Noncytotoxic silver nanoparticles as a new antimicrobial strategy

Drug-resistance of bacteria is an ongoing problem in hospital treatment. The main mechanism of bacterial virulency in human infections is based on their adhesion ability and biofilm formation. Many approaches have been invented to overcome this problem, i.e. treatment with antibacterial biomolecules, which have some limitations e.g. enzymatic degradation and short shelf stability. Silver nanoparticles (AgNPs) may be alternative to these strategies due to their unique and high antibacterial properties. Herein, we report on yeast Saccharomyces cerevisiae extracellular-based synthesis of AgNPs. Transmission electron microscopy (TEM) revealed the morphology and structure of the metallic nanoparticles, which showed a uniform distribution and good colloid stability, measured by hydrodynamic light scattering (DLS). The energy dispersive X-ray spectroscopy (EDS) of NPs confirms the presence of silver and showed that sulfur-rich compounds act as a capping agent being adsorbed on the surface of AgNPs. Antimicrobial tests showed that AgNPs inhibit the bacteria growth, while have no impact on fungi growth. Moreover, tested NPs was characterized by high inhibitory potential of bacteria biofilm formation but also eradication of established biofilms. The cytotoxic effect of the NPs on four mammalian normal and cancer cell lines was tested through the metabolic activity, cell viability and wound-healing assays. Last, but not least, ability to deep penetration of the silver colloid to the root canal was imaged by scanning electron microscopy (SEM) to show its potential as the material for root-end filling.


Material and methods
Reagents. Yeast  Microorganisms and cell lines. Saccharomyces cerevisiae (10,058/69 strain) were previously isolated from patient mouth, delivered by Department of Microbiology (Medical University of Wrocław) and genetically identified 20 . The YPD medium (10 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) was used for preculture and the yeasts were grown in flat-bottom flask at 28 °C with rotatory shaking (150 rpm). For antimicrobial assays, bacteria (Staphylococcus aureus ATCC 25,923, Pseudomonas aeruginosa ATCC 27,853, Escherichia coli PCM 2209) and fungi (Candida albicans ATCC 14,053) strains were employed.
Bacteria were precultured in LB medium at 37 °C with overnight shaking (150 rpm), while fungi in YPD medium at 30 °C. The cell lines used in this study were mouse embryonic fibroblasts (NIH 3T3, ATCC CRL-1658), human keratinocytes (HaCaT, ATCC CRL-2522), human osteosarcoma (U-2OS, ATCC HTB-96) and human non-small cell lung carcinoma (NCI-H1299, ATCC CRL-5803). They were grown and handled according to standard technique as described elsewhere 21 . Cultures were performed in 24-or 96-well plate (Corning) containing 1.5 mL and 200 µL of DMEM medium for wound-healing assay and MTT assay or AO/EB staining, respectively. Medium was supplemented with 1% of antibiotics solution and 10% of fetal bovine serum and cultures were incubated at 37 °C in a 5% CO 2 air saturated incubators. 24 h before the experiments, cells were seeded at a density 5 × 10 4 and 5 × 10 3 cells/well for 24-or 96-well plate, respectively.

