducing agents: A review, Reviews in Chemical Engineering. 34 (2018) 529–559. https://doi.org/10.1515/REVCE-2017-0005 /ASSET/GRAPHIC/J_REVCE-2017-0005_CV_003.JPG . F. Amin, Fozia, B. Khattak, A. Alotaibi, M. Qasim, I. Ahmad, R. Ullah, M. Bourhia, A. Gul, S. Zahoor, R. Ahmad, Green Synthesis of Copper Oxide Nanoparticles Using Aerva javanica Leaf Extract and Their Characterization and Investigation of in Vitro Antimicrobial Potential and Cytotoxic Activities, Evidence-Based Complementary and Alternative Medicine. 2021 (2021). https://doi.org/10.1155/2021/5589703 . H. Liu, G. Wang, J. Liu, K. Nan, J. Zhang, L. Guo, Y. Liu, Green synthesis of copper nanoparticles using Cinnamomum zelanicum extract and its applications as a highly efficient antioxidant and anti-human lung carcinoma, J Exp Nanosci. 16 (2021) 411–423. https://doi.org/10.1080/17458080.2021.1991577 . V. Balakrishnan, K. Thangaraj, M. Palani, M. Vaiyapuri, Green synthesis of copper oxide nanoparticles using Euphorbia hirta leaves extract and its biological applications, Https://Doi.Org/ 10.1080/24701556.2021.1952260 . 52 (2021) 809–818. https://doi.org/10.1080/24701556.2021.1952260. R. Rajamma, S.G. Nair, F.A. Khadar, B. Baskaran, Antibacterial and anticancer activity of biosynthesised CuO nanoparticles, IET Nanobiotechnol. 14 (2020) 833–838. https://doi.org/10.1049/IET-NBT.2020.0088 . A. Abbasi, K. Ghorban, F. Nojoomi, M. Dadmanesh, Smaller Copper Oxide Nanoparticles have More Biological Effects Versus Breast Cancer and Nosocomial Infections Bacteria, Asian Pac J Cancer Prev. 22 (2021) 893–902. https://doi.org/10.31557/APJCP.2021.22.3.893 . S. Tabrez, A.U. Khan, A.A. Mirza, M. Suhail, N.R. Jabir, T.A. Zughaibi, M. Alam, Biosynthesis of copper oxide nanoparticles and its therapeutic efficacy against colon cancer, Nanotechnol Rev. 11 (2022) 1322–1331. https://doi.org/10.1515/NTREV-2022-0081/PDF . S. Bhavana, C.G. Kusuma, V. Gubbiveeranna, C.K. Sumachirayu, H. Ravikumar, S. Nagaraju, Green route synthesis of copper oxide nanoparticles using Vitex altissima [L] leaves extract and their potential anticancer activity against A549 cell lines and its apoptosis induction, Https://Doi.Org/ 10.1080/24701556.2022.2081195 . (2022). https://doi.org/10.1080/24701556.2022.2081195. B.B. Sokmen, S. Ugras, H.Y. Sarikaya, H.I. Ugras, R. Yanardag, Antibacterial, antiurease, and antioxidant activities of some arylidene barbiturates, Appl Biochem Biotechnol. 171 (2013) 2030–2039. https://doi.org/10.1007/S12010-013-0486-6 . S.D. Sarker, L. Nahar, Y. Kumarasamy, Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals, Methods. 42 (2007) 321–324. https://doi.org/10.1016/J.YMETH.2007.01.006 . S. Gupta, K.K. Tejavath, Catalytic Reduction of Organic Dyes with Green Synthesized Silver Nanoparticles using Aloe vera Leaf Extract, Journal of Nanoscience Nanoengineering and Applications. (2019) 9–21. https://doi.org/10.37591/JONSNEA.V9I2.661 . A.E.D. Mahmoud, K.M. Al-Qahtani, S.O. Alflaij, S.F. Al-Qahtani, F.A. Alsamhan, Green copper oxide nanoparticles for lead, nickel, and cadmium removal from contaminated water, Sci Rep. 11 (2021). https://doi.org/10.1038/S41598-021-91093-7 . M. Bin Mobarak, M.S. Hossain, F. Chowdhury, S. Ahmed, Synthesis and characterization of CuO nanoparticles utilizing waste fish scale and exploitation of XRD peak profile analysis for approximating the structural parameters, Arabian Journal of Chemistry. 15 (2022). https://doi.org/10.1016/J.ARABJC.2022.104117 . M.N. Abdillah, W.S.B. Dwandaru, XRD Peak Shift and Enhancement of Repeated Mechanically Exfoliated SnO 2 Thin Films Synthesized from SnCl 2 Powder by Direct Heating, Nanoscience and Nanotechnology Research, Vol. 4, 2017, Pages 127–131. 4 (2017) 127–131. https://doi.org/10.12691/NNR-4-4-2 . S. Tavakoli, M. Kharaziha, S. Ahmadi, Green synthesis and morphology dependent antibacterial activity of copper oxide nanoparticles, Journal of Nanostructures. 9 (2019) 163–171. https://doi.org/10.22052/JNS.2019.01.018 . N. Ahmad, B.C. Ang, M.A. Amalina, C.W. Bong, Influence of precursor concentration and temperature on the formation of nanosilver in chemical reduction method, Sains Malays. 47 (2018) 157–168. https://doi.org/10.17576/JSM-2018-4701-19 . J. Penders, M. Stolzoff, D.J. Hickey, M. Andersson, T.J. Webster, Shape-dependent antibacterial effects of non-cytotoxic gold nanoparticles, Int J Nanomedicine. 12 (2017) 2457–2468. https://doi.org/10.2147/IJN.S124442 . F.J. Osonga, A. Akgul, I. Yazgan, A. Akgul, G.B. Eshun, L. Sakhaee, O.A. Sadik, Size and Shape-Dependent Antimicrobial Activities of Silver and Gold Nanoparticles: A Model Study as Potential Fungicides, Molecules. 25 (2020). https://doi.org/10.3390/MOLECULES25112682 . S. Sonia, N.D. Jayram, P. Suresh Kumar, D. Mangalaraj, N. Ponpandian, C. Viswanathan, Effect of NaOH concentration on structural, surface and antibacterial activity of CuO nanorods synthesized by direct sonochemical method, Superlattices Microstruct. 66 (2014) 1–9. https://doi.org/10.1016/J.SPMI.2013.10.020 . A. Chinnathambi, T. Awad Alahmadi, S. Ali Alharbi, Biogenesis of copper nanoparticles (Cu-NPs) using leaf extract of Allium noeanum, antioxidant and in-vitro cytotoxicity, Https://Doi.Org/ 10.1080/21691401.2021.1926275 . 49 (2021) 500–510. https://doi.org/10.1080/21691401.2021.1926275. Y. Guo, S. Zhao, H. Qiu, T. Wang, Y. Zhao, M. Han, Z. Dong, X. Wang, Shape of Nanoparticles as a Design Parameter to Improve Docetaxel Antitumor Efficacy, Bioconjug Chem. 29 (2018) 1302–1311. https://doi.org/10.1021/ACS.BIOCONJCHEM.8B00059 . D. Raghunandan, B. Ravishankar, G. Sharanbasava, D.B. Mahesh, V. Harsoor, M.S. Yalagatti, M. Bhagawanraju, A. Venkataraman, Anti-cancer studies of noble metal nanoparticles synthesized using different plant extracts, Cancer Nanotechnol. 2 (2011) 57–65. https://doi.org/10.1007/S12645-011-0014-8 . A. Fouda, S.E.D. Hassan, A.M. Eid, M.A. Awad, K. Althumayri, N.F. Badr, M.F. Hamza, Endophytic bacterial strain, Brevibacillus brevis-mediated green synthesis of copper oxide nanoparticles, characterization, antifungal, in vitro cytotoxicity, and larvicidal activity, Green Processing and Synthesis. 11 (2022) 931–950. https://doi.org/10.1515/GPS-2022-0080 /ASSET/GRAPHIC/J_GPS-2022-0080_FIG_008.JPG . D. Letchumanan, S.P.M. Sok, S. Ibrahim, N.H. Nagoor, N.M. Arshad, Plant-Based Biosynthesis of Copper/Copper Oxide Nanoparticles: An Update on Their Applications in Biomedicine, Mechanisms, and Toxicity, Biomolecules. 11 (2021). https://doi.org/10.3390/BIOM11040564 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3530645","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":248249375,"identity":"13033808-269d-4659-90d3-8f7be32b45a2","order_by":0,"name":"Saima Rafique","email":"data:image/png;base64,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","orcid":"","institution":"Air University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Saima","middleName":"","lastName":"Rafique","suffix":""},{"id":248249376,"identity":"9c722dc3-dc3e-40e3-902c-751009840663","order_by":1,"name":"Rizwan Akram","email":"","orcid":"","institution":"Air University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rizwan","middleName":"","lastName":"Akram","suffix":""},{"id":248249380,"identity":"be2b9bd7-b012-4252-bd5c-5a46fb632352","order_by":2,"name":"Tooba Javed","email":"","orcid":"","institution":"Air University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tooba","middleName":"","lastName":"Javed","suffix":""},{"id":248249381,"identity":"5be4600b-af30-4be1-a6cf-31e785313db6","order_by":3,"name":"Shazia Bashir","email":"","orcid":"","institution":"Pakistan Institute of Engineering and Applied Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shazia","middleName":"","lastName":"Bashir","suffix":""},{"id":248249383,"identity":"a6cadf14-8796-4398-866e-7b63e0374900","order_by":4,"name":"Zobia Noreen","email":"","orcid":"","institution":"COMSATS University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zobia","middleName":"","lastName":"Noreen","suffix":""}],"badges":[],"createdAt":"2023-11-01 03:59:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3530645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3530645/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46440314,"identity":"e7eb2cef-9112-4964-9b64-57a5396dd96e","added_by":"auto","created_at":"2023-11-14 18:45:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1059256,"visible":true,"origin":"","legend":"\u003cp\u003e(a) steps for preparation of aloe vera extract (b) schematic diagram of the synthesis of copper oxide CuO NPs for different precursor concentrations and aging time (c) chemistry involved in the synthesis of CuO NPs using aloe vera.