Biomaterials 30 (2009) 6333–6340
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Antifungal activity of silver nanoparticles against Candida spp. Alesˇ Pana´cˇek a, Milan Kola´rˇ b, Renata Vecˇerˇova´ b, Robert Prucek a, Jana Soukupova´ a, Vladimı´r Krysˇtof c, Petr Hamal b, Radek Zborˇil a, Libor Kvı´tek a, * a
University, Olomouc 771 46, Czech Republic Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc 775 20, Czech Republic Department Microbiology, Faculty of Medicine and Dentistry, Palacky c University, Olomouc 771 46, Czech Republic Laboratory of Growth Regulators, Faculty of Science, Palacky b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2009 Accepted 30 July 2009 Available online 20 August 2009
The antifungal activity of the silver nanoparticles (NPs) prepared by the modified Tollens process was evaluated for pathogenic Candida spp. by means of the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and the time-dependency of yeasts growth inhibition. Simultaneously the cytotoxicity of the silver NPs to human fibroblasts was determined. The silver NPs exhibited inhibitory effect against the tested yeasts at the concentration as low as 0.21 mg/L of Ag. The inhibitory effect of silver NPs was enhanced through their stabilization and the lowest MIC equal to 0.05 mg/L was determined for silver NPs stabilized by sodium dodecyl sulfate against Candida albicans II. The obtained MICs of the silver NPs and especially of the stabilized silver NPs were comparable and in some cases even better than MICs of the conventional antifungal agents determined by E-test. The silver NPs effectively inhibited the growth of the tested yeasts at the concentrations below their cytotoxic limit against the tested human fibroblasts determined at a concentration equal to 30 mg/L of Ag. In contrast, ionic silver inhibited the growth of the tested yeasts at the concentrations comparable to the cytotoxic level (approx. 1 mg/L) of ionic silver against the tested human fibroblasts. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Silver Nanoparticles Antifungal Fungicidal Yeasts Candida spp.
1. Introduction In recent years, severe fungal infections have significantly contributed to the increasing morbidity and mortality [1] of immunocompromised patients who need intensive treatment including broad-spectrum antibiotic therapy [2,3]. Candida spp. represent one of the most common pathogens which are responsible for fungal infections often causing hospital-acquired sepsis with an associated mortality rate of up to 40% [4]. Currently most of the available effective antifungal agents are based on polyenes (amphotericin B), triazoles (fluconazole, itraconazole, voriconazole, posaconazole) or echinocandins (caspofungin, micafungin and anidulafungin). However, administration of these antifungals is often accompanied by various complications such as amphotericin B toxicity and adverse effects of some azoles including toxicity and drug interactions [5–8] and yeast resistance to antifungal therapy [9,10]. Due to that, other options for effective antifungal therapy must be found to avoid the above-mentioned adverse effects. Silver nanoparticles (NPs), exhibiting very strong bactericidal activity against both gram-positive and gram-negative bacteria
* Corresponding author. Tel.: þ420 585634420; fax: þ420 585634425. E-mail address:
[email protected] (L. Kvı´tek). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.07.065
including multiresistant strains [11,12], can be considered as potential antifungal agent. The bactericidal effect of silver NPs as well as silver nanocomposites or silver nanoparticle-based materials has been intensively studied recently [13–17], mostly due to the growing bacterial resistance to common antibiotics. Antibacterial activity of silver-containing materials may help in reducing infections when treating burns [18] as well as in preventing bacterial colonization of prostheses and catheters [19,20]. The already published studies on bactericidal activity have proved that silver NPs kill bacteria at such low concentrations (units of mg/L) [11,14], which do not reveal acute toxic effects on human cells [21,22]. In addition, silver NPs have not been shown to cause bacterial resistance currently complicating antibiotic therapy of bacterial infections. This is presumably due to the fact that, unlike antibiotics, silver NPs do not exert their antibacterial effects in a single specific site but at several levels such as bacterial wall, proteosynthesis and DNA [12,14,23]. The antifungal effect of silver NPs has received only marginal attention and just a few studies on this topic have been published [13,24,25]. The only study dealing more specifically with their activity against clinical isolates and ATCC strains of Trichophyton mentagrophytes and Candida spp. was published as late as in 2008 [26]. However, it raises serious doubts about preparation of the dispersion of silver NPs and their physical and chemical properties.