Synthesis of AgNPs.
The Saccharomyces cerevisiae 10,058/69 strain was used for AgNPs synthesis due to their silver nitrate reduction properties. The production of AgNPs were performed with lower and higher concentration of AgNO 3 , 1 mM and 3 mM (for further analysis the abbreviations were as followed: AgNPs_L or AgNPs_H). Briefly, after 48 h of yeast culturing at optimal temperature 28 °C with shaking, the cell suspension, when reached the density atOD 600 ~ 14, was centrifuged. The obtained supernatant was removed, followed the yeast biomass was resuspended in sterile distilled water and cultured for next 48 h in 28 °C with shaking. The post-culturing water was collected by centrifugation and its pH was adjusted to 10. Consequently, the silver nitrate was added to the post-culturing water in final concentration 1 mM and 3 mM and placed into 60 °C for 24 h. The specific conditions during the synthesis (i.e. silver ions concentrations, time and pH of the synthesis) were chosen based on our preliminary results (data not shown). For the evaluation of the ability to penetrate the Physicochemical characterization of AgNPs. UV-Vis spectrum of AgNPs was recorded on TECAN spectrofluorometer (Infinite M200, Thermo Scientific) in the range of 330-900 nm. To determine the concentration of AgNPs, the microbalance technique using Radwag MYA 5.4Y balance was used. Briefly, 100 μL of the colloid suspension was placing on aluminum crucible with known mass and evaporating the solvent to a dry mass. Concentration values are given as mean and standard deviation of triplicate. The Dynamic Light Scattering (DLS) and the polydispersity index (PdI) were determined using the universal Nanoplus HD3 system (Particulate System/Micrometrics) equipped with 660 nm laser diode, as described elsewhere 22 . All the analysis were performed at 25 °C. Prior measurement, solutions of AgNPs were sonicated (310 W, 50 Hz, 100%, 10 min, Polsonic, SONIC-3). AgNPs were imaged with transmission electron microscopy (FEI Tecnai Osiris as described elsewhere 23 . Antimicrobial potential of AgNPs. The antimicrobial of synthesized AgNPs activity through the zone of inhibition was tested against different pathogens such as S. aureus, P. aeruginosa, E. coli and C. albicans, according to a modified standard protocol 24 . Briefly, this modification consisted in punching 5-mm diameter wells in the nutrient agar instead of using a soaked disc. 100 µL of the overnight culture of the strains suspensions were spread onto the agar plates. When dried, 50 µL of AgNPs_1 and AgNPs_3 were aseptically transferred into separate wells. The plates were incubated at 30 or 37 °C for 24 h, for yeast and bacteria respectively. The inhibition zones surrounding the wells were recognized as the ability of the NPs to inhibit the growth of cells. For further analysis, P. aeruginosa and E. coli strains have been chosen to evaluate the influence of AgNPs on the bacteria ability to create biofilm in the presence of nanoparticles. Here, two type of assays have been implemented, to determine if the AgNPs can (i) prevent biofilm formation or (ii) reduce the final biofilm biomass 25 . (i) To evaluate the inhibition of biofilm formation the overnight bacteria inoculums (~ 10 8 CFU) were mixed with the colloid of AgNPs in the range of concentrations of 0.125-2 mg/mL. Cells were then incubated for 24 h at 37 °C without agitation, to allow biofilm formation. Plates were washed thrice with PBS from non-adhered cells and dried for 15 min at room temperature. Biofilms were stained for 15 min by adding 0.4% of crystal violet (CV) solution to each tested well. Then after, cells were washed with PBS to remove excess of the dye. Finally, the crystals were solubilized with 30% acetic acid, followed by absorbance measurement at 595 nm using a microplate readers (Infinite M200, Thermo Scientific). Results are reported relative to untreated biofilm biomass, as an OD values. (ii) Degradation of pre-established biofilm was tested as described above with some alterations. Cells were seeded in 96-well plate in LB without adding AgNPs and incubated at 37 °C for 24 h to allow biofilm creating. After this time, the wells were rinsed and replaced with fresh one, containing different concentrations of AgNPs. After 24 h the CV staining as described above was performed. The results were expressed as a percentage of biofilm degradation compare to non-treated cells.

Cytotoxicity of AgNPs.
Cell metabolic activity. The 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay was used as an indicator of metabolism activity level of the cell after AgNPs solutions treatment. The assay was performed according to 23 . Briefly, cells were seeded on 96-well plate at density 5 × 10 3 cells/well, 24 h before the experiment. After this time, the medium was removed and replaced with the fresh one, containing different concentrations of AgNPs colloid (1 mM and 3 mM of precursor variants) in the range of 0.125-2 mg/ mL. Cells were cultured for 24 h at 37 °C in a humidified incubator in a 5% of CO 2 atmosphere. The medium was discarded from each well and 200 µL of fresh DMEM with MTT (0,5 mg/mL) was added into well. After an additional incubation of 2-3 h, to allow the formazans to form, 100 µL of DMSO were added per well to stop the reaction and dissolve crystals. The absorbance was finally measured at 565 nm wavelength on microplate reader (Tecan, Switzerland) and the metabolic activity results were expressed as a percentage of the control (untreated cells). All tests were performed at least in triplicate.
Cell viability. Examination of cell viability has been conducted by ethidium bromide (EB) and acridine orange (AO) staining to distinguish live and death cells, according to 26 . Briefly, confluent cells were incubated for 24 h in the presence of 0.5 or 1 mg/mL of AgNPs. After this time, plates were centrifuged (300 × g, 5 min) and supernatant was discarded. Cells were washed with PBS and were stained with a solution of acridine orange (100 μg/ mL in PBS) and ethidium bromide (100 μg/mL in PBS) at a volume ratio of 1:1 for 5 min. Under fluorescence microscope (Olympus, Japan) living cells were visualized as green while dead cell were stained in red. For cell viability determination, a total of 100 cells from image (at least six images from different wells were examined) were counted and dead cells were expressed as a percentage of the total number of the cells.
Wound-healing assay. The impact of AgNPs on cells migration were performed using wound-healing assay (scratch assay), according to 27 . Briefly, the cells were seeded at a density 5 × 10 4 cells/well in 24-well plate and incubated until reached 90% of confluence. Wells with confluent cells were scratched by using a P10 pipette tip in the diameter of the culture. Then after, the medium was removed, and the cells were washed with PBS to discard detached cells. Followed, the 0.5 mg/mL of AgNPs solution suspended in DMEM with 1% of FBS was added to each well and cultured for 24 and 48 h. Wound closures were periodically visualized (0, 24 and 48 h intervals) under inverted microscope. The ability of cell to migrate and wound closure after AgNPs treatment was calculated with using ImageJ software according to 28 . The results were expressed as a percentage of the scratch closure compared to scratch surface at 0 h time in each group. All tests were performed at least in duplicate. single-rooted human teeth with straight canals were taken, in which 2 μL of a solution of silver nanoparticles were injected. The tooth samples were prepared in the longitudinal direction of the root canal to visualize the lumen of the dentinal tubules. The study was performed using an electron scanning microscope (ESEM XL30, Philips, Netherlands).

