Method Article

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects

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DOI:

10.3791/66725

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September 27th, 2024

 ,  ,  ,  ,  ,  , 
1School of Integrative Engineering, Chung-Ang University, 2Feynman Institute of Technology, Nanomedicine Corporation, 3Department of Chemical Science and Engineering, Tokyo Institute of Technology, 4Department of Chemical Engineering, Kumoh National Institute of Technology

* These authors contributed equally

In This Article

Summary

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

Abstract

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

Introduction

Multidrug-resistant (MDR) bacterial infections pose a significant global threat to human health1. As these infections can be fatal in patients with underlying conditions, active research is attempting to address this issue2. Bacteria have evolved to evade the action of various drugs. Penicillin, widely known and credited with saving millions of lives worldwide, is a β-lactam antibiotic that inhibits the synthesis of the bacterial cell wall3. However, bacteria have evolved to neutralize the efficacy of drugs through various mechanisms such as efflux pumps, transpeptidase alterations, or decreased permeability4. Additionally, bacterial cells can transmit these resistance genes to the next generation, increasing the survival rates of the subsequent generation and strengthening the problem of resistant strains5.

The increase in antibiotic-resistant bacteria has led to the emergence of MDR bacteria, which commonly exhibit resistance to multiple antibiotics. MDR strains are most frequently encountered in hospital settings, where multiple bacterial strains are exposed to and consequently develop resistance to different antibiotics6. Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), is a gram-positive commensal bacterium that forms clusters on the skin of approximately 30% of humans7,8. MRSA, which was first identified in the 1960s, exhibits reduced sensitivity to β-lactam antibiotics, resulting in a sharp increase in infection rates since the 1990s9. Among gram-negative bacteria, Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent strains acquired in hospitals. This species, a facultative rod-shaped bacterium, causes opportunistic infections in humans10. Particularly, MDR strains that directly affect human health are responsible for over 50 % of healthcare-associated infections11. In this study, we utilized the most commonly encountered multidrug-resistant strains within hospitals, MRSA and P. aeruginosa.

The use of nanoparticles (NPs) for antimicrobial purposes has been extensively investigated to tackle the issue of antibiotic resistance. Metallic NPs, in particular, induce bacterial cell death through various mechanisms, offering a potential solution to the problem of drug resistance. Metallic NPs exert antimicrobial activity through multiple mechanisms, including the release of antimicrobial ions, generation of reactive oxygen species (ROS), and physical disruption of cells, among other means12. NPs composed of silver, copper, zinc oxide (ZnO), and titanium oxide possess high antimicrobial efficacy and are thus being actively researched13.

ZnO NPs have been approved by the U.S. Food and Drug Administration (FDA) for use in humans. Conversely, despite their high antimicrobial efficacy, the use of silver and copper NPs in humans is limited by their high cytotoxicity. However, ZnO NPs are commonly found in everyday life and are even present in widely used sunscreen formulations14. Of note, Zn2+ ions released from ZnO NPs are highly effective in bacterial treatment, inducing bacterial cell death through the generation of ROS and other physical damage mechanisms15.

This study outlines the protocol for synthesizing ZnO nanoparticles (NPs) using a precipitation method and introduces an antimicrobial testing approach using a microbroth dilution method with clinical samples of MRSA and P. aeruginosa. The precipitation method for ZnO NPs involves synthesizing insoluble solid ZnO NPs by adjusting pH and temperature using soluble precursors such as zinc acetate or zinc nitrate16. Along with relatively facile and rapid production, this method ensures repeatability in synthesis and facilitates control over particle size and morphology17. In this synthesis protocol, sodium hydroxide (NaOH), one of the most commonly used precipitation agents, was utilized to precipitate zinc acetate, and a small amount of hexadecyltrimethylammonium bromide (CTAB) was employed to inhibit the uncontrolled synthesis of the nanoparticles18. Among various antimicrobial tests, the antibacterial activity of ZnO nanoparticles was evaluated using the microbroth dilution method, which avoids optical interference from metal oxide nanoparticles and enables direct colony measurement for determining MIC19.

