The ability of yeast water extract to reduce silver ions in the reacting solution and formed the silver nanoparticles was monitored with UV–visible spectra, where specific surface plasmon resonance (SPR) should appear during nanoparticles formation29. SPR is the absorption of the visible electromagnetic radiation of the collective oscillations of surface electrons30. Indeed, the maximum absorption peak was observed at 420 nm, for both of the samples (Fig. 1A). This wavelength was identical as reported by Elamawi et al., who obtained silver NPs synthetized from the cell-free fungal extract of Trichoderma sp.31. And was also closely matched (410 nm) to those obtained from the cell-free filtrate of Aspergillus fumigatus32. According to the Mie’s theory only a single SPR band is expected in the absorption spectra of spherical metal nanoparticles33. We present that silver nanoparticles absorb blue light and exhibit one single peak, thus, they are spherical in shape. Moreover, morphology of the synthesized NPs was confirmed with transmission electron microscopy (Fig. 1B), where predominantly spherical shape was imaged.
Figure 1(A) UV–Vis absorption spectra of biosynthesized AgNPs_L (circle) and AgNPs_H (diamond). (B) Example of TEM image of AgNPs.
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For various bioapplications, physicochemical properties determine nanomaterial cellular uptake, transport and fate34. Considering this, here we evaluated some of the most important features of the nanomaterials, primarily the size, stability and surface chemistry of biomanufactured AgNPs. The dynamic light scattering (DLS) was used to study the size distribution and colloidal stability of AgNPs35. The synthetized silver nanoparticles presented a size with median value 20.1 nm and 17.5 nm, for the sample AgNPs_L and AgNPs_H, respectively (Fig. 2). The stability of the nanoparticles as colloid is very important, as unstable NPs will not be able to disperse homogenously, which may effect on their antibacterial properties and reducing the efficacy31,36. Therefore, the polidyspersity index (PdI) was used as a value which show the stability of the nanomaterial. The higher the PdI value is, the less monodispersed are the nanoparticles37. In this study, the PdI of the materials were equal to 0.107 and 0.397 after synthesis or 0.327 and 0.319, after 8 month storage, for the AgNPs_L and AgNPs_H, respectively (Fig. 2). Thus, this suggest the nanoparticles are stable as they do not exhibit any considerable aggregation. It is worth to mention that many papers reported on difficulties in the synthesis of stable solution of NPs due to their tendency to agglomerate38,39,40. According to Gorham et al. PdI of AgNPs increased due to oxidation-dependent processes. The authors reported that citrate-coated AgNPs are characterized by increasing of agglomeration level during 104-day storage, despite citrate use41. Similar effect was observed by Izak-Nau et al., who show that agglomeration of citrate-coated AgNPs can be delayed effectively about 6 months since being synthesized. After this time, the hydrodiameter size of NPs in tested solutions increased significantly42. Interestingly, our results clearly showed that using post-harvested yeast water during synthesis allow to obtain stable NPs solutions, as PdI is not increased significantly, even after 8-month-storage.
Figure 2Size of the AgNPs_L (A) and AgNPs_H (B) evaluated by hydrodynamic light scattering analysis. PdI, polidyspersity index.
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The morphology and elemental composition of the AgNPs were determined by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). Figure 3A depicts the HRTEM image of biosynthesized silver nanoparticles showing the lattice fringes clearly. The calculated inter planar distance was equal to 0.235 nm, confirmed occurrence of phase of Ag. According to the STEM-HAADF and EDS results (Fig. 3B-F) the synthesized nanomaterial is mainly constituted of Ag (Fig. 3D). Interestingly, the presence of sulfur precisely covering the nanoparticles was mapped (Fig. 3E). This may suggest that biocompounds reach in sulfur, which exists in the yeast water extract, may have the capping and stabilizing role. Many studies have revealed that the use of inorganic stabilizers such as citrate, PVP or PAA during synthesis allow to obtain stable NPs solutions43,44. However, some of them can influence negatively on human health e.g. PAA, which may cause the irritation of respiratory system after inhalation45. On the other hand, during the biological synthesis of nanoparticles, some biocompounds i.e. exopolysaccharides or proteins may exist as a stabilizing agent when nanoparticles are formed46,47. The sulfur which was imaged on Fig. 3E suggests the presence of some yeast proteins coating the surface of NPs. According to the above-mentioned literature, we suppose that during the synthesis, the Saccharomyces cerevisiae proteins or sulfur-rich biocompund coating the AgNPs surface and stabilize them, allowing to maintain stable while storage.
