Name: synthesis of multi-walled carbon nanotubes modified with silver nanoparticles and evaluation of their antibacterial activities and cytotoxic properties
Uploaded: 2018-05-10T12:30:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties at JoV

This study was supported by Chung-Ang University Research Grants (2016) and by the Nano-Material Technology Development Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (No. 2017M3A7B8061942).

1. MWCNT Oxidation
<ol>
	<li>Measure 30 - 50 mg of MWCNT into a 50-mL vial. Slowly add 8 mL of a H2SO4:HNO3 solution (90% initial concentration, 3:1 vol/vol) by pipette with 1 mL pipette tips. 
	CAUTION: This preparation must be conducted in a chemical fume hood.
	<ol>
		<li>Allow 30 min for the exothermic reaction to complete.</li>
	</ol>
	</li>
	<li>Sonicate the solution at 60 - 80 &#176;C and 160 W for 1 h until MWCNT settles at the bottom of the vial. 
	CAUTION: The water level in the sonicator should be lower than the top of vial so that water will not be introduced into the vial.</li>
	<li>Transfer the solution to a centrifuge tube.
	<ol>
		<li>With a micropipette, add 30 mL of distilled water dropwise to the MWCNT-COOH solution. 
		CAUTION: A strong exothermic reaction occurs during addition of distilled water. Add distilled water slowly and allow time for cooling.</li>
	</ol>
	</li>
	<li>Centrifuge the MWCNT-COOH solution at 12,000 x g and room temperature for 15 min. 
	CAUTION: When operating a centrifuge, ensure the centrifuge remains balanced by placing a tube with the same weight opposite the sample tube.</li>
	<li>Remove the supernatant with a pipette and 1 mL pipette tip. Add 5 mL of ethanol with a pipette and 1 mL pipette tip. Rinse the walls of the vial with additional pipetted ethanol. Centrifuge the solution at 12,000 x g and room temperature for 15 min.
	<ol>
		<li>Determine pH of supernatant with pH paper. Repeat washing and centrifuging until the supernatant pH is neutral.</li>
	</ol>
	</li>
	<li>Remove all supernatant. Add 1 mL of distilled water to precipitate and disperse MWCNT-COOH evenly in solution. Freeze-dry solution at -60 &#176;C under vacuum until dry.</li>
</ol>
2. Silver Nanoparticle Deposition on MWCNTs
<ol>
	<li>Place 10 mg of MWCNT-COOH in a 50-mL conical tube and dilute with 30 mL of distilled water. Sonicate solution at 60 &#176;C and 160 W for 1 h.</li>
	<li>Place 6 mL of 0.1 N AgNO3 in a 50-mL conical tube and dilute with 18 mL of distilled water. Sonicate solution at 60 &#176;C and 160 W for 1 h. 
	CAUTION: Add AgNO3 in proportion to the total mass of MWCNTs such that the total mass of Ag does not exceed 20%.</li>
	<li>Add the AgNO3 solution dropwise to the MWCNTs solution with a pipette and 1 mL pipette tip while sonicating the mixture at 60 &#176;C and 160 W. Continue sonicating for 1 h after adding the AgNO3.</li>
	<li>Centrifuge the solution at 12,000 x g and room temperature for 15 min and remove the supernatant. Centrifuge the solution and remove the supernatant two additional times.</li>
	<li>Add 1 mL of distilled water to the Ag-MWCNTs mixture and disperse the precipitate evenly in the solution. Add 5 mL of ethanol and allow the reaction to proceed at room temperature for 1 h.</li>
	<li>Centrifuge the solution at 12,000 x g and room temperature for 15 min and remove the supernatant.</li>
	<li>Determine pH of supernatant with pH paper. Repeat washing and centrifuging until supernatant pH is neutral.</li>
	<li>Add 1 mL of distilled water to Ag-MWCNTs and disperse evenly in solution. Freeze-dry the solution at -70 &#176;C under vacuum for 24 h.</li>
</ol>
3. Zone of Inhibition Test
<ol>
	<li>Mix 18.12 g of R2A agar and 1 L of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
	<li>Allow gel to cool at room temperature. Place 10 mL of gel in Petri dishes prior to hardening to prepare the solid medium.</li>
	<li>Place 100 &#181;L of bacteria on the solid medium. Spread the bacteria on the solid medium using a spreader. 
	CAUTION: This procedure must be conducted in a chemical fume hood lit with an alcohol lamp.</li>
	<li>Incubate the dishes at 30 &#176;C for 48 h.</li>
	<li>Mix 3.12 g of R2A broth with 1 L of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
	<li>Transfer the grown colonies to a liquid medium using a loop and needle and incubate in an incubator at 180 rpm and 30 &#176;C.</li>
	<li>Record the OD (Optical Density) value by UV spectrophotometry at 600 nm on an hourly basis to measure bacterial growth. 
