This research was supported by the Chung-Ang University research grant in 2022 (during Professor Jonghoon Choi&#39;s sabbatical leave).&#160;This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. RS-2023-00275006), and the Korea Environment Industry &#38; Technology Institute (KEITI) grant funded by the Korea Ministry of Environment (MOE) (No. 2022002980003). Clinical MRSA and P. aeruginosa were obtained from Chung-Ang University Hospital.

1. Preparation of the GO/Cu nanocomposites
NOTE: The size and morphology of Cu nanoparticles that grow on the GO nanosheets are determined by the degree of GO oxidation, the concentration of the Cu precursor, and the concentration of the reducing agent26.
<ol>
	<li>Prepare 10 mL of 1 mg/mL GO suspension in a glass bottle. Sonicate the GO suspension for 1 h until GO is well dispersed in distilled (DI) water.</li>
	<li>Prepare a 20 mM CuCl2 solution in a glass vial. Sonicate the CuCl2 solution until CuCl2 is well dispersed in DI water.</li>
	<li>Add 10 mL of the 20 mM CuCl2 solution to the GO solution and sonicate the mixture at 70 &#176;C for 1 h.</li>
	<li>Prepare a 20 mM NaBH4 solution. 
	CAUTION: This procedure must be conducted in a chemical fume hood. A redox reaction occurs as soon as the reducing agent NaBH4 is introduced into water. Prepare the NaBH4 solution immediately before the experiment.</li>
	<li>Preparing the mixture of NaBH4, GO, and CuCl2
	<ol>
		<li>Add 20 mL of the 20 mM NaBH4 solution to the GO and CuCl2 mixture while stirring with a magnetic bar at 200 rpm. After adding the NaBH4 solution, stir continuously for 30 min. 
		CAUTION: This procedure must be conducted in a chemical fume hood.</li>
	</ol>
	</li>
	<li>Centrifugation of the mixture
	<ol>
		<li>Transfer the mixture from step 1.5.1 to a centrifuge tube. Centrifuge the solution at 23,000 &#215; g for 10 min at room temperature. Discard the supernatant. 
		CAUTION: When operating the centrifuge, always maintain balance by placing tubes of equal weight.</li>
	</ol>
	</li>
	<li>Resuspend the GO/Cu nanocomposite mixture with 10 mL of DI water and sonicate them to enable the precipitate to disperse evenly throughout the solution.</li>
	<li>Centrifuge the solution at 23,000 &#215; g for 10 min at room temperature; then remove the supernatant.</li>
	<li>Repeat steps 1.7-1.8 once more to remove the unreacted chemicals.</li>
	<li>Add 1 mL of distilled water to the GO/Cu nanocomposites and sonicate the mixture to disperse the sediment evenly throughout the liquid phase. 
	NOTE: Collect all GO/Cu nanocomposite solutions in one conical tube.</li>
	<li>Freeze-dry the solution at &#8722;60 &#176;C under a vacuum overnight until the GO/Cu nanocomposites are completely dried and obtain GO/Cu nanocomposite powder.</li>
	<li>Store the GO/Cu nanocomposite powder at &#8722;20 &#176;C until use.</li>
</ol>
2. Preparation of bacteria for the antibacterial test
CAUTION: This procedure must be conducted in a biological safety cabinet with an alcohol lamp. Bacterial waste must be autoclaved before disposal. When handling antibiotic-resistant bacteria, gloves, gowns, and masks must be worn, and hands must be washed using soap or hand sanitizer after the experiment. Always thoroughly disinfect; if contamination occurs in the experimental area, immediately disinfect it with 70% ethanol.
<ol>
	<li>Bacterial culture media preparation
	<ol>
		<li>Mix 20 g of tryptic soy agar and 500 mL of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
		<li>Prepare the agar plate by pouring 15 mL of the agar solution into a Petri dish before the agar hardens. Store the agar plate at 4 &#176;C until use. 
		NOTE: This must be done quickly before the agar solution hardens. To prevent contamination, sterilize the agar plate with UV for 15 min during hardening.</li>
		<li>Mix 15 g of tryptic soy broth (TSB) and 500 mL of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