Protocol

The reagents and equipment used in this study are listed in the Table of Materials.

1. Preparation of zinc oxide nanoparticles

  1. Measure 200 mL of absolute ethyl alcohol and pour it into a glass round bottom flask.
  2. Place the round bottom flask on a heating mantle and maintain stirring at 25-40 °C.
  3. Measure 500 mg of CTAB in a 50 mL vial and add it to the ethyl alcohol in the flask. Stir until CTAB is completely dissolved.
  4. Add 1.4 g of zinc acetate into the solution and stir until it is completely dissolved.
  5. Raise the temperature of the solution by setting the heating mantle temperature to 70 °C.
  6. Add 25 mL of 0.5 M NaOH solution to the mixture and let it react for 1 h until the clear solution becomes white in color.
  7. Aliquot the solution to 50 mL conical tubes, centrifuge at 15000 × g for 15 min at room temperature, and then discard the supernatant.
  8. Add 10 mL of distilled water to one of the conical tubes and resuspend the nanoparticles by sonicating the solution. Transfer the suspended solution to a different conical tube containing the ZnO pellet and repeat until all ZnO solutions are collected in one conical tube.
  9. Wash the nanoparticles through centrifugation at 15000 × g for 15 min (room temperature), remove the supernatant, and resuspend in distilled water. Check the pH of the supernatant solution using pH test paper and repeat until the pH of the solution becomes neutral.
    NOTE: When the pH of the supernatant solution becomes neutral (pH = 7), discard the supernatant solution and do not resuspend with distilled water.
  10. Vacuum dry the sample pallet at 60 °C for 24 h and obtain the ZnO NP powder.

2. Antibacterial tests using MRSA and P. aeruginosa

  1. Bacterial culture
    NOTE: Clinical MDR bacterial strains were obtained from Chung-Ang University Hospital, Seoul, South Korea.
    1. Take out MRSA and P. aeruginosa bacterial strains stocked in Tryptic Soy Broth (TSB) from the deep freezer.
    2. After thawing the bacterial solutions, streak the solution onto a Tryptic Soy Agar (TSA) plate using a disposable inoculation loop. Place the streaked agar plates in the incubator and incubate for 24 h.
      NOTE: A single colony picked from the TSA plate was added to 10 mL of TSB media in a 50 mL conical tube using a bacterial inoculation loop. Bacteria were cultured for 24 h. Bacteria were cultured under aerobic culture conditions at 37 °C.
    3. To measure the concentration of the bacteria solution, dilute the cultured solution using 10-fold serial dilution to 10-6 using distilled water. Afterward, place 50 µL of the diluted solution onto TSA plates and spread the solution using a L-shaped spreader.
    4. Incubate the plates in the incubator for 24 h.
      NOTE: Colonies were optically counted, and the concentration of the cultured solution was calculated by multiplying the dilution factors by the number of colonies counted.
  2. Bacterial sampling
    1. To test a wide range of ZnO concentrations, prepare 2 mg/mL ZnO NPs solution using Dulbecco's Phosphate-Buffered Saline (DPBS) and perform 2-fold serial dilution to make different concentrations.
      NOTE: 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, and 62.5 µg/mL were tested.
    2. Add 100 µL for each of the tested concentrations of ZnO NPs into a 96-well plate.
      NOTE: Use double (2x) of the final desired concentration of ZnO NPs, as each sample in the well will be diluted with the addition of 100 µL of bacteria culture. Use a 2% Antibiotic-Antimycotic (A/A) solution as the positive control and DPBS as the negative control. A/A is a penicillin and streptomycin antibiotic complex, which is effective against gram-positive and gram-negative bacteria, respectively.
    3. Dilute the bacterial culture to 1 × 106 CFU/mL using TSB media and add 100 µL to each well containing different concentrations of ZnO NPs.
      NOTE: Initial MRSA and P. aeruginosa culture solution concentrations were 3 x 109 CFU/mL. Bacteria culture concentration 1 x 106 CFU/mL was made by 1/20 and 1/150 dilution of the culture solution. After mixing with 100 µL of ZnO NPs, the final concentration of bacteria will be 5 × 105 CFU/mL.
    4. Place the 96-well plate in a 37 °C incubator and incubate for 24 h.
  3. Bacterial spreading
    1. Pipette 100 µL from each well and prepare various 10-fold serial dilutions until 10-6.
      NOTE: Add 100 µL of ZnO NPs containing bacterial solution into 900 µL of sterilized distilled water and repeat 6 times.
    2. Pipette 50 µL from four diluted solutions and add to TSA media plates. Use an L-shaped cell spreader to spread the bacterial suspension onto the agar plate.
      NOTE: Use the 100, 10-2, 10-4, and 10-6 dilutions. Conduct all experiments in triplicate. Spread until all the bacterial solution is absorbed onto the agar plate.
    3. Place the agar plates into a 37 °C incubator and incubate for 24 h.
  4. Bacterial CFU counting
    1. Select a dilution factor that is countable for each group. Mark all the colonies in the countable dilution plate and recalculate so that the concentration becomes # of CFU/mL.
      NOTE: The countable dilution factor indicates plates that have 20-100 CFU on an individual plate. Initially counted concentration results # of Counted CFU/50 µL. Using the spread plate with the undiluted bacterial solution, determine the potential minimum bactericidal concentration.
    2. Use the obtained data to represent the percentage of live bacteria relative to those in the negative control.
      NOTE: Percentage of live bacteria (%) =   Bacterial analysis equation, percentage concentration calculation, scientific result representation.