Statistical analysis.
The results represent the mean ± SD from at least three independent experiments.

Results and discussion
Physicochemical characterization of AgNPs. 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 formation 29 . SPR is the absorption of the visible electromagnetic radiation of the collective oscillations of surface electrons 30 . 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 fumigatus 32 . According to the Mie's theory only a single SPR band is expected in the absorption spectra of spherical metal nanoparticles 33 . 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.
For various bioapplications, physicochemical properties determine nanomaterial cellular uptake, transport and fate 34 . 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 AgNPs 35 . 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 efficacy 31,36 . 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 nanoparticles 37 . 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 agglomerate [38][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 use 41 . 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 significantly 42 . 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. www.nature.com/scientificreports/ 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 solutions 43,44 . However, some of them can influence negatively on human health e.g. PAA, which may cause the irritation of respiratory system after inhalation 45 . 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 formed 46,47 . 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  Antimicrobial effect of AgNPs. Zone of inhibition. Antimicrobial potential of the silver nanoparticles is ascribed to their diverse mechanism of action, and it is believed as the multistep and multilevel process 48 .
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 strains 49 . 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 isolates 49 . Interestingly, Jalal et al. showed high antifungal properties of AgNPs (extracellularly synthetized by C. glabrata supernatant) towards six Candida species 50 . Similarly results were presented by Perween et al., who reported on potential usefulness of AgNPs in C. albicans infections, better than well-known antifungal xenobiotics 51 . 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.
Inhibition of biofilm formation. Bacterial colonization on abiotic or biotic surfaces may leads to biofilm formation and these microbial aggregates in biofilms produce a blockade that resists antimicrobial agents 48 . Thus, due to extremely small sizes of NPs, they may be useful for accomplishing antimicrobial actions and fighting intracellularly with bacteria 52 . 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 noticed 53 . Thus, it is suggest that AgNPs may become an antimicrobial agent on the multidrug-resistant strain, which is an ongoing problem in the medicine 53 . 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   Biofilm eradication. The presence of bacterial biofilms is an emerging problem in nowadays hospital infections, due to high resistance of these structures on antibiotic 56,57 . Many xenobiotics were tested as a potential anti-biofilm agents, however the highest efficiency of biofilm eradication was proved by AgNPs 58,59 . 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 biofilm 60 . Moreover, Ching-Yee et all. showed that citrate-coated AgNPs were characterized by high antibacterial properties against P. aeruginosa biofilm and caused its detachment 61 . Nevertheless, many literature data show that biofilm eradication ability of AgNPs is strict correlated with concentration -the higher AgNPs content, the more effective biofilm degradation 62,63 . The same tendency was observed in our results, proving that AgNPs usually act in dosedependent manner and can be useful in treatment of antibiotic-resistant bacterial strains.
Biological activity. Cell metabolic activity and viability. 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 enzyme 64 . 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 nanoparticles 65,66 . The cytotoxicity effect of these nanostructures is correlated to ROS generation, after uptake inside the cell 2 . According to Kumari et all. cancer cell lines are more resistant to oxidative stress generation, due to their adapting ability 67 . 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 toxicity 68 . 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 properties 69 . Many authors usually report on the cytotoxicity of the nanomaterials toward cells based on the only one method used for the analysis [70][71][72][73][74] . Considering only mitochondria activity, it may give the false positive results due  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). www.nature.com/scientificreports/ to cells exhibit some activity even in early and late apoptosis stadium 75 . 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 type 76 . 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.

Scientific
Wound healing assay. Cancer cell migration and invasion play a key role in disease progression 77 . 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 nanomaterials 78 . Herein, the strong inhibition efficacy of AgNPs on migration was observed in cancer cells, which were in line with other group 79,80 . Thus, this suggests that silver nanoparticles may have potential function in the inhibition of the metastasis.
Penetration of the root canal of the tooth by AgNPs. 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 fact 81 . 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 microflora 82 . 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, 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 nanomaterials 47,84 . 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 layers [85][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 cells 88,89 . Altogether, the presented method of AgNPs synthesis, is a simple, costeffective and efficient approach to obtain the nanomaterials.

Conclusions
The presented work was focused on the physicochemical and biological characterization of biosynthesized silver nanoparticles. It was found that manufactured AgNPs had spherical shape with the size range between 17-20 nm, depends on the concentration of the silver ions. TEM, DLS and PdI analysis pointed out that AgNPs were administered as a stable and a well dispersed single particles suspension. The EDX map of sulfur located around the nanoparticles suggests that some proteins or sulfur-rich biocompund coat the AgNPs surface and stabilize them. A series of assays with bacteria and fungi strains have confirmed the antimicrobial activity of the AgNPs. Importantly from the potential biomedical application, beside the pronounced antibacterial activity, the reduced cytotoxic effect towards mammalian somatic and tumoral cells was confirmed. Moreover, the ability to deep penetration of the silver colloid to the root canal, imaged by SEM, highlight its potential as the material for root-end filling. 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).