		<li>Aliquot 40-50 mL of the broth solution into a 50 mL conical tube. Store the broth at 4 &#176;C until use. 
		NOTE: To prevent contamination, sterilize the aliquoted broth with UV for 15 min.</li>
	</ol>
	</li>
	<li>Bacterial culture
	<ol>
		<li>Inoculate the MSRA or P. aeruginosa stock solution on the agar plate using a loop. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h.</li>
		<li>Inoculate one colony of the bacteria into 10 mL of the broth using a loop. Incubate the broth at 37 &#176;C using a shaking incubator at 200 rpm for 24 h.</li>
		<li>Repeat steps 2.2.1-2.2.2 once more to obtain bacteria with the same characteristics.</li>
		<li>After 24 h, serially dilute the bacterial solution tenfold using sterile distilled water. Inoculate 100 &#181;L of the diluted bacterial suspension onto the agar plate and spread it with a spreader. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h. 
		NOTE: The dilution level is dependent on the bacteria. Aim to prepare less than 100 colonies in an agar plate.</li>
		<li>Count the bacterial colonies after the 24 h incubation to determine the colony-forming unit (CFU) using equation (1). 
		<img alt="Equation 1" src="/files/ftp_upload/66762/66762eq01.jpg" style="margin: auto;" /> (1)</li>
		<li>Repeat steps 2.2.4-2.2.5 at least 3x to confirm whether the CFU of the bacteria is relatively constant.</li>
	</ol>
	</li>
</ol>
3. Antibacterial test using the microdilution broth method
<ol>
	<li>Day 1) Inoculate one colony of the bacteria from the agar plate into 10 mL of the broth using a loop. Incubate the broth at 37 &#176;C using a shaking incubator at 200 rpm for 24 h.</li>
	<li>Day 2) Prepare the GO/Cu nanocomposite mixture in at least three concentrations using Dulbecco&#39;s phosphate-buffered saline (DPBS). Sonicate the GO/Cu nanocomposite suspension until the GO/Cu nanocomposites are well dispersed in DPBS. 
	NOTE: In this protocol, 500, 250, 125, and 62.5 &#181;g/mL of the GO/Cu nanocomposite mixture were tested.</li>
	<li>Prepare the control solutions. The negative control is DPBS, and the positive control is 1% of a penicillin/streptomycin solution in DPBS that will kill the bacteria.</li>
	<li>Add 100 &#181;L of the GO/Cu nanocomposite suspension and the control solutions into 96-well plates. Add all samples in triplicate. 
	NOTE: Sterilize samples with UV for 15 min before applying them to bacteria.</li>
	<li>Based on the CFU after 24 h of incubation, dilute bacterial suspension using TSB to 1 &#215; 106 CFU/mL. 
	NOTE: The initial MRSA and P. aeruginosa culture suspension concentrations in our study, according to step 2.2.5, are 4.5 &#215; 109 CFU/mL and 3 &#215; 109 CFU/mL, respectively. Bacterial concentrations of 1 &#215; 106 CFU/mL are obtained by diluting the cultures 4,500x and 3,000x, respectively.</li>
	<li>Inoculate 100 &#181;L of the 1 &#215; 106 CFU/mL diluted bacterial suspension into the sample wells in the 96-well plates. Incubate the 96-well plates at 37 &#176;C using a shaking incubator at 200 rpm for 24 h. 
	NOTE: The final concentration of bacteria is 5 &#215; 105 CFU/mL after mixing with 100 &#181;L of sample.</li>
	<li>Day 3) Mix the sample and bacterial suspensions vigorously with a 200 &#181;L micropipette tip. Serially dilute the sample-bacteria mixture tenfold with sterile distilled water. 
	NOTE: The dilution level is dependent on the bacteria. Aim to prepare less than 100 colonies in an agar plate.</li>
	<li>Inoculate 100 &#181;L of the diluted bacterial suspension onto the agar plate and spread it with a spreader. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h.</li>
	<li>Day 4) Count the bacterial colonies and determine the CFU values to confirm the antibacterial activity of the GO/Cu nanocomposites using equation (2). 
	<img alt="Equation 2" src="/files/ftp_upload/66762/66762eq02.jpg" style="margin: auto;" /> (2)</li>
</ol>

