Este estudio fue apoyado por becas de investigación de la Universidad Chung-Ang (2016) y por el programa de desarrollo de la tecnología de Nano Material a través de la Fundación de investigación nacional de Korea(NRF) financiado por el Ministerio de ciencia y las TIC (Nº 2017M3A7B8061942).

1. MWCNT oxidación
<ol><li>Miden 30-50 mg de MWCNT en un frasco de 50 mL. Agregar lentamente 8 mL de H2hasta4: solución HNO3 (concentración inicial de 90%, 3:1 vol/vol) con una pipeta con puntas de pipeta de 1 mL. PRECAUCIÓN: Esta preparación debe realizarse en una campana de humos químicos.<ol>
<li>Deje 30 minutos para la reacción exotérmica completar.</li>
	</ol>
</li>
	<li>Someter a ultrasonidos la solución a 60-80 ° C y 160 W durante 1 hora hasta MWCNT se instala en la ...

     

<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>

Aquí, Divulgamos un método simple para la preparación de ello con nanopartículas de Ag depositadas. Este nanomaterial con plata demuestra actividad antibacteriana substancial y mínimo potencial de absorción incontrolada de nanopartículas de plata en el cuerpo. Demostramos que 30 μg/mL de Ag sintetizado-ello es un nivel eficaz de actividad antibacteriana contra Methylobacterium spp con la insignificante citotoxicidad a hepatocitos de mamífero. Aunque mejoras adicionales y evaluaciones de bioseguridad par...

El objetivo de este estudio es hacer medio ambiente nanomateriales antibacteriano que pueden inhibir el crecimiento de bacterias forma biofilms. Estos nanomateriales antibacterianos tienen el potencial para superar los problemas de resistencia de toxicidad y antibiótico de antibióticos compuestos químicos o de productos químicos comúnmente usados. Un biofilm es una sustancia polimérica extracelular hidratada (EPS) que se compone de polisacáridos, proteínas, ácidos nucleicos y lípidos1,2. Biopelículas protegen la entrada de sustancias extrañas y ayudan a las bacterias a crecer vigorosamente3,4. Biofilms provocan olor y enfermedades infecciosas crónicas5,6. Methylobacterium spp., por ejemplo, crece adhiriéndose a lugares donde el agua está siempre presente o donde es difícil garantizar la Erradicación bacteriana en una base continua, como intercambiadores de calor de aire acondicionado, duchas y sanitarios. Estos tipos de biofilms provocan olor y enfermedades infecciosas crónicas5,6.
Normalmente, se utilizan productos químicos o compuestos químicos antibióticos para inhibir el crecimiento de bacterias que forman biopelículas. La aparición de bacterias resistentes a los antibióticos y las preocupaciones sobre seguridad en vivo de los productos químicos están impulsando la necesidad de desarrollar nuevos materiales para prevenir la formación de biofilms y para inhibir el crecimiento de bacterias.
En este estudio, se sintetizan nanomateriales antimicrobianos que están libres de toxicidad y resistencia a los antibióticos. La plata es una sustancia antimicrobiana conocida, y los acontecimientos recientes en la Nanociencia y la nanotecnología han llevado a investigación sobre los efectos antimicrobianos de nanopartículas de metal7,8. Estudios recientes han informado que el pequeño tamaño y alta relación superficie a volumen de las nanopartículas como resultado aumento de la actividad antibacteriana9,10,11.
Los nanomateriales presentados combinan nanopartículas de plata con nanotubos de carbono con un alto cociente de aspecto, lo que aumenta el área superficial por unidad de volumen y aumento de propiedades antimicrobianas. El compuesto de plata fabricado en carbono nanopartículas nanotubos presenta importantes propiedades antimicrobianas y mínima toxicidad para las células humanas y animales. Los procesos sintéticos en estudios anteriores utilizan peligrosos agentes reductores como el NaBH4, formamida, dimetilformamida e hidracina. El proceso es complicado, peligroso y desperdiciador de tiempo. El proceso sintético registrado aquí utiliza etanol como agente reductor significativamente menos peligroso.
Se midieron la zona de inhibición y la concentración bactericida mínima (CBM) para el Ag-ello; Vivo/muerto y ensayos de azul de tripano fueron utilizados para medir la toxicidad y propiedades antibacterianas. Concentración mínima inhibitoria (CMI) y ensayos de toxicidad mitocondrial (MTT), no se realizaron debido al color inusual de los nanotubos de carbono que habría interferido con los ensayos. Finalmente, se determinó la concentración mínima para prevenir el crecimiento de Methylobacterium spp sin afectar a células de mamíferos.

