Evaluation of Antibacterial and Cytotoxicity Properties of Silver Nanowires and Their Composites with Carbon Nanotubes for Biomedical Applications.

In this work, we prepared silver nanowires (AgNWs) via the polyol method in the presence or absence of single wall carbon nanotubes (CNTs) and tested their physicochemical, antibacterial and cytotoxic properties. Results showed that the introduction of CNTs lead to the formation of AgNWs at lower temperature, but the final product characteristics of AgNWs and AgNWs-CNT were not significantly different. AgNWs exhibited antibacterial properties against all the studied bacterial species via the formation of oxygen reactive species (ROS) and membrane damage. Furthermore, AgNWs exhibited a dose-dependent and time-dependent toxicity at concentrations ≥ 10 µg/mL. Fibroblasts appeared to be more resistant than human colorectal adenocarcinoma (Caco-2) and osteoblasts to the toxicity of AgNWs. The cytotoxicity of AgNWs was found to be related to the formation of ROS, but not to membrane damage. Overall, these results suggest that AgNWs are potential antibacterial agents against E. coli, S. aureus, MRSA and S. saprophyticus, but their dosage needs to be adjusted according to the route of administration.

Different concentrations of freeze dried AgNWs, AgNWs-CNT and AgNWs-CNT-mix (0.1, 1, 1.5 mg/ml) were suspended in a 1 %w/v chitosan solution in lactic acid 0.1 M. The samples were vortex mixed for approximately 2 minutes, followed by sonication for a further 90 minutes. Each sample was then added drop wise on to SEM stubs. The samples were allowed to dry, under vacuum, for 48 hours, before being coated with gold, using a sputter coater (Polaron e500, Quoram Technologies, UK) and analyzed via SEM imaging (JEOL JSM-6060 LV Scanning Electron Microscope, UK). The images obtained are shown in Fig. S1. SEM images were analysed using ImageJ, each full wire present in the images was measured 3 times to determine the diameter and the length and size distribution graphs were obtained (Fig S2).

Effect of CNTs and AgNWs-CNT-mix on bacterial growth curves
CNTs and AgNWs-CNT-mix were used as control samples. CNTs (Fig. S3) did not have any effect on the growth of bacteria, while AgNWs-CNT-mix had some effect only at the highest concentration tested (

Effect of silvernanowires on lag time, rate of growth and stationary phase of bacteria
Bacterial growth curves were analyzed to determine the effect of silver nanowires on the lag time, rate of growth and maxium bacterial concentration at the stationary phase (Fig. S5).

Figure. S5
. Bacterial growth lag time (A, B); time to establish stationary phase (C, D) and max absorbance at stationary phase (E, F) of bacterial cultures treated with different concentrations (blue 100; green 50; red 25; yellow 12.5 and black 0 µg/ml) of AgNWs (A, C, E) and AgNW-CNTs (B, D, F). Data are reported as average ± S. D. (n=3). One way Anova was performed for all parameters for each bacteria, Tukey post-hoc test was also performed, symbols indicate significant difference £ compared to control; # compared to 25; *compared to 50; $ compared to 100 µg/ml

Confocal imaging of bacterial cells treated with silver nanowires
E. coli cultures were treated for 6 h with either AgNWs or AgNWs-CNTs (25 µg/ml), and then stained with the Live/Dead BacLightTM staining kit (Molecular ProbesTM, Life technologies), according to the manufacturer's instructions. Briefly, bacteria pellets were obtained by centrifugation, the supernatant was discarded and the pellet was resuspended in 1 ml of PBS (pH 7.4). Then, equal volumes (3 µl) of dye component A and B were added to 1 ml of bacterial suspension, and incubated in the dark at room temperature for 15 minutes. Finally, 5 µl of the stained bacterial suspension was placed on a microscope slide and covered with a coverslip; images were acquired with a fluorescence microscope (EVOS fl, AMG, Westover Scientific, Inc.). The fluorescence images of the control showed numerous viable green colored E. coli cells. On the contrary, cells treated with AgNWs showed in proportion more red staining. Moreover, as already shown by growth curves analysis, AgNWs appeared to interfere with the bacterial reproduction, leading to a decreased number of cells in the samples.