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In that case, the results suggesting antifungal activity are not reliable as there is no guarantee that silver NPs were involved. This study is aimed on the determination of the fungistatic and fungicidal effects of the silver NPs against selected pathogenic yeasts causing invasive life-threatening fungal infections in intensive care patients at the University Hospital Olomouc, Czech Republic. Additionally, potential increase in the antifungal activity of the silver NPs by their stabilization using polymers and surfaceactive agents was studied. The obtained results were compared with those achieved by the use of common antifungal agents. 2. Materials and methods 2.1. Chemicals For the synthesis of silver NPs, silver nitrate (99.9%, Safina), ammonia (p.a., 25% [w/w] aqueous solution, Lachema), sodium hydroxide (p.a., Lachema) and D(þ)-maltose monohydrate (p.a., Riedel–de Hae¨n) were used. The prepared silver NPs were stabilized using the following substances supplied by Sigma–Aldrich: sodium dodecyl sulfate (SDS; >98%), polyoxyethylene sorbitan monooleate (Tween 80; >98%) and surfactants from the Brij group – polyoxyethylene (23) lauryl ether (Brij 35; >98%), polyoxyethylene (20) cetyl ether (Brij 58; >98%), polyoxyethylene (10) oleyl ether (Brij 97; >98%) and polyoxyethylene (20) oleyl ether (Brij 98; >98%). Besides the above-mentioned surfactants, the following polymers were used as stabilizers: polyvinylpyrrolidone (PVP) of the following average molecular weights: 10,000; 40,000 and 360,000 (PVP 10; PVP 40; PVP 360) and polyethylene glycols (PEGs) with molecular weights of 1500; 4000; 10,000 and 35,000 (PEG 1500, PEG 4000, PEG 10000, PEG 35000; p.a., Fluka). 2.2. Biological material Antimycotic activity was tested using Candida albicans (I and II), Candida tropicalis and Candida parapsilosis strains isolated from the blood of those patients at the University Hospital Olomouc who had confirmed Candida sepsis. The yeasts were identified using conventional mycological procedures: appearance on CHROMagar Candida (CHROMagar Microbiology), micromorphology on the rice agar and by assimilation and fermentation tests including the ID 32C kit (bioMe´rieux). To determine cytotoxicity of ionic silver and silver NPs, human fibroblast (BJ) cell lines were used. 2.3. Preparation and stabilization of silver NPs The synthesis of 25-nm-sized silver NPs with narrow size distribution was performed according to a previously published procedure using a modified Tollens reaction [27]. Briefly, the complex cation [Ag(NH3)2]þ was reduced by D-maltose in alkaline media (pH ¼ 11.5). The initial concentrations of the reaction components were 1.103 mol/L and 5.103 mol/L for AgNO3 and NH3, respectively. The concentration of the used NaOH and maltose was 1.102 mol/L. The reduction was performed at laboratory temperature (w25 C) at vigorous stirring and the process was completed after a reaction time of 5 min. The concentration of silver NPs prepared this way was 108 mg/L. The average size and size distribution of the prepared silver NPs were determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS (Malvern). The nanodimensions of the synthesized silver NPs were confirmed by transmission electron microscopy (Fig. 1A) using the JEM-2010 (Jeol) and by UV–Vis
absorption spectroscopy (Fig. 1B) with the Specord S 600 spectrophotometer (Analytik Jena AG). In the UV–Vis absorption spectra of the prepared silver NPs, narrow surface plasmon absorption peak located at the wavelength of 420 nm indicates the presence of the nanometer-sized silver NPs. Subsequent stabilization of silver NPs was performed by adding the used stabilizer to the prepared dispersion of silver NPs in the final concentration of 1% (w/w). 