Figure 3(A) Representative HRTEM images of single silver nanoparticle with lattice fringes. Silver was identified by the inter-planes spacing d = 0.235 nm corresponding to the (111) plane of silver. Measurement of inter-planes spacing was done on FFT image. (B, C) HAADF STEM image of AgNPs with various magnification. (D–F) The chemical composition analysis of AgNPs. EDS elemental mapping images of silver (red), sulfur (blue), and an overlay of the Ag and S. All the EDS measurements are collected from image B (lower magn.) and C (higher magn).
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Antimicrobial potential of the silver nanoparticles is ascribed to their diverse mechanism of action, and it is believed as the multistep and multilevel process48. To evaluate this potential, the biosynthesized AgNPs (both of the tested variants) were analyzed against most pathogenic strains of bacteria: Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus and fungi Candida albicans, through the zone of inhibition assay. After 24 h of exposure, the growth was inhibited for each of the bacteria strains, whereas no inhibition was observed for yeast strain for both of the tested samples (Fig. 4). Kota et al. showed that AgNPs were characterized by high antibacterial properties, confirmed by analysis on sixteen pathological isolates from human, both Gram positive and Gram negative strains49. Moreover, the authors proved that 50 µg/µL concentration of these nanoparticles were able to increase zone of bacteria growth inhibition with the same efficiency in all tested isolates49. Interestingly, Jalal et al. showed high antifungal properties of AgNPs (extracellularly synthetized by C. glabrata supernatant) towards six Candida species50. Similarly results were presented by Perween et al., who reported on potential usefulness of AgNPs in C. albicans infections, better than well-known antifungal xenobiotics51. These conclusions are in the opposition of our results, which showed no effect on C. albicans growth inhibition of tested AgNPs solutions, which may be caused by higher diameter of tested AgNPs and/or sulfur coating of NPs surface.
Figure 4Antimicrobial activity of the AgNPs after 24 h of incubation against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans.
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Bacterial colonization on abiotic or biotic surfaces may leads to biofilm formation and these microbial aggregates in biofilms produce a blockade that resists antimicrobial agents48. Thus, due to extremely small sizes of NPs, they may be useful for accomplishing antimicrobial actions and fighting intracellularly with bacteria52. Herein, the antibiofilm efficacy of the silver nanoparticles was evaluated with the crystal violet staining assay, in a case of ability to inhibit biofilm formation. Different biofilm percentage of reduction was detected for inhibition biofilms when treated with different concentrations of AgNPs_L, however, in the concentration dependent manner. For the E. coli strain, the best reduction was achieved at the highest concentration 2 mg/mL, which causes reducing the OD biofilm from 1.3182 (control) to 0.2806 in the tested group (Fig. 5A). While P. aeruginosa exhibits decreasing of biofilm OD up to 0.1813 (control group 1.0831) after 24 h treatment of 2 mg/mL concentration of AgNPs_L (Fig. 5B). Various mechanisms of antibacterial properties of AgNPs are described in the literature. Among them the high level of ROS production and the failure to eliminate them by P. aeruginosa after silver NPs exposition was noticed53. Thus, it is suggest that AgNPs may become an antimicrobial agent on the multidrug-resistant strain, which is an ongoing problem in the medicine53. Similarly, our results showed the high antibacterial potential of AgNPs_L against E. coli and P. aeruginosa. Moreover, the presented results revealed the potential of tested AgNPs to prevent the creation of bacterial biofilm. Masurkar et al. highlighted that AgNPs were able efficiently to inhibit biofilm of Staphylococcus aureus, comparable to antibiotic-treated group54. Martinez-Gutierrez et al. presented a comprehensive report about negative impact of AgNPs on many clinically important bacteria strains i.e. S. aureus, S. epidermidis and A. baumanni which are considered to be problematic in hospital treatment55. Our results clearly showed that AgNPs synthetized with easy, cheap, fast and cost-effective way, may have high application value to treat pathogenic strains.
