Langmuir：单壁碳纳米管能杀灭大肠杆菌
To further verify the antimicrobial activity of SWNTs and the validity of the area-based determination of microbial viability, we prepared a SWNT-coated filter (details in Supporting Information). E. coli cells were gently filtered so that they would accumulate on the top of the SWNT-coated filter and could be enumerated as individual cells. Results showed that 86.8 ± 6.8% of the cells (number-based) on the SWNT-coated filter were stained with PI after the 60 min incubation (Figure 2d). This finding is in agreement with the previously described results of cells interacting with the suspended SWNT aggregates. The results also confirm that direct contact between E. coli and SWNTs is necessary for the inactivation of the model bacteria.
To further confirm the antimicrobial activity of SWNTs based on PI staining (indicative of cells with damaged or compromised membranes), we assessed the metabolic activity of cells exposed to SWNTs with 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC).19 We found that only 6.6 ± 4.7% of cells were stained with CTC (i.e., metabolically active) whereas the majority of cells (74.0 ± 7.3%) on the PVDF membrane with no SWNTs (serving as a control) were metabolically active.
We then determined changes in the morphology of E. coli cells exposed to SWNTs. The scanning electron microscopy (SEM) images showed that the cells in the control (without SWNTs) were intact and maintained their outer membrane structure (Figure 3a). In contrast, cells that were incubated with SWNTs lost their cellular integrity (Figure 3b). The morphological change of SWNT-treated cells is attributable to cell membrane damage as discussed below.
Figure 3 Scanning electron microscopy (SEM) images of E. coli. (a) Cells incubated without SWNTs for 60 min. Cells were filtered and observed via SEM on the filter. (b) Cells incubated with SWNTs for 60 min.
On the basis of the fluorescence dye tests (Figure 2) and SEM images (Figure 3), we conclude that E. coli cells in contact with SWNT aggregates or an SWNT-deposited layer suffered membrane damage that likely resulted in compromised membrane permeability. The apparent cell damage should have led to an efflux of cytoplasmic materials into the solution. We proved the efflux of cytoplasmic materials by two independent measurements, namely, the concentrations of DNA and RNA in solutions of E. coli cells incubated with and without SWNTs. Even without taking into account the adsorption of plasmid DNA and RNA to SWNTs, we measured more than a 5-fold increase of plasmid DNA and a 2-fold increase of RNA in solutions in contact with SWNTs. The significant increase in the concentration of plasmid DNA and RNA in the presence of SWNTs confirms the severe damage to cell membrane integrity.
Although our results show that the E. coli undergoes severe membrane damage and subsequent loss of viability due to SWNTs, very little information is currently available with regard to the cytotoxic mechanisms of SWNTs. Previous studies, mainly focusing on mammalian cells, have proposed three principal cytotoxic mechanisms: oxidative stress,9 metal toxicity,20 and physical piercing.6 Of these, our results showed that metal toxicity was not an important mechanism of SWNT toxicity because we were effectively able to purify the SWNTs, resulting in residual metal catalyst (mainly Co) measuring less than 0.8 wt % (which is equivalent to 40 ppb). Several studies have proposed oxidative stress as a direct cause of membrane damage with carbon-based nanomaterials, which is caused by lipid peroxidation within the mammalian cell.8,9,12 However, these studies centered around the presence of residual metals within CNTs, which may lead to oxidative stress.21 In fact, recent studies suggest that the physical interaction of carbon-based nanomaterials (fullerene) with cells, rather than oxidative stress, is the primary killing mechanism.22,23
Several recent studies suggest that peptide or lipid nanotubes can penetrate through cell membranes because of their cylindrical shape and high aspect ratio and lead to cell death.24,25
In summary, our study provides the first direct confirmation that pristine SWNTs can exhibit strong antimicrobial activity. By using highly purified, pristine SWNTs with a narrow diameter distribution, we demonstrate that direct cell contact with SWNTs can cause severe membrane damage and subsequent cell inactivation. Our finding suggests that SWNTs can be useful as building blocks for antimicrobial materials.
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