Synthesis of Uniform Zinc Peroxide Nanoparticles for Antibacterial Application

Ying Xiong; Song Shen; Z. Yi

doi:10.36468/pharmaceutical-sciences.spl.923

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Research Paper

Synthesis of Uniform Zinc Peroxide Nanoparticles for Antibacterial Application

Ying Xiong, Song Shen¹ and Z. Yi^*

Department of Pharmacy, Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, Yancheng, Jiangsu 224500,¹Jiangsu University, Zhenjiang, Jiangsu 212013, China

*Corresponding Author:: Z. Yi
Department of Pharmacy, Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, Yancheng, Jiangsu 224500, China
E-mail: j598shensong@163.com

This article was originally published in a special issue, “Emerging Therapeutic Interventions of Biopharmaceutical Sciences”

Indian J Pharm Sci 2024:86(3) Spl Issue “219-225”

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

DOI: 10.36468/pharmaceutical-sciences.spl.923

Abstract

Peroxides and metals have been emerged as alternative antibacterial agents. However, the application of peroxides or metals alone for antibacterial treatment cannot yield satisfactory results. Combined therapy based on peroxides and metals is expected to generate synergistic effects. In this work, uniform zinc peroxide nanoparticles with an average size of 60 nm were synthesized as antimicrobial agent. Zinc peroxide nanoparticles were relatively stable in a neutral environment and showed quick release of hydrogen peroxide and zinc ion in acidic environment. After incubation with Escherichia coli and Staphylococcus aureus, large amount of reactive oxygen species was generated. Zinc peroxide nanoparticles illustrated outstanding antibacterial activity for Escherichia coli and Staphylococcus aureus, holding a great potential to be a highly efficient antibacterial agent.

Keywords

Metal peroxides, antibacterial, reactive oxygen species, zinc peroxide, nanoparticles, Escherichia coli and Staphylococcus aureus

Treatment of bacterial infection is still a challenge due to the emergence of bacterial resistance, which leads to the exploration of alternative antibacterial and treatments[1,2]. Therefore, it is extremely urgent to synthesize novel materials with broadspectrum antimicrobial activity to enhance or replace traditional antibiotics. These drugs play crucial role in surgeries, immunotherapy, and other medical procedures, significantly reducing human mortality and morbidity rates[3]. The mechanism of antibiotic resistance is complex and bacteria generally reduce the effectiveness of antibiotics through several different mechanisms[4]. These include reducing their permeability[5], actively pumping antibiotics out of the cell[6], altering or modifying the antibiotic's target affinity[7], rendering the antibiotic inactive, or modifying it in some way[8,9].

Peroxides and metals hold great promise as alternatives to antibiotics. Peroxides, such as Hydrogen peroxide (H₂O₂) and Sodium hypochlorite (NaClO), can cause oxidation of bacteria and has been widely used as bactericide[10,11]. These compounds generate Reactive Oxygen Species (ROS), which can disrupt essential cellular functions and damage bacterial cells by oxidizing cellular components[12,13]. Metals kill bacteria by different mechanisms; when metals come in contact with bacterial cells, they disrupt the outer membranes and penetrate the cells. Inside the cells, these metal ions interfere with various cellular processes, such as depleting antioxidants, disrupting the normal functioning of proteins and enzymes, damaging the cellular membranes and interfering with the electron transport, thereby causing genotoxicity[14,15]. Considering different antibacterial mechanisms, it is reasonable to deduce that the combination of the peroxides and metals is a potential method to enhance the antimicrobial activity. Among different metal oxides, Zinc Oxide (ZnO) is a widely used antimicrobial and/ or antifungal agent due to its advantages such as lower cytotoxicity, better selectivity and higher stability[16,17]. Previous studies have shown that Zinc peroxide (ZnO₂) Nanoparticles (NPs) can release Oxygen (O₂) triggered by temperature or pH in dispersing media[18]. It can be proved that the nanoparticles continuously release O₂ for about 3 d at a pH<7.5 in an aqueous dispersion. The amount of O₂ released depends on the different samples and is controlled by the different compositions and selected pH values of the samples.

