Zinc Oxide Nanoparticles: Antimicrobial Properties, Mechanisms, and Applications

Abstract

Zinc oxide (ZnO) is a cornerstone in biomedicine, appearing in enzymes, sunscreens, and topical analgesics. Its wide band‑gap confers strong UVA/UVB absorption, and its biological impact is governed by morphology, size, exposure duration, concentration, pH, and biocompatibility. ZnO nanoparticles exhibit potent activity against a broad spectrum of bacteria and fungi, including Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Sarcina lutea, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Pseudomonas vulgaris, Candida albicans, and Aspergillus niger. Light activation triggers ZnO nanoparticles to penetrate bacterial membranes, where they disrupt cell envelopes and accumulate in the cytoplasm, interacting with biomolecules and initiating apoptosis that culminates in cell death, as confirmed by SEM and TEM imaging.

Background

Nanotechnology, defined by particles ≤100 nm, is integral to material science, agriculture, food, cosmetics, medicine, and diagnostics [1–10]. Nanoscale inorganic compounds exhibit pronounced antibacterial activity at minuscule concentrations due to their high surface‑to‑volume ratio and unique physicochemical properties [11]. They also demonstrate enhanced thermal stability [12] and, in many cases, are biocompatible, containing essential mineral elements [13]. Metallic and metal‑oxide nanoparticles—silver, gold, copper, TiO₂, and ZnO—are among the most potent antibacterial agents [14–15]. Zinc is indispensable for human health; it activates enzymes such as carbonic anhydrase, carboxypeptidase, and alcohol dehydrogenase. Unlike cadmium and mercury, zinc is non‑toxic and essential for eukaryotic physiology [16–17]. Bamboo salt, rich in zinc, has anti‑inflammatory properties via caspase‑1 modulation, and ZnO nanoparticles suppress inflammatory cytokine mRNA by inhibiting NF‑κB activation [18]. Bacterial infections pose a global health threat, amplified by antibiotic resistance and emergent pathogens. ZnO’s antibacterial legacy dates back to ancient Egypt, where it treated wounds and boils around 2000 BC [20]. Today, it remains a staple in sunscreens, supplements, photonic devices, cosmetics, and catalysis [21–25]. Despite annual production, only a fraction is utilized medicinally [26], yet the FDA classifies ZnO as safe (21 CFR 182.8991) [27]. Its photocatalytic and photo‑oxidizing properties render it effective against biochemicals [28]. In the EU, ZnO is classified as ecotoxic (N; R50‑53). While zinc is present in 2–3 g in the human body with a daily requirement of 10–15 mg [29,31], no carcinogenic, genotoxic, or reproductive toxicity has been reported in humans [32]. However, inhalation or ingestion of zinc powder can cause “zinc fever” (chills, fever, cough) [27]. ZnO nanoparticle morphology varies with synthesis: nanorods, nanoplates, nanospheres, nanoboxes, hexagons, tripods, tetrapods, nanowires, nanotubes, nanorings, nanocages, and nanoflowers [33–43]. Gram‑positive bacteria are generally more susceptible than gram‑negative counterparts. Foodborne pathogens such as Salmonella, Staphylococcus aureus, and E. coli threaten ready‑to‑eat foods; antimicrobial packaging incorporating ZnO nanoparticles can inhibit microbial growth without affecting food quality [44–48]. In vivo biodistribution studies reveal that oral or intraperitoneal ZnO nanoparticles accumulate in liver, spleen, kidneys, and bones, with dose‑dependent effects on body weight and enzyme activity [50–51]. Low concentrations (<50–500 mg kg⁻¹) exhibit minimal toxicity, while high doses (5000 mg kg⁻¹) increase organ weights and alter zinc‑metabolism genes [50]. Overall, ZnO nanoparticles are innocuous at therapeutic doses, stimulating beneficial enzymes while suppressing disease pathways [52]. This review consolidates current knowledge on ZnO nanoparticles as antibacterial agents, detailing their interaction mechanisms with diverse microbes.

