Biological synthesis of nanoparticles utilizing plants as bioreductants can be beneficial over other biological processes since the process can be scaled up appropriately for large-scale synthesis and does not require the maintenance of cell culture. In this study, ethanolic extracts of Azadirachta indica and Simmondsia chinensis were used as reactants to nucleate nanoparticles in solution. The synthesis of CuO NPs was confirmed by the change in the color of the reaction mixture and by UV‒visible spectroscopy. When plant extracts were added to the CuSO4 solution, CuO NP production was indicated by the change in color from pale blue to green to dark brown. The obtained results are intriguing since they can be used as a basis for identifying possible therapeutic plants for synthesizing CuO NPs. A. indica extract contains biomolecules that can reduce metal ions to metal NPs, including terpenoids, nimbaflavone, and polyphenols [22–24]. Our results are unique since this is the first study to mention the biosynthesis of CuO NPs by using Neem and Jojoba extracts to overcome multidrug resistance in Egypt.
The biosynthesis of CuO NPs was characterized by several techniques. The UV‒Vis spectroscopy results of the CuO NPs showed an absorption peak at 344–345 nm, which confirmed the formation of CuO NPs. According to the authors, the presence of copper oxide is indicated by an absorbance peak at 386 nm in the UV‒vis spectrum [25]. The FTIR results for the synthesized copper oxide nanoparticles are shown in Figs. 5D and 6D. The size, morphology, and form of the bio-CuO NPs were determined via transmission electron microscopy (TEM). TEM images confirmed the formation of nanocrystalline CuO Nps. In accordance with [26], CuO NPs with a spherical shape were observed.
FTIR spectroscopy is an efficient way to investigate the potential interactions of CuO NPs with different functional groups [27]. Other functional groups appear at various locations in the FTIR spectrum (Figs. 4d and 5d). The carbonyl group that forms nanoparticles is represented by the powerful bands at 1627 and 1669 cm − 1. Both the -OH groups in alcohols and the -NH2 groups in primary aromatic amines are responsible for another strong, intense band at 3128 cm − 1 [28]. Similarly, as indicated in Figs. 5D and 6D [29], the presence of bands at 611 cm − 1 in the FTIR spectra of the biogenic CuNPs might be attributed to the occurrence of CuO. The EDX profile of the CuNPs demonstrated substantial elemental signals corresponding to copper, confirming the presence of CuO NPs. The carbon and oxygen found in organic molecules that surround the nanoparticles, such as capping molecules and the tape used to mount the CuO NPs, make up the remaining weight percentage [30].
In vitro, the antimicrobial activity of the biosynthesized CuO NPs was evaluated against G + ve (MRSA) and G-ve (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia) bacteria using the agar diffusion method. The inhibition zone differed between Gram-negative bacteria (19 to 34 mm) and Gram-positive bacteria (28 to 32 mm). CuO NPs were effective against Acinetobacter spp. followed by Stenotrophomonas maltophilia. The most resistant bacteria to CuO NPs were E. coli and Pseudomonas aeruginosa. This difference is mainly due to the differences in the structure of the bacterial membranes. According to a previous study, nanoparticles inhibit the growth of bacteria by interacting with the phosphorous moiety in DNA. This inactivates DNA replication and consequently lowers the activity of enzymes [31–33]. Additionally, it can inhibit respiratory enzymes in bacteria and halt the synthesis of ATP, which kills cells. Additionally, several changes, including membrane detachment, cytoplasmic shrinkage, and eventually membrane rupture, are caused by the electrostatic interactions between the positive charge of the NPs and the negative charge of the bacterial surface [34, 35]. The total MICs for various extracts that resemble CuO NPs confirm that the addition of phytochemicals to various nanoformulations increases their activity. The results showed that even at low concentrations, the CuO NPs biosynthesized from the Neem and Jojoba ethanolic extracts prevented the growth of the tested bacteria. The MIC of CuO NPs biosynthesized by the ethanolic Neem and Jojoba extracts ranged from 62.5 to 125 µg/ml. These findings are consistent with those of Marzban et al. [36], who reported that the MICs of biosynthesized CuO NPs ranged from 150 to 250 µg/ml.
