Cancer drug 5-FU loaded onto Mn-doped ZnO nanocomposite boosts antibacterial activity

The work, published in Frontiers in Materials, was driven by the growing problem of antimicrobial resistance and by the fact that 5-fluorouracil, although it has intrinsic antibacterial activity, is limited clinically by dose-dependent toxicity, a short half-life, reduced bioavailability, poor absorption and rapid elimination.

The team prepared the Mn-doped ZnO nanocomposite by a co-precipitation method, dissolving zinc acetate dihydrate and manganese acetate tetrahydrate in deionized water at molar concentrations of 0.5 M and 0.05 M respectively, which corresponds to a nominal manganese content of 9.1 atomic percent of the total metal cations. A 0.5 M sodium hydroxide solution was added dropwise under continuous stirring until the pH reached 9, and the mixture was stirred for about two hours until precipitates formed. After repeated washing with ethanol and double-distilled water, the precipitates were dried at room temperature for 24 hours and then annealed in a furnace at 300 °C for two hours. Quantitative energy-dispersive X-ray analysis of the final material revealed an experimental manganese content of approximately 8.3 atomic percent, calculated as Mn/(Mn + Zn), corresponding to an Mn/Zn atomic ratio of about 0.091, closely matching the precursor ratio.

Loading of the drug was carried out by dissolving 5 mg of 5-fluorouracil in 25 mL of distilled water and mixing it with 25 mg of the nanocomposite dispersed in 50 mL of water, with continuous stirring for 24 hours at 25 °C while the pH was held at six. The loaded particles were separated by centrifugation at 10,000 rpm for 10 minutes, and the amount of drug remaining in the supernatant was measured by UV–visible spectroscopy at the characteristic 248 nm absorbance peak to estimate the drug-loading efficiency. The researchers then examined how the loaded material releases the drug by placing it in 50 mL of phosphate-buffered saline and sampling at intervals from 0 to 120 hours. The cumulative release reached approximately 40 percent within the first 12 hours and about 71 percent by 120 hours, a profile the authors attribute to an initial burst of 5-fluorouracil molecules loosely attached to or near the nanocomposite surface, followed by slower, sustained diffusion of more strongly retained drug molecules.

Optical measurements documented clear changes upon drug loading. The Mn-doped ZnO nanocomposite showed maximum absorbance at 281 nm, while the 5-FU-loaded material peaked at 249 nm, and the authors describe a red shift between the two spectra. Tauc plot analysis of the absorption data yielded optical band-gap energies of approximately 3.13 eV for the Mn-doped ZnO and 2.92 eV for the 5-FU-loaded nanocomposite, a reduction of about 0.21 eV. The team interprets this narrowing as evidence that 5-fluorouracil molecules interact with the nanocomposite surface, possibly introducing additional electronic states or modifying the surface electronic environment so that electronic transitions can occur at lower photon energies, which may also enhance light absorption in the visible or near-visible region.

Fourier-transform infrared spectroscopy identified the functional groups expected from both components. The spectra displayed characteristic bands assigned to Zn–O–Zn and Mn–O vibrations in the 450–620 cm⁻¹ region, N–H stretching near 3409 cm⁻¹, O–H stretching at 3335 cm⁻¹, C=O stretching at 1647 cm⁻¹, C–N stretching at 1250 cm⁻¹, C–F stretching at 1230 cm⁻¹ and C–H deformation at 825 cm⁻¹. X-ray diffraction confirmed the crystalline nature of the synthesized material: the Mn-doped ZnO pattern matched the standard reference JCPDS No. 96-901-2843 and was indexed to a tetragonal crystal system identified as hetaerolite with space group I41/amd, whereas the 5-FU-loaded material matched JCPDS No. 96-410-5959, corresponding to an orthorhombic system with a Pmmm space group, indicating that drug incorporation was associated with a change in crystalline structure.

Scanning electron microscopy revealed predominantly spherical or granular morphology for the Mn-doped ZnO nanocomposite, with average particle sizes of approximately 17 and 20 nm determined from particle-size distribution analysis. The EDX spectrum showed peaks for zinc, manganese, carbon and oxygen, with zinc the most intense, and no additional significant elemental peaks were detected, suggesting relatively high compositional purity. After drug loading, the particles took on a flaky or plate-like appearance and appeared closely packed, which the authors associate with deposition of 5-fluorouracil onto the nanocomposite surface. The EDX spectrum of the loaded material showed characteristic peaks for manganese, zinc, oxygen, carbon, fluorine and nitrogen, and the appearance of fluorine and nitrogen peaks is consistent with the presence of 5-FU. Elemental mapping visualized the spatial distribution of all constituent elements in both samples.

