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).

The reagents and equipment used in this study are listed in the Table of Materials.
1. Preparation of zinc oxide nanoparticles
<ol>
	<li>Measure 200 mL of absolute ethyl alcohol and pour it into a glass round bottom flask.</li>
	<li>Place the round bottom flask on a heating mantle and maintain stirring at 25-40 &#176;C.</li>
	<li>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.</li>
	<li>Add 1.4 g of zinc acetate into the solution and stir until it is completely dissolved.</li>
	<li>Raise the temperature of the solution by setting the heating mantle temperature to 70 &#176;C.</li>
	<li>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.</li>
	<li>Aliquot the solution to 50 mL conical tubes, centrifuge at 15000 &#215; g for 15 min at room temperature, and then discard the supernatant.</li>
	<li>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.</li>
	<li>Wash the nanoparticles through centrifugation at 15000 &#215; 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.</li>
	<li>Vacuum dry the sample pallet at 60 &#176;C for 24 h and obtain the ZnO NP powder.</li>
</ol>
2. Antibacterial tests using MRSA and P. aeruginosa
<ol>
	<li>Bacterial culture 
	NOTE: Clinical MDR bacterial strains were obtained from Chung-Ang University Hospital, Seoul, South Korea.
	<ol>
		<li>Take out MRSA and P. aeruginosa bacterial strains stocked in Tryptic Soy Broth (TSB) from the deep freezer.</li>
		<li>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 &#176;C.</li>
		<li>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 &#181;L of the diluted solution onto TSA plates and spread the solution using a L-shaped spreader.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Bacterial sampling
	<ol>
		<li>To test a wide range of ZnO concentrations, prepare 2 mg/mL ZnO NPs solution using Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) and perform 2-fold serial dilution to make different concentrations. 
		NOTE: 1000 &#181;g/mL, 500 &#181;g/mL, 250 &#181;g/mL, 125 &#181;g/mL, and 62.5 &#181;g/mL were tested.</li>
		<li>Add 100 &#181;L 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 &#181;L 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.</li>
		<li>Dilute the bacterial culture to 1 &#215; 106 CFU/mL using TSB media and add 100 &#181;L 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 &#181;L of ZnO NPs, the final concentration of bacteria will be 5 &#215; 105 CFU/mL.</li>
		<li>Place the 96-well plate in a 37 &#176;C incubator and incubate for 24 h.</li>
	</ol>
	</li>
	<li>Bacterial spreading
	<ol>
		<li>Pipette 100 &#181;L from each well and prepare various 10-fold serial dilutions until 10-6. 
		NOTE: Add 100 &#181;L of ZnO NPs containing bacterial solution into 900 &#181;L of sterilized distilled water and repeat 6 times.</li>
		<li>Pipette 50 &#181;L 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.</li>
		<li>Place the agar plates into a 37 &#176;C incubator and incubate for 24 h.</li>
	</ol>
	</li>
	<li>Bacterial CFU counting
	<ol>
		<li>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 &#181;L. Using the spread plate with the undiluted bacterial solution, determine the potential minimum bactericidal concentration.</li>
		<li>Use the obtained data to represent the percentage of live bacteria relative to those in the negative control. 
		NOTE: Percentage of live bacteria (%) =&#160; &#160;<img alt="Equation 1" src="/files/ftp_upload/66725/66725eq01.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
</ol>

 Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

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

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...

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 &#946;-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 &#946;-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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DLS</td>
			<td>Zetasizer Pro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, absolute</td>
			<td>DAEJUNG</td>
			<td>4023-2304</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader&#160;</td>
			<td>BioTeck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL JEM-F200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TSA</td>
			<td>DB difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>TSB</td>
			<td>DB difco</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>XRD</td>
			<td>NEW D8-Advance</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc acetate</td>
			<td>Sigma-Aldrich</td>
			<td>383317</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of zinc oxide nanoparticles and the evaluation of their antibacterial effects

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.