\u003c/p\u003e","description":"","filename":"floatimage118.png","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/8a85c0c7136e26786ccf1d18.png"},{"id":46440318,"identity":"7bc6ba6e-e232-46b4-9617-66e36c7a91d7","added_by":"auto","created_at":"2023-11-14 18:45:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":578153,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption spectra of aloe vera extract samples synthesized using (a-b) different weights of aloe vera and time optimization (c) temperature optimization (d) copper oxide nanoparticles synthesized with aloe vera extract with different concentrations and aging time.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/1fe11a7adab67f3d131a266d.jpeg"},{"id":46440313,"identity":"be621b90-f5a1-49f5-92e9-77e54580bb85","added_by":"auto","created_at":"2023-11-14 18:45:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27623,"visible":true,"origin":"","legend":"\u003cp\u003e(a) XRD graph CuO nanoparticles (b) shift in the peak position for 0.2, 0.4, and 0.6 M solutions with 4 and 24 hours aging time.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/ab0fd0ffec253bb66e328e35.png"},{"id":46440316,"identity":"76ba7812-0917-4cc1-8397-f377b9da7d8f","added_by":"auto","created_at":"2023-11-14 18:45:49","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1017081,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of different samples (a-b) of 0.2 M, (c-d) of 0.4 M and (e-f) of 0.6 M concentration of precursor with an aging time of 4 and 24 hrs at 500 nm scale. EDX shows the elemental composition and these EDX spectrums show no extra peak other than Cu and O elements. This confirms the high purity of CuO.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/fcd91df4cf3be4a0d94ffb1f.jpeg"},{"id":46440692,"identity":"ff22ed21-304c-44a2-99db-0a046aaa67f4","added_by":"auto","created_at":"2023-11-14 18:53:49","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":983450,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition of CuO nanoparticles (b) bar graph representation of zone of inhibition.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/6fcdbf6c1d9f54f7f469fa62.jpeg"},{"id":46440319,"identity":"3294119d-23ff-46e7-a3e2-5455e1731d0b","added_by":"auto","created_at":"2023-11-14 18:45:49","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1175624,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The cytotoxicity properties of the normal cervical epithelial cell line, the anticancer properties (b) for 12 hours, (c) 24 hours (d) 48 hours for Aloe Vera, 0.2 M/4 hr, 0.2 M/24 hr, and 0.4 M/2 hrs, respectively. Morphological changes in cancer cell (A) control (B) 0.4 M/2 hrs (C) 0.2 M/24 hr (D) 0.2 M/4 hr, green synthesized CuO NPs.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/fae7b1063db576c3c0a8fc04.jpeg"},{"id":48355458,"identity":"40d8cfb7-b3a0-47a2-ae86-37ae8a9a614d","added_by":"auto","created_at":"2023-12-17 20:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1475775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3530645/v1/7ae1ff98-cc8f-4af2-a8b5-58c078908208.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biosynthesized copper oxide nanoparticles using aloe vera leaves extract and their evaluation of antibacterial, anticancer in human Hela cancer cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRecently, nanoparticles have extensively been used in various fields such as medicine, diagnostics, gas sensing, etc. due to their distinct physical and chemical properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Nanoparticles come in a variety of sizes, shapes, and chemical compositions for human benefit [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nanoparticles are synthesized via physical, chemical, and biological processes. In the chemical synthesis approach some toxins and chemicals absorbed at the surface have a variety of negative impacts on medical and environmental applications. An intriguing area of nanotechnology is biologically synthesized nanoparticles using plant extract, which is environmentally benign compared to chemical and physical methods [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent reports describe the use of various plants including cucumis prophetarum [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], origanum majorana [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], moringa oleifera [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and cayratia pedata [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] to synthesize nanoparticles. Aloe vera is a significant medicinal plant that comes in two parts: the peel (leaf) and the gel. Numerous studies have shown that its leaf contains a wide variety of phenolic compounds, sterols, vitamins, saponins, proteins, lignin, flavonoids, enzymes, polysaccharides, and organic acids. All of these are crucial for the stabilization of synthesized nanoparticles and the reduction of ions to their constituent elements to form NPs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. There are over 75 potentially active components in aloe vera, and they have properties that can heal wounds or burns, suppress inflammation, and fight parasites [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this connection, metal or metal oxide nanoparticles are particularly important in the field of nanomedicine, where nanoparticles are used as effective therapeutic agents, drug delivery vehicles, and nano biosensors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These nanoparticles are strong stable, and highly effective against a variety of microbes. Besides this, copper oxide NPs have a shelf life that is too long compared to other organic antimicrobial agents. