In the recent years, metal nanoparticles have been widely applied in various fields consisting of electrical, magnetic, catalytic and biological activities due to their unique physical and chemical characteristics [1-3]. In particular, copper oxide nanoparticles (CuO-NPs) have been broadly studied in various fields including agriculture, fabrics, and especially in hospital and clinical applications due to their unique biological and antibacterial properties, with high efficiency for a broad spectrum of microorganisms, and fewer prices compared to noble metals with similar properties [4-6].
CuO-NPs can be synthesized using various approaches consisting of hydrothermal , solution combustion , precipitation , electrochemical , microwave assisted , chemical , force hydrolysis , sol-gel , reverse microemulsion  and sono-chemical  techniques. Most of these approaches are eco-hazardous limiting their applications in medicine and bioengineering. Therefore, attentions have focused to develop eco-friendly techniques such as biosynthesis method by employing microorganisms such as bacterial and fungi, as well as whole plants, tissues and extracts . Recently, the extracts of plants such as Geranium leaf, Catharanthus roseus and lemongrass have been applied to stabilize and reduce the metal ions such as ZnO , In2O3, FexOy , gold (Au) and silver (Ag)  nanoparticles. Between various kinds of the plant, Aloe vera plant has been widely applied to green synthesize various kinds of metal and metal oxides [22, 23]. Aloe vera comprises of about 75 potentially active constituents, which possess immunomodulatory, anti-inflammatory, antiparasitic and wound or burn-healing characteristics . Aloe vera extracts have recently applied in order to synthesize copper based nanomaterials such as CuO, Cu and etc. For instance, Kumar et al.  synthesized spherical like CuO nanoparticle with average crystallite size of 22 nm using Aloe vera extracts and evaluated their antibacterial characteristics against three bacterial fish pathogens.
Researches demonstrated that nanoparticles with various sizes, morphologies, chemical compositions and surface charges have different antibacterial characteristics which could be due to different ability to generation reactive oxygen species (ROS) and their corresponding ions from nanoparticles [26-29]. Results demonstrated that the antibacterial characteristics of nanoparticles often stems from the production of ROS from nanoparticles which result in oxidative damages to cell construct . For instance, Azamet al.  revealed the particle size-dependent antibacterial characteristics of chemically synthesized CuO nanoparticles against gram-positive and negative bacterial strains. In another study, Xiong et al.  studied the effects of various morphologies of Cu/Cu2O nanoparticles consisting of polyhedral, flower-like, and thumbtack-like on the antibacterial activities. They used chemical reducing agent to synthesize Cu/Cu2O nanoparticles. Peng et al.  applied glycine-mediated mixed-solvothermal approach to synthesize Cu2O with various morphologies from cubic to octahedral. They revealed that the antibacterial properties changed from general bacteriostasis to high selectivity by changing the morphology of nanoparticles from cubic to octahedral.
This work is aimed to find out for the first time how the biosythesize parameters affect on the size and morphology of CuO nanoparticles. Furthermore, the effects of these morphology changes on the antibacterial activities against various bacterial organisms are studied. In this study, we adopted a green chemistry approach for the synthesize of CuO-NPs with various morphologies and sizes using the extract plant of Aloe Vera as reducing agent and investigated the effects of biosynthesize parameters on the antibacterial activities against various bacterial organisms. Furthermore, the antibacterial properties of CuO-NPs were studied against a Gram negative, Escherichia coli (E. coli) and a gram-positive, Staphylococcus aureus (St. aureus) bacteria. Results of this study may provide a new strategy to gain effective antibacterial activities via at least price and eco-friendly approach.
MATERIALS AND METHODS
Copper (II) chloride salt (CuCl2.2H2O), Copper (II) sulfate (CuSO4.5H2O) and sodium hydroxide (NaOH) were purchased from Merck Co. and used as received without further purification. Double distilled (DI) water was used in all the sections of experiment.
Preparation of Aloe Vera leaf extract
Fresh leaves of two-year Aloe Vera were washed with DI water and then dried, completely. Washed, dried and cut leaves (25 g) were added to DI water (30 ml) and kept for 30 min at 110ºC in oven until the color of aqueous solution changed from watery to light yellow. After that, the mixture was stirred for 30 min at 110 ºC. As prepared solution was passed through a whatman filter paper to remove any solid particle. Finally, the solution was stored at 4 ºC as stock for the synthesis of copper oxide (CuO).