2.4. Fungistatic and fungicidal assay The fungistatic activity of both non-stabilized and stabilized silver NPs was performed by the modified microdilution method which enabled to determine the minimum inhibitory concentrations (MICs) of the silver NPs inhibiting the growth of the tested C. albicans, C. tropicalis and C. parapsilosis strains [28]. The dispersion of the non-stabilized and stabilized silver NPs was diluted in microtiter plates by the culture medium (Mueller Hinton Broth, Difco, France) in a geometric progression, from 2 to 2048 times. The obtained concentrations of silver in the dispersion ranged from 54 mg/L to 0.05 mg/L (54; 27; 13.5; 6.75; 3.38; 1.69; 0.84; 0.42; 0.21; 0.1; 0.05 mg/L). In the case of the stabilized silver NPs, the initial concentration of the used stabilizers (1% w/w), when diluting the dispersion of silver NPs by the culture broth, was not maintained constant but it decreased gradually with the dilution of the silver NP dispersion. Therefore, the concentrations of the used stabilizers decreased to the values from 0.5% to 4.9 104% w/w. After the silver NP samples were diluted, a standard amount of the tested yeast was inoculated onto microtiter plates so that the inoculum density in the wells was equal to 106 CFU/ml. After 36-h incubation at 37 C, the MIC was recorded as the lowest concentration of the agent inhibiting the visible growth of microorganisms. The minimum fungicidal concentration (MFC) was determined by inoculating the contents of all wells of the testing plate onto a new microtiter plate with culture broth without silver NPs. Following 72-h incubation, the MFC was recorded as the lowest concentration of the tested agent inhibiting the visible growth of microorganisms. The time-dependency of the inhibition of the yeast growth by silver NPs was determined by photometry using the EL 808 reader (BioTek Instruments, Inc.). The microtiter plate was inoculated in the same manner as in the case of MIC determination. Immediately after inoculation, the microtiter plate was covered with a clear tape to prevent the culture medium from drying out. Over 72 h, at 1-h intervals and after 3-min shaking, the absorbance of the sample was measured at the wavelength of 630 nm. The MICs of the currently available antifungals in the isolated yeasts were determined by the E-test strips (AB Biodisk, Solna,, Sweden) placed on the RPMI agar with 2% glucose [29]. The effects of itraconazole, fluconazole, amphotericin B, voriconazole and caspofungin were tested. For comparison purposes, fungistatic and fungicidal activities were determined for ionic silver as well. For that, a stock solution of silver nitrate at a silver concentration of 108 mg/L was prepared. The silver nitrate solution was then diluted with culture broth in a microtiter plate to the concentrations equal to those that were used when diluting the dispersion of silver NPs. As a control, fungistatic and fungicidal activities of all stabilizers used for stabilizing silver NPs were tested. Pure stock solutions of the used stabilizers were prepared at a concentration of 1% w/w. 2.5. Cytotoxic assay The ability of ionic silver and silver NPs to inhibit cell growth was determined in vitro with BJ cell lines. The cells, cultured in Dulbecco’s Modified Eagle’s Medium (supplemented with 10% fetal calf serum, 4 mM glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin) in a humidified CO2 incubator at 37 C, were redistributed
Fig. 1. TEM image (A) and UV–vis absorption spectra (B) of the prepared silver NPs with the average size of 25 nm.
´ cˇek et al. / Biomaterials 30 (2009) 6333–6340 A. Pana into 96-well microtiter plates at the appropriate densities for their respective cell sizes and growth rates. After 12-h preincubation, the solution of ionic silver and dispersion of the silver NPs were added in order to get the final concentrations ranging from 54 mg/L to 0.01 mg/L. Following 72 h of incubation, the cells were treated for 1 h with Calcein AM, and live-cell fluorescence was measured at 485 nm/ 538 nm (ex/em) with the Fluoroskan Ascent microplate fluorometer (Labsystems). LC100 values, the concentrations of silver lethal to 100% of the cells, were determined from the dose–response curves. The cytotoxic effects of surfactant-stabilized silver NPs were not determined because the pure solutions of the tested surfactants proved to be highly cytotoxic. As the used cell lines had no cell walls they could not adequately resist changes in the surface activity caused by the presence of the used surfactants.