Figure 5Biofilm inhibition after treatment of AgNPs in E. coli (A) and P. aeruginosa (B). Strains were incubated for 24 h in the presence of AgNPs. Post-treatment surface-associated biofilm was stained and the OD of biofilm biomass were presented. Mean values with standard deviation (error bars) with *, **, ***are statistically different from the respective control at P < 0.05, P < 0.01, and P < 0.001, respectively (one-way ANOVA, Tukey test).
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The presence of bacterial biofilms is an emerging problem in nowadays hospital infections, due to high resistance of these structures on antibiotic56,57. Many xenobiotics were tested as a potential anti-biofilm agents, however the highest efficiency of biofilm eradication was proved by AgNPs58,59. Therefore, herein the efficiency of AgNPs ability to E. coli and P. aeruginosa biofilm eradication was evaluated (Fig. 6). In both tested bacterial strains, AgNPs caused decreasing in biofilm biomass. The highest eradication was observed for 1 mg/mL and 2 mg/mL concentrations for both tested strains and reached 65% and 53% in E. coli or 44% and 36% in P. aeruginosa, respectively (Fig. 6). The degradative effect of AgNPs in dose-dependent manner was observed, providing by higher eradication of established biofilm of 2 mg/mL and 1 mg/mL concentrations in comparison to 0.5 – 0.125 mg/mL concentrations of tested AgNPs (Fig. 6) E. coli established biofilm was more sensitive to AgNPs presence than the P. aeruginosa one (Fig. 6). Similar antibacterial effect on E. coli biofilm was also proved by Goswami et al., who showed the AgNPs (synthetized by tea leaf extract) were able to eradicate biofilm up to 100%, similar properties of this AgNPs were also observed for S. aureus biofilm60. Moreover, Ching-Yee et all. showed that citrate-coated AgNPs were characterized by high antibacterial properties against P. aeruginosa biofilm and caused its detachment61. Nevertheless, many literature data show that biofilm eradication ability of AgNPs is strict correlated with concentration – the higher AgNPs content, the more effective biofilm degradation62,63. The same tendency was observed in our results, proving that AgNPs usually act in dose-dependent manner and can be useful in treatment of antibiotic-resistant bacterial strains.
Figure 6Biofilm eradication after treatment of AgNPs in E. coli (A) and P. aeruginosa (B). Created biofilms were treated with AgNPs for 24 h and the percentage of biofilm eradication in comparison to untreated bacteria was calculated. Mean values with standard deviation (error bars) with *, **, ***are statistically different from the respective control at P < 0.05, P < 0.01, and P < 0.001, respectively (one-way ANOVA, Tukey test).