According to Tomayo et al. (2015) [37], the bacteriolytic mode of copper ions involves switching the semipermeable membrane properties, which prevents bacterial cells from controlling transport through the plasma membrane and results in cell lysis and the release of cytoplasmic materials outside the membrane. CuO NPs were found in an aggregated form, as demonstrated by the TEM images (Figs. 9b and 9d). TEM micrographs, however, revealed that there was no direct contact between the different particles, which may be explained by the capping molecules that are present stabilizing the nanomaterial [38]. The bacterial ultrastructures of the control MRSA and Klebsiella pneumoniae cells were examined using TEM, which demonstrated that the cells were readable and that they did not appear to have been destroyed.
The antibiofilm activity of the CuO NPs had a range of effects on different bacterial isolates. The results showed that the CuO NPs biosynthesized by the ethanolic extract of Jojoba were better antibiofilm agents than were those synthesized by the ethanolic extract of Neem, and the inhibition percentages were 93.4 to 95% and 74.2 to 95.6%, respectively, at 125 µg/ml. Our results are similar to those of Agarwala et al. (2014) [39], who showed that the antibiofilm effects of CuO NPs on MRSA and E. coli were 120 and 160 µg/ml, respectively. Several factors, such as the antimicrobial activity of the material, its biosorption-dependent mechanism, its size, its penetration capabilities, and other chemical properties influencing the ability of the material to form biofilms, could also account for the variation in inhibitory activity [40].
Antioxidants combat reactive oxygen species (ROS), which are waste products of biological functions. Antioxidants can neutralize free radicals, which are responsible for several diseases [41]. The DPPH activity of nanoparticles increased in a dose-dependent way [42]. However, the CuO NPs biosynthesized by the Jojoba extract exhibited greater inhibition of DPPH scavenging activity than did the CuO NPs biosynthesized by the Neem extract. The antioxidant potential of CuO NP increased as the particle size decreased, supporting previous findings [43].
The viability of normal HBF4 cells treated with the biosynthesized CuO NPs was assessed using the MTT assay. The toxicity of CuO NPs synthesized from ethanolic Neem and Jojoba extracts was tested in mammalian cells. The HBF4 cells treated with CuO NPs biosynthesized by Jojoba had significantly greater proliferation activity (IC50 value of 383.41 ± 3.4 µg/ml), whereas the IC50 value of CuO NPs biosynthesized by neem was 402.73 ± 1.86 µg/ml. Nearly all normal human HBF4 cells were shown to survive up to 250 µg/ml of biosynthesized CuO NPs. The CuO NPs biosynthesized by the ethanolic Neem and Jojoba extracts exhibited 99.7 and 99.2% cell viability at 125 µg/ml, respectively. Cytotoxicity was lower than that of Shiravand and Azarbani (2017) [44], who reported that 88% of Ferula macrocolea flowers were biosynthesized at 100 µg/ml.
In silico, molecular docking is a type of bioinformatics modelling in which two or more molecules are combined to generate a stable adduct. they have been carried out on biosynthesized CuO NPs against PBP4 and OXA-48 beta-lactamase (PDB IDs: 1TVF and 7AUX, respectively). Molecular docking generates different possible adduct structures that are ranked and grouped using a scoring function in the software. The information obtained from the docking technique can be used to determine the binding energy, free energy, and stability of the ligand. The findings revealed that the biosynthesized CuO NPs effectively promoted the expression of essential bacterial proteins. The CuO NPs exhibited hydrogen bonds with the remaining material that had close interactions with the active sites [45]. The capacity of nanoparticles to interact with bacteria through electrostatic, van der Waals, or other hydrophobic interactions by stabilizing the binding proteins, inhibiting the growth of the targeted proteins, and determining their antibacterial efficacy [46, 47]. These biological screening investigations have been supported by molecular docking research.
All the results reported in the present study support the use of Neem and Jojoba extracts in place of hazardous chemical reductants to CuO NPs that can suppress the growth of various multidrug-resistant bacteria.