In this work, we synthesized uniform ZnO₂ NPs to further improve the antimicrobial activity (fig. 1). ZnO₂ NPs were synthesized by oxidizing zinc acetate with H₂O₂using Polyvinylpyrrolidone (PVP) as stabilizer[19]. We evaluated the reaction of ZnO₂ NPs in Phosphate Buffered Saline (PBS) at pH 5.5 and 7.4 and demonstrated that the Zinc (Zn²⁺) ions and hydrogen peroxide release rates are greatly accelerated in acidic environments. Simultaneously, we found that in the presence of ZnO₂ NPs, ROS can be produced in both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), laying the foundation for the antibacterial effect of ZnO₂NPs. The toxicity experiments were conducted to explore the antibacterial effect of ZnO₂ NPs, which opened up a new path for the future application of ZnO₂.

Fig. 1: Schematic diagram showing the dissolution of ZnO₂ nanoparticles and the formation of Zn²⁺ and H₂O₂ in acidic environment
Note: Zn²⁺ and H₂O₂ effectively damage the bacteria

Materials and Methods

We used Transmission Electron Microscopy (TEM) for characterization of the obtained ZnO₂ NPs. As shown in fig. 2a, the ZnO₂ NPs were uniform in spherical morphology with an average diameter of 60 nm; Dynamic Light Scattering (DLS) was used to determine the hydrated particle size by. The average size increased to 70 nm due to the inclusion of solvation shell (fig. 2b). X-Ray Diffraction (XRD) was utilized to detect the crystal structure of the ZnO₂ NPs. As shown in fig. 3a, the pattern of ZnO₂ with typical peaks at 31°, 37°, 53° and 63° which was supported by the Joint Committee on Powder Diffraction Standards (JCPDS) (having number: 00-076-1364), attributing to cubic zinc blende structure[20,21]. We have noticed that the diffraction peaks of ZnO₂ NPs were wide and weak, indicating relatively small size of the nanocrystals. Small sized crystals implied the capability of PVP in inhibiting the formation and growth of crystals.

Fig. 2: ZnO₂ NPs measured by dynamic light scattering, (a): TEM image and (b): Hydrodynamic diameter

PVP molecules play an essential role in the formation of the nanocrystals during the formation of ZnO₂NPs. PVP can chelate with metal ions and then assist the ions to form ZnO₂ nanocrystals in the presence of oxidants. From the TEM image of ZnO₂ NPs, we found that the ZnO₂ NPs were composed of many small nanocrystals. The formation of the larger nanocrystals aggregations was because of the tendency to reduce the high surface energy. Meanwhile, PVP can also prevent the sudden and random aggregation of the nanocrystals to help the formation of uniform ZnO₂ NPs owing to the steric hindrance effect of the PVP.

Results and Discussion

In neutral and acidic environment, ZnO₂ NPs could react with Water (H₂O) and Hydrogen ions (H⁺) to produce H₂O₂ by the reactions (1) and (2), respectively.

ZnO₂+2H+→Zn²++H^₂O^₂ (1)

ZnO₂+2H+→Zn²++H₂O₂ (2)

In acidic environment, ZnO₂ NPs reacted with H+ to generate H₂O₂ as per reaction (1). Thus generated H₂O₂ was determined by Potassium Permanganate (KMnO₄)-based colorimetric method.

3H⁺+2KMnO₄+5H₂O₂→2K⁺+2Mn²++5O₂+ 8H₂O (3)

Purple colored Permanganate (MnO₄ ^-) ion was reduced by peroxo groups to form colourless Manganese (Mn²⁺) ion. As shown in fig. 3b, the purple color of MnO₄ ^- in acidic solution disappeared after addition of H₂O₂ or ZnO₂ NPs, indicating the formation of H₂O₂ in the acidic medium.