<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>Methylobacterium spp.</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 (40kV, 100mA)</td>
		</tr>
		<tr>
			<td>JuLI Br</td>
			<td>NanoEnTek</td>
			<td>JULI-BRSC&#160;</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

En este estudio, los nanotubos de carbono multipared (ello) fueron tratados con una solución acuosa de ácido sulfúrico para formar un grupo funcional a base de oxígeno. Ello plata fueron preparado por la deposición reductiva de plata de una solución acuosa de AgNO3 en ello oxidado. Dado el color único de los CNTs, no era posible aplicar a la concentración mínima inhibitoria o ensayos de toxicidad mitocondrial para evaluar las características de toxicidad y antibacteriana, ya que pueden interferir con los ensayos. La zona de inhibición y la concentración bactericida mínima para la Ag-ello se midieron y análisis de azul de tripano y vivos/muertos se utilizaron para medir las características de toxicidad y antibacteriano sin interferir en el color de los CNTs.

Imágenes de microscopía electrónica (TEM) de transmisión confirman la formación de Ag-ello (figura 1A y 1B). Su síntesis exitosa fue confirmada por el cambio en la carga de superficie. Se calculó el tamaño de las partículas de Ag en el ello (figura 1). El tamaño de partícula promedio fue aproximadamente 3.83 nm. El patrón de DRX de la Ag-ello sintetizado como se muestra en la fig...

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

En este estudio, nanomateriales antimicrobianos fueron sintetizados por oxidación ácida de nanotubos de carbono pirólisis y posterior deposición reductiva de nanopartículas de plata. Se realizaron pruebas de actividad y la citotoxicidad antimicrobianas con los nanomateriales como preparado.

Síntesis de nanotubos de carbono multipared modificados con nanopartículas de plata y la evaluación de sus actividades antibacterianas y propiedades citotóxicas