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNanoparticles show new or better properties based on certain characteristics such as morphology, size, and distribution. Therefore in nanoparticle synthesis, the main goal is the formation of nanoparticles with different shapes, minimum particle size, and maximum stability. Haocong et al., synthesized copper oxide nanoparticles (CuO NPs) using cinnamomum zelanicum extract [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. They studied the antioxidant and anticancer activity for lung carcinoma. The synthesized nanoparticles have spherical morphology with size ranges of 19.55 to 69.70 nm. They came to the conclusion that there was no cytotoxicity on the normal cell line and that the copper nanoparticles exhibited very low cell viability and dose-dependent anti-lung cancer effects against different cell lines. Similarly, Vaitheeswari et al., used euphorbia hirta leaf extract for the synthesis of CuO NPs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and studied their antimicrobial and antioxidant activity. They synthesized spherical-shaped nanoparticles of uniform size. These nanoparticles showed prominent antibacterial activity and antioxidant activity. Resmi et al., synthesize CuO NPs using nilgirianthus ciliates plant extract [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. They prepared spherical particles with uniform morphology and particle size in the range of 20 nm which shows a 13 mm zone of inhibition against \u003cem\u003eE. coli\u003c/em\u003e. Similarly, Abbasi et al., studied the effect of the size of CuO NPs on antibacterial and anticancer properties [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. They found that 30 nm CuO NPs showed a strong antibacterial effect against \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e bacteria and more potential anticancer properties were obtained against 4T1 cell lines than 60 nm CuO NPs. Tabrez et al., used pumpkin seed extract to synthesize CuO NPs [I7] and evaluated their anticancer efficiency against the HCT-116 cell line. The data showed that spherical nanoparticles with an average size of 20 nm were formed. CuO NPs demonstrated 50% inhibitory concentration (IC50) against the HCT-116 cell line at 25 gml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Bhavana et al., uses vitex altissima leaf extract for CuO NPs synthesis and examined their anticancer activity against A549 cell lines. The synthesized CuO NPs were spherical and oval in shape [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The findings demonstrated that the CuO NP-treated cell lines A549 cells exhibited a considerable increase in the ratio of early to late apoptotic cells and a decrease in the percentage of viable cells.\u003c/p\u003e \u003cp\u003eIn the present study, we report the green synthesis of CuO NPs using an aloe vera leaf extract. The objective is to examine the morphology and size-dependent antibacterial and anticancer properties of CuO NPs by variation in aging time and concentration of precursor solution. The Agar Well-diffusion method was used to investigate the antibacterial impact of different shapes of CuO NPs on \u003cem\u003eEscherichia coli, and Staphylococcus aureus.\u003c/em\u003e Finally, we have determined the concentration, shape, and time-dependent effect of CuO NPs exposure in human cervical cancer (HeLa) cells.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe copper nitrate (Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) was purchased from Sigma Aldrich. The aloe vera was collected from Islamabad Pakistan. Double distilled (DI) water was used for the preparation of the extract and the solutions of metal salt.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Aloe vera leaf extract\u003c/h2\u003e \u003cp\u003eFully grown aloe vera leaves were washed with DI water, dried completely, and cut into small pieces. 100 ml of DI water and 40, 50, and 70 g of cleaned, dried, and chopped leaves were combined. The mixture was then placed in an oven at 110 \u003csup\u003eo\u003c/sup\u003eC for 30 minutes, or until the aqueous solution's color changed from transparent to light yellow. The mixture was then stirred for 30 minutes at 110 \u003csup\u003eo\u003c/sup\u003eC. The prepared solution was passed through a Whatman filter paper to remove any solid particles as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a). The solution was thereafter kept at 4 \u003csup\u003eo\u003c/sup\u003eC as a stock for the synthesis of copper oxide (CuO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Copper oxide nanoparticle (CuO NPs) synthesis\u003c/h2\u003e \u003cp\u003eCuO NPs with diverse morphologies were synthesized using copper nitrate as a precursor. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) shows the complete biosynthesizing process. The copper precursor aqueous solutions were combined with aloe vera extract, mixed for 30 minutes at 110\u0026deg;C, and then placed in an oven for 45 minutes at 100\u0026deg;C. In order to age the prepared solutions, they were kept for 4 and 24 hours (aging time). The obtained solutions were then turned brown by gradually adding an aqueous solution of NaOH. CuO NPs of different shapes and sizes were prepared by varying the precursor solution's concentration (0.2 M, 0.4 M, and 0.6 M) and the ageing period (4 hrs, 24 hrs). The as-precipitated CuO NPs were then removed after 10 minutes of centrifuging and subjected to analysis. The chemical processes used to synthesize CuO NPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c). For the preliminary evaluations, the CuO NPs were synthesized at room temperature and assessed using UV-Visible spectroscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antibacterial activity of copper oxide nanoparticles (CuO NPs)\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Test microorganisms\u003c/h2\u003e \u003cp\u003eA wide variety of multi-drug resistant pathogenic bacteria, such as gram-negative enteropathogenic Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus), were examined for the antibacterial activity of all the synthesized nanoparticles. The bacteria was obtained from Microbiology and Public Health Laboratory CIIT, Islamabad, Pakistan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Screening of nanoparticles for antibacterial activity\u003c/h2\u003e \u003cp\u003eThe Agar well diffusion method was used to screen the antibacterial activity of the nanoparticles [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, stock solutions of each synthesized nanoparticle were prepared at a concentration of 100 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMSO. Overnight culture of bacteria was spread on Muller Hinton Agar plates and 10 mm well were made by using a sterile corker borer. 100 \u0026micro;L of stock solutions of nanoparticles was added to each well and the petri plates were incubated at 37\u0026ordm;C for \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e for 24 hrs. The zone of inhibition was measured in mm after the specified incubation period Ampicillin was used as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Determination of minimum inhibitory concentration\u003c/h2\u003e \u003cp\u003eThe micro-titer well plate test was used to determine the minimum inhibitory concentration (MIC) of each of the synthesized nanoparticles. 2, 3, 5-Triphenyl tetrazolium chloride (TTC) was utilized as a metabolic indicator to determine if the bacterial cells were alive or dead after being exposed to the nanoparticles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Each synthesized nanoparticle's stock solution was made at a concentration of 100 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After that, a series of dilutions were produced by serially diluting these stock solutions, with concentrations ranging from 100 to 1.5 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Briefly, 180\u0026micro; L of Muller Hinton broth was used to inoculate each micro-titer plate well with overnight bacterial suspension (roughly 107 CFUmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Next, 20 \u0026micro;l of nanoparticles from each of the stock solutions were added, resulting in final concentrations of 10 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 5 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.5 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.25 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.625 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.325 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 0.15 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of synthesized nanoparticles. The 96 well plate was incubated at 37\u0026deg;C for 24 h \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. Untreated bacterial cells served as positive control and Ampicillin-treated cells as a negative control, respectively. While no color change was regarded as dead bacteria, any change from pink to pale red was noted as active bacteria.