Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties
May 10th, 2018
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.
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.
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Overview
This study focuses on the synthesis of antibacterial nanocomplexes using multiwalled carbon nanotubes (MWCNTs) and silver nanoparticles. The aim is to develop materials that exhibit strong antibacterial properties while maintaining low toxicity to humans.
Key Study Components
Area of Science
- Nanomaterials
- Antimicrobial agents
- Biocompatibility
Background
- Antimicrobial nanomaterials are essential in combating bacterial infections.
- Carbon nanotubes have unique properties that enhance the effectiveness of silver nanoparticles.
- Understanding the balance between antimicrobial activity and cytotoxicity is crucial for safe applications.
- This study explores a method to synthesize these nanocomplexes effectively.
Purpose of Study
- To synthesize antibacterial nanocomplexes using MWCNTs and silver nanoparticles.
- To evaluate the antimicrobial efficacy of the synthesized materials.
- To assess the cytotoxicity of these nanocomplexes on human cells.
Methods Used
- Acidic oxidation of MWCNTs using a sulfuric and nitric acid solution.
- Reductive deposition of silver nanoparticles onto the oxidized MWCNTs.
- Sonication of the mixture to ensure proper dispersion and reaction.
- Testing for antimicrobial activity and cytotoxicity of the final product.
Main Results
- The synthesized nanocomplexes demonstrated significant antibacterial activity.
- Low levels of cytotoxicity were observed in human cell tests.
- The method proved effective for producing stable and functional nanomaterials.
- Results indicate potential for further development in medical applications.
Conclusions
- The study successfully synthesized antibacterial nanocomplexes with MWCNTs and silver nanoparticles.
- These materials show promise for use in medical applications due to their low toxicity and high efficacy.
- Further research is needed to explore their full potential in clinical settings.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables the synthesis of silver nanoparticle-functionalized multi-walled carbon nanotubes (Ag-MWCNTs) for antibacterial applications, addressing the need for antimicrobial materials with reduced cytotoxicity in early-stage therapeutic development. The method supports target validation in antimicrobial discovery by providing a tunable nanocomplex platform that demonstrates dose-dependent bacterial inhibition while maintaining biocompatibility thresholds. Such nanocomplexes offer mechanistic de-risking value in preclinical screening pipelines by combining nanomaterial delivery with metallic antimicrobial activity, facilitating hypothesis testing around pathogen-specific efficacy and host tolerance.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of silver-based antimicrobial mechanisms through controlled nanoparticle deposition on carbon nanotube scaffolds.
- Operational Value: Provides a reproducible synthesis route for generating standardized Ag-MWCNT dispersions at defined concentrations (e.g., 10–50 µg/mL) for consistent bioactivity screening.
- Predictive Value: Supports assessment of structure-activity relationships by correlating silver loading (approx. 3.83 nm particle size) with bacteriostatic and bactericidal outcomes against model organisms like Methylobacterium.
Screening & Assay Development
- Assay Readiness: Utilizes zone of inhibition and minimum bactericidal concentration (MBC) measurements to quantify antibacterial potency without spectral interference from nanomaterial color.
- Quantitative Outputs: Employs Live/Dead and Trypan Blue assays to generate cytotoxicity and viability data compatible with high-throughput screening formats.
- Scalability Consideration: Includes repeated centrifugation and washing steps to achieve pH-neutral supernatants, ensuring batch-to-batch consistency for assay reproducibility.
Translational & Preclinical Research
- Translational Continuity: Demonstrates a clear progression from synthesis to functional evaluation, supporting go/no-go decisions based on observed MBC at 30–40 µg/mL and absence of significant cytotoxicity at 30 µg/mL.
- Risk-Adjusted Advancement: Enables identification of a therapeutic window where antibacterial efficacy (e.g., 100,000-fold reduction at 30 µg/mL) is separable from cytotoxic effects observed at higher concentrations.
- Mechanistic De-risking: Facilitates evaluation of nanocomplex behavior in biologically relevant conditions, including sterility maintenance via autoclaving and UV vacuum drying, critical for preclinical translation.
Pipeline & Workflow Integration
The method fits within the antimicrobial discovery continuum, linking nanomaterial synthesis to functional validation in early lead identification, particularly for topical or surface-directed antibacterial strategies where localized delivery minimizes systemic exposure.
- Discovery Biology: Supports hypothesis testing around nanoparticle-mediated antimicrobial action by enabling precise dosing and controls (e.g., methanol reference, blank disks) in agar-based assays.
- Screening: Delivers standardized, dispersible nanocomplexes suitable for zone of inhibition and MBC assays, key outputs for comparing lead candidates.
- Analytics: Generates quantitative endpoints including optical density monitoring, colony counting, and viability staining, enabling data-driven prioritization.
- Translational Research: Connects synthesis to preclinical relevance through biocompatibility assessment using Trypan Blue exclusion, a proxy for membrane integrity in mammalian cells.
- Enterprise Reuse: Establishes a modular platform where MWCNT functionalization and metal deposition can be adapted to other metals or targeting ligands for broadened application in antimicrobial or antibiofilm programs.
Operational & Enterprise Impact
- Scientific Value: Provides mechanistic insight into nanocomplex-mediated antibacterial activity while minimizing false positives from assay interference.
- Operational Value: Ensures reproducibility through standardized purification (acid wash, ethanol rinse, pH monitoring) and stabilization (freeze-drying under vacuum).
- Strategic Value: Informs risk-benefit profiling by decoupling antimicrobial potency from cytotoxicity, supporting safer candidate selection.
- Portfolio Impact: Enables data-driven advancement decisions based on concentration-dependent efficacy and safety thresholds observed in parallel assays.
Implementation Considerations
- Requires expertise in nanomaterial handling, sonication protocols, and sterile microbiological techniques.
- Depends on access to centrifugation equipment (12,000 G), sonicators (60–80°C, 160W), and freeze-dryers (-60 to -70°C under vacuum).
- Necessitates standardization across teams for dispersion protocols, particularly for achieving consistent nanoparticle deposition and avoiding aggregation.
- Involves adaptation considerations when shifting from Methylobacterium to other bacterial strains or biofilm models, requiring re-optimization of inoculation and incubation parameters.
- Practical limitation: The dark color of carbon nanotubes necessitates alternative assays (zone of inhibition, Live/Dead, Trypan Blue) over colorimetric methods like MTT, increasing assay complexity.
Biopharma FAQs
Chapters in this video
0:04
Title
0:39
Multi-walled Carbon Nanotube (MWCNT) Oxidation
2:34
Silver Nanoparticle Deposition on MWCNTs
4:59
Zone of Inhibition Test
7:35
Antibacterial Test
9:20
Results: Characterization and Evaluation of Antibacterial Activity and Cytotoxicity of Ag-MWCNTs
10:50
Conclusion