	NOTE: R2A agar was used in this test, rather than the standard Mueller-Hinton agar, because R2A is the preferred agar for growth of Methylobacterium spp.</li>
	<li>Place 10 mL of prepared R2A agar in a Petri dish to prepare a bottom agar.
	<ol>
		<li>Mix 3.12 g of R2A broth and 8 g of agar with 1 L of distilled water. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min. Place 10 mL of this gel on the bottom agar.</li>
	</ol>
	</li>
	<li>Load 50 &#181;L of Ag-MWCNTs solution on sterile paper disc with a micropipette.
	<ol>
		<li>Dry and sterilize Ag-MWCNTs sample in a vacuum chamber under UV light for 30 min.</li>
	</ol>
	</li>
	<li>Place Ag-MWCNTs sample on agar.</li>
	<li>Incubate the agar plate at 30 &#176;C for 48 h.</li>
	<li>Measure zone of inhibition12. 
	NOTE: Instructions for software used are included in the references.</li>
</ol>
4. Antibacterial Test
<ol>
	<li>Repeat steps 3.1 to 3.7 to prepare bacterial culture.</li>
	<li>At maximum OD, inoculate 100 &#181;L of bacteria into 3 mL of fresh liquid medium with a pipette and 100-&#181;L micro pipette tip.</li>
	<li>Prepare five 50 &#181;L samples of the control solution in 15-mL conical tubes. Add the following to each tube: 1) methanol; 2) 1000 &#181;g/mL MWCNT-COOH; 3) 50 &#181;g/mL Ag-MWCNTs; 4) 40 &#181;g/mL Ag-MWMWCNTs; 5) 30 &#181;g/mL Ag-MWCNTs.</li>
	<li>Incubate at 180 rpm and 30 &#176;C until the control is maximally active as determined by UV spectrophotometry as described above.</li>
	<li>Measure the OD value of each sample and calculate the MIC concentration. The OD value is directly related to the number of bacterial cells in the sample broth.</li>
	<li>Spread the cultured bacteria on a solid medium with a spreader.</li>
	<li>Incubate the dish at 30 &#176;C for 48 h.</li>
	<li>Count bacterial colonies and the measure CFU values to determine antibacterial activity12. 
	NOTE: Instructions for software used are included in the references.</li>
</ol>
5. Viability Assay
<ol>
	<li>After removing the culture cells from incubator, suction the medium and wash three times with a dropwise wash of 5 mL of PBS.</li>
	<li>Add 1 mL of trypsin-EDTA in PBS to the washed cell and suction after 1 min.
	<ol>
		<li>Tap the plate to remove stuck cells.</li>
	</ol>
	</li>
	<li>Add 5 mL of DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium) and then remove the cells. Centrifuge the samples at 200 x g for 3 min and remove the supernatant.
	<ol>
		<li>Add 1 mL of DPBS and mix.</li>
		<li>Count cells in 10 &#181;L of the solution with an automated cell-counting machine. 
		NOTE: Instructions for cell-counting machine used are included in the references13.</li>
	</ol>
	</li>
	<li>Place 1 &#215; 105 of AML12 cells per well in a six-well plate containing DMEM solution. The medium contains 10% (v/v) fetal bovine serum and 1% sterile antibiotic.</li>
	<li>Incubate plate for 8&#160;- 12 h at 37 &#176;C in a 5% CO2/95% air environment.</li>
	<li>Suction the supernatant from wells with an aspirator.
	<ol>
		<li>Add each sample comprised of control, NMS-DMSO (dimethyl sulfoxide) and 30-40 &#181;g/mL Ag-MWCNT to 1 mL of DMEM.</li>
		<li>Incubate at 37 &#176;C in a 5% CO2/95% air environment for 8 h.</li>
	</ol>
	</li>
	<li>Remove supernatant and wash samples with 5 mL of PBS.</li>
	<li>Add 1 mL of prepared live/dead kit (20 &#181;L of 2 mM EthD-1 with 5 &#181;L of 4 mM calcein AM in DMSO in 10 mL of PBS) dropwise to the cell.</li>
	<li>Incubate at room temperature for 30 - 45 min or 37 &#176;C for 10 min. Measure fluorescence with standard fluorescence microscopy at 100X or 300X.</li>
</ol>
6. Trypan Blue Assay
<ol>
	<li>Prepare samples according to steps 5.1 through 5.6.2 above.</li>
	<li>Remove supernatant.</li>
	<li>Wash plate with 1 mL of 1x DPBS and decant.</li>
	<li>Add 1 mL of trypsin to plate and incubate for 3 min to detach the cells from culture. Use cell culture medium to wash plate and collect cells. Place collected cells in a 15-mL tube.</li>
	<li>Centrifuge tube at 200 x g and 4 &#176;C for 3 min.</li>
	<li>Discard supernatant. Add 1 mL of fresh medium to cell pellet and re-disperse the cells.</li>
	<li>Add 10 &#181;L of trypan blue with 10 &#181;L of dispersed cells and count the cells with an automated cell-counting machine. 
	NOTE: Instructions for cell-counting machine used are included in the references.</li>
</ol>