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibiotic+resistance.">Molecular mechanisms of antibiotic resistance.</a> Nat Rev Microbiol. 13 (1), 42-51 (2015).</li><li>S&#225;nchez-L&#243;pez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-based+nanoparticles+as+antimicrobial+agents:+An+overview.">Metal-based nanoparticles as antimicrobial agents: An overview.</a> Nanomaterials (Basel). 10 (2), 292 (2020).</li><li>Makabenta, J. M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterial-based+therapeutics+for+antibiotic-resistant+bacterial+infections.">Nanomaterial-based therapeutics for antibiotic-resistant bacterial infections.</a> Nat Rev Microbiol. 19 (1), 23-36 (2021).</li><li>Liang, X., Govindaraju, S., Yun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+applicability+of+polyaniline+coated+gold+nanorods:+A+study+of+antibacterial+and+redox+activity.">Dual applicability of polyaniline coated gold nanorods: A study of antibacterial and redox activity.</a> BioChip J. 12 (2), 137-145 (2018).</li><li>Ni&#241;o-Mart&#237;nez, N., Salas Orozco, M. F., Mart&#237;nez-Casta&#241;&#243;n, G. A., Torres M&#233;ndez, F., Ruiz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+bacterial+resistance+to+metal+and+metal+oxide+nanoparticles.">Molecular mechanisms of bacterial resistance to metal and metal oxide nanoparticles.</a> Int J Mol Sci. 20 (11), 2808 (2019).</li><li>Kim, J., Choi, Y., Park, J., Choi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin-gallic+acid+microcomplexes+release+go/cu+nanomaterials+to+eradicate+antibiotic-resistant+microbes+and+their+biofilm.">Gelatin-gallic acid microcomplexes release go/cu nanomaterials to eradicate antibiotic-resistant microbes and their biofilm.</a> ACS Infect Dis. 9 (2), 296-307 (2023).</li><li>Jang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+antibiofilm+nanocomposites:+Ag/cu+bimetallic+nanoparticles+synthesized+on+the+surface+of+graphene+oxide+nanosheets.">Development of antibiofilm nanocomposites: Ag/cu bimetallic nanoparticles synthesized on the surface of graphene oxide nanosheets.</a> ACS Appl Mater Interfaces. 12 (32), 35826-35834 (2020).</li><li>Jang, J., Choi, Y., Tanaka, M., Choi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+silver/graphene+oxide+nanocomposites+for+antibacterial+and+antibiofilm+applications.">Development of silver/graphene oxide nanocomposites for antibacterial and antibiofilm applications.</a> J Ind Eng Chem. 83, 46-52 (2020).</li><li>Bankier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+antibacterial+effects+of+metallic+nanoparticle+combinations.">Synergistic antibacterial effects of metallic nanoparticle combinations.</a> Sci Rep. 9 (1), 16074 (2019).</li><li>Mendes, C. 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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.

Herein, we report a low-cost and simple method for preparing GO nanosheets deposited with Cu nanoparticles, which would be a potentially efficient method for eradicating antibiotic-resistant bacteria. The critical step in synthesizing GO/Cu nanocomposites is thoroughly dispersing GO and CuCl2 in the solution and maintaining an elevated temperature while mixing them. Further, the redox step must be conducted quickly because the reducing agent causes a reduction reaction as soon as it comes in contact with water...