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The potential effect of AgNPs on cell metabolic activity was tested using MTT assay which measures the cell mitochondrial activity through NAD(P)H-dependent cellular oxidoreductase enzyme64. The toxicity of various concentrations of the silver nanoparticles (in the range of 0.125–2 mg/mL) toward four different cell lines: mouse embryonic fibroblasts (NIH 3T3), human keratinocytes (HaCaT), human osteosarcoma (U-2OS) and human non-small cell lung carcinoma (NCI-1299) is shown on Fig. 7. Generally, cell metabolic activity was decreased in a dose-dependent manner for human fibroblasts, keratinocytes and osteosarcomas (Fig. 7A-C). While, NCI-1299 cell line exhibits similar level of toxicity no matter on the concentration of the NPs (Fig. 7D). The highest significant (***P < 0.05) decrease was obtained at 2 mg/mL for each tested line, for both type of sample (AgNPs_L and AgNPs_H) compared to the nontreated cells. Comparing the results for cancer and normal cell lines, after their exposure toward two highest concentrations of AgNPs (2 and 1 mg/mL), it is shown that the cancer cells exhibit higher level of metabolic activity in the range from 48 to 73% (compared to control), respectively for U-2OS and NCI H1299 cell lines, contrary to normal cells (HaCaT and NIH 3T3), which metabolic activity was in the range from 37 to 43%, in comparison to control (Fig. 7). It is known that AgNPs are one of the most reactive metal nanoparticles65,66. The cytotoxicity effect of these nanostructures is correlated to ROS generation, after uptake inside the cell2. According to Kumari et all. cancer cell lines are more resistant to oxidative stress generation, due to their adapting ability67. Our results showed higher toxicity of AgNPs in normal keratinocytes and fibroblasts than in cancer ones, which confirm the higher resistance of these cell types to AgNPs-dependent oxidative stress (Fig. 7). On the other hand, Garvey et al. proved higher toxicity in lung carcinoma cells in comparison to normal human keratinocytes. However, the authors used chemically synthetized AgNPs citrate-coated, which are well-known of their high toxicity68. Capping agent attached to the surface of nanomaterial may have an impact on their biological activity. Indeed, our EDS results (Fig. 3D-F) showed that silver nanoparticles have been coated with sulfur-rich molecules, which act as a stabilizers. Therefore, we supposed these compounds decrease direct contact of high-reactive AgNPs surface with cells and decrease their toxic effect. This is in line with Senthil et al. who reported on the green synthetized AgNPs and showed less cytotoxicity effect on HaCaT cells, but higher antibacterial properties69.
Figure 7Cell metabolic activity of the NIH 3T3 (A), HaCaT (B), U-2OS (C), and NCI-1299 (D) after 24 h exposure to various concentration of AgNPs_L and AgNPs_H. The values are the means (n = 6) with standard deviation (error bars). The statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.005 according to one-way ANOVA, Tukey test.
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Many authors usually report on the cytotoxicity of the nanomaterials toward cells based on the only one method used for the analysis70,71,72,73,74. Considering only mitochondria activity, it may give the false positive results due to cells exhibit some activity even in early and late apoptosis stadium75. Therefore, in this study, either the AO/EB staining or MTT assays have been implemented to evaluate the ratio of the live and dead cells or metabolic activity, respectively. The dual acridine orange/ethidium bromide (AO/EB) staining assay was used to discriminate the live and dead cells after exposure to silver nanoparticles. Confluent cells were incubated with 0.5 and 1 mg/mL of AgNPs_L or AgNPs_H for 24 h and were labeled by AO/EB. For the higher NPs concentration (1 mg/mL), the number of dead cells was significantly increased for each type of cell line (Fig. 8A-D). Contrary, the number of live cells exposure to 0.5 mg/mL of AgNPs was still around 95%. Thus, this confirms the previous results and exhibits no or low toxicity in the range of 0.125–0.5 mg/mL of AgNPs. Representative images of the cells stained with AO/EB are shown on Fig. 8a-l, where red and green colors are dead and live cells, respectively. Based on this result, for further wound healing assay the AgNPs_L sample was used. Sambale et al. used the LDH release level as an indicator of AgNPs toxicity in lung carcinoma (A549) and proved that tested nanostructures did not significantly change the LDH level in the medium, highlighting that cytotoxicity effect of AgNPs is related to stabilizer and cells type76. Similarly, our results allow to concluded that the tested nanostructures (in the lower concentration of NPs) were more toxic for normal cell lines than for cancer ones.
Figure 8Cell viability of the NIH3T3 (A), HaCaT (B), U2OS (C) and NCI-1299 (D) after exposure to AgNPs_L and AgNPs_H. Cells after NPs treatment were stained with acridine orange and ethidium bromide, and were imaged with inverted fluorescence microscope (representative images—right panel). Dead cells were scored per 100 total cells analyzed and expressed as % (graphs).