<ol>
	<li>L&#246;ndahl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+Biological+Properties+of+Bioaerosols.">Physical and Biological Properties of Bioaerosols.</a> Bioaerosol Detection Technologies. , 33-48 (2014).</li><li>Jennings, S., Moran, A., Carroll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioaerosols+and+biofilms.">Bioaerosols and biofilms.</a> Biofilms in medicine, industry and environmental biotechnology. , 160-178 (2003).</li><li>Flemming, H. -. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nature Reviews Microbiology. 8 (9), 623-633 (2010).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrobial agents and chemotherapy. 45 (4), 999-1007 (2001).</li><li>Doronina, N. 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=Methylobacterium+suomiense+sp.+nov.+and+Methylobacterium+lusitanum+sp.+nov.,+aerobic,+pink-pigmented,+facultatively+methylotrophic+bacteria.">Methylobacterium suomiense sp. nov. and Methylobacterium lusitanum sp. nov., aerobic, pink-pigmented, facultatively methylotrophic bacteria.</a> International Journal of Systematic and Evolutionary Microbiology. 52 (3), 773-776 (2002).</li><li>Seo, Y., 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=Antibacterial+activity+and+cytotoxicity+of+multiwalled+carbon+nanotubes+decorated+with+silver+nanoparticles.">Antibacterial activity and cytotoxicity of multiwalled carbon nanotubes decorated with silver nanoparticles.</a> International Journal of Nanomedicine. 9, 4621-4629 (2014).</li><li>Chen, X., Schluesener, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosilver:+a+nanoproduct+in+medical+application.">Nanosilver: a nanoproduct in medical application.</a> Toxicologyletters. 176 (1), 1-12 (2008).</li><li>Singh, M., 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=Nanotechnology+in+medicine+and+antibacterial+effect+of+silver+nanoparticles.">Nanotechnology in medicine and antibacterial effect of silver nanoparticles.</a> Digest Journal of Nanomaterials and Biostructures. 3 (3), 115-122 (2008).</li><li>Morones, J. R., 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=The+bactericidal+effect+of+silver+nanoparticles.">The bactericidal effect of silver nanoparticles.</a> Nanotechnology. 16 (10), 2346 (2005).</li><li>Martinez-Castanon, G., 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=Synthesis+and+antibacterial+activity+of+silver+nanoparticles+with+different+sizes.">Synthesis and antibacterial activity of silver nanoparticles with different sizes.</a> Journal of Nanoparticle Research. 10 (8), 1343-1348 (2008).</li><li>Lok, C. -. N., 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=Silver+nanoparticles:+partial+oxidation+and+antibacterial+activities.">Silver nanoparticles: partial oxidation and antibacterial activities.</a> JBIC Journal of Biological Inorganic Chemistry. 12 (4), 527-534 (2007).</li></ol>

The authors have nothing to disclose.