Synthesis of copper oxide nanoparticles (CuO-NPs)
In order to synthesize CuO-NPs with various morphologies, two copper precursors were applied: CuCl2.2H2O and CuSO4.5H2O. The biosynthesize process is depicted in Fig. 1. Aloe Vera extract was added to the aqueous solutions of cupper precursor, stirred at 110 ºC for 30 min and then kept at 100 ºC for 45 min in oven. As prepared solutions were maintained for 72 hr (aging time) to change their color to yellow for samples C and D, and green for samples A, B and E. Afterwards, the aqueous solution of NaOH was added drop by drop to change the color of as-prepared solutions to brown. In order to prepare CuO-NPs with various morphologies and size, the concentration of solution and aging time were changed (as in Table 1). Finally, after centrifuging for 10 min, as-precipitated CuO-NPs were taken out for characterization.
Characterization of copper oxide nanoparticles (CuO-NPs)
Phase structure analysis of CuO-NPs was carried out by X-ray diffractometer (XRD, Philips X-pert) using Ni filtered Cu Kα (λ CuKα=0.154186 nm, radiation at 40 kV and 30 mA) over the 2θ range of 30–80° (time per step: 2.5 sec and step size: 0.05°). The average crystallite size was estimated using broadening XRD peaks and Scherrer equation (Eq. (1)):
where λ is the wavelength (0.15406 nm), θ is the Bragg angle, k is a constant (0.9), and t is the crystallite size (nm). For this purpose, three diffraction peaks of , (111) and , which have the advantages of being well-separated and high intensities, were chosen for the determination of crystallite size. Sigma plot software was also applied to calculate the half-widths of peaks.
The morphology of the powders was investigated by scanning electron microscopy (SEM Phillips XL 30: Eindhoven, The Netherlands). Before imaging, the powders were sputter-coated with a thin layer of gold and the SEM images were utilized to determine the average particle sizes (n=20) using (NIH) Image J software.
Antibacterial activity of copper oxide nanoparticles (CuO-NPs)
The antibacterial activity of CuO-NPs was studied against a Gram negative bacteria (E. coli) and a gram-positive bacterium (St. aureus) using well-diffusion agar assay. Bacterial strains were obtained from Persian Type Culture Collection (PTCC) and maintained on Mueller Hinton (MH) slants. A loopful of slant culture was inoculated into Tryptic Soy Broth (Merck, Germany) and incubated at 37 ºC for 18 hr (Memert, Germany) until the population of bacteria reached 108 CFU/mL. 100 μL of broth culture were poured into sterile petri dishes and then 25 mL of molten Mueller Hinton Agar Media (Merck, Germany) (45 ºC) was added to each petri dish and the plates were gently agitated until the bacteria were spread through whole culture media. The wells were made using the bottom of sterile pasture pipets and the special concentration of nanoparticles suspension containing 10 μg of CuO-NPs was loaded into the wells. The plates were incubated at 37 ºC for 24 hr. Finally, the zone of growth inhibition was measured as antibacterial activity of samples. Three replicates were maintained for each sample and the data presented as mean ± SD (standard error of the mean).
GraphPad, Prism Software (V.5) was used to determine statistical significance in particle and crystallite size results using one-way ANOVA analysis followed by Tukey’s multiple comparison. P≤ 0.05 was considered statistically significant. Furthermore, analysis of variance in antibacterial activity evaluation was performed by SASTM 9.2 software (SAS institute, USA) to compare mean values using LSD test at P≤ 0.05 significance level.