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

The overall goal of this procedure is to produce antibacterial nanocomplexes consisting of carbon nanotubes with silver nanoparticles synthesized on the nanotube's surfaces. This method can be used in the development of novel bacterial nanomaterials that have low toxicity to humans while having strong septic properties to diverse kinds of bacteria and biofilms. The protocol describes the evaluation of nanocomplexes in eradicating bacteria while maintaining their bio-comparability with humans.To begin the procedure, place 30 to 50 milligrams of MWCNTs in a 50 milliliter vial. Using a micropipette fitted with one milliliter pipette tips, slowly add eight milliliters of a three-to-one by volume solution of sulfuric acid and nitric acid to the MWCNTs. Wait 30 minutes for the exothermic reaction to finish, then sonicate the solution at 60 to 80 degrees celsius and 160 watts until the MWCNTs settle at the bottom of the vial, which usually takes about one hour.Transfer the mixture to a centrifuge tube. Use a micropipette to carefully add 30 milliliters of distilled water drop-wise to the carboxylic acid functionalized MWCNTs, pausing to allow the mixture to cool as needed. Then, centrifuge the mixture at 12, 000 G for 15 minutes.Remove the supernatant with a pipette equipped with a one milliliter pipette tip. Add five milliliters of ethanol to the solid and rinse the walls of the vial with additional ethanol. Centrifuge the mixture again with the same settings and check the pH of the supernatant with pH paper.Continue washing the solids by centrifugation in ethanol until the supernatant pH is neutral, then remove the supernatant and add one milliliter of distilled water to the precipitate. Disperse the carboxylic acid functionalized MWCNTs in the water and freeze dry the dispersion at minus 60 degrees celsius under vacuum. Place 10 milligrams of freeze-dried carboxylic acid functionalized MWCNTs in a 50 milliliter conical centrifuge tube.Add 30 milliliters of distilled water to the tube. Combine six milliliters of 0.1 normal silver nitrate and 18 milliliters of distilled water in another 50 milliliter conical tube. Sonicate both mixtures at 60 degrees celsius and 160 watts for one hour.Then, while still sonicating the MWCNT dispersion at 60 degrees celsius and 160 watts, add the silver nitrate solution drop-wise to the MWCNT dispersion using a pipette equipped with a one milliliter pipette tip. Once addition is complete, continue sonicating the mixture for one hour, then centrifuge the mixture for 15 minutes at 12, 000 G at room temperature and remove the supernatant. Re-disperse the silver MWCNTs in 30 milliliters of distilled water by sonication at 24 degrees celsius for 30 minutes.Repeat the centrifugation in 30 milliliters of distilled water and removal of the supernatant twice. Next, disperse the washed silver MWCNTs in one milliliter of distilled water by sonication for five minutes. Add five milliliters of ethanol to the dispersion and stir the mixture at room temperature for one hour.Then centrifuge the mixture for 15 minutes at 12, 000 G at room temperature and check the pH of the supernatant. Remove the supernatant and re-disperse the mixture in five milliliters of ethanol by vortexing. Continue washing the precipitate in ethanol by centrifugation until the supernatant pH is neutral.Finally, disperse the washed silver MWCNTs in one milliliter of distilled water. Freeze dry the dispersion at minus 70 degrees celsius under vacuum for 24 hours and store the dispersion in a sealed container. To begin preparing the test, mix and autoclave 18.12 grams of R2A agar and one liter of distilled water.Allow the gel to cool at room temperature. When the gel is ready to pour, place 10 milliliters of gel in 90 millimeter Petri dishes and allow the agar to harden. In a fume hood, use a loop and needle sterilized in alcohol flame to streak 100 microliters of a methylobacterium species mixture on the surface of the culture medium.Incubate the dishes at 30 degrees celsius for 48 hours, then mix 3.12 grams of R2A broth with one liter of distilled water and autoclave the medium at 121 degrees celsius for 15 minutes. Use a sterilized loop and needle to transfer the colonies to the R2A growth medium. Incubate the sample in a shaking incubator at 30 degrees celsius and 180 RPM.Measure the optical density at 600 nanometers hourly until the optical density is greater than 0.7. Next, prepare another Petri dish containing 10 milliliters of R2A agar. Then mix 3.12 grams of R2A broth and eight grams of agar with one liter of distilled water.Autoclave the mixture at 121 degrees celsius for 15 minutes. Mix 100 microliters of the methylobacterium culture into the agar mixture. Pour 10 milliliters of the methylobacterium-containing gel over the bottom layer of R2A agar in the Petri dish, then prepare a dispersion of the desired concentration of freeze-dried silver MWCNTs in distilled water.Use a micropipette to load 50 microliters of this dispersion onto a six-by-one millimeter sterile paper disk. Apply distilled water to another sterile paper disk as a control. Dry and sterilize both disks in a vacuum chamber under UV light for 30 minutes.Then place the silver MWCNT sample and the control on the agar and incubate the agar plate at 30 degrees celsius for 48 hours. Measure the zone of inhibition around the silver MWCNT sample when finished. Prior to the test, autoclave triple-distilled water at 121 degrees celsius for 15 minutes.Prepare a 1000 microgram per milliliter dispersion of carboxylic acid functionalized MWCNTs and 50 microgram per milliliter, 40 microgram per milliliter, and 30 microgram per milliliter dispersions of silver MWCNTs in this sterile water. To begin the antibacterial testing procedure, prepare a methylobacterium culture as previously described and monitor its optical density at 600 nanometers. When the optical density is at maximum, innoculate 100 microliters of the methylobacterium culture into three milliliters of fresh R2A culture medium.Place 500 microliters of this solution in each of the five 15 milliliter conical tubes. Add 100 microliters of methanol to one of the tubes to prepare the methanol reference sample. Prepare a sample for each of the four dispersions in the same way.Incubate the samples and the remaining methylobacterium control culture at 180 RPM and 30 degrees celsius until the optical density of the control indicates maximal activity. Use a sterilized spreader to spread each sample on solid R2A agar gel in Petri dishes. Incubate the dishes at 30 degrees celsius for 48 hours.Count the resultant bacterial colonies and determine the minimum bacteriocidal concentration. Silver MWCNTs were successfully synthesized using the described procedure. The average deposited silver particle size was approximately 3.83 nanometers.Methylobacterium growth was not observed when cultured with 50 microgram per milliliter or 40 microgram per milliliter dispersions of silver MWCNTs. The bacterial population was reduced 100, 000 times relative to the control sample when cultured with a 30 microgram per milliliter dispersion of silver MWCNTs. A zone of inhibition was observed around a sample prepared from a 10 microgram per milliliter dispersion of silver MWCNTs.No zone of inhibition was observed for a sample prepared from a one microgram per milliliter dispersion. Some cytotoxicity was observed when a 40 microgram per milliliter dispersion of silver MWCNTs was added. No significant cytotoxicity was observed when a 30 microgram per milliliter dispersion was added.A trypan blue assay confirmed that there was minimal cytotoxicity when a 30 microgram per milliliter dispersion of silver MWCNTs was added. This protocol would help researchers in preparing nanocomplexes that have metal nanoparticles that act directly on carbon nanomaterials. Following this protocol, researchers would also be able to evaluate the antibacterial properties of the prepared nanocomplexes.