Fig. 3: Typical peaks demonstrating the presence of peroxo groups in ZnO₂ NPs, (a): XRD patterns of the synthesized NPs and (b): Colorimetric analysis
Note: ( Equation ): Control; ( ): H₂O₂ and ( ): ZnO₂

Most metal oxides are unstable and will hydrolyze in H₂O. However, the hydrolysis rate is quite different. Calcium peroxide (CaO₂) NPs hydrolyze quickly in aqueous solution, while the hydrolysis of Copper peroxide (CuO₂) was much slower[22,23]. We have evaluated the reaction of ZnO₂ NPs at pH 5.5 and 7.4 Phosphate Buffer Saline (PBS) and determined the release rates of Zn²⁺ and H₂O₂. As shown in fig. 4a and fig. 4b, both Zn²⁺ and H₂O₂ were gradually released from the ZnO2 NPs at pH 5.5, showing a cumulative release >60 % at 4 h. In contrast, the release rates were slower at pH 7.4 PBS that only 5 % was released after 6 h. These results indicate that the ZnO₂ NPs are relatively stable in neutral environment and will decompose quickly in acidic medium. The bacteria acidic microenvironment also facilitates the release of zinc and hydrogen peroxide.

Fig. 4: Release profiles of Zn²⁺ and H₂O₂ from ZnO₂ NPs at a pH of 5.5 and 7.4 of PBS, (a): Zn²⁺ and (b) H^₂O₂
Note: ( Equation ): pH 5.5 and ( ): pH 7.4

It is reported that bacterial infection will form weak acidic microenvironment. Hence, H₂O₂ is expected to generate after incubation of the bacteria with ZnO₂ NPs. The intracellular ROS was detected using 2,7-Dichloro-dihydro-Fluorescein Diacetate (DCFH-DA) as an indicator, which is nonfluorescent and can be oxidized by ROS to form fluorescent Dichlorodihydrofluorescein (DCF).

As shown in fig. 5a and fig. 5b, barely visible fluorescence was observed in E. coli and S. aureus without the treatment of ZnO₂ NPs. In contrast, bacteria treated with ZnO₂ NPs illustrated bright green fluorescence, indicating the generation of ROS in bacteria. We also noticed that the strength of the fluorescence was time dependent. The fluorescence intensities of E. coli and S. aureus at 2 h were 1.58 and 2.15 times to those of 0.5 h, respectively (fig. 6a and fig. 6b). It is necessary to search appropriate incubation time to enhance the antibacterial effects of ZnO₂ NPs. The generated ROS will cause oxidative stress to bacterial cells and result in the damage to protein, membrane lipids and Deoxyribonucleic Acid (DNA).

Fig. 5: Fluorescence intensity of E. coli, (a): DCFH-DA stained E. coli showing intracellular ROS after treated for 0.5 and 2 h and (b): Corresponding fluorescence intensity
Note: ( Equation ): Control and ( ): ZnO₂

Fig. 6: Fluorescence intensity of S. aureus, (a): DCFH-DA stained S. aureus showing intracellular ROS after treated for 0.5 and 2 h and (b): Corresponding fluorescence intensity
Note: ( Equation ): Control and ( ): ZnO₂

In addition to the evaluation of size, morphology, crystallinity, generation of ROS and the O₂ release performance of ZnO₂ NPs were also studied. Previous studies have indicated that O₂ release from ZnO₂NPs was related to temperature or pH values. We investigated its O₂release in aqueous solutions with different pH values. ZnO₂ NPs dissociates into Zn²⁺ ions and H₂O₂ in acidic aqueous media, while H₂O₂ can be catalyzed by the metal on the NPs surface to produce into H₂O and O₂. We measured the time-dependent release of O₂ from ZnO₂NPs in different pH media using an O₂meter.