RSULTS AND DISCUSSION
Characterization of CuO-NPs
XRD patterns demonstrated while pure CuO-NPs were synthesized in samples A, B and E, the secondary phase of Cu(OH)2 could be detected in samples C and D (Fig. 2(a)). However, the major peaks of CuO located at 2Ө= 35.70° and 38.65° (indexed as and (111) planes based on JCPDS no. 05-0667) could be detected in the samples C and D confirming incomplete formation process of CuO based on following equations:
Based on these equations, Cu (OH)2 synthesized during the process, could act as nuclei for the growth of CuO-NPs. Therefor, when Cu2+ and OH- concentrations reached the crucial value, CuO nuclei formed spontaneously in the aqueous solution. Based on our data, the low concentrations of CuCl2 (6 mM) and NaOH (1 M) were not enough to complete the equations 2 and 3 leading to the formation of Cu (OH)2 as the secondary phase in samples C and D.
The average crystallite size of CuO-NPs was estimated in the range of 9-23 nm, depending on the precursor conditions (Fig. 2(b)). Samples C and D revealed significantly bigger crystallite size than samples A and B (P<0.05) demonstrating the crucial role of NaOH concentration on the controlling the crystallization of CuO-NPs. When NaOH to CuCl2 concentration ratio increased (sample C compared to B), the crystallite size of the CuO-NPs enhanced. Based on these results, through choosing the appropriate Cu2+/OH- concentration ratio, the crystallite growth of CuO-NPs could be controlled. Similar result was reported in previous report .
SEM micrograph of the synthesized CuO-NPs demonstrated the effective roles of precursor concentration and type as well as aging time on the morphology and size of CuO-NPs (Fig. 3 (a-e)). While the morphology of particles synthesized via A, D and E conditions were spherical, samples B and C consisted of platelet particles with sharp edges and rod shape, respectively. Additionally, sample E consisted of spherical plate-particles with smooth surface. The average particle sizes determined using image J software (n=20) are presented in Fig. 3(f). The particle sizes varied from 55 to 512 nm depending on the experimental parameters. Between these samples, conditions A and B resulted in significantly reduced particle size (P<0.05) revealing the effective role of precursor concentration (CuCl2.2H2O) on the particle size.
Based on our results, increasing the aging time from 0 to 72 hr resulted in changing the morphology of CuO-NPs from platelets with sharp edge to spherical and rod forms. Similar result was reported by Zhou et al.  who worked on other kinds of precursors (Cu(NO3)2·٣H2O and Cu(CH3COO)2·H2O). It might be due to the reduction of surface energy in order to be thermodynamically stabilized. Longer aging time could provide enough time to reduce the surface energy of system via changing the morphology of particles from platelets to spherical type. Furthermore, the morphology of CuO-NPs can be altered via changing the type of precursor (Fig. 3(a) vs. (e)). Similar result was reported, recently . Like as the crystallite size, Cu2+/OH- concentration ratio also resulted in changing the morphology of particles from rod to spherical shape. While the morphology of CuO-NPs synthesized using 15 M NaOH (Fig. 3(c)) was rod-like with length of 514±180 nm and thickness of 190±80 nm, less NaOH concentration (1 M, Fig. 3(d)) resulted in the formation of spherical morphology with diameter of 148±28 nm. This result was reported similarly for copper particles synthesized using copper (II) dodecyl sulfate (Cu(DS)2) precursor . Increasing the concentration of Cu (DS)2resulted changing the particle morphology from elongated to spherical form . Since OH- concentration strongly related to the reactions, which led to the growth of CuO nanostructures (equations 2 and 3), NaOH concentration revealed crucial role in the formation of CuO nanoparticle. The primary product of NaOH incorporation was Cu(OH)2 clusters which acted as nuclei for the growth of CuO nanoparticle. Based on our results, when the concentrations of Cu2+ and OH- reached the critical value, CuO nuclei formed spontaneously in the aqueous solution via decomposition of Cu(OH)2. As the molecules at the surface of particles are vigorously less stable than the ones in the interior, in order to reduce the interfacial free energy, CuO nuclei combined together. Therefore, it could be concluded that, the kinetic factors in aqueous solution growth of CuO could be determined by thermodynamic factors as well as the concentration of OH- and Cu precursor.