3. Results 3.1. Fungistatic and fungicidal activities of non-stabilized silver NPs The fungistatic activity of the silver NPs with an average diameter of 25 nm against the tested yeasts was determined by the standard microdilution method, with the silver NPs being diluted in Mueller Hinton Broth to the final concentrations of silver ranging from 54 mg/L to 0.05 mg/L. The obtained results showed that the non-stabilized silver NPs inhibited all of the tested Candida spp. at very low concentrations and the inhibition was dependent on the yeast species tested (Table 1). The lowest MIC of silver NPs, at the silver concentration of 0.21 mg/L, was obtained against C. albicans II. The growth of C. albicans I and C. tropicalis was inhibited at MICs equal to 0.42 mg/L and 0.84 mg/L, respectively. In comparison to those yeast species, C. parapsilosis was less sensitive, with the MIC value of the silver NPs reaching 1.69 mg/L. The antifungal activity of silver NPs was compared with that of ionic silver as a reference sample. The MICs of ionic silver against the tested yeasts were identical to those of silver NPs, with the exception of C. albicans II which was inhibited at higher concentration of ionic silver equal to 0.42 mg/L when compared with silver NPs. Simultaneously with the study of the fungistatic activity of silver NPs, their fungicidal activity against the tested yeasts was assessed. The obtained MFCs are considerably higher in comparison to MICs. Silver NPs killed all of the tested yeasts at the concentration of 27 mg/L. The MFCs of ionic silver, as a reference sample, were determined to be equal to 13.5 mg/L for all of the tested yeasts. 3.2. Fungistatic and fungicidal activities of stabilized silver NPs The silver NPs were stabilized by surfactants and polymers in order to increase their antifungal effects by increasing the aggregate stability of the silver NPs in the used culture medium. The tests of the antifungal activity of the stabilized silver NPs confirmed better antifungal effects following stabilization by both surfaceactive agents (SDS, Brij, Tween 80) and polymers with the exception of polyethylene glycols (Table 1). Considering polymers, the highest increase in antifungal activity was achieved when PVP 360 was applied. In this case, the MICs were lower by one or two dilution degrees 0.84 mg/L for C. parapsilosis and 0.42 mg/L for
Table 1 Minimum inhibitory concentrations of the non-stabilized and stabilized silver NPs against the tested yeasts. Tested yeasts
MIC (mg/L) Ionic silver
C. C. C. C.
albicans I albicans II parapsilosis tropicalis
0.42 0.42 1.69 0.84
Non-stabilized silver NPs
Stabilized silver NPs SDS
Tween 80
Brij
PVP 360
0.42 0.21 1.69 0.84
0.052 0.1 0.84 0.42
0.1 0.21 0.84 0.42
0.1 0.21 0.84 0.42
0.1 0.21 0.84 0.42
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C. tropicalis. In C. albicans I, the MIC decreased to 0.1 mg/L and in C. albicans II, the MIC remained the same as in the case of nonstabilized silver NPs. The NPs stabilized by other polyvinylpyrrolidones (PVP 10 and PVP 40) showed better antifungal effects by one dilution degree only against C. albicans I. Silver NPs stabilized by polyethylene glycols showed no increase in antifungal activity (data not shown). Unlike the polymer-stabilized nanoparticles, silver NPs stabilized by surface-active agents showed in all cases higher antifungal activity. The silver NPs stabilized by Tween 80 and agents from the Brij group revealed the identical MICs values as silver particles stabilized with PVP 360, i.e. lower by one or two dilution degrees. The most significant increase in the antifungal activity of the silver NPs stabilized by surfactant was observed for SDS stabilizer, with the MIC of 0.052 mg/L for C. albicans I, and MICs in other tested yeast decreased to one-half when compared with the MICs of the non-stabilized silver NPs (Table 1). Additionally, the impact of stabilization of the silver NPs by polymers and surfactants on their fungicidal effects was assessed. The stabilized silver NPs did not reveal any higher fungicidal activity when compared to the non-stabilized nanoparticles, with the only exception represented by SDS stabilization. The MFC of the SDS-stabilized silver NPs was much lower compared to non-stabilized silver NPs and was determined at a concentration equal to 3.38 mg/L of Ag.