The determinations were performed in degassed PBS in nitrogen atmosphere to influence of atmospheric O₂ on quantification. PBS solution without ZnO₂ nanoparticles determined in identical conditions was used as control. The experiment was performed at room temperature (T=25°) in neutral and weakly acidic PBS with pH values of 7.4 and 6.5, respectively. The results indicated that there is a constant O₂ release for at least 96 h and then reached a plateau resulting in a saturation curve. Since PBS solution was degassed and placed under a nitrogen atmosphere, there was negligible O₂ present during the determination. After dispersing the NPs in PBS, O₂ was constantly released, causing the increase of O₂ concentration in H₂O. When the reaction of NPs with H₂O was completed, O₂ concentration reached the plateau. Considering the high solubility of O₂ in H₂O (8.7- 8.4 mg/l at 25°)[24,25], the plateau was because of the nanoparticle deactivation. The determinations of the ZnO₂ nanoparticles in different pH value i.e., at 6.5 revealed that the amount of released O₂ was dependent on the pH. As shown in fig. 7, the O₂ release increased as the decrease of pH value. The amount of released O₂ increased from 0.73 to 2.1 mg/l with changing pH value from 7.4 and 6.5. These results were in line with the expectations because the acidic environment accelerates the O₂ release by accelerating the decomposition of the ZnO₂ NPs.

Fig. 7: Time dependent O₂ release measurements in aqueous media at different pH values
Note: ( Equation ): pH 7.4 and ( ): pH 6.5

ZnO is a transition metal oxide and semiconductor with wide band gap (3.3 eV). Oxidizing character and oxidizing sites would be created once there is radiation with energy larger than the band gap of the ZnO. Thus, formed oxidizing sites are capable of oxidizing water molecules and hydroxide anions to form strong oxidizing species and generate antibacterial effect. For ZnO₂ NPs, can react with H₂O and H⁺ to release ROS and are expected to cause better antibacterial effect. The antibacterial activity of the ZnO₂NPs was then evaluated using E. coli and S. aureus as model bacteria by colony counting assay method. To comprehensively understand the antibacterial efficiency, ZnO NPs were used as control. As presented in fig. 8a and fig. 8b, ZnO₂ NPs reduced the number of E. coli and S. aureus by about 40 % and 50 %, respectively. The toxicity of ZnO₂ NPs towards the E. coli and S. aureus was much stronger than the ZnO NPs. The increased toxicity of ZnO₂ NPs should be attributed to the synergistic effect of H₂O₂ and Zn²⁺. The effect of ZnO₂NPs was comparable to H₂O₂. Although ZnO NPs are considered as a bacteriostatic agent, their effect could be further improved by forming peroxides. For the antibacterial mechanism, it is reported that metal NPs will attach to the surfaces of bacteria by the adsorption and electrostatic interaction, resulting in damage to the cell membrane. Meanwhile, the released Zn²⁺ ions can chelate with teicoic and lipoteichoic acids, and then be carried by passive diffusion across membrane proteins[16].

Fig. 8: Antibacterial effects of H₂O₂, ZnO NPs and ZnO₂ NPs by colony count assay (n=3), (a): E. coli and (b): S. aureus

The Minimum Inhibitory Concentration (MIC) values of the ZnO₂NPs were further studied by micro-broth dilution method; MICs against E. coli and S. aureus were 90 and 60 μg/ml, respectively. In contrast, MICs of ZnO NPs for E. coli and S. aureus were 350 and 200 μg/l, respectively. These results were in agreement with the results obtained by colony counting assay method, suggesting the enhancement of the bacterial viability inhibition effect.

In summary, ZnO₂ NPs with uniform size were successfully synthesized with the help of PVP through a facile method. NPs could release H₂O₂ and Zn²⁺ quickly in acidic environment. ZnO₂ NPs showed outstanding antibacterial activity, owing to the synergistic effect of released H₂O₂ and Zn²⁺. Although the ZnO₂ NPs were relatively stable in H₂O, it is difficult to store for long-time storage in aqueous medium. For clinic application, dispersing the ZnO₂ NPs in non-aqueous media, such as glycerin, may be a feasible method to enhance the stability.