The antibacterial performance was tested using the agar well diffusion method against Staphylococcus aureus, a Gram-positive strain, and Escherichia coli, a Gram-negative strain, both obtained from the Department of Biosciences at the University of Wah. Bacterial lawns were prepared on Mueller–Hinton agar, four 6 mm wells were cut into each plate, and the nanocomposites, diluted from a 10 mg/mL stock, were added at 10 and 15 µL. Amoxicillin at 1 mg/mL served as the positive control and deionized water as the negative control, and all experiments were performed in triplicate and reported as mean ± standard deviation. The 5-FU-loaded Mn-doped ZnO nanocomposite produced inhibition zones of 13.8 mm at 10 µL and 16.8 mm at 15 µL against S. aureus, and 12.5 mm at 10 µL and 14.8 mm at 15 µL against E. coli, exceeding both the unloaded Mn-doped ZnO nanocomposite, which gave 7.5 and 8.5 mm against S. aureus and 5.3 and 6.5 mm against E. coli, and amoxicillin, which produced 10.5 and 12.8 mm against S. aureus and 8.6 and 10.8 mm against E. coli at the same volumes.

Activity increased with concentration for all tested samples, indicating a concentration-dependent effect, and the greater inhibition against S. aureus points to comparatively higher susceptibility of the Gram-positive strain. The negative control showed no appreciable inhibition zone, indicating that the solvent itself did not contribute to the observed activity. The authors suggest that the enhanced response of the physical mixture may reflect the complementary actions of the two components, with Mn-doped ZnO capable of interacting with bacterial cell surfaces through electrostatic and coordination interactions, potentially disrupting membranes and contributing to oxidative stress, while 5-fluorouracil can interfere with nucleic acid metabolism by inhibiting thymidylate synthase.

Under suitable irradiation, photogenerated electron–hole pairs in ZnO may react with water molecules or hydroxide ions to produce reactive oxygen species, which the authors propose could damage bacterial cell membranes and other cellular components. The authors propose that manganese doping may modify the electronic and optical properties of ZnO and potentially influence charge separation and ROS generation, while the loaded 5-fluorouracil may add a second mechanism through its interaction with bacterial nucleic acid synthesis. The authors note, however, that because their MTT cytotoxicity assay did not directly quantify specific mechanisms such as ROS generation or apoptosis, these mechanisms remain proposed rather than experimentally confirmed.

Safety was assessed in vitro using the MTT assay in NIH-3T3 and HEK-293 cell lines, with 5,000 cells per well seeded into 96-well plates and exposed to the nanocomposite for 24, 48 and 72 hours. Cell viability remained relatively high at lower concentrations but declined markedly as concentration increased, reaching approximately 40 to 43 percent at 100 µg/mL, and decreased only modestly as exposure time increased, indicating a predominantly concentration-dependent cytotoxic response. The authors conclude that combining 5-fluorouracil with Mn-doped ZnO yields a multifunctional nanocomposite with sustained drug-release characteristics and enhanced, concentration-dependent antibacterial activity, and they position the system as a candidate nanostructured antimicrobial delivery platform for further investigation, one that could potentially enhance the antimicrobial utility of 5-FU while reducing the drug dose required.

Subject of Research: Synthesis and antibacterial evaluation of a 5-fluorouracil-loaded Mn-doped ZnO nanocomposite

Article Title: 5-fluorouracil-loaded Mn-doped ZnO nanocomposite: synthesis, characterization, and antibacterial activity

Article References: Zafar, R., Jamil, I., Bibi, S., Nawaz, F., Hassan, S. W., & Hoque, M. E. (2026). 5-fluorouracil-loaded Mn-doped ZnO nanocomposite: synthesis, characterization, and antibacterial activity. Frontiers in Materials, 13, Article 1911252. https://doi.org/10.3389/fmats.2026.1911252

Image Credits: AI Generated

DOI: 10.3389/fmats.2026.1911252

Keywords: 5-fluorouracil, Mn-doped ZnO, nanocomposite, antimicrobial resistance, antibacterial activity, co-precipitation, drug delivery, Staphylococcus aureus, Escherichia coli, reactive oxygen species, band gap, agar well diffusion