Here, we report a simple method for the preparation of MWCNTs with deposited Ag nanoparticles. This silver-containing nanomaterial demonstrates substantial antibacterial activity and minimal potential for uncontrolled absorption of silver nanoparticles in the body. We demonstrate that 30 &#181;g/mL of synthesized Ag-MWCNTs is an effective level of antibacterial activity against Methylobacterium spp. with negligible cytotoxicity to mammalian liver cells. Though additional improvements and biosafety assessments for Ag-MWCNTs are required before expansion in the commercial sector, using ethanol as a reducing agent could help produce environmentally-friendly and inexpensive Ag-MWCNTs.
The following points are demonstrated. It is possible to destroy various types of bacteria with the developed nanomaterials. It is possible to engineer multiple sites on a nanotube surface upon which silver nanoparticles can be deposited. Antibacterial properties can also be tailored by controlling the size and number of silver nanoparticles that are deposited on the surface. The developed nanomaterials are toxic to bacterial cells but are nontoxic to human and animal cells in appropriate concentrations.
The following mechanism is proposed for the antibacterial activity. The Ag-MWCNT nanostructures directly contact the bacterial cell surface, damage the cell wall, and cause secondary oxidation of reactive oxygen species; these processes result in oxidative stress. Ag-MWCNT nanostructures have been confirmed to release silver ions that inhibit the quorum sensing of Methylobacterium spp., thereby inhibiting the expression of the genes that govern the formation of biofilms.
The limitations of the current protocol would include the undetermined maximum amount of silver nanoparticles and Ag-MWCNTs allowable in human trials. In this protocol, the amount of silver nanoparticles included in the 30 &#181;g/mL sample of Ag-MWCNTs was estimated as 0.4 &#181;g/mL, on average. This concentration is regarded as biocompatible with the mammalian cells as reported in previous studies6,7,8,9. While the mechanism for cytotoxicity at the concentration of 40 &#181;g/mL of Ag-MWCNTs is undetermined, it is proposed that the non-attachment of blood cells to the bottom of culture plates may increase the likelihood of Ag-MWCNT contact during culture. In the future, detailed toxicity studies of silver nanoparticles with a variety of cell types under different culture conditions can be performed. These studies may provide additional information on the mechanism of Ag-MWCNT interaction with cells.

The ultimate goal of this study is to make environmentally friendly antibacterial nanomaterials that can inhibit the growth of bacteria that form biofilms. These antibacterial nanomaterials have the potential to overcome the toxicity and antibiotic resistance problems of commonly used chemicals or antibiotic chemical compounds. A biofilm is a hydrated extracellular polymeric substance (EPS) that is composed of polysaccharides, proteins, nucleic acids, and lipids1,2. Biofilms prevent the intrusion of foreign substances and help bacteria grow vigorously3,4. Biofilms cause odor and chronic infectious diseases5,6. Methylobacterium spp., for example, grows by adhering to places where water is always present or where it is difficult to ensure bacterial eradication on a continual basis, such as air conditioner heat exchangers, shower rooms, and medical devices. These types of biofilms cause odor and chronic infectious diseases5,6.
Typically, chemicals or antibiotic chemical compounds are used to inhibit the growth of bacteria that form biofilms. The emergence of antibiotic resistant bacteria and concerns about in vivo safety of chemicals are driving the need to develop new materials to prevent the formation of biofilms and to inhibit the growth of bacteria.
In this study, antimicrobial nanomaterials are synthesized that are free from antibiotic resistance and toxicity. Silver is a well-known antimicrobial substance, and recent developments in nanoscience and nanotechnology have led to active research into the antimicrobial effects of metal nanoparticles7,8. Recent studies have reported that the small size and high surface-to-volume ratio of the nanoparticles result in increased antibacterial activity9,10,11.
The nanomaterials presented herein combine silver nanoparticles with increased antimicrobial properties and carbon nanotubes with a high aspect ratio, thereby increasing the surface area per unit volume. The fabricated silver nanoparticle-carbon nanotube composite exhibits substantial antimicrobial properties and minimal toxicity to human and animal cells. The synthetic processes in previous studies use hazardous reducing agents such as NaBH4, formamide, dimethylformamide, and hydrazine. The process is complicated, dangerous, and time-consuming. The synthetic process reported here uses ethanol as a significantly less hazardous reducing agent.
The inhibition zone and minimum bactericidal concentration (MBC) for the Ag-MWCNTs were measured; Live/Dead and Trypan Blue assays were used to measure toxicity and antibacterial properties. Minimum inhibitory concentration (MIC) and mitochondrial toxicity (MTT) assays were not performed due to the unusual color of the carbon nanotubes which would have interfered with the assays. Finally, the minimum concentration to prevent the growth of Methylobacterium spp. without affecting mammalian cells was determined.