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell proliferation test to assess anticancer efficacy\u003c/h2\u003e \u003cp\u003eThe MTT assay and direct microscope strategy have opted to study the in vitro anticancer activity of synthesized CuO NPs.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Cell line and cell culture\u003c/h2\u003e \u003cp\u003eThe HeLa cervical cancer cells (ATCCCCL-2TM) were grown in a DMEM medium supplemented with 5% fetal bovine serum (FBS) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Hela cells were cultured at a density of 2 \u0026times;10\u003csup\u003e4\u003c/sup\u003e cells into 96 well microplates. Next, the cell was treated with synthesized CuO NPs at a fixed concentration of 0.5 \u0026micro;gml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 12 hrs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 MTT assay methodology\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe vitality of cells was evaluated using MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-DiphenyltetrazoliumBromide). After incubating the cells with MTT at 37\u0026deg;C, the formazan crystals produced were dissolved with HCl. An ELISA plate reader was used to measure the absorbance at 540 nm in order to assess the vitality of the cells. The percentage of cell viability was evaluated by using the following equation:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{%} \\text{v}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}= \\frac{Average OD of sample}{Average OD of control} \\times 100$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Characterization of CuO NPs\u003c/h2\u003e \u003cp\u003eDifferent characterization techniques were used to investigate the structural, morphological, and optical properties of synthesized CuO NPs. The structural evaluation of CuO NPs was conducted using the Panalytical X'pert Pro. The FEI-Nova Nano SEM 430 scanning electron microscope was used to examine the morphology of composite materials. The FT-IR, JASCO 6600 was used to capture the FTIR spectra in the range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Parameter optimization for plant extract preparation\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the UV spectra of aloe vera leaf extract for different optimization conditions. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a-b) represents the UV results for weight optimization and time optimization (1 and 3 hr) and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c) for temperature optimization (70, 80, and 110℃), respectively. The extract had two significant resonances in every sample, one between 265 and 285 nm and another weak but wide resonance between 330 and 380 nm. It might be as a result of the polyphenolic components found in the leaf extract, which suggest the formation of aloe vera leaf extract [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Whereas the peak at 280 nm is due to the presence of flavonoids compound of plant extract. The highest intensity of UV spectra is obtained for 70 g which was kept on stirring and heating for 3 hrs at 80 ℃. This may be due to the fact that at low temperatures, time is insufficient to produce a homogeneous mixture. The primary metabolites will change into secondary metabolites when the temperature rises, and at that specific temperature, the phenolic molecule has acquired. With a further increase in temperature to 110 ℃, the water from the solution evaporates quickly which does not form a homogenous mixture. Therefore, 70 g of aloe vera heated at 80 ℃ for 3 hrs will be used for further experimentation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization of copper oxide nanoparticles (CuO NPs)\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Ultra-violet visible spectroscopy (UV-vis)\u003c/h2\u003e \u003cp\u003eThe presence of phytochemicals in aloe vera plant extracts is responsible for the creation of complexes with copper salt, which reduces the ions to synthesize the nanoparticles. Since the color change in the produced solutions could be seen, UV-vis spectroscopy was carried out in the 200\u0026ndash;700 nm range. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d) shows UV spectra of green synthesized CuO NPs with a different molar ratio of copper nitrate (0.2 M, 0.4 M, and 0.6M) with an aging time of 4 and 24 hours. In all samples, a prominent peak at 288 nm is observed that is the characteristic of CuO nanoparticles arising due to the interband transition of the core electrons of the CuO NPs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. One sign that the narrow-band gap CuO NPs were effectively synthesized is the green synthesised CuO NPs' strong absorbance throughout the visible wavelength range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. X-Ray diffraction (XRD) analysis\u003c/h2\u003e \u003cp\u003eX-ray powder diffraction was used to analyze the phase composition and crystallite structure of the synthesized CuO NPs. CuO NPs XRD pattern is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a). It shows diffraction peaks at 2θ\u0026thinsp;=\u0026thinsp;29.66\u0026deg;, 32.72\u0026deg;, 35.78\u0026deg;, 38.97\u0026deg;, 46.60\u0026deg;, 49.10\u0026deg;, 53.81\u0026deg;, 58.78\u0026deg;, 61.88\u0026deg;, 66.70\u0026deg;, 68.52\u0026deg;, 72.80\u0026deg;, and 75.47\u0026deg; having miller indices (200), (110), (-111), (111), (-112), (-202), (020), (202), (-113), (-311), (222), (311) and (004) respectively. These crystal planes and the 2θ values were in very close agreement with the JCPDS card no 01-074-1021. This shows that it has a monoclinic structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The peak at 29.66\u0026deg; belongs to the cubic phase of Cu\u003csub\u003e2\u003c/sub\u003eO with miller indices (200) matched with card no (JCPDS # 00-002-1067). All the diffraction peaks correspond to typical monoclinic structure and no other phase was observed.\u003c/p\u003e \u003cp\u003eIt can be observed that CuO NPs synthesized at 0.4 M with 4 and 24 hrs aging time give the highest peak intensity and for CuO NPs prepared at 0.2, 0.4 M with 4 hrs aging time, the value was the lowest. Additionally, it was noted that as the concentration from 0.2 to 0.4 M and aging time, The strength of the major diffraction peaks was raised, as was the degree of crystallinity of CuO NPs. It was observed that by increasing the concentration from 0.2 M to 0.4 M the full width at half the maximum intensity increased. With a further increase in concentration (0.6 M), it decreases as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b).\u003c/p\u003e \u003cp\u003eIt can be observed that in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c) increasing the aging time for 0.2 M concentration makes the peaks tend to shift to smaller diffraction angles. Whereas for 0.4 M, 4, and 24 hrs aging time no shift was observed. With further increase of concentration, first blue shift is observed for 4 hrs aging time and redshift for 24 hrs aging time. These peaks shift via XRD diffraction; the left side shift demonstrates lattice relaxation, whereas the right side shift reveals lattice strain [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These variations in lattice characteristics, as indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, also affect the crystalline size by causing it to contract or shrink (right side) and expand (left side).\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003e\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003c/div\u003e \u003c/caption\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eTable 1 Lattice parameters (a, b, c), average crystallite size and full width at half the maximum intensity, β for CuO NPs synthesized with 0.2, 0.4 and 0.6 M concentration and aging time 2 and 24 hours.