Results

The successful synthesis of ZnO NPs was confirmed using transmission electron microscopy (TEM), as shown in Figure 1A. The obtained ZnO NPs were observed to be round in shape, with an average particle size of 35.35 nm and a standard deviation of 6.81 nm. The precipitation of these nanoparticles was observed through a double-displacement reaction by adding NaOH solution to zinc acetate, where Zn2+ ions underwent hydrolysis.

Using dynamic light scattering (DLS), the average size and zeta potential of the synthesized nanoparticles were determined to be 130.4 nm and 28.92 mV, respectively, as shown in Figure 1B. The discrepancy in the size of ZnO NPs measured using DLS compared with that obtained from the TEM image was attributed to the aggregation of bare nanoparticles. The positive zeta potential indirectly confirmed the acquisition of ZnO NPs, which can interact electrostatically with bacterial cell surfaces, potentially causing physical damage. The magnitude of zeta potential indicates the potential stability of a colloidal system. When all particles in a suspension have large positive or negative zeta potentials, there is a tendency for them to repel each other, preventing aggregation. Particles with zeta potentials greater than +30 mV or less than -30 mV are generally considered stable. The synthesized ZnO NPs exhibited a zeta potential of +28.92 mV, indicating relative stability in water20.

The absorption spectra of ZnO NPs were examined using a microplate reader, revealing a specific absorption peak for ZnO at 360 nm (Figure 1C). The precursor zinc acetate does not have a unique peak, whereas ZnO NPs are known to have a unique peak at 360-370 nm. The synthesis was confirmed by the presence of the 360 nm unique peak in the synthesized ZnO NPs21. These specific UV absorption characteristics confirmed the direct synthesis of ZnO NPs. Furthermore, X-ray diffraction (XRD) analysis (Figure 1D) revealed distinct crystalline peaks that are characteristic of ZnO. When compared to the representative wurtzite structure of ZnO NPs (JCPDS No. 36-1415), it was observed that all the planes (1, 0, 0), (0, 0, 2), (1, 0, 1), (1, 0, 2), (1, 1, 0), (1, 0, 3), (2, 0, 0), (1, 1, 2), and (2, 0, 1) were in alignment22.