3.3. Time-dependent growth inhibition of yeasts Antifungal activity of the silver NPs was also studied by means of the yeast growth observation and measurements of the optical density of the sample during the 72-h cultivation. The rate and extent of growth inhibition can be determined from the timedependency of the recorded growth of the tested yeasts. The growth inhibition of the yeasts by ionic silver and silver NPs, recorded as a function of time, suggested slight differences in antifungal activity between ionic silver and silver NPs (Fig. 2). To make the figure clear, growth inhibitions of the tested yeasts are not shown for all the silver concentrations used. Therefore, only representative concentrations of silver from the lowest used concentration (0.05 mg/L) to the first concentration leading to the continuous inhibition of growth of the tested yeasts during 72-h cultivation have been used. As seen from the growth inhibition rates in Fig. 2, ionic silver has a stronger inhibitory effect than silver NPs. For example, the rate of the growth inhibition of C. albicans I caused by ionic silver at concentration of 1.69 mg/L is higher than the one caused by silver NPs at the same concentration. Such stronger antifungal effects of ionic silver, when compared with silver NPs, were observed for all of the tested Candida spp., excluding only C. albicans II. The time-dependence study of the growth inhibition of yeasts confirmed higher sensitivity of C. albicans II to silver NPs than to ionic silver as determined by the standard microdilution method. PVP 360, Tween 80 and SDS revealed a positive effect on the antifungal activities of the silver NPs, which was also demonstrated by the study of the time-dependent inhibition of the yeast growth (Fig. 3). Increased rates of inhibition of yeast growth caused by surfactant-/polymer-stabilized silver NPs was manifested in all the tested concentrations of stabilized NPs. The growth curves in Fig. 3 suggest that the stabilized silver NPs, at the representative concentration of 0.1 mg/L, are better at inhibiting yeast growth than non-stabilized NPs at the same concentration. In addition, the graphs show that the growth inhibition rates in yeasts are comparable no matter whether ionic silver or the stabilized silver NPs are used.
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Fig. 2. Growth curves of C. albicans I (A), C. albicans II (B), C. parapsilosis (C) and C. tropicalis (D) inhibited by ionic silver (left side) and non-stabilized silver NPs (right side).
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Fig. 3. Comparison of yeast growth inhibited by ionic silver, non-stabilized silver NPs and silver NPs stabilized by SDS, Tween 80 and PVP 360 having the concentration of silver equal to 0.1 mg/L, against C. albicans I (A), C. albicans II (B), C. parapsilosis (C) and C. tropicalis (D).
4. Discussion The obtained results of the antifungal activity clearly reveal that the growth of yeasts is inhibited at concentrations as low as 0.21 mg/L using the non-stabilized silver NPs (against C. albicans II) and 0.05 mg/L for SDS-stabilized silver NPs (against C. albicans I). Based on the comparison of MICs of both stabilized and nonstabilized silver NPs with MICs of the selected clinically available antifungal agents (as determined by the E-test), silver NPs could be pronounced to have either comparable or even better effects (Fig. 4). Silver NPs stabilized by polymers and surfactants exhibited reasonably high antifungal activity as the result of their enhanced aggregate stability. Moreover, surfactant activity disrupts the cell walls of yeasts, which increases their sensitivity to silver NPs. The highest antifungal activity of the silver NPs was determined when PVP 360, Tween 80, Brij, and SDS stabilizers were used. Such results are in a good agreement with the previously published paper considering the impact of the stabilization of silver NPs on their antibacterial activity [30]. The antibacterial activity of the stabilized silver NPs was most significant when PVP 360, Tween 80, and especially SDS stabilizers were used, which correspond with extraordinary stabilizing ability of silver NPs. The results, including the impact of the performed stabilization of the silver NPs on their antimicrobial activity, clearly show that the antibacterial and antifungal activities are affected by aggregate stability of the silver NPs. Also, if surfactants are used for stabilizing silver NPs, better antifungal effects can be attributed not only to higher stability of silver NPs but also to the disruption of the cell walls by the surfactants. The best fungistatic properties were observed in the case of the SDS-stabilized silver NPs and particularly against C. albicans I and C. albicans II. Such enhancement of the fungistatic
activity of the silver NPs stabilized by SDS in comparison with other used surfactants results from the antifungal effect of the SDS stabilizer itself which was not proved for the other surfactants (Tween 80, the Brij group). Pure solution of SDS proved to inhibit the growth of all of the tested yeasts at concentrations up to 0.016%, equal to 64-fold dilution of the pure SDS solution. Unlike the nonstabilized silver NPs, the particles stabilized by SDS had very low values of MIC, decreasing from 0.42 mg/L to 0.052 mg/L in the case of C. albicans I. Such low values of MIC of the SDS-stabilized silver nanoparticles result from the synergistic effect of the fungistatic activities of silver NPs and the effect of the SDS stabilizer itself. The synergistic effect of the SDS stabilizer and silver NPs was also observed in the case of the fungicidal activity of silver NPs. Pure SDS solution showed fungicidal effects at concentrations up to 0.125% (4.3 103 mol/L), equal to 8-fold dilutions of the sample. The non-stabilized silver NPs showed fungicidal activity at the concentration of 27 mg/L, equal to 4-fold dilutions of the sample. The fungicidal effect of the SDS-stabilized silver NPs was observed at the 32-fold dilutions of the sample, i.e. the silver concentration of 3.38 mg/L (0.03% w/w SDS). The antifungal activity of SDS against Candida spp. has not been studied in such detail and the currently available literature sources do not offer any MFC values obtained by standardized testing methods. Probably only the work by Hrenovic et al., in which they determined the EC50 value of SDS equal to 3.03 104 mol/L against Saccharomyces cerevisiae, can be used for the qualitative comparison [31]. However, the testing method used for the evaluation of EC50 value does not correspond exactly with the conditions of the herein testing of the antifungal activity of SDS surfactant. Therefore the results published by Hrenovic et al. can be used only as a confirmation of the fungicidal properties of SDS against yeasts.