Antimicrobial Activity of Zinc Oxide Nanoparticles

ZnO nanoparticles impede bacterial growth by permeating cell membranes, inducing oxidative stress that damages lipids, proteins, DNA, and carbohydrates [53]. Lipid peroxidation disrupts membrane integrity, impairing essential cellular functions [54], a mechanism confirmed in E. coli [55]. Bulk ZnO suspensions generate H₂O₂, contributing to antibacterial activity [56]. The release of Zn²⁺ ions, due to ZnO’s amphoteric nature, also plays a role; Zn²⁺ readily binds proteins and carbohydrates, halting vital bacterial processes. Comparative toxicity studies using Vibrio fischeri show ZnSO₄·7H₂O is six times more toxic than ZnO nanoparticles, reflecting the higher solubility and bioavailability of Zn²⁺ ions [57]. Bulk ZnO and ZnO nanoparticles are 40–80‑fold less toxic than ZnSO₄, underscoring the importance of solubility and ion release [58]. Contact alone, without internalization, can suffice for toxicity; a high nanoparticle concentration can envelope bacterial cells, restricting nutrient uptake [59]. ZnO exhibits superior antibacterial activity compared to CuO, Al₂O₃, La₂O₃, Fe₂O₃, SnO₂, and TiO₂ against E. coli [49]. However, zinc ions may enhance viral replication in certain contexts, such as a 69.6 % increase in infectious pancreatic necrosis virus at 10 mg L⁻¹ Zn [46]. SEM/TEM imaging confirms ZnO nanoparticles damage bacterial membranes, increase permeability, and accumulate intracellularly, suppressing proliferation [62–63]. Recent studies demonstrate that 15 μg mL⁻¹ ZnO nanoparticles inhibit Staphylococcus aureus, E. coli, Salmonella typhimurium, and Klebsiella pneumoniae, with K. pneumoniae requiring as little as 5 μg mL⁻¹ [63,64]. Growth inhibition escalates with concentration; 45 μg mL⁻¹ over 4–5 h strongly suppresses bacterial proliferation, and extended exposure further amplifies the effect [63]. ZnO nanoparticles first compromise the bacterial cell membrane, then permeate, and may generate H₂O₂ as an alternative antibacterial mechanism [65,66]. At physiological pH (2–5 in the stomach), ZnO can release Zn²⁺, activating digestive enzymes (carboxypeptidase, carbonic anhydrase, alcohol dehydrogenase) and protecting against E. coli [65]. The minimal cytotoxicity of ZnO nanoparticles at low doses (<5 μM) aligns with their therapeutic window [67–70].

Solubility and Concentration‑Dependent Activity of Zinc Oxide Nanoparticle

Nanoparticles serve as drug carriers; ZnO nanoparticles remain safe to normal cells at concentrations ≤100 μg mL⁻¹, offering an antibiotic alternative. A 500–1000 μg mL⁻¹ dose eradicates 90 % of bacterial colonies within 6 h. Even drug‑resistant strains (S. aureus, Mycobacterium smegmatis, Mycobacterium bovis) are significantly suppressed when combined with low‑dose rifampicin (0.7 μg mL⁻¹). At 1000 μg mL⁻¹ for 24 h, these pathogens are eliminated, suggesting repeat dosing could achieve clinical cure. Nanoparticle sizes between 50 and 500 nm exhibit comparable antibacterial potency [71–73]. Cytotoxicity assays reveal that ZnO nanoparticle toxicity is concentration‑ and solubility‑dependent; 125 mg L⁻¹ suspensions release 6.8 mg L⁻¹ Zn²⁺, yet the antibacterial effect primarily stems from nanoparticle–microbe interactions rather than free ions [75]. In acidic environments (pH 4.5), ZnO nanoparticles dissolve rapidly, releasing Zn²⁺ that binds intracellular biomolecules and disrupts growth [76,77]. Transcriptomic analyses show upregulation of metallothionein genes upon ZnO nanoparticle exposure, indicating zinc homeostasis disruption [78]. Smaller nanoparticles (≤19 nm) reach higher blood concentrations than larger ones (>100 nm) [79]. Overall, the antibacterial efficacy hinges on medium chemistry, ion release, and cell penetration.

Size‑Dependent Antibacterial Activity of Zinc Oxide Nanoparticles

Azam et al. demonstrated that antibacterial activity against both gram‑negative (E. coli, P. aeruginosa) and gram‑positive (S. aureus, B. subtilis) bacteria increases as particle size decreases, due to higher surface‑to‑volume ratios. The largest inhibition zone (25 mm) was observed against B. subtilis (Figure 1) [82]. Multiple studies confirm that smaller ZnO nanoparticles (≤10–14 nm) are internalized by bacteria and damage membranes (TEM images) [63,84]. Conversely, particles >100 nm show minimal inhibitory effects [89]. The antibacterial potency correlates inversely with size and directly with concentration [88]. Photocatalytic activation under sunlight or diffused light can further enhance ROS generation, although H₂O₂ production may not be the sole driver [88].