\u003c/p\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ec (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ (\u0026deg;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAverage crystallite size (nm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.2 M, 4 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.2 M, 24 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.4M, 4 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.4 M, 24 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.6 M, 4 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.6 M, 24 hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Scanning electron microscopy analysis\u003c/h2\u003e \u003cp\u003eThe precursor concentration and aging time play an effective role in the size and morphology of CuO NPs. The morphology of the synthesized samples was studied using FESEM shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a-f). For 0.2 M concentration and 4 hrs aging time, the triangular\u0026thinsp;+\u0026thinsp;cube shaped (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a)) morphology of particles was observed. With the increase in aging time to 24 hrs, cube and rod-like shapes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b)) are observed. When the concentration is increased to 0.4 M and for 4 hrs aging time, the particle morphology was spherical (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c)). As the aging time increases to 24 hrs, morphology changes to leaf/spherical (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)) like structure. As the concentration is increased to 0.6 M, the particle size increases and gets agglomerated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(e)) showing spherical plate-particles with a smooth surface. Whereas the morphology changes to cube/spherical (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(f)) structure when aging is increased to 24 hrs.\u003c/p\u003e \u003cp\u003eAccording to the findings, increasing the aging time from 4 to 24 hours resulted in a change in the morphology of CuO NPs. It might be because surface energy was reduced in order to achieve thermodynamic stability. A longer aging period might give adequate time for the system's surface energy to be reduced by switching the particle morphology from cube to rod and leaf/spherical type [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides this, for fixed aging time, while the concentration has increased an aggregation/ increment in the size of nanoparticles was clearly observed when the concentration increased from 0.2 M to 0.6 M. The high NP aggregation/agglomeration may be induced by the polarity and electrostatic attraction of CuO NPs as a result of the higher concentration of the salt precursor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, it could be concluded that the aging time and precursor concentration both affect the size and shape of CuO NPs.\u003c/p\u003e \u003cp\u003eEnergy dispersive X-ray (EDX) spectroscopy was used to analyse the elemental makeup of the synthesized nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(g) displays the copper oxide nanoparticles' EDX spectrum. The outcomes show that the reaction product is made up of very pure CuO NPs, which is consistent with the XRD result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The weight composition of the normalized spectrum obtained from EDS analysis was Cu (75.62%) and oxygen (24.38%). Additionally, nonstoichiometric CuO NPs containing oxygen vacancies were discovered by EDS, which may improve their antibacterial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe potential antibacterial applications of CuO NPs produced under different conditions were also studied. Antibacterial activity of the synthesized CuO NPs of different shapes was determined using Gram-positive bacteria \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and Gram-negative pathogenic bacteria- \u003cem\u003eEscherichia coli\u003c/em\u003e following the disc diffusion method. The CuO NPs were subjected to these bacteria and then the diameter of the zone of inhibition was measured. The inhibition zone (ZOI) obtained is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It can be seen that all the samples exhibit effective bacterial inhibitory activity. However, the size and shape of the CuO NPs had a substantial impact on the growth of the two studied bacteria. The CuO NPs synthesized at 0.2 M/24 hr revealed the highest antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, sample0.6M/24 hr showed minimum inhibition. In the present work, cube-shaped, rod-like, and spherical particles showed the highest inhibition effect against \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIt can be seen that the ZOI for \u003cem\u003eE. coli\u003c/em\u003e increased from 22 to 27 mm as 0.2 M and 4 hr is not enough to synthesize enough amount of particles but as the aging time increased the particles are grown to rod-like and their antibacterial activity increased to 27 mm. As the concentration is increased to 0.4 M and 4 hr aging time the concentration of particles (average particle size 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm) is increased which gives a high value of ZOI of 26 mm. However, as the concentration of precursor is increased to 0.6 M the size of particles increased which affects the antibacterial activity as well. These results are in good agreement with the literature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. So, the antibacterial activity depends on the size and shape of CuO NPs. The minimum inhibition concentration (MIC) obtained was 0.325 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE.coli\u003c/em\u003e at 0.2 M and 24 hr aging time (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From here three sets of CuO NPs synthesized at 0.2 M/4 hr, 0.2 M/24 hr, and 0.4 M/2 hrs were selected to examine the anticancer activity.\u003c/p\u003e \u003cp\u003eIn addition to their size and morphology, CuO-NPs antibacterial activity was also influenced by the type of microbe. Gram-negative was more vulnerable to CuO NPs, whereas Gram-positive was more resistant to them. Similar results were reported by Sania et al., [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] demonstrating that the action of CuO-NPs is more pronounced against Gram-negative than Gram-positive bacterial strains. The different cell membrane composition and structure could be the cause of the differential in the activity against these two different types of bacteria. Compared to Gram-negative bacteria, Gram-positive bacteria have thicker peptidoglycan wall, so CuO NPs are unable to break this wall and showed weak antibacterial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimum inhibition concentration (MIC) against E. coli and S. aureus\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eMIC (mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2 M/2hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2 M/24hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4 M/2hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.4 M/24hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6M/2hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.6M/24hrs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Anti-cancer activity of CuO NPs\u003c/h2\u003e \u003cp\u003eIn the anticancer assay, several parameters such as size, shape, texture, and type of surface functions affect the anticancer activity. According to earlier studies, small-size particles have more anticancer effectiveness