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 are 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. 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 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. 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 1 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 serial dilutions until 10 to the negative six.

Transfer 50 microliters from four dilutions to tryptic soy agar or 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.