Morphological analysis in bacteria treated with AgNWs and AgNWs-CNT by SEM imaging
Bacterial suspensions were prepared as described in 4.3.1. Then, bacteria were diluted to an OD600 of 0.01 and they were treated with different concentrations of nanoparticles (50 and 800 µg/ml) and incubated for 8 hours. The experiment was carried out in 96 well plates (150 µl). The samples were then aseptically removed and centrifuged at 5000 x g for 10 min, the medium was removed and 800 µl of 0.2 M cacodylate was added to suspend the pellet. Then, 50 µl of cell suspension was added to a sterile silicon wafer (shiny side up) (Agar Scientific) and it was allowed to settle for 40 min. Glutaraldehyde (0.5 ml, 2%) in 0.2 M cacodylate was gently added to each silicon wafer, making sure the specimens were completely immersed. The samples were fixed for 1 hour, at 4°C. Then, the samples were washed once with buffer and were dehydrated through a series of ethanol solutions (30, 50, 70, 90, 95 and 100%) for 5 min each at room temperature. Finally, samples were covered with acetone (until evaporation) and in 100% hexamethyldisilazane (HMSD) before being allowed to air-dry overnight. Images were taken by a scanning electron microscope (Zeiss EVO MA10), after having sputter coated the samples with gold and palladium (Polaron e500, Quoram Technologies, UK). Images of the 4 bacterial strains were acquired by SEM (Fig. S8) to detect potential morphological changes, 8h after treatment with AgNWs and AgNWs-CNT. AgNPs were noted to deposit on the surface of bacteria and with cell surface damage evident. The ability of AgNPs to cause big gaps formation in E. coli membrane has already been shown by Das et al. [1].

Mitochondrial membrane potential
Mitochondrial membrane potential (ΔΨm) was measured using a mitochondrial permeability detection kit, containing JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) (Abcam, Cambridge, UK). When the mitochondrial membrane (MM) is hyperpolarized, JC-1 enters the mitochondria and aggregates yielding a red colored emission (ca. 590 nm), whereas when the MM is depolarized, JC-1 is mainly a monomer that yields green fluorescence (ca. 529 nm). Briefly, 1,500 cells/well were plated in black 96 well plates and allowed to attach overnight. The day after, cells were treated with the nanomaterials for 24 h. Then, they were rinsed three times with PBS and stained with 20 µM JC-1 for 20 min at 37°C. Finally, the cells were washed twice with PBS and the fluorescence was measured (ex 475 nm; em 530 and 590 nm). Figure S10. Mitochondrial membrane potential of osteoblasts, fibroblasts and Caco-2 cells treated with AgNWs, Ag-NWs-CNT and CNTs at different concentrations. Data are reported as mean ± SD (n=3). Data were analyzed by one-way Anova and Tukey's multicomparison test.
Loss of mitochondrial transmembrane potential (ΔΨM) is a significant signal of early phase apoptosis. Healthy mitochondria generate an electrochemical proton gradient across their membrane to produce ATP. On the other hand, apoptotic mitochondria present opened permeability pores and loss of electrochemical gradient. In our study, we have used a lipophilic dye, called JC-1, which is an indicator of the MM's condition, generating a fluorescent green signal in normal cells and a red one in injured ones. As reported by Sivandzase et al. [2], the red/green fluorescence ratio of the dye in the mitochondria can interpreted as a direct assessment of the health of the mitochondria: the higher the red/green ratio, the healthier the cells and vice-versa. In this study, results indicate that both AgNWs and AgNWs-CNT incubation for 24 hours reduced the MMP for all the cell types in a dose-dependent manner. These findings are in good agreement with previous literature that has highlighted a connection between an increased ROS production (by silver nanowires, too) with mitochondrial membrane damage [3,4]. Therefore, the results suggest that the production of ROS by AgNWs could have triggered the formation of MM pores.