The antimicrobial efficacy of the synthesized ZnO NPs was evaluated using a microbroth dilution test against clinical samples of P. aeruginosa and MRSA obtained from the Chung-Ang University Hospital in Seoul, South Korea. Images of bacterial cultures were captured for analysis. To visually assess the antimicrobial efficacy of the nanoparticles using the same dilution factor, spread plates with undiluted bacterial solution were used (Figure 2A,B). Spread plates with the original solution were employed to determine the potential minimum bactericidal concentration. Since bacterial colonies were observed even at the highest concentration for both strains, a complete bactericidal effect was not achieved. The bacterial concentrations in each group were calculated using countable dilution factors. When comparing the survival rates of each treatment group to the negative control group, the antimicrobial effects of ZnO NPs were evident in both P. aeruginosa and MRSA strains. Considering the researched cytotoxicity range of ZnO NPs, different ZnO concentrations were tested, starting from the highest concentration known to induce toxicity, which is 1000 µg/mL, down to the non-toxic range of 62.5 µg/mL, through serial dilution23,24. In the case of P. aeruginosa, the antimicrobial activity of ZnO NPs was increased in a concentration-dependent manner (Figure 2A). However, a visible decrease in the number of P. aeruginosa bacterial colonies from the starting undiluted (100) bacterial culture was not evident.

Conversely, ZnO NPs exhibited high antimicrobial activity against the gram-positive bacterium MRSA, with a noticeable decrease in bacterial colony-forming units (CFU), as confirmed by comparing images of diluted bacterial cultures to that of the starting undiluted (100) bacterial culture. This result confirmed that the synthesized ZnO NPs displayed antimicrobial activity against both bacterial strains, particularly showing increased efficacy against the MRSAstrain.

Nanoparticle characterization diagram; TEM images, size distribution, DLS, zeta potential, XRD analysis.
Figure 1: Characterization of zinc oxide nanoparticles. (A) Transmission electron microscopy images of ZnO NPs under different magnifications. (B) Size (left) and zeta potential distribution (right) by DLS analysis. (C) Absorbance spectrum of ZnO NPs using a microplate reader. (D) XRD analysis of ZnO NPs and their crystalline peaks. Please click here to view a larger version of this figure.

P. aeruginosa, MRSA inhibition by ZnO NPs; bar graph and petri dish images; concentration effect.
Figure 2: Antibacterial properties of ZnO NPs tested on 5 x 105 CFU/mL bacterial strains. (A) Antibacterial test against the P. aeruginosa strain. (B) Antibacterial test against the MRSA strain. N = negative control (DPBS), P = positive control (A/A). Asterisks denote the statistically significant difference compared with the controls, ****p ≤ 0.0001. Data presented as mean ± SD of three independent experiments performed in triplicates. Please click here to view a larger version of this figure.

Discussion

The synthesis of ZnO NPs via precipitation is relatively simple and straightforward. To successfully synthesize ZnO NPs using this method, stirring is crucial to ensure that the precursor (zinc acetate) is fully dissolved in the solvent. Moreover, increasing the temperature helps to induce a successful double-displacement reaction. In the synthesis of ZnO NPs, there are many factors that determine the size and shape, including the precipitation agent, the concentration of the precipitation agent, and the surfactant. The use of precipitation agents other than NaOH can change the particle shape. As reported by Gharpure et al.25, when ammonium hydroxide (NH4OH) was used to precipitate zinc acetate with different surfactants, particles were mostly synthesized in cone and triangle shapes. Even when synthesized using potassium hydroxide (KOH), it was difficult to see spherical particles26. This observation confirmed that precipitation with NaOH facilitated the synthesis of ZnO nanoparticles with rounded shapes.

In this study, we synthesized ZnO NPs using a small amount of surfactant and NaOH due to their potential application in biomedical applications and the development of a rapid synthesis method. As a result, even though the synthesis of round-shaped nano-scale particles was possible, the synthesis of homogenous spherical particles was limited without additional modifications27. ZnO NPs are known to disperse well in nonpolar solvents or mixed polar and nonpolar solvents, leading to limited stability and aggregation issues in water28,29. Since the ZnO NPs were dispersed in DPBS, aggregation phenomena were observed, which was evident from the difference in size between TEM images and DLS measurements. The introduction of capping agents and coupling agents such as 3-aminopropyltriethoxysilane (APTES) is expected to enhance dispersion in water, although no modifications were conducted in this study to evaluate the intrinsic antimicrobial properties of ZnO NPs30.