due to their improved capacity to penetrate cell lines [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, it is also reported that different shapes of the nanoparticles showed different anticancer activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The cytotoxicity of copper nanoparticles (synthesized at 0.2 M/4 hrs, 0.2 M/24 hrs, and 0.4 M/2 hrs) was investigated in this study by using the MTT assay for 12 hr on Hela cancer cell lines. The interaction is expressed as cell viability (%) was observed at different CuO NPs concentrations (0-180 ngml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with the cell line and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a-d). When CuO NPs were used to treat the Hela cell line, cytotoxicity increased in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003eMTT assay has shown that CuO NPs (0.2 M/4 hrs) significantly decreased the viability in the concentration range of 60\u0026ndash;180 ngml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. CuO spheres exerted the smallest cytotoxic effect compared to other analyzed nanoparticles. The cell viability decrease with an increase in time.\u003c/p\u003e \u003cp\u003eComparing the irregularly shaped CuO NPs to the rod-like and spherical CuO NPs, the current study demonstrates that the irregularly shaped CuO NPs have the greatest anti-cancer effect on the cell line. The irregularly shaped nanoparticles can move through unevenly distributed neoplastic cells with more ease; it is plausible to conclude that these unique unpredictable cube/triangle forms have a stronger anti-proliferative effect [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, after incubation for 12, 24, and 48 hours, the morphological alterations caused by treatment with the CuO NPs (180 ngml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were observed. As can be seen, the untreated (control) cells are widely dispersed and have adherent epithelial cells as their morphology under the inverted microscope. At high temperatures, the epithelial monolayer sheets are destroyed partially or entirely.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, several morphological modifications were seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A-D). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A-D) represents the morphological changes observed for control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), 0.4 M/2 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), 0.2 M/24 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), and 0.2 M/4 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) for 48 hours of incubation. Furthermore, compared with the untreated cells several morphological changes like rounding, migrating, buoyancy, shrinkage, and granulations are observed. In the case of 0.2 M/4 hrs synthesized CuO NPs, apoptotic and dead cells were found in large numbers throughout the cultivated plates, causing significant changes in cell structure and quantity.\u003c/p\u003e \u003cp\u003eCuO NPs ability to inhibit the growth of cancer cells may be directly related to their ability to produce ROS [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. When a cell is exposed to CuO NPs, it dissociates to generate Cu\u003csup\u003e2+,\u003c/sup\u003e which binds to important macromolecules such as proteins, nucleic acids, and ribosomes. This causes the molecules to become distorted or broken down, which ultimately causes cell death. Moreover, CuO-NPs in the cell interact with the lysosome's acidic environment or the mitochondria, producing ROS that harms the cells. According to studies that have been published, cancer cells produce more ROS than healthy cells do as a result of their high metabolic activity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and this characteristic is advantageous for the use of NPs in the treatment of cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe present work reports a green synthesis approach by co-precipitation method using aloe vera leaf extract in the production of copper oxide nanoparticles. The CuO NPs were characterized by UV Visible, XRD, and SEM. The SEM analysis showed that the morphology of CuO NPs changes from spherical to agglomerated flake-like form as the concentration of precursor and aging time changes. The crystallite size for 4 hr aging time was 22.35, 19.53, and 28.35 for 0.2 M, 0.4 M, and 0.6 M respectively. However, for 24 hr aging time, the crystallite size changed from 27.35, 20.79, and 22.18 for 0.2 M, 0.4 M, and 0.6 M respectively. The elemental composition analysis showed that the prepared CuO NPs of high purity as no other compound was found in EDX. The prepared CuO NPs were used to examine the antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The maximum zone of inhibition (28 mm) attained by CuO NPs 0.2 M with an aging time of 24 hr against multidrug-resistant \u003cem\u003eE.coli\u003c/em\u003e. The dose-dependent cytotoxicity of CuO NPs against human Hela cells points to the possibility of using these particles as an anticancer treatment. Results of this study point to the possibility of using biosynthesized CuO NPs as antibacterial and anticancer products for commercial and medicinal uses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or other conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific \u003cem\u003egrant\u003c/em\u003e from any \u003cem\u003efunding\u003c/em\u003e agency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Functional Material Laboratory (FML), Air University, Islamabad, provided the synthesis facilities, for which the authors are grateful. The Pakistan Institute of Engineering and Applied Sciences (PIEAS) Islamabad and the Microbiology and Public Health Laboratory at CIIT in Islamabad, Pakistan, are also acknowledged by the authors for providing characterization facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. The main concept was designed by Dr. Saima Rafique. Material preparation and data collection were performed by Ms. Tooba Javed. The analysis was performed by Dr. Shazia Bashir and Dr. Rizwan Akram. The biological test were performed by Dr. Zobia Noreen and Ms. Zeenat Haq. The first draft of the manuscript was written by Dr. Saima Rafique and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eY. Khan, H. Sadia, S.Z. Ali Shah, M.N. Khan, A.A. Shah, N. Ullah, M.F. Ullah, H. Bibi, O.T. Bafakeeh, N. Ben Khedher, S.M. Eldin, B.M. Fadhl, M.I. Khan, Classification, Synthetic, and Characterization Approaches to Nanoparticles, and Their Applications in Various Fields of Nanotechnology: A Review, Catalysts 2022, Vol.\u0026nbsp;12, Page 1386. 12 (2022) 1386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/CATAL12111386\u003c/span\u003e\u003cspan address=\"10.3390/CATAL12111386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN. Baig, I. Kammakakam, W. Falath, I. Kammakakam, Nanomaterials: a review of synthesis methods, properties, recent progress, and challenges, Mater Adv. 2 (2021) 1821\u0026ndash;1871. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D0MA00807A\u003c/span\u003e\u003cspan address=\"10.1039/D0MA00807A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Hadji, K. Bouchemal, Effect of micro- and nanoparticle shape on biological processes, J Control Release. 342 (2022) 93\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JCONREL.2021.12.032\u003c/span\u003e\u003cspan address=\"10.1016/J.JCONREL.2021.12.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Pandit, A. Roy, S. Ghotekar, A. Khusro, M.N. Islam, T. Bin Emran, S.E. Lam, M.U. Khandaker, D.A. Bradley, Biological agents for synthesis of nanoparticles and their applications, J King Saud Univ Sci. 34 (2022) 101869. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JKSUS.2022.101869\u003c/span\u003e\u003cspan address=\"10.1016/J.JKSUS.2022.101869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHemlata, P.R. Meena, A.P. Singh, K.K. Tejavath, Biosynthesis of Silver Nanoparticles Using Cucumis prophetarum Aqueous Leaf Extract and Their Antibacterial and Antiproliferative Activity Against Cancer Cell Lines, ACS Omega. 5 (2020) 5520\u0026ndash;5528. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACSOMEGA.0C00155\u003c/span\u003e\u003cspan address=\"10.1021/ACSOMEGA.0C00155\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.T. Yassin, F.O. Al-Otibi, A.A.F. Mostafa, A.A. Al-Askar, Facile Green Synthesis of Silver Nanoparticles Using Aqueous Leaf Extract of Origanum majorana with Potential Bioactivity against Multidrug Resistant Bacterial Strains, Crystals (Basel). 12 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/CRYST12050603/S1\u003c/span\u003e\u003cspan address=\"10.3390/CRYST12050603/S1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.S. Moodley, S.B.N. Krishna, K. Pillay, Sershen, P. Govender, Green synthesis of silver nanoparticles from Moringa oleifera leaf extracts and its antimicrobial potential, Advances in Natural Sciences: Nanoscience and Nanotechnology. 9 (2018) 015011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/2043-6254/AAABB2\u003c/span\u003e\u003cspan address=\"10.1088/2043-6254/AAABB2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Jayachandran, A. T.R., A.S. Nair, Green synthesis and characterization of zinc oxide nanoparticles using Cayratia pedata leaf extract, Biochem Biophys Rep. 26 (2021) 100995. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.BBREP.2021.100995\u003c/span\u003e\u003cspan address=\"10.1016/J.BBREP.2021.100995\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Fardsadegh, H. Jafarizadeh-Malmiri, Aloe vera leaf extract mediated green synthesis of selenium nanoparticles and assessment of their in vitro antimicrobial activity against spoilage fungi and pathogenic bacteria strains, Green Processing and Synthesis. 8 (2019) 399\u0026ndash;407. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/GPS-2019-0007/MACHINEREADABLECITATION/RIS\u003c/span\u003e\u003cspan address=\"10.1515/GPS-2019-0007/MACHINEREADABLECITATION/RIS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Surjushe, R. Vasani, D. Saple, ALOE VERA: A SHORT REVIEW, Indian J Dermatol. 53 (2008) 163\u0026ndash;166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/0019-5154.44785\u003c/span\u003e\u003cspan address=\"10.4103/0019-5154.44785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ. Vaseghi, A. Nematollahzadeh, O. Tavakoli, Green methods for the synthesis of metal nanoparticles using biogenic reducing agents: A review, Reviews in Chemical Engineering. 34 (2018) 529\u0026ndash;559. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/REVCE-2017-0005\u003c/span\u003e\u003cspan address=\"10.1515/REVCE-2017-0005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/ASSET/GRAPHIC/J_REVCE-2017-0005_CV_003.JPG\u003c/span\u003e\u003cspan address=\"http:///ASSET/GRAPHIC/J_REVCE-2017-0005_CV_003.JPG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF. Amin, Fozia, B. Khattak, A. Alotaibi, M. Qasim, I. Ahmad, R. Ullah, M. Bourhia, A. Gul, S. Zahoor, R. Ahmad, Green Synthesis of Copper Oxide Nanoparticles Using Aerva javanica Leaf Extract and Their Characterization and Investigation of in Vitro Antimicrobial Potential and Cytotoxic Activities, Evidence-Based Complementary and Alternative Medicine. 2021 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2021/5589703\u003c/span\u003e\u003cspan address=\"10.1155/2021/5589703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Liu, G. Wang, J. Liu, K. Nan, J. Zhang, L. Guo, Y. Liu, Green synthesis of copper nanoparticles using Cinnamomum zelanicum extract and its applications as a highly efficient antioxidant and anti-human lung carcinoma, J Exp Nanosci. 16 (2021) 411\u0026ndash;423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17458080.2021.1991577\u003c/span\u003e\u003cspan address=\"10.1080/17458080.2021.1991577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV. Balakrishnan, K. Thangaraj, M. Palani, M. Vaiyapuri, Green synthesis of copper oxide nanoparticles using Euphorbia hirta leaves extract and its biological applications, Https://Doi.Org/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/24701556.2021.1952260\u003c/span\u003e\u003cspan address=\"10.1080/24701556.2021.1952260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 52 (2021) 809\u0026ndash;818. https://doi.org/10.1080/24701556.2021.1952260.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Rajamma, S.G. Nair, F.A. Khadar, B. Baskaran, Antibacterial and anticancer activity of biosynthesised CuO nanoparticles, IET Nanobiotechnol. 14 (2020) 833\u0026ndash;838. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1049/IET-NBT.2020.0088\u003c/span\u003e\u003cspan address=\"10.1049/IET-NBT.2020.0088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Abbasi, K. Ghorban, F. Nojoomi, M. Dadmanesh, Smaller Copper Oxide Nanoparticles have More Biological Effects Versus Breast Cancer and Nosocomial Infections Bacteria, Asian Pac J Cancer Prev. 22 (2021) 893\u0026ndash;902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.31557/APJCP.2021.22.3.893\u003c/span\u003e\u003cspan address=\"10.31557/APJCP.2021.22.3.893\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Tabrez, A.U. Khan, A.A. Mirza, M. Suhail, N.R. Jabir, T.A. Zughaibi, M. Alam, Biosynthesis of copper oxide nanoparticles and its therapeutic efficacy against colon cancer, Nanotechnol Rev. 11 (2022) 1322\u0026ndash;1331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/NTREV-2022-0081/PDF\u003c/span\u003e\u003cspan address=\"10.1515/NTREV-2022-0081/PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Bhavana, C.G. Kusuma, V. Gubbiveeranna, C.K. Sumachirayu, H. Ravikumar, S. Nagaraju, Green route synthesis of copper oxide nanoparticles using Vitex altissima [L] leaves extract and their potential anticancer activity against A549 cell lines and its apoptosis induction, Https://Doi.Org/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/24701556.2022.2081195\u003c/span\u003e\u003cspan address=\"10.1080/24701556.2022.2081195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2022). https://doi.org/10.1080/24701556.2022.2081195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB.B. Sokmen, S. Ugras, H.Y. Sarikaya, H.I. Ugras, R. Yanardag, Antibacterial, antiurease, and antioxidant activities of some arylidene barbiturates, Appl Biochem Biotechnol. 171 (2013) 2030\u0026ndash;2039. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S12010-013-0486-6\u003c/span\u003e\u003cspan address=\"10.1007/S12010-013-0486-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.D. Sarker, L. Nahar, Y. Kumarasamy, Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals, Methods. 42 (2007) 321\u0026ndash;324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.YMETH.2007.01.006\u003c/span\u003e\u003cspan address=\"10.1016/J.YMETH.2007.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Gupta, K.K. Tejavath, Catalytic Reduction of Organic Dyes with Green Synthesized Silver Nanoparticles using Aloe vera Leaf Extract, Journal of Nanoscience Nanoengineering and Applications. (2019) 9\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.37591/JONSNEA.V9I2.661\u003c/span\u003e\u003cspan address=\"10.37591/JONSNEA.V9I2.661\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.E.D. Mahmoud, K.M. Al-Qahtani, S.O. Alflaij, S.F. Al-Qahtani, F.A. Alsamhan, Green copper oxide nanoparticles for lead, nickel, and cadmium removal from contaminated water, Sci Rep. 11 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/S41598-021-91093-7\u003c/span\u003e\u003cspan address=\"10.1038/S41598-021-91093-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Bin Mobarak, M.S. Hossain, F. Chowdhury, S. Ahmed, Synthesis and characterization of CuO nanoparticles utilizing waste fish scale and exploitation of XRD peak profile analysis for approximating the structural parameters, Arabian Journal of Chemistry. 