<table><tbody><tr><td>0.1 N silver nitrate</td><td>SIGMA-ALDRICH</td><td>1090811000</td><td></td></tr><tr><td>Carbon nanotube, multi-walled</td><td>Tokyo Chemical Industry Co., LTD</td><td>308068-56-6</td><td></td></tr><tr><td>R2A agar</td><td>MBcell</td><td>MB-R1129</td><td></td></tr><tr><td>R2A broth</td><td>MBcell</td><td>MB-R2230</td><td></td></tr><tr><td>&lt;em&gt;Methylobacterium spp.&lt;/em&gt;</td><td>KCTC</td><td>12618</td><td>from Korea Collection for Type Cultures Daejeon Korea 12618, Daejon, Korea</td></tr><tr><td>LIVE/DEAD Cell imaging Kit</td><td>ThermoFisher SCIENTIFIC</td><td>R37601</td><td></td></tr><tr><td>AML12</td><td></td><td></td><td>from Chungnam University, Dajeon, Korea</td></tr><tr><td>human PBMC</td><td>ATCC</td><td>PCS-800-011</td><td></td></tr><tr><td>TEM</td><td>JEOL</td><td>JEM-2100F</td><td></td></tr><tr><td>XRD</td><td>Rigaku</td><td>D/MAX 2500</td><td>Cu K photon source (40&lt;em&gt;k&lt;/em&gt;V, 100mA)</td></tr><tr><td>JuLI Br</td><td>NanoEnTek</td><td>JULI-BRSC&amp;nbsp;</td><td></td></tr></tbody></table>

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.

Transmission Electron Microscopy (TEM) images confirm the formation of Ag-MWCNTs (Figure 1A and 1B). Their successful synthesis was confirmed by the change in surface charge. The size of the Ag particles deposited on the MWCNTs was calculated (Figure 1C). The average particle size was approximately 3.83 nm. The XRD pattern of the as-synthesized Ag-MWCNTs is shown in Figure 1D. The peak at 20 - 30&#176; corresponds to MWCNT; the remaining peaks correspond to Ag. Antimicrobial activity data is shown in Figure 2. Bacterial colony populations were confirmed by Methylobacterium control (Figure 2A); addition of methanol reduced the population 103 times (Figure 2B), and addition of MWCNT-COOH reduced the population 108 times (Figure 2C). Colonies could not be identified in the 50 &#181;g/mL Ag-MWCNT (Figure 2D) and 40 &#181;g/mL Ag-MWCNT (Figure 2E) samples. In the 20 &#181;g/mL Ag-MWCNT sample the population was reduced 105 times (Figure 2F). In the zone of inhibition test against Methylobacterium (Figure 3), the zone of inhibition was identified when 10 &#181;g/mL was added. A zone of inhibition was not observed at a concentration of 1 &#181;g/mL. In the cytotoxicity test, the Live/Dead assay (Figure 4) confirmed the absence of cytotoxicity in the negative control (Figure 4A) and the presence of cytotoxicity in the positive control (Figure 4B) when methanol was used. The addition of 40 &#181;g/mL Ag-MWCNTs (Figure 4C) revealed some cytotoxicity, however the addition of 30 &#181;g/mL Ag-MWCNTs (Figure 4D) did not reveal a significant amount of cytotoxicity. A trypan blue assay was performed (Figure5) for the control (Figure 5A), methanol (Figure 5B), 40 &#181;g/mL Ag-MWCNTs (Figure 5C) and 30 &#181;g/mL Ag-MWCNTs (Figure 5D) samples. The results confirmed that there was very little cytotoxicity at 30 &#181;g/mL Ag-MWCNTs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57384/57384fig1.jpg" /> 
Figure 1: Physicochemical characterization of Ag-MWCNTs. (A) and (B) TEM images of Ag-MWCNTs; (C) size distribution of Ag-MWCNTs as assessed via TEM (n = 100); (D) XRD pattern of Ag-MWCNTs. <a href="/files/ftp_upload/57384/57384fig1large.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/57384/57384fig2.jpg" /> 
Figure 2: Antibacterial evaluation of Ag-MWCNTs relative to Methylobacterium spp. (A) control, (B) methanol, (C) MWCNT-COOH, (D) 50 &#956;g/mL of Ag-MWCNTs, (E) 40 &#956;g/mL of Ag-MWCNTs, and (F) 30 &#956;g/mL of Ag-MWCNTs. <a href="/files/ftp_upload/57384/57384fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57384/57384fig3.jpg" /> 
Figure 3: Zone of inhibition test of Ag-MWCNTs.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57384/57384fig4.jpg" /> 
Figure 4: Cell viability assays for Ag-MWCNTs. Photomicrographs of AML12 that are fluorescently labeled for dead (red) and live (green) cells. (A) control, (B) methanol, (C) 40 &#956;g/mL of Ag-MWCNTs, and (D) 30 &#956;g/mL of Ag-MWCNTs. <a href="/files/ftp_upload/57384/57384fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57384/57384fig5.jpg" /> 
Figure 5: Trypan blue assays for 30 and 40&#956;g/mL Ag-MWCNTs. (A) control, (B) methanol, (C) 40 &#956;g/mL of Ag-MWCNTs, and (D) 30 &#956;g/mL of Ag-MWCNTs. (Red: dead cells; blue: live cells) <a href="/files/ftp_upload/57384/57384fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties at JoV