The synthesis method of ZnO NPs presented in this study differs from microwave-assisted or solvothermal techniques in that it does not require high pressure or heat, and does not involve any linker other than a small amount of surfactant31,32. With a synthesis reaction time of only 2 h, it is easily accessible to anyone. From a biomedical application perspective, this study evaluated the antibacterial properties of ZnO NPs using MDR clinical samples obtained from Chung-Ang University, enabling the determination of the necessary concentration of ZnO for eradicating bacteria in real-world environments. Compared to the study by Khan et al., where most bacteria were eradicated in the concentration range of 60 µg/mL against P. aeruginosa, it was found that the MDR clinical strains used in this study exhibited higher survival rates33.

The antimicrobial efficacy of the synthesized ZnO NPs was assessed against P. aeruginosa and MRSA strains. Given the short doubling time of bacteria during the experiment, first placing the nanoparticle solution in a 96-well plate, and then adding the bacterial solution promptly is crucial. Moreover, employing various dilutions for culturing is crucial for accurately determining the antimicrobial activity of nanoparticles. Thorough mixing of the bacterial solution during the generation of the 10-fold serial dilutions is also crucial; therefore, careful pipetting and vortexing are necessary. However, one must note that this experimental procedure is time-consuming. Thus, a preliminary assessment using absorbance measurements at 600 nm can provide an approximate estimation of antimicrobial activity before culturing.

The synthesized ZnO NPs exhibited higher antimicrobial activity against the MRSA strain, likely due to differences in the bacterial structure. Gram-negative bacteria, such as P. aeruginosa, differ from MRSA in their cell membrane structure, specifically in the formation of a double membrane containing lipopolysaccharides, as opposed to a single-layered thick peptidoglycan membrane of MRSA. This double-membrane structure potentially hinders the penetration of the antimicrobial Zn2+ ions, increasing the survival rate of P. aeruginosa. ZnO NPs are utilized for their antimicrobial properties and are also actively used in sunscreen and skin regeneration. Thus, diverse studies utilizing ZnO NPs are anticipated to continue in the future.

ZnO NPs can be applied in various fields, through surface modification and various material conjugations. ZnO holds potential applications in drug delivery, antibacterial coatings, cancer therapy, and wound healing, among others34. ZnO NPs can be applied in drug delivery by conjugating targeting molecules such as antibodies, enabling targeted delivery to specific cells or tissues35. Additionally, through the generation of reactive oxygen species, ZnO NPs can target not only bacterial cells but also cancer cells. Furthermore, the release of Zn2+ ions from ZnO nanoparticles and the generation of hydrogen peroxide (H2O2) due to reactive oxygen species can promote wound-healing processes at the wound site36. It is anticipated that these potentials will lead to numerous research exploring the use of ZnO NPs across different fields.

Disclosures

Dr. Jonghoon Choi is the CEO/Founder, and Dr. Yonghyun Choi is the CTO of the Feynman Institute of Technology at the Nanomedicine Corporation.

Acknowledgements

This research was supported by the Chung-Ang University Graduate Research Scholarship in 2022 (Ms. Gahyun Lee). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2020R1A5A1018052) and by the Technology development Program (RS202300261938) funded by the Ministry of SMEs and Startups (MSS, Korea).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DLSZetasizer Pro
Ethyl alcohol, absoluteDAEJUNG4023-2304
Microplate reader BioTeck
Sodium HydroxideSigma-Aldrich221465
TEMJEOL JEM-F200
TSADB difco236950
TSBDB difco211825
XRDNEW D8-Advance
Zinc acetateSigma-Aldrich383317

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Nanoparticle SynthesisPrecipitation MethodTransmission Electron MicroscopyDynamic Light ScatteringX Ray DiffractionBroth Microdilution TestMultidrug Resistant BacteriaColony Forming Units

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