Bacterial infections have a significant impact on public health. Pathogenic bacteria, in particular, can evade the body&#39;s protective mechanisms and cause diseases1. Antibiotics are widely used to treat bacterial infections. However, the inappropriate use of antibiotics has precipitated the emergence of antibiotic-resistant bacteria. Currently, nosocomial infections attributable to antibiotic-resistant bacteria have caused notable complications in healthcare facilities2. Unfortunately, bacteria have resistance mechanisms for all current antibiotics3. Therefore, developing new antibiotics is essential,&#160;though&#160;there is also a high probability that resistance mechanisms will emerge.
Metal nanoparticles have emerged as promising agents for combating antibiotic-resistant bacteria because of their effective antibacterial properties4,5,6. It is difficult for bacteria to develop resistance mechanisms against metal nanoparticles because they do not bind to any specific bacterial receptors7. In particular, silver, copper, and zinc oxide nanoparticles are the focus of extensive research because of their remarkable antibacterial efficacy8,9,10,11,12,13. The toxicity of metal nanoparticles toward bacteria is attributed to three primary mechanisms: 1) the release of metal ions, 2) the generation of reactive oxygen species (ROS), such as &#8226;OH and &#8226;O2&#8722;, and 3) physical interaction and attachment4,14.
In this study, graphene oxide/copper (GO/Cu) nanocomposites were developed as an antimicrobial agent. Copper (Cu) efficiently kills pathogens that encounter its surface and interferes with bacterial gene replication. However, it is an essential microelement in the human body, and it is less toxic to mammalian cells because it has homeostatic mechanisms that regulate the concentrations of Cu inside the cells4. When Cu nanoparticles are oxidized, they generate Cu ions, which have a relatively high affinity for negatively charged bacteria15,16. Cu ions bind to the cellular structures (e.g., proteins, membranes, and DNA), disrupting cell functions17. By synthesizing Cu nanoparticles on the surface of graphene oxide (GO), the release rate of metal ions can be controlled18,19,20. GO also shows an antibacterial effect by preventing bacterial adhesion with a rough surface through oxidative stress or by forming ROS21,22. Moreover, GO/Cu nanocomposites release more ROS than GO through the chemical action of Cu2+, which damages bacterial proteins and DNA, leading to the death of bacteria23,24.
This paper outlines the protocol for synthesizing GO/Cu nanocomposites and introduces an antimicrobial testing approach against clinical bacteria MRSA and P. aeruginosa, the most common multidrug-resistant bacteria strains that cause nosocomial infections25. This protocol aims to introduce a facile chemical reduction method for synthesizing GO/Cu nanocomposites that can impede the growth of antibiotic-resistant bacteria. We use copper(II) chloride (CuCl2) and sodium borohydride (NaBH4) as a precursor and reducing agent, respectively. The protocol also describes the specifics of applying these nanocomposites to bacteria using the microdilution broth method. To exclude errors due to the absorption interference of metal nanoparticles and make accurate evaluations, the antibacterial ability is evaluated using the colony counting method.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical MDR bacterial strains&#160;</td>
			<td>Chung-Ang University Hospital (Seoul, South Korea)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) chloride dihydrate</td>
			<td>Duksan</td>
			<td>10125-13-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Field Emission Scanning Electron Microscope (FE-SEM)</td>
			<td>Carl Zeiss</td>
			<td>SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphene oxide</td>
			<td>Sigma</td>
			<td>796034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Borohydride</td>
			<td>Sigma</td>
			<td>71320</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>JEOL</td>
			<td>JEM-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>BD difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>BD difco</td>
			<td>211825</td>
			<td></td>
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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.

Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) analyses were performed to confirm the formation of GO/Cu nanocomposites. Figure 1A,B show that heterogeneous Cu nanoparticles were grown on the GO sheets. As shown in Figure 1C, the EDS mapping confirmed that the particles on the GO sheet were Cu nanoparticles.
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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.

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 ...

fabrication-of-antibacterial-graphene-oxidecopper-nanocomposites

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Research

JoVE Journal

Bioengineering

676 Views.  Chung-Ang University. 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.

Video: Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...