Shape, Composition, and Cytotoxicity of Zinc Oxide Nanoparticles

ZnO nanoparticle cytotoxicity varies with concentration, cell type, and morphology [90–91]. Tetrapod ZnO, synthesized via flame transport, exhibit lower mammalian fibroblast toxicity compared to spherical particles, likely due to oxygen vacancy tuning and UV‑induced structural changes that impede viral entry [92–94]. In vivo inhalation studies reveal pulmonary inflammation and oxidative stress induced by ZnO nanoparticles, surpassing the effects of carbon or SiO₂ particles [95]. Cytotoxicity is shape‑dependent, with spherical particles showing higher membrane disruption than other morphologies [95].

Polymer‑Coated Nanoparticles

Coating ZnO nanoparticles with hydrophilic polymers—PVP, PVA, PGA, PEG, chitosan, dextran—improves stability and reduces toxicity while maintaining antibacterial activity [97–99]. A hydrogel composite of N‑isopropylacrylamide and ZnO nanoparticles exhibits potent activity against E. coli at 1.33 mM, with no mammalian cytotoxicity over one week, making it suitable for biomedical coatings [96].

Effect of Particle Size and Shape of Polymer‑Coated Nanoparticles on Antibacterial Activity

PEG‑coated ZnO nanoparticles (401 nm–1.2 µm) display enhanced antibacterial activity with decreasing size and increasing concentration; effective doses exceed 5 mM. SEM imaging reveals significant morphological changes in E. coli after exposure [84]. While PEG‑coated nanorods are cytotoxic to osteoblast cancer cells above 100 µM, they are harmless to healthy cells [87].

In Vivo and In Vitro Antimicrobial Activity for Wound Dressing

Chitosan hydrogel bandages embedded with 70–120 nm ZnO nanoparticles effectively treat burns, wounds, and diabetic ulcers, eliminating pathogens such as P. aeruginosa and S. intermedicus within three weeks in murine models [100]. The degradation products—D‑glucosamine and glycosamine glycan—are naturally present in the body, ensuring biocompatibility.

Effect of Doping on Toxicity of Zinc Oxide Nanoparticles

Iron doping reduces ZnO nanoparticle toxicity, aligning the Zn²⁺ concentration required for 50 % microbial viability with that of free Zn²⁺ [101,102]. Coatings with mercaptopropyl trimethoxysilane or SiO₂ also lower cytotoxicity [103]. In BEAS‑2B cells, uptake of ZnO nanoparticles drives zinc accumulation and biomolecule binding; dissolution to Zn²⁺ is a key toxicity pathway [104].

Interaction Mechanism of Zinc Oxide Nanoparticles

ZnO nanoparticles exert antibacterial effects through multiple pathways: membrane disruption, DNA damage, ROS generation, and ion release. Figure 2 illustrates the core mechanisms: photonic activation, water splitting, ROS formation, and intracellular accumulation.

ZnO is less toxic than silver nanoparticles across concentrations (20–100 mg L⁻¹) with an average size of 480 nm [55,62,63]. Metal oxides compromise bacterial membranes and DNA; ROS generation via photocatalysis further enhances cell death [112]. UV‑Vis spectra of ZnO suspensions peak between 370 and 385 nm, indicating strong light absorption [113]. ROS species—hydroxyl radicals, superoxides, hydrogen peroxide—react with proteins, lipids, and DNA, triggering apoptosis [114].

ZnO nanoparticles produce ROS when exposed to UV‑Vis light, generating H₂O₂ that permeates bacterial cells, while negatively charged radicals remain on the surface, compromising membrane integrity [116,117]. TEM images confirm nanoparticle interaction with Salmonella typhimurium membranes (Figure 4) [115].

In 18 h incubations with MIC ZnO nanoparticles, E. coli cells exhibit wall rupture and cytoplasmic leakage, with nanoparticles adhering to and penetrating the outer membrane (Figure 5) [120].

The interaction sequence involves ZnO absorption of sunlight, water splitting to produce superoxide and hydroxyl radicals, and subsequent ROS-mediated membrane damage. While the exact oxygen production mechanism requires further validation, evidence supports a dose‑dependent antibacterial effect, with 5 μg mL⁻¹ as an effective minimal inhibitory concentration.

Conclusions

Zinc is a critical trace element in human physiology, constituting bones, teeth, enzymes, and proteins. While essential in trace amounts, excessive zinc or its nanoparticulate form can be toxic to fungi, viruses, and bacteria. Genetic deficiencies in zinc‑binding proteins lead to conditions such as acrodermatitis enteropathica. Although contradictory reports exist regarding nanoparticle safety, properly engineered ZnO nanoparticles, especially when polymer‑coated, can serve as effective antimicrobial agents, wound dressings, and drug carriers with minimal host toxicity. Their ROS‑mediated antibacterial action can complement or replace antibiotics, yet chronic high‑dose exposure may pose health risks, underscoring the need for careful dosage regulation.