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Fig. 4. Comparison of MICs of non-stabilized and stabilized silver NPs with MICs of the commonly used antifungals against the tested isolates of Candida spp.
The fungicidal activity of the SDS-stabilized NPs results from the synergistic effect of the fungicidal activities of silver NPs and the SDS stabilizer. SDS penetrates the permeability of the cell wall and cytoplasmic membrane through it bonding on lipids and proteins already at low concentrations. This primarily realized action enables better accessibility of the silver NP into the cell, which results in more intensive toxic action. Higher concentrations of SDS proved to have a toxic effect itself, which is based on the disruption of the cell wall and cytoplasmic membrane resulting in the cell lysis. Except for the interaction with proteins and lipids present in the cell wall, SDS inhibits the activity of various enzymes such as ATPase activity of P-glycoprotein or lecitin/cholesterol acyltransferase [32]. Such a high fungicidal activity of the SDS-stabilized silver NPs against Candida spp. is comparable or even better than the fungicidal activity of some antifungals such as amphotericin B (MFC around 2–16 mg/L) [33], posaconazole (MFC around 8 mg/L) [34], itraconazole and voriconazole (MFC more than 10 mg/ L) [35] or caspofungin (MFC around 1 mg/L) [36]. A comparative study of the fungistatic and fungicidal activities of the two forms of silver (ionic and nanoparticulate) by means of time-dependency assay (rate of fungistatic activity) as well as by the determination of MFCs proved a higher antifungal effect of ionic silver than of the non-stabilized silver NPs. This is due to higher toxicity of ionic silver when compared to the silver NPs against eukaryotic cells as proved by cytotoxic assay. Ionic silver was cytotoxic against the tested eukaryotic cell lines at LC100 ¼ 1 mg/L whereas the silver NPs showed a cytotoxic effect only at concentrations higher than 30 mg/L. Similar results were obtained when the ionic and nanoparticulate silver toxic effects against Paramecium caudatum were tested in our previous study [37]. The ionic form of silver caused immediate death of this eukaryotic organism at the concentration equal to 0.4 mg/L while the silver NPs displayed an acute toxic effect against Paramecium spp. at concentrations higher than 25 mg/L [37]. However, the difference between the fungicidal activity of the ionic and nanoparticulate silver was not so remarkable as in the case of the toxic effects against the tested human fibroblasts and P. caudatum. The values of cytotoxic concentration of ionic silver (LC100 ¼ 1 mg/L), herein presented, is
10 times lower than minimum fungicidal concentration of ionic silver (MFC ¼ 13.5 mg/L) against the tested yeasts while the cytotoxic concentration and MFC of the silver NPs are not significantly different. These obtained results represent completely new view point on the development of the antifungal substances based on the silver compounds that have not been published so far. Similar relations between cytotoxicity and antibacterial activity of ionic and nanoparticulate silver have been already published for the nanoparticulate silver bone cement, which proved to be highly effective against multiresistant bacteria and was free of in vitro cytotoxicity [38]. The use of ionic silver-containing materials, as a fungicidal agent, represents a serious complication connected with high cytotoxicity of ionic silver against eukaryotic cells. On contrary, the cytotoxic effect of the silver NPs was not so pronounced as the effect of ionic silver. The cytotoxic concentration of the silver NPs to human fibroblasts was comparable to the concentration of minimum fungicidal concentration against the tested yeasts. Additionally, the fungistatic activity of the silver NPs was determined at the concentrations 100 times lower than their cytotoxic level and therefore the use of silver NPs as the antifungal agents is less complicated in terms of their cytotoxicity to human eukaryotic cells when compared to ionic silver. Comparing the antifungal activity of ionic silver with the activity of the stabilized silver NPs, the polymer- and surfactant-stabilized silver NPs show higher fungistatic and, in case of SDS-stabilized silver NPs, fungicidal activities. The stronger fungistatic effect of the stabilized silver NPs was confirmed by determination of the MICs using the microdilution method for all of the used stabilizers. The inhibition rate of stabilized silver NPs is comparable to that of ionic silver, as it can be seen from the time-dependency inhibition of yeasts growth. Comparing the fungicidal activities of the polymerand surfactant-stabilized silver NPs and ionic silver, lower MFCs were recorded only for SDS-stabilized silver NPs, as a result of both the stabilization effect and the synergistic fungicidal effect of silver NPs and SDS. Despite of the fact that the stabilized silver NPs display a stronger inhibiting effect against yeasts, when compared to the non-stabilized silver NPs, they do not exhibit stronger cytotoxic