Antibacterial activity of CuO-NPs
CuO-NPs synthesized through various conditions were also investigated with respect to the potential antibacterial applications. The antibacterial activities of the CuO-NPs were assessed against the growth of two Gram negative and positive bacteria. To determine the antibacterial activity of prepared samples against bacteria, Well-Diffusion assay was used. In this assay, the antimicrobial effects of compounds were determined by measuring the diameter in which microbial growth was inhibited (inhibition zone) reflecting the magnitude of sensitivity of the microorganism . Higher extent of inhibition zone represented more active antimicrobial compound. The results of Well-Diffusion tests are shown in Table 2 (as in Table 2) and Fig. 4. All samples showed the effective bacterial inhibitory behavior. However, the magnitude of inhibitory effect on the growth of both tested bacteria was significantly related to the shapes and sizes of the particles. In this study, while samples C and E revealed the highest inhibitory effects on St. aureus and E. coli, sample D showed at least ones. Based on our results, rod-like and plate-like particles (samples C and E) resulted in the highest efficacy in inhibiting the growth of bacteria. Previously reports also demonstrated the morphology-dependent antibacterial activity in other particles [36, 37]. For instance, Wang et al.  showed the effects of morphology on the antibacterial characteristics of Ag2O. Based on these studies and our results, the morphology-dependent catalytic influences can be due to higher surface active sites and surface energy of various structural morphologies [36, 38].
The antibacterial effect of CuO-NPs depended on not only their morphology and size, but also the type of microorganism. While St. aureus was more resistant to CuO-NPs, E. coli was more susceptible to them (Fig. 4 and as in Table 2). Our results were in agreement with the result of Sonia et al.  confirming CuO-NPs effect is more noticeable against Gram-negative bacterial strains than Gram-positive bacterial strains. The difference in the activity against these two types of bacteria could be attributed to the structural and compositional differences of the cell membrane. Gram-positive bacteria have thicker peptidoglycan membranes compared to the Gram-negative bacteria . So it was unbreakable for CuO-NPs to penetrate it and led to a low antibacterial effect .
Various mechanisms have been recommended to explain the antibacterial activity of nanomaterials, such as the production of physical damage  and release of metal ions . Physical damage is an efficient mechanism in the inhabitation of bacterial growth recommended by Akhavan and Ghaderi . Based on this mechanism, the bacterial cell membrane was damaged by interrelating with the sharp edges of the nanomaterials [36, 37]. Perelshtein et al.  demonstrated that the antibacterial activity of nano-silica silver nanocomposite could be related to this mechanism. Release of soluble ions from nanostructured metal oxide has also been suggested as an important inhibitory bacterial growth mechanism . The induction of ROS after exposure to nano-metal oxides has also been demonstrated in numerous studies, leading to DNA damage and subsequently cell death [40, 42, 44]. Previous reports demonstrated the simultaneously contribution of Cu2+ ion release and ROS generation in the antimicrobial activity of CuO-NPs [32, 45]. Therefore, various interface characteristics have diverse chemical–physical adsorption–desorption abilities, which resulted in different antibacterial activities . Based on this theory, higher surface area of the interface between bacteria and nanoparticles could be more efficient in the production of toxic copper ions resulted in superior antibacterial activity of the rod-shape particles . In contrary to rod-shape and platelet-like morphologies, spherical particles provided less exposed facets resulting less inhibitory effects on the antibacterial activity. Our results are in agreement with results of Azam et al.  who worked on the antibacterial activity of CuO synthesize by using a gel combustion method by cupric nitrate trihydrate and citric acid and reported that the antibacterial activity of CuO-NPs is size-dependent.
In this work, we presented an eco-friendly method for synthesis CuO-NPs with various morphologies and particle sizes by using Aloe Vera leaf extract with minimum energy consuming and cost. Furthermore, the antibacterial activity of CuO-NPs against E. coli and St. aureus was investigated. Based on our results, CuO-NPs with rod, spherical and platelet-like shapes and different sizes were synthesized via changing the copper precursor type and concentration as well as aging time. Results demonstrated the formation of pure CuO-NPs with average crystallite size of 9-23 nm, depending on the functional parameters. Additionally, results demonstrated the microbial sensitivity to the CuO-NPs depended on the microbial species and also nanoparticle properties. Based on our data, CuO-NPs with rod and platelet shape morphology with bigger surface area were more effective bacterial retardant behavior.
The authors are grateful for support of this research by Isfahan University of Technology.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest regarding the publication of this manuscript.