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

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 humans^7,8. 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 (%) =   

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...

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 surfact...

Materials

Name	Company	Catalog Number	Comments
DLS	Zetasizer Pro
Ethyl alcohol, absolute	DAEJUNG	4023-2304
Microplate reader	BioTeck
Sodium Hydroxide	Sigma-Aldrich	221465
TEM	JEOL JEM-F200
TSA	DB difco	236950
TSB	DB difco	211825
XRD	NEW D8-Advance
Zinc acetate	Sigma-Aldrich	383317