The research team, led by Anas Mohamed Ali of Kampala International University and Hormuud University, together with Ibrahim Garba Wawata, Ritah Nakayinga, Bazil Okello, Vidya Sankarapandian, Mai Abdalla Ali, and Ibrahim Ntulume, set out to address a growing crisis: the overuse and abuse of antibiotics have fueled an upsurge in multidrug-resistant (MDR) bacteria, which are now a leading cause of death globally. The consequences include increased mortality and morbidity, lengthened hospital stays, and substantial financial loss. Against this backdrop, the team turned to green nanotechnology—the synthesis of nanoparticles using plant extracts—as a cheaper, less toxic, and more scalable alternative to chemical or microbial methods.

The choice of Aloe vera was deliberate. While previous studies have used dried Aloe vera leaves to synthesize copper oxide nanoparticles, the researchers argued that fresh leaves offer distinct advantages: they contain high levels of labile, water-soluble phytochemicals such as polysaccharides (notably acemannan), polyphenols, flavonoids, vitamins, and enzymes. These compounds act as natural reducing, capping, and stabilizing agents during nanoparticle synthesis, functioning as bio-templates and surfactants that aid size control and prevent agglomeration of metal oxide particles. Fresh leaves also enable rapid synthesis, improve purity, reduce processing costs, and are available year-round.

The synthesis process itself was straightforward. Fresh Aloe vera leaves collected from Rukararwe botanical gardens in Bushenyi district, western Uganda, were cleaned, finely diced, and boiled at 80 degrees Celsius for five minutes in deionized water. The resulting extract was filtered twice through 0.2-micrometer Whatman filter paper and stored at 4 degrees Celsius. The researchers then combined 600 milliliters of this extract with 50 milliliters of a 10 millimolar aqueous solution of copper sulfate (99.99 percent pure) and maintained continuous agitation between 100 and 120 degrees Celsius on a shaker set at 100 rpm. Over 24 hours, the deep blue reaction mixture turned colorless, then brick red, and finally dark crimson—visual evidence of nanoparticle formation. The mixture was centrifuged at 10,000 rpm, washed, dried overnight, and heated in a muffle furnace at 500 degrees Celsius for three hours to yield the final copper oxide powder.

Characterization of the resulting nanoparticles revealed a well-defined crystalline structure. X-ray diffraction (XRD) showed eleven strong peaks at 2-theta values of 28.44, 29.44, 35.59, 38.5, 40.65, 43.20, 48.62, 58.02, 61.5, 67.29, 68.14, and 75.5 degrees, corresponding to the (110), (100), (111), (200), (020), (−202), (202), (113), (311), (220), and (204) planes of monoclinic CuO (tenorite), in conformity with the standard JCPDS 00–048–1548 reference. The average crystallite size, calculated using the Debye-Scherrer equation, was 25 ± 2.4 nanometers. Transmission electron microscopy (TEM) depicted plate-like, faceted hexagonal structures with a length of approximately 500 nanometers, indicating polycrystalline nature and a high surface area with exposed reactive sites. Energy-dispersive X-ray spectroscopy (EDS) confirmed the elemental composition: copper at 58.37 percent by weight and oxygen at 41.63 percent, agreeing with the chemical formula of copper(II) oxide.

Fourier transform infrared (FTIR) spectroscopy provided insight into the phytochemicals involved in reducing copper ions and capping the nanoparticles. The extract revealed a broad band at 2860 cm−1 attributed to aliphatic C–H stretching, unique anthraquinone-associated C=O/C=C overtones at 2475 and 2420 cm−1, and carbonyl, amide, and carboxylate bands at 1086, 1129, 1305, 1435, and 1543 cm−1. Pronounced C–O/C–O–C polysaccharide signals appeared within the 1000–1200 cm−1 range, consistent with the presence of polyphenols, flavonoids, proteins, and sugars—particularly acemannan—in Aloe vera. In the spectra of the CuO nanoparticles, these bands exhibited anticipated alterations: a reduction or absence of C–H (shifted and broadened from 2860 to 3050 cm−1) and carbonyl/phenolic bands (shifting from 2475 to 2359 cm−1 and 2420 to 2328 cm−1, with diminished intensity), indicative of consumption during the reduction of Cu2+ ions. The persistence and alteration of C=O/amide and C–O bands signified their role as adsorption capping agents, while the emergence of a new Cu–O vibration at 640 cm−1 confirmed the formation of monoclinic CuO. Ultraviolet-visible spectroscopy showed a broad absorption peak at 359 nanometers, attributed to the surface plasmon resonance of the CuO nanoparticles, alongside peaks at 240, 299, and 379 nanometers assigned to the phytochemicals in the plant extract.

