Name: preparation of zinc oxide nanoparticles and the evaluation of their antibacterial effects
Uploaded: 2024-09-27T13:05:00.000+00:00
Duration: 6 min 42 s
Description: Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...

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 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 &#181;g/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.

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><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&amp;nbsp;</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.

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 &#181;g/mL, down to the non-toxic range of 62.5 &#181;g/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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66725/66725fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66725/66725fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66725/66725fig02.jpg" /> 
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 &#8804; 0.0001. Data presented as mean &#177; SD of three independent experiments performed in triplicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66725/66725fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.4K Views.  Chung-Ang University. Antibioticaresistente bacteriële infecties zijn momenteel in opkomst als een wereldwijd probleem. Om dit probleem aan te pakken, hebben we ons gericht op de ontwikkeling van antibacteriële metalen nanodeeltjes, waarbij verschillende mechanismen worden gebruikt om bacteriële cellen uit te roeien. Zinkoxide nanodeeltjes staan bekend om hun uitstekende antibacteriële eigenschappen.Omdat ze zijn goedgekeurd door de FDA, is bekend dat ze biocompatibel z...

Video: Auteur in de schijnwerpers: onderzoek naar de antibacteriële effecten van zinkoxide-nanodeeltjes bij het overwinnen van antibioticaresistentie

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent years. However, studies that elucidate the underlying mechanisms for ZnO NP-induced toxicity are scanty. We determined the toxicity profiles of ZnO NPs using both in vitro and in vivo experimental models. A significant decrease in cell viability was observed in ZnO NP-exposed MRC5 lung fibroblasts, showing that ZnO NPs exert cytotoxic effects. Similarly, interestingly, gut exposed to ZnO NPs exhibited a dramatic increase in reactive oxygen species levels (ROS) in the fruit fly Drosophila. More in-depth studies are required to establish a risk assessment for the increased usage of ZnO NPs by consumers.

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

We describe a detailed protocol for evaluating the toxicological profiles of zinc oxide nanoparticles (ZnO NPs) in particular, the type of cell death in human MRC5 lung fibroblasts and ROS formation in the fruit fly Drosophila.

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent ...

Immunology and Infection

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...

Bioactive ZnO Nanoparticles from Eucommia ulmoides for Wound Healing Enhancement

Formulation of Zinc-Based Nanomaterials using the Eucommia ulmoides Bark Extract and their Wound Healing Potential

The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here aims to explore the preparation of zinc oxide (ZnO) nanoparticles using the Eucommia ulmoides bark-mediated polyisoprene-rich aqueous extract. Meanwhile, the proposed protocol is associated with the preparation of wound healing material by easing the process. In addition, the wound-healing potential of the synthesized nanoparticles (Eu-ZnO-NPs) was evaluated using a simple scratch assay on a human umbilical vein endothelial cell (HUVEC) monolayer. After 24 h of treatment with Eu-ZnO-NPs, the cell proliferation and migration of HUVEC cells were assessed. At the end of the study, cell proliferation and migration were observed in scratched monolayer treated with different concentrations of Eu-ZnO-NPs, whereas poor cell migration and proliferation rates were observed in control cells. Of the chosen concentrations, 20 &#181;g/mL Eu-ZnO nanomaterials showed better cell migration and enhanced wound healing potential.

Here, we present a protocol to synthesize the zinc oxide (ZnO) nanoparticles using the polyisoprene-rich aqueous extract obtained from Eucommia ulmoides tree bark. The wound-healing potential exhibited by the synthesized ZnO nanoparticles on human umbilical vein endothelial cells (HUVECs) was evaluated using scratch assay, a simple, cost-effective, and efficient method.

The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here ...

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

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