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effects. The lethal concentrations of the polymer-stabilized silver NPs, against the tested human fibroblast were the same as those determined for the non-stabilized silver NPs, i.e. LC100 ¼ 30 mg/L. It means that the stabilization by polymers increases the inhibition effect of the silver NPs but the cytotoxic effects to human fibroblasts (this study) and against P. caudatum [37] remain unchanged. It was impossible to evaluate the cytotoxic concentration of the surfactants-stabilized silver NPs because the pure solutions of these stabilizing agents exhibit high cytotoxicity against the tested cell lines. These cell lines have no cell walls and are thus unable to counter the effects of the used surfactants effectively. The comparison of the antifungal activity of the silver NPs with their antibacterial activity clearly showed that the silver NPs inhibit yeast growth at lower concentrations than in the case of the bacterial growth inhibition. However, the inhibition effect of the silver NPs against bacteria and Candida spp. can be considered comparable no matter how evolutionary different both of the organisms are. The variability of the values of the inhibition activity of the silver NPs against bacteria can be ascribed to strain differences of the used bacteria. The difference in the sensitivity of the bacterial strains can be observed also in the case of the sensitivity against commonly used antibiotics [39,40]. Therefore the MICs of the silver NPs against bacteria were obtained values of 1.69 mg/L against Escherichia coli and also values several times higher (6.75 mg/L against Enterococcus faecalis). In the case of the tested Candida spp. the strain dependency of MIC of the silver NPs was comparably observed in the similar concentration extent (from 0.21 mg/L for C. albicans II up to 1.69 mg/L for C. parapsilosis) no matter that all of the tested Candida spp. were species related. However, the fungicidal activity of the silver NPs is significantly lower than their bactericidal effects. While the non-stabilized silver NPs killed yeasts at the concentration as high as 27 mg/L, the minimum bactericidal concentrations ranged from 1.69 mg/L to 6.75 mg/L, depending on the types of the tested bacteria [30]. The differences in the minimal bactericidal and fungicidal concentrations of the silver NPs probably result from the differences between the bacterial and yeast cell type. Due to their less complex structure, evolutionarily older prokaryotic types of bacteria are unable to fight the toxic effects of the silver NPs as effectively as the eukaryotic yeast cells that can resist higher concentrations of silver thanks to their better cell organization and structure, and better detoxification system. This selective toxic effect against prokaryotic and eukaryotic organisms has been already confirmed for the ZnO NPs. These particles proved to be toxic against prokaryotic organisms at the concentrations of units of mM/L while ZnO NPs had minimal effects on eukaryote human T-lyphocytes cell viability at concentrations toxic to tested bacteria [41]. 5. Conclusions Silver NPs exhibit high antifungal activity against pathogenic Candida spp. at the concentrations around 1 mg/L of Ag as was proved in this study. Antifungal activity of the silver NPs is comparable with those of ionic silver; however, ionic silver remains cytotoxic at those concentrations that inhibit the growth of the tested yeasts. On contrary, the silver NPs inhibit growth of the yeasts at very low concentrations that are comparable to those of common antifungals. Furthermore, the silver NPs exhibit no cytotoxic effects on human fibroblasts at these concentrations. Based on the previously reported high antibacterial activity and the herein proved antifungal activity, it can be concluded that the silver NPs constitute an effective antimicrobial agent against common pathogenic microorganisms. In the previously published papers concerning antifungal activity of silver NPs, the MICs were not as low as those reported in this systematic study.
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