With the nanoparticles fully characterized, the team turned to the critical question: how effective are they against drug-resistant bacteria? The researchers tested the CuO nanoparticles against two clinical isolates of MRSA (MRSA-A001 and MRSA-A002) and two isolates of MDR K. pneumoniae (MDR K. pneumoniae-A001 and MDR K. pneumoniae-A002), along with their respective reference strains (S. aureus ATCC 29213 and K. pneumoniae NCTC 9633). All test isolates were previously obtained from septic wound infections, urine, and nasal swabs, and were re-screened using Kirby-Bauer disk diffusion assays against a panel of twelve antibiotics, including cefotaxime, gentamicin, meropenem, cefoxitin, erythromycin, and ampicillin. Notably, K. pneumoniae-A002 exhibited phenotypic resistance even to meropenem, a last-resort antibiotic, while both S. aureus strains showed resistance to cefoxitin. The isolates retained their resistance genes, including the tet(A), mecA, and blaCMY genes previously genotyped by uniplex polymerase chain reaction.

The antibacterial results were striking. Using the agar well diffusion method, the researchers applied 50 microliters of the CuO nanoparticle suspension at concentrations of 125, 250, and 500 micrograms per milliliter to Mueller-Hinton agar plates inoculated with the test bacteria. The nanoparticles exhibited broad-spectrum, concentration-dependent activity, with significantly higher inhibitory effects at 500 micrograms per milliliter compared to lower concentrations. At 125 micrograms per milliliter, the nanoparticles only exhibited inhibitory effects against the two MRSA strains (inhibition zones of 10.00 ± 0.33 and 12.33 ± 0.33 millimeters), while the MDR K. pneumoniae strains showed maximum resistance at this concentration. The positive control, gentamicin, exhibited a statistically significant inhibitory effect compared to the nanoparticles, though there was no significant difference between gentamicin and the nanoparticles against MRSA-A002. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, performed in 96-well round-bottom microtiter plates using conventional broth microdilution techniques in brain heart infusion broth, revealed lower MIC values against MRSA strains (31.25–62.5 micrograms per milliliter) than against MDR K. pneumoniae strains (62.5 micrograms per milliliter). The MBC values were 125 micrograms per milliliter against both MRSA strains and 500 micrograms per milliliter against both MDR K. pneumoniae strains, indicating strong bactericidal activity.

The researchers attribute the nanoparticles’ efficacy to several factors. Their small size enhances stability compared to larger particles, while the polyhedral morphology observed by TEM increases surface energy from different exposed crystal planes, ultimately generating more copper ions and releasing reactive oxygen species (ROS) within a short time. The antimicrobial mechanism of copper-containing nanoparticles includes the production of ROS that destroy cell walls and cell membranes, and subsequently react with proteins and DNA. The abundant bioactive compounds within the aqueous Aloe vera extract—including aloe-emodin and its derivatives (75.75 percent), aloesin, aloe-emodin-diglucoside, 2′-p-methoxycoumaroylaloeresin, aloins, aloinosides, 8-O-methyl-7-hydroxyaloin, cinnamic acid derivatives, anthracene compounds, and flavonoids—may also contribute to the overall antimicrobial effect, as several of these phytochemicals have documented antibacterial efficacy against gram-negative bacteria such as MDR K. pneumoniae and Pseudomonas aeruginosa. Aloe vera aqueous extract has also been reported to rapidly destroy biofilms formed by the majority of MDR K. pneumoniae and MRSA.

The study’s authors acknowledge limitations, including a lack of cytotoxicity data on human cells and the inability to confirm whether the antibacterial activity is due to the release of copper ions, disruption of the bacterial cell wall or membrane by the nanoparticle itself, or purely ROS generation. They recommend future work to characterize nanoparticles synthesized from organic solvent extracts such as ethanol and methanol, to test the nanoparticles against other resistant bacteria including MDR Pseudomonas aeruginosa, Enterococcus species, Streptococci species, and Proteus species, and to conduct in vivo efficacy and safety experiments to validate the nanoparticles for human use. Nonetheless, the green-synthesized copper oxide nanoparticles derived from fresh Aloe vera leaf aqueous extract represent a promising, low-cost, and environmentally friendly alternative in the development of novel antimicrobial agents for the treatment of antibiotic-resistant bacteria—an urgent need as the world grapples with the rising tide of superbug infections.

Subject of Research: Synthesis of copper oxide nanoparticles using Aloe vera leaf extract and their antibacterial activity against methicillin-resistant Staphylococcus aureus and multi-drug resistant Klebsiella pneumoniae

Article Title: Synthesis of copper oxide nanoparticles using Aloe vera leaf extract and their antibacterial activity against methicillin-resistant Staphylococcus aureus and multi-drug resistant Klebsiella pneumoniae

Article References: Ali, A. M., Wawata, I. G., Nakayinga, R., Okello, B., Sankarapandian, V., Ali, M. A., & Ntulume, I. (2026). Synthesis of copper oxide nanoparticles using Aloe vera leaf extract and their antibacterial activity against methicillin-resistant Staphylococcus aureus and multi-drug resistant Klebsiella pneumoniae. Frontiers in Drug Discovery, 6, Article 1745824. https://doi.org/10.3389/fddsv.2026.1745824

Image Credits: AI Generated

DOI: 10.3389/fddsv.2026.1745824

Keywords: Synthesis, copper, oxide, nanoparticles, Aloe, vera, leaf, extract, antibacterial, activity, against, methicillin-resistant