Author Spotlight: Exploring the Antibacterial Effects of Zinc Oxide Nanoparticles in Overcoming Antibiotic Resistance

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

Antibiotic-resistant bacterial infections are currently emerging as a global issue. To address this problem, we have focused on development of antibacterial metallic nanoparticles, employing various mechanisms to eradicate bacterial cells. Zinc oxide nanoparticles are known for their excellent antibacterial capabilities.As they're approved by the FDA, they're known to be biocompatible to the human body. The major advantage of our zinc oxide nanoparticle synthesis method lies in its simplicity and relatively short synthesizing time compared to other protocols, owing to its precipitation-based synthesis approach. This makes it a highly effective method, particularly for mass production from a commercialization perspective, and additionally, its chemical synthesis without the need for specialized equipment adds to its appeal as well.Our research team plans to further develop antibacterial therapy using zinc oxide nanoparticles or inorganic nanoparticles, and integrate them with drug delivery for real-world applications. To achieve more effective bacteria-specific treatment, we will utilize antibodies and lectins for targeting bacteria, minimizing general cell toxicity of particles, and conducting research on delivering particles to pathogens in actual infection environments.

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...

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.

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...

preparation-zinc-oxide-nanoparticles-evaluation-their-antibacterial

Research

JoVE Journal

Bioengineering

Preparation of Zinc Oxide Nanoparticles Using Precipitation Method for Antibacterial Testing 

To begin, measure 200 milliliters of absolute ethyl alcohol and pour it into a glass round-bottom flask. Place the flask on a heating mantle and maintain stirring at 25 to 40 degrees Celsius. Weigh 500 milligrams of CTAB in a 50 milliliter vial and add it to the ethyl alcohol in the flask.Keep stirring the solution until CTAB is completely dissolved, then add 1.4 grams of zinc acetate to the solution. Set the heating mantle temperature to 70 degrees Celsius to increase the temperature of the solution. Add 25 milliliters of 0.5 molar sodium hydroxide solution to the mixture and let it react for one hour until the clear solution becomes white in color.Aliquot the solution to 50-milliliter conical tubes, then centrifuge it at 15, 000 G for 15 minutes at room temperature. Discard the supernatant. Next, add 10 milliliters 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 zinc oxide pellet. Centrifuge the nanoparticles at 15, 000 G for 15 minutes at room temperature, then remove the supernatant. Resuspend the nanoparticles in distilled water.Check the pH of the supernatant solution using pH test paper. Fugation until the pH of the solution becomes neutral. Then discard the supernatant and vacuum dry the sample pellet at 60 degrees Celsius for 24 hours to obtain the zinc oxide nanoparticle powder.The successful synthesis of zinc oxide nanoparticles was confirmed using transmission electron microscopy. The obtained nanoparticles were round. Dynamic light scattering showed that the synthesized nanoparticles had an average size of 130.4 nanometers and a zeta potential of 28.92 millivolts.The obtained zeta potential indicates the relative stability of the nanoparticles in water. The absorption spectra of zinc oxide nanoparticles showed a specific absorption peak for zinc oxide at 360 nanometers confirming the synthesis of the nanoparticles. X-ray diffraction analysis revealed distinct crystalline peaks that are characteristic of zinc oxide.

Antibacterial Testing of Zinc Oxide Nanoparticles Using MRSA and Pseudomonas aeruginosa

To begin, prepare two milligrams per milliliter of zinc oxide nanoparticle solution using DPBS. Perform twofold serial dilution to make different concentrations. Add 100 microliters of each tested zinc oxide nanoparticles concentration into a 96-well plate.Dilute the bacterial culture to one million CFU per milliliter with tryptic soy broth or TSB media. Add 100 microliters to each well containing different concentrations of zinc oxide nanoparticle solution. Incubate the plate at 37 degrees Celsius for 24 hours.Pipette 100 microliters of zinc oxide nanoparticles with bacterial solution from each well and prepare various tenfold cereal dilutions until 10 to the negative six. Transfer 50 microliters from four dilutions to tryptic soy agar TSA media plates. Incubate the agar plates at 37 degrees Celsius for 24 hours.After selecting 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 number of CFU per milliliter. Use the obtained data to represent the percentage of live bacteria relative to those in the negative control. The antibacterial properties of zinc oxide nanoparticles were tested on 0.5 million CFU per milliliter Pseudomonas aeruginosa strain.The antimicrobial activity of the nanoparticles increased in a concentration-dependent manner. However, a visible decrease in the number of bacterial colonies from the starting undiluted bacterial culture was not evident. When tested against a 0.5 million CFU per milliliter MRSA strain, these nanoparticles exhibited increased antibacterial activity, leading to a significant reduction in bacterial colony formation.

796 Views.  Chung-Ang University.  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. 

Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

Summary