15 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ARABJC.2022.104117\u003c/span\u003e\u003cspan address=\"10.1016/J.ARABJC.2022.104117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.N. Abdillah, W.S.B. Dwandaru, XRD Peak Shift and Enhancement of Repeated Mechanically Exfoliated SnO \u0026lt; SUB \u0026gt; 2\u0026lt;/SUB \u0026gt; Thin Films Synthesized from SnCl \u0026lt; SUB \u0026gt; 2\u0026lt;/SUB \u0026gt; Powder by Direct Heating, Nanoscience and Nanotechnology Research, Vol.\u0026nbsp;4, 2017, Pages 127\u0026ndash;131. 4 (2017) 127\u0026ndash;131. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12691/NNR-4-4-2\u003c/span\u003e\u003cspan address=\"10.12691/NNR-4-4-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Tavakoli, M. Kharaziha, S. Ahmadi, Green synthesis and morphology dependent antibacterial activity of copper oxide nanoparticles, Journal of Nanostructures. 9 (2019) 163\u0026ndash;171. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22052/JNS.2019.01.018\u003c/span\u003e\u003cspan address=\"10.22052/JNS.2019.01.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN. Ahmad, B.C. Ang, M.A. Amalina, C.W. Bong, Influence of precursor concentration and temperature on the formation of nanosilver in chemical reduction method, Sains Malays. 47 (2018) 157\u0026ndash;168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17576/JSM-2018-4701-19\u003c/span\u003e\u003cspan address=\"10.17576/JSM-2018-4701-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Penders, M. Stolzoff, D.J. Hickey, M. Andersson, T.J. Webster, Shape-dependent antibacterial effects of non-cytotoxic gold nanoparticles, Int J Nanomedicine. 12 (2017) 2457\u0026ndash;2468. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S124442\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S124442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF.J. Osonga, A. Akgul, I. Yazgan, A. Akgul, G.B. Eshun, L. Sakhaee, O.A. Sadik, Size and Shape-Dependent Antimicrobial Activities of Silver and Gold Nanoparticles: A Model Study as Potential Fungicides, Molecules. 25 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/MOLECULES25112682\u003c/span\u003e\u003cspan address=\"10.3390/MOLECULES25112682\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Sonia, N.D. Jayram, P. Suresh Kumar, D. Mangalaraj, N. Ponpandian, C. Viswanathan, Effect of NaOH concentration on structural, surface and antibacterial activity of CuO nanorods synthesized by direct sonochemical method, Superlattices Microstruct. 66 (2014) 1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SPMI.2013.10.020\u003c/span\u003e\u003cspan address=\"10.1016/J.SPMI.2013.10.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Chinnathambi, T. Awad Alahmadi, S. Ali Alharbi, Biogenesis of copper nanoparticles (Cu-NPs) using leaf extract of Allium noeanum, antioxidant and in-vitro cytotoxicity, Https://Doi.Org/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21691401.2021.1926275\u003c/span\u003e\u003cspan address=\"10.1080/21691401.2021.1926275\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 49 (2021) 500\u0026ndash;510. https://doi.org/10.1080/21691401.2021.1926275.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY. Guo, S. Zhao, H. Qiu, T. Wang, Y. Zhao, M. Han, Z. Dong, X. Wang, Shape of Nanoparticles as a Design Parameter to Improve Docetaxel Antitumor Efficacy, Bioconjug Chem. 29 (2018) 1302\u0026ndash;1311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.BIOCONJCHEM.8B00059\u003c/span\u003e\u003cspan address=\"10.1021/ACS.BIOCONJCHEM.8B00059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Raghunandan, B. Ravishankar, G. Sharanbasava, D.B. Mahesh, V. Harsoor, M.S. Yalagatti, M. Bhagawanraju, A. Venkataraman, Anti-cancer studies of noble metal nanoparticles synthesized using different plant extracts, Cancer Nanotechnol. 2 (2011) 57\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S12645-011-0014-8\u003c/span\u003e\u003cspan address=\"10.1007/S12645-011-0014-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Fouda, S.E.D. Hassan, A.M. Eid, M.A. Awad, K. Althumayri, N.F. Badr, M.F. Hamza, Endophytic bacterial strain, Brevibacillus brevis-mediated green synthesis of copper oxide nanoparticles, characterization, antifungal, in vitro cytotoxicity, and larvicidal activity, Green Processing and Synthesis. 11 (2022) 931\u0026ndash;950. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/GPS-2022-0080\u003c/span\u003e\u003cspan address=\"10.1515/GPS-2022-0080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/ASSET/GRAPHIC/J_GPS-2022-0080_FIG_008.JPG\u003c/span\u003e\u003cspan address=\"http:///ASSET/GRAPHIC/J_GPS-2022-0080_FIG_008.JPG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Letchumanan, S.P.M. Sok, S. Ibrahim, N.H. Nagoor, N.M. Arshad, Plant-Based Biosynthesis of Copper/Copper Oxide Nanoparticles: An Update on Their Applications in Biomedicine, Mechanisms, and Toxicity, Biomolecules. 11 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/BIOM11040564\u003c/span\u003e\u003cspan address=\"10.3390/BIOM11040564\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanoparticles, CuO, antibacterial activity, anticancer activity, Hela cell line","lastPublishedDoi":"10.21203/rs.3.rs-3530645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3530645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research work assessed the shape-dependent anti-cancer activity of copper oxide nanoparticles (CuO NPs) synthesized from aloe vera leaf extract on the human HeLa cell line (ATCCCCL-2TM) and their antimicrobial action against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) and \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e). The CuO NPs were synthesized by varying the concentration of precursor and aging time to obtain different shapes of nanoparticles. The synthesized particles had rod-like, cube-shaped, triangular, and spherical morphologies. The CuO NPs were examined against gram-positive and gram-negative bacteria. The results showed that NPs inhibit the growth of these bacteria and antibacterial activity depends on the size and shape of CuO NPs. The minimum inhibition concentration obtained was 0.325 mgml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE. coli\u003c/em\u003e at 0.2 M and 24 hrs aging time. A dose-dependent reduction in cell viability was observed using CuO NPs. CuO NPs caused significant morphological alterations in Hela cell lines, including shrinkage, detachment, and distorted shape. These findings imply that different-shaped CuO NPs may inhibit bacterial growth, elevate oxidative stress, and induce apoptosis in addition shows cytotoxic effects on cancer cells.\u003c/p\u003e","manuscriptTitle":"Biosynthesized copper oxide nanoparticles using aloe vera leaves extract and their evaluation of antibacterial, anticancer in human Hela cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-14 18:45:43","doi":"10.21203/rs.3.rs-3530645/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be06c266-2eaa-4882-a642-f43ff94d50f0","owner":[],"postedDate":"November 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-17T19:59:13+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-14 18:45:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3530645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3530645","identity":"rs-3530645","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}