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Cancer cell migration and invasion play a key role in disease progression77. Therefore, we further examine the impact of the silver nanoparticles on the behavior of the cancer and normal cells through the scratch assay. The motility capacities of the cells were performed on human keratinocytes and osteosarcoma cells. After 48 h either HaCaT or U2OS control cells (without AgNPs treatment) were able to migrate and close the scratch (Fig. 9). While, after exposure to AgNPs this ability was inhibited. Although, the migration ability of both of the tested cell line decreased after nanomaterial exposure, there is a difference among each type of cell. AgNPs inhibited more the migratory capability of the human osteosarcoma cell, compared to keratinocytes. After 48 h the % of scratch closure compare to time 0 was equal to 54% and 15% for normal and cancer cells, respectively. Many researchers emphasize on cytotoxicity of the metal NPs and highlight the migration of tumor cell and metastasis-related ability may be impacted by nanomaterials78. Herein, the strong inhibition efficacy of AgNPs on migration was observed in cancer cells, which were in line with other group79,80. Thus, this suggests that silver nanoparticles may have potential function in the inhibition of the metastasis.
Figure 9Cell migration ability in response to AgNPs. Comparison of migration in both HaCaT (A) and U-2OS (B) cell lines by taking images at different time intervals (0, 24, 48 h). Results are represented by marking the scratch with parallel lines and visually displaying the number of cells migrated in to the scratch area. The widths were measured using Image J software, and the data were analyzed using Prism 5.0. The values are the means with standard deviation (error bars). The statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.005 according to one-way ANOVA, Tukey test (compared to respective control), and #P < 0.05 are statistically different from respectively tested group (HaCaT vs. U-2OS).
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As can be seen from the obtained photomicrographs, the walls of the root canal are covered with silver nanoparticles (Fig. 10A). In addition, the penetration of nanoparticles into the dentin structure is observed. The depth of free penetration of silver nanoparticles in the dentinal tubule is about 20 μm (Fig. 10B), which is an extremely important experimental fact81. From a physical point of view, the dentin-nanoparticle system tries to reach thermodynamic balance. Nanoparticles tend to occupy a position that corresponds to their minimum energy costs. Such conditions have been found in the developed system of dentinal microcanals, penetrating and lingering in them at a certain depth. Thereby causing a deep bactericidal effect on the pathogenic microflora82. We observe that the size of silver inclusions distributed at different depths in the microtubules is preferably slightly larger than the stated 15 nm sizes of nanoparticles (Fig. 10B). This is due to their tendency over time to agglomerations and clustering. This fact can play another, no less important role. Agglomerates of nanoparticles, the size of which acquires the measurement of the diameter of the microtubules, reliably block the return of bacteria from the periphery of the dentin to the macrocanal of the root43,83. Thus, the binary action of nanoparticles actually significantly enhances the bactericidal effect of silver.
Figure 10The wall of the macrocanal covered with silver nanoparticles (A) and the micrograp of the penetration of silver nanoparticles into the dentinal canal (B) with agglomerates of nanoparticles (inset).
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The green synthesis of silver NPs is usually based on either the whole yeast cells or cell extract. In case of metal nanoparticles are formed with using yeast biomass, they can accumulate inside the cells in response to exposure to metal ions and additional steps are demand to extract the nanomaterials47,84. So far, there is lack of the data where the nanoparticles biomanufacturing is performed with the post-culturing water. In that way, the yeast biomass can be easy obtained as the waste product, and may be used many times for preparing the nanomaterials. Moreover, the low-cost of their production will take place in case of culturing in the huge bioreactors at the industrial scale, which does not require the complicated down-stream process for the recovery of the silver nanoparticles. What more, due to antibacterial effect these materials may be useful for various biomedical application, i.e. as antimicrobial and disinfect agent, or to prepare the antiseptic layers85,86,87. Simultaneously, the same nanomaterial, depends on the concentration used, could be a great platform for targeted drug delivery, as well as to combat with cancer cells88,89. Altogether, the presented method of AgNPs synthesis, is a simple, cost-effective and efficient approach to obtain the nanomaterials.
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