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 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.
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.
1. MWCNT Oxidation
2. Silver Nanoparticle Deposition on MWCNTs
3. Zone of Inhibition Test
4. Antibacterial Test
5. Viability Assay
6. Trypan Blue Assay
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° 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 µg/mL Ag-MWCNT (Figure 2D) and 40 µg/mL Ag-MWCNT (Figure 2E) samples. In the 20 µg/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 µg/mL was added. A zone of inhibition was not observed at a concentration of 1 µg/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 µg/mL Ag-MWCNTs (Figure 4C) revealed some cytotoxicity, however the addition of 30 µg/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 µg/mL Ag-MWCNTs (Figure 5C) and 30 µg/mL Ag-MWCNTs (Figure 5D) samples. The results confirmed that there was very little cytotoxicity at 30 µg/mL Ag-MWCNTs.
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. Please click here to view a larger version of this figure.
Figure 2: Antibacterial evaluation of Ag-MWCNTs relative to Methylobacterium spp. (A) control, (B) methanol, (C) MWCNT-COOH, (D) 50 μg/mL of Ag-MWCNTs, (E) 40 μg/mL of Ag-MWCNTs, and (F) 30 μg/mL of Ag-MWCNTs. Please click here to view a larger version of this figure.
Figure 3: Zone of inhibition test of Ag-MWCNTs.
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 μg/mL of Ag-MWCNTs, and (D) 30 μg/mL of Ag-MWCNTs. Please click here to view a larger version of this figure.
Figure 5: Trypan blue assays for 30 and 40μg/mL Ag-MWCNTs. (A) control, (B) methanol, (C) 40 μg/mL of Ag-MWCNTs, and (D) 30 μg/mL of Ag-MWCNTs. (Red: dead cells; blue: live cells) Please click here to view a larger version of this figure.
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 µg/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 µg/mL sample of Ag-MWCNTs was estimated as 0.4 µg/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 µg/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 authors have nothing to disclose.
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).
0.1 N silver nitrate | SIGMA-ALDRICH | 1090811000 | |
Carbon nanotube, multi-walled | Tokyo Chemical Industry Co., LTD | 308068-56-6 | |
R2A agar | MBcell | MB-R1129 | |
R2A broth | MBcell | MB-R2230 | |
Methylobacterium spp. | KCTC | 12618 | from Korea Collection for Type Cultures Daejeon Korea 12618, Daejon, Korea |
LIVE/DEAD Cell imaging Kit | ThermoFisher SCIENTIFIC | R37601 | |
AML12 | from Chungnam University, Dajeon, Korea | ||
human PBMC | ATCC | PCS-800-011 | |
TEM | JEOL | JEM-2100F | |
XRD | Rigaku | D/MAX 2500 | Cu K photon source (40kV, 100mA) |
JuLI Br | NanoEnTek | JULI-BRSC |