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

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 ...

synthesis-multi-walled-carbon-nanotubes-modified-with-silver

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K Views.  Korea Institute of Science and Technology. In deze studie werden antimicrobiële nanomaterialen gesynthetiseerd door zure oxidatie van multiwalled koolstof nanotubes en de daaropvolgende reductieve afzetting van zilveren nanodeeltjes. Antimicrobiële activiteit en cytotoxiciteit tests werden uitgevoerd met de als-bereid nanomaterialen.

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

Evaluating Plasmonic Transport in Current-carrying Silver Nanowires

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information support1,2,3. In this context, metal nanowires are especially desirable for realizing dense routing networks4. A prerequisite to operate such shared nanowire-based platform relies on our ability to electrically contact individual metal nanowires and efficiently excite surface plasmon polaritons5 in this information support. In this article, we describe a protocol to bring electrical terminals to chemically-synthesized silver nanowires6 randomly distributed on a glass substrate7. The positions of the nanowire ends with respect to predefined landmarks are precisely located using standard optical transmission microscopy before encapsulation in an electron-sensitive resist. Trenches representing the electrode layout are subsequently designed by electron-beam lithography. Metal electrodes are then fabricated by thermally evaporating a Cr/Au layer followed by a chemical lift-off. The contacted silver nanowires are finally transferred to a leakage radiation microscope for surface plasmon excitation and characterization8,9. Surface plasmons are launched in the nanowires by focusing a near infrared laser beam on a diffraction-limited spot overlapping one nanowire extremity5,9. For sufficiently large nanowires, the surface plasmon mode leaks into the glass substrate9,10. This leakage radiation is readily detected, imaged, and analyzed in the different conjugate planes in leakage radiation microscopy9,11. The electrical terminals do not affect the plasmon propagation. However, a current-induced morphological deterioration of the nanowire drastically degrades the flow of surface plasmons. The combination of surface plasmon leakage radiation microscopy with a simultaneous analysis of the nanowire electrical transport characteristics reveals the intrinsic limitations of such plasmonic circuitry.

Silver nanowires can simultaneously transport electrons and optical information in the form of surface plasmons. A procedure is described here to realize such a shared circuitry and the limitations at propagating both information carriers are evaluated.

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information ...

Engineering

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

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 ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Immunology and Infection

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

Biology

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 ...

 Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

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

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

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

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

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

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 ...

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

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